Sunit K. Singh and Daniel Růžek: RNA Viruses and Retroviruses

Edited by
Sunit K. Singh and Daniel Růžek

Neuroviral
Infections
RNA Viruses and Retroviruses
Neuroviral
Infections
Edited by
Sunit K. Singh and Daniel Růžek
Boca Raton London New York
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Contents
Preface.......................................................................................................................ix
Acknowledgement......................................................................................................xi
Editors..................................................................................................................... xiii
Contributors.............................................................................................................. xv
Section I RNA Viruses
Chapter 1 Alphavirus Neurovirulence................................................................... 3

Katherine Taylor and Slobodan Paessler
Chapter 2 Neurological Chikungunya: Lessons from Recent Epidemics,

Animal Models, and Other Alphavirus Family Members.................. 21
Vincent G. Thon-Hon, Shiril Kumar, Duksha Ramful,
Stephanie Robin, Marie Christine Jaffar-Bandjee,
and Philippe Gasque
Chapter 3 Arenaviruses and Neurovirology........................................................ 39

Larry Lutwick and Jana Preis
Chapter 4 Bunyaviruses....................................................................................... 67
Patrik Kilian, Vlasta Danielová, and Daniel Růžek
Chapter 5 Human Coronaviruses: Respiratory Pathogens

Revisited as Infectious Neuroinvasive, Neurotropic, and
Neurovirulent Agents.......................................................................... 93
Marc Desforges, Dominique J. Favreau, Élodie Brison,
Jessica Desjardins, Mathieu Meessen-Pinard, Hélène Jacomy,
and Pierre J. Talbot
Chapter 6 Nonpolio Enteroviruses, Polioviruses, and Human

CNS Infections.................................................................................123
Anda Baicus and Cristian Baicus
v
vi Contents
Chapter 7 Neurovirulence of the West Nile Virus............................................. 149

Kim-Long Yeo and Mah-Lee Ng
Chapter 8 Murray Valley Encephalitis Virus.................................................... 167

Natalie A. Prow, Roy A. Hall, and Mario Lobigs
Chapter 9 Japanese Encephalitis Virus and Human CNS Infection.................. 193

Kallol Dutta, Arshed Nazmi, and Anirban Basu
Chapter 10 Tick-Borne Encephalitis.................................................................... 211

Daniel Růžek, Bartosz Bilski, and Göran Günther
Chapter 11 St. Louis Encephalitis....................................................................... 239

Luis Adrian Diaz, Lorena I. Spinsanti, and Marta S. Contigiani
Chapter 12 Powassan Virus................................................................................. 261

Laura D. Kramer, Alan P. Dupuis II, and Norma P. Tavakoli
Chapter 13 Neurological Dengue......................................................................... 289

Aravinthan Varatharaj
Chapter 14 Influenza Virus and CNS Infections................................................. 325

Jun Zeng, Gefei Wang, and Kang-Sheng Li
Chapter 15 Human Paramyxoviruses and Infections of the

Central Nervous System.................................................................... 341
Michael R. Wilson, Martin Ludlow, and W. Paul Duprex
Chapter 16 Rabies Virus Neurovirulence............................................................ 373

Claire L. Jeffries, Ashley C. Banyard, Derek M. Healy,
Daniel L. Horton, Nicholas Johnson, and Anthony R. Fooks
Chapter 17 Rubella Virus Infections................................................................... 395

Jennifer M. Best, Susan Reef, and Liliane Grangeot- Keros
Contents vii
Section II Retroviruses
Chapter 18 Human T-Lymphotropic Virus.......................................................... 431

Motohiro Yukitake and Hideo Hara
Chapter 19 Human Immunodeficiency Virus Neuropathogenesis...................... 457

Ritu Mishra and Sunit K. Singh
Preface
Neurovirology is an interdisciplinary field that represents a melding of virology, clini-
cal neuroscience, molecular biology, and immunology. Apart from clinical neurosci-
ence, neurovirology includes molecular virology, biochemical virology, diagnostic
virology, and molecular pathogenesis and is inextricably bound to the field of immu-
nology. Neurovirology became an established field within the past 30 years. Since
then, there has been a tremendous explosion of information related to viral infections
of the central nervous system, and, also, several new viruses have been discovered.
The aim of this book is to present an up-to-date overview on major RNA viruses
and retrovirus-mediated neuroviral infections to virologists, specialists in infectious
diseases, teachers of virology, and postgraduate students of medicine, virology, neu-
rosciences, or immunology. We hope that it will serve as a useful resource for all
others interested in the field of viral infections of the central nervous system.
An inclusive and comprehensive book such as this is clearly beyond the capacity
of an individual’s effort. Therefore, we are fortunate and honored to have a large
panel of internationally renowned virologists as chapter contributors, whose detailed
knowledge on viral neuroinfections have greatly enriched this book.
We conceptualized this book in two sections. Section I includes the major RNA
virus chapters and Section II concludes with the specific information pertinent to
individual major retroviruses and their diseases. Each chapter consists of a review
on the classification, epidemiology, clinical features, and diagnostic and therapeutic
approaches of one or a group of related viruses.
The professionalism and dedication of executive editor Barbara Norwitz and
senior project coordinator Jill Jurgensen at CRC Press greatly contributed to the
final presentation of the book. Our appreciations extend to our families for their
understanding and support during the compilation of this book.
Sunit K. Singh
Daniel Růžek
ix
Acknowledgment
This book is dedicated to a magnanimous group of virologists, whose willingness to
share their in-depth knowledge and expertise has made this extensive overview on
viral neuroinfections possible.
xi
Editors
Dr. Sunit Kumar Singh completed his bachelor’s degree
program from GB Pant University of Agriculture and
Technology, Pantnagar, India, and master’s degree program
from the CIFE, Mumbai, India. After receiving his mas-
ter’s degree, Dr. Singh joined the Department of Paediatric
Rheumatology, Immunology, and Infectious Diseases,
Children’s Hospital, University of Wuerzburg, Wuerzburg,
Germany, as a biologist. Dr. Singh completed his PhD
degree from the University of Wuerzburg in the area of
molecular infection biology. Dr. Singh has completed his
postdoctoral trainings at the Department of Internal Medicine, Yale University, School of
Medicine, New Haven, Connecticut, USA, and the Department of Neurology, University
of California Davis Medical Center, Sacramento, California, USA, in the areas of vector-
borne infectious diseases and neuroinflammation, respectively.
He has also worked as visiting scientist at the Department of Pathology, Albert
Einstein College of Medicine, New York, USA, Department of Microbiology, College
of Veterinary Medicine, Chonbuk National University, Republic of Korea; and the
Department of Arbovirology, Institute of Parasitology, Ceske Budejovice, Czech
Republic. Presently, he is serving as a scientist and leading a research group in the
area of neurovirology and inflammation biology at the prestigious Centre for Cellular
and Molecular Biology, Hyderabad, India. His main areas of research interest are
Neurovirology and Immunology.
There are several awards to his credit, including the Skinner Memorial Award,
Travel Grant Award, NIH-Fogarty Fellowship, and Young Scientist Award. Dr. Singh is
associated with several international journals of repute as associate editor and editorial
board member.
Dr. Daniel Růžek is a research scientist at the Institute of

Parasitology, Academy of Sciences of the Czech Republic,
and an assistant professor at the Department of Medical
Biology, Faculty of Science, University of South Bohemia.
He received his PhD in the field of molecular and cellular
biology and genetics from the Academy of Sciences of the
Czech Republic and the University of South Bohemia. He
had postdoctoral training at the Department of Virology
and Immunology, Texas Biomedical Research Institute
(formerly Southwest Foundation for Biomedical Research),
San Antonio, Texas, USA. His primary field is virology with research emphasis on
vector-borne viruses, especially tick-borne encephalitis virus, Omsk hemorrhagic
fever virus, dengue virus, West Nile virus, and so forth. In 2009, he was awarded with
a prestigious international Sinnecker–Kunz Award for young researchers.
xiii
Contributors
Anda Baicus Bartosz Bilski
Microbiology Department, University of Medical Sciences Poznań
“Cantacuzino” Poznań, Poland
University of Medicine and Pharmacy
“Carol Davila” Élodie Brison
Viral Enteric Infections Laboratory Laboratory of Neuroimmunovirology
National Institute of Research and Institut National de la Recherche
Development for Microbiology and Scientifique
Immunology Université du Québec
Bucharest, Romania Laval, Québec, Canada
Cristian Baicus Marta S. Contigiani

Department of Internal Medicine Laboratorio de Arbovirus
Colentina University Hospital Instituto de Virología “Dr. J. M.
University of Medicine and Pharmacy Vanella”
“Carol Davila” Universidad Nacional de Córdoba
Bucharest, Romania Córdoba, Argentina
Ashley C. Banyard Vlasta Danielová

Wildlife Zoonoses and Vector Borne Centre of Epidemiology and
Disease Research Group Microbiology
Department of Virology National Institute of Public Health
Animal Health and Veterinary Prague, Czech Republic
Laboratories Agency
Surrey, United Kingdom Marc Desforges
Laboratory of Neuroimmunovirology
Anirban Basu Institut National de la Recherche
Cellular and Molecular Neuroscience Scientifique
Division Université du Québec
National Brain Research Centre Laval, Québec, Canada
Haryana, India
Jessica Desjardins
Jennifer M. Best
Institut National de la Recherche
King’s College London
Scientifique
London, United Kingdom
Université du Québec
Laval, Québec, Canada
xv
xvi Contributors
Luis Adrián Diaz and

Laboratorio de Arbovirus University of Liverpool
Instituto de Virología “Dr. J. M. National Consortium for Zoonosis
Vanella” Research
Universidad Nacional de Córdoba Neston, United Kingdom
and
Instituto de Investigaciones Biológicas y Philippe Gasque
Tecnológicas Immunopathology and Infectious
Consejo Nacional de Investigaciones Disease Research Group
Científicas y Técnicas University of la Reunion
Córdoba, Argentina St. Denis, Reunion, France
W. Paul Duprex Liliane Grangeot-Keros

Departments of Microbiology and Virology Department
Neurology Antoine Béclère Hospital
and National Reference Laboratory for
National Emerging Infectious Disease Rubella
Laboratories Clamart, France
Boston University
Boston, Massachusetts Göran Günther
Department of Medical Sciences
Alan P. Dupuis II Uppsala University
Department of Zoonotic Diseases Uppsala, Sweden
New York State Department of Health
Albany, New York Roy A. Hall
Australian Infectious Diseases Research
Kallol Dutta Centre
Cellular and Molecular Neuroscience University of Queensland
Division St Lucia, Australia
National Brain Research Centre
Manesar, Haryana, India Hideo Hara
Department of Internal Medicine
Dominique J. Favreau Saga University
Laboratory of Neuroimmunovirology Saga, Japan
Institut National de la Recherche
Scientifique Derek M. Healy
Université du Québec Wildlife Zoonoses and Vector Borne
Laval, Québec, Canada Disease Research Group
Department of Virology
Anthony R. Fooks Animal Health and Veterinary
Wildlife Zoonoses and Vector Borne Laboratories Agency
Disease Research Group Surrey, United Kingdom
Department of Virology
Animal Health and Veterinary
Laboratories Agency
Surrey, United Kingdom
Contributors xvii
Daniel L. Horton Laura D. Kramer

Wildlife Zoonoses and Vector Borne Department of Zoonotic Diseases
Disease Research Group New York State Department of Health
Department of Virology and
Animal Health and Veterinary Department of Biomedical Sciences
Laboratories Agency State University of New York
Surrey, United Kingdom Albany, New York
Hélène Jacomy Shiril Kumar

Laboratory of Neuroimmunovirology Immunopathology and Infectious
Institut National de la Recherche Disease Research Group
Scientifique University of la Reunion
Université du Québec St. Denis, Reunion, France
Kang-Sheng Li
Marie Christine Jaffar-Bandjee Department of Microbiology and
Immunopathology and Infectious Immunology
Disease Research Group Shantou University Medical College
University of la Reunion Shantou, Guangdong, China
St. Denis, Reunion, France
Mario Lobigs
Claire L. Jeffries Australian Infectious Diseases
Wildlife Zoonoses and Vector Borne Research Centre
Disease Research Group University of Queensland
Department of Virology St Lucia, Australia
Animal Health and Veterinary
Laboratories Agency Martin Ludlow
Surrey, United Kingdom Departments of Microbiology and
Neurology
Nicholas Johnson and
Wildlife Zoonoses and Vector Borne National Emerging Infectious Disease
Disease Research Group Laboratories
Department of Virology Boston University
Animal Health and Veterinary Boston, Massachusetts
Laboratories Agency
Surrey, United Kingdom Larry Lutwick
Veterans Affairs New York Harbor
Patrik Kilian Health Care Center
Institute of Parasitology and
Biology Centre of the Academy of State University of New York
Sciences of the Czech Republic Downstate Medical School
and Brooklyn, New York
Faculty of Science
University of South Bohemia
České Budějovice, Czech Republic
xviii Contributors
Mathieu Meessen-Pinard Duksha Ramful

Laboratory of Neuroimmunovirology Immunopathology and Infectious
Institut National de la Recherche Disease Research Group
Scientifique University of la Reunion
Université du Québec St. Denis, Reunion, France
Susan Reef
Ritu Mishra Centers for Disease Control and
Laboratory of Neurovirology and Prevention
Inflammation Biology Atlanta, Georgia
Centre for Cellular and Molecular
Biology Stephanie Robin
Council of Scientific and Industrial Immunopathology and Infectious
Research Disease Research Group
Hyderabad, India University of la Reunion
Arshed Nazmi
Cellular and Molecular Neuroscience Daniel Rů žek
Division Institute of Parasitology
National Brain Research Centre Biology Centre of the Academy of
Manesar, Haryana, India Sciences of the Czech Republic
České Budějovice, Czech Republic
Mah-Lee Ng
Sunit K. Singh
Department of Microbiology
Laboratory of Neurovirology and
National University of Singapore
Inflammation Biology
Singapore
Centre for Cellular and Molecular
Biology
Slobodan Paessler Council of Scientific and Industrial
Department of Pathology Research
University of Texas Hyderabad, India
Galveston, Texas
Lorena I. Spinsanti
Jana Preis Laboratorio de Arbovirus
Veterans Affairs New York Harbor Laboratorio de Arbovirus
Health Care Center Instituto de Virología “Dr. J.M. Vanella”
and Universidad Nacional de Córdoba
State University of New York Córdoba, Argentina
Down State Medical School
Brooklyn, New York Pierre J. Talbot
Natalie A. Prow Institut National de la Recherche
Australian Infectious Diseases Scientifique
Research Centre Université du Québec
University of Queensland Laval, Québec, Canada
St Lucia, Australia
Contributors xix
Norma P. Tavakoli Michael R. Wilson

Department of Genetics Departments of Microbiology and
New York State Department of Health Neurology
and and
Department of Biomedical Sciences National Emerging Infectious Disease
State University of New York Laboratories
Albany, New York Boston University
Boston, Massachusetts
Katherine Taylor
Department of Pathology Kim-Long Yeo
University of Texas Department of Microbiology
Galveston, Texas National University of Singapore
and
Vincent G. Thon-Hon NUS Graduate School for Integrative
Immunopathology and Infectious Sciences and Engineering
Disease Research Group Centre for Life Sciences
University of la Reunion Singapore
Motohiro Yukitake
Aravinthan Varatharaj Division of Neurology
Brain Infections Group Department of Internal Medicine
Department of Clinical Infection, Saga University
Microbiology and Immunology Saga, Japan
University of Liverpool
Liverpool, United Kingdom Jun Zeng
Department of Microbiology and
Gefei Wang Immunology
Department of Microbiology and Shantou University Medical College
Immunology Shantou, Guangdong, China
Shantou University Medical College
Shantou, Guangdong, China
Section I
RNA Viruses
1 Alphavirus
Neurovirulence
Katherine Taylor and Slobodan Paessler
CONTENTS
1.1 Introduction....................................................................................................... 3
1.2 Alphavirus......................................................................................................... 4
1.3 Interference with Antiviral Transcription.........................................................6
1.4 Changes in Cellular Tropism............................................................................. 8
1.5 Host-Antiviral Response....................................................................................9
1.6 Interferon......................................................................................................... 10
1.7 Innate Immune Response................................................................................ 11
1.8 Adaptive Immune Response............................................................................ 12
1.9 Host Response and Vaccine Development....................................................... 13
1.10 Conclusion....................................................................................................... 16
References................................................................................................................. 16
1.1 INTRODUCTION
Due to their parasitic, obligate, intracellular nature, evolution favors viruses with
the ability to evade the barriers and impediments the host organism utilizes to limit
their ability to replicate or cause cellular dysfunction. Avoidance of host defense
mechanisms and successful replication often leads to virulence, the ability to cause
fatal disease. Neurovirulent viruses alter the highly sensitive nature and critical
functioning of the central nervous system (CNS) leading to fatal encephalitis or, in
the event of recovery, severe neurological sequelae. With 20 viruses known to cause
human encephalitis, arboviruses (arthropod-borne viruses) represent a significant
public health threat as emerging infectious diseases both in the United States and
worldwide. The focus of this review, arboviruses in the Alphavirus genus in the
family Togaviridae, contains three viruses capable of causing human encephalitis:
Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis virus
(EEEV), and western equine encephalitis virus (WEEV). No specific therapy or
vaccine is currently available against these viruses.
For the encephalitic alphaviruses, virulence reflects the severity of neurologi-
cal disease and is determined by the efficiency of host and viral factors. Peripheral
replication, viremia, neuroinvasiveness, and neurotropism combined and interacting
together lead to neurovirulence and subsequent disease (Griffin 2007). Each indi-
vidual component is influenced by the overall molecular character of the infecting
3
4 Neuroviral Infections: RNA Viruses and Retroviruses
virus as well as the subsequent response, genetic background, age, and sex of the
infected host (Griffin 2007). The balance of the characteristics of the host and virus
ultimately determines outcome to infection, and minor changes in either can drasti-
cally impact disease etiology, leading to lethality, persistence, or abortive infection.
Thus, each stage or step of viral pathogenesis can be considered a struggle by the
virus against host defense to obtain the evolutionary ideal of a relative equilibrium
between the host and the virus.
In this context, two experimental paradigms are utilized to determine the com-
ponents of neurovirulence: (1) the use of mutated viruses to define the molecular
changes capable of altering pathogenesis and (2) the study of host variables utiliz-
ing analysis of viral pathogenesis in hosts where key immunological components
are missing or altered (Fleming 1988). In considering these two experimental
approaches, it is important to remember that minor variability and alterations in
the viral genome can result in significant changes in neuropathogenesis. Pathogenic
alphaviruses can be roughly grouped by a combination of phylogenetics, geographi-
cal circulation, and disease manifestation into two groups: Old World and New World
viruses. Old World viruses differ significantly from New World viruses in tropism
and disease manifestations in humans, causing arthralgia, malaise, or rash, whereas
New World alphaviruses result in a flu-like syndrome that may progress to encepha-
litis. Two Old World viruses, Sindbis and Semliki Forest, have been used exten-
sively in small animal models as prototypical encephalitic alphaviruses through the
use of either neuroadapted or the rare naturally encephalitic strains, respectively.
Although valuable knowledge regarding neuropathogenesis has been derived from
these models, experimental approaches utilizing Old World alphaviruses to represent
naturally encephalitic strains need to be confirmed using New World alphaviruses
(Atasheva et al. 2008; Charles et al. 2001; Garmashova et al. 2007a,b; Kolokoltsov
et al. 2006a,b; Yin et al. 2009). Thus, this review will first seek to understand how
changes in viral replication of the primary encephalitic New World viruses, EEE,
WEE, and VEE alter pathogenesis followed by analysis of how the host response
potentially contributes to the disease.
1.2 ALPHAVIRUS
The encephalitic alphaviruses spread in a biphasic manner through the mammalian
host. Viral-host interactions and the viability of the virus as a neurological agent
require entry and successful replication at the site of infection. Evidence from exper-
imental models indicates that as the mosquito feeds on the experimental host, virus
is deposited from infected saliva extravascularly into the tissues (Turell et al. 1995).
Virus then replicates at the site of inoculation, typically skeletal muscle or immune
cells such as Langerhan’s cells of the skin (Grimley and Friedman 1970; Johnston
et al. 2000; Liu et al. 1970; Murphy and Whitfield 1970). As the infected immune
cells carry the virus to the draining lymph nodes, the virus is delivered to the vas-
cular system, where it spreads to other target tissues, initiating the secondary phase
of infection (Griffin 2007). Griffin (2007), in Fields Virology, defines peripheral
replication and viremia as key components of neurovirulence. In the initial, periph-
eral phase, efficient replication at the site of inoculation enhances neuroinvasiveness
Alphavirus Neurovirulence 5
through viremia although sustained, high levels of blood-borne virus may not be
required for neuroinvasion.
Following replication in lymphoid and/or myeloid tissues, the virus enters the
CNS, resulting in a second phase of replication in the brain and spinal cord neurons.
Entry to the CNS is critical to neurovirulence, but the precise mechanism of entry
remains unknown. However, literature supports a model of nasal mucosa infection
leading to the infection of the olfactory neurons and neuronal spread to through the
CNS (Charles et al. 2001; Steele et al. 1998).
The initiation of inflammation leads to damage to the vascular integrity of the
blood–brain barrier (BBB), enabling further entry of the virus to the compromised
CNS (Schafer et al. 2009, 2011). The multifocal nature of the infection in the brain
by 5 or 6 days postinfection indicates damage to the BBB and free crossing of viral
particles across the typically sealed barrier (Charles et al. 2001; Jackson et al. 1991;
Roy et al. 2009; Vogel et al. 2005; Zlotnik et al. 1972). Damage to the vascular
integrity of the BBB as a result of inflammation has additional support from a range
of studies examining viral entry to the CNS (Cook and Griffin 2003; Lossinsky and
Shivers 2004; Wang et al. 2004).
Disease symptoms in patients mimic the biphasic nature of both the immune
response and viral replication. In patients, as the initial immune response and viral
propagation exponentially grow, a fever develops. As the secondary spread of virus
begins, patients may develop another fever that coincides with development of neu-
rological symptoms. This phase results in neuropathology and, in some cases, fatal
encephalitis. Regardless, in the majority of infected individuals, the host response
appears to be sufficient to prevent CNS damage and results in asymptomatic infec-
tions. This serves to emphasize how little is known regarding the susceptibility of
particular individuals to neurovirulent viruses. For instance, in WEE epidemics,
only 1 of 1000 infected individuals actually develop clinical encephalitis with fatal-
ity resulting in only 3% of these cases (Rennels 1984). Similar statistics are observed
for VEE and EEE, although mortality in these infections is significantly higher.
Nevertheless, the question remains why the viruses that are apparently intrinsically
of low virulence cause severe disease in some patients and what factors account for
occasional severe neurovirulence in some individuals? This is reflected in experi-
mental situations where variability between inbred strains of mice results in altera-
tions in outcomes (Ludwig et al. 2001). In human infections, there is undoubtedly an
age-associated susceptibility to the encephalitic alphaviruses, with neurovirulence
and associated mortality increasing at the extremes of the age spectrum in pediatric
and elderly patients; however, the underlying causes for increased susceptibility in
these populations remain poorly defined. The immature CNS may explain the sus-
ceptibility in pediatric patients, as WEE is more cytopathic in immature human neu-
ronal cells than mature cells. Additionally, mature neuronal cells are more sensitive
to type I IFN requiring less to reduce viral cytopathology and replication (Castorena
et al. 2008).
The ability of the immunocompentent host to prevent neuroinvasion is uncer-
tain and viral invasion without associated symptomatic illness has been reported
in the literature in experimental models. Indicative of the importance of immune
host control at the site of initial replication, intracerebral or intranasal inoculation of
many alphaviruses causes fatal disease in experimental models, whereas subcutane-

ous or intraperitoneal infection results in either asymptomatic disease or a clinical
syndrome without lethality or severe neuropathology (Griffin 2007). Although the
development of encephalitis and neurological disease in animal models is readily
apparent as gauged by seizures or measurable paralysis, the utility of these models
in evaluating the human host response is unclear as the disease development may be
significantly different. Gaining a clearer understanding of the effects viral infection
has on the host is a major component of drug development and vaccine platforms
(Holbrook and Gowen 2008).
The two-wave model of infection is supported by a series of studies using VEE
viral replicon particles (VRP), which are capable of targeting and infecting the same
cells as VEE but unable to propagate beyond the first infected cell, to model early
events in neuroinvasion. Direct intracranial infection of VRP resulted in VEE-like
encephalitis in mice and was associated with a robust and rapid innate immune
response that resulted in compromised BBB integrity. These initial results estab-
lished a model for the identification of host and viral factors that contribute to the
invasion of the brain (Schafer et al. 2009). More recent studies show that replication
of the VRP in the nasal mucosa induced the opening of the BBB allowing peripher-
ally administered VRP to enter the brain (Schafer et al. 2009, 2011). Subsequent
inhibition of the initial opening of the BBB resulted in a delay in viral neuroinvasion
and pathogenesis. Thus, initial entry into the CNS through the olfactory pathways
initiates viral replication in the brain, inducing the opening of the BBB and allowing
for a secondary wave of virus from the periphery to enter the brain (Schafer et al.
2011). Further supporting initial replication and entry through the olfactory epithe-
lium and neurons, the administration of the toxin tunamycin damages the ultrastruc-
ture of the BBB. However, entry to the CNS still appears to occur via the olfactory
system despite the prior damage BBB induced by pretreatment with the toxin. An
increased viral load in the brain with both virulent and attenuated strains of virus is
observed in mice treated with tunamycin to induce artificial BBB breakdown (Steele
et al. 2006).
1.3 INTERFERENCE WITH ANTIVIRAL TRANSCRIPTION

All three encephalitic alphaviruses are highly susceptible to the effects of type I
IFN (IFN-α and IFN-β), and as such, resistance to type I IFN signaling and pro-
duction is associated with increasing neurovirulence of the virus (Aguilar et al.
2008a; Armstrong et al. 1971; Jahrling et al. 1976; Jordan 1973; Julander et al.
2007). Closely related virulent and avirulent strains of virus and indeed virulent
New World and less virulent Old World alphaviruses demonstrate significant differ-
ences in their ability to interfere with gene transcription associated with viral attenu-
ation linked to enhanced susceptibility to the effects of type I IFN. Comparison of
IFN-sensitive avirulent strains and IFN-resistant virulent strains has led to com-
prehensive knowledge of the genes responsible for controlling IFN resistances and
subsequent neurovirulence. Research supports the underlying cause of differences in
disease presentation and incidence in humans between North and South American
isolates of EEE results from alterations to IFN sensitivity. Comparison of replication
of NA and SA strains in IFN pretreated Vero cells showed a suppressive effect only
on the replication of the less virulent SA strains. However, no differences in induc-
tion of IFN in vivo were observed (Aguilar et al. 2005). Similar results were found
for VEE with attenuation in enzoonotic strains limiting the ability of the virus to
interfere with the type I IFN pathways, and this may partially explains the absence
of disease symptoms following infection with enzoonotic strains (Anishchenko et al.
2004; Jahrling et al. 1976; Simmons et al. 2009). Conversely, epizootic and epidemic
strains are able to limit the host production of type I interferon.
Comparison of both naturally and experimentally attenuated viruses to virulent
strains has generated valuable knowledge regarding the basis for IFN resistance and
subsequent neurovirulence. Priming neurons with IFN before infection with either
VEEV or SINV further demonstrates VEEV’s resistance to an established antiviral
state compared with SINV as VEEV continues to replicate and produce progeny
virion in primed cells in contrast to more sensitive SINV. VEEV resistance was
attributed to partial blockade of phosphorylation of IFN signaling pathway mole-
cules, STAT1 and STAT2, mediated by expression of nonstructural proteins. VEEV
also inhibits interferon signaling genes (ISG) through structural protein expression
(Yin et al. 2009).
Furthermore, comparison of an IFN-sensitive SA EEE strain to a resistant NA
EEE strain identified both structural and nonstructural genes as important in IFN
sensitivity (Aguilar et al. 2008a,b). Additional data derived from the genetic manipu-
lation of virulent viruses has contributed to the understanding of the mechanisms
underlying virulence associated with IFN resistance. Artificial attenuation of viru-
lent EEE results in a marked increased in sensitivity to type I IFN and is associ-
ated with decreased virulence of the virus (Aguilar et al. 2008b). Attenuations in
WEE and VEE reflect similar results (Anishchenko et al. 2004; Aronson et al. 2000;
White et al. 2001). The significant differences in the an antihost response for New
World alphaviruses compared with the Old World likely play a role in the increased
neurovirulence of the former with the ability to down-regulate the cellular antivi-
ral machinery increases neurovirulence. The recombination events leading to the
formation of WEE from SINV and EEV-like ancestors allowed WEEV to acquire
capsid protein function to inhibit the transcription of antiviral factors and thereby
effectively evade the antiviral effects of type I IFN. Thus, the acquisition of type I
IFN evasion led to the emergence of WEEV as a pathogenic virus (Garmashova et
al. 2007b; Hahn et al. 1988).
The ability of EEEV and VEEV to interfere with cellular transcription and
induce subsequent cytopathic effect is controlled by a N-terminal 35-amino acid
long peptide fragment of the capsid protein. One domain is critical in balancing the
presence of protein in the cytoplasm and nucleus, and the downstream peptide may
contain nuclear localization signals. These domains determine the intracellular dis-
tribution of the VEEV capsid and are essential for protein function in the inhibition
of transcription. The cytopathologic effects are reduced and infection is attenuated
in vivo without effecting viral replication by replacing the N-terminal fragment of
the VEEV capsid with the Old World alphavirus, SINV (Garmashova et al. 2007a).
The pathogenic effect of the capsid protein appears to work via inhibition of mul-
tiple receptor-mediated nuclear import pathways leading to down-regulation of the
cellular antiviral machinery. Again, the capsid protein of SINV had no effect on
nuclear import (Atasheva et al. 2008). Interestingly, the Old World alphaviruses,
SINV and SFV, are both able to interfere with cellular transcription. However, dif-
ferent virus-specific proteins are utilized to cause this effect. For the Old World,
alphaviruses transcriptional shutoff depends on nsP2, whereas for the New World
alphaviruses, it depends on VEEV and EEEV (Garmashova et al. 2007a,b).
Thus, changes to the E2 envelope glycoprotein are significant attenuators of the
neurovirulent virus. Early studies comparing TC83 structural proteins to virulent
parent strain VEE-TRD found that the two strains differed at 12 nucleotides with no
alterations in the nonstructural proteins or the open reading frame coding the viral
polyprotein. Only nine of these changes occurred in the dominant population of the
RNAs from plaque-purified viruses. Significantly, six of the nine mutations appeared
in the E2 surface glycoprotein, and all five of the nucleotide changes producing non-
conservative amino acid substitutions were located here (Johnson et al. 1986). Two
mutations in E1 were found: one silent and one that did not alter the character of the
protein. An additional nucleotide difference was found in the noncoding region pre-
ceding the 5′ end of the 26S mRNA. This early publication determined that both E2
and the noncoding regions were candidates for the molecular determinants of VEE
neurovirulence. In the early 1990s, the proof of attenuation of VEEV through serial
cell-culture passage that resulted in TC83 is encoded in the 5′ noncoding region and
the E2 envelope glycoprotein was confirmed. Studies showed E2-120 appears to be
the major structural determinant of attenuation, but genetic markers composed of
genome nucleotide position 3 in the 5′ noncoding region were also significant to the
attenuated phenotype (Kinney et al. 1993). The biological effect of the attenuating
mutation in the 5′ untranslated region during murine infection was ultimately traced
to increased sensitivity to IFN-α and IFN-β (White et al. 2001).
1.4 CHANGES IN CELLULAR TROPISM

The molecular changes responsible for changes in cellular tropism between the
encephalitic alphaviruses are poorly defined; however, viral tropism for the cells
of the periphery varies among the three encephalitic alphaviruses. The altered tro-
pism for the cells of the CNS (neurons, oligodendria, microglia, and astrocytes) does
not appear to account for the changes in neurovirulence between the closely related
strains, as both virulent and avirulent strains are capable of productive infection of
neurons. However, the efficiency of replication in neurons differs dramatically with-
out necessarily effecting replication in the cells of the periphery. The molecular basis
for enhanced replication appears to be targeted to specific changes in the coding and
noncoding regions of the genome, ultimately leading to enhanced neurovirulence
(Griffin 2007).
However, altered tropism for other cells may impact the level of peripheral repli-
cation, viremia, and subsequent neuroinvasion and virulence. In the case of EEEV
and VEEV, such changes are reflected in altered cell tropism. Indeed, the infection
of immune cells by viruses may have a significant effect on a pathogenesis as evi-
denced by differences in tropism between EEE and VEE. In the case of EEEV and
VEEV, such changes are reflected in altered cell tropism. Although both viruses
cause severe morbidity and mortality in equines and humans, VEEV infects the
DCs and macrophages of the lymphoid tissues, whereas EEEV replicates poorly in
lymphoid tissues and preferentially infects muscle and fat cells. Both viruses rep-
licate efficiently in mesenchymal cell lineages. The inability of EEEV to replicate
in myeloid cell lineages is due to interferon-independent inhibition of EEEV trans-
lation. VEEV-infected mice display higher levels of serum IFN and result in IFN
up-regulation in more animals compared with EEEV infection. Interestingly, the
altered tropism of EEEV may help the virus to evade systemic IFN induction in vivo
enhancing EEEV neurovirulence and contributing to differences in disease etiology
(Gardner et al. 2008).
1.5 HOST-ANTIVIRAL RESPONSE

Viral replication at the site of inoculation, level of viremia, and subsequent spread
to secondary sites are controlled by viral clearance after the initiation of the host
response. Given the close relation and ability of the innate immune response to
modulate the later adaptive immune response needed to effectively clear the virus,
an early, robust, and properly directed innate immune response is essential. In the
biphasic model of alphavirus infection, as the primary phase of viral replication is
initiated, the host begins responding to the pathogenic changes at the site of inocu-
lation by inducing robust production of cytokines, particularly IFN (Griffin 2007).
Cytokine production also results in recruitment of other immune effectors capable
of generating positive feedback producing an antiviral environment in the host. The
infection of immune cells serves to propagate the virus but also results in the infil-
tration of antigen-presenting cells to the nearest lymph node, introducing antigen to
naive B and T cells in the lymph nodes and initiating the adaptive immune response.
Given the primary tropism of the encephalitic alphaviruses for dendritic cells, it
would be unsurprising if alterations in antigen presentation and subsequent modifi-
cation of an efficient adaptive immune response occurred; however, little work has
been done to determine the effect of such tropism in these infections. These early
mechanisms are also required to limit peripheral replication, viremia, and spread to
secondary sites of infection before the development of an effective adaptive immune
response.
Immunocompentent mice infected with VEEV develop a typical biphasic illness
with an early lymphoid phase, characterized by ruffling of fur and progression to
hunching, and a later fatal CNS phase, characterized by progression from ataxia to
severe paralysis. In the lymphoid phase, peripheral serum viremia and replication in
organs is resolved concurrent with production of IgM at 3 days postinfection, which
steadily increases to the time of death as well as with rapid, robust production of type
I IFN by 18–24 h postinfection that rapidly waned after 24-h postinfection. However,
mice still developed fulminate encephalitis, and the mean time to death is 7.8 days
(Charles et al. 2001).
In contrast, severely immunocompromised SCID mice fail to develop early signs
of disease and symptoms develop at 6–7 days postinfection marked by aggression
and agitation with death occurring at 8.9 days postinfection. Animals fail to develop
hallmark hind-limb paralysis and become ataxic and progressively less responsive,
indicating alterations of the neurological disease in the immunocompromised host.

Organ tropism differed in these animals with persistent viral replication at or near
peak titer appearing in peripheral organs until the death of the animal without the
resolution seen by 120 h after infection in sera and lymphoid tissues in competent
animals. Unsurprisingly, patterns of antibody and type I IFN production vary from
the immunocompentent host with a complete lack of neutralizing antibody pro-
duction and lower levels of IFN that increased slowly and were below the limit of
detection by 48 h postinfection. The time of death is delayed in these mice, with an
average survival time of 8.9 days (Charles et al. 2001).
Given the alteration in clinical symptoms, the pathologies in the brain present as
a severe spongiform encephalopathy different from that of the immunocompetent
hosts’ fulminate encephalitis, indicative of a significant immunological component
to CNS pathology. Charles et al. (2011) does not attribute the proximal cause of death
to brain lesions in either case. Thus, the establishment of a functional host response
in the periphery requires a competent immune system but is unable to prevent death.
The changes in peripheral cellular tropism occur more rapidly than can be explained
by the adaptive response and likely involve innate nonspecific response to viral
pathogens, particularly the lack of IFN in SCID mice, preventing the establishment
of the antiviral state (Charles et al. 2001).
Unfortunately, little is known about the specific mechanisms of host defense apart
from an early, antiviral role for type I IFN and a correlation of neutralizing antibody
production with peripheral clearance.
1.6 INTERFERON
All three of the encephalitic alphaviruses are highly susceptible to the effects of type
I IFN and, as mentioned previously, have developed effective evasion tactics to avoid
the antiviral effect. In fact, in the absence of type I IFN signaling in receptor knock-
out mice, even attenuated strains of VEE typically unassociated with illness can
cause complete mortality (White et al. 2001). However, prophylactic and, in some
cases, early therapeutic treatment with type I IFN or compounds capable of inducing
type I IFN provides protection, indicating the importance of the early generation of
an immune environment conducive to host protection.
Beginning with the discovery that EEE replication was suppressed in the pres-
ence of type I IFN, Wagner (1961, 1963) showed peak viral production and high levels
of cytopathogenicity in chick embryos and L-cells correlated with a high-level pro-
duction of IFN. In vivo serum levels of type I IFN are low compared with those of
VEE-infected mice and likely reflect the inability of EEE to infect cells of the myeloid
lineages because the ability of EEE to antagonize type I IFN induction is cell-dependent
(Burke et al. 2009). By artificially increasing the serum type I IFN before infection by
administration of a TLR3 agonist, poly-IC, Aguilar et al. (2005) demonstrated a dose-
dependent IFN-mediated protection of mice to EEE infection. Similar results are seen
for WEE infections, where pretreatment of hamsters with either a consensus type IFN-α
or a stimulator of type I IFN signaling, Ampligen, resulted in the complete survival of
the animal. The antiviral effect of type I IFN levels was reflected in decreased clinical
symptoms and weight loss associated with a significantly lower viral load in the brain at
4 days postinfection (Julander et al. 2007). Complete survival and depression of clini-
cal symptoms is also associated with transiently expressed, artificially high levels of
IFN-α in mouse models of WEE infection. Therapeutic treatment up to 7 days before
infection provides complete protection. Early prophylactic elevation of IFN-α at 6 h
postinfection results in increased survival rates but fails to provide complete protec-
tion (Wu et al. 2007a,b). Unsurprisingly, VEE responds similarly to the early induction
of type I IFN with artificial induction of signaling through administration of a TLR3
agonist or prophylactic administration of pegylated IFN-α, resulting in delayed time
to death and increased survival in mouse models (Julander et al. 2008b). Although the
early administration of IFN demonstrates some prophylactic effects in decreasing time
to death or disease symptoms, in the case of intranasal or aerosol exposure, the rapid
entry of the virus to the CNS may limit or alter the effectiveness of early innate immune
mechanisms such as IFN or other unexplored factors. The substantial difference in the
effect of therapeutic and prophylactic administration of type I IFN or inducers of IFN
production indicates the importance of modulating the specific immune response in the
CNS to create a distinct antipathogenic environment after viral spread and replication.
The CNS of immunologically normal mice is still invaded in the presence of very
high circulating levels of IFN, indicating that the cells that comprise the CNS may
be less sensitive to the presence of IFN or have slower kinetics for the establishment
of an effective antiviral state (Charles et al. 2001).
Due to the greater stability of IFN-α, most studies use its modified forms, and
to the authors’ knowledge, no studies examining the effects of IFN-β have been
performed to date. Interestingly, IFN-β treatment of the CNS disorder, multiple scle-
rosis, helps control the disease in some patients and indicates that mechanisms other
than the antiviral effect of the type I interferons may be important in control of CNS
damage (Galligan et al. 2010; Plosker 2011).
1.7 INNATE IMMUNE RESPONSE

Pretreatment and, in some cases, therapeutic administration (up to 12 h pi) of cationic-
liposome DNA complexes (CDLCs) in mice infected with WEE results in significant
protection. This protection was associated with changes in the host immune response
due to CDLC administration. Treated mice had significantly increased serum
IFN-γ, TNF-α, and IL-12, indicative of a strong TH1-biased antiviral activation of
the immune system. In infected animals large increases in IFN-γ, TNF-α, IL-12,
MCP-1, and IL-10 in the brain were observed by 72 h pi, as expected, with neuroin-
vasion and viral replication in the CNS (Schafer et al. 2009). Similar cytokine pro-
files are found in the brain homogenates of C3H/HeN mice lethally infected with the
vaccine strain of VEEV, TC83. In these animals, IL-1a, IL-1b, IL-6, IL-12, MCP-1,
IFN-γ, TNF-α, MIP1-α, and RANTES were significantly elevated over time with
peak cytokine levels at six to seven days postinfection. Depression in cytokine levels
occurs immediately before death around 8 to 10 days postinfection. Interestingly,
treatment with IFN-α B/D or TLR3 agonist significantly improved cytokine levels
and mean day to death, indicating a connection between mortality and the early
host response (Julander et al. 2008a,b). Thus, robust, nonspecific activation of the
innate immune response, while necessary to influence the phenotype of the adaptive
immune response, requires careful modulation in the CNS to elicit significant pro-
tective immunity against rapidly lethal strains of encephalitic alphaviruses (Schafer
et al. 2009).
1.8 ADAPTIVE IMMUNE RESPONSE

The second phase of the immune response to alphavirus infection is characterized by
the waning of type I IFN and the development of a robust cell-mediated immunity
that theoretically limits CNS damage and clears the virus from circulation and sites
of replication.
Of the factors in the cell-mediated immune response responsible of resolving
infection, the role of B-cells and antibody production is well-defined. The develop-
ment of antigen specific B-cells capable of antibody production play a key role in
reducing peripheral replication and removing virus from the blood stream. Thus, an
efficient antibody response is integral to preventing or limiting neurovirulence from
the earliest phase. However, the time lag between peripheral infection and antigen-
specific antibody production permits spread and replication at secondary sites of
infection as mentioned previously, and additional cell-mediated mechanisms may be
required to resolve infections once they reach the CNS (Griffin 2007).
In the lymphoid phase of murine infection (CB17), high-titer serum viremia is
associated with the production of VEE-specific IgM antibody at 3 days postinfec-
tion, with titers increasing until the time of death. Animals failed to develop VEEV-
specific IgG or IgA. However, fulminate encephalitis still develops and animals
survive only an average of 6.8 days postinfection (Charles et al. 2001). Further recon-
stitution of SCID mice and depletion of immunologically normal mice identified
production of VEE-specific IgM antibody, produced in the absence of T-cell help,
as the significant factor determining immune mediated clearance in the periphery.
Given the paralysis and death of these animals following infection, it appears that
peripheral production of neutralizing antibody does not play a significant role in pre-
venting lethal encephalitis once virus reaches the CNS (Charles et al. 2001).
Once the virus becomes neuroinvasive, the utility of peripherally produced
antibody is uncertain, and T cells, particularly CD4+ T cells, are integral in clear-
ance of CNS infection. Indicative of the role of T cells in resolving viral invasion
of the CNS, α/β T-cell receptor knockout mice deficient in both CD4+ and CD8+ T
cells develop lethal VEE encephalitis following vaccination protective to the wild-
type counterpart. Specifically, reconstitution of the CD4+ T cells, but not CD8+ T
cells, from vaccinated wild-type donors resulted in recovery following vaccination
and challenge in α/β T-cell receptor knockout mice, indicating an integral role for
CD4+ T cells in preventing lethal encephalitis (Paessler et al. 2007; Yun et al. 2009).
In the absence of a competent T-cell compartment, α/β T-cell receptor knockout
mice also have impaired antibody production in the absence of CD4+ T-cell help.
However, passive transfer of HIAF antibody failed to induce a protective response
in vaccinated animals indicating either a direct role for CD4+ T cells in the CNS or
an indirect, B-cell, antibody-independent mechanism of action (Yun et al. 2009).
Studies using B-cell-deficient uMT mice infected with an attenuated strain of VEEV
resulted in the development of severe, but ultimately, asymptomatic encephalitis. An
antibody-independent Th1-biased response characterized by CD4+ T-cell production

of IFN-γ was implicated in the control of viral replication and survival of the ani-
mals (Brooke et al. 2010).
Charles et al. (2001) demonstrated that treatment of CD-1 mice with T-cell-
depleting factors before infection results in a disease such as that of SCID mice with
absent clinical symptoms early in disease and no development of paralysis. However,
infection in the absence of T cells had no effect on clearance of virus from the serum
and did not prevent production of IgM (Charles et al. 2001). Reconstitution of SCID
mice with T cells results in the reversion to fulminant encephalitis seen in wild-type
mice, as does the inverse scenario, with the depletion of wild-type Cd-1 in mice lead-
ing to spongiosis and vaculation of the neuropil as seen in SCID mice. Analysis of
inflammatory infiltrates in the brain indicated that T cells represent the majority of
infiltrates and the predominant phenotype was CD8+ (Charles et al. 2001). Treatment
of murine splenocytes with both B- and T-cell mitogens increases the susceptibility
of VEE infection and indicates that the activation state of lymphocytes may be criti-
cal to lymphoid pathogenesis of VEEV (Charles et al. 2001).
Animal models of infection have provided some insight into the role of IFN-γ
in protection. For VEE, the priming of the immune response via the vaccination of
mice with a deficiency in the type II IFN (IFN-γ) receptor is only partially protec-
tive, unlike the complete protection seen in wild-type animals following vaccination,
indicating that type 2 IFN signaling may play some role in preventing the develop-
ment of encephalitis. However, unlike type I IFN signaling, type 2 IFN signaling is
not absolutely required for effective protection (Paessler et al. 2007). Additionally,
IFN-γ signaling does not appear to be significant in controlling EEE infection, as
IFN-γ-receptor-deficient animals demonstrate equivalent levels of viremia and mor-
tality rates similar to wild-type animals (Aguilar et al. 2008a).
1.9 HOST RESPONSE AND VACCINE DEVELOPMENT

Neurovirulence is considered the standard for vaccine candidates derived from vir-
ulent neurotropic viruses (Arya and Agarwal 2008; Fine et al. 2008). Intracranial
injection of susceptible mice is routinely used for vaccine safety studies, and the
mice are evaluated based on the occurrence of pathological processes in the neu-
rological tissues. Intracranial viral infection causes damage to neuronal and glial
cell populations in addition to inducing migration of potentially harmful immuno-
logically active cells in to the perivascular space and brain parenchyma. However,
in the case of live vaccine candidates, such as TC83, the current IND vaccine for
VEE, these studies fail to take into account the effects of neuroinvasion in the
absence of detectable pathologies. The ability of RNA viruses to persist in the
CNS and the tropism of live vaccine candidates for the cells of the CNS must also
be considered in developing viable vaccines. Arya et al. points out that for practi-
cal use of vaccines derived from neurotropic viruses, a close examination of the
CNS for subtle lesions is required, particularly neurological developments after
vaccination for other such vaccines as seen with poliovirus vaccine lots in the
1960s (Arya and Agarwal 2008; Cristi and Dalbuono 1967). To evaluate potential
CNS damage, experimental animals should be observed for much longer periods
to examine the occurrence and location of brain lesions after vaccination (Arya
and Agarwal 2008).
The current key requirements for the development of a VEEV vaccine are (a) a
high level of immunogenic response in mice and hamster, which are both sensitive
to infection, and (b) a protective response in the NHP model when challenged with
virulent virus (Rao et al. 2006). Unfortunately, the sole parameter for immunogenic
response in small animal models is the production of neutralizing antibody that may
not necessarily correlate with protection once infection reaches the CNS.
In addition to neurovirulence as a safety parameter, vaccine studies use neutral-
izing antibody production as a correlate for the efficacy of vaccination. In such cases,
antibody is used as a measure of protection; however, the ability of peripherally pro-
duced antibody to provide protection and complete clearance of virus once the virus
invades the peripheral nervous tissue or the olfactory tissue via olfactory nerve tracts
remains poorly determined (Arya and Agarwal 2008; Fine et al. 2008).
Alterations in pathogenesis between closely related strains can be demonstrated
from vaccination studies using attenuated strains of virus. These studies also derived
important data regarding the host response and indicate that additional parameters to
antibody production may be necessary when evaluating safety and efficacy of vac-
cine or therapeutic candidates.
A series of studies beginning in small animal models and progressing to NHPs
compared the current live, attenuated IND vaccine strain, TC83, to candidate V3526,
a live attenuated virus derived from an infectious clone of VEEV, TrD. Intracranial
inoculation of both TC83 and V3526 results in the replication in the brain, with
V3526 replicating at lower levels. Although neither strain was lethal in BALB/c
mice, those infected with TC83 developed symptomatic illness, and the infection of
C3H/HeN mice resulted in complete lethality, demonstrating the differential suscep-
tibility of different inbred mice strains as shown previously by Steele et al. (Ludwig
et al. 2001; Steele et al. 1998). V3526, despite its ability to replicate in the CNS,
was “avirulent” in both strains and did not cause symptomatic illness. Interestingly,
pathological changes were found in the brains of mice infected with either strain, al
though, correlating with viral load, changes in V3526 inoculated animals were less
severe and of shorter duration than TC83-inoculated animals (Ludwig et al. 2001).
Comparison of wild-type virus to attenuated, vaccine strain TC83 and V3526
showed similar results following aerosol exposure: while the attenuated virus does
not necessarily cause symptomatic disease or mortality, they can be neuroinvasive
and cause lesions throughout the CNS, which, however, are readily resolved. All
three strains infected the brains and induced encephalitis. However, viral spread
varied, with a gradient occurring from complete invasion of all regions of the brain
with TrD, to sparing the caudal regions with TC83, to the involvement of only the
neocortex and diencephalon with V3526. TrD infection resulted in uniform mortal-
ity with significant peripheral dissemination between mouse strains. Despite altera-
tions in viral spread through the CNS, TC83 still induced 100% mortality in C3H/
HeN animals but not BALB/c mice. Interestingly, significant differences between
inbred mouse strains exist, and TC83, which is avirulent, so to speak, in BALB/c
mice, causes complete lethality in C3H/HeN animals. V3526 caused no mortal-
ity in either strain. Neither attenuated strain extended beyond the infection of the
olfactory epithelium (Steele et al. 1998). Thus, viral replication and spread do not
necessarily correlate with mortality as seen in the case of the more limited spread
of TC83 to caudal regions compared with TRD and equivalent mortality in C3H/
HeN mice.
Manifestations of disease were found in rhesus macaques inoculated intratha
lamically/intraspinally with both V3526 and TC83, although a greater percentage
of TC83 infected animals developed clinically significant signs. All symptomatic
disease resolved by 3 weeks postinfection. Interestingly, one of seven animals
infected with attenuated V3526 showed extensive brain lesions similar to all four
wild-type animals at D18. Six of seven infected macaques showed scattered lesions
throughout the CNS as did four of seven TC83-infected animals at D18. All lesions
were resolved by termination of the study at D181. Thus, clinical symptoms do not
necessarily correlate with viral invasion, replication, and pathogenesis in the CNS
(Atasheva et al. 2008; Fine et al. 2008).
Studies evaluating the propagation-defective VEEV replicon particles in mice
resulted in weight loss and inflammatory changes in the brain. However, changes
are less severe than those caused by TC83, the current IND vaccine. Peripheral
inoculation demonstrated minimal neurovirulence and lack of neuroinvasive poten-
tial (Kowalski et al. 2007). Despite the rapid and transient nature of CNS lesions
following vaccination, little is known about the degree of magnitude required to
induce potentially undetectable but significant pathogenic alterations to the delicate
homeostasis of the CNS. Although peripheral inoculation routes with propagation
defective particles are likely unable to reach the CNS, this may not be true for live,
attenuated vaccine candidates that may in fact reach, replicate, or even persist in the
CNS at undetectable levels. The biological relevance of low level replication in the
CNS is uncertain, but the immune-privileged nature of the CNS makes any altera-
tion of concern.
Intracranial infection with VRP results in a VEE-like encephalitis. The use of
a VRP-mRNP tagging system to distinguish the response of infected cells from
bystander cells showed the initiation of a robust and rapid innate inflammatory
response in the CNS by both infected neurons and uninfected bystander cells that
led to an adaptive immune response characterized by proliferation and activation of
microglia and infiltration of inflammatory monocytes as well as CD4+ and CD8+ T
lymphocytes. Thus, the ability of the naive CNS to induce a robust innate immune
response and activate local professional antigen-presenting cells that can, in turn,
activate primed cells may be crucial to the outcome of infection by determining the
composition and dynamics of the adaptive immune response and ultimate noncyto-
pathic or lethal attempts to clear the virus (Schafer et al. 2009).
A formalin-inactivated form of TC83, C-84, is currently used as a booster if vac-
cinated individuals fail to develop a response to TC83. The low efficacy and undesir-
able side effects have led to additional vaccination strategies and vaccine platforms.
One such approach utilizes microspheres to encapsulate the vaccine and induce a
more robust response. These spheres are composed of dl-lactide-co-glycolide (dl-
PLG). Microencapsulating the vaccine increased the primary circulating IgG and
resulted in a rapid increase in antibody activity when boosted with a second vac-
cination. Interestingly, circulating anti-VEE virus antibody response was lower, with
nonformalin-fixed virus utilizing this platform. Following systemic challenge with

virulent VEE, the microencapsulated virus was more effective at inducing a protec-
tive immune response (Greenway et al. 1995).
1.10 CONCLUSION
Alphaviruses represent a significant public health threat. A better understanding of
the mechanisms both virus and host use to control infection and prevent neuroinva-
sion are required for development of safe and effective vaccines and therapeutics.
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2 Neurological
Chikungunya
Lessons from Recent
Epidemics, Animal Models,
and Other Alphavirus
Family Members
Vincent G. Thon-Hon, Shiril Kumar,
Duksha Ramful, Stephanie Robin,
Marie Christine Jaffar-Bandjee, and Philippe Gasque
CONTENTS
2.1 Introduction: Chikungunya, a Paradigm Shift From “Old” to “New”
World Alphavirus?........................................................................................... 21
2.2 Neurological Chikungunya in Human Neonates.............................................24
2.3 Neurological Manifestations in Pediatric Patients..........................................25
2.4 Neurological Chikungunya in Adults..............................................................26
2.5 Neurological Chikungunya: Routes to CNS Infection and Tissue Injury.......26
2.6 Neuroinfection by “Old World” Sindbis Virus................................................ 30
2.7 Neuroinfection by New World Alphaviruses: EEEV, VEEV, WEEV............. 30
2.8 Conclusion....................................................................................................... 32
References................................................................................................................. 33
2.1 INTRODUCTION: CHIKUNGUNYA, A PARADIGM SHIFT

FROM “OLD” TO “NEW” WORLD ALPHAVIRUS?
Infections of the central nervous system (CNS) by viruses are relatively uncommon
yet virtually devastating (Bruzzone et al. 2010). Viral invasion and successful infec-
tion of the CNS is an important step in the life cycle of many neurotropic viruses
such as poliovirus, rabies, and measles (Griffin 2003). Viruses that invade the brain
(and peripheral nerve tissues) have been postulated to cause diseases but through
very diverse mechanisms with a unique mechanism of entry, replication, defense
21
against the immune system, and dissemination to preferred target cells, and devel-
opmental stages of infection (van den Pol 2006). Each of these aspects needs to be
carefully addressed to understand the physiopathology of a given virus with neuro-
logical complications.
Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family transmit-
ted by mosquitoes of the Aedes (Ae) genus (Pialoux et al. 2007; Weaver and Barrett
2004). CHIKV was first isolated in 1952 in Tanganyika now Tanzania (Robinson
1955). Recurrent epidemics have been reported primarily in Africa and Asia (Powers
and Logue 2007). The largest epidemic of CHIKV disease ever recorded took place
in 2004–2011 and was associated with the emergence of CHIKV that were efficiently
transmitted by Ae. albopictus a vector that has seen a dramatic global expansion
in its geographic distribution (Charrel et al. 2007). The epidemic began in Kenya,
spread across the Indian Ocean Islands to India (with an estimated 1.4–6.5 million
cases) and South East Asia (Ng and Hapuarachchi 2010). Remarkably, CHIKV has
accumulated key mutations (such as E1-A226V and E2-I211T) that probably contrib-
uted to the recently changed epidemiology and with possible clinical impacts, yet
to be fully ascertained (Schuffenecker et al. 2006). The first autochthonous infec-
tions in Europe occurred in Italy in 2007 (less than 250 cases) and few reported
cases in France in 2010 (Grandadam et al. 2011; Jaffar-Bandjee et al. 2010; Schwartz
and Albert 2010). With increased and faster human transportation in the shrinking
world, viruses can potentially move into new geographical locations and expand
their geographical range. Thus, imported CHIKV cases have now been reported in
nearly 40 countries including the United States, Japan, and several European coun-
tries (Powers 2011).
The alphavirus group comprises 29 viruses, six of which are called Old World
alphaviruses, and they can cause human joint disorders (arthralgia evolving to
arthritis). This is the case for CHIKV, o’nyong-nyong virus (ONNV), Semliki forest
virus (SFV), Ross River (RRV), Sindbis virus (SINV), and Mayaro virus (MAYV).
The acute phase of the disease with Old World alphaviruses is highly symptomatic
(>90%) and is characterized mostly by fever, generalized myalgia, and arthralgia
(Borgherini et al. 2008, 2007). Arthralgia and crippling arthritis are symptoms that
can persist for years (Simon et al. 2011; Sissoko et al. 2009).
In contrast, the so-called New World alphaviruses such as the eastern equine
encephalitis virus (EEEV) and Venezuelan equine encephalitis virus (VEEV) are
mostly known for their profound neuropathological activities. These alphaviruses
were isolated from diseased horses in California, Virginia, and New Jersey and from
humans in Venezuela. Since then, these viruses have been isolated from infected
mosquitoes (Culex), horses, humans, and other vertebrate species, predominantly
birds and rodents.
Due to their worldwide emergence/reemergence as well as potential agents of
bioterrorism, EEEV, VEEV, and CHIKV have been declared high priority pathogens
by the National Institutes of Health.
Remarkably, it has long been known that CHIKV can contribute to neuropathol-
ogy but by mechanisms largely ill-characterized (Carey et al. 1969; Chastel 1963;
Chatterjee et al. 1965; Hammon et al. 1960; Jadhav et al. 1965; Nimmannitya et
al. 1969; Thiruvengadam et al. 1965). The attack rate for CHIKV disease can be
Neurological Chikungunya 23
TABLE 2.1
Neurological Chikungunya in Human and Animal Model
Neonate/
Symptoms Species Adult Infant References
Headache Human Yes – (Lemant et al. 2008;
Lewthwaite et al. 2009;
Robin et al. 2008)
Reduced consciousness Human Yes – (Rampal et al. 2007; Robin
et al. 2008)
Febrile seizures Human Yes Yes (Lewthwaite et al. 2009;
Ramful et al. 2007; Robin
et al. 2008)
Flaccid paralysis Human/mouse Yes – (Singh et al. 2008)
Meningeal syndrome Human Yes Yes (Robin et al. 2008)
Guillain-Barré syndrome Human Yes – (Economopoulou et al.
2009; Robin et al. 2008;
Wielanek et al. 2007)
Epileptic seizures Human Yes Yes (Economopoulou et al.
2009; Ramful et al. 2007)
Acute encephalopathy Human Yes Yes (Gerardin et al. 2008;
Lemant et al. 2008; Robin
et al. 2008)
Neuropathy Human Yes – (Chandak et al. 2009)
Myeloneuropathy Human Yes – (Chandak et al. 2009;
Kashyap et al. 2010)
Myelomeningoencephalitis Human Yes – (Economopoulou et al.
2009)
Encephalomyeloradiculitis Human Yes – (Ganesan et al. 2008)
Meningoencephalitis Human/macaque Yes – (Economopoulou et al.
2009; Robin et al. 2008)
Optic neuritis Human Yes – (Rampal et al. 2007)
Encephalomyelitis Human Yes – (Rampal et al. 2007)
Meningitis Human Yes – (Lewthwaite et al. 2009)
Encephalitis Human Yes Yes (Casolari et al. 2008;
Chandak et al. 2009;
Economopoulou et al.
2009; Kashyap et al. 2010;
Rampal et al. 2007; Robin
et al. 2008)
Encephalitis Mouse Yes (Wang et al. 2008)
very high; a survey on Grande Comoros Island in 2005 suggested an attack rate of
about 50% (Sergon et al. 2007), and in Reunion Island, 266,000 cases were reported
(38% of the population) during 2005–2006. Hence, it is certainly the unprecedented
incidence rate in the Indian Ocean with efficient clinical facilities that allowed a
better description of CHIKV cases with severe encephalitis, meningoencephalitis,
peripheral neuropathies, and deaths among neonates (mother-to-child infection), and

infants as well as in elderly patients (Economopoulou et al. 2009; Gerardin et al.
2008; Lemant et al. 2008; Lewthwaite et al. 2009; Ramful et al. 2007; Robin et al.
2008; Tandale et al. 2009) (see Table 2.1).
2.2 NEUROLOGICAL CHIKUNGUNYA IN HUMAN NEONATES

Mother-to-child transmission of CHIKV with an estimated prevalence rate of 0.25%
was first reported during the 2005–2006 outbreak in La Réunion Island, and this
novel mode of transmission occurring during the peripartum maternal infection was
responsible for a high rate of neurological morbidity (Gerardin et al. 2008; Ramful
et al. 2007). Almost 50% of the neonates were infected from mother with intra-
partum viremia. In a population-based series of 47 cases of perinatal mother-to-
child CHIKV infection in La Réunion island, neonates developed illness from day
2 to day 10 (mean, 4 days) after birth and 16 (34%) presented severe complications
including encephalopathy with seizures in 9 cases in the acute phase of the disease.
Chikungunya alphavirus neurological involvements (human and animal models)
Perivascular white Meningoencephalitis

matter lesions
Arachnoid membrane
Ventricles Pial membrane
Demyelination Choroid
plexus
Neuronal necrosis Encephalitis

Neuronal apoptosis
Cerebellum
Purkinje cells
Perivascular infiltrates Peripheral nerve
Encephalomyeloradiculitis
Perivascular hemorrhages
Optic neuritis
Guillain-Barré
FIGURE 2.1 Chikungunya neuropathology. CHIKV is not classically considered as a true

neurotropic virus, but there is substantial recent evidence from neonates and elderly severe
CNS cases and together with data from experimental infections (mice and macaque) using
different CHIKV strains. CHIKV can infect neurons and cause apoptosis together with mild
gliosis (astrocytes and microglia). Activated glial cells may contribute to the inflammatory
response potentially contributing to reported demyelination and perivascular white matter
lesions. Cells of the adaptive immune response (T and B cells) recruited at the site of injury
may have a double-edged sword activity to either protect from infection or promote further
neurotoxicity and demyelination. Of critical note, CHIKV and several other alphaviruses can
infect the epithelial cells of the choroid plexus and the ependymal cells of the brain ventricles
next to the stem cell niche. Peripheral neuropathies have also been reported in acute CHIKV
infection in humans.
Mechanical ventilation was needed in 25% of patients due to apneic spells, status
epilepticus, or hemodynamic instability, and one neonate died because of necrotiz-
ing enterocolitis. Pathological brain MRI was noted in 17 of 30 patients with brain
swelling, scattered white matter lesions in the supratentoriel regions, including the
corpus callosum and the periventricular and subcortical areas, parenchymal hemor-
rhages (hematomas and petechias), and early cytotoxic edema on diffusion-weighted
sequences evolving toward vasogenic edema in the subsequent course of the dis-
ease (Gerardin et al. 2008; Samperiz et al. 2007). Head ultrasound was unspecific
with sometimes lenticulothalamostriatal vasculitis. CHIKV RNA was detected in
spinal fluid even in apparently uncomplicated cases (23 of the 26 patients tested)
even if biochemical and cellular characteristics of the cerebrospinal fluid (CSF) were
often unremarkable. Preliminary data concerning long-term clinical follow-up of the
infected neonates confirm poor outcome with a mean developmental quotient of 86
(51% of the cases <85) compared with 100 in the control group (p < 0.001) at 2 years
old (D. Ramful, personal communication). Gerardin et al. (2008) described persis-
tent disabilities, with cerebral palsy, behavioral deficiencies, epilepsia, and language
delay in 4 patients (of 9 with neonatal encephalopathy), where long-term sequelae in
imaging studies sometimes included parenchymal cavitations secondary to hemato-
mas and cerebral subcortical atrophy. Conversely, in maternal infection occurring
far from delivery, there was no propensity to prematurity, growth restriction, fetal
deaths, stillbirths, or congenital anomalies, and newborns seemed to be healthy at
birth with no detectable IgM antibody at birth (Fritel et al. 2010) (Figure 2.1).
2.3 NEUROLOGICAL MANIFESTATIONS IN PEDIATRIC PATIENTS

CHIKV fever is usually benign in children and neurological manifestations are rare.
However, the two deaths reported in La Réunion Island in children (excluding the
neonate mentioned before) were associated with severe neurological presentation
of the disease including a case of acute disseminated encephalomyelitis and one
case of coma associated with an acute hemorrhagic shock syndrome (Robin et al.
2008). In a hospital-based study in La Réunion Island, Robin et al. (2008) described
a case series of 30 pediatric patients with neurological manifestations associated
with CHIKV infection ranging from simple (n = 4) and complex febrile seizures
(n = 6) to meningeal syndrome (n = 4), acute encephalopathy (n = 4), diplopia, acute
disseminated encephalomyelitis (n = 1), and encephalitis (n = 11) with often unre-
markable CSF findings and unspecific electroencephalography. Cerebrospinal fluid
pleocytosis was found in only 4% of examined cases, although CSF RT-PCR was
positive in 61%. Similar findings were subsequently described in infants in India and
Mayotte Island (Le Bomin et al. 2008; Lewthwaite et al. 2009).
Risk factors for residual neurological deficit (20% of the patients) in the La
Réunion cohort included young age (neonatal infection), severe initial clinical pre-
sentation (encephalitis), and initial pathological MRI findings. Neurological clinical
manifestations of CHIKV infection in the pediatric population added growing evi-
dence to the potential neurovirulence of this arboviral disease with an age-dependent
condition (neonatal infection) and consistent laboratory (CHIKV RNA in CSF of
patients) and imaging features (pathological MRI). These findings are consistent
with the below mentioned mouse models, where young age is a risk factor for severe
disease involving the CNS.
2.4 NEUROLOGICAL CHIKUNGUNYA IN ADULTS

CHIKV is highly symptomatic (arthralgia, myalgia) over a period of days to weeks,
and most patients will recover. However, neurological manifestations described in
adults requiring hospitalization involved cases of encephalopathy frequently associ-
ated with lymphopenia, thrombocytopenia, elevated CRP, and the presence of IgM
anti-CHIKV in the CSF (Lemant et al. 2008). It was evident that these patients had
severe comorbidities (e.g., diabetes, renal impairment) and with a mortality of almost
50% probably not directly related to CHIKV (Lemant et al. 2008). Similar neuro-
logical complications and fatalities were reported by several groups looking at severe
hospitalized CHIKV cases from India (Chandak et al. 2009; Rampal et al. 2007;
Tandale et al. 2009). Other neurological complications associated with CHIKV were
seizures, encephalitis, Guillain-Barré, encephalomyeloradiculitis, and rare deaths
(Chandak et al. 2009; Economopoulou et al. 2009; Ganesan et al. 2008; Lebrun et
al. 2009; Lemant et al. 2008; Tournebize et al. 2009; Wielanek et al. 2007). Febrile
seizures were found to be associated with both adults and neonates (simple febrile
seizures) (Lewthwaite et al. 2009; Ramful et al. 2007; Robin et al. 2008), whereas
epileptic seizures were observed in 12 of 610 patients (Economopoulou et al. 2009).
Three cases of Guillain-Barré syndrome with associated symptoms of facial palsy
and weakness in the hand, feet, or both were observed in La Réunion (Wielanek
et al. 2007). Encephalomyeloradiculitis was observed after CHIKV infection by
neuroimaging data in two patients and brain autopsy in one patient. In both cases,
the symptoms were associated with neck rigidity, drowsiness, and extensor plantar
response (Ganesan et al. 2008). Of critical note, these reports should not be general-
ized, given that they are hospital-based studies and were obtained from sick patients
with high proportions of complications.
CHIKV infection in adults was also associated to bilateral frontoparietal white
matter lesions with restricted diffusion, which are described as an early sign of viral
encephalitis (Ganesan et al. 2008). Focal perivascular lymphocytic infiltrates were
also present in areas of active demyelination, and some degree of microglial activa-
tion was also noted in the gray matter, which may contribute to bystander neuronal
loss. Regrettably, this is one of the rare histopathological studies substantiating the
contribution of CHIKV to brain damage and most of the evidence comes from in
vitro studies and animal models (in mice and macaques).
2.5 NEUROLOGICAL CHIKUNGUNYA: ROUTES TO

CNS INFECTION AND TISSUE INJURY
Viruses can instigate neurological injuries not only by direct cytolytic actions on
neurons or glia (oligodendrocytes, astrocytes) but also by inducing apoptosis, dis-
rupting of the protective blood–brain barrier (BBB), polarizing resident innate
immune cells (microglia) to produce proinflammatory cytokines, initiating (auto)
immune attack on specific cells, expressing viral genes and inhibiting cellular genes,
altering neuronal migration, attenuating neural progenitor replication, and blocking
CSF generation and flow. The multiple mechanisms of viral induction of CNS neu-
roinfection and dysfunction further complicate our understanding of viral agents in
brain disease.
Although data are still scarce, the number of recent human cases with CNS
involvement appears to support the neurotropic/neuroinfectious activity of CHIKV
(Ganesan et al. 2008; Gerardin et al. 2008; Ramful et al. 2007). This unique CNS
infection illustrated by subventricular white matter lesions, and intraparenchy-
mal hemorrhages have been described experimentally and in clinical settings for
other alphaviruses such as SFV, RRV, EEEV, and SINV (Deresiewicz et al. 1997;
Fazakerley et al. 2006; Jackson et al. 1987; Mims et al. 1973) (see Table 2.2).
CHIKV was shown to infect mouse brain and to replicate in primary culture of
neurons and glial cells (Chatterjee and Sarkar 1965; Das et al. 2010; Precious et al.
1974). CHIKV can also replicate in a human neuroblastoma cell line, SH-SY5Y,
and cause cytopathic activities (Dhanwani et al. 2011; Solignat et al. 2009). Further
evidence comes from experimental infections where mice were inoculated with
CHIKV (clinical isolates and genetic clones). Interestingly, CNS infection is par-
ticularly described in young mice (outbred CD1, ICR) and recapitulating the human
clinical disease (Ziegler et al. 2008). Infected mice showed signs of illness sugges-
tive of human clinical pathology such as loss of balance, difficulty of walking, drag-
ging of the hind limbs, skin lesions but with rare mortality. No definite histological
evidence of tropism to neurons was reported in these two mouse models, and the
CNS infection seemed to be tightly controlled by ill-characterized antiviral mecha-
nisms given that the viral titer was reduced to basal levels at day 10 postinfection.
BALB/c mice infected intranasally developed neuronal infection and tissue necrosis
in the anterior olfactory lobe (Powers and Logue 2007). Weaver and colleagues also
used intranasal injection of CHIKV but the Ross strain selected because of its exces-
sive mouse passage history and which may have increased its neurovirulence (Wang
et al. 2008). The 5-week-old C57BL/6 mice developed encephalitis 7 days postinfec-
tion with severe multifocal infection and liquefactive necrosis in the cerebral cortex.
Immunohistochemistry techniques revealed that neurons were infected and induced
to apoptosis while a prominent microgliosis and perivascular cuffs were distributed
throughout the parenchyma. Moreover, the authors reported neuronal degeneration
in the hippocampus and multifocal lymphocytic leptomeningitis. In mice deficient
in the IFN-α signaling pathway (KO for the IFN-α receptor), CHIKV neuroinfec-
tion was particularly severe and targets the leptomeninges, the choroid plexus, and
ependymal cells lining the subventricular zone (SVZ) also known as the neural stem
cell niche (Hauwel et al. 2005a,b). Of critical note, RRV was also shown to infect
ependymal cells and lead to cortical thinning and hydrocephalus (Mims et al. 1973).
To what extent CHIKV infection could affect the SVZ niche and subsequently the
stem cells is currently unknown. There is also little evidence about the cellular and
molecular mechanisms of brain tissue injury, which can be direct (neurotoxicity) or
indirect through the mobilization of glial cells. To promote host survival, infected
cells may undergo apoptosis, which can be qualified as an “altruistic” suicide in
response to viral infections; however, neurons have limited capacity for regeneration.
TABLE 2.2
Cellular Targets, Immune Response, and Long-Term Consequences Following
CNS Alphavirus Infection
CNS Immune
Alphavirus CNS Target Cell Response Long-Term Consequences
SINV (mice) Neurons (Griffin and Production of IL-1β, Hind limb paralysis, death (Griffin
Johnson 1977; IL-4, IL-6, IL-10, and Johnson 1977; Jackson et al.
Johnson 1965) TNF-α, LIF, and 1987)
Purkinje cells TGF-β (Wesselingh et Acute encephalomyelitis (Jackson
(Johnson 1965) al. 1994) et al. 1987), Kyphoscoliosis
Meningeal cells Production IFN-γ by (Jackson et al. 1988)
(Johnson 1965) CD4+ and CD8+ Swelling of lumbar and thoracic
Ependymal cells (Binder and Griffin neurons (Jackson et al. 1988)
(Jackson et al. 2001) Death (Griffin and Johnson 1977;
1987; Jackson et al. Apoptosis of infected Jackson et al. 1987)
1988; Johnson neurons (Levine et al.
1965) 1993; Nava et al.
1998)
Bcl-2 protects against
fatal encephalitis
(Levine et al. 1996)
EEEV Neurons (mouse Pleocytosis Large spectrum of general
(mouse and model and human) (Deresiewicz et al. neurological complications
human) (Deresiewicz et al. 1997) (confusion, somnolence, focal
1997; Griffin 2003) In the CSF weakness, epileptiform
(Deresiewicz et al. discharges, seizures, stupor,
1997): altered mental status,
- Elevated protein paresthesia accompanying
concentrations paresis, hemiparesis)
- Elevated red blood (Deresiewicz et al. 1997); lesions
cell counts of the basal ganglia and cortex,
Leukocytosis encephalomalacia, focal
(Deresiewicz et al. intraparenchymal perivascular
1997) hemorrhage in the caudate
Hyponatremia nucleus and putamen,
(Deresiewicz et al. microglial nodules, meningeal
1997) enhancement, leptomeningeal
vascular congestion, brain
hemorrhage, neuronal
destruction, neuronophagia,
cranial nerve palsies, focal
necrosis, spotty demyelination)
(Deresiewicz et al. 1997); coma
and death (Deresiewicz et al.
1997)
TABLE 2.2 (Continued)

Cellular Targets, Immune Response, and Long-Term Consequences Following
CNS Alphavirus Infection
CNS Immune
Alphavirus CNS Target Cell Response Long-Term Consequences
VEEV Neurons (Griffin Meningeal infiltrates General neurological diseases
(human) 2003) composed of (somnolence, confusion,
lymphocytes, disorientation, mental
mononuclear cells, depression, convulsions,
and neutrophils seizures, ataxia, paralysis)
(Steele and Twenhafel (Steele and Twenhafel 2010);
2010) predominant CNS pathologies
(edema, congestion,
hemorrhages, vasculitis,
meningitis, encephalitis) (Zacks
and Paessler 2010); common
neurological sequelae (Zacks and
Paessler 2010); coma and death
(Steele and Twenhafel 2010)
WEEV* In the brain (Reed et Monocytic Demyelination and myelitis in
(macaque al. 2005) inflammation in the white matter areas (Reed et al.
and human) - Microglia CNS expanding 2005)
- Purkinje cells perivascular spaces General neurological diseases
In the spinal cord (Reed et al. 2005) (partial left seizures, deep
(Reed et al. 2005) Occasional infiltrates consciousness depression,
- α-motor neurons of lymphocytes, hyperreflexia, bilateral Babinski
plasma cells, PMN sign) (Delfraro et al. 2011)
leukocytes Severe CNS disorders
surrounding (multifocal necrosis in the deep
arterioles in the gray matter, dilation of the
cerebral cortex temporal ventricles and
(Anderson 1984) compression of the peritroncal
and sylvian cisterns) (Anderson
1984; Delfraro et al. 2011)
Coma and death (Delfraro et al.
2011)
* Italicized, NHP model; boldface, human studies.
CHIKV can cause programmed cell death through extrinsic apoptosis (death recep-
tor/caspase 8 pathway), intrinsic apoptosis (cytochrome C/caspase 9 pathway) or
autophagy of many cell types including neuroblastoma cells (Krejbich-Trotot et al.
2011) and unpublished observations. This will need to be confirmed using primary
cultures, but it should be stressed that this is also salutary to the CNS tissue to limit
virus spreading.
Astrogliosis and microgliosis have been reported in human and animal models of
CHIKV neuroinfection, and these responses may be essential to ward off the infec-
tious challenge through the production of interferon and interferon-stimulated gene
(for a review, see Ryman and Klimstra 2008). The immune response to CNS infection
has double-edged sword activity, which protects from infections, on the one hand, and
promotes further tissue injury if uncontrolled, on the other (Hauwel et al. 2005a). It is
essential to have a better understanding of the plausible role of innate immune effec-
tors such as cytokines and complement proteins produced at the site of injury. This
is largely unknown in the case of CHIKV neuroinfection, and important information
should be obtained from other alphaviruses affecting brain cells and functions.
2.6 NEUROINFECTION BY “OLD WORLD” SINDBIS VIRUS

SINV was named after its first isolation in Sindbis health district near Cairo (Egypt)
from a pool of Culex pipiens and Cx univittatus mosquitoes (Taylor et al. 1955).
Other synonyms or subtypes of SINV have been described in Sweden (Ockelbo
disease), Finland (Pogosta disease), and Russia (Karelian fever) in the early 1980s,
according to the region involved (Brummer-Korvenkontio and Kuusisto 1981; Lvov
et al. 1982; Skogh and Espmark 1982). SINV can cause fever, rash, and arthralgia in
humans (Turunen et al. 1998) and encephalomyelitis in mice (Jackson et al. 1987).
Neuroadapted SINV (NSINV) is a neurovirulent strain that have been developed by
serial passage of the original isolate AR339 of SINV from mouse brain (Griffin and
Johnson 1977). Infection of weanling mice with NSINV induced an acute encepha-
lomyelitis with high mortality rate, and animals can develop kyphoscoliosis, which
is an abnormal curvature of the spine, and hind-limb paralysis (Jackson et al. 1987,
1988). Swelling of lumbar and thoracic neurons has also been documented in NSINV
infection (Jackson et al. 1988). SINV is known to infect the Purkinje cells of the
cerebellum and meningeal and ependymal cells in mouse (Griffin and Johnson
1977; Jackson et al. 1987, 1988; Johnson 1965). The maturity of infected neurons
determines their susceptibility to SINV. Immature neurons replicate SINV to high
titers and undergo apoptosis, whereas mature neurons are more resistant to SINV
replication and survive viral infection (Binder and Griffin 2001; Burdeinick-Kerr
and Griffin 2005). It was shown that infection of neonatal mice with a SINV chi-
mera expressing bcl-2, an anti-apoptotic gene, induced a lower mortality rate (7.5%)
(Levine et al. 1996). Hence, controlling neuronal apoptotosis protects mice against
fatal SINV encephalitis. After intracerebral inoculation of SINV in mice, transcripts
of IL-1β, IL-4, IL-6, IL-10, TNF-α, leukemia inhibitory factor (LIF), and TGF-β
were produced by brain cells in response to infection and may contribute to neuronal
loss (Wesselingh et al. 1994).
2.7 NEUROINFECTION BY NEW WORLD

ALPHAVIRUSES: EEEV, VEEV, WEEV
EEEV, VEEV, and WEEV are New World alphaviruses whose symptoms can be
similar to those described for SINV, but as their names suggest, they can frequently
cause severe encephalitis in humans and horses (Zacks and Paessler 2010). They
are naturally maintained through enzootic cycles involving arthropods as vectors
with ensuing amplification in small mammals or birds, and epizootic cycles between
bridging mosquitoes vectors and large mammals such as horses and humans, which
are dead-end hosts, as they are not viremic enough to infect mosquitoes and propa-
gate the cycle. Among the three, EEEV seems to be the most virulent in humans,
causing mortality in approximately 40% of symptomatic cases (Deresiewicz et al.
1997). In survivors, permanent neurological sequelae can occur, and some with
severe impairment die within a few days.
EEEV was first isolated in Virginia and New Jersey from infected horses in 1933
(Giltner and Shahan 1933; TenBroeck and Merrill 1933) and was first recognized to
infect humans in 1938 after an outbreak in Massachusetts (Feemster 1938). Three chil-
dren died and two had encephalitis. An interesting coincidence was that those five
cases occurred in essentially the same area as the equine disease. The eastern strain
of the equine encephalomyelitis virus had next been isolated from human brain tis-
sue of the first case (Feemster 1938). Enzootic transmission cycle of EEEV involves
ornithophilic mosquitoes (mainly Culiseta melanura) and passerine birds (Komar and
Spielman 1994; Scott and Weaver 1989). EEE usually begins abruptly and rapidly
instigate fever, chills, myalgia, and arthralgia; after a few days, neurological signs may
appear. EEEV has not been as well studied in animals as VEEV; nonetheless, a variety
of animal models have been described, such as mice, hamsters, guinea pigs, and non-
human primates (NHP), which are the best studied (Zacks and Paessler 2010). In mice
and human, EEEV is known to infect neurons (Deresiewicz et al. 1997; Griffin 2003).
VEEV was first isolated in Venezuela from the brain of an encephalitic horse in
1938 (Kubes and Rios 1939) and first recognized to infect humans in 1943 (Casals
et al. 1943). VEEV is efficiently amplified during a cycle involving equids and mos-
quitoes that occurs in an agricultural area (Aguilar et al. 2011). In human, VEEV is
usually an acute and often mild systemic disease. Clinical signs can be characterized
by fever, chills, generalized malaise, severe headache, photophobia, and myalgia
mainly localized in the legs and lumbosacral region. Very young or elderly patients
are more likely to develop severe infections. In adults, cases of encephalitis and
fatality are scarce (Zacks and Paessler 2010). In mice, neurons are cellular targets
for VEEV (Griffin 2003). CNS infection by VEEV has been shown to induce the
infiltration of lymphocytes, mononuclear cells, and neutrophils in the meninges. In a
minority of cases, these inflammatory cells extended into the Virchow-Robin spaces,
which are tiny fluid-filled canals surrounding arteries and veins in brain parenchyma
(Steele and Twenhafel 2010). In humans, VEEV-induced fatality can occur in one
third of children and 10% in adults of cases. Nevertheless, neurological diseases
including disorientation, ataxia, mental depression, and convulsions can reach 14%
of cases, mainly in children. The severity of neurological complications can range
from somnolence and mild confusion to seizure, ataxia, paralysis, and coma (Steele
and Twenhafel 2010). The predominant CNS pathological findings in deadly VEEV
human cases comprise edema, congestion, hemorrhages, vasculitis, meningitis, and
encephalitis (Zacks and Paessler 2010).
WEEV was first isolated in California from the brain of horses suffering from
encephalitis in 1930 (Meyer et al. 1931). In 1938, the virus had been recovered for
the first time from the brain of a 20-month-old boy in California (Howitt 1938).
WEEV is maintained in an enzootic cycle between passerine birds, which are its
natural host and culicine mosquitoes, with a variety of mammals as incidental hosts.
WEEV is usually asymptomatic and much milder than EEE. The disease gener-
ally appears abruptly and potentially includes fever, chills, headache, nausea, vom-
iting, anorexia, malaise, and occasional respiratory signs. WEEV appears to be a
recombinant virus originated from a recombination between an EEEV-like and an
SINV-like ancestor giving rise to a new virus with encephalogenic properties of
EEEV and the antigenic specificity of SINV (Hahn et al. 1988). After aerosol expo-
sure to WEEV in the NHP model, abundant microglia and neurons in the cerebral
cortex were WEEV-positive. In addition, a low amount of cerebellar Purkinje cells
and alpha-motor neurons in the spinal cord gray matter showed WEEV antigens
(Reed et al. 2005). From clinical and radiographic data of 38 patients infected by
EEEV, Deresiewicz et al. (1997) found biological abnormalities including pleocy-
tosis (97% of cases), elevated protein concentrations and red blood cell counts in
CSF (94% and 77%, respectively), leukocytosis (69%), and hyponatremia (60%).
They reported a wide spectrum of neurological complications such as confusion,
somnolence, focal weakness, epileptiform discharges, seizures, stupor, altered men-
tal status, paresthesia accompanying paresis, and hemiparesis. Lesions of the basal
ganglia and cortex were observed in a 14-year-old boy who died from EEE and for
whom autopsy revealed a diffuse encephalomalacia, marked perivascular chronic
inflammatory changes, and focal intraparenchymal perivascular hemorrhage in the
caudate nucleus and putamen. Several microglial nodules were noticed. Deresiewicz
et al. (1997) also reported meningeal enhancement, leptomingeal vascular conges-
tion, brain hemorrhage, neuronal destruction, neuronophagia, cranial nerve parlsies,
focal necrosis, spotty demyelination, encephalomalacia, coma, and death.
After aerosol exposure to WEEV of NHP, Reed et al. (2005) demonstrated a pro-
nounced encephalitis characterized by monocytic inflammation expanding in peri-
vascular spaces and infiltrating into the surrounding neutrophils. They found that
infection of WEEV in the CNS of NHP resulted in multifocal areas of demyelination
in the white matter of the brain and spinal cord, occasionally associated with inflam-
mation. In humans, Anderson (1984) reported a fatal case in a 75-year-old woman
infected with WEEV and presenting perivascular infiltrates and multifocal necrosis
in the deep gray matter. Recently, a fatal human case was reported in which the
14-year-old boy experienced partial left seizures, consciousness depression, hyper-
reflexia, and bilateral Babinski sign (Delfraro et al. 2011). Depression of conscious-
ness progressed at a deeper level, and the brain showed dilatation of the temporal
ventricles and compression of the peritroncal and sylvian cisterns. The level of coma
progressed until the patient died (Delfraro et al. 2011).
2.8 CONCLUSION
As highlighted throughout this review, our understanding of neurological and
potentially encephalitic alphavirus is still in its infancy. CHIKV in addition to its
profound arthritogenic activity also has encephalitic potential particularly in new-
borns and elderly patients with severe comorbidities. Moreover, CHIKV is known
to persist in tissue sanctuaries (not in the brain as far as we know) and contrib-
utes to chronic diseases. Some CHIKV elderly patients can experience rheumatism
5 years postinfection and with long-term brain development defects in neonates.
Although cell permissiveness and reactivity has been studied in great depth, the
mechanisms of CHIKV persistence and associated tissue injuries remains largely
ill-characterized. Therefore, with a proven potential to spread globally, it is now
critical to devise strategies to circumvent infection of populations at risk and new
epidemics. In the absence of a specific antiviral therapy, treatment remains sup-
portive. Recently, the development of polyvalent immunoglobulins, purified from
human plasma samples of convalescent patients that exhibited in vitro and in vivo
neutralizing activities could be of benefit to neonates born from viremic mothers at
delivery and to encephalitic patients at the initial stage of the disease (Couderc et
al. 2009). Moreover, protection against mosquito bites remains essential in CHIKV
prevention. Parental training about prevention of mosquito bites during the peri-
natal period and distribution of impregnated mosquito nets should be carried out
during outbreaks. DEET (N,N-diethyl-m-toluamide, now called N,N-diethyl-3-
methylbenzamide) is considered as the most effective insect repellent for personal
protection but is not recommended for young children, pregnant, and lactating
women due to potential neurotoxic effects.
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3 Arenaviruses and
Neurovirology
Larry Lutwick and Jana Preis
CONTENTS
3.1 Introduction..................................................................................................... 39
3.2 History............................................................................................................. 41
3.3 Biological Properties....................................................................................... 42
3.4 Clinical Presentation.......................................................................................44
3.4.1 Lymphocytic Choriomeningitis Virus.................................................44
3.4.1.1 Rodent Infection....................................................................44
3.4.1.2 Human Infection...................................................................44
3.4.1.3 Normal Host..........................................................................44
3.4.1.4 Immunocompromised Host..................................................46
3.4.1.5 Congenital Infection............................................................. 47
3.4.2 Hemorrhagic Fever Arenaviruses........................................................ 49
3.4.2.1 Rodent Infection.................................................................... 49
3.4.2.2 Human Viral Hemorrhagic Fever Infection.......................... 50
3.4.3 Lassa Fever.......................................................................................... 50
3.4.4 South American Hemorrhagic Fevers................................................. 51
3.5 Diagnosis......................................................................................................... 52
3.6 Epidemiology................................................................................................... 53
3.7 Treatment......................................................................................................... 54
3.7.1 Immune Plasma Therapy..................................................................... 54
3.7.2 Antiviral Therapy................................................................................ 54
3.7.2.1 Ribavirin............................................................................... 54
3.7.2.2 Nonribavirin.......................................................................... 55
3.8 Preventive Measures........................................................................................ 56
3.8.1 Vaccines............................................................................................... 56
3.9 Antiviral Postexposure Prophylaxis................................................................ 57
3.10 Future Perspectives.......................................................................................... 58
References................................................................................................................. 59
3.1 INTRODUCTION
The family Arenavirdiae represents a unique genus (Arenavirus) containing more than
30 viruses classified into two groups of agents based on their antigenic properties.
The Old World (Eastern Hemisphere) group also referred to as the Lassa-
lymphocytic choriomeningitis (LCM) serocomplex contains viruses indigenous
39
to Africa and LCM (Table 3.1). LCM is the only arenavirus to have a worldwide
distribution because of the ubiquitous distribution of its rodent reservoirs (primar-
ily Mus musculus and M. domesticus), whereas each of the other arenaviruses has
a limited geographic distribution directly related to the range of its specific rodent
reservoir. The New World (Western Hemisphere) group also called the Tacaribe
serocomplex (Table 3.2) is divided into three lineages (clades) designated as A, B,
and C.
Representatives of each of the groups are clearly causes of zoonotic infections of
man, that is, infections primarily of animals that can be passed directly or indirectly
to man. Many of the groups, however, are rare or unproven causes of human infec-
tion. In many ways, LCM is one of the ancestors of neurovirology because it was
the first clearly documented cause of viral meningitis and much of this chapter will
be devoted to the classical agent and the manifestations of disease that it may cause.
Human infections caused by other members of Arenaviridae are primarily viral
hemorrhagic fevers including Lassa fever in parts of Africa and primarily members
of clade B of the New World group including the Guanarito, Junin, Machupo, and
Sabia viruses.
TABLE 3.1
Old World Arenaviruses Relevant to Human Infection
Lymphocytic Choriomeningitis Virus
Disease Lymphocytic choriomeningitis
Location Wide distribution especially Europe and
Americas
Reservoir Mus musculus, M. domesticus (house mouse),
Mesocricetus auratos (Syrian hamster)
Reservoir habitat Domestically, grasslands
Seasonality September, October
Human acquisition Peridomestically
Lassa Virus
Disease Lassa fever
Location West Africa, especially Sierra Leone, Nigeria,
Liberia, Guinea
Reservoir Mastomys natalensis (multimammate mouse)
Reservoir habitat Savannah, forest clearings
Seasonality January to April
Note: Other viruses with unclear or little human significance include Mopeia virus (Southern
Africa), Mobala virus (Central African Republic), Ippy virus (Central African
Republic), Lujo virus (Zambia), Merino Walk virus (South Africa), and Morogoro
virus (Guinea).
Arenaviruses and Neurovirology 41
TABLE 3.2
New World Arenaviruses Relevant to Human Infection
Guanarito Virus (Clade B)
Disease Venezuelan hemorrhagic fever
Location Central Venezuela Portuguesa State and adjacent regions of Barinas
state
Reservoir Zygodontomys brevicauda (cane mouse)
Reservoir habitat Agricultural areas
Seasonality November to January
Human acquisition Agriculturally
Junin Virus (Clade B)

Disease Argentine hemorrhagic fever
Location Argentine provinces of Buenos Aires, Córdoba, Santa Fe, and La Pampa
Reservoir Calomys musculinus (corn mouse), Akondon azalae (grass field mouse),
Bolomys obscurus (dark field mouse)
Reservoir habitat Cultivated fields, grasslands
Seasonality February to May
Human acquisition Agriculturally
Machupo Virus (Clade B)

Disease Bolivian hemorrhagic fever
Location Northeastern Bolivia, especially Beni Department
Reservoir C. callosus (vesper mouse)
Reservoir habitat Brush, peridomestically
Seasonality April to July
Note: The Sabia virus (clade B) from Brazil (Brazilian or Sao Paolo hemorrhagic fever) has caused one
natural infection and several laboratory infections. Other virus of unclear or little human rele-
vance, some of which have caused human infection, include the Allpahuayo virus (Peru), Parana
virus (Paraguay), and Pinchinde virus (Colombia) in clade A; the Amapari virus (Brazil) and
Tacaribe virus (Trinidad) in clade B; and the Latino virus (Bolivia, Brazil), Oliveros virus
(Argentina), and others of unclear taxonomic status such as U.S. isolates of Whitewater Arroyo
virus, Big Brushy Tank virus, Tonto Creek virus, and Bear Canyon virus in clade C.
3.2 HISTORY
In 1934, reports (Armstrong and Lillie 1934; Armstrong and Wooley 1935) began
to appear of a previously undescribed virus causing neurological disease in man.
The original isolation was discovered during passage, in monkeys, of an agent from
brain tissue of an individual who died in St. Louis, Missouri, during an encephalitis
epidemic in 1933. In monkeys and subsequently in mice, the pathology found was
a lymphocytic meningitis, most prominent in the choroid plexus structure in the
ventricles of the brain where cerebrospinal fluid is produced, consisting of modified
ependymal cells. Because of the histopathologic appearance, the agent was called
lymphocytic choriomeningitis virus. Although fatal cases of infection were well
represented in the initial cases, the most common disease state recognized due to
LCM was a benign, self-limited meningitis.
As LCM was the first agent isolated in cases of what became referred to as acute
aseptic meningitis in Wallgren’s (1925) original description, lymphocytic meningitis
(Hughes 1937) or benign lymphocytic meningitis (Dummer 1937), for a substantial
amount of time, LCM and aseptic meningitis were regarded as identical (Roebroek
et al. 1994). Subsequently, numerous other etiologies of the aseptic meningitis syn-
drome have been elucidated including many other viruses but LCM will be indelibly
associated with the disease.
The Lassa fever virus was first recognized in a nosocomial setting when an
American health-care worker, at a mission health care station in the town of Lassa
in northeast Nigeria, became sick and precipitated a chain of health-care-associated
infections locally and involved laboratory workers in the United States.
3.3 BIOLOGICAL PROPERTIES

Arenaviruses possess negatively stranded RNA genomes with a bisegmental orga-
nization (two segments) (Charrel and de Lamballere 2003). The larger genomic seg-
ment (referred to as L) consists of about 7200 nucleotides that code for the virus’
RNA-dependent RNA polymerase and a small RING finger protein Z. The smaller
(S) segment is about 3500 nucleotides in length and codes for two structural pro-
teins, a nucleoprotein (NP), and a glycoprotein precursor (GPC). Each gene on the
segments (Emonet et al. 2006) is separated by an intergenic noncoding region (IGR)
with a potential to be able to form one or more hairpin configurations, and the gene
reading frames are arranged in opposite polarities referred to as ambisense. NP and
the polymerase are transcribed into complementary mRNA, whereas GP and Z are
from a replicative intermediate (Emonet et al. 2009). The 5′ and 3′ ends of each
of the RNA segments possess reasonably well-conserved reversed complementary
sequences of 19 nucleotides at each extremity.
GPC is posttranslationally cleaved into the G1 and G2 envelope proteins of the
virus as well as a small (58-amino-acid) stable signal peptide (SSP) (Emonet et al.
2009). The cleavage is mediated by the SK1/S1P cellular protease (Kunz et al. 2003).
It has been shown that the protease has a strong preference to arenaviral sequences
resembling its autoprocessing sites. The Lassa fever virus resembles the protease
C-site, whereas the Junin virus has a similarity to the B-site (Pasquato et al. 2011).
The authors suggested that arenaviral GP complexes have evolved to mimic the pro-
tease’s autoprocessing sites to ensure efficient cleavage.
SSP appears to be unique as the protein remains associated with the GP complex
after cleavage by the signal protease and helps trafficking of the complex in the
cell (Eichler et al. 2003). The surface glycoproteins associate as trimers to form the
spikes mediating host cell interactions with GP1 on the distal end of the spike. GP1
contains both the receptor binding and the antibody neutralization sites and remains
noncovalently bound to GP2. GP2 contains a transmembrane region that serves to
anchor the GP complex to the lipid bilayer of the virus (Burns and Buchmeirer 1991).
Both glycoproteins contain N-glycosylations and the pattern of glycosylation is well
preserved among arenaviruses. They are important in fusion of the virus to the host
cell and also serve as a “cloud” that can mask epitopes on the GP complex from neu-
tralizing antibodies (Bonhomme et al. 2011).
The Z protein is the major factor involved in newly formed viral release from an
infected cell (Strecker et al. 2003), which is a process mediated by the areas on the
Z protein that are proline-rich, the way many enveloped negative-stranded RNA
viruses accomplish with the matrix protein bridging ribonucleoprotein and GP and
plays a major role in controlling viral RNA production by locking the viral RNA
polymerase in a promoter-bound state and ensuring polymerase packaging during
virus maturation (Kranzusch and Whelan 2011).
Arenavirus virions are spherical to pleomorphic in shape with diameters rang-
ing between 50 and 300 nm, with the average diameter of the spherical forms about
120 nm. The virions possess dense lipid-containing viral envelopes that are cov-
ered with 8- to 10-nm-long club-like projections of the GP complex. Ribosomes,
20–25 nm (acquired from the host cell and do not appear to be required for viral
replication (Leung and Rawls 1977)), are found inside the virions and are the deriva-
tion of the name arenavirus, as the structures give the virion a “sandy” appearance.
The Latin word harena means sand, a sandy place or seashore, or a place of combat,
literally “a place strewn with sand.” The virions are rapidly inactivated by ultraviolet
or gamma wave irradiation and by a pH below 5.5 or above 8.5. Temperatures above
56°C also quickly inactivate the agents.
The diversity and ancestry of LCM, as the prototypic and most widely spread are-
navirus, has been studied (Albariño et al. 2010). In analyzing the RNA of 29 strains
from a variety of geographic sources (including some of the earliest 1935 isolates),
it was found that the strains are highly diverse with several apparent lineages but
without correlation with time or place of isolation. Bayesian analysis estimated that
the most recent common ancestor was 1000 to 5000 years old, consistent with the
complex phylogeographic relationships observed.
As discussed in the sections regarding rodent and human infection, the manifesta-
tions of LCM infection can be quite varied, especially in rodents whether self-limited
or chronic infection occurs. Quite subtle changes in the glycoprotein of LCM have
been shown to be part of the cause of persistence of LCM, as studied in dendritic
cells. Dendritic cells are present in tissues in contact with the external environment,
such as the skin (where there is a specialized dendritic cell type called Langerhans
cells), and the inner lining of the nose, lungs, stomach, and intestines. As an example,
LCM clone 13 infection, which results in persistence, and differs from the standard
Armstrong strain only by few nucleotides, three of which result in coding changes
(Sullivan et al. 2011), two in GP1 and one in the RNA-dependent RNA polymerase.
The GP1 changes (especially F260L) mediate exceptionally strong binding affinity to
the LCM cellular receptor, alpha-dystroglycan. This effect on dendritic cells results in
decreased amounts of costimulatory ligands, an inability to fully prime T cells, up-
regulation of T-cell inhibitory receptors, and difficulties in viral clearance of LCM.
Alternative receptors have also been described for LCM virus (Kunz et al. 2004),
which do not produce immune suppression. A number of the New World arenaviruses
can use human transferring receptor 1 as a cellular receptor (Radoshitzky et al. 2007).
LCM entrance into host cells, after receptor binding, appears to be mediated via
viropexis in large smooth-walled vesicles followed by a pH-dependent fusion event
inside the cell (Borrow and Oldstone 1994). Unlike classical phagocytosis, LCM
uptake is a microfilament-independent process, not related to the direct fusion with
host cell plasma membrane.
3.4 CLINICAL PRESENTATION

3.4.1 Lymphocytic Choriomeningitis Virus
3.4.1.1 Rodent Infection
Wild house mice are the natural reservoir for LCM virus. Laboratory or pet rodents
such as hamsters and guinea pigs can be infected from exposure to feral mice in a
breeding facility, pet store, or home. It is unclear how relevant other rodent species
such as chinchillas, dwarf hamsters, and gerbils are in infecting humans with LCM.
3.4.1.2 Human Infection

Transmission of LCM from the rodent reservoirs to man is generally indirect
through contact with dried rodent urine or droppings in the environment. The expo-
sure can be related to aerosolization of particles contaminated with rodent urine
or saliva, ingestion of contaminated food, or by direct contact with rodent excreta
with abraded or broken skin. The kind of incidental contact depends on the habits
of both the rodent vehicle and man. As an example, if the infected rodent species is
one that prefers a field locale, human infection is associated with agricultural work.
Alternatively, if the rodent (like the house mouse) inhabits the urban setting, the
arenavirus can be acquired in a domestic setting. Human-to-human transmission
of LCM virus does not occur except in intrauterine infection and related to organ
transplantation as discussed below.
3.4.1.3 Normal Host

In man, the manifestations of LCM virus infection outside the neonatal period can
range from the asymptomatic state (in about a third of cases) to an influenza-like
illness without neurological symptomatology (in about half of the cases) to the clas-
sical aseptic meningitis, which more rarely can be an overt meningoencephalitis,
potentially fatal in outcome.
Initially, LCM manifests with fever and headache that can be also associated
with a nonspecific lymphadenopathy (enlarged lymph glands) and a maculopapular
rash. The symptom complex can also include malaise, sometimes striking muscle
aches, nausea and/or vomiting, and loss of appetite and dysesthesias, particularly
hyperesthesias. A history of exposure to rodents or the excreta of these mammals
may be elicited to have occurred 1–3 weeks prior to the onset of illness. These initial
symptoms may resolve in 3 to 5 days, and in those with subsequent neurological ill-
ness, illness recurs after 2 to 4 days with additional illness. The manifestations of the
second part of the classical (but not pathognomonic) biphasic or hump-back illness
include increasing headache with meningismus (stiff neck), photophobia, and mild-
to-moderate lethargy. Persistent lethargy, overt confusion or coma, and seizures
indicate the development of meningoencephalitis. Other manifestations that may
occur during this second phase of illness include arthralgia/arthritis of the shoulders
and particularly the small joints of the hands, parotitis, and unilateral orchitis. The
biphasic may not always be recognizable. A similar biphasic illness associated with
rodent exposure can occur with leptospirosis, the classical maladie des porchers, a
spirochetal illness that also is associated with chronic urinary excretion in some
rodents.
The clinical manifestations of LCM virus in man has been clearly documented
(Lepine et al. 1937). In a study performed in the prehuman use committee era, inves-
tigators inoculated individuals with tertiary syphilis paretic patients with a mouse
brain-derived suspension of LCM virus and observed that after an incubation period
of 2–3 days, an influenza-like illness developed and was followed by mild, self-
limited lymphocytic meningitis without sequelae.
A typical case of LCM aseptic meningitis illness is one described by Roebroek
et al. (1994) from the Netherlands. A patient, a previously healthy painter with mice
frequently seen in his home, was admitted to the hospital with fever of 38.7°C, mal-
aise, cough, vomiting, a frontally localized throbbing headache, irritability, and
cutaneous hyperesthesia for 6 days. Two weeks earlier, he had a nonspecific febrile
illness that lasted 5 days. Cerebrospinal fluid examination (Table 3.3) revealed a
lymphocytic pleocytosis with a mildly elevated protein and somewhat low glucose
(hypoglycorrhachia). Acute and convalescent sera obtained 3 weeks apart revealed
a significant rise in LCM virus antibody from 1:8 to 1:32. He slowly recovered over
a 4-week period without any sequelae. CSF analysis generally reveals between 100
and 400 cells/mm3, usually >90% lymphocytes, a protein between 50 and 300 mg/
dL (normal, <40 mg/dL), and a normal to slightly low glucose (normal, 50%–70% of
blood glucose). Patients tend to have prominent leucopenia and thrombocytopenia in
the peripheral blood.
Sporadic cases of LCM infection is generally linked to exposure to mice in the
household environment, but outbreaks of the infection tend to be associated with
exposure to rodents, especially Syrian hamsters or tissue cell lines derived from
them, in a workplace setting such as a laboratory or having hamsters as pets. One of
the earliest outbreaks (Lewis et al. 1965; Baum et al. 1966) involved 10 laboratory
personnel at the National Institutes of Health in Bethesda, Maryland. The episode
TABLE 3.3
Typical CSF Pattern in LCM Virus Meningitis
Hospital Day
Day 4 Day 13 Day 26
Cells/mm3 108 63 48
% lymphocytes 95 99 99
CSF glucose 47 49 52
Serum glucose 124 95 63
CSF protein 116 123 76
Source: Roebroek, R.M.J.A. et al., 1994, Clin. Neurol. Neurosurg.

96, 178–180. With permission.
was noteworthy in that it was the first hamster-associated epidemic ever reported,
and the source was contaminated tumor transplants into hamsters and then transmis-
sion among infected to uninfected rodents. All 10 had influenza-like illnesses, and
although none developed meningitis, 2 had subsequent arthritis and 3 had orchitis. In
3 cases that occurred following a single exposure to infected material, the incubation
periods ranged from 9 to 14 days. A similar outbreak (Hotchin et al. 1974; Hinman
et al. 1975) occurred in the early 1970s among staff members of the University of
Rochester Medical Center and involved 48 cases with the initial source also was
contaminated tumor cells (from a different supplier). Of note, personnel infection
occurred not only through direct contact with infected animals but also from the
mere presence in the room where the animals were housed. A cluster of LCM infec-
tion in researchers was also reported associated with nude (athymic) mice (Dykewicz
et al. 1992). The significant risk factors found in the infected workers were cleaning
the cages of the nude mice and changing their bedding or water. The source of the
virus was also LCM-infected tumor lines. The increased use of nude mice in the
laboratory likely precipitated the outbreak as the immunosuppressed rodents shed
virus of higher titer especially in their urine.
Pet hamsters are also a clear source for human LCM virus (Deibel et al. 1975;
Maetz et al. 1976; Biggar et al. 1975). In a 1975 report from the New York State
Department of Health (Deibel et al. 1975), 60 individuals with LCM virus infection
were diagnosed, 12 with a physician diagnosis of CNS disease (8 meningitis and 4
meningoencephalitis), and 48 with other illnesses (mostly influenza-like illness). Of
the 60, 55 had pet hamsters in their households and 4 were employees of hamster
wholesalers or retailers. Infection rates within families with infected pet hamsters
varied with the location and type of hamster cage (Biggar et al. 1975). Open cages
and cages situated in common living areas were associated with the highest infec-
tion rates. Of note in this study, the severity of human illness was not associated with
more direct contact with the hamster reservoir.
3.4.1.4 Immunocompromised Host

One of the earliest reports of the manifestations of LCM virus infection in an immu-
nocompromised host was that of Horton et al. (1971) in which MP virus (a strain
of LCM) was injected into three far-advanced lymphoma patients under informed
consent. Some information had been previously reported (by those who had isolated
the strain) (Molomut and Padnos 1965) that the virus might have had some effect
in suppressing certain murine tumors. No observable effectiveness occurred, and
all three patients subsequently died. Two of the three were persistently viremic and
febrile until death, although the role that the virus played in the deaths was unclear.
The third patient had 15 days of viremia, which subsequently cleared with the devel-
opment of antibody to LCM. No changes in the CSF were reported. Much more
recently, an adolescent with T-cell lymphoma was found to have LCM virus men-
ingitis by serological testing after presenting with lymphocytic meningitis without
encephalopathy or leukemic cells in the CSF in the setting of possible exposure to
mice in her home (Al-Zein et al. 2008). She subsequently developed hydrocephalus,
requiring CSF shunting but was reported to be infection- and leukemia-free 4 years
after the completion of chemotherapy for the leukemia.
LCM infection has also occurred through solid organ transplantation, resulting
in rapidly progressive, high-mortality rate disease. Two clusters of cases have been
described (Fischer et al. 2006) involving two donors and eight recipients receiving
cadaveric kidney, liver, or lung, of which all but one died, between 9 and 76 days
after transplantation, despite lowering immunosuppressive therapy in some recipi-
ents. The posttransplantation courses were characterized by altered mental status,
fever, graft dysfunction, pulmonary infiltrates, and abdominal pain with thrombo-
cytopenia, increased aminotransferase levels, and coagulopathy. Seizures, renal fail-
ure, diarrhea, and a peri-incisional rash were also variably noted. Only one of the
patients was found to have meningoencephalitis at postmortem examination.
It is important to note that one of the donors had no known history of rodent
exposure and no direct evidence of donor infection with LCM virus was found by
immunohistochemical analysis, cell culture, RT-PCR, or viral serologies. A mem-
ber of the second donor’s household had adopted a pet hamster 3 weeks prior to the
donor’s death, but the donor was not the primary caretaker of the hamster and was
seronegative for IgM and IgG antibody to LCM virus without any reported symp-
toms referable to LCM. The viral isolations from the recipients and the hamster were
identical. A trace-back analysis of the hamster (Amman et al. 2007) was able to fol-
low the hamster back from a Rhode Island pet store to a distribution center in Ohio
to the parent facility in Arkansas. Virus from hamsters at both facilities were discov-
ered to be phylogenetically linked to the index hamster and the transplant recipients.
The organ recipient who survived was treated with ribavirin and decreasing
immunosuppressant beginning 26 days after transplantation once the identification
of LCM infection in corecipients was made. Treatment resulted in decreasing illness
and normalizing laboratory abnormalities, and seroconversion to IgM anti-LCM
occurred on day 63 after transplantation. No active infection was found on day 311
with continued immunosuppression. It should be noted that the immunosuppres-
sion as reflected by a functional cell-mediated immunity assay remained prominent
despite reduction of medication, suggesting an effect caused directly by LCM, which
was reconstituted with clearance of the virus (Gautam et al. 2007). Corneas were
transplanted from one of the donors to individuals who did not require systemic
immunosuppression, and neither patient developed any sign of infection or had graft
loss.
Another arenavirus has subsequently been linked to fatal disease in three
Australian recipients of organs from a single donor (Palacios et al. 2008). The donor
had died 10 days after returning from a 3-month trip to the former Yugoslavia, and
all three died within 29 and 36 days after transplantation of illnesses characterized
by fever, encephalopathy, and pulmonary infiltrates. Postmortem histopathology of
the patients was not elaborated. Arenavirus was identified using high-throughput
sequencing identified an Old World arenavirus which was found to be related to
LCM virus and specific PCR found presence of the unique virus in blood, liver, and
CSF.
3.4.1.5 Congenital Infection

The first report of LCM infection as an intrauterine disease appeared in 1955 in
the United Kingdom regarding a neonate who died of meningitis on day 12 of life
(Komrower et al. 1955). It is apparent that fetal infection due to LCM has been an
underrecognized event that can present as a spontaneous abortion (Barton and Mets
2001) or with a clinical complex similar to other congenital infections due to organ-
isms such as toxoplasmosis, rubella virus, cytomegalovirus, or syphilis. As urged
(Barton and Mets 2001), the public and medical communities need to be aware of the
risk that laboratory, pet, and wild rodents can pose to pregnant women.
In a report of 20 children with congenital LCM (Bonthius et al. 2007a), all had
chorioretinitis and structural brain abnormalities, but the presenting clinical mani-
festations including the severity of visual disturbance and the location, character,
and severity of the neuropathology varied quite significantly. The combination of
microencephaly and periventricular calcifications was the most common finding on
imaging, and all these children manifested profound intellectual retardation, cere-
bral palsy, and seizures. Other imaging findings included hydrocephalus, cerebellar
hypoplasia, and porencephalic or periventricular cysts. Isolated cerebellar hypopla-
sia occurred with jitteriness and ataxia as the only long-term dysfunction. In a com-
panion article, clinical and pathological diversity was assessed in an animal model
(Bonthius et al. 2007b). Host age at the time of infection profoundly affected the
cellular targets of infection, maximal viral titers, immune response to the viral infec-
tion, and the severity, nature, and location of the neuropathology. All of the patholog-
ical changes observed in children with congenital LCM infection were reproduced in
the rat model by infecting the rat pups at different ages.
In this rat pup model (Bonthius et al. 2007b), host age was clearly an important
variable. Infection on postnatal day 1 resulted in prominent infection of astrocytes
and neurons, whereas when infection was induced 3 days later, neurons were spared,
and if infection was begun on day 21 neither astrocytes nor neurons could be infected.
Ependymal cells and the olfactory bulb cells, however, remained susceptible.
Infection on postnatal day 1 (Bonthius et al. 2007b) induced cerebellar hypoplasia
manifest by a small but normal cytoarchitectured cerebellum, whereas infection on
day 4 or 6 did not cause cerebellar hypoplasia but rather induced a neuronal migra-
tion problem (Bonthius et al. 2007c) where cerebellar granular cells did not migrate
appropriately into the ventral lobules and encephalomalacia ensued. Furthermore,
infection induced on day 21 did not cause any cerebellar pathology but continued
the susceptibility of the olfactory bulb and the ventricular lining ependymal cells,
resulting in olfactory bulb destruction and hydrocephalus.
Glia, the supportive cells in the central nervous system that do not conduct elec-
tric impulses and consist of astrocytes, oligodendrocytes, and microglia, seem to
play an important role in LCM virus infection of the developing rat brain. It has been
demonstrated (Bonthius et al. 2002) that, in the neonatal rat brain, these cells were
the first to be infected and that the spread occurred via glial cells to other glial cells
and neurons, specifically neurons in the cerebellum, olfactory bulb, dentate gyrus
of the hippocampus, and the periventricular area. The manifestations of neuronal
involvement were different based on the location of the neuron involved as shown in
Table 3.4. It was thought that the specificity of neuronal involvement may be related
to the higher metabolic rate of these neurons (Bonthius et al. 2002).
The neuropathology related to LCM infection in developing rat pup brain is
caused by a combination of noncytolytic viral damage and the innate immune
TABLE 3.4
Neuropathology of LCM Virus-Affected Neuronal Regions
Region Neuropathology
Cerebellum Dorsal lobules—acute immune-mediated destruction
driven by CD8+ lymphocytes
Ventral lobules—neural migration pathology causing
permanently ectopic located cerebellar granular cells
within the molecular level
Olfactory bulb Acute hypoplasia of the bulb due to a reduced production
of granule cells. This effect is transient and the bulb can
be normal sized by adulthood
Hippocampus Despite a substantial viral burden, initially the dentate
gyrus appears histologically normal but several months
later the previously infected cells begin to die resulting
in a delayed, prominent loss of dentate granule cells
Periventricular area No pathological changes despite neuronal infection by
LCM virus
Source: Bonthius, D.J. et al., 2002, J. Virol. 76, 6618–6635. With permission.
system response. As an example, cerebellar damage appears to be immune-mediated

(Bonthius et al. 2002) as shown by the lack of destruction in both the nude (athymic)
mouse without a functional cellular immune system and rats treated with antilym-
phocyte serum.
It is important to note that even if LCM virus has been cleared, progressive dete-
rioration of the previously infected neurons can continue to occur. The mechanism
of this effect is not totally clear but may be related to virus-induced hyperexcitability,
especially of the GABAergic inhibitory neural circuits (Pearce et al. 1996, 2000).
3.4.2 Hemorrhagic Fever Arenaviruses

3.4.2.1 Rodent Infection
With the exception of the Tacaribe virus that is endemic in fruit-eating bats of the
Artibeus genus, rodents are the reservoir for these agents. An arenavirus-like virus, the
first described in a non-mammal, is noted below. In general, each virus is carried by a
single rodent species. Infection rates among the target rodent population are very vari-
able between areas and over time. In the so-called hot spot areas for the Junin virus, the
prevalence rate can be 5%–10%, whereas outside these foci, rates are much lower or
the virus is completely absent. The viruses can be carried lifelong in seemingly healthy
rodents as overt clinical disease is not reported in naturally infected animals.
Laboratory rodents can be infected with the hemorrhagic fever arenaviruses
experimentally but have not been found naturally infected. The infections, unlike
with LCM, have not been observed in pet rodents, but it is conceivable that expo-
sure to a potentially susceptible rodent may occur if an imported, infected, exotic
rodent is exposed in a pet shop, animal “swap meet,” or distribution center to other
species, as was observed in the monkeypox imported in the United States in the giant
pouched Gambian rat and transmitted to American prairie dogs that were subse-
quently sold as pets (Reed et al. 2004). A non-mammal arenavirus-like pathogen has
recently been described in snakes associated with inclusion body disease (Stenglein
et al. 2012). This multisystem disease causes behavioral changes in the snakes and,
intestingly, has some characteristics of filoviruses as well.
3.4.2.2 Human Viral Hemorrhagic Fever Infection

Human exposure to these pathogens mostly through dried rodent urine can be influenced
by the host reservoir’s behavior. As an example, the Junin virus rodent hosts tend to
aggregate along linear habits such as fence lines and roadsides. Additionally, the Lassa
fever reservoir, although found throughout much of sub-Saharan Africa, can exhibit local
species population dynamics that can affect the tendency of the rodent to enter houses.
Although uncommon, secondary human-to-human cases of Lassa fever can occur
generally related to direct exposure to virus-containing body fluids rather than the
aerosol route. As reported (Bausch et al. 2010), at least 25 imported cases of Lassa
fever have occurred in the developed world, involving at least 1500 cumulative
contacts since 1969 and only a single putative and asymptomatic secondary case
occurred. Aerosol transmission may have caused human-to-human cases during a
nosocomial outbreak of Machupo virus (Bolivian hemorrhagic fever) in 1971 involv-
ing 5 persons (Peters et al. 1974).
3.4.3 Lassa Fever

Following an incubation period of 7–18 days, infected individuals can develop fever,
weakness, and malaise that can be associated with cough, severe headache, sore
throat, nausea, vomiting, and diarrhea. The spectrum of disease can range from
asymptomatic infection to fatal disease with hemorrhage. Severe cases will early on
display evidence of increased vascular permeability such as facial edema and pleural
effusion. Deterioration may occur quickly, within 6 to 10 days after illness onset,
associated with pulmonary edema, respiratory distress, shock, and signs of encepha-
lopathy (Moraz and Kunz 2011). Hepatic disease manifests as multifocal hepatocel-
lular necrosis without cellular infiltration or hepatic failure. Liver failure, however,
is not uncommon in the rare cases of Sabia virus infection (Brazilian hemorrhagic
fever) and has been described in a few human infections with Whitewater Arroyo
virus in the United States (CDC 2000). Signs of bleeding occur in 15%–20% of
cases but are not prominent and do not contribute to the hypotension. Disseminated
intravascular coagulation is rarely seen.
Neurological signs in Lassa fever (Cummins et al. 1992) include confusion, rap-
idly followed by tremor, grand mal seizures, abnormal posturing, and coma (Table
3.5). Neither focal neurological signs nor increased intracranial pressure occur. The
development of encephalopathy was not found to correlate with the presence of virus
or antibody directed at the virus in the CSF. Despite significant encephalopathy, con-
sistent lesions in the brain of fatal cases are not found (Walker et al. 1980).
Sensorineural hearing loss (SNHL) is clearly recognized as a usually late mani-
festation of Lassa fever. A prospective study of 49 patients with Lassa fever in Sierra
TABLE 3.5
Manifestations of Lassa Fever Encephalopathy,
n=9
Symptom Number Affected
Confusion 9
Tremor 7
Grand mal seizures 7
Abnormal posturing 3
Coma 8
Death 8
Source: Cummins, D. et al., 1992, J. Trop. Med. Hyg. 95, 197–

201. With permission.
Leone (Cummins et al. 1990) found 14% of cases developed SNHL with no cases in
febrile controls. Additionally, 17.6% (9 of 51) people who had evidence of previous
Lassa infection had SNHL, and 26 of 32 locals who had previously developed SNHL
had serological evidence of past infection as compared with 6 of 32 controls. Other
studies have reported substantially lower risks of SNHL of about 4% (McCormick
et al. 1987a).
3.4.4 South American Hemorrhagic Fevers

The South African hemorrhagic fever viruses cause quite similar illnesses, regard-
less of the specific virus involved. Unlike LCM virus, human-to-human infection
can occur with inadequate barrier protection especially with Machupo virus where
aerosolization of infectious material can occur (CDC 1994). Such high-dose inocula
can decrease the usual incubation period of 5–21 days to as short as 2–3 days.
Presenting with the gradual onset of fever and malaise, these illnesses can also be
associated with muscle aches, headache, back pain, and dizziness. Hyperesthesias
of the skin similar to LCM virus infection also may occur. Hemorrhagic manifesta-
tions include skin petechiae, and gum, vaginal, and gastrointestinal bleeding start at
about day 4 of illness and herald the onset of shock. Similar to the process in dengue
virus infection, despite some hemorrhage, increasing vascular permeability causes
a hemoconcentration state. Disseminated intravascular coagulation can occur with
prominent thrombocytopenia.
Neurological manifestations of the South American hemorrhagic fevers may begin
with hand tremors and the inability to swallow or speak clearly and can progress to
grand mal seizures, coma, and death even without clear-cut signs of vascular leak
syndrome or significant bleeding. The mortality rate of these illnesses is 15%–30%,
but survival generally occurs without residuae (Emonet et al. 2009). Despite the
prominent encephalopathy associated with the South African hemorrhagic fevers,
the CSF examination is generally normal. Beginning with moderate to severe confu-
sion, irritability, prominent ataxia, and tremors may progress to delirium, grand mal
seizures, and coma. Intercurrent bacterial infections such as pneumonia and bactere-
mia occur at this time and contribute to the mortality rate.
A convalescence period of several months may occur with substantial irritability,
prominent asthenia, and memory loss. Human immune plasma can be used therapeu-
tically, especially in Argentine hemorrhagic fever (Maiztegui et al. 1979; Enria et al.
1984). Increased survival occurs if given at adequate titer, but it is well reported that
about 10% of survivors treated in this way, after a period free of symptoms, develop
what is referred to as late neurological syndrome (LNS). This syndrome does not seem
to occur at all in those who recover in the absence of immune therapy. LNS presents
with fever, cerebellar signs, cranial nerve palsies, and an abnormal CSF examination,
with a lymphocytic pleocytosis, normal CSF glucose, and a high ratio of CSF/serum
anti-Junin virus antibodies. Several animal models have been used to study LNS,
including marmosets (Avila et al. 1987) and guinea pigs (Kenyon et al. 1986).
3.5 DIAGNOSIS
These agents can be diagnosed by the use of serologies, detecting antibodies raised
against the offending agent. The usual antibody testing using an enzyme-linked immu-
nosorbent assay (ELISA) or indirect fluorescent antibody testing (IFA) can be used. In
these assays, a fourfold increase in antibody should be found in assays of acute and con-
valescent sera run in parallel. Alternatively, a specific IgM can be tested. In these assays,
there is substantial cross-reactions between viruses, but with the knowledge of where
the index may have been exposed can suggest the arenavirus involved. Virus neutraliza-
tion testing is highly specific to each virus but requires live virus for testing, creating
more laboratory hazard. Unfortunately, in the acute situation where a finite diagnosis is
needed, antibody levels may not yet have risen and the neutralizing antibody may not
appear into convalescence. A single non-IgM-positive antibody test can also reflect past
infection and not be related to the acute illness being evaluated.
Likewise, in the animal reservoir (Charrel and de Lamballerie 2010), finding anti-
bodies specific for an arenavirus in a rodent is a poor indication of active infection
because of a poor correlation with whether a live virus is present. In various situ-
ations related to the virus and age of the rodent, persistent viral infection can be
present with or without detectable antibodies. The presence of antibodies reflects
circulation of virus in the rodent cohorts and indicates the need for more specific
testing by molecular means.
Measurement of viral antigens is certainly useful in the early detection of arena
virus infection. Jahrling et al. (1985c) found antigens in the first serum available
in patients, and antibody positivity did not occur for at least 3 days afterward. The
development of antibodies coincided with a declining antigenemia. The antigen test-
ing could be done safely using beta-propiolactone-inactivated sera. Similarly, testing
was compared in 305 suspected cases of Lassa fever (Bausch et al. 2000). Using
virus isolation with a positive reverse transcriptase-PCR as a gold standard, they
found Lassa fever virus antigen and IgM ELISA were 88% sensitive (95% confidence
interval 77%–95%) and 90% specific (95% confidence interval 88%–91%) for acute
infection. Antigens specific for a virus can also be detected in situ by immunoper-
oxidase staining of surgical or autopsy tissue.
Direct measurement of circulating RNA of the viruses can be done as well. Vieth
et al. (2007) developed an assay using the polymerase domain of the L RNA segment
that used segments of the Old World arenaviruses and found the assay to be appli-
cable as a complimentary diagnostic test for Lassa virus and LCM virus, to identify
new Old World arenaviruses, and to screening potential rodent hosts for Old World
arenaviruses. Similar assays have been developed for New World arenaviruses to be
used as complementary diagnostic assays for the New World arenaviruses (Lozano
et al. 1993; Vieth et al. 2005).
The arenaviruses can be isolated in Vero cells. Attempts at such isolations should
be done in an appropriate laboratory, one with biosafety level 4 containment because
of the risk of laboratory-acquired infection.
3.6 EPIDEMIOLOGY
Lymphocytic choriomeningitis virus is widely distributed in the Americas and in
Europe but has been found in rodents in Asia and Africa as well. The following are
examples of seroprevalence of humans and/or rodents and illustrate differences in
areas with regard to risk groups.
In Baltimore, Maryland, 4.7% (54/1149) inner-city residents were found to have
LCM virus antibodies (Childs et al. 1991). Antibody prevalence increased with age
without gender or ethnic differences. Seropositivity was rare as compared with the
rates of contact with house mice. The seropositivity of house mice in Baltimore
revealed an overall positivity of 9.0% (Childs et al. 1992), with the highest preva-
lence (13.4%) in the inner-city area where positive mice were identified to be sig-
nificantly clustered within blocks and households and correlated with mouse density
within individual blocks. In Birmingham, Alabama, the overall incidence of anti-
body was 3.5% (56/1600) (Park et al. 1997), with, as in Baltimore, increased age was
significantly correlated with seropositivity (age: <30 years, 0.3%; >30 years, 5.4%)
and was negatively linked to socioeconomic status. These authors suggested that,
given the age-related data, human LCM infection may be decreasing over the past
30–40 years, presumably related to less rodent contact.
In Nova Scotia, Canada, a seroprevalence of 4% has been reported (Marrie and
Saron 1998), with a prominent shift (17 of the 20 seropositives) in females. Few,
if any, cases of LCM have been reported from this Canadian province. The rate
compared with that of 2.38% in over 7000 males in Santa Fe Province, Argentina
(Ambrosio et al. 1994). In and around Madrid, Spain (Liedo et al. 2003), 1.7% of 400
human sera were seropositive with no statistical difference related to age or urban
residence; 9% of rodent sera were seropositive.
In nonclassical areas of human LCM infection, no seroprevalence of LCM virus
was found in Upper Egypt, but other areas have rodent seropositivity ranging from
1.8% to 11.5% (el Karamany and Imam 1991). Additionally, some mice captured at
the Yokohama port in Japan were found to be positive on certain piers but not others
and was present in some years and not in others, suggesting introduction from ships
without clear persistence (Morita et al. 1991).
Lassa fever is a substantial febrile illness in West Africa that has been estimated
to be the cause of as many as 15% of adult admissions to the hospital in adults and as
many as 40% of nonsurgical deaths (McCormick et al. 1987a). The annual number
of cases ranges from 100,000 to 300,000 with 5000 to 10,000 deaths and 30,000 left
with deafness (McCormick et al. 1987; Birmingham and Kenyon 2001). The virus
is associated with a high mortality rate during pregnancy as well. The disease has
been periodically imported into Europe, North America, and Japan by travelers from
the endemic area (CDC 2004; Schmitz et al. 2002). Nosocomial spread in the devel-
oped world can occur but is a lower risk than in Africa because of the availability of
adequate barrier protection.
Argentine hemorrhagic fever was first recognized in 1943, and the first isolation
of the Junin virus was in 1958. A range of 300–600 cases per year are generally
reported primarily in individuals involved with agricultural activities in the pampas
of the country (Maiztegui et al. 1986). The areas of endemicity have extended, espe-
cially northward, but overall, there are hot spots of infection in those areas (Mills et
al. 1991).
Bolivian hemorrhagic fever was recognized in 1959 among patients in the town of
San Joaquin in the Beni department of northeast Bolivia involving about 1000 cases
(Tesh et al. 1999) and the Machupo virus first isolated in 1963. Several outbreaks of
the disease occurred in the 1960s, but subsequently, the disease has been recognized
only sporadically in the endemic area.
Venezuelan hemorrhagic fever was recognized in the towns of Guanarito and
Guanare, Portuguesa State, Venezuela, in 1989 during an outbreak that originally
was thought to be dengue (de Manzione et al. 1998). The endemic areas involve the
south and southwest parts of the Portuguesa state as well as the adjacent areas of the
state of Barinas in the central plains of the country.
3.7 TREATMENT
3.7.1 Immune Plasma Therapy
For Argentine hemorrhagic fever (Junin virus), passive transfer of immune plasma
can decrease the case fatality rate from 30% to 1% (Enria et al. 1984). Immune
plasma may be of benefit in Lassa fever if the plasma has a high titer of neutralizing
antibody and is matched to the infecting strain (Jahrling and Peters 1984; Jahrling et
al. 1985a). A minimal improvement of case-fatality rates have been found with other
arenaviruses, possibly due to the lower titers of antibodies in the preparations.
3.7.2 Antiviral Therapy
3.7.2.1 Ribavirin
Ribavirin is a guanosine (1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide) that
has been used, with variable effectiveness, for Lassa, Junin, and Machupo viruses.
In Lassa fever, if intravenous ribavirin is begun within the first 6 days of illness,
the mortality rate of severe Lassa fever can be decreased substantially (McCormick
et al. 1986). This study identified two distinct risk factors for a high case-fatality
rate, elevation of serum aspartate aminotransferase (AST), and significant viremia.
Breakdown of the results can be found in Table 3.6. With Lassa fever, the addition
TABLE 3.6
Case-Fatality Rate of Severe Lassa Fever with and without Ribavirin Use
CFR
Risk Factor No Ribavirin Ribavirin (≤6 days) Ribavirin (>6 days)
Serum AST >150 IU/L 33/60 (55%) 1/20 (5%) 11/43 (26%)
High viremia 35/46 (76%) 1/11 (9%) 9/19 (47%)
Source: McCormick, J.B. et al., 1986, N. Engl. J. Med. 314, 20–26. With permission.
of immune plasma did not reduce case-fatality rates from the levels with or without
ribavirin.
Bolivian hemorrhagic fever due to Machupo virus was anecdotally studied due
to the less common nature of the infection as compared with Lassa fever. In the
report (Kilgore et al. 1997), the two patients treated with immune plasma survived.
Likewise, some reports exist that ribavirin may be useful in man with Argentine
hemorrhagic fever (Enria and Maiztegui 1994) and is underscored by the effective-
ness in primate model (Weissenbacher et al. 1986). Lymphocytic choriomeningitis
virus is inhibited in vitro by ribavirin, but there are little data to assess its efficacy
in substantial LCM disease but it seemed to be effective in LCM-associated organ
transplant infection (Fischer et al. 2006).
The substantial toxicity of ribavirin in man is likely tied to its effect on guanosine
biosynthesis. Among the side effects found include hemolytic anemia, headache,
irritability and anxiety, depression, muscle and joint aches, loss of appetite, and dif-
ficulty sleeping as well as being teratogenic for pregnant women.
3.7.2.2 Nonribavirin
T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide, or favipiravir) is a viral RNA-
dependent RNA polymerase inhibitor with the likelihood of less toxicity given its
mode of action. It has been found to have a broad range of activity among RNA
viruses including influenza viruses, flaviviruses, and bunyaviruses. In a cell culture
model, it disrupted steps in LCM virus replication that could be rescued by the addi-
tion of purine bases or nucleosides (Mendenhall et al. 2011a). In a guinea pig model
using an adapted strain of Pichinde virus (Mendenhall et al. 2011b), favipiravir
appeared to be highly effective even if begun 1 week after virus challenge when the
animals were quite ill. Its effectiveness was also reflected by a more rapid recovery
than that of animals treated with ribavirin. Similar data were reported using a ham-
ster model, in which no additional benefit was reported with favipiravir and ribavirin
together (Gowen et al. 2008).
The arenavirus Z protein has been studied as an antiviral target. Garcia et al.
(2006, 2010) have investigated an aromatic disulfide NSC 20625 that inactivated
viral particles. These virions retained GP complex functions for binding and entry
into host cells but were unable to replicate viral DNA. NSC 20625-treated virions
had abnormal Z protein electrophoretic patterns.
High-throughput screening of small-molecular-weight compounds that inhibit

arenavirus entry into cells by blocking binding has been recently reviewed (Lee
2010). Considering that endemic areas of most arenavirus, with the major exception
of LCM virus, are in countries with low levels of public health infrastructure, the
development of orally dosed, heat-stable, and low-cost compounds is a high priority.
A number of compounds have been found that may offer potential using these crite-
ria, which function as inhibitors of arenavirus fusion.
Another target being investigated for anti-arenavirus activity is the cellular pro-
tease SKI-1/S1P, which acts on the arenavirus GP precursor protein to a produce
fusion-ready GP complex. As a host cell protein, the protease is involved processing
a number of host proteins including the sterol regulatory element-binding proteins,
activating transcription factor 6, and in parts of lipid metabolism (Lee 2010). Other
viral envelopes proteins are processed using SKI-1/S1P include the highly patho-
genic, tick-borne bunyavirus Crimean Congo hemorrhagic fever virus (Vincent et al.
2003). Because SKI-S1P performs important host cellular functions, inhibitors need
to be found that have high effects in processing arenavirus and as low as possible
in its effect upon host cell function. As it is likely that the length of therapy of these
inhibitors will be short and the mortality of these viruses is high, compounds such
as PF-429242 are being investigated. This aminopyrrolidine amide compound has
been able to prevent glycoprotein processing of LCM virus and Lassa fever virus in
a cell culture model (Urata et al. 2011).
3.8 PREVENTIVE MEASURES

As LCM is quite widespread in mice, prevention of laboratory-acquired infection
involves institution of policies to prevent introduction into the animal population.
These policies include seromonitoring of rodents from both commercial suppliers
and intramural breeding colonies as well as testing of tumors to be implanted into
the mice for virus. Even with these monitoring procedures, introduction of LCM into
a laboratory murine facility can occur (Smith et al. 1984). In this introduction, which
did not involve human cases, the source was not recognized but may have been from
a feral mouse and resulted in depopulation of the facility.
The classical public health principles of infection control should be facilitated in any
patient with an illness and appropriate epidemiology compatible with viral hemorrhagic
fever. Isolation includes parenteral and droplet precautions, but it is likely that strict uni-
versal precautions are likely to be effective (Fisher-Hoch 1993). Small-particle-aerosol
precautions using an HEPA filter mask are used in procedures that can generate aerosols,
such as endotracheal intubation. Disinfection of items that had direct contact with the
patient, including chemical or heat inactivation of human waste, is also suggested.
3.8.1 Vaccines
Despite the ubiquitous distribution of LCM virus and the significant number of are-
navirus hemorrhagic fevers, until the late 1990s, no vaccine had truly been successful
in preventing these viral infections. In 1998, Maiztegui et al. reported a prospective,
randomized, and double-blind, placebo-controlled efficacy study of Candid1, a live,
attenuated Junin virus vaccine. In a study involving 6500 male agricultural workers
in the disease endemic area, 23 study individuals developed laboratory-confirmed
Argentine hemorrhagic fever. Of these, all but one had placebo (protective efficacy
95% with a 95% confidence interval of 82%–99%). Three individuals in each group
developed mild infections that did not reach the case definition of hemorrhagic fever.
These additional cases produced a protective efficacy for the prevention of any ill-
ness linked to Junin virus infection of 84% (95% confidence limits, 60% to 94%).
The vaccination was well tolerated, with only 1% reporting adverse effects tempo-
rally related to the vaccine administration.
The vaccine strain differed from wild-type by only 6 amino acids, and the attenu-
ation appeared to be related to a single amino acid substitution in the transmembrane
domain of the G2 glycoprotein transmembrane domain (Albariño et al. 2011). The
mutation (F427I) produces a destabilization of the glycoprotein metastable confir-
mation. Additionally, the mutation produces an increased dependence on the trans-
ferring receptor type 1 for entrance to host cells, affecting the tropism of the vaccine
strain (Droniou-Bonzom et al. 2011). Initially produced in the United States, an
Argentine-manufactured Candid1 vaccine is now available and appears to be equiva-
lent to the original biologic (Enria et al. 2010).
Several prototypic vaccines have been studied in the search for a vaccine against
Lassa fever. These include a recombinant vesicular stomatitis virus vector expressing
Lassa viral glycoprotein, which was able to elicit a protective immune response in
nonhuman primates (Geisbert et al. 2006); a recombinant yellow fever vaccine virus
(the 17D strain) expressing Lassa GP, which protected guinea pigs (Bredenbeek et al.
2006), and an attenuated recombinant Lassa/Mopeia virus, which can protect both
guinea pigs and nonhuman primates (Lukashevich et al. 2008).
As reviewed by Shedlock et al. (2011), numerous candidate vaccines have been
studied against the LCM virus. These include infectious vaccine platforms including
recombinant viruses such as vaccinia, adenovirus, and influenza and recombinant
bacterial platforms including Salmonella and Listeria as well as subunit vaccines
involving GP, NP, and virus DNA. Shedlock et al. (2011) reported on a highly opti-
mized DNA vaccine that conferred complete protection against a high-dose lethal
LCM virus challenge in mice. The magnitudes of both cellular and humoral immune
responses in the mice were robust, approaching that found in natural LCM infection.
Additionally, a multivalent vaccine has been studied against arenaviruses associ-
ated with human disease. The technique utilized well-conserved epitopes and a number
of epitopes specific for the multiple arenavirus species used combined into a vaccinia
virus platform. Mice immunized with this “cocktail” vaccine produced T-cell responses
against LCM virus, Lassa, Junin, Guanarito, Machupo, Sabia, and Whitewater Arroyo
viruses and protected mice against challenge (Kotturi et al. 2009).
3.9 ANTIVIRAL POSTEXPOSURE PROPHYLAXIS

Suggested postexposure prophylaxis (PEP) for arenaviruses aims primarily for
Lassa fever although can be applied to the other hemorrhagic fever viruses empiri-
cally. Bausch et al. (2010) recommends using ribavirin only in the event of a real
high-risk exposure (Table 3.7), especially in the setting of exposure to patients with
TABLE 3.7
Definitive High-Risk Exposures to High Viremic Lassa Fever Infection
1. Prolonged (for hours) and continuous contact in an enclosed space to a patient with Lassa fever
without the use of personal protective equipment (PPE).
2. Participation in emergency procedures such as suctioning, intubation, and resuscitation after
cardiac arrest without the use of appropriate PPE.
3. Penetration of skin by a contaminated sharp instrument such as a blood-contaminated needle.
4. Contamination of mucosal surfaces or broken skin with blood or body fluids such as a blood
splash in the eyes or mouth.
Source: Bausch, D.G. et al., 2010, Clin. Infect. Dis. 51, 1435–1441. With permission.
severe symptomatology that occurs late in the disease with the highest levels of vire-
mia. There are no suggestions of using PEP during the incubation period or after
recovery. The only exception to late PEP use is in the setting of sexual transmission
as significantly delayed times of viral eradication (although no more than 3 months)
can occur in the gonads (WHO 2000). An oral regimen of a 35-mg/kg loading dose
(maximum dose 2500 mg) is given to be followed with 15 mg/kg (maximum dose
1000 mg) 3 times daily for 10 days. A usual adult dose is 2400 mg followed by
1000 mg three times daily. The general form of oral ribavirin consists of a 200-mg
tablet. Even if not totally effective, oral PEP should substantially decrease viremia
and, as a consequence, lower morbidity and mortality. Dosing requires downward
adjustment in persons with creatinine clearances of less than 50 mL/min.
3.10 FUTURE PERSPECTIVES

New arenaviruses continue to be recognized with a rate of one every 1–2 years. A
majority of the isolates are from rodents without known human infection. Many other
arenaviruses remain unrecognized, and especially as mankind invades areas where
some of these agents are endemic, the distinct possibility of new zoonotic infections
exists either in sporadic or outbreak form. Human behavior such as deforestation
replacing wooded lands with agricultural fields and pastures with adjacent human
habitats is a classical scenario for the type of ecology change to produce such infec-
tions. As stated by Tesh (2002), “The arenaviruses are very old and probably evolved
with their rodent hosts; but when people change the ecology, allowing an increase in
the abundance of the rodent hosts, the risk of human–virus contact increases.”
Additionally, it is well recognized that, as occurs with influenza viruses, genetic
reassortments of arenaviruses can occur with the two single-stranded RNAs. Rivere
and Oldstone (1986) studied this process in LCM virus and found that reassortants
can cause lethal infection in mice, whereas neither the parent strains nor the recip-
rocal reassortants did. The authors suggest that such alterations could account for
unanticipated outbreaks of virulence by an arenavirus. This seems to have occurred
in the natural outbreak of Lassa fever with reassortment in vivo of different iso-
lates of the virus (Jahrling et al. 1985b). Whether reassortment can occur between
arenaviruses is not as clear. It is important to note that depending upon the diagnostic
assay used, such viruses may not be easily differentiated from the wild type strains.
Finally, on a darker aspect, certain arenavirus hemorrhagic fever (Junin,
Machupo, and Lassa) agents have been deemed category A bioterrorism pathogens.
As discussed by Bausch and Peters (2009), although there is no record of deploy-
ment of a hemorrhagic fever virus as a weapon, research and development aimed to
weaponize these agents is reported to have taken place in the former Soviet Union
and the United States prior to the abolition of these activities. It is not known that
any country is covertly developing these agents as weapons, but real concerns per-
sist regarding clandestine activity using stocks produced during the Soviet era that
may not have been destroyed with the fall of the former Soviet Union. Additionally,
the Japanese cult group that successfully released neurotoxic gas on the Tokyo sub-
way and attempted to release aerosols of anthrax spores attempted to acquire Ebola
virus, a filovirus causing hemorrhagic fever. Possible scenarios for the dissemination
of these agents, either singly or in combination, include (Bausch and Peters 2009)
implantation of infected humans in the community or hospitals to initiate person-to-
person transmission, release of an infected reservoir into a “virgin” environment, and
direct dissemination of the virus through artificially produced aerosols or fomites.
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4 Bunyaviruses
Patrik Kilian, Vlasta Danielová, and Daniel Růžek
CONTENTS
4.1 Introduction..................................................................................................... 67
4.2 General Properties of Bunyaviruses................................................................ 68
4.2.1 Structure of the Viral Particle............................................................. 68
4.2.2 Large Genomic Segment..................................................................... 69
4.2.3 Medium Genomic Segment................................................................. 70
4.2.4 Small Genomic Segment..................................................................... 71
4.3 Replication in the Host.................................................................................... 72
4.3.1 Antiviral Response of Infected Cells................................................... 74
4.3.2 Replication in Mosquito Vector........................................................... 74
4.3.3 Pathogenesis of Orthobunyavirus Infection........................................ 75
4.3.3.1 Genetic Determinants of Virulence and Infectivity............. 77
4.4 Orthobunyavirus: Ecology and Epidemiology................................................ 78
4.4.1 Ecology................................................................................................ 78
4.4.1.1 Serogroup California............................................................ 78
4.4.1.2 Serogroup Simbu..................................................................80
4.4.2 Epidemiology.......................................................................................80
4.4.2.1 Serogroup California............................................................80
4.4.2.2 Serogroup Simbu.................................................................. 82
4.4.2.3 Serogroup C.......................................................................... 82
4.5 Phleboviruses: Ecology and Epidemiology..................................................... 82
4.5.1 Toscana Virus...................................................................................... 83
4.5.2 Rift Valley Fever Virus: Ecology and Epidemiology.......................... 83
4.6 Conclusions...................................................................................................... 85
References................................................................................................................. 85
4.1 INTRODUCTION
The family Bunyaviridae is one of the largest viral families. At present, more than 350
different bunyaviruses are known to infect animals. Members of the Bunyaviridae
family are divided into five genera. Viruses belonging to the Orthobunyavirus,
Nairovirus, and Phlebovirus genera are typical arboviruses (i.e., viruses transmit-
ted by arthropods), whereas members of the Hantavirus genus are so-called robo
viruses (rodent-borne viruses, since they are transmitted through rodent excrement).
The last genus in the Bunyaviridae family, Tospovirus, includes viruses that infect
plants and these are transmitted mainly by thrips. The family also includes 41 unas-
signed viruses with an unclear taxonomic classification. Uukuvirus, which was
67
previously assigned to a separate genus, is now included in the Phlebovirus genus.

Although there are several bunyaviruses that can infect humans, only a few of them
are associated with infections of the central nervous system (CNS). This chapter
mainly focuses on members of the Orthobunyavirus genus and to a lesser extent the
Phlebovirus genus, which are both known to cause human neuroinfections.
4.2 GENERAL PROPERTIES OF BUNYAVIRUSES

4.2.1 Structure of the Viral Particle
The structure of the viral particle is analogous across the genera of the Bunyaviridae
family. The majority of the data that are available focuses on the virus Bunyamwera
(BUNV), a prototype virus of the Bunyaviridae family. The virions have a spherical
shape and a diameter that is approximately 100 nm in length (Figure 4.1) (Obijeski et
al. 1977). Under the electron microscope, typical spikes of approximately 10 nm in
size can be seen; these are formed by two viral glycoproteins, Gn and Gc (formerly
G2 and G1 according to their migration in acrylamide gel). The viral genome con-
sists of three segments of single-stranded RNA of negative polarity. Thus, the viral
RNA cannot be directly translated into proteins and is not infectious. Each genomic
segment is named according to its size: S (small), M (medium), and L (large). The
sizes of the segments differ between genera (Table 4.1). The 5′ and 3′ termini of
each segment consist of noncoding sequences (NCR; noncoding region) of various
lengths. Approximately 11 terminal bases from both ends are highly conserved and
Gn and Gc
glycoproteins
(a) (b) Large Medium Small
segment segment segment
Region
1 NSs
protein
Gn
protein
Region
2
N protein
pre A
NSm
Polymerase module
L protein
protein
L segment A
B
C
D
E
Gc
protein
Conserved
region
M segment
ent
S seg m
Nucle
ocaps
id p
Unknown
rotein function
FIGURE 4.1 (a) Schematic drawing of bunyavirus particle; (b) coding region of each
genomic segment with regard to the genus Orthobunyavirus.
Bunyaviruses 69
TABLE 4.1
Differences in Length of Genomic Segments
Genus Segment Approximate Length (Bases)
Orthobunyavirus S 960
M 4460
L 6875
Nairovirus S 1712
M 4890
L 12,230
Hantavirus S 1690
M 3920
L 6530
Phlebovirus S 1720
M 3230
L 6430
Tospovirus S 2915
M 4820
L 6890
complementary to each other (Elliott 1990). These complementary sequences allow

for the formation of the characteristic panhandle structures shown by the individ-
ual genomic segments. These structures can be observed by electron microscopy
(Obijeski et al. 1976). In addition, the genomic segments are closely associated with
molecules of the structural protein N, and together with the RNA-dependent RNA
polymerase (RdRp; called also L protein) they form ribonucleoprotein particles
(RNP) (Elliott 1990). The segmented form of the genome facilitates the rapid evolu-
tionary change shown by bunyaviruses due to genomic reassortment.
4.2.2 Large Genomic Segment

The large genomic segment contains one long open reading frame that encodes one
protein (L protein; RdRp). The molecular weight of the L protein is about 259 kDa
(in phleboviruses, 241 kDa). The enzyme activity is multifunctional and crucial for
the life cycle of the virus. Endonuclease function and possible RNA helicase activity
have been revealed. However, most of the active sites on this protein have not been
identified so far. Several structural motifs in the RdRp sequence have been identified
using in silico methods. The first motifs to be identified, A, B, C, and D (Poch et al.
1989), were thought to represent the core of the polymerase active site. This role was
subsequently confirmed experimentally (Jin and Elliott 1992). Two other structural
motifs were identified in close proximity to the N terminus of the L protein by Müller
et al. (1994). Two additional polymerase module motifs, preA and E, were also iden-
tified (Figure 4.1). The function of the conserved region close to the middle of the L
protein is unknown (Aquino et al. 2003), and the endonuclease domain was identi-
fied within Region 1 (Reguera et al. 2010). The endonuclease activity is dependent
on metal ions (especially manganese) and on catalytic lysine 95. No other conserved
regions or potential functional domains were described in the second part of the L
protein near its C terminus. However, mutants containing a tag in the C terminus of
the L protein exhibit restricted growth kinetics in Vero cells and are producers of
smaller plaques when compared with the wild type (Shi and Elliott 2009). Recently,
it was revealed that the L protein of BUNV is able to repair single nucleotide dele-
tions or insertions in the 5′ and 3′ NCRs (Walter and Barr 2010).
In comparison to other genera of the Bunyaviridae family, nairoviruses have a
much larger L genomic segment and have a different composition. The L protein of
nairoviruses is almost twice as large, reaching 459 kDa when compared with other
bunyaviruses, and exhibits a wide range of enzymatic activity. Interestingly, the
amino terminus is predicted to harbor a conserved ovarian tumor (OTU-like) pro-
tease, which shows autoproteolytical activity. The major function of the OTU-like
domain seems to be autoproteolytical cleaving of the L protein to yield a polymerase
and a helicase. Other activities such as deubiquitation have also been proposed
(Honig et al. 2004).
4.2.3 Medium Genomic Segment

The size of the medium (M) genomic segment varies between genera of the
Bunyaviridae family (Table 4.1). The segment has a single open reading frame,
which encodes a polyprotein, and is contranslationally cleaved into two structural
proteins Gn and Gc. Orthobunyaviruses and tospoviruses encode one additional
nonstructural protein, NSm. In orthobunyaviruses, the sequence for NSm is located
between the Gn and the Gc genes, whereas in tospoviruses the NSm sequence is
encoded in the antisense form (Nichol et al. 2005). A short signal sequence precedes
the sequences of each of the proteins. The signal sequence is recognized and cleaved
by a host protease (Elliott 1990).
Gc and Gn proteins are type I transmembrane proteins. The N termini of the
Gc and Gn proteins are projected onto the surface of the viral particle and the
C terminus is integrated into the viral envelope. After translation, both proteins
undergo several modifications and accumulate in the membrane of the Golgi appa-
ratus (GA) where virus assembly occurs. The bunyavirus particle does not contain
a matrix protein joining RNP with a lipid envelope, but instead a 78-aa-long cyto-
plasmic tail (CT) of Gn protein that is considered to be crucial for interaction with
RNP and further packaging (Överby et al. 2007). The CT plays an important role
(probably together with the CT of the Gc protein) during membrane fusion between
the host endosome and the viral envelope (Shi et al. 2007). The transmembrane
domain (TMD) of the Gn protein is shorter and contains about 18 aa. The TMD
contains a signal sequence for the targeting and retention of the Gc–Gn heterodi-
mer in the GA (Shi et al. 2004). Using a reverse genetics approach, the N terminal
half of the Gc protein was shown to be dispensable (Shi et al. 2009). Both Gn and
Gc proteins are modified by N-linked glycosylations. Deletion of the glycosylation
sites in the Gn protein completely alters its folding and transportation to the GA.
Glycans attached to the Gc protein play an enhancement role during virus replica-
tion (Shi et al. 2005).
Bunyaviruses 71
The nonstructural protein NSm is a transmembrane protein of approximately

11 kDa and is translocated together with Gc and Gn into the membrane of the GA
(Fuller and Bishop 1982; Nakitare and Elliott 1993). Using a hydrophobicity profile
prediction, three hydrophobic (I, III, V) and two hydrophilic (II, IV) domains were
identified. Although the internal part of the molecule (mainly domain III, but also
part of domains II and IV) is dispensable for the viral life cycle, domain I is impor-
tant for virus maturation. In infected cells, the NSm is found inside unusual tubes
associated with the GA (Shi et al. 2006). Members of Tospovirus genus use the NSm
to facilitate cell-to-cell transport of the virus through plasmodesmata (Storms et al.
1995).
4.2.4 Small Genomic Segment

The small (S) genomic segment encodes the structural nucleocapsid protein N.
In the case of Orthobunyavirus, Phlebovirus, Tospovirus, and Hantavirus genera,
the S segment also encodes one additional protein: the nonstructural protein NSs
(Elliott 1990; Jääskeläinen et al. 2007). However, the ORF for this protein is not
present in the S segment of Anopheles A, Anopheles B, or the Tete group of the
Orthobunyavirus genus (Mohamed et al. 2009).
Nucleocapsid protein N contains approximately 235 aa. The primary function of this
protein is to encapsidate both viral RNA (vRNA) and complementary RNA (cRNA) to
protect their secondary structure and promote transcription by the L protein. The N pro-
tein binds to the RNA on the 5′ end but also binds nonspecifically to other parts of the
RNA. The N protein forms long multimers and in this way encapsulates the entire RNA
molecule (Leonard et al. 2005; Osbourne and Elliott 2000). In addition to protecting
RNA molecules, the N protein also facilitates the formation of the panhandle structure
of the RNA and is considered to be an RNA chaperone (Mir and Panganiban 2006).
The N protein also interacts with other structural proteins (L, Gc, and Gn) and enables
the formation of the ribonucleoprotein and assembly of the viral particle.
The function of the nonstructural protein NSs (12 kDa) is not completely under-
stood. Although NSs is not encoded by all members of the Bunyaviridae family and
is not important for virus viability, it can play an important role during the pathogen-
esis of the infection (Janssen et al. 1986; Bridgen et al. 2001). Sequence homology
between the N terminus of NSs and the proapoptotic protein Reaper from Drosophila
has been reported (Colón-Ramos et al. 2003). In vitro experiments showed that NSs
induces the release of cytochrome C from the mitochondria of the host cells through
binding to the Scythe protein; this triggers caspase activation and ultimately leads
to cell apoptosis. Moreover, both NSs protein and Reaper block translation of the
cell proteins. In vivo experiments with the recombinant Sindbis virus (Togaviridae,
Alphavirus) that was modified to express NSs showed induction of neuronal apopto-
sis when inoculated intracerebrally into suckling mice. However, other experiments
with the Bunyamwera virus and La Crosse Virus (LACV) suggested that the NSs
protein had an antiapoptotic effect (Kohl et al. 2003; Blaquori et al. 2007). There
is evidence to suggest that the NSs protein serves as an important inhibitor of both
interferon α and β in infected cells rather than as a modulator of apoptosis (Bridgen
et al. 2001; Blaquori et al. 2007).
4.3 REPLICATION IN THE HOST

As is typical for most RNA viruses, the replication of bunyaviruses takes place in the
cytoplasm of the host cell (Figure 4.2). The exact manner that is used for bunyavirus
cell entry is still not fully understood. Nevertheless, basic similarities to other envel-
oped viruses can be found. The first step requires the virus particle to attach itself to
a specific receptor on the surface of the host cell. Orthobunyaviruses are thought to
use protein Gc for this step in both mammalian and insect cells (Hacker et al. 1995),
although there are some data to suggest that the Gn protein is involved as a receptor
ligand in insect cells (Ludwig et al. 1991). Based on experiments with the Uukuniemi
virus, it is known that viral binding to the cell surface is specific but quite inefficient.
Receptors on either insect or mammalian cells remain completely unknown. After
the virus attaches to the receptor, endocytosis occurs very rapidly (Lozach et al.
2010). The Uukuniemi phlebovirus is internalized by clathrin independent endo-
cytosis, but orthobunyaviruses are internalized by clathrin-coated vesicles (Santos
et al. 2008; Lozach et al. 2010). After endocytosis, the virus quickly reaches the
endosome. As a consequence of the acidification inside the endosome, Gn and Gc
proteins change their conformation and trigger the fusion of the viral envelope with
the endosomal membrane, then RNP is released into the cytoplasm of the host cell
(Hacker and Hardy 1997). During the membrane fusion, a dominant role is played by
the Gc protein, although the interaction with the Gn protein is also important (Jacoby
et al. 1993; Plassmeyer et al. 2007; Shi et al. 2007).
(a) (b)
1. Cleaving of the primer together with 7 mG cap 1. Binding to receptor, endocytosis
from host cell mRNA
10–18 nt G host cell mRNA
7 mG 3´ 9. Exocytosis
N Endosome
N N N L protein
2. Membrane
2. Hybridization of the primer with vRNA fusion and releasing
and synthesis of beginning of mRNA 8. Transport of viral
of RNP particle to cell surface
vRNA
3´-AUC AUC AUC UG 5´ 3. Primary 5. Replication of RNA GA
transcription
7 mG G L protein
10–18 nt
NN N
N 4. Translation Rought ER
3. Realign
vRNA
3’-AUC AUC AUC UG 5´ Nucleus
7 mG G UAG UAG L protein
10–18 nt
NN N 7. Forming of viral
N 6. Transport of Gc and Gc particle
heterodimers to GA
4. Synthesis of mRNA according to vRNA template
vRNA
3´-AUC AUC AUC UC 5´
7 mG G UAG UAG L protein
10–18 nt Viral mRNA
NN N
N
FIGURE 4.2 (a) Basic steps in the initiation of transcription; (b) general overview of bunya-
virus replication (in black: replication steps; in gray: cell organelles: ER, endoplasmic reticu-
lum; GA, Golgi apparatus).
Bunyaviruses 73
Once RNP is released into the cytoplasm, the panhandle structure of the RNA is
relaxed and the N proteins dissociate from the 3′ terminus of the RNA. Primary tran-
scription of the vRNA to mRNA occurs in the cytoplasm through the cap-snatch mecha-
nism. The endonuclease activity of the L protein is responsible for cleavage of the 5′ cap
together with several nucleotides from selected host mRNAs (Figure 4.2b). The cleaved
cap subsequently associates with the vRNA, and this initiates synthesis of the comple-
mentary strand. The viral protein N plays an important role in this process as it serves as
a cap binding protein (similar to the eukaryotic eIF4E), and helps the L protein to cleave
host mRNA (Panganiban and Mir 2009). Some specific host mRNAs are cleaved pref-
erentially; for instance, LACV in mosquito cells utilizes preferable mRNA coding for
proteins that are similar to apoptosis inhibitors from Drosophila (Borucki et al. 2002). To
provide sufficient mRNAs for cleavage, hantaviruses have developed effective strategies
for their storage in the cell. After the binding of N protein to the 5′ cap, the mRNAs are
translocated to the so-called P-bodies where the mRNA is protected from degradation
by binding to a viral nucleocapsid protein (Mir et al. 2008). At the 3′ end of the synthe-
sized RNA, a couple of nucleotides are missed. These missed nucleotides are added by
a mechanism called realign, when the L protein slides back to the start of the template
vRNA after transcribing several nucleotides (Figure 4.2b) (Jin and Elliott 1993; Garcin
et al. 1995). Only vRNA that is associated with an N protein can serve as a template for
mRNA transcription (Dunn et al. 1995).
Transcription of the S and L genomic segments is terminated by at least two
terminating sequences, which are localized in the 5′ NCR. However, no analogous
sequences were found in the M segment (Barr et al. 2006). Immediately after tran-
scription, the translation of viral proteins begins. The translation starts when the N
protein associates with the 5′ cap of the mRNA. The N protein mimics the activity
of eIF4G (eukaryotic translation initiation factor 4 gamma), which activates the host
cell’s 43S preinitiation complex and thus facilitates translation (Panganiban and Mir
2009). Replication of the viral genome is started upon primary transcription and
occurs through a cRNA intermediate with a positive polarity. Nevertheless, cRNA
lacks the 5′ cap and its transcription is primer independent. The mechanism by which
the L protein starts the primer independent transcription is poorly understood. It may
have some association with the attachment of the N protein (Schmaljohn and Nichol
2007). Viral replication is a complex process and takes place in so-called viral facto-
ries. The viral factory is formed by the GA, mitochondria, and rough endoplasmatic
reticulum. The key part of the viral factory is an unusual tubular structure that con-
sists of the viral NSm and actin from the host cell that form a connection between
the different parts of the replicating complex. Both viral L protein and N protein are
held in a globular domain that provides protection to the newly synthesized RNAs,
and allows for the formation of RNPs. The RNPs are then transported to the GA
along the fibrous structures (Fontana et al. 2008). The glycoproteins Gc and Gn form
heterodimers, which are transported via vesicles to the GA, where they wait for the
RNPs. After the interaction between the N protein (RNPs) and the glycoproteins, the
complex buds into the GA and forms an immature viral particle called an intracel-
lular annular virus. The glycans that are attached to Gc and Gn proteins are further
modified during the passage through GA. The first maturation step takes place in
the trans-GA and produces a dense, intracellular particle. The particles are then
transported in vesicles toward the host cell membrane where they undergo a second
maturation step that results in the formation of extracellular dense virus particles.
After that, the particles are released from the host cell by exocytosis (Salanueva et al.
2003). Interestingly, the lipid envelope of plant tospoviruses is formed by wrapping
Golgi membranes around RNPs (Kikkert et al. 1999).
4.3.1 Antiviral Response of Infected Cells

Bunyaviruses have successfully adapted to counteract cell immunity; this is mainly
due to the viral NSs protein, which acts as an inhibitor of the interferon (INF)
response. In BUNV infected cells, NSs interacts with part of the cell mediator pro-
tein, MED8. As a basic component of RNA polymerase II machinery (RNAP II),
the mediator protein plays a key role in all transcriptional processes that are per-
formed by RNAP II. The interaction between the C-terminus of the NSs and MED8
inhibits the phosphorylation of serine 2 within RNAP II, which is crucial for mRNA
elongation. This mechanism inhibits mRNA transcription in the host cell, includ-
ing synthesis of INF (Thomas et al. 2004; Leonard et al. 2006). However, different
mechanisms of INF response inhibition have been observed in bunyaviruses. The
NSs protein of the LACV directly causes RNAP II degradation in a proteasome-
dependent manner (Verbruggen et al. 2011). Rift Valley fever virus (RVFV) inhibits
the INF response by direct interaction of NSs protein with the host cell transcription
factor TFIIH, and this decreases the amount required for mRNA transcription.
The accumulation of viral proteins, maturation and budding of viral particles
leads to Golgi apparatus fragmentation and a breakdown of the cell secretory path-
way (Salanueva et al. 2003). These processes result in cell death. However, in mos-
quito cells the situation is completely different. Here, the massive synthesis of viral
proteins in the primary phase is followed by a recession, and the infection goes into
a persistent phase (Scallan and Elliott 1992). During the primary phase of the infec-
tion in mosquito cells in vitro, the cells become highly mobile and form projections
that connect to other cells. Structures such as microtubules, mitochondria, GA, and
lysozymes can be seen in the projections. The viral nonstructural protein NSm was
also observed inside these structures. However, it seems that NSm does not enter un
infected cells via the projections. The most probable function of the structures is to
deliver warning signals to uninfected cells. Generally, the formation of complexes
consisting of N and L viral proteins is considered to be the first phase of persistent
infection (López-Montero and Risco 2011). A similar situation can be observed in
mammalian cells when the interferon-induced antiviral protein MxA combines with
viral protein N and formed complexes are accumulated in the perinuclear area. Since
N protein is required for viral replication, its aggregation limits its utilization for viral
multiplication (Kochs et al. 2002). Nevertheless, similar proteins that are responsible
for the aggregation of L and N proteins in mosquito cells have not yet been identified.
4.3.2 Replication in Mosquito Vector

Perhaps, all of the encephalitis bunyaviruses are transmitted by mosquitoes. The
mechanism by which viruses multiply in the mosquito body is well understood
Bunyaviruses 75
thanks to studies performed by Dr. Danielová on the Ťahyňa virus (Danielová 1962,
1968). It is important that the virus not only persists in the mosquito but that it repli-
cates without affecting its vector (Elliott and Wilkie 1986; Scallan and Elliott 1992).
Approximately 24 h after the ingestion of an infectious blood meal by the mos-
quito, the titer of the virus dramatically decreases and the minimum level is reached
about 3 or 4 days postfeeding. During this so-called eclipse phase, the titer of the
virus is at the minimal level and it is not possible to transmit the infection to the host.
Subsequently, the virus starts to multiply in mosquito midgut cells but is still detect-
able only in the abdomen. After multiplying in the midgut cells, the virus spreads
into the hemocoel and is delivered via the hemolymph to other organs including the
salivary glands, legs, ovaries, and Malpighian tubes. In these organs, the virus is
detectable after 7 days after feeding. In case of the Ťahyňa virus, the mosquito is able
to transmit the virus about 1 week after the infection. The highest viral titer in the
mosquito is seen around 14 days after infection, and the titer is stable (after a slight
decrease) for at least 30 days. In the Aedes aegypti mosquito, infection was observed
51 days postfeeding and is considered to be lifelong.
However, not all of the mosquitoes that ingest infectious blood became infected. The
ability of the vector to biologically transmit the virus is called vector competence and is
dependent on several factors on both sides: mosquito and virus. From the mosquito side,
there are many barriers that the virus needs to overcome before reaching the salivary
glands for horizontal transfer or ovaries for vertical transmission (Figure 4.3). The per-
meability of individual barriers varies between mosquito species and may be connected
to different enzymes that are present in the mosquito midgut or the presence of specific
cell receptors. The basal lamina of the mosquito midgut represents an important barrier.
The pores in this noncellular structure are just 10 nm in diameter, but it is not clear how
viral particles are able to cross it (Mellor 2000). From the side of the virus, especially
products of the M genomic segment (Gn, Gc, and NSm) determine successful per os
infection of the mosquito (Beaty et al. 1982).
Interestingly, it has been shown that infection with the LACV affects the behavior of
infected mosquitoes. In a laboratory experiment, infected females were able to mate ear-
lier and more frequently than uninfected ones (Gabitszch et al. 2006; Reese et al. 2009).
4.3.3 Pathogenesis of Orthobunyavirus Infection

Most of the studies on the pathogenesis of orthobunyavirus infection have been done
using the LACV in laboratory mice. Mice are considered to be a good laboratory animal
model, mimicking the course of the infection in humans. There are variations in the dis-
ease outcome that depend on a number of factors, including the viral dose inoculated, the
virulence of the virus strain, genetic background, or the age of the infected individual.
In the case of California encephalitis infections, young individuals have a higher risk of
contracting the severe form of the disease than older people. On the other hand, in the
case of the Jamestown Canyon virus, older individuals are more susceptible than younger
ones (Rust et al. 1999). Suckling mice are highly susceptible to subcutaneous infection,
whereas adult individuals do not develop high viremia but are susceptible to intracerebral
inoculation. Thus, suckling mice are more useful models for studies that focus on the
extraneural phase of the disease, and adults for the neural phase of infection (Janssen
Infected
blood
Midgut infection barrier
Midgut
Midgut escape barrier
Hemocoel
Dissemination barrier
Salivary gland infection barrier
Salivary gland Legs
Ovaries infection barrier
Ovaries
Salivary gland escape barrier
Saliva
Vertical transmission
Horizontal transmission
FIGURE 4.3 Barriers that the virus needs to overcome for the infection of the mosquito
vector, and successful transmission.
et al. 1984). Mosquito saliva is important for a successful infection because it promotes
virus transmission, probably via the inhibition of an early interferon response at the mos-
quito feeding site (Borucki et al. 2002).
During the extraneural phase of the infection, the virus primarily replicates
in striated muscles and to a lesser extent in smooth or heart muscles. The virus is
then thought to penetrate the lymphatic system and access the blood, then viremia
appears. During the viremic phase, the virus crosses the blood–brain barrier and
invades the CNS. Replication in the CNS is highly age-dependent. In suckling mice,
there is a pancellular infection, whereas in adult mice the virus primarily replicates
in neurons (Griot et al. 1993). The LACV also induces apoptosis of the neurons
(Pekosz et al. 1996). Death occurs approximately 3–4 days after infection. However,
most of the infections do not progress to the CNS phase. The basic steps taken by
California encephalitis viruses are depicted in Figure 4.4. An alternative model of
Bunyaviruses 77
Encephalitis, death
Neuroreplication
CNS
Neurons apoptosis
Blood–brain barrier
Blood
viremia
Olfactory neurons
Afferent lymphatics
Neuroinvasion
Striated, Nasal turbinates

heart muscle
Subcutaneous infection
Lymphatics, blood
FIGURE 4.4 Classical (bold arrows) and alternative (dashed arrows) steps of pathogenesis
for the California serogroup virus infection.
orthobunyavirus pathogenesis has been published by Bennett et al. (2008). In this

case, i.e. an intraperitoneal infection of a weanling Swiss Webster mouse with the
LACV, the virus initially replicated in tissues near the site of inoculation and then
unidentified cells in nasal turbinates became infected via the blood stream. In this
case, olfactory neurons facilitated virus entry into the CNS. Together with the previ-
ous observation, it seems that the virus can use more than one method for entry into
the CNS. The observed differences in the course of the infection may be due to the
use of different strains of mice or virus used in each study or due to different sites
of inoculation.
4.3.3.1 Genetic Determinants of Virulence and Infectivity

Bunyaviruses, like other animal viruses, can differ in virulence in experimental ani-
mals. From experiments with laboratory mice, several determinants of virulence
have been identified. As suckling mice are highly susceptible, they are the perfect
model for studying neuroinvasiveness, whereas adults are used for neurovirulence
experiments. These tests suggest that major determinants of the neuroinvasiveness,
i.e., the ability to replicate in peripheral tissues and reach the CNS, are probably
located in the M genomic segment, which encodes viral glycoproteins. Thus, periph-
eral attenuation is possibly related to the interaction of the viral glycoproteins with
the host cell receptors and also to fusion activity. However, in the case of neuroviru-
lence, it seems that major determinants are encoded in the L genomic segment and
this is linked to the virus’ ability to replicate in neurons in the adult mice. Although
genetic determinants of neurovirulence and neuroinvasiveness are mapped to the L
and M segments, respectively, it is possible that the products of the S genomic seg-
ment influence virulence as well (Janssen et al. 1986; Endres et al. 1991; Griot et al.
1993). A recent study that compared the sequences of the M and S segments of the
biologically different strain of Ťahyňa virus did not identify unambiguous mutations,
which can alter virulence after subcutaneous or intracerebral inoculation (Kilian et
al. 2010). It is likely that the biological properties of the virus are determined by
several accidental mutations or by several substitutions that occur independently in
all three genomic segments.
4.4 ORTHOBUNYAVIRUS: ECOLOGY AND EPIDEMIOLOGY

4.4.1 Ecology
Orthobunyaviruses are arboviruses that are distributed throughout the world. There
are more than 150 viruses belonging to this genus, and all of those that affect humans
are transmitted by mosquitoes. Only viruses from the Simbu serogroup, including the
epidemiologically notable Oropouche virus, are transmitted by Ceratopogonidae.
Viruses circulate between their vectors and natural hosts, mostly mammals and
birds, quite independently of the presence of humans. Although the viral infection of
vectors and natural hosts do not reveal any apparent symptoms, human infection can
range from an unapparent infection to a very severe disease. In most cases humans
are dead-end hosts without the possibility of further virus transmission; there is only
one exception, i.e., the urban cycle of the Oropouche virus. A high level of virus–
vector specificity is often developed, but on occasion, a wide range of susceptible
mosquito species are available. Nevertheless, their vector competence highly differs
and is virus-specific.
4.4.1.1 Serogroup California

Viruses of this serogroup, named after the California virus, the first isolated virus
from this group, were isolated in North and South America, Asia, Africa, and Europe.
Because of its association with a severe human disease, the ecology of the LACV,
isolated in the United States, has been studied more extensively than other members
of the California serogroup in North America. The primary vector of this virus,
Aedes triseriatus, is a mosquito that uses tree-hole breeding and can be found in
woodland. Nevertheless, this mosquito species can also be found in suburban loca-
tions where discarded containers (tires, cans, etc.) are used as a substitute for natural
breeding places. This way, the virus can invade distant localities. LACV is transmit-
ted by this mosquito transovarially (Watts et al. 1973), which allows hibernation of
the virus in its eggs. It can also be transmitted venereally from an infected male mos-
quito to a female; this is another way to horizontally transmit the virus (Thompson
and Beaty 1977, 1978). Transovarial transmission has been demonstrated in eight
California serogroup viruses; this probably serves as the primary method of viral
maintenance during periods of vector inactivity. As demonstrated experimentally,
transovarial transmission of the California virus in Aedes dorsalis can provide a
stable infection in a mosquito population for several consecutive generations and
maintains the virus during unfavorable conditions (Turell and Le Duc 1983). This
Bunyaviruses 79
suggests that vertical transmission is more important for maintenance of a virus

focus than it is for amplification through horizontal transmission. In the horizontal
transmission cycle, chipmunks, tree squirrels, cottontail rabbits, and foxes have been
found as principal natural hosts, providing high viremia that lasts 2–5 days and thus
enables mosquito infection. Some other susceptible hosts were found by serological
surveys (white-tailed deer, woodchuck, etc.) (Yuill 1983).
The California encephalitis virus was primarily isolated from Aedes melanimon,
but Aedes dorsalis appears to be the principal vector (Crane et al. 1977). The pri-
mary vector of the Jamestown Canyon virus is Culiseta inornata, although Aedes
triseriatus also plays an important role. Culiseta inornata also appears to be the
principal vector of the Snowshoe hare virus together with several Aedes species
(Turell and Le Duc 1983). In addition to North America, this virus has been found
in northern and Asian Russia.
It is an epidemiological question, whether Aedes albopictus, a synanthropic spe-
cies, which spread from Asia to other continents including North America in half of
the 1980s and which is susceptible to the California group viruses, will be integrated
into their circulation cycle. Under laboratory conditions, a stable infection of the San
Angelo virus (California group) in Aedes albopictus has been demonstrated (Turell
and Le Duc 1983).
Ťahyňa virus (TAHV), the first arbovirus isolated from mosquitoes in Europe, was
recovered in the former Czechoslovakia in 1958 (Bárdoš and Danielová 1959). Later,
it was isolated in many European countries including European and Asian Russia,
Middle Asian republics, China, Turkey, Northern and northeastern Africa and as
a Lumbo variety also in Central, West, and South Africa (Hubálek and Halouzka
1996; Zhi Lu 2009). Its ecology was studied very extensively. Aedes vexans appears
to be the primary vector for TAHV. Virus isolations from mosquitoes collected in
the field and laboratory experiments have shown other species that can serve as vec-
tors: Aedes cantans, Aedes caspius, Aedes dorsalis, Aedes cinereus, Aedes sticticus,
and Culiseta annulata. Nevertheless, most of the TAHV isolated strains originated
from Aedes vexans (Danielová 1992). Moreover, its infection threshold is the lowest
of all other examined species (Danielová 1966, 1972b), and it can transmit TAHV
transovarially (Danielová and Ryba 1979) enabling virus hibernation. Virus hiber-
nation has also been shown in hibernating females of Culiseta annulata (Danielová
and Minář 1969). However, it has been shown that active virus circulation is associ-
ated with the breeding wave of Aedes vexans following late spring or summer flood-
ing, although other species susceptible to TAHV infection may already be active
(Danielová 1972a). Hares, rabbits, hedgehogs, and pigs are considered to be primary
hosts (Málková 1980). It was found that hedgehogs, which are heterotherm animals,
can play a role in virus hibernation shortly before their overwintering. The viremia
then persists for up to a couple of days after their awakening (Málková et al., unpub-
lished data).
The Inkoo virus, first isolated in Finland, is another member of the California
serogroup in Europe (Brummer-Korvenkontio et al. 1973). It is distributed mainly in
northern Europe and Russia, transmitted by Aedes communis and Aedes punctor in
Scandinavia; in Russia it was also isolated from Aedes hexodontus and Aedes punc-
tor (Mitchell et al. 1993).
4.4.1.2 Serogroup Simbu

Viruses from this serogroup are distributed globally and are transmitted by culi-
coids. From the epidemiological aspect, the Oropouche virus is the most important
member of this serogroup. although it was first isolated in Trinidad, it is most widely
distributed in Brazil and Peru, predominantly in the Amazon basin regions (Pinheiro
et al. 1981). This virus reveals the distinctiveness of its ecology. It circulates and
perpetuates in a sylvatic cycle among forest primates, sloths, birds, and an uniden-
tified vector, but it can be introduced into urban settings by infected people or by
Culicoides paraensis, which initiates an urban ecological cycle. In the urban cycle,
Culicoides paraensis plays the role of vector, and humans serve as amplifier hosts.
Unlike most other arboviruses, the anthropophilic Culicoides paraensis can trans-
mit the virus to the human population without a wild animal host, as humans develop
a high enough viremia for the infection of the vector. Although the infection rate of
Culicoides paraensis is rather low, vast amounts can be found in rural regions where
it breeds in decaying waste from agricultural products. Outbreaks occur when there
is a large concentration of both vectors and susceptible humans (Pinheiro et al. 1981).
4.4.2 Epidemiology
The Orthobunyavirus genus includes more than 150 viruses, which have been
divided into 18 antigenic groups. Currently, 48 virus species are known. However,
serogroups are still frequently used as taxonomical units. As is commonly seen with
other arboviral infections, the unapparent forms of the disease predominate and their
presence can be revealed by serological surveys only. Most of these viruses cause
febrile illness or influenza-like symptoms, but some of them have the ability to cause
a severe disease. Neither a vaccine nor a specific treatment is yet known and there-
fore the treatment is only symptomatological. Unless there has been an outbreak, the
actual number of people who have contracted the disease is mostly underreported.
From an epidemiological point of view, the most important serogroups are the
California group found in North and South America, Europe, Africa, and Asia; the
Simbu group, found in South and Central America, Africa, Asia, and Australia;
group C occurring in South and Central America and the Bunyamwera group dis-
tributed predominantly in Africa but also in Europe, Asia, and North America.
4.4.2.1 Serogroup California

The California encephalitis virus is the prototype member of this serogroup. The
virus was isolated in 1941 and is thought to be a cause of human encephalitis in
California (Hammon and Reeves 1952). Later, it was discovered that the LACV,
isolated from the brain of a 4-year-old girl in 1964 (Thompson et al. 1965), is respon-
sible for severe disease, predominantly in children younger than 16 years of age, and
that it appears more frequently. The incidence of LACV infection can be up to 20–30
cases per 100,000 inhabitants in endemic areas.
Like the two mentioned above, the Jamestown Canyon virus occurs in North
America. Unlike the LACV, the Jamestown Canyon virus was shown to be a cause
of human encephalitis that is predominant and more severe in adults (Grimstad et
al. 1986). These three viruses all cause similar encephalitic diseases that differ with
Bunyaviruses 81
respect to age dependence and severity of symptoms. It seems that the LACV and
the Jamestown Canyon virus cause encephalitis more frequently than the California
encephalitis virus (Campbell et al. 1992).
The Snowshoe hare virus is another virus within the California serogroup found
in North America that causes meningitis and encephalitis in humans. This virus is
distributed across the northern United States, Canada, and Alaska as far as the arctic
(Fauvel et al. 1980). Many cases of clinical illness caused by the Snowshoe hare
virus frequently go unrecognized.
In terms of causing human disease, the LACV is the most significant member of
this serogroup. According to the CDC, approximately 80–100 LACV neuroinvasive
disease cases are reported each year in the United States, less severe cases being
significantly underdiagnosed and underreported. In the past, most cases of LACV
neuroinvasive disease have been reported in the upper Midwestern states. Recently
however, more cases have been reported from the mid-Atlantic and southeastern
states. Most of the cases appear between July and September, but in subtropical
endemic areas (e.g., the Gulf States), rare cases can occur in winter as well. People
living in or visiting woodland habitats and those who work outside or participate in
outdoor recreational activities in areas where the disease is common are at risk of
infection from the LACV (thus the higher frequency in men 1:1.5 is explained).
After a 5- to 15-day incubation period, 2–3 days of fever follow (temperature
ranges from 38°C to 41°C). Other symptoms include headaches, nausea, vomiting,
fatigue, and lethargy. Severe neuroinvasive disease occurs most frequently in chil-
dren under the age of 16. Although seizures are common, fatal cases are rare (<1%)
and most patients recover completely. In some cases neurologic sequelae (recurrent
seizures, hemiparesis, and neurobehavioral abnormalities) of varying duration have
been reported. In addition, approximately 10% of children develop epilepsy, and less
than 2% could have some learning dysfunction or cognitive disorder (Soldan and
Gonzáles-Scarano 2005). A temporal lobe abnormality similar to that seen in herpes
simplex encephalitis was observed in a 10-year-old child (Sokol et al. 2001). As there
is no specific antiviral treatment for clinical LACV infection available, symptomato-
logical treatment is used. No vaccine against the LACV yet exists.
Diagnosis of a LACV infection is performed serologically by the detection of
LACV-specific IgM antibodies in serum or by CSF IgG seroconversion. Positive tests
should be confirmed by neutralizing antibody testing of acute- and convalescent-
phase serum specimens. As cross-reactivity between California serogroup viruses
occurs sympatrically, a less specific diagnostic method could lead to misleading
results. At present, molecular biology techniques (RT-PCR, SSCP) are available for
accurate diagnosis.
As the primary vector of the Ťahyňa virus is thought to be Aedes vexans, which
breeds rapidly after floods and is able to seek hosts from a long distance, high num-
bers of humans are seropositive, mostly after an unapparent infection. The sero
prevalence reaches up to more than 50% of inhabitants in endemic areas. Clinical
manifestation of the Ťahyňa virus infection was serologically diagnosed in many
cases; in several of them, the virus was isolated from the viremic blood of an infected
individual. The incubation period is short, only 1–2 days. After that, the majority of
apparent clinical cases of Ťahyňa virus infection manifest as a sudden febrile onset
associated with a headache and a combination of mostly catarrhal symptoms in the

pharynx and respiratory tract, conjunctivitis, sore throat, anorexia, gastrointestinal
disorders, weakness, malaise, myalgia, arthralgia, and sometimes bronchopneumo-
nia. The disease usually lasts from 3 to 8 days, but rarely longer. Aseptic meningitis
and other neurological symptoms including coma have been observed, predomi-
nantly in children. Every seventh influenza-like disease and every fifth case with
neurological symptoms in children is thought to be caused by the Ťahyňa virus in
South Moravia (the Czech Republic) during the summer season. Fatal cases were
not reported (Šimková 1980; Bárdoš et al. 1975, 1980). Besides Czechoslovakia, the
infections caused by the Ťahyňa virus have been observed in other European and
Asian countries (Janbon et al. 1974; Kolobukhina et al. 1990; Lvov et al. 1977; Zhi
Lu 2009).
Clinical manifestation of the Inkoo virus is similar to that of the Ťahyňa virus,
including the neurological symptoms (Kolobukhina et al. 1990; Lvov et al. 1996).
4.4.2.2 Serogroup Simbu

Unlike other orthobunyaviruses, humans infected with the Oropouche virus develop
viremia levels capable of infecting Culicoides paraensis, which serves as a vector
for this virus. It has been demonstrated that the urban cycle of the Oropouche virus
involves a man-to-man cycle maintained by Culicoides paraensis. Thus, humans
appear to serve as amplifier hosts in the urban cycle. This fact, together with the high
abundance of the anthropophilic Culicoides paraensis promotes epidemic situations.
Over the past 45 years, many outbreaks of Oropouche fever have been reported, with
approximately 500,000 of these cases in the Americas. Oropouche virus has been
isolated in Trinidad, Panama, Peru, and Brazil. During the past 40 years, Oropouche
fever has emerged as a public health problem in tropical areas of Central and South
America (Azevedo et al. 2007). Clinical manifestation appears after a 4- to 8-day
incubation period with a sudden onset of fever, chills, headache, myalgia, arthralgia,
sometimes rash, meningitis or encephalitis, but no fatal cases or permanent sequelae
have been reported. On the other hand, prostration is common (Le Duc and Pinheiro
1988).
4.4.2.3 Serogroup C
Several viruses (Apeu, Caraparu, Ossa, Madrid, Marituba, Murutucu, Restan,
Nepuyo, Itaqui, Oriboca) associated predominantly with the tropical forests in Central
and South America have been isolated from febrile humans, monkeys, rodents, mar-
supials, fruit bats, and mosquitoes. No large epidemics have been recorded. Sporadic
infections of people are characterized by fever, rigors, photophobia, conjunctivitis,
tachycardia, myalgia, arthralgia, prostration, leucopenia, and occasionally jaundice
(Swanepoel 2004).
4.5 PHLEBOVIRUSES: ECOLOGY AND EPIDEMIOLOGY

Although members of the Phlebovirus genus are not typical representatives of neuro-
virulent viruses, there are at least two viruses that should be mentioned. The first is
Toscana virus which affects mainly inhabitants and travellers in the Mediterranean
Bunyaviruses 83
region. The second is RVFV, which circulates mainly on the African continent and
can also cause encephalitis in humans.
4.5.1 Toscana Virus
Like most bunyaviruses, Toscana (TOSV) is an arthropod-borne virus. The first
isolation of the virus was reported in 1971 in Tuscany, Italy, from the mosquito
Phlebotomus perniciosus. The main vector for this virus is the sand fly belonging to
the Phlebotominae family, mainly the Phlebotomus, Lutzomyia, and Sergentomyia
genera that colonize humid habitats in the Mediterranean region (Verani et al.
1988). TOSV is circulating in Algeria, Spain, Portugal, Cyprus, Greece, and Turkey
(Valassina et al. 2003; Depaquit et al. 2010). Very little is known about the verte-
brate host of TOSV because serological studies revealed no antibody prevalence
among domestic and wild animals. However, isolation of TOSV from the brain of a
bat (Pipistrellus kuhli) indicates some possible ecological importance to these ver-
tebrates (Verani et al. 1988). However, no conclusive evidence has been reported.
Nevertheless, it has been proposed that transmission in nature is possible in the
absence of a vertebrate host. In this case, the vector itself can be considered as a
reservoir. Subsequently, TOSV is transmitted transovarialy and even venerealy from
an infected male to an uninfected female during copulation. In winter, the virus may
persist in diapausing phlebotomus larvae (Tesh et al. 1992).
TOSV causes a predominantly influenza-like infection but during the summer
months it is frequently associated with encephalitis and meningitidis (Braito et al.
1998). During a three year investigation, it was shown that 81% of aseptic menin
gitides in summer are caused by TOSV (Valassina et al. 2000). In another study, sero-
prevalence of anti-TOSV antibodies in a high risk group, such as forestry workers
in Italy, reached more than 72% (Valassina et al. 2003). Among other residents,
seroprevalence ranges from 3% to 22% in Italy, 5% to 26% in Spain, and about
12% in France (Depaquit et al. 2010). TOSV infection can affect both children and
adults, and the highest number of reported cases is in August. As mentioned previ-
ously, TOSV infections are mainly asymptomatic or influenza-like. However, some
neurological symptoms can occur. A typical TOSV infection manifests as high fever,
headache, sore eyes, and photophobia. Rarely, aseptic meningitides can be present.
The disease is often milder in children. Laboratory diagnosis is based primarily on
detection of the viral nucleic acid by RT-PCR or by isolating the virus from the blood
of an infected individual. Detection of rising IgM antibodies may also help.
4.5.2 Rift Valley Fever Virus: Ecology and Epidemiology

Since its first isolation in 1930 in Kenya, East Africa, the RVFV has been found in
many countries on the African continent and even in Madagascar and Saudi Arabia
(Daubney et al. 1931; Andriamandimby et al. 2010). RVFV mainly affects livestock
such as goats, camels, or sheep. Although RVFV infections in animals are mainly
mild or unapparent, large outbreaks periodically emerge and cause heavy mortality
among newborn animals or abortion in pregnant females, which is associated with
high economic losses. For humans who have been infected because they work with
sick animals or have been bitten by an infected mosquito, disease is mainly associ-
ated with acute febrile illness, hepatitis, retinitis, renal failure, hemorrhagic fever
and rarely encephalitis or meningoencephalitis (Bouloy and Weber 2010).
RVFV can be transmitted by a variety of mosquitoes including those of the Aedes,
Anopheles, Culex, Mansonia, and Eretmapoites genera. Two different types of viral
circulation in nature can be distinguished: the epizootic and the interepizootic cycles.
The epizootic cycle mainly prevails during the heavy rain season when the mosquito
population is increasing and the virus can be transmitted from mosquitoes to mam-
mals and then to the uninfected vector. The interepizootic (or survival) cycle relies on
transovarial transmission of the virus, so it must survive in mosquito eggs until the next
rainfall occurs and a sizeable amount of infected mosquitoes hatch. Newborn mosqui-
toes infect new animals and trigger the start of a new epizootic cycle. Infected livestock
develop a high viremia, which allows infection of other mosquitoes. Depending on a
number of factors such as the availability of susceptible hosts and abundance of effi-
cient vectors, large outbreaks can occur (Sall et al. 1998). Additionally, the virus can be
transmitted in the eggs of mosquitoes “by contaminated wind” to a new region (Sellers
et al. 1982). The main mode of transmission to humans is by close contact with infected
animal body fluids during slaughter or handling of aborted animals. Humans can also
be infected after a mosquito bite or drinking raw milk.
The infection in humans is mainly asymptomatic or manifests as common flu-like
symptoms. The incubation period is very short; approximately 2–5 days. After that,
patients develop moderate fever, headaches, weakness, nausea, and muscle pain. The
fever usually lasts for one week and most patients recover completely. Further com-
plications have been observed in less than 5% of cases and are associated with CNS
manifestation such as confusion, lethargy, convulsion, or coma. Encephalitis symp-
toms may last for about 4 weeks and most patients recover completely. The others
can suffer from neurologic sequelae such as hemiparesis. In less than 1% of affected
people, the infection can lead to highly lethal hemorrhagic fever. An increased fatal-
ity rate reaching almost 14% was observed during an outbreak in Saudi Arabia (Pepin
et al. 2010). The infection in animals takes a similar course to that in humans; how-
ever, mortality is much higher, reaching up to 30% in adult animals and even 100%
among newborn individuals. The infection often leads to abortion or a decrease in
milk production (Balkhy and Memish 2003; LeBeaud et al. 2010).
The laboratory diagnosis of an RVFV infection relies on the detection of IgM
antibodies in a single specimen or on increasing tendencies of IgG antibodies. In a
patient with the developed encephalitis form, IgM antibodies can also be detected
in cerebrospinal fluid. In an appropriately equipped BSL-3 or BSL-3+/4 laboratory,
isolation of the virus from the blood of patients in the acute phase can be performed.
Further, molecular biology techniques such as PCR or qPCR are also available
(Garcia et al. 2001; Sall et al. 2001).
The treatment of diseases caused by RVFV is symptomatic only, and no specific
treatment is known. Thus, prevention programs are highly encouraged. To date, both
live attenuated and inactivated vaccines are available. However, both of these are
only for veterinary use; no vaccines for human use have been registered. Several
new generation vaccines such as recombinant viral vectors or DNA vaccines are also
under development (Ikegami and Makino 2009; Boshra et al. 2011).
Bunyaviruses 85
4.6 CONCLUSIONS
Bunyaviruses represent a unique group of viruses that can infect vertebrates, inver-
tebrates or plants. Only a few of the Orthobunyavirus and Phlebovirus genus are,
however, associated with neuroinfections in humans. Although diseases caused
by bunyaviruses are often mild, their importance should not be underestimated.
Vector-borne diseases, including those caused by bunyaviruses, are being studied by
scientists all over the world. Climate change, together with changes in land usage,
increase vector populations and allow them to spread to new areas. Together with
their vector, bunyaviruses can be introduced to an immunologically naive population
and cause severe outbreaks, e.g., as shown by RVFV, which was introduced to Saudi
Arabia. However, bunyaviruses possess another feature that provides them with
some epidemiological advantages: genomic segment reassortment. Thanks to this
mechanism, bunyaviruses can change their properties very rapidly. Ngari virus is
an example of an orthobunyavirus that has caused outbreaks in Somalia and Kenya.
Ngari genomic segments originated from the Bunyamwera and Batai orthobunya
viruses. Although considerable progress has been made in bunyavirus research over
recent years, there is no vaccine or specific treatment as yet. This, combined with
their global distribution, means that bunyaviruses present a considerable worldwide
public health challenge.
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5 Respiratory Pathogens
Human Coronaviruses
Revisited as Infectious
Neuroinvasive, Neurotropic,
and Neurovirulent Agents
Marc Desforges, Dominique J. Favreau,
Élodie Brison, Jessica Desjardins,
Mathieu Meessen-Pinard,
Hélène Jacomy, and Pierre J. Talbot
CONTENTS
5.1 Introduction.....................................................................................................94
5.2 Viruses and Human Neurological Diseases.................................................... 95
5.3 The Coronaviruses: An Overview...................................................................96
5.4 Nonhuman Coronaviruses Infecting the CNS.................................................97
5.4.1 Mouse Hepatitis Virus.........................................................................97
5.4.2 Swine Coronaviruses (PHEV)............................................................. 98
5.4.3 Feline Coronaviruses (FCoV).............................................................. 98
5.5 Human Coronaviruses Infecting the CNS.......................................................99
5.6 Human Coronaviruses: Epidemiology of Respiratory Pathogens...................99
5.7 Respiratory Human Coronaviruses Invading the CNS................................. 100
5.7.1 Human Coronaviruses Other than SARS-CoV:
Possible Association with Neurological Diseases in Humans........... 103
5.7.2 SARS-CoV: Possible Association with Neurological
Diseases in Humans........................................................................... 105
5.8 Possible Mechanisms of Human Coronavirus-Induced Neuropathogenesis........105
5.9 Conclusion and Significance.......................................................................... 111
Acknowledgement.................................................................................................. 112
References............................................................................................................... 112
93
5.1 INTRODUCTION
The central nervous system (CNS) is a highly complex biological system that main-
tains life, and insures its quality. However, like the rest of the organism, it is not
immune to microbial infection in general and viral infection in particular. The
viruses that penetrate the CNS therefore possess neuroinvasive properties and are
usually also neurotropic, meaning that they infect neural cells (neurons and glial
cells) and by doing so may also become neurovirulent as they can participate in the
development of neurological diseases.
Neurological diseases are diverse and often remain not well understood, but
one constant fact is that they usually end up in loss of neuronal cells because a
portion of these precious cells will eventually die by different mechanisms. These
pathologies may be genetically determined or caused by environmental factors or
be provoked or perpetuated through a combination of both genes and environment.
Among the several environmental factors presumed or known to be involved are
the viruses, which possess potential neuropathogenic properties. Indeed, a viral
origin of neurological diseases is often suspected, but causes of neurological syn-
dromes of viral origin have often been difficult to identify, since CNS viral infec-
tions are often thought to represent complications of systemic viral illnesses. Some
of these neurovirulent viruses are able to infect neurons and/or glial cells, caus-
ing either an acute disease (e.g., rabies-induced death; reviewed by Hankins and
Rosekrans 2004) or chronic disease (e.g., measles virus-associated subacute scle
rosing panencephalitis; SSPE; reviewed by Young and Rall 2009). Several diseases
once described as degenerative, such as SSPE, are actually slow viral infections
with long asymptomatic incubation periods and prolonged durations of overt clin-
ical illness. Neuropathology can be a direct result of the infection, which will alter
cell functions or an indirect consequence of the infection, for example through the
action of proinflammatory mediators (neuroinflammation) that have direct detri-
mental effects on neural cells or attract inflammatory leukocytes to the site of
infection or both.
Neurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s d isease
(AD), amyotrophic lateral sclerosis (ALS), or multiple sclerosis (MS) have all been
postulated to have an infectious origin and described to comprise an inflammatory
component that could be triggered or enhanced by a viral infection. Among these
different neurological diseases, MS represents the archetype of scientific uncertain-
ties toward a CNS affliction of unknown etiology: both genes and environment have
been suspected, and several genes as well as several environmental factors may con-
tribute to neuropathogenesis. Several viruses have been implicated in MS etiology
or propagation in the last five decades, although not one has so far withstood the test
of time or closer scrutiny, perhaps because several different viruses may be involved
(Cook and Dowling 1980; Gilden 2005; Kakalacheva et al. 2011; Talbot et al. 2001).
However, the idea that ubiquitous viruses to which a genetically determined aber-
rant response is made and leads to relatively rare neurological diseases has garnered
increasing support.
In the long list of viruses that are neuroinvasive, neurotropic, and potentially
neurovirulent, the coronaviruses, from the order Nidovirales (reviewed by Siddell
Human Coronaviruses 95
and Snijder 2008), are prevalent and have been occasionally associated with neuro-
degenerative diseases of the CNS including MS, PD, and even AD. In humans,
these viruses are recognized respiratory pathogens involved in upper respiratory
tract infection in the population in general and in lower respiratory tract infection
associated with pneumonia and asthma exacerbation in more vulnerable populations
such as infant, elderly, and immunocompromised persons. Human coronaviruses
(HCoVs) other than SARS-CoV co-circulate during seasonal outbreaks, and they are
distributed worldwide, whereas SARS-CoV has not been detected in humans since
2003 (Vabret et al. 2009). Over the years, some coronaviruses have been associated
with neurological diseases in animals and among the five different coronaviruses
able to infect humans, at least three strains are neuroinvasive and neurotropic (Xu et
al. 2005; Gu et al. 2005; Arbour et al. 2000).
5.2 VIRUSES AND HUMAN NEUROLOGICAL DISEASES

Viral infection of the human CNS does occur and often induces acute encepha
litis, which can even lead to death depending on the tropism of the different viruses
involved (Whitley and Gnann 2002). Rabies virus (Hankins and Rosekrans 2004),
herpes simplex virus (HSV) (Aurelian 2005), as well as arthropod-borne flaviviruses,
including West Nile virus (WNV), Japanese encephalitis virus (JEV), and dengue
virus (Mackenzie et al. 2004), all represent well characterized viral agents that can
induce encephalitis in humans.
Long-term human neurological diseases may also be linked to viral infection. In
HIV dementia, the human immunodeficiency virus (HIV) induces the degeneration
of neurons in the hippocampus (reviewed by Mattson et al. 2005) with such neuro-
degeneration resulting in motor dysfunction and possibly cognitive impairment
(Nath and Berger 2004; Berger and Arendt 2000). Progressive multifocal leuko-
encephalopathy (PML) represents a human demyelinating disease where severe and
prolonged immunosuppression leads to the reactivation of the latent polyoma JC
virus (JCV; reviewed by Weissert 2011; Roberts 2005). Subacute panencephalitis
sclerosis (SSPE), a progressive fatal neurological disease, is also clearly linked to
a viral cause: the persistence of measles virus within the CNS (Rima and Duprex
2005).
However, in several long-term human neurological diseases, it is very hard
to ascertain a role of any given virus, due to the difficulty of establishing the
time point at which this (or these) virus(es) become(s) involved. In this regard,
Giovannoni et al. (2006) have elaborated a series of new criteria, adapted from Sir
Austin Bradford Hill’s for causation (Hill 1965). According to these authors, these
“new” criteria should replace Koch’s postulate when one wants to evaluate the
relevance of any given virus in relation to multiple sclerosis etiology (Giovannoni
et al. 2006). It is obvious that these criteria could also more efficiently fulfill the
requirements for other long-term human neurological diseases potentially related
to a viral infection as well. The HSV-1 and HHV-6 viruses were proposed as poten-
tial infectious agents causing or exacerbating AD (Itzhaki et al. 2004). Influenza A
was described as a factor that may increase the risk of PD (Takahashi and Yamada
1999) and viral respiratory infections in general were also described as a possible
risk factor in the development of PD (Tsui et al. 1999). Epidemiological studies
have also revealed an association between the risk of ALS and herpesviruses and
echoviruses infection (Cermelli et al. 2003). Finally, psychiatric disorders were
also investigated as a possible consequence of Borna disease virus (BDV) infection
(Waltrip et al. 1995).
5.3 THE CORONAVIRUSES: AN OVERVIEW

The Coronaviruses are members of the family Coronaviridae within the order
Nidovirales. They are classified in three different genera, namely α-, β-, and
γ-coronaviruses (de Groot et al. 2012) and are ubiquitous mainly respiratory and
enteric pathogens, with neurotropic and neuroinvasive properties in various hosts
including humans, cats, pigs, and rodents. Coronaviruses form a group of envel-
oped viruses that have the largest genome among RNA viruses. This 30-kb posi-
tive single-stranded polyadenylated RNA possesses 4 or 5 genes encoding structural
proteins (S, E, M, N; HE for the genus β-coronavirus) and several genes encoding
nonstructural proteins (Figure 5.1).
The spike protein (S) is a large type 1 transmembrane glycosylated protein
responsible for the recognition of the cellular receptor used by the virus to infect
a susceptible cell (Cavanagh 1995). The larger portion of the S protein is exposed
outside the surface of the viral envelope where it forms a homotrimer (Delmas and
Laude 1990), visible by electron microscopy, and which gives the virus its “crown-
like” (corona in Latin) morphology from what its name is derived (Figure 5.1a).
The envelope (E) protein is a small structural protein anchored in the viral enve-
lope, which has a role in the assembly of the virion and which appears responsible
for the adequate curving of the viral envelope (Liu et al. 2007). The membrane
(M) protein possesses three transmembrane domains and interacts with all the
other structural proteins of the virus and therefore helps to shape and maintain the
structure of the virion (Hogue and Machamer 2008). The nucleocapsid (N) pro-
tein associates with the viral genome and plays an essential role in encapsidating
it in a helical nucleocapsid within the viral particle (Hogue and Machamer 2008;
Macneughton and Davies 1978). The hemagglutinin-esterase (HE) is only present
in most species of the β-coronavirus genus. Like the S protein, it is a type 1 trans-
membrane protein that forms homodimers (Hogue and Machamer 2008) and that
interacts with different types of sialic acid associated with an apparent role in hem-
agglutination. It also possesses an acetyl-esterase function, which may be impor-
tant early during infection or during the release of viral particles from the infected
cells at the end of the replication cycle of the β-coronaviruses (Rottier 1990). The
larger part of the genome, the ORF1a and 1b, encode two polyproteins (pp1a and
pp1ab) which are cleaved by two viral proteases to yield 15 to 16 nonstructural
proteins (nsp), which all play a role in the replication of the virus (Gorbalenya et al.
2006; Lai and Cavanagh 1997). The other nonstructural (ns) proteins are accessory
and appear to mainly play a role in pathogenesis and in the virus–host interaction
(Narayanan et al. 2008).
(a)
(b) S (trimer)
E
HE (dimer)
N (associated with RNA)
(c) ORF1ab: 20–22 kb 7−10 kb
ns2 ns12.9
Leader ORF 1a ORF 1b HE S E M N PolyA
5´
Nonstructural proteins Structural proteins
FIGURE 5.1 Overview of the coronaviruses: electron microscopic appearance (a; bar =
100 nm) and schematic diagram of the viral particle (b) and of the 30-kb RNA genome (c).
The viral particle and the genome of the β-coronavirus HCoV-OC43 are represented as an
example, but the overall organization of all the coronaviruses is the same, with the excep-
tion of the HE gene, which encodes a hemagglutinin-esterase protein, only present as a fifth
structural protein in the viral envelope of some coronaviruses. The nonstructural (ns) proteins
are specific to each genus and their number varies between the different coronaviruses. ORF1
encodes two large polyproteins designated pp1a and pp1ab, which are cleaved by viral prote-
ases to yield 15 to 16 nonstructural proteins (nsp).
5.4 NONHUMAN CORONAVIRUSES INFECTING THE CNS

5.4.1 Mouse Hepatitis Virus
The first coronavirus shown to be neurotropic was the mouse hepatitis virus (MHV),
a member of the β-coronavirus genus, as early as 1949 when the JHM strain was iso-
lated from mice with disseminated encephalomyelitis and extensive demyelination
(Cheever et al. 1949; Bailey et al. 1949).
MHV represents the best characterized coronavirus. It infects mice and rats and
some strains are neurotropic and neuroinvasive, causing a large spectrum of dis-
eases from hepatitis to encephalitis and chronic demyelination. It is the subject of
several good reviews, which describe every aspects of its implication in neurological
diseases and which highlight the importance of both viral and host factors in the
process (reviewed by Bender and Weiss 2010; Cowley and Weiss 2010; Hosking
and Lane 2010). Briefly, MHV can invade the CNS using the transneuronal route
through the olfactory nerve (Barnett and Perlman 1993; Lavi et al. 1988). During
the acute phase of infection of the CNS, MHV induces encephalitis and appears to
infect different type of cells, which appear to vary for the different strains (Bender
and Weiss 2010). The virus spreads throughout the brain and rapidly reaches the
spinal cord, and an important up-regulation of cytokines, chemokines, and matrix
metalloproteinases (MMP) occurs as part of the antiviral innate immune response
(Hosking and Lane 2010). The MHV can also persist within the CNS and induce a
chronic demyelinating disease, which is partially immune-mediated, similar to what
is observed in multiple sclerosis in humans (Hosking and Lane 2010). Furthermore,
using the C57BL/6 murine model, it was shown that the moderately neurovirulent
MHV-A59 strain induced a modulation in expression of different types of genes
within the CNS, including several immunity-related, and that this modulation in
transcriptomic profile was accompanied by the activation of autoreactive T cells spe-
cific to myelin basic protein (Gruslin et al. 2005).
5.4.2 Swine Coronaviruses (PHEV)

The porcine hemagglutinating encephalitis virus (PHEV) was demonstrated to
induce disease ranging from gastroenteritis to encephalomyelitis in piglets (Siddell
et al. 1983; Andries and Pensaert 1980). The virus was isolated from the brains of
suckling pigs suffering from encephalomyelitis several years ago (Greig et al. 1962),
and the disease could be experimentally reproduced in piglets following intranasal
inoculation (Alexander 1962). Moreover, using murine models, the neuroinvasive-
ness and neurotropism of the virus were demonstrated (Hirano et al. 2004; Yagami
et al. 1986). PHEV induced a poor inflammatory reaction in CNS and infected cells
showed no cytopathological changes (Hirano et al. 2004).
5.4.3 Feline Coronaviruses (FCoV)

Feline infectious peritonitis (FIP) is a common cause of death in cats, caused by a
highly virulent variant of the feline coronavirus (FCoV), called FIPV, which either
represents a naturally distinct circulating virulent form of FCoV (Brown et al. 2009)
or which emerges from the less virulent virus feline enteric coronavirus (FECV) after
acquiring mutations (Rottier et al. 2005; Vennema et al. 1998). The intestine was
identified as the major site of persistence (Meli et al. 2004; Foley et al. 1997), but the
virus also persists in macrophages of healthy cats (Kipar et al. 2010). These infected
macrophages disseminate systemically and trigger immunological responses, which
result in microgranuloma formation, vasculitis, organ failure, and death (Vennema et
al. 1998; Poland et al. 1996; Pedersen and Boyle 1980). Neurological FIP may occur
in about one cat out of three with FIP disease (Foley et al. 1998; Kline et al. 1994).
The neurological FIP appears partially immune-mediated and may result in
uncontrolled inflammation in different parts of the brain that leads to diverse patho-
logical manifestations including meningitis (Slauson and Finn 1972) and even spinal
cord involvement (Legendre and Whitenack 1975). During neurological FIP, there
is often a small amount of virus present in FIP-affected brain tissue (Foley et al.
1998), but inflammatory cells are nevertheless recruited to the brain and appear to
contribute to disease, in part, through uncontrolled secretion of cytokines (Foley et
al. 2003).
5.5 HUMAN CORONAVIRUSES INFECTING THE CNS

Coronaviruses are all molecularly related in structure and mode of replication (Brian
and Baric 2005; Lai and Holmes 2001). Therefore, historically, the close structural
and biological relatedness of HCoV to the neurotropic animal coronaviruses has led
to speculation about the possible involvement of HCoV in neurological diseases. Up
until today, no clear specific association was made with any known human neuro-
pathology. However, HCoV-229E and HCoV-OC43 (Arbour et al. 2000; Arbour et al.
1999a,b; Bonavia et al. 1997), as well as SARS-CoV (Xu et al. 2005; Gu et al. 2005),
were shown to be neuroinvasive and neurotropic. The significance of the presence
of HCoVs in the human CNS and the related possible implications for neurological
diseases is discussed in the next sections.
5.6 HUMAN CORONAVIRUSES: EPIDEMIOLOGY

OF RESPIRATORY PATHOGENS
HCoV were first isolated in the mid-1960s from patients with upper respiratory tract
disease (McIntosh et al. 1967; Hamre and Procknow 1966; Tyrrell and Bynoe 1965),
and up until the fall of 2002, the only two known serological groups were repre-
sented by strains OC43 and 229E, which are respiratory pathogens responsible for
10 to 35% of common colds (Myint 1995). Over the last decade, several new corona-
viruses were identified, including the human HCoV-NL63 (van der Hoek et al. 2004),
HKU1 (Woo et al. 2005a), and the SARS-CoV, the causative agent of the severe
acute respiratory syndrome (SARS) (Drosten et al. 2003; Fouchier et al. 2003).
Therefore, HCoVs are now represented by five different strains; HCoV-229E,
-OC43, -NL63, -HKU1, and SARS-CoV. HCoV other than SARS-CoV are primar-
ily associated with upper and lower respiratory tract disease worldwide (Vabret et
al. 2009). However, even before the SARS epidemic of 2002, the HCoV were regu-
larly associated with severe respiratory distress in newborns (Gagneur et al. 2002;
Sizun et al. 1993) and as important trigger of acute asthma exacerbations (El-Sahly
et al. 2000; Johnston et al. 1995; Nicholson et al. 1993). More recently, HCoVs
were associated with acute lower respiratory tract infection, including pneumonia,
in both infants and immunocompromised patients (Gerna et al. 2006; Woo et al.
2005b). As summarized by Vabret and collaborators, two epidemiologic pictures of
HCoV infections have to be distinguished today. HCoVs other than SARS-CoV co-
circulate during seasonal outbreaks, and they are distributed worldwide, even though
a “regional” distribution may vary according to the geographic area and season.
On the other hand, the SARS-CoV, which was responsible for the first emerging
infectious disease pandemic of the 21st century, 8096 probable cases of SARS were
reported with a fatality of about 10% between the fall of 2002 and summer of 2003,
has stopped circulating in July of 2003 with the help of drastic public health policy
around the world (Vabret et al. 2009). Only a few additional sporadic cases were
reported between the fall of 2003 and the spring of 2004 in China and Singapore,
most of them being laboratory-related infections (Gu and Korteweg 2007; http://
www.who.int/csr/don/archive/disease/severe_acute_respiratory_syndrome/en/index
.html). Since the end of the SARS outbreak, it has been confirmed that bats are the
natural reservoir of the SARS-CoV (Li et al. 2005).
Among the five HCoV strains, at least HCoV-229E and HCoV-OC43, as well as
SARS-CoV, possess neuroinvasive properties as viral RNA can be detected in the
human brain (Xu et al. 2005; Gu et al. 2005; Arbour et al. 2000).
5.7 RESPIRATORY HUMAN CORONAVIRUSES INVADING THE CNS

As reviewed by Talbot and collaborators for HCoVs other than SARS-CoV (Talbot
et al. 2008) and for SARS-CoV (Nicholls et al. 2008; Gu and Korteweg 2007), coro-
naviruses that infect humans are primarily respiratory pathogens and they usually
target first epithelial cells from the respiratory tract (Miura and Holmes 2009).
One factor that influences a virus-induced pathogenesis is the type of cell suscep-
tible to this virus in a tissue or a specific organ. Binding of the virus to its target cell
through a receptor is a critical early step in infection. HCoV-229E uses aminopepti-
dase N (APN), also called CD13 (Yeager et al. 1992). SARS-CoV (Li et al. 2003) and
HCoV-NL63 (Hofmann et al. 2005) use angiotensin-converting enzyme 2 (ACE2);
SARS-CoV can also use L-SIGN (CD209L) (Jeffers et al. 2004). However, the cel-
lular receptor for HCoV-HKU1 and HCoV-OC43 remains to be identified. Sialic acid
in the form of N-acetyl-9-O-acetylneuraminic acid was identified as a ligand for
the S protein of HCoV-OC43 (Kunkel and Herrler 1993) and as a receptor determi-
nant during infection (Krempl et al. 1995), and it is now known that this particular
sialic acid is essential for infection of susceptible human epithelial and neuronal cells
(Desforges 2011). The major histocompatibility complex (MHC) class I C molecule
(HLA-C) was recently identified as an attachment factor that facilitates the infection
of susceptible cells by HCoV-HKU1 (Chan et al. 2009). All of the known receptors
mentioned above have been shown to be expressed in different cell types within the
respiratory tract and other tissues, including the CNS.
HCoVs are recognized respiratory pathogens, however, infectious particles, anti-
gens or RNA, were detected in tissues other than the respiratory tract, including the
CNS. To be neuroinvasive, viruses such as HCoV-229E and -OC43 and SARS-CoV
may use two different routes from the periphery. The first, called the hematogenous
route, involves the presence of a given virus in the blood where it can either remain
free for a period of time before it can infect the endothelial cells of the blood–brain
barrier (BBB) or infect leukocytes that will become some sort of viral reservoir.
Both situations occur during HIV infection of the CNS. Indeed, HIV-infected leuko
cytes migrating through the BBB (called the Trojan horse hypothesis; reviewed by
Kim et al. 2003) is one of the routes, and direct infection of the endothelial cells
from the BBB is also possible even though the viral replication is low (Argyris et al.
2007). A second form of any viral spread toward the CNS is through neuronal dis-
semination, where a given virus infects neurons in periphery and uses the machinery
of transportation within those cells in order to gain access to the CNS.
Infection of human leukocytic cell lines and of monocytes/macrophages by HCoV-
229E and HCoV-OC43 was reported (Desforges et al. 2007; Collins 2002), and
infection by HCoV-229E of peritoneal macrophages (Patterson and Macnaughton
1982) and murine dendritic cells expressing the human APN (Wentworth et al. 2005)
suggests that HCoVs may use these cells to disseminate to other tissues, where they
could be associated with other types of pathologies. SARS-CoV was also shown
to be able to infect human monocytes/macrophages (Nicholls et al. 2006; Gu et al.
2005), which produced a small amount of infectious particles (Yilla et al. 2005).
Moreover, monocyte-derived dendritic cells are also susceptible to a low-level pro-
ductive infection by SARS-CoV (Spiegel et al. 2006).
Both the human monocytic cell line THP-1 and human primary monocytes are
activated to produce TNF-α and MMP-9 following infection by HCoV-229E in cell
culture (Desforges et al. 2007). As activated monocytes eventually become macro-
phages as they invade tissues, this activation suggests that HCoV-229E-infected
monocytes would become activated in vivo, thus facilitating their passage toward
other tissues, especially in immunocompromised individuals, as this was observed
for murine cytomegalovirus (MCMV) (Reuter et al. 2004). The fact that HCoV-
229E could only infect partially immunocompromised transgenic mice (Lassnig et
al. 2005) suggests that HCoV-229E could take advantage of an immunosuppressed
environment and disseminate to different organs within susceptible individuals. The
establishment of a persistent infection in a human leukocytic cell line (Desforges et
al. 2007) is also consistent with the possibility that monocytes/macrophages serve
as a reservoir and vector for HCoV-229E toward other tissues, including the CNS
for this neuroinvasive HCoV (Arbour et al. 2000). The same situation may occur
for SARS-CoV, which infects monocytes-macrophages (Nicholls et al. 2006; Gu et
al. 2005) as a study with a mouse model suggests that after an intranasal infection,
the virus primarily replicate in the lungs before going into the brain (McCray et al.
2007). Furthermore, considering the fact that SARS-CoV is able to modulate the
innate immunity in dendritic cells (Spiegel et al. 2006), one can also speculate that
these cells could also serve as an eventual reservoir for this virus in order to reach
and maintain itself in the CNS. Our results indicate that HCoVs were also shown to
infect human endothelial cells of the BBB in culture (unpublished data), and it has
been speculated that SARS-CoV could do the same after viremia (Guo et al. 2008),
as both ACE-2 and CD209L are expressed on the endothelial cells of the human
BBB (Li et al. 2007). Therefore, the neuroinvasive HCoVs could use the hematoge
nous route to penetrate the CNS as illustrated in Figure 5.2.
On the other hand, after an intranasal infection, both HCoV-OC43 (Jacomy and
Talbot 2003) and SARS-CoV (McCray et al. 2007) were shown to infect the lungs
in mice and to be neuroinvasive as HCoV-OC43 (Butler et al. 2006; St-Jean et al.
CCL5/CXCL10/CXCL11
aT
Macrophage
MMP-9 CCL2/CCL5/CXCL12
TNF-α Astrocyte
ICAM-1
ICAM-1
TNF-α
Monocyte
Oligodendrocyte
Neuron
Blood Endothelial cells (BBB) CNS
FIGURE 5.2 Hematogenous route of neuroinvasion and possible mechanism of neuroviru-

lence of HCoV. Human monocytes are susceptible to infection by HCoVs including SARS-
CoV and are activated after infection. This activation involves, among other factors, the
production of MMP-9, which increases the permeability of the BBB, and of TNF-α, which
up-regulates the adhesion molecule ICAM-1 on endothelial cells of the BBB, facilitating
the passage of infected monocytes into the central nervous system (CNS). Viruses may also
directly infect endothelial cells to gain access to the CNS, where they can infect neurons.
Once in the CNS, the infected and activated monocytes produce proinflammatory cytokines,
such as TNF-α that can damage the myelin-synthesizing oligodendrocytes and/or neurons.
Infected monocyte-derived macrophages that entered the CNS (or microglia) may produce
chemokines, such as CCL5, CXCL10, or CXCL11, which will chemoattract activated T cells
and/or other monocytes into the CNS. These cells may then mediate an immune-mediated
pathogenesis after infection. Moreover, after sensing the infection, astrocytes may produce
chemokines, such as CCL2, CCL5, and CXCL12, that will also participate in the recruitment
of more infected leukocytes. Thus, coronaviruses can initiate a neuroinflammatory loop lead-
ing to neuropathology.
2004) and SARS-CoV (Netland et al. 2008) were detected in the CNS of susceptible
mice. Therefore, these two coronaviruses may use both the hematogenous and the
transneuronal route through the olfactory nerve toward the CNS. Furthermore, as
shown here in Figure 5.3, once in the brain, HCoV-OC43 is able to disseminate in
the cortex and medulla but the cerebellum remains uninfected. The hippocampus
represents another specific structure infected by HCoV-OC43 in the brain. Once in
(a) (b)
Cx
Cb
OB
M
(c) Cx
Cb
M
OB
(d) (e) (f )
CA1
DG
CA3
FIGURE 5.3 Transneuronal route of neuroinvasion through the olfactory nerve and spread
into the CNS of HCoV. (a) After intranasal infection of susceptible mice, HCoV-OC43 is
able to get into the brain through the olfactory nerve and replicate in the CNS. (b) At 3
days postinfection, viral antigens are detected in neuronal cells in the olfactory bulb (OB).
The right panel presents a higher magnification of the insert on the left panel. (c) At 7 days
postinfection, virus is still present in the OB, and spread is observed into the cortical area
(Cx) and the medulla (M). The cerebellum (Cb) is spared from infection. The right panel pre
sents a higher magnification of the insert on the left panel. (d) The hippocampus is infected by
HCoV-OC43 as shown by the presence of viral antigens in numerous neurons of the dentate
gyrus (DG) and in CA3 pyramidal neurons. The low number of infected neurons in the CA1
pyramidal layer (insert magnified in e) suggests that the virus uses the Schaffer’s collaterals
from CA3 cells before spreading to CA1 cells. This illustrates the transneuronal spread of
HCoV. (f) Hippocampal neuron infected by HCoV-OC43 illustrating that infection occurs in
the whole dendritic tree and axonal extension.
this region of the brain, the virus appears to propagate by a transneuronal route as
also illustrated and described in Figure 5.3.
5.7.1 Human Coronaviruses Other than SARS-CoV: Possible

Association with Neurological Diseases in Humans
Traditionally, the four Koch’s postulates have been applied to establish whether a
particular infectious agent causes a specific disease (Koch 1942). However, as beauti-
fully presented by Fredericks and Relman (1996), there are situations where Koch’s
postulates have to be reconsidered. Several viral infections, and especially the slow
viral infections related to diseases that are rare manifestations of a particular infec-
tion, represent this kind of situation where Koch’s postulate should be replaced.
Two well-known examples of the latter situation are related to Epstein-Barr virus
(EBV), where only a minority of individuals will develop Burkitt’s lymphoma and to
the human T-cell lymphotrophic virus (HTLV-1), which will cause the progressive
tropical spastic paraparesis/HTLV-1-associated myelopathy (PTSP/HAM) in only

1% of infected individuals (reviewed by Giovannoni et al. 2006).
The presence of HCoV-229E and HCoV-OC43 has been detected in different
neurological diseases in humans. An association that was made with PD stems from
a report of antiviral antibodies (Fazzini et al. 1992) and HCoV RNAs (Cristallo et al.
1997) in the cerebrospinal fluid (CSF) of PD patients. Moreover, detection of viral
RNA in human PD brains revealed that three out of three patients were positive for
HCoV-229E and one of them also for HCoV-OC43 (Arbour et al. 2000). Like many
neurodegenerative diseases, genetic and environmental factors seem to be involved
in the etiology of PD (Olanow and Tatton 1999). Influenza A was described as a
factor that may increase the risk of PD (Takahashi and Yamada 1999), and viral
respiratory infections in general were also described as a possible risk factor in the
development of PD (Tsui et al. 1999).
Multiple sclerosis (MS) represents another human neurological disease where an
infectious agent or agents may play a triggering role, with viruses the most likely cul-
prit in genetically predisposed individuals (Kurtzke 1993). There is a presumption that
several neurotropic viruses could be involved in MS pathogenesis but that they may
do so through similar direct and/or indirect mechanisms (reviewed by Kakalacheva
et al. 2011; Gilden 2005; Talbot et al. 2001; Johnson 1985; Cook and Dowling 1980).
However, research has not isolated or directly linked any specific virus with MS.
Association of coronaviruses with MS was suggested by their isolation from the CNS
of two patients (Burks et al. 1980). Other reports include intrathecal antibody synthesis
(Salmi et al. 1982) and ultrastructural observation (Tanaka et al. 1976). One report
demonstrated a significant association of colds with MS exacerbation and a significant
association of HCoV-229E infection in MS patients (Hovanec and Flanagan 1983) and
another report on the association of viral infections and MS (Sibley et al. 1985) com-
mented that seasonal HCoV infection patterns do fit the observed occurrence of MS
exacerbations. Acute disseminated encephalomyelitis (ADEM) is a neurological dis-
order characterized by inflammation of the brain and spinal cord caused by damage to
the myelin and is seen most frequently after nonspecific upper respiratory tract infec-
tions. Even though the etiological agent remains unknown, HCoV-OC43 was detected
in the CNS of a child with ADEM (Yeh et al. 2004).
As previously stated, although any direct correlation between HCoV-229E and
HCoV-OC43 and neuropathology in humans remains to be investigated, the detec-
tion of RNA in human brains does confirm that they are truly neuroinvasive (Arbour
et al. 2000). Furthermore, the case of these HCoVs in the CNS may represent a new
example where the traditional Koch’s postulate should be replaced by the previously
cited adapted Hill’s criteria (Giovannoni et al. 2006).
Even though HCoV-NL63 and HKU1 have never been detected in human CNS, a
recent report suggests that they may represent a comorbid risk factor in individuals
with serious mental disorders. The authors of this report are careful in the conclu-
sions they draw, as they are aware of the highly circumstantial nature of this associa-
tion of HCoV with recent psychotic symptoms, which is based on high titers of IgG
specific to these viruses in the serum of patients (Severance et al. 2011). Nevertheless,
studies to evaluate the extent that HCoV-NL63 and HKU1 can be neuroinvasive are
warranted.
5.7.2 SARS-CoV: Possible Association with

Neurological Diseases in Humans
The neuroinvasive properties of SARS-CoV were first suspected when viral RNA
was detected in the CSF of a 32-year-old female patient in Hong Kong in 2004 (Lau
et al. 2004). The year after, SARS-CoV neuroinvasive properties were indeed dem-
onstrated. The virus was isolated from brain tissue of a SARS patient who presented
neurological symptoms and a neuropathology associated with necrosis of neuronal
cells and glial cell activation. Moreover, the chemokine CXCL9/Mig (monokine
induced by γ-interferon) was expressed by glial cells in association with infiltration
of T cells and macrophages (Xu et al. 2005). The same year, another report indicated
that SARS-CoV RNA was also detected in the brain of eight different patients who
died from SARS, as the presence of the genomic RNA was detected in the cyto-
plasm of numerous hypothalamic and cortical neurons. Furthermore, edema and
scattered red degeneration were observed in the brain of six out of the eight autopsied
brains (Gu et al. 2005). Therefore, SARS-CoV is neuroinvasive, neurotropic, and
could be associated with the development of a neurological disease. Furthermore, the
involvement of SARS-CoV in CNS infections was underscored by the findings that
made use of transgenic mouse models expressing the human angiotensin-converting
enzyme 2 (ACE-2), which is the cellular receptor used by the SARS-CoV to infect
susceptible cells. Indeed, using these mice, it was shown that the SARS-CoV could
invade the CNS after an intranasal infection primarily through the olfactory bulb
(Netland et al. 2008) or even after an intraperitoneal infection (Tseng et al. 2007),
with concomitant neuronal loss (Netland et al. 2008; Tseng et al. 2007).
5.8 POSSIBLE MECHANISMS OF HUMAN CORONAVIRUS-

INDUCED NEUROPATHOGENESIS
Viral infection of oligodendrocytes could lead to demyelinating disease through the
alteration of their normal function or cytolysis as is the case for reactivated JCV, which
induces the progressive lysis of oligodendrocytes during PML (Roberts 2005; Sweet
et al. 2002). The release of myelin components could also provide targets for auto-
immune attack. Infection or activation of astrocytes and microglia could lead to release
of inflammatory mediators that could damage oligodendrocytes (Hovelmeyer et al.
2005; Gonzalez-Scarano and Baltuch 1999; Miller et al. 1997; McLarnon et al. 1993).
Several years ago, HCoV-OC43 was shown to productively infect cultured mouse
CNS cells and human fetal glial cells were also susceptible to HCoV-OC43 infec-
tion, although no infectious virus was detected (Pearson and Mims 1985). Over the
years, our own studies have shown that cell lines representative of the human CNS
are susceptible to productive infection by HCoV-229E and HCoV-OC43, including
long-term viral persistence (Arbour et al. 1999a,b), and that primary cultures of fetal
and adult human astrocytes and adult microglia are susceptible to infection by both
HCoV strains (Bonavia et al. 1997) with preliminary results consistent with infec-
tion of adult oligodendrocytes and human brain endothelial cells (unpublished data).
Furthermore, making use of a different mouse model, we also showed that
HCoV-OC43 induces an acute vacuolating encephalitis (Jacomy and Talbot 2003)
and viral persistence in the CNS associated with motor disabilities (Jacomy et al.
2006), suggesting that respiratory pathogens, like neurotropic and neuroinvasive
HCoVs, could be associated with neurodegenerative disease in susceptible individ-
uals. Like the mouse coronavirus MHV (the murine counterpart of HCoV-OC43),
which is able to induce a chronic white matter pathology characterized by focal
demyelinating lesions in brain and spinal cord in mice that survived acute encephali-
tits (Weiner 1973; Lampert et al. 1973) associated with immunopathological mech-
anism (Houtman and Fleming 1996; Wang et al. 1990), the HCoV-OC43 appears
to be able to establish itself in the CNS where it could eventually participate in
the development of a chronic demyelinating disease resembling MS. Furthermore,
we have demonstrated that MHV activates myelin basic protein-reactive T-cells
(Gruslin et al. 2005) and identified HCoV-myelin T-cell cross-reactivity in MS
patients (Boucher et al. 2007; Talbot et al. 1996). Moreover, variants of HCoV-OC43
harboring mutations in the Spike protein (S), acquired after a persistent infection
in human neural cells, are able to induce a long-term demyelination in the spinal
cord of susceptible mice (Jacomy et al. 2010) resembling the lesions observed in MS
patients. This suggests that persistence in neural cells has allowed HCoV-OC43 to
acquire mutations that may modify the capacity of the virus to spread in the CNS
correlating with demyelination in the spinal cord, which ends up in a modification in
the resulting neuropathology it causes in susceptible hosts.
We have shown that HCoV-OC43-infected astrocytes and microglia are activated
to produce proinflammatory mediators (Edwards et al. 2000). Activation of CNS glial
cells, astrocytes, and especially microglia is now recognized as a hallmark of neuro-
logical disorders (reviewed by Raivich and Banati 2004; Nelson et al. 2002), includ-
ing MS (Sriram and Rodriguez 1997) and AD (Barger and Harmon 1997). Viruses
that enter the CNS, such as HCoV (Arbour et al. 2000; Bonavia et al. 1997) are
thus prime candidate mediators of some of this neuropathologically relevant activa-
tion involving release of proinflammatory molecules such as cytokines, chemokines,
nitric oxide (NO), and reactive oxygen intermediates (ROS; reviewed by Bilzer and
Stitz 1996), as well as local antigen presentation to infiltrating T lymphocytes (Aloisi
et al. 2000). Studies of the interaction of viruses with microglial cells may hold keys
to understanding some neuropathogenic mechanisms. Even though HIV appears to
be able to infect neurons in very young children (Canto-Nogues et al. 2005), in the
adult CNS, HIV does not infect neurons, and it is therefore believed that, the damage
induced to neurons in HIV dementia is initiated by either cellular soluble mediators
such as MMP released from infected infiltrating macrophages (Zhang et al. 2003) or
by HIV proteins released from infected glial cells (Mattson et al. 2005). Our results
showing that HCoV-OC43 induces neuronal apoptosis in murine primary cultures and
in vivo (murine model), with apoptotic cells being infected or not, suggests that, like it
is the case for HIV, soluble mediators released by glial cells surrounding neurons may
be involved in the neuronal degeneration that leads to neuropathology (Jacomy et al.
2006). As stated above, HCoV-229E and HCoV-OC43 have the potential to infect dif-
ferent cells in the CNS. However, we have shown that the neurons are the main target
cell of HCoV-OC43 in the CNS of susceptible mice (Jacomy and Talbot 2003) and
in mixed primary cultures from the murine CNS (Jacomy et al. 2006) (Figure 5.4a)
and in co-cultures of human neurons and astrocytes (Figure 5.4b) obtained from the
(a) (b)
(c) (d)
8
HCoV-OC43
6
TCID50/ml
4
2
0
0 24 48 72 96 5 10 15 20 25
Hours p.i. Days p.i.
MAP1b
FIGURE 5.4 (See color insert.) The main target of HCoV-OC43 infection is the neuron in
mouse and human cell cultures. (a) Mixed primary cultures from the murine CNS. (b) Cocultures
of human neurons and astrocytes obtained from differentiated human NT2 cell line using a
protocol, which gives rise to a mixture of neurons and astrocytes. In both type of cultures,
HCoV-OC43 primarily targets the neuron for infection leading to axonal beading (white arrows
in a and b). The +viral S protein is in green in infected neurons and red represents the glial
fibrillary acidic protein (GFAP) in activated astrocytes. The blue signal is the nucleus detected
by the DNA-specific dye DAPI. (c) NT2-N cells (95% pure human neuronal culture) infected by
HCoV-OC43. The viral S protein is in red in infected neurons and green represents the micro
tubule associated protein 1b (MAP1b) expression in differentiated neurons. (d) HCoV-OC43
can establish a long term infection of the NT2-N cells for up to 25 days postinfection even
though cell death occurred in a portion of the NT2-N cells after acute infection.
differentiation of the human NT2 cell line using a modified protocol that give rises to
a mixture of neurons and astrocytes (Sandhu et al. 2003).
The NT2 cell line may also be differentiated as a 95% pure neuronal culture
named NT2-N, which expresses different markers of human neuronal cells (Pleasure
et al. 1992). Even though cell death was induced in a portion of the NT2-N cells
after infection by HCoV-OC43, these cells were able to sustain a long time pro-
ductive infection by HCoV-OC43 for at least 25 days (Figure 5.4d). When analyz-
ing this transcriptome modulation of the infected NT2-N cells (Figure 5.5) and by
confirming the results by RT-PCR, it became clear that, like other coronaviruses
(a) 24 h.p.i. 48 h.p.i. 72 h.p.i.
108
(Down-regulated/Up-regulated) (Down-regulated/Up-regulated) (Down-regulated/Up-regulated)

Neurotransmission/Neurogenesis 3/2 17/9 25/8
Mobility/Adhesion 0/2 3/8 6/7
Biosynthesis/Degradation 0/5 9/25 16/23
Immunity 2/0 4/14 5/19
Transcription/Replication 0/3 7/15 9/10
Cellular signaling 2/0 10/10 13/12
Metabolism 0/3 11/13 14/10
Cytoskeleton 2/0 7/1 7/1
Apoptosis/Survival 1/1 7/13 6/15
(b) (c) (d) Apoptosis/

Apoptosis/ Apoptosis/ Survival
Survival Survival 6 genes
1 genes 7 genes Neurotransmission/
Neurogenesis Cytoskeleton
17 genes 7 genes Neurotransmission/
Cytoskeleton Neurogenesis
Neurotransmission/ 7 genes
Neurogenesis 25 genes
Cytoskeleton Metabolism
2 genes 3 genes Mobility/Adhesion 14 genes
Metabolism 3 genes
11 genes Mobility/Adhesion
6 genes
Biosynthesis/ Cellular signaling
Degradation 13 genes
Cellular signaling 9 genes Biosynthesis/
Immunity Degradation
Cellular signaling 2 genes 10 genes
2 genes Transcripton/ 16 genes
Immunity
Transcripton/ 4 genes Replication
Replication 9 genes Immunity
7 genes 5 genes
(e) (f) Neurotransmisson/ (g) Neurotransmisson/
Apoptosis/ Neurogenesis Neurogenesis
Survival Cytoskeleton 9 genes 8 genes
1 genes 1 genes Apoptosis/
Neurotransmission/ Cytoskeleton Apoptosis/
Survival Mobility/ Mobility/
Neurogenesis 1 genes Survival
13 genes Adhesion Adhesion
2 genes 15 genes
8 genes 7 genes
Metabolism
3 genes Metabolism Metabolism
Mobility/Adhesion 13 genes
2 genes Biosynthesis/ 10 genes Biosynthesis/
Degradation Degradation
Cellular signaling 25 genes Cellular signaling 23 genes
10 genes 12 genes
Transcription/ Biosynthesis/
Replication Degradation
3 genes 5 genes Transcripton/ Transcripton/
Replication Immunity Replication Immunity
15 genes 14 genes 10 genes 19 genes
Neuroviral Infections: RNA Viruses and Retroviruses
(Bechill et al. 2008; Chan et al. 2006) in different cell types, HCoV-OC43 induces

the modulation of expression of several genes related to the unfolded protein response
(UPR) in infected human NT2-N and LA-N-5 neuronal cell lines (Favreau et al.
2009). The UPR is associated with the induction of ER (endoplasmic reticulum)
stress and represents a process that serves in cell homeostasis but which can become
deleterious to the cell when the stimulus is too strong or when it remains for a long
period of time (Ron and Walter 2007). Moreover, ER stress and impaired UPR has
been associated with human neurological diseases (Lindholm et al. 2006; Paschen
2003). Using two different variants of HCoV-OC43, we were able to relate the level
and duration of the UPR induction in neuronal cells with the S protein. Indeed, com-
pared to the wild type HCoV-OC43 virus, a variant harboring two-point mutations
in the putative receptor binding domain of the S protein, acquired during a persistent
infection of human neural cells, induced a stronger UPR, which eventually led to
more activation of caspase 3 and neuronal cell-death (Favreau et al. 2009). This is
of particular interest as we previously showed that caspase 3 activation and apparent
apoptosis was induced in the brain of infected mice and in mixed primary cultures
from the murine CNS (Jacomy et al. 2006).
The analysis of the transcriptomic modulation in the NT2-N neuronal cells
infected by the HCoV-OC43 also indicates that the level of expression of different
families of genes that can be related to diverse metabolic pathways is either up- or
down-regulated after the infection (Figure 5.5). For instance, the genes related to
the induction of the UPR are compromised in the “biosynthesis and degradation”
family. The modulation of expression of several genes in this family was directly
correlated with an ER stress and effective UPR in HCoV-OC43-infected neuronal
cells (Favreau et al. 2009). Therefore, one can argue that evaluating the complete
transcriptome of infected cells will certainly help to decipher the more complete
neuronal cell response after infection. In this regard, recent results from our labora-
tory clearly indicate that the “apoptosis/cell survival” (Favreau et al. 2012) and the
“neurotransmission/neurogenesis” family (Brison et al. 2011) identified in Figure
5.5 represent important pathways in the neuron response to HCoV-OC43 infection.
Neurotransmission between adjacent neurons is an essential process in the CNS
where glutamate is the major excitatory neurotransmitter involved in several func-
tions. In physiological conditions, glutamate is synthesized by neurons and released
in the synaptic cleft. Two types of ionotropic transmembrane receptors for glutamate
that mediates synaptic transmission exist in the central nervous sytem (CNS). The
FIGURE 5.5 Virus-induced modulation of the neuronal transcriptome in the NT2-N model
of human neurons. Infection of NT2-N cells by HCoV-OC43 induces a modulation of the
whole neuronal transcriptome. Exhaustive analysis on a genome-wide scale revealed that the
level of expression of several different genes encoding cellular proteins involved in diverse
metabolic pathways was significantly modulated. The genes were classified within nine dif-
ferent functional families, as shown in panel a, where the number indicates the number of
genes that are either down- or up-regulated at 24, 48, and 72 h postinfection (hpi). The rela-
tive proportion of the different gene families that are down-regulated are represented in pie
charts: (b) 24, (c) 48, and (d) 72 hpi, and the relative proportion of the different gene families
that are up-regulated are represented in pie charts: (e) 24, (f) 48, and (g) 72 hpi.
first is named the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor

(also known as AMPAr) and the second N-methyl-d-aspartate receptor (NMDAr)
(reviewed by Watkins and Jane 2006). Activation of the downstream AMPA receptor
allows the entry of sodium ions in the postsynaptic neuron in order to mediate the
neuronal membrane depolarization. Using another type of glutamate receptor, called
the glutamate-transporter 1 (GLT-1), astrocytic cells clear the excess of extracellular
glutamate in the synaptic cleft by uptake through special receptors that are high-
affinity tansporters of glutamate (reviewed by Kuzmiski and Bains 2010). A disrup-
tion of glutamate homeostasis may induce neuronal degeneration and eventual cell
death by an excitotoxic process (Mark et al. 2001), which is an excessive stimulation
by the neurotransmitter glutamate on its specific receptors (AMPAr and NMDAr)
(Olney 1969; Figure 5.6), This is of particular interest considering that since then,
Macrophage/Microglia
Presynaptic
IL-1/IL-6/TNF-α
neuron
GLT-1
K+ GLT-1
AMPAr
NMDAr
GLT-1
Na+ Ca2+
Postsynaptic
neuron
Excitotoxicity Astrocyte
FIGURE 5.6 HCoV infection induces neuronal death by excitotoxicity. Glutamate serves as
the primary excitatory neurotransmitter in the mammalian CNS. In physiological conditions,
glutamate is mainly synthesized by neurons and released into the synaptic cleft. Activation of
AMPA receptors (AMPAr) allows entry of sodium ions into the postsynaptic neuron, which is
responsible for depolarization of the neuronal membrane. This leads to activation of NMDA
receptors (NMDAr) that allow an entry of calcium ions into the postsynaptic neuron. Over-
stimulation of glutamate receptors (AMPAr and NMDAr) leads to neuronal injury by excito-
toxicity. In pathological conditions, regulation of glutamate release and uptake may be altered
by HCoV-OC43 infection, which induces an overwhelming stress in infected neurons of the
spinal cord that may lead to an increased synthesis/release of glutamate into the synaptic
cleft, leading to high level of calcium influx and excitotoxicity where neurons could be dam-
aged and die following excessive stimulation of glutamate on its specific receptors. In this
model, virus infection of neurons is detected by macrophage/microglia, which produce high
level of pro-inflammatory cytokines, which will in turn down-regulate the expression of the
glutamate receptor GLT-1 on astrocytes, which is responsible for the uptake of glutamate.
Therefore, these astrocytes will no longer be able to recapture the excess of glutamate and
neuronal cells will undergo excitotoxicity and eventual degeneration and death.
glutamate excitotoxicity was shown to be involved in several viral infections such

as WNV, Sindbis virus, JEV, HIV and HSV (Blakely et al. 2009; Carmen et al.
2009; Golembewski et al. 2007; Mishra et al. 2007; Haughey et al. 2001). The loss
of neuronal subpopulations in the brain during HIV dementia was also related to an
indirect mechanism conjugating glial activation, cytokines released and excitotoxic
transmission (Alirezaei et al. 2008; Masliah et al. 1996). Using a mouse model, we
recently showed that during infection by HCoV-OC43, hippocampal neurons died in
part by this glutamate excitotoxicity as they were partially protected from degenera-
tion in mice that were treated with the AMPA receptor antagonist (GYKI-52466)
(Brison et al. 2011). Therefore, HCoV infection of the CNS may also involve excito-
toxicity, which could account for the neurological disease associated with the infec-
tion. From a scientific point of view, it is interesting to note that this process has
over the years been associated with diverse human neurological diseases such as
Huntington’s disease, AD, PD, and ALS, as well as MS (Lau and Tymianski 2010;
Haeberlein and Lipton 2009).
Less work has been done in relation to the potential neurovirulence of the other
coronaviruses that can infect humans. However, some very interesting and relevant
data have been gathered on the underlying possible mechanisms related to the SARS-
CoV. Following infection of the mouse CNS, SARS-CoV was detected in a large
number of neurons in the cerebrum, thalamus, and brain stem although the olfactory
bulb and the cerebellum appeared relatively spared. Death of mice occurred before
significant infiltration of immune cells in the CNS, suggesting that the rapid infec-
tion of neurons was the cause of death in the absence of encephalitis (McCray et al.
2007). Moreover, it was shown only the year after that SARS-CoV could enter the
brain via the olfactory bulb and that it was able to spread very fast by a transneuronal
route to connected areas of the brain. Moreover, mice infected by SARS-CoV likely
died from death or dysfunction of infected neurons in particular regions of the brain,
including the cardiorespiratory centers of the medulla (Netland et al. 2008). This fact
is of high interest as SARS-CoV can be neuroinvasive in humans and that in those
cases, infected neurons underwent necrosis (Xu et al. 2005).
5.9 CONCLUSION AND SIGNIFICANCE

The presence of coronaviruses in the human CNS is now a recognized fact. Indeed,
they appear to be part of a viral flora of the brain, with potential neuropathological
consequences in genetically susceptible individuals with or without additional
environmental insults. Even though these respiratory pathogens could induce an
acute encephalitis as established using a murine model, they may also be associated
with the induction or exacerbation of other types of neurological diseases for which it
is very hard to ascertain a role to any given virus due to the difficulty of establishing
the time point at which this virus becomes involved.
In that regard, the “new” Hill’s criteria elaborated by Giovannoni and collabo
rators may represent a highly relevant tool in order to evaluate the relevance of these
recognized human respiratory pathogens as a factor that will influence the develop-
ment or the exacerbation of a long-term human neurological disease potentially
related to a viral infection. The examples of neurological diseases once described
as degenerative and which are slow viral infections with long-term asymptomatic
incubation periods related to different human viruses are potent indicators that it
may be relevant to associate the recognized presence of HCoVs in the CNS with
human neurological diseases. Therefore, more in-depth studies on neuroinvasive,
neurotropic, and potentially neurovirulent HCoVs are warranted in order to better
understand how they influence the neural cell functions and eventual destiny in con-
junction with genetic factors of the host.
ACKNOWLEDGEMENT
Work from our laboratory was supported by Operating Grant No. MT-9203 from
the Institute of Infection and Immunity (III) of the Canadian Institutes of Health
Research (CIHR) and Discovery Grant No. 42619-2009 from the Natural Sciences
and Engineering Research Council of Canada (NSERC) to P.J.T., who is the holder
of the Tier-1 (Senior) Canada Research Chair in Neuroimmunovirology award. E.B.
acknowledges a graduate studentship from the Multiple Sclerosis Society of Canada.
J.D. acknowledges a graduate studentship from NSERC. M.M.-P. acknowledges a
doctoral studentship from the Fondation Armand-Frappier. D.J.F. acknowledges a
doctoral studentship from the Fonds de la recherche en santé du Québec (FRSQ).
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6 Nonpolio Enteroviruses,
Polioviruses, and Human
CNS Infections
Anda Baicus and Cristian Baicus
CONTENTS
6.1 Introduction................................................................................................... 123
6.1.1 Classification, Morphology, Genome Structure, Organization,
and Protein Functions........................................................................ 124
6.1.1.1 Biological Properties........................................................... 124
6.1.1.2 Morphology......................................................................... 125
6.1.1.3 Genome Structure, Organization, and Protein Functions... 125
6.1.2 Cell Biology of Enterovirus Infection............................................... 126
6.2 Clinical Presentations.................................................................................... 127
6.2.1 Neurological Diseases....................................................................... 128
6.2.2 Skin and Eye Diseases....................................................................... 129
6.2.3 Fetal and Neonatal Infections............................................................ 130
6.2.4 Infections in Immunocompromised Patients..................................... 130
6.2.5 Nonneurological Aspects................................................................... 131
6.2.5.1 Cardiovascular Diseases..................................................... 131
6.2.5.2 Pleurodynia......................................................................... 131
6.2.5.3 Autoimmune Diseases........................................................ 131
6.2.5.4 Epidemiology...................................................................... 131
6.3 Pathogenesis................................................................................................... 133
6.4 Diagnosis....................................................................................................... 134
6.4.1 Laboratory Diagnosis........................................................................ 134
6.4.2 Conventional Techniques................................................................... 135
6.4.3 Molecular Techniques........................................................................ 136
6.5 Prognosis, Prevention, and Treatment........................................................... 137
6.6 Conclusions and Future Perspectives............................................................ 139
References............................................................................................................... 139
6.1 INTRODUCTION
Human enteroviruses (HEVs) are members of the Enterovirus genus in the
Picornaviridae family. This family consists of the following genera: Aphthovirus,
Cardiovirus, Enterovirus, Hepatovirus, Parechovirus, Erbovirus, Kobuvirus, and
Teschovirus (Carsten and Ball 2009).
123
Poliovirus, the prototype strain of the Enterovirus genus, is the etiological agent
of an acute paralytic disease, poliomyelitis. The studies on poliovirus began in 1908,
when Landsteiner and Popper transmitted the disease to monkeys (Landsteiner and
Popper 1908). Flexner supposed that poliovirus was strictly neurotropic, in 1910
(Flexner and Lewis 1910), and Enders et al., in 1949, cultured poliovirus strains in
nonneuronal tissue culture, opening the way for the production of viral vaccines
(Enders et al. 1949).
In 1948, the large group of viruses to which the polioviruses belong was discov-
ered. The pathogenesis of the infection in human and in experimental suckling mice
was at the origin of the classification of human enteroviruses (HEV) into four clus-
ters: (i) polioviruses (PV), which cause acute flaccid paralysis (AFP) (poliomyelitis)
in humans but not in mice; (ii) coxsackieviruses A (CVA), which cause myositis,
diseases of the central nervous system (CNS), exanthems and herpangina in humans,
and acute flaccid paralysis and myositis in mice; (iii) coxsackieviruses B (CVB),
which cause myocarditis and dilated cardiomyopathy, muscle disorders in humans,
and spastic paralysis and focal and limited myositis in striated muscles, in mice
(Godman et al. 1952); (iv) enteric cytopathogenic human orphan (ECHO) viruses,
which were not associated at the beginning with human or mice diseases.
Traditionally the enteroviruses are divided into five clusters, based on the dif-
ferences in host range and pathogenic potential: poliovirus, human enterovirus A
(HEV-A), human enterovirus B (HEV-B), human enterovirus C (HEV-C), and human
enterovirus D (HEV-D). Different viral serotypes were included within each of these
clusters on the basis of their antigenicity. The recent isolates of HEVs received con-
secutive numbers starting with HEV68. On the basis of phylogenetic analyses, HEVs
are classified into four species of enteroviruses (HEV-A, HEV-B, HEV-C and HEV-
D), and three species of rhinoviruses. The three poliovirus serotypes belong to the
human enterovirus C species (Brown et al. 2003). HEV isolates should be classified
as the same serotype if they diverge in the VP1 region less than 25%, and 12% within
corresponding nucleotide and amino acid sequences (Oberste et al. 1999; Caro et
al. 2001). The species HEV-A consists of coxsackieviruses CVA2–8, 10, 12, 14, 16,
human enteroviruses HEV71, 76, 89–92, 114; the species HEV-B consists of cox-
sackieviruses CVA9, CVB1–6, echoviruses (ECHO) (1–7, 9, 11–21, 24–27, 29–33),
HEV69, 73–75, 77–88, 93, 97, 98, 100, 101, 106, 107, the species HEV-C consists of
PV1–3, coxsackieviruses A1, 11, 13, 17, 19, 20, 21, 24, Human enteroviruses HEV95,
96, 99, 102, 104, 105, 109, 113, and the species HEV-D consists of human enterovi-
ruses HEV68, 70, 94, 111.
6.1.1 Classification, Morphology, Genome Structure,

Organization, and Protein Functions
6.1.1.1 Biological Properties
Enteroviruses are small particles, about 30 nm in diameter, with a positive sense,
single stranded RNA within a nonenveloped icosahedral symmetric protein capsid.
The genome is approximately 30% of the virion mass. Enteroviruses are resistant to
alcohol, phenol, quaternary ammonium ether, chloroform, and sodium deoxycholate,
Nonpolio Enteroviruses, Polioviruses, and Human CNS Infections 125
detergents that destroy other viruses (e.g., orthomyxoviruses) and are acid stable. All
enteroviruses have a density of about 1.34 g/mL in caesium chloride, and a sedimen-
tation coefficient of about 156S. The viral particle is inactivated by drying, ultra-
violet light, treatment with 0.3% formaldehyde, 0.1 N HCl, or free residual chlorine
at a level of 0.3–0.5 ppm, and heating at 50°C for 30 min. Their inactivation at all
temperatures tested is inhibited by magnesium chloride (a concentration of 1 mol/L
hinders inactivation). This property has led to the use of MgCl2 as a stabilizer of oral
polio vaccine (WHO 1997/2004).
6.1.1.2 Morphology
The viral capsid is formed of 60 identical units, protomers, and copies of the four
capsid proteins VP1, VP2, VP3, and VP4. The surface of the virion is formed by
the viral proteins, VP1, VP2, VP3, each containing about 250 amino acids. The
inner surface is formed by the smaller and relatively unstructured viral protein VP4
(70 amino acids), in conjunction with the amino (N-) terminal extension of VP1
and VP2. The core structures of the proteins VP1–VP3 are the same topologically,
each consisting of eight-strand antiparallel β barrel. The aspect of β barrel is wedge-
shaped, and the β strands are joined at one end by four short loops. The major struc-
tural differences among VP1, VP2, and VP3 are in the conformation and size of the
loops and in the sequences of N- and C-terminal extensions. The antigenic sites of
the virus are determined by these loops, and the N-terminal extensions contribute to
its stability. The three-dimensional structures of the capsid proteins of some entero-
viruses have been determined by X-ray crystallography and cryoelectron micros-
copy (Hendry et al. 1999; Hogle et al. 1985; Hogle and Filman 1989; Muckelbauer et
al. 1995). It has been shown that the surface of enterovirus has a star shaped peak at
the five fold axis of symmetry, surrounded by a channel of 2.4 nm deep and 1.2 to 3.0
nm wide (the canyon), and another protrusion at the three fold axis of symmetry. The
canyon is the attachment site for the enterovirus receptor. A lipid factor is located
in a hydrophobic pocket, beneath the floor of the canyon. By interaction of the virus
with the receptor, this lipidic factor must be displaced before the conformational
changes in the capsid. This pocket has been used as a target for antiviral compounds
that filled it and prevented conformational rearrangements associated with uncoating
and releasing of RNA (Rossmann et al. 2002).
6.1.1.3 Genome Structure, Organization, and Protein Functions

The enterovirus genome is a positive sense, single stranded RNA of about 7500 nt
in size. It is infectious because it functions as mRNA, and is directly translated into
a polyprotein in a cap-independent manner by the host cell ribosomes. The RNA
genome is covalently linked at its 5′ end to a virus encoded proteine Vpg (22-amino
acid long), and has a polyadenylated 3′ end (poly A). The genome is monocistronic,
with a 5′ untranslated region (UTR) (about 10% of the genome, ranging between
742 and 750 nt), followed by the single open reading frame (ORF), which encodes
a polyprotein of about 250 kDa, and by a 3′UTR (about 1% of the genome, ranging
between 70 and 100 nt).
The secondary structure of the 5′UTR genome looks like a tRNA-like structure.
It contains a cloverleaf structure playing a role in the initiation of negative strand
RNA synthesis, and an internal ribosomal entry site (IRES), which mediates the
cap independent translation of the viral RNA. The 3′UTR has a secondary structure
(pseudoknot) involved in RNA replication, and it is highly conserved among the
enteroviruses. The 3′ poly A end has a role in infectivity (Hellen and Wimmer 1995).
6.1.2 Cell Biology of Enterovirus Infection

Enterovirus replication takes place in the cell cytoplasm. The enterovirus infection
begins with the attachment of the virus to a specific receptor on the surface of the
host cell membrane. Many picornaviruses have receptor molecules belonging to the
integrin (e.g., αvβ3, αvβ6), SCR (Short Consensus Repeat) like (e.g., DAF [decay accel-
erating factor], CD 55) and the immunoglobulin superfamilies (e.g., CD155, ICAM-1
intercellular adhesion molecule-1). The poliovirus uses only one receptor (CD155)
for attachment and entry into the cell (He et al. 2000), but some enteroviruses (CAV
21) have been shown to use a cellular receptor (DAF, CD 55) for attachment, and a
coreceptor (ICAM-1) for entry into the cell (Shafren et al. 1997). Coxsackievirus B3
(CVB3) recruits CAR (coxsackievirus adenovirus receptor) to the site of infection by
binding to a second receptor DAF that is expressed in the epithelial cells (Coyne and
Bergelson 2006). The extracellular region of the receptors from the immunoglobulin
superfamily comprises 2 to 5 amino-terminal immunoglobulin-like domains. The
amino-terminal domain (D1) of these molecules is involved in the binding with the
conserved amino acid residues of the picornavirus canyon, which can trigger viral
instability and uncoating.
After binding to the receptor, the virus protein shell is removed and its genome
enters the cytoplasm. The interaction between the cell receptor sequences and the resi-
dues of the canyon floor displaces residues at the protomer interface and below the
canyon floor. This conformational change results in externalization of myristoylated
capsid protein VP4 and the N-terminus of the capsid protein VP1. An altered A viral
particle (135S) results, with a higher affinity for lipid membranes than native particles.
The insertion of the externalized proteins into membrane binds the virus particle to
the cellular membrane in a receptor-independent manner. Thus, the pores and channels
are created in the cell membrane through which the viral RNA may enter the cyto-
plasm. By using a combination between the imaging of fluorescent PV in live cells and
biochemical assays, it was demonstrated that PV enters the cell by independent clas-
sical clathrin- and caveolin-mediated pathways, sense an energy-, actin-, and tyrosine
kinase dependent endocytic mechanism. This process is followed by low pH-mediated
exposure of hydrophobic residues in an early endocytic vesicle (Thorley et al. 2010).
In the cytoplasm of the infected cell, a cellular phosphodiesterase removes the
Vpg prior to RNA translation. The cellular enzymes are responsable for RNA trans-
lation to polyprotein. The 40S ribosomal subunit associates with the IRES and scans
the RNA to the initiation codon AUG, where a 60 S ribosomal subunit joins the com-
plex, and elongation of translatation polyprotein occurs. The cleaveage of the eukary-
otic translation initiation factor (eIF4G), which is a factor of the initiation complex
(eIF4F) by the viral 2A protease, is responsible for the inhibition of cap-dependent
initiation of translation. The viral polyprotein is clevead by virus-encoded proteases
(2A, 3C, 3CD) into 3 intermediate proteins, P1, P2, and P3. Protease 2A separates
the structural protein P1 from the nonstructural proteins P2 and P3. Protease 3CD
cleaves P1 into VP0, VP3, and VP1. The cleavage of VP0 into VP4 and VP2 pro-
teins occurs during viral maturation, and it may be linked to the encapsidation of the
RNA. The nonstructural proteins precursors P2 and P3 are separated by 3C/3CD
protease into 2A, 2B, 2C, 3A, 3B (Vpg), 3C, and 3D. The viral 2A and 3C proteases
mediate the inhibition of cellular RNA synthesis. 3D is an RNA-dependent RNA
polymerase necessary for viral RNA synthesis and 2B-C, 3A-B proteins play differ-
ent roles in viral multiplication.
The replication involves transcription of the positive stranded RNA into a com-
plementary negative stranded RNA that is used as a template for many new single
positive strands. The process begins with a VPg uridylation and synthesis of comple-
mentary negative-strand RNA molecules via the transcription of poly(A) by the RNA
dependent RNA polymerase 3D. The replication takes place in the cytoplasm of host
cell endoplasmic reticulum-derived rosette-like membranous structures that act as
scaffold for assembly of the replication complex and protect the RNA from nucleases.
Other cellular and viral factors are involved in RNA synthesis, cellular RNA bind-
ing proteins, viral proteins 2A, 2B, 2C, 3AB, 3C, and 3CD. The RNA-dependent
RNA polymerase lacks proofreading activities, and this results in rapid accumulation
of mutations upon replication. The new positive RNA strand synthesis is linked to
the encapsidation or it could be a template for translation. The time required for a
simple replication cycle ranges from 5 to 10 h and it depends on the serotype, mul-
tiplicity of infection, pH, and temperature. In the enterovirus assembly, the earliest
component is the 5S protomer, which consists of one copy of each of VP0, VP3, and
VP1. The protomer is the precursor of the 14S pentamer, with the composition (VP0-
VP3-VP1)5. 12 pentamers form, by self association, 75S procapsids (empty capsids)
(VP0-VP3-VP1)60. By insertion of the newly synthesized RNA into the procapsid is
formed the provirion 150S, that is not infectious. The cleavage of VP0 into VP4 and
VP2 is responsible for conversion of the provirion into the virion, making the viral
assembly irreversible. In the infected cells, the positive stranded genome is ampli-
fied through a negative stranded intermediary to about 50000 copies /cell, but only
0.1%–2% of them are infectious. The progeny viruses are released from the cell by cell
lysis. The morphological changes developed by the cells infected with HEV strains
include cytopathic effects, condensation of chromatin, nuclear blebbing, proliferation
of membranous vesicles, changes in membrane permeability, leakage of intracellular
components, and shrivelling of the entire cell (Racaniello 2006).
6.2 CLINICAL PRESENTATIONS

The clinical manifestations in enterovirus infections are determined by factors such
as viral serotype, infecting dose, tissue tropism, portal of entry, patient’s age, gender,
and immune status. The ability of these viruses to multiply in the gastrointestinal tract
gave their name, even if they are not responsible for enteric disease. Most enteroviral
infections are asymptomatic or are associated with undifferentiated febrile illness,
associated with milder respiratory illness or a rashes disease. The nonspecific lower
respiratory illnesses caused by the HEVs can results in bronchitis, bronchiolitis, and
pneumonia. HEV strains including HEV68, 71, coxsackieviruses A9, A21, B2, B4,
and echovirus 9, 11, 22 have been isolated from samples of the patients with severe
or fatal viral bronchopneumonia (Chang et al. 1999; Jacques et al. 2008; Oberste et
al. 2004). Less commonly, some infections cause severe illness such as viral menin-
gitis, encephalitis, acute flaccid paralysis, acute hemorrhagic conjunctivitis (AHC),
neonatal sepsis-like disease, myocarditis, and pleurodynia. Severe chronic diseases
such as dilated cardiomyopathy, neuromuscular diseases, and type 1 diabetes could
be associated with enterovirus infections. The rates of these infections are higher in
infants, compared with adults.
6.2.1 Neurological Diseases
Viral infection of the CNS can involve the meninges (meningitis), the brain (encepha-
litis), the spinal cord (myelitis), spinal roots (radiculitis), or a combination of sites
(meningoencephalitis, encephalomyelitis, or myeloradiculitis). HEV infections are
more frequently associated with viral meningitis, but infrequently associated with
encephalitis. In aseptic meningitis, there is clinical and laboratory evidence for men-
ingeal inflammation, with negative bacterial culture. The etiologies of aseptic menin-
gitis include viruses (enteroviruses, herpes simplex virus, human immunodeficiency
virus, West Nile virus, varicella-zoster virus, mumps, and lymphocytic choriomen-
ingitis virus), bacterial infections (mycobacteria, spirochetes), parameningeal infec-
tions, brain abscess, medications, and malignancy. The clinical symptoms of aseptic
meningitis are similar to those of bacterial meningitis: fever that ranges from 38°C
to 40°C, headache, no change in mental status, no seizures, stiff neck, photophobia,
occasionally anorexia, nausea, and vomiting. Over 90% of aseptic meningitis cases
in infants are due to HEV, and the most common symptoms are fever and irritabil-
ity. The outbreaks of meningitis are caused by certain serotypes of HEV-B species:
coxsackievirus B5, echoviruses 6, 9, 30, whereas coxsackievirus A9, B3, and B4 are
mostly endemic (Lee and Davies 2007). The children recover completely within 3 to
7 days of onset, but symptoms often persist in adults for longer (Rotbart et al. 1998).
In acute viral encephalitis, direct invasion of the brain occurs as an extension
of viral meningitis or via retrograde spread through the peripheral nerves. In this
disease, an altered level of consciousness, often with seizure, and focal neurologi-
cal signs, occur. In pure encephalitis, photophobia and nuchal rigidity are usually
absent, but they often occur in meningoencephalitis. 11% to 22% of all cases of viral
encephalitis are caused by HEV strains, most often coxsackievirus types A9, B2, B5,
and echovirus types 6 and 9.
HEV71 has been recognized as a highly neurotropic virus. Aseptic meningitis,
encephalitis, flaccid paralysis, and rhombencephalitis occur as complications in
children younger than 5 years. The necessity to improve surveillance for HEV71-
associated HFMD (hand, foot, and mouth disease) outbreaks, even for adults, has
been demonstrated by several studies (Chan et al. 2003; Hamaguchi et al. 2008; Ooi
et al. 2010). The distribution of viral lesions in the HEV71 infections involves the
pyramidal and extrapyramidal tracts of the CNS. The neurological recovery is poor,
and the risk of mortality is increased for the patients with diffuse cerebral edema or
intractable seizures. The recovery is rapid for the patients with self-limited seizure
activity (Fowlkes et al. 2008).
Poliomyelitis is an acute flaccid paralysis (AFP) caused by three poliovirus (PV)

serotypes. Most of poliovirus infections are asymptomatic (90%) or are associated
with minor illness (abortive poliomyelitis, 4%–8%) characterized by fever, headache,
sore throat, malaise, listlessness, nausea, and vomiting. In patients with persistent
viremia (1%–2%), the virus enters the nervous system by crossing the blood–brain
barrier or by axonal transportation from a peripheral nerve and attacks the anterior
horn of the spinal cord, leading to inflammation and possible cell death. About one
third of cases with CNS infections are limited to aseptic meningitis. In 0.1%–1% of
cases, poliovirus infection is associated with paralytic poliomyelitis. The persist-
ing asymmetric weakness (flaccid paralysis), which results from lower motor neuron
damage is the characteristic sign of poliomyelitis. The severity of this disease ranges
from flaccid paralysis of a single extremity to tetraplegia, with development of the
paralysis over 2 to 3 days. In the affected extremities, decreased or absent tendon
reflexes without sensory loss occur. The proximal limb muscle is more involved
than the distal, and the legs are more involved than the arms. Most recoveries occur
within 6 months, but a long time (2 years) may be required for complete remission.
The involvement of the motor cranial nerves (most common the VIIth, IXth, and
Xth) occurs in bulbar poliomyelitis, which is characterized by dysphagia, dysarthria,
difficulty in handling secretions, respiratory compromise, and cardiovascular dys-
function. During the 1950s, before vaccination programs, iron lungs were used to
assist the breathing of patients with such polio disease (Melnick 1996).
In the differential diagnosis enters the polio-like illnesses, which can be caused by
nonpolio enteroviruses (CVA7, HEV70, 71), West Nile Virus, and Borrelia burgdor-
feri. Other diseases produce acute paralysis associated with other clinical features,
such as spinal cord disorders (transverse myelitis, infarction, compression), periph-
eral neuropathy (Guillain-Barré syndrome, acute intermittent porphyria, infectious,
and toxic neuropathies), disorders of the neuromuscular transmission (myasthenia
gravis, botulism, tick paralysis), and disorders of the muscles (inflammatory myopa-
thy and rhabdomyolysis) (Simmons 2010).
Postpoliomyelitis syndrome (PPS) is a neurological disorder that occurs in per-
sons who had a period of partial or complete functional recovery after acute paralytic
poliomyelitis, followed by an interval (usually 15 years or more) of stable neurologi-
cal function. The symptoms are new or increased muscle weakness and/or atrophy,
muscle and joint pain, increased muscular fatigability, fasciculations, cramps, gen-
eral fatigue, and cold intolerance (Farbu et al. 2006). The distal degeneration of the
enlarged postpoliomyelitis motor units is the most likely cause (Grimby et al. 1998;
Lin and Lim 2005). Contributing factors to PPS may be aging with motor unit loss,
overuse, and disuse. The excessive metabolic effort on remaining motor neurons
over years leads to atrophy of the orphaned muscle fibers, with progressive weakness
in the previously affected muscles, followed by fatigue and pain.
6.2.2 Skin and Eye Diseases

The hand, foot, and mouth disease (HFMD) is a self-limiting childhood disease char-
acterized by fever, vesicular lesions on the buccal mucosa and tongue, and small,
tender cutaneous lesions on the hands, feet, and buttocks. Herpangina is a disease
characterized by an onset with fever and odynophagia associated with vesicular or

ulcerative lesions of the tonsils, uvula, and soft palate (Keels 2010). HEV71, CVA16,
CVA10 cause HFMD and herpangina. The illness usually resolves in 2 to 3 days with-
out complications (Pallansch and Ross 2001). The main complications of HFMD are
encephalitis and a polio-like disease. The infection with CVA16 is not associated with
neurological disease, but the rash it causes is indistinguishable from that caused by
HEV71. In children younger than 5 years, the exanthem caused by enteroviruses man-
ifest as rubelliform or roseola-like rashes on the face, neck, and trunk. The petechial
and purpuric rash caused by infection with echovirus 9 or CVA9 could create a con-
fusion with meningococcemia if aseptic meningitis occurs simultaneously. A highly
contagious acute hemorrhagic conjunctivitis (AHC) is commonly caused by HEV70
and a new antigenic variant CVA24v and adenovirus (Leveque et al. 2010; Sane et al.
2008). This ocular infection is characterized by pain, periorbital swelling, red eyes
with conjunctival hemorrhage and excessive tearing, usually with involvement of the
second eye within 24 to 48 h. The illness is self-limited and resolves within 10 days
without complications. When AHC is caused by HEV70, the CNS diseases can occur.
6.2.3 Fetal and Neonatal Infections

Maternal enteroviral infection during pregnancy can have a transplacental spread to
the fetus, increasing the risk of early spontaneous abortions or in rare cases of intra-
uterine fetal death (Johansson et al. 1992). Most neonates of infected mothers are
unaffected, because the transplacental passage of virus does not occur easily (Amstey
et al. 1988). Clinical presentations of the neonate infections that occur in the perinatal
period vary from nonspecific febrile illness, to severe life-threatening disease. The
severe enteroviral infections in neonates are caused by echovirus 6, 11, 20, 21, 31,
and CVB types 2 and 5. Coxsackieviruses B1 and A9 have been reported to occur,
but very rarely (Lu et al. 2005). Approximately 50% of severe cases have meningo-
encephalitis, 25% have myocarditis, and 25% have a sepsis-like disease. Neonatal
myocarditis due to coxsackievirus infection is often accompanied by encephalitis,
and sometimes by hepatitis (Lin 2003). The rate of mortality is low (10%) for the
group with meningoencephalitis and high (100%) for the group with sepsis-like dis-
ease (most deaths occur within 1 week of onset) (Burchett and Dalgic 2008).
6.2.4 Infections in Immunocompromised Patients

In patients with acquired or hereditary defects in B lymphocytes, enterovirus infec-
tions result in chronic infections of the CNS, skeletal muscles, and the gastrointestinal
system. Enterovirus infections occur also in children with X-linked agammaglobulin-
emia, with severe immunodeficiency syndrome (Fischmeister et al. 2000; Chakrabarti
et al. 2004). When these persons are exposed to oral polio vaccine viruses, the virus
multiplication for a long time in the bowel is followed by a high risk for the emergence
of neurovirulent poliovirus strains. The persistent nonpolio enterovirus infections
in CNS have been caused especially by echoviruses, and isolate by CVA4, CVA11,
CVA15, and CVB2 and CVB3. The chronic progressive meningoencephalitis associ-
ated with a dermatomyositis-like syndrome and chronic hepatitis is characteristic for
infection. The disease is progressive and the disorder is usually fatal because of the
gliosis of gray and white matter and focal loss of neurons.
6.2.5 Nonneurological Aspects
6.2.5.1 Cardiovascular Diseases
Cardiac involvement of enterovirus infection occurs in the form of myopericarditis.
The most frequent pathogens identified in myopericarditis include CVB and echovi-
ruses (Kuhl et al. 2005). Inside the myocyte, the viral protease 2A of CVB cleaves a
cytoskeletal protein dystrophin, leading to disruption of the dystrophin-glycoprotein
complex that is essential for normal cardiac function (Badorff et al. 1999; Xiong et
al. 2007). In neonates, enterovirus myocarditis is a rare and severe disease, and often
results in chronic cardiac sequelae or leads to death (Freund et al. 2010).
6.2.5.2 Pleurodynia
Pleurodynia (Bornholm disease, devil’s grippe) is an acute illness caused by CVB
viruses (mainly CVB3 and CVB5), which are responsible for severe muscular
pain in the chest and abdomen, sometimes mimicking serious surgical conditions.
Symptoms as fever, headache, anorexia, nausea, and emesis are associated.
6.2.5.3 Autoimmune Diseases

Coxsackieviruses have been linked to the induction of autoimmune diseases such
as chronic autoimmune myocarditis and type 1 diabetes. While myocarditis is con-
sidered to be a clinical precursor to dilated cardiomyopathy (DCM) (Richer and
Horwitz 2009), type 1 diabetes is a disease characterized by a defect in insulin pro-
duction caused by selective destruction of islet β cells (80%–90%) of the pancreas. A
recent systematic review showed a clinically significant association between entero-
virus infection and type 1 diabetes (T1D), with a more than nine times the risk of
infection in cases of diabetes and three times the risk in children with autoimmunity
(Yeung et al. 2011). CVB viruses (mainly CVB4) can play a role in the early phase
of the disease through the infection of β cells and the activation of innate immunity
and inflammation (Hober and Sauter 2010).
6.2.5.4 Epidemiology
Enterovirus infections are quite prevalent worldwide as sporadic infections or epi-
demic outbreaks. More than 50% of nonpolio enterovirus infections and more than
90% of poliovirus infections are asymptomatic. Only a minority of infections are
associated with specific clinical syndromes. These infections occur throughout the
year in the tropics, but in temperate climates, the rates of infection are highest in
the summer and fall. The viruses spread mainly by the fecal-oral route, but can
also be transmitted by respiratory droplets or indirectly via contaminated water or
fomites. The transmission is higher in poor sanitation conditions and in crowded
living conditions. Most of the infections occur in young children that are shedders
of enteroviruses and are usually the index cases in family outbreaks. Host factors
such as immunodeficiency, especially a deficient humoral immunity, and age, can
predispose to severe infections.
Depending on environmental conditions, HEVs can remain viable at room temper-

ature for several days and in contaminated soil and water for weeks or even months
(Cohen 2006). Sewage contamination of water supplies or swimming in natural waters
in areas where there is limited sewage treatment can result in enterovirus outbreaks
(Begier et al. 2008). The information concerning circulations of HEV serotypes dur-
ing any given year are important because changes in the predominant serotype can be
accompanied by disease outbreaks (Leveque and Laurent 2008). The environmental
surveillance takes place when there is the risk of reintroduction of wild poliovirus or
the risk of the circulation of vaccine-derived poliovirus strains in population.
Enteroviral meningitis outbreaks with echovirus types 4, 9, 11, 30 involved many
communities throughout Europe and also other continents (CDC 2000; Dalwai et al.
2010; Leveque et al. 2010; Wang et al. 2002; Yamashita et al. 1994). In the United
States, between 1970 and 2005 was reported that echovirus 9 was the most com-
monly isolated enterovirus from cases with aseptic meningitis (Khetsuriani et al.
2006), whereas in 2007 CVB1 was implicated in an outbreak of severe neonatal
infections (CDC 2008). Before 2000, sporadic cases of aseptic meningitis with echo-
virus type 13 were recorded worldwide. Since 2000, a number of countries, includ-
ing Germany (Diedrich et al. 2001), the United States (CDC 2001), Spain (Avellon
et al. 2003), France (Archimbaud et al. 2003), Belgium (Thoelen et al. 2003), Israel
(Somekh et al. 2003), and Japan (Iwai et al. 2010) reported aseptic meningitis out-
breaks associated with this serotype.
Since the first report about HEV71 isolation in 1969 in California from the stool
sample of an infant with encephalitis (Schmidt et al. 1974), outbreaks of HEV71 dis-
ease have been reported worldwide (Alexander et al. 1994; Chan et al. 2003; Gilbert
et al. 1998; Ho et al. 1999; Podin et al. 2006; Tu et al. 2007; Zhang et al. 2009).
HEV71 circulation appears to be increasing in the United States (Perez-Velez et
al. 2007). A 2–3 year cyclical pattern of these outbreaks was recorded in Malaysia
(Podin et al. 2006), Japan (Ang et al. 2009), and the United Kingdom.
The earliest descriptions of epidemic poliomyelitis by using basic epidemiological
methods were recorded in the United States between 1893 and 1894 (Caverly 1894;
Putnam and Taylor 1893) and in Sweden in 1905 by Wickman. At the beginning of
the 20th century, the polio outbreaks began to be more frequent, and widespread
throughout Europe and the United States. The disease was controlled by using two
vaccines: the formalin-inactivated vaccine (IPV) accomplished by Jonas Salk (1953),
and live-attenuated vaccines (OPV) accomplished by Albert Sabin (1956) (Sabin
1985; Salk 1955). Both vaccines contain three components, one for each serotype of
poliovirus. IPV, obtained by formaldehyde inactivation of tissue culture-propagated
selected wild poliovirus strains, namely Mahoney (Salk type 1), MEF-1 (Salk type 2),
and Saukett (Salk type 3), induces less mucosal immunity, in the intestin than OPV,
requires a booster to achieve lifelong immunity, and poses no risk of vaccine-related
disease. The OPV strains of Sabin consists of three live attenuated Sabin poliovi-
rus strains obtained by sequential in vitro and in vivo passages of the wild strains,
and it has now become the main instrument for the wild-type-poliovirus eradication
program in the developing countries. The risks of OPV vaccination is the potential
reversion to neurovirulence of the attenuated strains during their replication in the
human intestine, with the emergence of virulent vaccine-derived poliovirus strains
(VDPVs) and vaccine-associated paralytic poliomyelitis (VAPP) (Ward et al. 1988).

Vaccine-associated paralytic poliomyelitis may occur in recipients of OPV at a fre-
quency of 1 case per 750,000 primary vaccinations and 1 per 1.2 million recipients
of two vaccine doses (Nkowane et al. 1987; Prevots et al. 1994). VAPP occurs in both
OPV recipients and their unimmunized contacts. The paralysis is most frequently
associated with type 3 in vaccine recipients, and with type 2 among contacts of
cases. The VDPV isolates have more than 1% nucleotide divergence in VP1 coding
region as opposed to the original vaccine Sabin strains. These isolates can spread in
populations with gaps in immunization (circulant cVDPV), and could be responsible
for outbreaks. Such cVDPV outbreaks have occurred in countries such as Nigeria,
Ethiopia, Congo, Myanmar, Niger, Indonesia, Madagascar, China, the Philippines,
Dominican Republic, and Haiti since 2000 (WHO 2009). In most situations, these
cVDPV strains were recombinants between OPV and HEV-C strains in the non-
structural coding region, excepting those from China (CDC 2006; Jiang et al. 2006).
In developing countries, poliovirus infection in children at an early age has less often
been associated with paralysis. In developed countries, poliovirus infection in older
children and adults unprotected by vaccination could be followed by paralysis.
Genetic recombination appears to be an integral part of poliovirus evolution.
Recombinant genomes are detected among OPV strains excreted by healthy vac-
cinated individuals, their contacts in the community (Blomqvist et al. 2003), and in
patients with VAPP (Georgescu et al. 1997).
6.3 PATHOGENESIS
Transmission of enteroviruses occurs especially by ingestion of fecally contami-
nated material. The clinical manifestations of the diseases are due by the differ-
ences in tissue tropism and the cytolytic capacity of the viruses. According to the
clinical syndrome, the incubation period varies between 3 and 5 days. From the port
of entry (the mouth), viral multiplication takes place in the lymphoid organs of the
oropharynx and in the small intestine. A transient minor viremia occurs, and the
virus spreads to the reticuloendothelial system. Many of the enterovirus infections
are asymptomatic and are limited at this stage. Further replication of the virus in the
reticuloendothelial system is associated with minor illness. The virus spreads hema-
togenously to lymphoid tissue throughout the body. The virus replication at these
sites, particularly the liver and spleen, produces a major viremia, which coincides
with the onset of symptoms. In patients with persistent viremia, the virus spreads to
target organs such as the CNS. Genomic differences among enterovirus serotypes
might explain the tendency of some strains to cause aseptic meningitis and encepha-
litis. Poliomyelitis is characterized by a biphasic pattern with a nonspecific febrile
illness occurring three to five days after exposure, followed by a period of relative
well being, and then a recurrence of fever with CNS manifestations 9 to 12 days after
exposure. In patients with persistent viremia, the poliovirus enters the nervous sys-
tem by crossing the blood–brain barrier or by axonal transportation from a periph-
eral nerve (Ohka et al. 1992; Ren and Racaniello 1992). The poliovirus multiplies in
the motor neurons of the anterior horn of the spinal cord, followed by denervation
of the associated skeletal musculature (spinal poliomyelitis) or it multiplies in the
neurons from the brain stem (bulbar poliomyelitis) (Modlin 2005; Muller 2005).
After viral replication in the oropharynx and intestine, poliovirus is eliminated in
oropharyngeal secretions for 1–3 weeks and in the stool for 1 or 2 months, until the
virus is completely out of the body. During reinfection, the virus is eliminated in the
stool within 3 weeks (Heymann 2004). The period of maximum communicability is
probably the first two weeks after enterovirus infection. The potential for prolonged
replication is higher in patients with immunodeficiency syndromes.
6.4 DIAGNOSIS
The diagnostic in suspected enterovirus infections is based on medical history and
examination of a patient, followed by CSF analysis, and identification of the serotype
by polymerase chain reaction amplification and serology. The history of a patient
with suspected viral meningitis includes the presence of classic symptoms. Important
aspects of the clinical examination include signs of meningeal inflammation (nuchal
rigidity, Kernig and Brudzinski’s signs), assessment of mental status (Glasgow
coma scale), and findings associated with specific viruses (e.g., conjunctivitis, rash,
herpangina, hand, foot, and mouth disease). The important distinguishing feature
between encephalitis and meningitis is the brain function. Electroencephalography
(EEG) is an indicator of cerebral involvement during the early stage of the disease.
The presence of focal neurological signs is suggestive for encephalitis. In patients
with signs or symptoms of increased intracranial pressure, computed tomography
(CT) is recommended as a screening examination. Magnetic resonance imaging is
more sensitive and specific than computed tomography (CT) for evaluation of the
brain inflammation, and is useful before lumbar puncture (Fleischer 2006; Logan
and MacMahon 2008; Steiner et al. 2010).
6.4.1 Laboratory Diagnosis

In aseptic meningitis and encephalitis with enteroviruses, the diagnosis requires
identification of a viral pathogen from cerebrospinal fluid specimen (CSF) or other
patient samples (e.g., throat swab, stool). The virus can be identified in a CSF sample
only in the acute phase of infection. In infections with poliovirus or HEV71, weak-
ness or paralysis may occur. The protocol for laboratory investigation of suspected
cases of paralytic poliomyelitis includes two stool specimens and two throat swabs
collected every 24 h at least, within 14 days since onset, a blood specimen for com-
plete blood count, a cerebrospinal fluid specimen (CSF) for chemical and cytologic
analysis, and acute and convalescent serum specimens, collected at an interval of at
least 2–4 weeks for detection and titration of the neutralizing antibodies to the three
poliovirus serotypes. The collection of CSF and throat swabs is not recommended
by the WHO for the surveillance because the polioviruses are rarely detected in CSF
and the titer of the virus in throat swabs is 10 fold lower than in stool specimens.
The laboratory tests show a peripheral leukocytosis and a pleocytosis in CSF
[increase of white blood cell (WBC) count, but less than 250/mm3 with predomi-
nance of neutrophils in early stage of infection, and shift from neutrophils to lym-
phocytes 8 h later in aseptic meningitis and few days later in poliomyelitis], an
elevated protein concentration (usually less than 150 mg/dL in aseptic meningitis
that may increase to 300 mg/dL for several weeks in poliomyelitis), and a normal
level of glucose (Melnick 1996). In children, the enteroviral meningitis frequently
occurs in the absence of either CSF pleocytosis or elevated protein levels. In this
situation, the CSF profile alone cannot distinguish between enteroviral and bacterial
meningitis, and the enteroviral polymerase chain reaction (PCR) must be performed
as an additional diagnostic test (Graham and Murdoch 2005). The phenotypic and
molecular laboratory techniques are important to rule out or confirm the diagnosis of
infection with enteroviruses. The correct virological diagnosis depends on the timely
collection, transportation, and storage of the specimens.
6.4.2 Conventional Techniques
Traditionally, cell culture has been employed for the isolation of enteroviruses from
clinical samples. Primary cells (primary monkey kidney cells) and suckling mice are
unavailable for routine diagnosis of HEVs as a result of the international standards
related to the care and management of experimental animals. The main cell cultures
used today for HEV isolation are RD (a human rhabdomyosarcoma derived cell line
recommended by the WHO), BGMK (Buffalo green monkey kidney cells), A549 (a
human lung adenocarcinoma epithelial cell line), MRC-5 cells (derived from normal
lung tissue of a 14-week-old male fetus), and HEp-2c cells (derived from a human
larynx epidermoid carcinoma). A genetically engineered mouse cell line expressing
the human poliovirus receptor PVR, L20B, recommended by the WHO is suscep-
tible only to poliovirus infection (Pipkin et al. 1993). Enterovirus isolation needs
inoculation of each specimen (directly or after pretreatment) onto continuous cell
lines that are available for use in different laboratories. For poliovirus isolation, RD,
HEp-2c, and L20B cell lines are recommended by the WHO. The cell lines must be
examined daily, and once the complete cytopathic effect (CPE) occurs, the infected
cells must be kept frozen (at –20°C) until viral identification. The time interval for
enterovirus isolation and characterization must be at least 10 days (minimum of 5
days postinoculation and minimum of 5 days postpassage) before a reported negative
test (WHO 2007). Rapid degeneration of the cell culture or cell death could appear
in the nonspecific toxicity of the specimen or in microbial contamination of the
culture maintenance medium (2% fetal calf serum), respectively. Characteristic CPE
progresses from rounding, refractory individual cells within the monolayer to the
detachment of the infected cell from the tissue culture tube.
The HEV identification and typing are carried out by seroneutralization with pools
of antisera or by indirect immunofluorescence assay. The Lim Benyesh-Melnick
(LBM) pools and the WHO enteroviral antisera pools are available for HEVs typ-
ing. The A-H and J-P pools from the LBM schedule identifies 42 serotypes including
PV1–3 (Melnick et al. 1973) and 19 CVA strains, most of which can only be isolate in
suckling mice (Melnick et al. 1997). The pools of polyclonal antisera against CVA9
and 20 echoviruses, CVB1–6, and PV1–3 have been developed by the National Institute
of Public Health and the Environment (RIVM), Bilthoven, the Netherlands, and are
supplied free of charge to WHO Polio Laboratory Network laboratories by WHO.
The typing by seroneutralization with these pools has limitations because the newly
discovered and circulating HEV strains are not recognized and these pools may not
even recognize the progeny of previously identified HEV strains due to the antigenic
drift that occurred over the years. Monospecific antisera have been used for confirma-
tion of the serotype. The methods recommended by WHO for intratypic differentiation
of poliovirus isolates are currently in use (van der Avoort et al. 1995). An enzyme-
linked immunosorbent assay (ELISA) method developed by RIVM detects antigenic
differences between wild and vaccine-related strains. The utilization of type-specific
neutralizing monoclonal antibodies developed by the Pasteur Institute, Paris, and the
National Institute for Biological Standards and Control, Potters Bar, was accepted, but
it is not currently supported by the WHO Global Polio Laboratory Network.
The commercial Light DiagnosticsTM Pan Enterovirus reagent (Millipore,
Chemicon, Temecula, CA) is used for the preliminary identification of enteroviruses
from cell culture by indirect immunofluorescence assay (IFA). The reagent contains
a mixtures that recognize specific groups of HEV: the PV mixture for PV types 1,
2, and 3 detection, the enterovirus mixture for HEV70, 71, CVA16 detection, the
Echo mixture for echovirus types 4, 6, 9, 11, 30 detection, and the CVB mixture for
CVB types 1–6. The monovalent antibodies are available for each serotype included
in the mixtures and for CVA9 and CVA24. By using of the Super E-Mix™ cell line
combined with the D3 IFA enterovirus test (Diagnostic Hybrids, USA), the time
for isolation and identification of the enteroviruses decrease as low as 16 h. Super
E-Mix™ is a mixed cell monolayer in shell vials which contains human lung car-
cinoma (A-549) cells together with buffalo green monkey kidney (BGMK) cells,
which have been genetically modified to produce large amounts of human decay-
accelerating factor (DAF) on the cell surface. The D3 IFA enterovirus reagent uses
a blend of Enterovirus VP1 antigen-specific murine monoclonal antibodies (MAbs)
conjugated with a fluorescein isothiocyanate labelled antimouse antibody. Lin et al.
(2008) described the development of an in-house indirect immunofluorescence assay
(IFA) for rapid detection of CVA types 2, 4, 5, 6, and 10.
Antibodies to nontyped enteroviruses are measured from serum and CSF by
enzyme immunoassay (EIA) tests. The presence of specific IgM in the CSF indicates
CNS disease. The microneutralization test has limited utility in the routine diagnosis
of nonpolio enterovirus infections because it is serotype specific, but it may be help-
ful in the diagnosis of paralytic poliomyelitis according to the vaccine history of the
patient (the number of doses received, the time after the last vaccine dose received)
and to the type of virus isolated. A 4-fold rise in serum antibody titer between the
acute and convalescent serum is significant for diagnosis.
6.4.3 Molecular Techniques
For CNS infections, rapid identification of a viral pathogen by molecular diagnostic
tests and prompt initiation of the therapy are potentially lifesaving, reduce hospital-
ization, and avoid the antibiotic use (King et al. 2007). The evidence of a microorgan-
ism in CSF, spinal, and brain tissue, which are normally sterile body sites, is probably
an infection (usually monomicrobial). The CSF has no inhibitors of polymerase chain
reaction (PCR) assays such as heme, endonucleases, and exonucleases. Nucleic acid
amplification methods are often more sensitive than conventional culture-based or
antigen detection methods. The molecular typing methods for HEVs are amplifica-
tion (PCR) and sequencing targeting to specific coding regions for VP1 or VP2. The
noncapsid encoding sequences are not highly conserved and may recombine with
other enteroviruses (Lukashev et al. 2003). A small volume of sample of about 150–
250 µl is enough for nucleic acid extraction. PCR allows exponential amplification
of short DNA sequences within a longer double stranded DNA molecule by using
a pair of primers (about 20 nucleotides in length, complementary to a sequence on
each of the two strands of the DNA) and a DNA polymerase. The enterovirus RNA
genome is converted initially into complementary DNA (cDNA), reverse transcrip-
tion (RT) using the enzyme reverse transcriptase, followed by amplification of the
correct amplicon using specific sets of primers and Taq polymerase, a thermostable
DNA polymerase. The performance of the RT-PCR assays depends on optimization
and standardization of the viral genome extraction, amplification, and detection. The
manual extraction method using the Qiagen QIAamp viral RNA kit based on silica
membrane columns (Qiagen GmbH, Hilden, Germany) is rapid and does not require
the utilization of hazardous materials as phenol or chloroform. There were a few stud-
ies that compared the manual with the automated nucleic acid extraction (Knepp et al.
2003; Dundas et al. 2008). Amplification efficiency of nucleic acids extracted by auto-
mated methods was similar to that by the manual methods. The molecular techniques
for amplification and detection of specific regions from HEV genome evolved from
the RT-PCR (Chapman et al. 1990; Rotbart 1990) to the real-time RT-PCR assays
(r-RT-PCR) (Archimbaud et al. 2009; Sofer et al. 2011; Baicus 2011). The primers
targeting different genomic regions of nonpolio enteroviruses and polioviruses are
reported by Balanant et al. (1991), Caro et al. (2001), Guillot et al. (2000), Kilpatrick et
al. (1998, 2004, 2009), Oberste et al. (1999, 2002), and Yang et al. (1991), respectively.
The relationships between isolates and enterovirus transmission could be detected by
VP1 sequence analysis (Sambrook et al. 1989). The amplicons could be sequenced by
the dideoxynucleotide method with the Big Dye Terminator Cycle Sequencing Ready
Reaction kit (the procedure recommended by Applied Biosystems, Perkin-Elmer) and
an ABI Prism automated sequencer (Applied Biosystems) using primers used for the
PCR reaction. The alignment and comparison of the sequences can be done with a
software (e.g., Clustal X version 2.0 or CLC Main Workbench software version 6.0.1).
The MARSH assay (Microarrays for resequencing and sequence heterogeneity) can
be applied to detect the point mutations present at a low level in heterogeneous popu-
lations and mixtures of different virus strains (Liu et al. 2007), and it has been used
for studying the VDPV strains (Cherkasova et al. 2003).
6.5 PROGNOSIS, PREVENTION, AND TREATMENT

Most enterovirus infections are self-limited and do not require specific therapy. The
prevention of the spreading of infection with enteroviruses is through measures
of hygiene, such as hand washing. There are no vaccines available for protection
against infections with nonpolio enteroviruses. For patients with enteroviral menin-
gitis, acute encephalitis supportive therapy is required. The therapy with intravenous
immune globulins has been useful in patients with agammaglobulinemia, chronic
meningitis, or meningoencephalitis because the clearance of HEV by the host is
primarily antibody mediated. Although there is no approved therapy, a variety of

antiviral agents have shown activity against enteroviruses in vitro, and in early clini-
cal trials. The capsid inhibiting class of drugs act by preventing the HEV uncoating
or viral binding to a cellular receptor. Pleconaril is one of these drugs, an orally
administered drug with a favorable pharmacokinetic and toxicity profile reaching
higher concentrations within the CNS than in serum. In limited clinical studies on
patients with enterovirus meningitis, pleconaril did not demonstrate overall clinical
benefit (Abzug et al. 2003; Rotbart et al. 2001; Tormey et al. 2003), and the drug is
not currently available for clinical use (Desmond et al. 2006). Aged and immuno-
deficient patients with aseptic meningitis may be considered for empiric therapy for
48 h until the diagnosis of viral meningitis is sure.
Treatment of poliomyelitis is supportive. Mechanical ventilation and close moni-
toring of cardiovascular status are necessary in patients with respiratory failure and
bulbar poliomyelitis. The poliomyelitis has been virtually eliminated in most coun-
tries by the widespread immunization with the formalin-inactivated polio vaccine
(IPV) or/and oral live-attenuated polio vaccine (OPV). After 22 years since the deci-
sion of the World Health Organization to globally eradicate poliomyelitis, circulation
of wild poliovirus types 1 and 3 continues in 4 countries, India, Nigeria, Pakistan,
and Afghanistan, because of underutilization of vaccine, vaccine failure, and the epi-
demiologic context (el-Sayed et al. 2008; Grassly et al. 2006; Jenkins et al. 2008).
The indigenous wild type 2 poliovirus was eradicated in 1999 but a type 2 circulating
vaccine-derived poliovirus (cVDPV) has persisted in northern Nigeria since 2006
(Adu et al. 2007). The supplementary immunization with monovalent strains of OPV
(mOPV) type 1 (Grassly et al. 2007) or type 3 has been introduced (CDC 2009) in
those regions where the virus has been difficult to control. A new bivalent oral polio-
vaccine bOPV (containing types 1 and 3 poliovirus) has been introduced into all 4
persistently endemic countries, beginning in 2009 with Afghanistan (WHO 2010).
Low vaccination coverage, and the extent of immunization gaps increase the potential
risk of emergence and circulation of VDPV strains (Nathanson and Kew 2010).
The WHO assists countries with decision-making on polio vaccination schedules
and vaccines, given their risk of poliovirus importations and the probable transmis-
sion potential for polioviruses in their country. In a given country, once wild poliovi-
rus was eliminated through the use of OPV, the public health authorities could decide
continuation of OPV immunization or transition from OPV to IPV (Chumakov et al.
2007; Ehrenfeld et al. 2008; Fine and Ritchie 2006). Global vaccination with IPV is
not yet possible because there are still financial, logistic, and scientific problems. The
IPV was introduced to prevent VAPP, to close existing immunity gaps, or to optimize
the administration of other antigens. A Poliovirus Antiviral Initiative (PAI) was pro-
posed in 2006 by the Advisory Committee on Poliomyelitis Eradication (ACPE) and
by the Task Force for Childhood Survival and Development (Task Force). The devel-
opment of polio specific antiviral drugs, used with either IPV or OPV, will improve
the control of outbreaks in the future (De Palma et al. 2008; Collett et al. 2008; Thys
et al. 2008). Pleconaril was shown to be safe and effective in treating diseases caused
by nonpolio enteroviruses (Bauer et al. 2002; Hayden et al. 2002; Utzig et al. 2003)
but has shown little or no activity against polioviruses. V-073 was found to have
potent, broad-spectrum antipoliovirus activity in cell culture (Thibaut et al. 2011).
6.6 CONCLUSIONS AND FUTURE PERSPECTIVES

The early clinical recognition and the rapid diagnosis by molecular techniques of the
neurological infections are among the most important problems in medicine. Rapid
detection of nonpolio enteroviruses in CSF within the first 24 h of hospitalization is
followed by cessation of antibiotic administration and shortening of hospitalization
time. The surveillance of the cocirculation and evolution of polioviruses and nonpo-
lio enteroviruses must be increased by the fast detection of the emergence of new
epidemic strains. The evaluation made in different scenarios concerning the risk and
costs of the polio eradication was changed because the ability of VDPV strains to
produce outbreaks. In April 2010, the first importation of wild PV type 1 from India
was detected in Tajikistan, in the WHO European Region, since it was certified polio-
free in 2002 (WHO 2010). In a polio-free region, the risk of importation and subse-
quent transmission of the poliovirus remains until polio is completely eradicated. In
countries with otherwise adequate levels of vaccine coverage, the groups that are at
high risk are the subpopulations that refuse immunization or with gaps in immunity
(Alexander et al. 2009; Combiescu et al. 2007). Many countries have switched the
schedule of vaccination against polio by using IPV instead of OPV to eliminate the risk
of vaccine-associated paralytic poliomyelitis (VAPP). The barriers for the global intro-
duction of IPV are its cost, the need for intramuscular injection, its inability to produce
optimal intestinal immunity, and the biocontainment required for its production. A
planning for the cessation of routine OPV immunization against type 2 polioviruses
is taken into account (WHO 2011). A research program for obtaining an affordable
IPV for developing countries was initiated and financed by GPEI/WHO (WHO 2009).
A number of strategies are followed including a schedule reduction, a reduction of
the antigen dose by intradermal administration (Mohammed et al. 2010; Resik et al.
2010), by using adjuvants (Baldwin et al. 2011), optimization of production processes,
and development of an inactivated poliovirus vaccine produced from Sabin strains
(Kreeftenberg et al. 2006; Simizu et al. 2006). Global Polio Eradication Initiative
(GPEI) developed new tools for maintaining the comprehensive AFP surveillance, for
achieving certification and containment of wild poliovirus, for preparing for VAPP and
VDPV elimination and for the post-OPV era (GPEI 2009). A target of 90% routine
vaccine coverage in low-income countries by 2010 has been an objective of the GPEI
and the Global Alliance for Vaccines and Immunization (GAVI) (Fine and Griffiths
2007). After global cessation of OPV administration, the immunization by using IPV
through the first 5 to 10 years is necessary (Aylward and Yamada 2011).
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7 Neurovirulence of
the West Nile Virus
Kim-Long Yeo and Mah-Lee Ng
CONTENTS
7.1 Introduction................................................................................................... 149
7.1.1 Molecular Biology of West Nile Virus.............................................. 149
7.2 History........................................................................................................... 150
7.3 Transmission.................................................................................................. 151
7.4.1 West Nile Fever.................................................................................. 152
7.4.2 West Nile Neuroinvasive Disease...................................................... 153
7.4.3 West Nile Meningitis......................................................................... 153
7.4.4 West Nile Encephalitis....................................................................... 153
7.4.5 West Nile Poliomyelitis..................................................................... 154
7.5 Diagnosis....................................................................................................... 155
7.6 Risk Factors................................................................................................... 155
7.7 Neuroinvasiveness of West Nile Virus.......................................................... 156
7.8 Viral Clearance.............................................................................................. 157
7.9 Treatment....................................................................................................... 158
7.10 Prevention...................................................................................................... 159
7.11 Conclusion..................................................................................................... 159
References............................................................................................................... 159
7.1 INTRODUCTION
The West Nile virus (WNV) is an arbovirus that can cause significant and devas-
tating neurological-related disease in infected persons. It was introduced into the
Western hemisphere in 1999 and has remained endemic ever since. Although our
understanding of the virus has expanded greatly in the past decade, treatment and
prevention of WNV infection are still in progress. This chapter provides an introduc-
tion to the biology of the virus, its history and transmission, clinical symptoms and
pathology of infection, and recent developments in treatment strategies.
7.1.1 Molecular Biology of West Nile Virus

The West Nile virus is an arthropod-borne virus that is closely related to approxi-
mately 70 other members of the family Flaviviridae. Specifically, it belongs to the
149
Japanese encephalitis serocomplex of the genus Flavivirus (Brinton 2002). The virus’
small positive-sense, single-stranded genome of about 10.8 kilobases is contained
within a capsid made of the viral capsid (C) protein. A 4-nm-thick host-derived lipid
membrane further surrounds the capsid. Inserted into this lipid membrane are 180
copies of the virus-encoded envelope (E) and premembrane (prM) glycoproteins.
Homodimers of the E protein lie in a herringbone-like arrangement that covers the
lipid bilayer, whereas the prM protein forms homotrimers that cap the external tip of
the E protein. Mature WNV particles have a distinctly smooth spikeless outer sur-
face, are about 50 nm in diameter, and have isocahedral symmetry (Mukhopadhyay
et al. 2003).
The infectious genome serves as a messenger RNA (mRNA) with a single long
open reading frame (ORF) that encodes for a 3443-amino acid polyprotein. Both
host and viral proteases cleave this polyprotein to form the C, prM, E, and 7 other
nonstructural (NS) proteins (Brinton 2002). The NS proteins are named in the order
that they are found in the genome NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5.
Whereas NS1 and N4 possess virus-antihost immunological-related functions, NS2,
together with NS3, act as viral protease to cleave the viral polyprotein into its indi-
vidual functional units. The largest NS protein, NS5, is the virus’ RNA-dependent
RNA polymerase. It synthesizes new copies of the viral genome from a negative-
stranded viral RNA in an asymmetrical manner (Brinton 2002).
7.2 HISTORY
The virus was first isolated from a febrile female patient in the West Nile region of
Uganda in 1937 (Smithburn et al. 1940), and is associated with periodic epidemics
of febrile illness throughout Africa, Southwest Asia, and Eastern Europe (Murgue
et al. 2002). Historically, WNV was found to cycle between Culicine mosquitoes
and native birds. Birds served as the natural amplifying host, and humans were acci-
dental secondary hosts. Symptoms of infection included dengue-like illness such as
fever, malaise, lymphadenopathy, and rash. Most infections were self-limiting and
resolved without much sequelae (Goldblum et al. 1954). In the 1990s however, fatal
cases of encephalitis became a significant feature of WNV epidemics in Romania,
Russia, and Israel. Approximately 60% of hospitalized patients had West Nile neuro-
invasive disease (WNND) with a 4% to 7% mortality rate (Klein et al. 2005).
In 1999, WNV appeared for the first time in North America. This was marked
by the simultaneous occurrence of an unusual number of deaths of exotic birds and
crows in the New York City Metropolitan Area (Nash et al. 2001). Introduction of the
virus has been largely attributed to infected migratory birds (Rappole et al. 2000) or
possibly illegally imported exotic birds. A human source was ruled out since humans
are dead-end hosts (Brinton 2002); this is with the exception of blood transfusion,
an organ transplantation, or transplacental infection. There is also a small possibil-
ity that the virus was introduced via infected mosquitoes unintentionally carried by
airplanes or other carriers (Davis et al. 2006). The notion that migratory birds were
the origin of the virus in New York was supported by the fact that comparison of the
nucleotide sequence of the envelope gene of the New York City virus (WNV-NY99)
showed more than 99% homology in amino acid sequence to a virus isolated from
Neurovirulence of the West Nile Virus 151
a goose in Israel in 1998 (Lanciotti et al. 1999; Brinton 2002). This also suggested
that WNV-NY99 originated from the Middle East or Eastern Europe, where a simi-
lar virus is circulating.
More recent genome sequencing of the E protein of the virus has revealed 2 dis-
tinct lineages of the virus (Lanciotti et al. 1999; Brinton 2002). Lineage 1 includes
pathogenic strains from North America, Europe, Australia, Africa, and Asia,
whereas lineage 2 includes strains from Africa and Madagascar. Lineage 1 viruses
are further subdivided into four clades. In general, lineage 1 viruses are widespread
and have caused recent epidemics of human encephalitis throughout western Africa,
the Middle East, Eastern Europe, and more recently, North America. All North
American isolates have so far been classified as lineage 1, clade B, and are closely
related to strains from Israel (Lanciotti et al. 1999). Lineage 2 viruses, on the other
hand, are associated with sporadic and endemic human cases of a less severe febrile
illness without involvement of the central nervous system.
The initial outbreak in New York resulted in 62 cases of encephalitis in humans.
Seven deaths occurred as a result of encephalitis (Nash et al. 2001). Over the past
decade, WNV has shown to have an increased tendency to cause WNND. From
1999 to 2005, 19,506 cases of human WNV diseases were reported in the US. This
included 8362 cases that were characterized as WNND, 782 of which resulted in
death (Hayes et al. 2005; CDC). The geographic range and burden of disease has
also greatly expanded to the 48 adjoining states of the US, as well as seven Canadian
provinces, Mexico, the Carribean islands, and Colombia (Granwehr et al. 2004;
Tyler 2004; Davis et al. 2005; Hayes et al. 2005; Public Health Agency of Canada).
As such, it has become the most common cause of epidemic meningoencephalitis in
the region.
7.3 TRANSMISSION
West Nile virus is maintained in nature by cycling between more than 200 species of
birds and many species of mosquitoes (van der Meulen et al. 2005). The virus enters
the mosquito via infected blood, penetrates the gut, and replicates in tissues includ-
ing the nervous system and salivary glands. Infection of the mosquito is noncyto-
pathic and persists for the life of the insect (Girard et al. 2005). Infected mosquitoes
then infect susceptible birds by injecting about 104 plaque-forming units (PFU) of
virus while feeding (Vanlandingham et al. 2004). Birds are the major amplifying
host for WNV and can have viremia lasting for more than 100 days (Komar et al.
2003). This allows for repeated cycles of mosquito infection. The highest titer vire-
mia of more than 1010 PFU has been reported (Komar et al. 2003). Viremia of this
magnitude leads to subsequent transmission to more than 80% of biting mosquitoes
(Turell et al. 2000). This is since the capacity to transmit infection increases dramati-
cally as the level of viremia increases in the host.
In the United States, transmission of WNV to humans often results from an
infected mosquito of the Culex species feeding on a human host (Campbell et al.
2002; Turell et al. 2002). This results in a dead end infection. Other modes of WNV
transmission in humans have also been reported. Twenty-three blood transfusion
recipients were infected with WNV in 2002 after given blood products obtained
from viremic donors (Pealer et al. 2003; Centers for Disease Control and Prevention
[CDC] 2004). This led to widespread screening of blood products using WNV-
specific nucleic acid amplification tests. However, very low levels of viremia escape
detection and contribute to transfusion-associated transmission. Human to human
transmission has also occurred through transplantation of an organ harvested from a
viremic donor (Iwamoto et al. 2003). Lastly, transplacental infection was confirmed
in 2002 when an infant was found to have WNV-specific IgM antibody in the blood,
and WNV nucleic acid was found in placental and umbilical cord tissue. The expect-
ing mother had developed WNND in her 27th week of pregnancy, and gave birth
to an infant with chorioretinitis and cystic cerebral lesion (CDC 2002; Alpert et al.
2003). Surveillance for WNV in pregnant women has thus been stepped up in light
of these findings.
However, during the typical mode of infection, the biting mosquito injects saliva-
containing WNV intradermally. The virus initially replicates in Langerhans den-
dritic cells (DC) (Chambers and Diamond 2003) before the infected DC migrates
to draining lymph nodes (LNs). Viral replication then occurs within the lymphoid
tissue (Johnston et al. 2000). Viremia peaks between 2 to 4 days after infection, and
prior to illness onset in healthy persons (Southam and Moore 1952, 1954; Hayes and
O’Leary 2004). Persistence of virus in immunocompromised persons may be more
prolonged (Southam and Moore 1952; Iwamoto et al. 2003). The virus can be detected
in blood within 1 to 2 days of a mosquito bite, and termination of viremia coincides
with production of neutralizing IgM antibodies. The antibody response is the pri-
mary method by which WNV is cleared from the infected person. Interestingly,
onset of illness coincides with IgM antibody production and a reduction in viremia.
Delayed onset of illness in infected persons may thus be indicative that the immu-
nological response of the individual plays a more important role in development of
symptoms.

It has been estimated that more than 80% of infected persons remain asymptom-
atic, and 80%–90% of symptomatic patients develop a self-limiting febrile illness
known as West Nile fever (WNF). Remaining persons can develop severe WNND-
like aseptic meningitis (WNM), encephalitis (WNE), or an acute poliomyelitis-like
syndrome (WNP) (Campbell et al. 2002). However, it should be noted that clini-
cal classification of West Nile infection is often not clear-cut, as it can be hard to
clinically distinguish among WNF, WNM, WNE, and WNP. For instance, a patient
presenting fever, headache, and “neck stiffness” may not be subjected to lumbar
puncture to demonstrate pleocytosis. As a result, a diagnosis of WNM may not be
reported. Regardless, description of clinical syndrome in WNV-infected persons is
reliable based on clinical grounds.
7.4.1 West Nile Fever

The predominant clinical syndrome seen in most infected persons is WNF. Although
WNF can develop in all ages, its incidence decreases with age (Brown 2004). After
an incubation of about 2–14 days, the infected person can experience a sudden onset
of fever, headache, fatigue, myalgia, and development of a transient maculopapular
rash (Campbell et al. 2002; Anderson et al. 2004; Watson et al. 2004; Del Giudice
et al. 2005; Ferguson et al. 2005; Gorsche and Tilley 2005). Rash development usu-
ally begins about 5 days after onset of illness and can last for about a week (Watson
et al. 2004). Rash is more frequently observed in younger persons than in older
persons (Ferguson et al. 2005). Although most patients experience complete and
unremarkable recovery from WNF, some may experience fatigue lasting up to 36
days (Watson et al. 2004). Profound fatigue in particular can interfere with work or
school activities (Gottfried et al. 2005).
7.4.2 West Nile Neuroinvasive Disease

In contrast to WNF, the incidence of WNND increases with age. It is significantly
higher in the elderly and immunocompromised patients (Pepperell et al. 2003;
O’Leary et al. 2004; Hayes and Gubler 2006). Although WNND is rarely reported
in children or individuals younger than 30 years, 317 cases of WNV infection in
children were categorized as WNND from 2002 to 2004 (Yim et al. 2004). However,
this is not indicative of an increased incidence of WNND in younger individuals.
7.4.3 West Nile Meningitis

Development of WNM is similar to other viral meningitides. There is a sudden onset
of fever, headache, and meningeal signs such as nuchal rigidity, photophobia, and
phonophobia. Examination of the cerebral spinal fluid (CSF) often presents modest
pleocytosis of less than 500/mm3. Although pleocytosis is frequently lymphocytic
in nature, CSF obtained during early onset of illness is predominantly neutrophilic
(Crichlow et al. 2004; Sejvar et al. 2005). Other neurological features are usually
absent and WNM is often associated with a good prognosis. However, some patients
can experience persistent headache, fatigue, and myalgia (Sejvar et al. 2003a).
7.4.4 West Nile Encephalitis

West Nile encephalitis is more common in the elderly over the age of 55 (Nash et al.
2001; Weiss et al. 2001; O’Leary et al. 2004; Bode et al. 2006). During the 1999 out-
break, 88% of those affected were older than 50 years old, and 51% were older than
70 years of age (Nash et al. 2001). Immunocompromised individuals, for instance,
organ transplant recipients taking immunosuppresive drugs, may be at high risk for
WNE as well (Agamanolis et al. 2003; Armali et al. 2003; Ravindra et al. 2004).
The severity of WNE can range from a mild, self-limited confusional state to that
of severe encephalopathy, coma, and death. Almost 100% of patients with WNE
were reported to have fever, 42%–85% with fatigue, 47%–90% with headache, and
46%–74% with altered mental status (Davis et al. 2006). Patients also often develop
a coarse postural tremor in the upper extremities during kinetic or rest (Sejvar et al.
2003a; Burton et al. 2004). Myoclonus was reported to occur in one-third of cases in
a series and can be indicative of WNND since it is distinctly unusual in nonflaviviral
encephalitis (Sejvar et al. 2003a). Parkinsonian-like features such as rigidity, bra-

dykinesia, and postural instability have also been reported in up to two-thirds of
WNND cases (Sejvar et al. 2003a).
Abnormalities noted above usually resolve over time, but persistence of tremor and
parkinsonism have been observed in some patients recovering from severe enceph-
alitis. More than 65% of encephalitic patients who underwent neuropsychological
testing after being admitted to an acute rehabilitation center still present persisting
cognitive deficits at discharge (Arciniegas and Anderson 2004). These included lan-
guage/social communication deficits, memory impairment, executive dysfunction,
and defects in attention and concentration. Approximately 25% of the patients had
slowed processing speed. Emotional dysregulation such as depression, anxiety and
irritability, and psychomotor abnormalities such as apathy and agitation, were found
in more than 75% of these patients (Arciniegas and Anderson 2004).
Besides cognitive impairment, patients with WNE have been reported to develop
a wide range of additional clinical signs and symptoms. These reflect the ability of
WNV to affect areas as diverse as the cerebral cortex, basal ganglia, brainstem, and
spinal cord. Patients with WNE who develop visual problems have also been increas-
ingly recognized. They often describe blurry vision, trouble seeing, and photophobia
(Anninger and Lubow 2004; Khairallah et al. 2004).
Fatality following an episode of WNE has ranged between 10% and 20% among
patients with severe WNE. Although mortality is higher among the elderly and
immunocompromised, independent risk factors for fatal outcome remain to be
known (Kleinschmidt-DeMasters et al. 2004). Evidently, outcome after WNE is
heterogeneous.
7.4.5 West Nile Poliomyelitis

Since WNV can infect neurons in the spinal cord, it can result in true poliomyelitis
(Doron et al. 2003; Leis et al. 2003; Sejvar et al. 2003b, 2006). Recent data have
suggested that WNP results from viral infection of the lower motor neurons in the
anterior horn cells of the spinal cord (Glass et al. 2002; Leis et al. 2002; Jeha et al.
2003; Li et al. 2003; Sejvar et al. 2003b, 2006). Patients who develop WNP are usu-
ally younger than those who develop WNE (Sejvar et al. 2003b; Sayao et al. 2004;
Sejvar et al. 2006).
Development of WNP can be abrupt and progress rapidly to weakness in one
or more limbs, especially the legs (Jeha et al. 2003; Leis et al. 2003; Sejvar et al.
2003a,b, 2006). Weakness may develop concomitantly with signs of meningitis or
encephalitis or several days after their onset (Leis et al. 2003; Sejvar et al. 2003a).
It should be noted that clinical features of WNP are characteristic and dramatic in
comparison to general muscle weakness experienced by many persons with WNV
infection. Thus, WNP should be easily differentiated from general extreme fatigue.
Weakness tends to be asymmetric and may often result in monoplegia. Some
patients with severe involvement may develop a more symmetric dense quadriplegia
(Jeha et al. 2003; Leis et al. 2003; Sejvar et al. 2003a,b, 2006). Frequently, bilateral
central facial weakness may also be seen (Li et al. 2003; Sejvar et al. 2006). Although
numbness or sensory loss is usually absent, some patients experience intense pain
in the affected limbs prior to or during the onset of weakness. The limb pain may
become persistent in some affected patients (Sejvar et al. 2006).
Respiratory muscle innervation, leading to diaphragmatic and intercostal muscle
paralysis, may also lead to respiratory failure in some persons (Fan et al. 2004;
Sejvar et al. 2006). Affected persons would require emergent endotracheal intubation
(Fan et al. 2004). Development of respiratory failure may be due to involvement of
the lower brain stem, including the motor nuclei of the vagus and glossopharyngeal
nerves (Agamanolis et al. 2003; Doron et al. 2003). Respiratory involvement in WNP
is often associated with high morbidity and mortality. For survivors, prolonged ven-
tilatory support may be required (Sejvar et al. 2006).
In addition to WNP, other forms of acute flaccid paralysis similar to Guillain-
Barré syndrome have also been associated with WNV infection (Ahmed et al. 2000;
Park et al. 2003). However, these appear to be less common than WNP and can be
differentiated based on clinical and electrophysiological features.
Recovery of limb strength from WNP is variable. Generally, less profound initial
weakness is associated with more rapid and complete strength recovery (Cao et al.
2005; Sejvar et al. 2006). In the short term, however, affected patients experience per-
sistent weakness and associated functional disability. Prolonged physical and occu-
pational therapy may be required to ensure complete recovery (Sejvar et al. 2006).
7.5 DIAGNOSIS
As with most viral infections, isolation of WNV from biological specimens such as
blood, serum, CSF, or histology tissues is the gold standard for diagnosis of infec-
tion. However, this is rarely done due to the need for proper bio-safety containment
facilities and bio-safety concerns. In addition, the virus is usually absent at the onset
of illness (Lanciotti et al. 2000). Although nucleic acid based platforms are available
for the detection of WNV nucleic acid in clinical samples, the technique has limited
usefulness due to limited sensitivity and the absence of virus during onset of illness
(Lanciotti et al. 2000). Thus, diagnosis is often done by demonstrating the pres-
ence of WNV-specific IgM antibodies in CSF or paired serum samples. Detection
of WNV-specific IgM in the CSF of a patient, together with clinically compatible
illness as mentioned above, is considered confirmatory.
However, confirmation of an acute infection requires demonstration of a four-fold
increase in antibody titers using functional assays such as neutralization and he
magglutination inhibition. This is since WNV-specific IgM antibodies can persist in
the serum for over a year (Kapoor et al. 2004). Diagnosis can also be complicated
by serologic cross-reactivity between WNV and other closely related flaviviruses.
This is especially so in areas where several flaviviruses such as WNV and St. Louis
encephalitis coexist (Martin et al. 2002). In light of such complications, the plaque-
reduction neutralization assay test is most widely utilized to circumvent the problem.
7.6 RISK FACTORS

Undoubtedly, the most important risk factor for acquiring WNV infection is expo
sure to infected mosquitoes (Hayes et al. 2005). People older than 50 years, especially
those between 60 and 89 years, have a 20-fold increase for developing WNM
(Petersen et al. 2002; O’Leary et al. 2004; Hayes et al. 2005). Recipients of organ
transplant who are under immunosuppressive therapy have up to a 40-fold increased
risk for developing WNND (DeSalvo et al. 2004; Kumar et al. 2004). Disease devel-
opment is also often more severe in these patients as compared with immunocompe-
tent individuals (Kleinschmidt-DeMasters et al. 2004). Although both children and
adults are equally susceptible to WNV infection, WNND is less common in children
(Yim et al. 2004).
A genetic determinant of WNV resistance, the 1B isoform of 2′–5′ oligoadenylate
synthetase (OAS) gene, has been found in mice (Mashimo et al. 2002; Perelygin et
al. 2002). The 2′–5′ OAS family of enzymes is activated in the presence of interferon
(IFN) and viral double stranded RNA (dsRNA). Activated 2′–5′ OAS synthesizes
oligoadenylates, which, in turn, bind to and activate RNase L. Activated RNase L
degrades viral RNA (Justesen et al. 2000; Mashimo et al. 2002; Perelygin et al.
2002; Lucas et al. 2003; Kajaste-Rudnitski et al. 2006). Mice resistant to WNV
infection have normal OAS genes while susceptible mouse strains have a truncated
form of the gene (Kajaste-Rudnitski et al. 2006).
However, the role of the OAS system in human susceptibility to WNV is still
unclear. A recent study demonstrated that there were differences in the frequency
distribution of at least one polymorphism in OAS genes in hospitalized patients with
WNV infection, as compared with controls (Yakub et al. 2005). This raised the pos-
sibility that the OAS system may play a role in human susceptibility to WNV infec-
tion. More work would need to be done to confirm this finding.
7.7 NEUROINVASIVENESS OF WEST NILE VIRUS

The frequency of invasion of the central nervous system (CNS) by WNV
increases with age and in immunocompromised individuals (Iwamoto et al. 2003;
Kleinschmidt-DeMasters et al. 2004; Hayes and Gubler 2006). This suggests that a
delayed production of neutralizing antibodies and a high viral load may be contribu-
tory factors to CNS infection (Iwamoto et al. 2003). However, the exact mechanism
by which WNV enters the CNS in humans remains unknown.
Murine models of infection have suggested that arrival of WNV in the LN trig-
gers a series of events that determine either asymptomatic or neuroinvasive and fatal
infection. While WNV-infected DCs secrete type I IFN to inhibit viral infection in
the peripheral tissues (Scholle and Mason 2005; Liu et al. 2006), and also to aid in
inducing adaptive immune response by presenting viral antigen to B lymphocytes,
they can produce cytokines that affect the permeability of the blood–brain barrier
(BBB). Cytokines such as tumor necrosis factor (TNF)-α (Diamond et al. 2003) and
macrophage migration inhibitory factor (MIF) (Arjona et al. 2007) have been shown
to contribute to WNV neuroinvasion. Similarly, WNV infection of toll-like receptor
(TLR) 3 knockout mice (TLR-3 –/–) that have decreased levels of circulating inflam-
matory cytokines, show reduced permeability of their BBB (Wang et al. 2004).
In addition, neurotropic flaviviruses can enter the CNS after direct infection of
endothelial cells in the cerebral mircrovasculature or after viremic dissemination to
the olfactory bulb. The virus then spreads via the neurons to the CNS (Chambers
and Diamond 2003). Although passive transfer of WNV-specific antibodies to mice

lacking functional B cells prevented viremia, the procedure could not completely
block viral spread to the CNS. This suggested that other nonhematogenous pathways
such as retrograde axonal transport from infected peripheral neurons might contribute
to CNS entry (Engle and Diamond 2003).
In the CNS, WNV has a propensity to infect neurons. Glial cells in the cerebral
cortex, basal ganglia, brain stem, and spinal cord are less often infected (Guarner et
al. 2004). In vitro experiments show that both cultured neurons and astrocytes sup-
port WNV infection with differing kinetics (Cheeran et al. 2005). Although microg-
lia do not support WNV growth, its infection leads to release of pro-inflammatory
cytokines such as interleukin 6 (IL-6) and TNF-α. Chemokines such as CXCL-10,
CCL-2, and CCL-5 are also produced. Both infected astrocytes and neurons also
produce CXCL-10 and CCL-5. Production of these chemokines is essential for
recruiting virus-specific T cells to clear the virus from the CNS (Cheeran et al.
2005; Glass et al. 2005).
7.8 VIRAL CLEARANCE

Although clearance of WNV from the brain and other organs is primarily antibody-
mediated, WNV-specific CD8+ cytotoxic T lymphocytes (CTL) have been shown to
be crucial to prevent mortality in mice (Wang et al. 2003; Shrestha and Diamond
2004). Mice lacking CTL or the major histocompatibility (MHC) class 1a antigen
have a 1000-fold increase in viral titer in the CNS. They also exhibit increased mor-
tality after footpad viral challenge (Wang et al. 2003; Shrestha and Diamond 2004).
Production of CXCL-10 by infected neurons facilitates recruitment of CXCR-3
receptor-bearing CTL to the CNS (Klein et al. 2005). Mice that lack CXCR-3
exhibit enhanced viral titer in the CNS and mortality after infection with WNV
(Klein et al. 2005). This phenotype was similar in both CD8- (Klein et al. 2005)
and CCR-5-deficient mice (Glass et al. 2005) that show defective T-cell recruitment
into the CNS. Altogether, this demonstrates the underlying importance of CTL in
clearing WNV from the CNS. However, studies have also suggested that CTL may
be responsible for pathological outcome by injuring WNV-infected neurons (Wang
et al. 2003).
Although the adaptive immune response plays a major role in viral clearance, the
innate immune response is crucial in mounting an initial anti-viral response. Infected
cells recognize and respond to WNV via endosomal dsRNA sensors such as TLR-3
(Wathelet et al. 1998; Alexopoulou et al. 2001; Iwamura et al. 2001; Wang and Fikrig
2004; Daffis et al. 2008; Wilson et al. 2008) and cytoplasmic dsRNA sensors such
as retinoic-acid-inducible gene (RIG) 1 and melanoma-differentiation-associated
gene (MDA) 5 (Andrejeva et al. 2004; Yoneyama et al. 2004; Rothenfusser et al.
2005; Yoneyama et al. 2005; Cárdenas et al. 2006; Chang et al. 2006; Fredericksen
and Gale 2006). Binding of viral dsRNA to these pathogen-associated recognition
receptors (PRR) induces phosphorylation and activation of downstream transcrip-
tion factors such as interferon regulatory factor (IRF) 3 (Lin et al. 1998; Sato et al.
1998a; Weaver et al. 1998; Yoneyama et al. 1998; Suhara et al. 2000) and IRF-7
(Marié et al. 1998; Sato et al. 1998b). Phosphorylated forms of IRF-3 and IRF-7
homodimerize and heterodimerize to translocate to the nucleus to up-regulate pro-

duction of type I IFN (Marié et al. 1998; Lin et al. 2000). Binding of secreted type
I IFN to surface IFN-α/β receptor (IFN-α/βR) up-regulates production of various
IFN-stimulated genes (ISG) such as 2′ 5′ OAS-2 and protein kinase R (PKR) to
inhibit virus replication. While 2′ 5′ OAS-2 activates latent RNAses to degrade viral
RNA (Justesen et al. 2000; Mashimo et al. 2002; Perelygin et al. 2002; Lucas et al.
2003; Kajaste-Rudnitski et al. 2006), viral dsRNA-induced dimerization and auto-
phosphorylation of PKR activates it to phosphorylate eukaryotic initiation factor 2
(eIF-2α) and inhibit viral protein translation (Samuel et al. 2006).
Besides up-regulating various ISG with antiviral properties, type I IFN produced
strongly augments its own production by establishing a positive feedback loop.
Binding of type I IFN to IFN-α/βR up-regulates both IRF-1 and IRF-7 to further
enhance the production of type I IFN (Marié et al. 1998; Sato et al. 1998b; Lin et al.
2000; Zhou et al. 2000). This establishes a strongly antiviral state to further inhibit
viral progression. The importance of type I IFN in limiting viral infection was dem-
onstrated in mice lacking the IFN-α/βR. Besides enhanced viremia and elevated
viral titers in both the brain and peripheral organs, these mice had increased mortal-
ity after WNV infection (Samuel and Diamond 2005). Treatment of cells with type
I IFN has been shown to reduce viral titer and inhibit cytopathicity (Anderson and
Rahal 2002; Samuel and Diamond 2005).
7.9 TREATMENT
Despite extensive investigations studying essential antiviral immune responses to
WNV in murine models, an efficacious treatment for WNV infection in humans
remains elusive. The fact that viremia is usually short and precedes illness further
compounds the problem. Thus, therapeutic agent(s) would have to be effective at
reducing both intracellular concentration of virus and the inflammatory response
to infection. Several agents have been tested recently to assess their potential as a
therapeutic. These include antivirals, nucleic acid-based agents, and immunomodu-
lating compounds.
The antiviral agent, ribavarin, is a guanosine analogue that can inhibit WNV rep-
lication and cytopathic effect in vitro (Jordan et al. 2000; Anderson and Rahal 2002).
Although it demonstrated efficacy against WNV infection in vitro, its effectiveness
in vivo was found wanting. Hepatitis C patients under a ribavarin treatment regime
developed WNV infection despite presence of the agent (Hrnicek and Mailliard
2004). During an outbreak in Israel in 2000, patients treated with ribavarin fared
worse than untreated patients. However, the unfavorable results could be biased by
the fact that more seriously ill patients were selected for treatment (Chowers et al.
2001).
A proprietary antisense oligomer construct made by AVI BioPharma, AVI-4020,
inhibited viral replication, and was found to be safe in a small pilot phase I human
clinical trial (Deas et al. 2005). However, trials have since been terminated due to
a limited pool of eligible WNV patients. Other RNA interference (RNAi)-based
constructs have been studied in vitro but have yet to show efficacy as a therapeutic.
Use of immunomodulating compounds such as IFN have also showed limited

efficacy. Although IFN treatment before WNV infection reduced mortality in mu
rine models of infection, no such protection was observed when IFN was given
2 days after infection (Morrey et al. 2004). Efficacy of IFN treatment in humans is
unclear due to the absence of data from randomized, placebo-controlled clinical
trial. However, the use of an intravenous immunoglobulin containing high titers of
anti-WNV IgG (Omr-IgG-am) has shown promising results (Shimoni et al. 2001;
Agrawal and Petersen 2003; Diamond et al. 2003). Phase II trials have just been
completed in March 2011, and Phase III trials are ongoing.
7.10 PREVENTION
In the absence of an effective therapeutic, prevention remains the key viable option
against WNV infection. Effective prevention involves both the individual and the
community. Reducing outdoor exposure during peak mosquito biting periods at dusk
and dawn can mitigate risk of exposure to infected mosquitoes. Covering exposed
skin and using mosquito repellent when outdoors for more than 30 minutes has been
found to reduce the risk of WNV infection by 50% during an epidemic (Loeb et al.
2005). Community-wide mosquito eradication programs can also reduce the avail-
ability of transmitting vectors.
Currently, vaccines for WNV are being developed. Such vaccines include inac-
tivated subunit vaccines, attenuated WNV vaccines, DNA-based vaccines, and chi-
meric vaccines. A phase II clinical trial in 200 subjects with a chimeric vaccine
containing the WNV prM and E genes using the vaccine strain of Yellow Fever virus
as a backbone has just been completed in February 2011. Results from the trial are
pending (NIH Clinical Trials).
7.11 CONCLUSION
Arrival of WNV in the Western hemisphere has raised considerable awareness
against this emerging pathogen. Despite extensive studies, an effective therapeutic
antiviral agent is yet to become available. Recent vaccine developments hold great
promise for future use to prevent WNV infection. In the meantime, it would be most
prudent for persons most at risk for developing symptomatic WNV infection to take
adequate personal precaution.
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8 Murray Valley
Encephalitis Virus
Natalie A. Prow, Roy A. Hall, and Mario Lobigs
CONTENTS
8.1 Introduction................................................................................................... 167
8.2 Structure and Replication.............................................................................. 168
8.3 Epidemiology................................................................................................. 169
8.4 Ecology.......................................................................................................... 171
8.5 Seroprevalence............................................................................................... 172
8.6 Clinical Presentation..................................................................................... 172
8.7 Laboratory Diagnosis of MVEV Infections.................................................. 173
8.7.1 Serological Tests................................................................................ 173
8.7.2 Viral Proteins Targeted in Serological Assays.................................. 174
8.8 MVEV Infection in Animals......................................................................... 174
8.8.1 Mice................................................................................................... 174
8.8.2 Veterinary Species and Wildlife........................................................ 176
8.9 Genetic Heterogeneity among Natural Isolates............................................. 176
8.10 Molecular Determinants of Virulence........................................................... 177
8.11 Immunobiology.............................................................................................. 178
8.11.1 Innate Immunity................................................................................ 178
8.11.2 Humoral Immunity............................................................................ 179
8.11.3 CD4+ T-Cell Immunity...................................................................... 180
8.11.4 CD8+ T-Cell Immunity...................................................................... 181
8.12 Vaccination.................................................................................................... 182
References............................................................................................................... 183
8.1 INTRODUCTION
Murray Valley encephalitis (MVE) is an important mosquito-borne viral disease of
Australia that causes annual, sporadic cases and occasional epidemics of potentially
fatal encephalitis in man. Although human cases of the disease are most commonly
reported in the tropical areas of Northern Australia, ecological factors and climatic
conditions occasionally result in cases appearing in more southerly areas of the
country, sometimes involving large-scale outbreaks of the disease. As there are no
virus-specific vaccines or treatment options currently available, this vector-borne
viral disease continues to represent a major public health threat in Australia. In this
chapter we discuss properties of the virus, cellular infection, clinical disease and the
167
host immune response to infection. We also describe recent advances in diagnosis of

the disease and strategies for disease prevention.
8.2 STRUCTURE AND REPLICATION

MVE virus (MVEV) is a spherical, enveloped particle with a positive strand,
nonsegmented RNA genome of about 11,000 nucleotides. The virus is a member
of the flaviviruses, family Flaviviridae. The replication and molecular biology
of the flaviviruses have been reviewed in detail by others (Chambers et al. 1990;
Fernandez-Garcia et al. 2009; Lindenbach and Rice 2003; Mukhopadhyay et al.
2005). Briefly, the viral RNA is translated into a polyprotein precursor, which is
cleaved into at least 10 viral proteins. These include three structural (capsid [C],
envelope [E], and premembrane/membrane [prM]), and seven nonstructural proteins
(NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The structural proteins together
with the viral genomic RNA assemble into the virion particle by a process that
involves budding across the membranes of the endoplasmic reticulum. The virion
surface consists of 180 copies of the envelope (E) protein, which are arranged in
icosahedral organization consisting of 30 rafts, each of which is composed of 3 E
protein dimers oriented in parallel and lying flat on the virus surface. Structural
studies show that the flavivirus E protein is organized into 3 domains: the central
structural domain (domain I) is flanked on one side by an elongated dimerization
domain (domain II) and on the other by an immunoglobulin-like domain (domain
III), which makes the highest protrusion from the otherwise smooth particle. The
fusion peptide is located at the distal end of domain II, and the receptor-binding
sites are thought to be located on domain III. The MVEV E protein is modified
by a single glycan linked to residue 154 on domain I (Dalgarno et al. 1986), which
facilitates virus morphogenesis and is a virulence determinant (see Section 8.10).
Most flavivirus-neutralizing antibodies recognize epitopes on the E protein: in
murine infections, serotype-specific neutralizing antibodies are mostly elicited by
epitopes on domain III and are thought to inhibit virus infectivity by preventing
virus binding to cell surface receptors; antibodies that recognize epitopes in domain
II are often cross-reactive with other flaviviruses, and can have neutralizing activity
due to inhibition of membrane fusion, while antibodies against epitopes in domain I
are generally nonneutralizing (Nybakken et al. 2005; Oliphant et al. 2007; Oliphant
et al. 2006). Studies in humans suggest that the antibody response against West Nile
virus (WNV) is biased to epitopes on domain II, especially the fusion loop, rather
than against domain III (Throsby et al. 2006).
Virus particles are transported from the intracellular site of assembly via the
secretory pathway to the cell surface to be released into the extracellular milieu.
During this process, virus maturation occurs, which requires the furin-catalyzed
cleavage of the small, virion surface prM to M protein. This triggers a conformational
rearrangement in E protein required for activation of the fusion function (reviewed
by Harrison 2008). A specific host receptor for MVEV has not been identified;
given the wide host and vector tropism of the virus, it is likely that it can employ
multiple cell surface receptors for establishment of productive infection. Heparan
sulfate proteoglycans are sulphated polysaccharides ubiquitous found on cellular
Murray Valley Encephalitis Virus 169
surfaces and extracellular matrices, and have been shown to facilitate attachment/
entry of MVEV in cell culture (Lee and Lobigs 2000). However, in mice variants
of MVEV and other flaviviruses selected for high binding-affinity to these cell
surface molecules display a complete loss of neuroinvasiveness due to a mechanism
involving rapid virus clearance from the blood stream, thereby preventing virus
dissemination (see Section 8.10). This suggests that heparan sulfate proteoglycans
do not function as uptake receptors in the transmission cycle of the virus, but
may still play a role in initial low-affinity attachment to cellular surfaces. MVEV
enters the cell via receptor-mediated endocytosis. Fusion of the viral membrane
with the endosomal membrane occurs after exposure to low pH in the endosomal
compartment, allowing viral RNA release into the cytoplasm of the newly infected
cell.
8.3 EPIDEMIOLOGY
MVEV is the main cause of arboviral encephalitis in Australia (reviewed by
Doherty 1974; French 1973; Mackenzie and Broom 1995; Marshall 1988) and was
first isolated from fatal human cases of encephalitis during an epidemic in 1951
in the Murray Valley in the south-east of the continent (French 1952; Miles et
al. 1951). The virus is a member of the Japanese encephalitis virus (JEV) sero-
logical complex within the genus Flavivirus, family Flaviviridae (Thiel et al.
2005). MVEV circulates in Australia, Papua New Guinea (PNG), and probably
on islands in the eastern part of the Indonesian archipelago (Mackenzie et al.
1994). The virus is thought to be enzootic in tropical northern Australia, mainly
the Kimberley region of Western Australia and the “Top End” of the Northern
Territory, and to a lesser extent in northern Queensland (Mackenzie et al. 1994;
Mackenzie and Williams 2009). Intermittent virus activity occurs in the Pilbara
region of Western Australia, as indicated by sentinel chicken seroconversion, virus
isolations from mosquitoes, and cases of MVE (Broom et al. 1989). MVEV is
sporadically found in central, south-eastern and south-western regions of Australia,
where epidemics of viral encephalitis can occur (Figure 8.1). It has been proposed
that during extended periods of above average rainfall MVEV may be transmitted
from enzootic northern regions to temperate zones by the southward movement
of viremic birds (Johansen et al. 2007; Lobigs et al. 1986, 1988). Such climatic
conditions also result in the surge in abundance of vertebrate hosts (water birds)
and mosquito vectors necessary for the appearance of clinical disease in humans
and horses.
Several large epidemics of encephalitis were observed in eastern Australia during
1917, 1918, 1922, and 1925 and were designated Australian X disease (Anderson
1954). In 1951, 45 cases of encephalitis were attributed to MVEV, where all but two
cases from the epidemic originated from the Murray Valley. The next major epidemic
occurring in 1974 resulted in MVE spreading to all mainland states with 54 cases,
including the first recorded West Australian case. From 1975 to 1999, all cases of
MVE (48 cases) were acquired in northern Australia with the majority from the
Kimberley region (Burrow et al. 1998; Mackenzie et al. 1993). An MVEV epidemic
in 2000 resulted in 15 human cases of encephalitis, 9 in Western Australia, 3 in the
Annual
endemic
activity
Recent epidemic
Occasional activity
epidemic
activity
Rare epidemic
activity
FIGURE 8.1 MVEV activity in Australia.
Northern Territories, and one in South Australia, the latter being the first recorded
case of MVE in the dry inland region of central Australia in 26 years (Brown et al.
2002). Also remarkable in this epidemic was the acquisition of MVE as close as 315
km north of the Western Australian capital, Perth. During the 2000 outbreak, West
Australian patients ranged in age from 10 months to 79 years, were predominately
male, and were largely non-Aboriginal adult visitors or residents. Nine patients
developed encephalitis and one died. From 2001 to 2008 there were 11 human cases
of MVE recorded from Western Australia, the Northern Territories, Queensland,
and New South Wales (Johansen et al. 2008). This marked the reappearance of MVE
in New South Wales after an absence of 24 years, and although there was evidence
that MVEV activity occurred in previous years, as indicated by seroconversions in
sentinel chickens and mosquito isolates (Doggett et al. 2008), no cases of human
disease were reported in the intervening years. In 2011, a significant outbreak of
arboviral encephalitis occurred in humans and horses in south-eastern Australia.
Although more than 1000 cases of equine disease were reported (Frost et al. 2012)
only a handful of human cases occurred. Interesting, the vast majority of confirmed
equine cases were caused by infections with a new strain of Kunjin virus (KUNV),
a subtype of WNV, while all confirmed human cases were shown to be associated
with MVEV infection. This was the first report of human cases in south-eastern
Australia since 1974. During the first five months of 2011, there have been a total
of 14 notifications of MVE (National Communicable Diseases surveillance report,
Fortnight 09 2011; http://www.health.gov.au/internet/main/publishing.nsf/Content/
cdnareport-fn9-11.htm.)
8.4 ECOLOGY
Culex annulirostris is the major mosquito species involved in MVEV transmission
(Kay et al. 1989). It is a widely distributed, highly adaptable, freshwater mosquito that
inhabits permanent and semipermanent water bodies, and has the ability to colonize
rain water pools within a day of their formation (Doherty et al. 1963). In addition,
the virus has also been isolated from numerous other mosquito species in Australia
(Broom and Whelan 2005; Broom et al. 1989; Kay and Carley 1980; Russell 1998;
van den Hurk et al. 2010). Ardeid water birds of the order Ciconiiformes, particularly
the rufous night heron (Nycticorax calendonicus), and other herons and egrets are
thought to be the major vertebrate hosts for MVEV (Anderson 1952; Boyle et al.
1983). Other avian and mammalian vertebrates have shown serological evidence
of infection and may play a role in virus transmission, although this is yet to be
confirmed (Kay et al. 1985a,b).
MVEV co-circulates with several other flaviviruses in Australia, including
KUNV. In northwestern Australia, MVEV is more frequently isolated from mos-
quitoes than KUNV, while in Queensland and south-eastern Australia KUNV is
isolated more frequently (Mackenzie et al. 1994). The vector competence of different
regional populations of Cx. annulirostris may contribute to the varying prevalence
of the viruses in the different locations (Kay et al. 1984, 1989), in addition to host
biology. Carver et al. (2009) have reviewed mechanisms by which hosts influence
MVEV and related virus transmission, and have identified five areas: host immunity,
cross-protective immunity and antibody-dependent enhancement, host abundance,
host diversity, and pathogen spill-over and dispersal.
Widespread transmission of JEV in the Torres Strait of northern Australia and
subsequent virus isolation on the Australian mainland have raised the possibility
that JEV would become enzootic in Australia, where suitable host and vector species
are thought to be abundant (reviewed by van den Hurk et al. 2009). This scenario
would greatly increase the complexity of the Australian flavivirus ecology with the
likely concurrent circulation of MVEV with JEV, the most important human and
veterinary pathogen of the JEV serocomplex. Both viruses coexist in PNG, which
shows that the two closely related viruses can be maintained in the same ecosystem.
However, it appears that JEV has, so far, not become established in natural trans-
mission cycles on the Australian mainland. Possible reasons for this include cross-
protective immunity to JEV in susceptible hosts, suboptimal vector competence of
the lineages of Cx. annulirostris to JEV, and the propensity of Cx. annulirostris to
feed on marsupials (which do not produce high levels of viremia) and not pigs (an
important amplified host for JEV) (van den Hurk et al. 2009).
Global warming and associated climate change have been postulated to lead to
increased activity of vector-borne diseases, such as MVEV (reviewed by Colwell
et al. 1998; Mackenzie and Williams 2009). Warmer, wetter, and more humid
conditions could lead to increases in both mosquito abundance and distribution.
These conditions could also lengthen the seasonal activity of some mosquito vectors.
Rising sea levels may lead to extensive flooding, leading to increases in mosquito
numbers (Mellor and Leake 2000). However, Russell et al. (2009) have suggested
that any evaluation of the potential effects of climate change will need a detailed
examination of at least site-specific vector and host factors and other aspects likely
to influence the outcomes of virus activity on human health. The authors suggest
that climate change, as currently projected, is unlikely to significantly change the
distribution and transmission of endemic arboviruses and is not likely to provide
cause for public health concern regarding mosquito-borne diseases in Australia.
8.5 SEROPREVALENCE
Antibody levels within a population provide information about the frequency of
infection in a community. Calculation of antibody seroprevalence rates of MVEV is
problematic due to the sporadic nature of epidemics and cases of disease throughout
Australia. Furthermore, the inability to serologically distinguish between MVEV
and KUNV in early epidemics added further ambiguity to the determination of
prevalence rates. Seroprevalence of MVEV and KUNV differ based on geographical
location (Hawkes et al. 1985, 1993). Antibody prevalence rates were higher for
KUNV than for MVEV in sera collected in New South Wales during 1981–1982
(Hawkes et al. 1985). In a second study of the same regions from 1981 to 1991, KUNV
antibody was detected, whereas MVEV antibody was relatively uncommon (Hawkes
et al. 1993). In Western Australia, MVEV and KUNV antibody prevalence tends to
decrease geographically from the north of the State to the south. The highest MVEV
seroprevalence (52.6%) was reported from the southeast Kimberley region, where
MVEV and KUNV are considered to be enzootic (Broom et al. 2002). A serosurvey
of human samples collected between 1999 and 2001 from the mid-west region of
Western Australia, where MVEV activity is epizootic, showed that only 2.3% of
samples contained antibodies against MVEV (Sturrock 2009). The seroprevalence
of MVEV antibodies in humans tends to increase with increasing age in enzootic
areas, as MVEV-specific antibodies are life-long (Broom et al. 2002); there is little
or no difference between antibody seroprevalence in males and females (Broom et
al. 2002; Hawkes et al. 1985).

MVEV commonly infects humans without producing apparent illness. It has been
estimated that only about 1 in 1000 infections results in clinical disease (reviewed
by Burrow et al. 1998; Mackenzie et al. 1993; Marshall 1988). Cases are reported
mostly among resident Aboriginal children and interstate or international travellers
to regions where the virus is endemic, indicating that natural exposure provides
long-lived immunity. The clinical aspects of MVEV infection (previously called
Australian encephalitis, which also included a rare disease caused by KUNV) have
been described for the 1951 (Robertson and McLorinan 1952) and 1974 (Bennett
1976) epidemics in south-eastern Australia, and in studies of more recent cases in
Western Australia (Mackenzie et al. 1993) and the Northern Territories (Burrow
et al. 1998). MVEV infection involves a prodromal stage with fever in all patients,
commonly with features such as severe frontal headache, nausea, vomiting,
dizziness, increasing confusion, and convulsions frequent among children. Within a
period of ~5 days of onset, this progresses to obvious neurological disease resulting
in encephalitis of variable severity. Signs of brain dysfunction such as drowsiness,

irritability, confusion, fitting, ataxia, neck stiffness, speech disturbance, cranial nerve
palsies, and movement disorders, which include parkinsonism and other tremors,
indicate onset of encephalitis. In severe cases, this will progress to respiratory
failure, coma, and death. The case fatality rate is high (15%–32%), while significant
neurological sequelae with physical and/or intellectual handicap are seen in ~50%
of survivors (Burrow et al. 1998; Mackenzie et al. 1993). Burrows et al. observed
four clinical patterns of disease: (i) relentless progression to death, (ii) prominent
spinal cord involvement, (“poliomyelitis-like”), (iii) cranial nerve/brainstem involve
ment and tremor, and (iv) encephalitis with complete recovery (Burrow et al. 1998).
Flaccid paralysis with rapid progression to respiratory failure is a predictor of poor
neurological outcome and death, and electrophysiological studies in these cases
indicate extensive anterior horn cell involvement (Burrow et al. 1998; Einsiedel
et al. 2003). Magnetic resonance imaging (MRI) reveals extensive and bilateral
thalamic injury, and may prove helpful in assessing the clinical long-term outcomes
of patients infected with MVEV (Burrow et al. 1998; Douglas et al. 2007; Einsiedel
et al. 2003; Kienzle and Boyes 2003): mild cases of MVE have no apparent MRI
changes with an excellent outcome, while more severe cases have striking changes
and poor outcome. In one case report, MRI findings showed typical temporal lobe
changes, suggesting that MVE can mimic herpes simplex encephalitis clinically and
radiologically (Wong et al. 2005). An accurate and detailed travel history of patients,
where there is a risk of exposure to MVEV, combined with serological testing are
therefore important for correct diagnosis and patient management. Notably, MVE is
after herpes simplex encephalitis, the second most serious acute viral encephalitis
to be encountered in Australia, and the most common cause of viral encephalitis in
tropical Australia (Burrow et al. 1998).
8.7 LABORATORY DIAGNOSIS OF MVEV INFECTIONS

8.7.1 Serological Tests
Diagnosis of clinical MVEV infections in humans is based on the signs and symp
toms at presentation (see above), but must be supported by laboratory confirmation
using serological and/or molecular tests. Standard assays used to diagnose arbo
virus infections in previous decades (e.g., haemagglutination inhibition and virus
neutralization) have largely been replaced by ELISA, immunofluorescence assays,
and more recently microsphere immunoassays (MIA) (Johnson et al. 2005). A
diagnostic requirement for detection of virus-specific IgM in patient serum and
preferably a 4-fold rise in virus-specific IgG between presentation (acute sample)
and follow-up testing (convalescent sample) has been recommended for conclusive
diagnosis (Mackenzie et al. 1993). Detection of MVEV-specific IgM in CSF samples
has also been used successfully for laboratory diagnosis. Similar symptoms can
present with infections in related flaviviruses that occur in Australia (e.g., MVEV,
JEV, KUNV) and the high level of serological cross-reactivity observed between
these viruses. It is highly recommended that samples must be tested against a
complete panel of antigens prepared from local flaviviruses and at least a four-fold
differential in antibody titer be determined to identify the infecting virus (Taylor et

al. 2005).
8.7.2 Viral Proteins Targeted in Serological Assays

Currently, high throughput serological assays used for the routine diagnosis of infections
with MVEV and other flaviviruses are based on virion antigens in direct binding assay
formats such as ELISA and MIA. These antigens have been successfully produced in
large quantities from infected cell culture fluid by chemical inactivation of the virion with
binary ethyleneimine and partial purification and concentration by lateral flow filtration
(Pyke et al. 2004). However, the relatively high level of antigenic conservation of the
envelope (E) protein between members of the JEV serogroup often results in a cross-
reactive primary response or original antigenic sin compounded by previous infections
with heterologous flaviviruses (Crill and Chang 2004).
More recently, serum antibody to other viral proteins such as NS1 (Blitvich et al.
2003a,b; Hall et al. 1995), prM (Hobson-Peters 2010; Setoh et al. 2011) or subdomains
and peptides of the E protein (Estrada-Franco et al. 2003; Hobson-Peters et al. 2008a,b;
Hobson-Peters 2010) or NS5 have also been suggested as more specific markers of
infection with JEV serogroup viruses. Recombinantly expressed peptides or domains of
E (DI and DIII), NS1, prM, and NS5 are currently being assessed by our laboratory and
our collaborators to differentiate between members of the JEV serogroup in multiplexed
MIAs (Johnson et al. 2005).
Blocking ELISAs have been developed to specifically identify antibodies of
MVEV and KUNV in sentinel birds based on monoclonal antibodies to immuno
genic epitopes on the E and NS1 proteins (Hall et al. 1995; Hawkes et al. 1990).
To some extent these assay have also been useful for screening for infections in
horses; however, their use for human diagnosis has been limited due to inability
to differentiate IgM from IgG responses and confounding results from previous
infections with heterologous flaviviruses (Hall et al. 1995; Spicer et al. 1999).
The development of high throughput RT-PCR technology has enabled the use of
molecular testing for RNA in viral infections on a routine basis. McMinn et al. described
the use of RT-PCR to detect MVEV RNA in the serum of a 4-year-old boy suffering
viral encephalitis at days 4 and 7 postonset (McMinn et al. 2000). This was crucial in the
diagnosis of the disease as virus-specific IgM was undetectable and cross-reactive IgG
responses due to a prior KUNV infection rendered the serological diagnosis inconclusive.
Consistent with previous reports, virus could not be isolated from the PCR positive
samples. These findings suggested that RT-PCR analysis of acute phase specimens is a
useful complement to serological testing. More recently, Pyke et al. (2004) have described
sensitive qRT-PCR protocols to test for MVEV, JEV, and KUNV in clinical specimens.
8.8 MVEV INFECTION IN ANIMALS

8.8.1 Mice
Mice provide an excellent animal model for MVE in humans. MVEV is neurotropic
in mice; when small virus doses are injected directly into the brain it grows to high
titers and uniformly causes fatal encephalitis (Licon Luna et al. 2002; MacDonald
1952a). In contrast, and similar to human infections, MVEV does not grow to
detectable virus titers in extraneural tissues of adult immunocompetent mice
following peripheral inoculation of the virus (Licon Luna et al. 2002; Lobigs et al.
2009; MacDonald 1952b), and often fails to produce morbidity or mortality over a
wide dose-range (up to 106 PFU) (Licon Luna et al. 2002). This dose-independence
of mortality and average survival time in peripherally infected adult mice has been
reported for other flaviviruses (Larena et al. 2011; Wang et al. 2003), and suggests
that equalizing factors exist: one such factor could be interferon (IFN) and other
innate immune responses, the magnitude of which may inversely correlate with the
virus dose inoculated.
The disease outcome in mice infected with MVEV is strongly age-dependent,
where the animals are highly susceptible to a low-dose peripheral virus inoculum
until the age of ~3 weeks (Lobigs et al. 1988; MacDonald 1952a; McMinn et al.
1996). In the weanling mouse model, MVEV is first detected in the lymph node
draining the inoculation site at 24 h after inoculation into the footpad. Further
replication at these sites generates a viremia between 2 and 3 days postinfection (pi)
prior to entry of the virus into the CNS at 4 days pi, where peak virus titers occur
between 6 and 9 days pi; the virus appears to enter the CNS via the olfactory lobes
and spreads throughout the brain in the following 3 to 4 days, producing neuronal
necrosis in the presence of inflammatory infiltrates, particularly noticeable in
regions of the hippocampus (Matthews et al. 2000; McMinn et al. 1996). While
the olfactory neuroepithelium is not protected by the blood-brain-barrier and is
richly supplied with capillaries having fenestrated endothelia, thereby providing a
potential route for virus entry into the CNS, other mechanisms by which MVEV
may breach the blood-brain-barrier have also been canvassed, such as (i) virus
infection of vascular endothelial cells of capillaries in the brain and release of virus
into the brain parenchyma (Dropulic and Masters 1990; Licon Luna et al. 2002) or
(ii) by diffusion of virus between capillary endothelial cells in individuals displaying
leakiness of the blood-brain-barrier due to factors unrelated or secondary to the virus
infection (reviewed by Mullbacher et al. 2003). The magnitude of viral load in the
circulation, while mostly below the detection limit in adult immunocompetent mice,
is a factor that contributes to virus infection of the CNS, based on two observations:
extraneural infection of adult mice with a high dose (108 PFU) of MVEV results
in early appearance of signs of encephalitis and high mortality (Colombage et al.
1998; Licon Luna et al. 2002) and infection of type I IFN response-defective mice
produces high viremia, which correlates with infection of the CNS in all infected
animals (Lobigs et al. 2003b).
Genetic resistance of wild and some inbred strains of mice to disease induced
by MVEV and other flaviviruses has been described (Brinton and Perelygin 2003;
Sangster et al. 1993, 1998; Silvia et al. 2004). Resistance is flavivirus-specific and
is controlled by a single dominant autosomal gene. Resistant animals are infected
productively, but produce significantly lower virus titers in brain relative to
susceptible mice. The resistance gene (Flv) has been identified as an IFN-inducible
gene encoding 2′-5′-oligoadenylate synthetase (Mashimo et al. 2002; Perelygin et al.
2002).
8.8.2 Veterinary Species and Wildlife

Among domestic animals, potentially lethal nervous system disease associated with
MVEV infection of horses is of concern. Limited reports of naturally acquired
or experimental infections have documented nervous system disease in horses
following an outbreak of MVEV in 1974 in south-eastern Australia, and only trace
amounts of virus in blood in the absence of clinical signs other than a transient
rise in temperature, respectively (Gard et al. 1977; Kay et al. 1987). Thus, MVEV
infection of horses resembles that of humans in terms of low apparent to non-
apparent infection ratio, and low or undetectable viremia, but high morbidity and
mortality in clinical cases. Although currently a low incidence disease, demand for
a vaccine against MVEV for use in horses may arise with increased agricultural
development and human activity in MVEV endemic regions in northern Australia
or altered frequency/severity of epidemic outbreaks of MVEV in south-eastern
and Western Australia as a possible consequence of climate change. Indeed, recent
incidents of fatal equine encephalitis in 2008 and 2011 have been associated with
MVEV infection, as determined by detection of viral RNA or isolation of virus
from necroscopy brain samples, and represent the first laboratory confirmed cases
of MVE in horses (Gordon et al. 2012; Holmes et al. 2012). No clinical signs have
been observed in a range of domestic animals and wildlife infected with MVEV,
although some species (rabbits, gray kangaroos, native birds, pigs, dogs, and
chicken) show evidence of moderate to high viremia (Kay et al. 1985a,b; Sanderson
1968).
8.9 GENETIC HETEROGENEITY AMONG NATURAL ISOLATES

Molecular epidemiological investigations on MVEV show only limited genetic
differentiation of the majority of virus isolates over time and geographic distance in
Australia (Lobigs et al. 1986, 1988; Coelen et al. 1988; Johansen et al. 2007). This
observation that most MVEV isolates belong to a single genetic lineage has provided
the strongest evidence, so far, for the hypothesis that the virus moves from enzootic
foci in northern Australia to areas further south under the influence of particular
climatic conditions. A second lineage of MVEV detected only in the Kimberley
region of Western Australia may have been introduced from outside Australia
(Johansen et al. 2007). In contrast to the overall genetic homogeneity of MVEV in
Australia, virus strains from PNG display marked genetic divergence both between
isolates and from the Australian genotype (Johansen et al. 2007; Lobigs et al. 1986).
A major question relating to MVEV infections of humans is the reason for the
high ratio of subclinical to clinical infection. One possibility is that certain strains
are inherently more virulent than others; however, this is most likely not the case,
(i) given the absence of genetic differences between virus isolated from fatal human
cases and from other sources (mosquitoes or birds) and (ii) in view of their identical
virulence profile in a mouse model of encephalitis (Lobigs et al. 1988). A naturally-
occurring subtype of MVEV, Alfuy virus, also circulates in Australia but has neither
been associated with human disease nor is it neuroinvasive in weanling mice even at
high doses (May et al. 2006; Thiel et al. 2005).
8.10 MOLECULAR DETERMINANTS OF VIRULENCE

Field isolates of MVEV do not show significant variation in virulence in mouse
models of MVE (Lobigs et al. 1988). In contrast, virus propagation in the laboratory
and reverse genetics with the use of full-length infectious cDNA clones of MVEV
(Hurrelbrink et al. 1999; Lee and Lobigs 2000) has allowed the generation of vari-
ants with altered virulence phenotypes. Studies on these variants are instrumental in
structure–function analyses of virus replication and in vivo pathogenesis, and have
identified molecular determinants of virulence attenuation with potential applica-
tion in live vaccine development. The latter should involve mutations in the viral
genome, which are not deleterious for replication, per se, but prevent disease in the
mammalian host. We have shown that host cell adaptation of MVEV by serial pas-
sage in mammalian cell culture (human adenocarcinoma [SW13] and baby hamster
kidney [BHK] cells) rapidly selects for variants with single amino acid changes in E
protein domain III, which give rise to a high level of virulence attenuation (Lobigs
et al. 1990). The substitutions in E protein (at residue 390) involve the acquisition of
an increase in net positive charge on the virion surface that accounts for augmented
binding affinity to heparan sulfate proteoglycans (Lee and Lobigs 2000). While this
change in receptor usage benefits growth of the host cell-adapted variants in cell
culture, it dramatically reduces their capacity to spread into the CNS from an extra-
neural site of infection in the mouse (Lee et al. 2004; Lee and Lobigs 2002). This
loss of neuroinvasiveness is due to rapid clearance of variant virus from the blood
as a result of nonproductive binding to glycosaminoglycans in tissues such as liver
and on extracellular matrices. This in vivo mechanism of virulence attenuation also
accounts, at least in part, for the loss of viscerotropism of yellow fever 17D vaccine
(Lee and Lobigs 2008) and the attenuation of a JEV live vaccine (SA-14-14-2 strain)
widely used in China (Lee and Lobigs 2002).
The E protein glycan of MVEV is conserved among all natural isolates and is
an important marker of virulence: removal of the glycosylation site in the protein
results in a marked reduction in neurovirulence and is associated with a reduced
level of virus in the blood (Prow et al. 2011). Our studies and those of others show
that virulence attenuation is due to less efficient secretion of the nonglycosylated
than glycosylated virus from infected cells (Beasley et al. 2005; Hanna et al. 2005;
Lee et al. 2010; Shirato et al. 2004; Setoh et al. 2012). Interestingly, this defect does
not reduce the efficiency of virus growth in mosquito cells, where loss of the glycan
markedly enhances infectivity of MVEV and other flaviviruses, albeit at the expense
of efficient virus release (Lee et al. 2010).
Studies by McMinn and colleagues (Hurrelbrink and McMinn 2001; McMinn
et al. 1995) and in our laboratory (Prow et al. 2011) have identified residues in the
flexible hinge region of the MVEV E protein (residues 273–277) that are critical for
virulence in mice. Amino acid substitutions introduced in this region are thought to
inhibit pH-dependant rearrangement of the E protein required for efficient fusion
triggered by mildly acidic pH, thereby reducing virus infectivity. Nevertheless, wild-
type and hinge region variants of MVEV grow equally well in both vertebrate and
invertebrate cell cultures, indicating that the reduced neuroinvasiveness of the latter
is not due to a major abnormality of replication (Prow et al. 2011).
In contrast to the virulence variants described above, we have also identified

molecular determinants of virulence attenuation, which impact on efficient virus
replication. A single amino acid substitution in NS1 (residue 250) eliminates dimer-
ization of the protein, but allows virus replication to continue; however, growth in
Vero cells of the variant is retarded and levels of viremia and virulence in mice
are markedly lower than those for wild-type MVEV (Clark et al. 2007). Similarly,
amino acid changes at the junction of the prM and E proteins of MVEV, which
uncouple the cleavage coordination between the two proteins required for efficient
virus assembly, reduce growth in mammalian cell culture by ~10-fold (Lobigs and
Lee 2004), and markedly reduce neuroinvasiveness in weanling mice (Lobigs et al.
2010a). Interestingly, a compensatory mutation in the prM signal peptide, which
restores virus growth in cell culture to wild-type levels, does not repair the growth
and virulence phenotype of the mutant virus in mice (Lobigs et al. 2010a). This dem-
onstrates the greater sensitivity of animal models to reflect the impact of mutations
that reduce the efficiency of virus replication that is found in cell culture.
The ability of flaviviruses to replicate efficiently in the mammalian host is
dependent on their ability to evade, at least in part, the innate immune responses,
particularly the antiviral activity of type 1 IFNs (Section 8.11.1). Recent studies on
WNV and JEV have identified critical motifs in the viral nonstructural proteins
that are responsible for enhanced resistance to the effects of IFN (Audsley et al.
2011; Daffis et al. 2011; Laurent-Rolle et al. 2010; Lin et al. 2008). Although similar
studies have, so far, not been performed with MVEV, it is assumed that this virus also
encodes IFN-resistance markers, which are important determinants of virulence.
8.11 IMMUNOBIOLOGY
8.11.1 Innate Immunity
Among the innate immune responses, type I IFN is of critical importance in
recovery from infection with MVEV (Lobigs et al. 2003b). In response to cytosolic
viral infection, activation of RIG-1-like receptors triggers IFN production, while
specialized immune cells (dendritic cells and macrophages) can also produce type
I IFNs following extracellular stimuli of viral origin by toll-like receptor (TLR)
engagement. Type I IFNs (IFN-α and -β) then induce immediate antiviral effects in
infected and neighboring cells and thereby limit viral spread. Mice that are deficient
in IFN-α/β responses show sustained viremia, fulminant disease, rapid virus entry
into the brain, and 100% mortality following administration of a low dose of MVEV
by the intravenous route (Lobigs et al. 2003b). Given their exquisite sensitivity to
infection with MVEV, type I IFN response-defective mice serve as an excellent
model for virulence testing of attenuated variants of the virus (Clark et al. 2007; Lee
and Lobigs 2002; Lobigs et al. 2010a; May et al. 2006). The therapeutic potential of
recombinant IFN in human cases of MVE has not been investigated, although in the
case of the closely related JEV, IFN therapy did not improve the outcome of patients
with encephalitis (Solomon et al. 2003).
IFN-γ is made exclusively by natural killer (NK) and T cells, and has important
immunoregulatory functions as well as antiviral activity. The latter is mostly mediated
by nitric oxide (NO) synthesized by monocytes, following induction of the enzyme

NO synthase by the cytokine. IFN-γ knock-out mice show a marginal increase in
susceptibility to MVEV relative to wild-type mice, which is reflected in a small increase
in virus titers in spleen, more rapid virus spread into the CNS and increased mortality
(Lobigs et al. 2003b). The marginally protective role of IFN-γ against MVEV appears
to be, at least in part, mediated by NO, given that mice deficient in NO synthase 2
also display a slight increase susceptibility to infection with MVEV in comparison to
congenic control mice (Lobigs et al. 2003b). In contrast to this investigation in adult
mice, a pathological contribution of NO production in the CNS following neutrophil
infiltration has been described in the weanling mouse model for MVE (Andrews et al.
1999). This discrepancy on the role of NO in recovery from MVEV infection probably
reflects the increased level of immunopathology in infected weanling relative to adult
mice as a consequence of not yet fully competent regulation of innate immune responses
of the developing immune system in the former, in combination with the higher viral
burden in the young relative to adult mice.
NK cells are part of the innate immune response and have been implicated in
the early defense against numerous viral infections (reviewed by Biron 2010). They
are activated by type I IFNs, are important producers of IFN-γ, and mediators of
cytotoxicity by causing apoptotic lysis of virally infected cells. While the induction
of classical NK cell cytolytic activity as a result of flavivirus infection has been
reported in mice (Mullbacher et al. 2003), it is thought that flaviviruses employ a
strategy to evade the NK cell-mediated host defense, which involves up-regulation
of major histocompatibility class I (MHC-I) molecules at the surface of infected
cells (Lobigs et al. 1996, 2003a; Momburg et al. 2001). MHC-I engages with NK
cell inhibitory receptors and can thereby down-regulate the NK cell response. To
assess whether NK cell cytotoxicity plays a role in recovery of MVEV infection, we
infected adult beige mice, which are deficient in the cytolytic function of NK cells
(Roder and Duwe 1979), with a low dose of MVEV (102 PFU), intravenously. No
significant difference in susceptibility of mice to the virus infection relative to wild-
type mice (60% and 46% mortality, respectively [n = 10]) was observed (Licon Luna
and Lobigs, unpublished).
8.11.2 Humoral Immunity
To investigate whether the adaptive immune responses are required in resistance
against MVEV, we compared the susceptibility of mice genetically deficient of both
B and T cells (RAG-1–/– mice) to that of wild-type mice (Table 8.1). A low dose (102
PFU) intravenous infection with MVEV resulted in almost complete mortality of
RAG-1–/– mice, while ~50% of congenic wild-type mice did not develop signs of
encephalitis and survived. Interestingly, the average time to death of RAG-1–/– mice
was significantly delayed relative to that in groups of wild-type mice, demonstrating
an immunopathological contribution of the adaptive cellular immune responses to
mortality with MVEV, which is T-cell-mediated (Licon Luna et al. 2002) (and see
below). In a second experiment we show that virus-immune B cells but not T cells
are required for the control of infection with MVEV (Table 8.1). Thus, transfer of
MVEV-immune B cells completely protected against challenge with a ~50% lethal
TABLE 8.1
Role of Adaptive Immune Responses in Recovery from Infection with MVEV
Mouse Strain and Treatmenta Nob % Mortalityc ATD ± SEMd
Experiment 1
B/6 (wt) 17 47 11.5 ± 0.6
RAG-1– /– 23 91 (P = 0.003) 17.1 ± 2.2
Experiment 2
B/6 (wt) mock-treated 13 46 10.5 ± 1.0
B/6 (wt) + MVEV-immune B cells 5 0
B/6 (wt) + MVEV-immune T cells 6 67 13.0 ± 0.8
Source: R. M. Licon Luna (2004). On the role of cell-mediated cytotoxicity in a mouse model of flavivi-
rus encephalitis. PhD thesis. The Australian National University, Canberra.
a Six-week-old C57Bl/6 (B/6) or congenic recombinant activating gene (RAG) 1 knock-out mice
(Mombaerts et al. 1992) were used. RAG-1-/- mice fail to produce mature B or T cells. Mice were chal-
lenged with 102 PFU MVEV by the intravenous route. For isolation of MVEV-immune B or T cell-
enriched splenocytes, donor B/6 mice were infected with 102 PFU MVEV, i.v., and spleens collected at
6 days pi. B and T cell-enriched fractions (78 and 73% pure, respectively) were isolated by nylon wool
separation and transferred (4 × 107 cells) into recipient mice at 3 days after challenge with MVEV.
b Number of infected mice/group.
c Difference in survival ratio relative to control mice was assessed using Fisher’s exact test.
d Average time to death, calculated in days, ± standard error of the mean (SEM).
dose of MVEV; in contrast, transfer of immune T cells failed to provide a survival

advantage in comparison to a group of mock-treated mice infected with MVEV. This
highlights the critical importance of the humoral immune response in recovery from
infection with MVEV.
Mechanistically, antibodies elicited against infection with flaviviruses exhibit
their action directly by neutralization of virus infectivity or indirectly by antibody-
mediated cytotoxicity, Fc-γ-receptor-mediated clearance of virus/antibody
complexes or complement-mediated cytotoxicity (Pierson et al. 2008). Neutralizing
antibodies predominantly target the E protein, and several groups have isolated
neutralizing monoclonal antibodies against MVEV and mapped the corresponding
epitopes on the E protein (Hall et al. 1990; Hawkes et al. 1988; McMinn et al. 1995).
Passive transfer of neutralizing monoclonal antibody offers significant protection
in mice from otherwise lethal intraperitoneal virus challenge (Hawkes et al. 1988).
Monoclonal antibodies against the MVEV NS1 protein have also been described
(Clark et al. 2007; Hall et al. 1990); these can control flavivirus infection by their
complement-mediated cytolytic potential (Hall et al. 1996; Schlesinger et al. 1986;
Schlesinger et al. 1985).
8.11.3 CD4+ T-Cell Immunity

There are two predominant lineages of peripheral T cells that can be differenti
ated on the basis of their co-receptor expression (CD4+ vs. CD8+). CD4+ T cells
contribute to the control of infection by various mechanisms, including antiviral and

immunoregulatory cytokine production, antibody class switching, direct cytotoxicity
and maintenance of CD8+ T-cell activity (Zhu et al. 2010). They recognize MHC-II/
peptide complexes on antigen presenting cells (dendritic cells, macrophages, and
B cells), and following activation proliferate and mature into one of three T helper
subsets, Th1, Th2, or Th17, depending on the cytokine milieu. Similar to most
viruses, infection with MVEV induces a strong Th1 response, which is reflected
in the mouse in antibody isotype switching toward the biased production of IgG2a
and IgG2b antibodies, while antibodies of the IgG1 isotype are rare (Colombage
et al. 1998). While production of early IgM antibodies in flavivirus infection is T
cell-independent, the sustained antibody response essential for virus clearance and
immunological memory requires CD4+ T-cell help (Larena et al. 2011). Two early
studies have mapped the source of peptide determinants recognized in association
with MHC-II by MVEV-immune CD4+ T cells to the E protein and show cross-
reactivity of the responses with related flaviviruses (Mathews et al. 1991, 1992).
8.11.4 CD8+ T-Cell Immunity

CD8+ T cells destroy cells that they recognize as virally infected via two distinct
mechanisms: ligation of the death receptor, Fas, on the target cell by FasL on the
T cell or via exocytosis of specialized granules that contain the pore-forming
protein perforin and the pro-apoptotic granzymes (reviewed by Chowdhury and
Lieberman 2008; Voskoboinik, Smyth, and Trapani 2006). In addition, CD8+ T
cells secrete IFN-γ and tumour necrosis factor (TNF) α in response to cognate
antigen. Infection of mice with MVEV elicits functional, virus-specific, CD8+ T-cell
responses characterized by in vitro cytotoxicity and ex vivo IFN-γ production (Licon
Luna et al. 2002; Lobigs et al. 1994; Lobigs et al. 1997; Regner et al. 2001a,b,c).
However, the memory cytotoxic T-cell response against MVEV exhibits the unusual
feature that restimulation of MVEV-primed splenocytes with virus, after lengthy
periods following priming, triggers high lysis of uninfected target cells; in contrast,
re-stimulation with MVEV peptide-pulsed target cells boosts a potent virus-specific
response (Mullbacher et al. 2003; Regner et al. 2001c). Accordingly, memory MVEV-
immune CD8+ T cells are clearly generated as a consequence of a primary infection
or immunization with recombinant subunit vaccines; however, the protective value
of CD8+ T cells against secondary MVEV infection is probably relatively poor, given
the high self-reactive component among the memory cytotoxic T cells.
To investigate the contribution of CD8+ T cells to recovery from primary
infection in a mouse model of MVE, Licon Luna et al. compared the pathogenesis
and mortality of mice with genetic defects in either or both of the cytolytic pathways
relative to that of congenic wild-type mice, using low-dose, extraneural infection
with MVEV (Licon Luna et al. 2002). This study showed that the cytolytic effector
functions of CD8+ T cells facilitate invasion of MVEV into the brain and that MVEV-
immune CD8+ T cells are detrimental in disease progression: mice deficient in either
the granule-exocytosis or Fas-mediated pathway of cytotoxicity show delayed and
reduced mortality, while mice deficient in both pathways are resistant to infection.
While the mechanism that allows MVEV to breach the blood-brain-barrier remains
uncertain, the above finding suggests that CD8+ T-cell cytotoxicity may induce
local breakdown (by killing virus-infected vascular endothelial cells of capillaries
in the brain) and thereby facilitate virus entry into the CNS. CD8+ T cells also
increase pathology due to the inflammatory response in the infected brain. This is
reflected in prolonged survival of mice defective in both granule exocytosis- and
Fas-mediated pathways of cytotoxicity relative to immunocompetent mice, follow
ing peripheral infection with a high dose (108 PFU) of MVEV, which results in
uniform and rapid virus entry into the CNS in both mouse strains (Licon Luna et
al. 2002).
Regions in the viral polyprotein encompassing epitopes recognized in association
with MHC-I by mouse MVEV-immune CD8+ T cell have been mapped. Similar
to other flaviviruses, the determinants are almost exclusively derived from the
nonstructural proteins and are clustered in the region from NS3 to NS4B (Lobigs
et al. 1994; Lobigs et al. 1997; Regner et al. 2001c). Two immunodominant H-2K k-
restricted peptides (MVE1785: REHSGNEI and MVE1971: DEGEGRVI) have been
described (Lobigs et al. 1994; Regner et al. 2001c). The response against these
determinants is broadly flavivirus cross-reactive, and paradoxically recognizes
disparate epitopes from corresponding regions in NS3 from other flaviviruses but
ignores more similar peptides from “self ” and other virus families (Regner et al.
2001a). This suggests that primary sequence homology is not always the crucial
factor in peptide recognition in the cross-reactive cellular immune responses against
flaviviruses.
8.12 VACCINATION
There are no vaccines or antiviral agents available against MVEV and, given
the relatively small number of human cases of encephalitic disease, there is no
commercial interest for development of an MVEV-specific vaccine. However,
it should be anticipated that the incidence of MVE increases as a consequence
of ongoing industrial and agricultural development in northern Australia and
associated increase of the “at-risk” population in regions of seasonal MVEV activity.
Furthermore, it is unclear whether climate change will impact on the frequency and/
or severity of epidemic outbreaks of MVEV in southeastern and western Australia.
Consequently, a public health demand for vaccination against the virus may arise
and justify production of an MVEV-specific vaccine, most likely in public-private
partnership. Given the vast worldwide expertise in vaccine research and development
against the closely related JEV (reviewed by Beasley et al. 2008; Monath 2002), it is
almost certain that manufacture of an effective and safe MVEV vaccine is feasible
within a relatively short period of time. The induction of potent and durable memory
B cells that produce high-affinity, neutralizing antibody against the E protein should
be considered as the prime criterion for efficacy of a putative MVEV vaccine, based
on our understanding of the immunological correlates for protection against the virus
(Section 8.11). Several studies in mice illustrate that experimental vaccines encoding
the viral prM and E proteins, which are secreted in the form of highly immunogenic
subviral particles (Lobigs 1993), induce protective humoral immunity; these include
DNA-based, alphavirus-vectored and vaccinia virus-vectored delivery of the MVEV
structural proteins (Colombage et al. 1998; Hall et al. 1996; Kroeger and McMinn
2002; Lobigs et al. 2003c).
An alternative approach to vaccination against MVEV is the use of available
vaccines against JEV, which can protect against the former (reviewed in Lobigs and
Diamond 2012). It has been known for many years that at least in animal models, live
viral infection with one virus belonging to the JEV serocomplex will induce cross-
protective immunity against other members of the serocomplex (Fang and Reisen 2006;
Goverdhan et al. 1992; Hammon and Sather 1956; Tesh et al. 2002; Williams et al. 2001).
Consistent with this observation, preclinical vaccine trials in mice and horses have shown
potent cross-protective immunity against MVEV induced by live (ChimeriVax-JE) or
inactivated (Advax-ccJE) candidate vaccines against JEV (Lobigs et al. 2009, 2010b).
ChimeriVax-JE is constructed from yellow fever virus cDNA by replacement of the
prM-E proteins with those of an attenuated JEV strain and has undergone phase 2 and
phase 3 trials for safety and efficacy in humans (reviewed by Appaiahgari and Vrati
2010); Advax-ccJE is a cell-culture-grown JEV antigen formulated with a carbohydrate-
based adjuvant that potently stimulates vaccine immunogenicity without the increased
reactogenicity seen with other adjuvants (Petrovsky 2008).
Vaccine efficacy in terms of magnitude and/or quality of the vaccine-elicited immune
response is most likely the critical property of the novel JEV vaccines for successful
vaccination against MVEV. The first internationally licensed but recently discontinued
JE vaccine (JE-VAX; Biken Institute) does not efficiently cross-protect, and the poor
immunity induced with JE-VAX can result, under specific experimental conditions, in
infection-enhancement in mice following challenge with MVEV (Broom et al. 2000;
Lobigs et al. 2009, 2003c; Wallace et al. 2003). The phenomenon of antibody-mediated
enhancement of infection was discovered in studies using MVEV and related flaviviruses
(Hawkes 1964). The mechanism for this finding is thought to involve the enhanced uptake
into Fc receptor-bearing cells of virus, when bound to an antibody that fails to neutralize,
resulting in an increase in viral burden and disease severity. While the remote risk of
immune enhancement was a consideration against recommendation of the emergency
use of JE-VAX in the face of an epidemic of MVEV (Marshall 1988), the significantly
enhanced immunogenicity of adjuvanted and live, recombinant candidate JEV vaccines
(Lobigs et al. 2009, 2010b) strongly indicates that protection against multiple viruses
belonging to the serocomplex can be achieved with the one vaccine. An additional public
health consideration for the availability of JEV vaccines with cross-protective value
against MVEV in Australia is the ongoing threat of emergence of JEV on the Australian
mainland (Mackenzie et al. 2004). This scenario would necessitate extensive vaccination
against JEV, where the choice of vaccine should be guided by its immunogenic potency
to prevent the remote risk of immune-enhancement of infection with MVEV with the
benefit of protection against the endemic pathogen.
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9 Japanese Encephalitis
Virus and Human
CNS Infection
Kallol Dutta, Arshed Nazmi, and Anirban Basu
CONTENTS
9.1 A Brief History of Japanese Encephalitis...................................................... 193
9.1.1 Introduction....................................................................................... 193
9.1.2 Disease Vectors in JE........................................................................ 194
9.1.3 Enzootic Life Cycle of the JE Virus.................................................. 194
9.1.4 Origin, Spread, and Current Geographic Realm of JE..................... 195
9.1.5 Molecular Architecture of the JE Virus............................................ 195
9.2 Human Infections.......................................................................................... 197
9.2.1 Virus Transmission from the Periphery to the CNS.......................... 197
9.2.2 Neuropathology Associated with JEV Infections.............................. 198
9.2.3 Clinical Features of JE...................................................................... 199
9.2.4 Diagnosis, Prophylaxis, and Therapy................................................ 203
References...............................................................................................................204
9.1 A BRIEF HISTORY OF JAPANESE ENCEPHALITIS

9.1.1 Introduction
The antecedents of Japanese encephalitis (JE) can be traced back to the days of
the “Yoshiwara cold” (Miyake 1964) even though cases of summer-fall disease out-
breaks bearing symptomatic similarity to JE was reported from Japan since 1871.
The largest outbreak was reported in 1924 in which there were more than 6000
reported cases of which about 3600–4000 were fatal (Hiroyama 1962). The first
clinical isolate of the Japanese encephalitis virus was prepared in 1933 from human
brain tissue in rabbits (Hayashi 1934) and was initially referred to as Japanese “B”
encephalitis virus to indicate its association with the “B” or summer type of epidemic
encephalitis in Japan. Von Economo’s encephalitis lethargica, prevalent throughout
the world in the years immediately following World War I, was type “A,” which had
different clinical and epidemiologic characteristics (Rosen 1986).
193
9.1.2 Disease Vectors in JE
JE is maintained in nature by extra-human hosts. Human beings are incidental hosts
and are known not to play any role in perpetuating the virus. The role of mosquitoes
as vectors for this disease was suggested when the virus was isolated from Culex
tritaeniorhynchus in 1938. Since then, this virus has been isolated from several other
culicine mosquitoes such as Culex fuscocephala, Culex vishnui, Culex sitiens, Culex
annulirostris, Culex gelidus, Culex bitaeniorhynchus, Culex epidesmus, Culex
pseudovishnui, and Culex whitmorei, four species of anophelines Anopheles annu-
laris, Anopheles barbirostris, Anopheles hyrcanus, and Anopheles subpictus, and
five species of other mosquito genera Armigeres subalbatus, Mansonia annulifera,
Mansonia bonneael dives, Mansonia uniformis, and Aedes vigilax (Muangman et
al. 1972; Reid et al. 2006; Rosen 1986; Trosper et al. 1980; Vythilingam et al. 1994).
Even though JEV has been reported to effectively replicate in other arthropod hosts
when infected parentally (Hurlbut and Thomas 1969), isolation of the virus from
arthropods other than mosquito in nature has been reported only twice; the first case
was from midges, Lasiohelea taiwana, collected while biting humans in China (Wu
and Wu 1957), and the second case was from ixodid ticks, Haemaphysalis japonica,
in the erstwhile USSR (Lvov 1978).
9.1.3 Enzootic Life Cycle of the JE Virus

The virus is able to replicate within the salivary glands of mosquitoes. Mature JE
virions remain entrapped in intracellular vacuoles and are later released into the api-
cal cavity of salivary gland cells through the fusion of these vacuoles with the apical
plasma membrane. This process is associated with primary re-synthesis of saliva
in mosquitoes following blood feeding activity. Another type of shedding involves
virus particles, either singly or in mass, being released directly through the api-
cal plasma membrane (Takahashi and Suzuki 1979). Components of the mosquito
saliva may also modulate infection by altering the local cytokine milieu. Feeding
by mosquitoes of Culex sp or administration of sialokinin-I, a mosquito salivary
protein, has been found to down-regulate IFN-γ production and up-regulate the TH2
cytokines, IL-4 and IL-10 (Zeidner et al. 1999).
Large perennial lakes, swamps, and rice fields provide a wintering and staging
ground for several migratory waterfowl; such areas also favor breeding and sur-
vival of mosquitoes. Human infections are mainly spread by Culex tritaeniorrhyn-
chus, which breeds in pools of stagnant water such as rice paddy fields (Innis 1995).
Because the rice paddy is unavoidable, the majority of the population in rural Asia
has been infected with the virus by early adulthood (Solomon 2003). Wading ardeid
water birds, particularly the black-crowned night heron (Nycticorax nycticorax) and
the Asiatic cattle egret (Bubulcus ibis coromandus), and bats serve as virus reser-
voirs or maintenance hosts, but the virus regularly spills over into pigs, members of
the family of equidae (e.g., horses, donkeys), and humans. Interestingly, the Asiatic
cattle egret’s range dramatically expanded across Asia in the 19th century following
changing agricultural practices (Hancock and Kushlan 1984), which coincides with
the evolution and spread of the more recent JEV genotypes. Pigs are considered as
Japanese Encephalitis Virus and Human CNS Infection 195
the main amplifications hosts as viremia results with a high titer. Due to the close
proximity of pigs with human dwellings these animals are considered main com-
ponents in the transmission cycle with respect to human infection (Ghosh and Basu
2009). JEV infection in other domestic animals does not result in high viremia and
thus they are not expected to transmit the virus to humans.
9.1.4 Origin, Spread, and Current Geographic Realm of JE

Traditionally, Africa is considered to be the cradle of all emerging pathogens.
However, a closer look at the various genotypes of JEV (I, II, III, IV, and V) showed
that the virus could have evolved from the present day Indonesia-Malaysia region
(also known as the Malay Archipelago). This tropical climate and great diversity
of insect and vertebrate life may also facilitate the emergence and rapid evolution
of viruses (Solomon et al. 2003). This is supported by the fact that all the five geno-
types including the most divergent types IV and V, which are believed to represent
the oldest lineages, are found in this region. From there, the virus has spread to the
islands of Japan and subsequently spread over the entire eastern and south-east Asian
region. Currently, the JE endemic region extends from the islands of Japan in the east
to Pakistan, in the west (Igarashi et al. 1994) and parts of Russia (former USSR) in
the north (Grascenkov 1964) to northern parts of Australia in the south (Hanna et
al. 1996, 1999).
Analysis of the different strains of JEV isolated from different countries has sug-
gested shifts in circulating genotypes (Fulmali et al. 2011; Nga et al. 2004). This
may be possible due to several factors such as bird migration, new irrigation projects,
and increasing animal husbandry (Innis 1995; Pfeffer and Dobler 2010; van den
Hurk et al. 2009). Wind-blown mosquitoes caught in air currents during the typhoon
season have been suggested to play a role in viral transmission from one country to
another (Mackenzie et al. 2001; Ritchie and Rochester 2001). This has been found to
be the case for the introduction of JEV to the Torres Strait region of Australia from
Papua New Guinea (Hanna et al. 1996). Due to these reasons, it is possible that in the
not so distant future, the geographic realm of JE may expand from its current state
and may even cross the Pacific to enter the United States (Nett et al. 2009). A recent
serosurvey from various bird species in Spain has confirmed the presence of viruses
belonging to the JEV antigenic group (Garcia-Bocanegra et al. 2010). This shows
that parts of the European continent can also come under the radar of this disease.
9.1.5 Molecular Architecture of the JE Virus

The JE virion consists of a single strand of positive-sense RNA of around 11 kb,
inside a nucleocapsid, and is surrounded by a glycoprotein-containing envelope. The
RNA comprises a short 5′ untranslated region (UTR), a longer 3ʹ UTR, and with a
single open reading frame between them. It codes for a single polyprotein, which
is translationally and posttranslationally cleaved by viral and host proteases into
three structural proteins (core-C, premembrane-PrM, and envelope-E), and seven
nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).
The C protein (12–14 kDa) is highly basic and combines with the RNA to form
the nucleocapsid (Chang et al. 1999). The prM is closely associated with the E pro-
tein, forming a heterodimer, and is thought to act as a “chaperone” to it, impairing
its function until after virion release. Immediately prior to virion release, the prM
protein (18–19 kDa) is cleaved to its mature M protein (8–9 kDa) form (Figure 9.1).
This allows the formation of E protein homodimers, which are thus “activated.” The
prM protein of JEV contains a single N-linked glycosylation site, which is highly
conserved among the JEV strains. Researchers indicated that this highly conserved
N glycosylation motif in prM is crucial for multiple stages of JEV biology; prM bio-
genesis, virus release, and pathogenesis (Kim et al. 2008). Depending on about 12%
genetic divergence in the C-prM region, the virus is classified into the four genotypes
(Chen et al. 1990). The E protein is the largest structural protein (53–55 kDa), with
up to two potential gylcosylation sites. It is the major target for the humoral immune
response, and is thought to be important for viral entry into host cells. It is worth
mentioning that low pH is extremely important for viral entry into the cell to trig-
ger viral membrane fusion with host endosomal membrane, thereby releasing the
nucleoplasmid in the cytosol.
~10.8 kb
5ĆAP (I)
7mGpppAm Open reading frame OH
5ŃCR 3ŃCR
5ĆS
3´SL
RCS2 CS2 CS1
~100 nt
~400–700 nt
Structural genes Nonstructural genes
–
~3400 aa
NS2B NS4A
NH3 PROT HEL MTase RdRP COOH
C prM E NS1 NS2A NS3 NS4B NS5
PROT
pr M 2Aα Cleaved NS3
FIGURE 9.1 JEV genome structure and expression. The viral genome is depicted with the
structural and nonstructural protein coding regions, the 5′ cap, open reading frame and the
5′ and 3′ noncoding regions (NCR). Models of functionally important secondary and tertiary
structures within the 5′ and 3′ NCR and the coding region are shown with predicted hairpin
loops. Boxes below the genome indicate precursors and mature proteins generated by the
proteolytic processing cascade. Structural proteins are in grayscale, whereas nonstructural
(NS) proteins are white. The proposed topology of the flavivirus polyprotein cleavage prod-
ucts is also depicted. The proteins are arranged in order (left to right) of their appearance in
the polyprotein.
9.2 HUMAN INFECTIONS

Owing to the highly effective neuroinvasive nature of the JEV, it has been difficult to
study the virus peripherally in clinical cases, as a result of which a lot remains unan-
swered. How the virus reaches the central nervous system (CNS) following periph-
eral inoculation or how it evades the hosts’ immune system still remains enigmatic.
Of the nearly 10,000 cases that are fatal out of 30,000–50,000 reported cases of JE
per year, most are children belonging to age groups younger than 15 years. Adults
living in the epidemic zones are not prone to be symptomatic for this disease; how-
ever, it is reported that they may become so in cases of spread to virgin territories.
Although human beings are considered as incidental dead-end hosts of the JEV,
mother to child transmission of the virus has been reported in the past (Chaturvedi
et al. 1980; Mathur et al. 1981). However, the clinical impact of such could not be
established. JEV persists in the human brain for 8 to 15 years after the onset of
encephalitis (Shiraki 1970); however, whether it reactivates during immunosuppres-
sion or has any long-term neuropathological effect is unknown.
Neurovirulence of the JEV involves a combination of infection and dysfunction of
neurons, caused by direct viral damage, and indirect damage mediated by the genera-
tion of an inflammatory milieu in the CNS, and other mechanisms such as apoptosis.
In the following sections, we shall try to link available laboratory research reports
along with clinical case studies so as to gain a better understanding of this disease.
9.2.1 Virus Transmission from the Periphery to the CNS

Human infections of Japanese encephalitis are common throughout the endemic region.
Despite the enormity of the disease, not enough is known about the neuropathogen-
esis of this disease, including the exact mechanism of spread to the CNS (Myint et al.
2007). However studies regarding other flaviviral infections or in vitro studies have
shown that following intracutaneous inoculation by mosquito bite, the virus enters the
Langerhan’s dendritic cells in the skin, which carry the virus into the nearest draining
lymph nodes (Johnston et al. 2000). From there it is carried out into the general circu-
lation via the thoracic duct, where the virus probably infects cells of myeloid lineage.
From the general circulation, the JEV is hypothesized to enter the CNS, but the exact
mechanism/s is yet to be elucidated. There are three possible mechanisms by which
the virus is expected to enter the CNS by crossing the blood brain barrier (BBB)—
passive transport across the endothelium, by active replication in endothelial cells or
by a “Trojan horse” mechanism in which the virus is carried into the brain by infected
peripheral inflammatory cells (Diamond 2003). Investigations in mice models (Dutta
et al. 2010a) and study of human autopsy samples (Miyake 1964; Mukherji and Biswas
1976) have confirmed that the JEV infects and is able to replicate in peripheral organs
such as lymph nodes, spleen, kidney, and lungs before crossing the BBB. Hematogenous
spread of the virus from the periphery to the CNS is supported by the observation that
in intranasally JEV inoculated monkeys, virus replication was widespread in the CNS,
but not always identified in the olfactory bulb (Myint et al. 1999). The hematogenous
route is also supported by observations that led to isolation of the virus from blood clots
collected during the acute phase of infection (Sapkal et al. 2007).
Perivascular cuffing is a common occurrence in human infections of JEV. This

leads to invasion of peripheral inflammatory cells into the CNS parenchyma.
Inflammatory cells invading the parenchyma are shown to be predominantly macro
phages with small numbers of T cells (Johnson et al. 1985), though the role of these
cells in transporting the virus from periphery into the CNS remains ambiguous.
Some in vitro studies have reported that JEV is capable of surviving within cells of
monocyte/macrophage lineage for prolonged time periods (Aleyas et al. 2009; Dutta
et al. 2010b). Although human data are lacking, studies in the mouse model of JE
has shown that there is up-regulation of the cellular adhesion molecules ICAM and
VCAM in the brain (Mishra et al. 2009a), which may be important in initiating adhe-
sion and migration of neutrophils and macrophages. This was also associated with
elevated levels of MMP-9 in the brain which could also contribute to increased BBB
permeability. Taken together, these findings could indicate a “Trojan horse” role of
these cells at a later stage of infection.
The disruptive role of cytokines on BBB has also been reported. In a mice model,
macrophage-derived neutrophil chemotactic factor was shown to alter the BBB per
meability in a dose dependent manner (Mathur et al. 1992). Tumor necrosis factor α
(TNF-α) and interleukin 8, which is involved in polymorphonuclear cell recruitment,
have also been reported to be elevated in cerebrospinal fluid (CSF) and serum of humans
with JE and are higher in fatal than nonfatal cases (Ravi et al. 1997; Singh et al. 2000).
How the virus actually gains entry to infect the neurons is not well understood.
JEV is believed to enter target cells through receptor-mediated endocytosis involv-
ing both clathrin-dependent and caveola-dependent pathways, low pH-triggered
membrane fusion and then replicate in intracellular membrane structures (Nawa et
al. 2003). The host membrane may play critical roles in various stages of the viral life
cycle—from entry, replication, assembly, and egress of the virus particles. A recent
study has claimed the involvement of host membrane lipid rafts in JEV entry and life
cycle into neural stem/progenitor cells (Das et al. 2010).
9.2.2 Neuropathology Associated with JEV Infections

JEV causes neuronal death. Time and again; various investigations involving human
autopsy samples or animal models or even in vitro studies with neuronal cell lines,
have shown conclusively that JEV infection leads to massive neuronal loss. Human
autopsy studies as early as 1933 identified severe damage to nerve cells and the brain
parenchyma including minute necroses, softening, and perivascular cuffing. The
majority of the lesions were observed in the diencephalon and the mesencephalon.
The overall inflammatory changes in the CNS were identified with marked increase
in the number of glial cells. This led early investigators to believe that Japanese
encephalitis was a “generalized toxic inflammation.” However, it should be kept in
perspective that this was before the isolation of the causative agent for the disease,
i.e., the JEV. Later, histopathological studies on autopsy samples by Drs. M. Miyake
and T. Ogota concluded that the disease was associated with “perivascular cell infil-
tration or cuffing, glial proliferation, glial nodules, chromolysis of Nissl’s bodies,
neuronophagia, haemorrhages, necrosis, softening and calcification” in the CNS
(Miyake 1964). These observations were confirmed from investigations of several
autopsy samples in the early 1960s. It was observed that these changes were scat-
tered widely from the cerebrum, cerebellum, and brain stem to the spinal cord and
most prominently in the cerebral cortex, thalamus, and substantia nigra.
JEV infection has been reported to initiate apoptotic death in neurons. The tumor
necrosis factor receptor (TNFR)-associated death domain (TRADD) has been sug-
gested to be the crucial signal adaptor that mediates all intracellular responses from
TNFR-1. Using an in vitro approach it has been shown that the altered expression
of TNFR-1 and TRADD following JEV infection regulates the downstream apop-
totic cascades (Swarup et al. 2007, 2008). However, even though the infected neu-
rons eventually die, recent evidences suggests that a possible intracellular innate
immune response against the virus is mounted following viral recognition through
the retinoic-acid-inducible gene I (RIG-I) (Nazmi et al. 2011).
Even in the early days of investigations it was known that this disease was accompa-
nied with inflammation in the CNS. The CNS is a unique organ where the movement
of cells or molecules is restricted by the BBB. Even though immune cells from periph-
ery do infiltrate into the CNS at different stages of the disease, the initial inflammation
is due to the activity of resident immune cells. The microglia and the astrocytes have
been reported to play extensive roles following JEV infection. In animal models as
well as in vitro models of JE, it has been reported that there is microglial activation
characterized by distinct morphological changes along with heightened release of pro-
inflammatory cyto/chemokines such as TNF-α, IL-6, MCP-1, IFN-γ, and IL-1β and
other mediators. A region specific analysis showed that these releases were the highest
from the hippocampus region (Ghoshal et al. 2007). This inflammatory milieu in the
brain has a severe detrimental effect on neurons, leading to their death. Neuronal death
also acts as a stimulator for further microglial activation, thereby creating a vicious
cycle. Even though it is difficult to ascertain the extent of direct viral killing or the
‘bystander’ death, the net effect of JEV infection remains neuronal death. Astrocytes,
on the other hand, also respond to the infection by increasing cytokine production,
lactic acid release, and glucose mobilization (Chen et al. 2000) even though it does not
confer significant neuroprotection (Mishra et al. 2007).
9.2.3 Clinical Features of JE

Infection with JE virus (JEV) may be asymptomatic or manifest as a mild febrile ill-
ness, aseptic meningitis, or classic severe meningomyeloencephalitis. The case fatal-
ity rate is approximately 25%, with 50% having neuropsychiatric sequelae and 25%
recovering fully. Long term sequelae in survivors include weakness, ataxia, trem-
ors, athetoid movements, paralysis, memory loss, and abnormal emotional behavior
(Simpson and Meiklejohn 1947).
Based on the clinical observations of development and progression of this disease,
it can be conveniently divided into three stages:
1. A prodromal stage preceding signs of involvement of the CNS.

2. An acute encephalitic stage marked by CNS signs and continuing fever.
3. Late stage marked by recovery or the persistence of cognitive dysfunction
as a result of irreversible CNS damage.
The essential features of the prodromal stage are general malaise, headache, and
fever. The onset of the illness is usually acute and is heralded with fever. Headache
is often accompanied by vomiting. However, these symptoms are common to vari-
ous other diseases that are not even related to flaviviral infections. Thus, a clinical
diagnosis at this stage is absolutely impossible. A good example is the characteriza-
tion of some U.S. military personnel serving during the Korean conflict, who were
suffering from war neurosis when they were actually infected with JEV (Solomon and
Vaughn 2002). The onset of this stage may be abrupt (1–6 h), acute (6–24 h), or sub-
acute (2–5 days). In more than 75% of patients, the onset is subacute. Although sponta
neous recovery (the so-called abortive encephalitis) is known following this stage, the
disease usually progresses to the acute encephalitis phase (Gourie-Devi et al. 1995).
The acute encephalitis stage is marked by continuous fever, nuchal rigidity, convul-
sions, and altered sensorium, progressing in many cases to coma, focal CNS signs,
polymorphonuclear leucocytosis in the peripheral blood, and CSF changes marked
by pleocytosis with a normal or raised glucose or protein content. Seizures occur
in approximately 85% of children and 10% of adults with JE (Kumar et al. 1990).
Continuous unremitting seizure lasting longer than 30 minutes (status epilepticus) or
multiple recurrent seizures are common in JE. Also, subtle motor status epilepticus,
in which the only clinical manifestation might be the twitching of a finger or eyebrow,
is important in JE (Solomon et al. 2002). Approximately 50% of the patients with JE
suffer from high CSF opening pressure. Brain swelling is a common feature that is
observed during autopsy, although herniation is not reported (Johnson et al. 1985).
Multiple uncontrolled seizures may be associated with this raised intracranial pressure.
Movement disorders are common in JE, both in the acute encephalitis stages
and also in survivors with neuropsychiatric sequelae. The characteristic features
include mask-like faces, abulia, tremors, and cogwheel rigidity that bear similarity
to Parkinson’s disease. Other movement disorders include generalized rigidity, jaw
dystonias, opisthotonus, choreoathetosis, orofacial dyskinesias (involuntary tongue
protrusions), oromandibular dystonia, myoclonic jerks, and opsoclonus myoclonus
(Kalita et al. 2011; Misra and Kalita 1997b). These clinical features grossly cor-
relate with changes observed by MRI scans of patients. The role of basal ganglia,
particularly the thalamus and the substantia nigra, have long been considered to be
significant in eliciting such responses. MRI reveals prominent changes in thalamus,
basal ganglia, substantia nigra, cerebellum, pons, cerebral cortex, and spinal cord.
These MRI lesions are generally hypointense on T1 and hyperintense on T2 and
fluid attenuation inversion recovery (FLAIR) sequence. The thalamic lesions may
be of mixed intensity on T1 and T2 in the subacute stage and may suggest hemor-
rhagic changes (Figure 9.2). Follow-up MRI after several months reveals shrinkage
of acute lesions which are hypointense on T1 and T2 sequences (Misra and Kalita
2010). In a comparative study of CT and MRI, the CT scan was abnormal in 55.3%;
MRI was abnormal in all the patients and revealed thalamic lesions in 94%, basal
ganglia in 35%, midbrain in 58%, pons in 26%, and cerebellum and cerebral cortex
in 19% each (Kalita and Misra 2000a,b). In JE, involvement of the temporal lobe
has also been reported in approximately 17% of the patients, but all of them had tha-
lamic and substantia nigra involvement (Handique et al. 2006). Diffusion-weighted
brain magnetic resonance imaging demonstrated abnormal high intensity lesions
(a) (b)
Fp1–A1
F7–A1
T3–A1
T5–A1
O1–A1
Fp2–A2
F8–A2
T4–A2
T6–A2
100 μV TC = 0.3 s
HF = 70 HZ
1 sec
(c) (d) (e)
FIGURE 9.2 Representative EEG and MRI patterns observed in JE. Typical electroencepha
lographic (EEG) and MRI patterns observed from JE patients are shown here. EEG of a
patient with secondary generalized seizure shows epileptiform (spike and wave) discharges
mainly on the left side (a). T1 sequence of cranial MRI of the same patient shows hemorrhagic
lesions on the left frontoparietal and bilateral thalami (b). Characteristic cranial MRI changes
on T2 sequence showing bilateral thalamic lesion (c), thalamic and basal ganglia lesions (d),
and substantial nigra (e) involvement on the right side are observed. Bilateral hyperintense
thalamic lesion are seen in T1 sequence (f) which are also hyperintense in T2 sequence (g)
suggesting subacute hemorrhage. (Reprinted from Prog. Neurobiol. 91(2), Misra, U. K. and
Kalita, J., Overview: Japanese encephalitis, pp. 108–20. Copyright (2010), with permission
from Elsevier.)
in the bilateral pulvinar and gray matter, with an abnormal appearance mimicking
pulvinar sign (Toshio et al. 2011). Single photon emission computed tomography
(SPECT) analysis of JE patients show thalamic hyperperfusion in the acute stage,
which is replaced by hypoperfusion in the subacute or chronic stage (Kalita et al.
1999; Kimura et al. 1997). EEG recordings during the acute stage were found to be
grossly abnormal. The outstanding features are diminution of electrical activity, dys-
rhythmia, and slowing with periodic lateralized epileptiform discharges (PLEDS).
(f ) (g)
T1 T2
FIGURE 9.2 (Continued)
In some patients, intention tremors and ataxia that are indicative of the cerebel-
lar involvement are observed. Other focal neurological signs include cranial nerve
palsies, upper motor neuron weakness (in 30%–50% of patients), and flaccid limb
weakness, with reduced or absent reflexes, which is often associated with respira-
tory or bulbar paralysis (Misra and Kalita 1997a). This disease is also referred to as
encephalomyelitis. The combination of upper and lower motor neuron damage can
lead to bizarre mixtures of clinical signs that can change hourly during the acute
stage (Solomon et al. 2007). JEV can also cause a poliomyelitis-like acute flaccid
paralysis in fully conscious patients. Acute retention of urine, due to an atonic blad-
der, may be an early clue that paralysis is due to a flavivirus (Solomon et al. 1998).
The late stage of the disease begins when active inflammation is at an end, i.e.,
when body temperature is normal and the neurological signs are stationary or tend-
ing to improve. When the encephalitic stage is short, recovery occurs rapidly and
the patient becomes normal within 2–4 weeks of the onset of illness. However, a
prolonged encephalitic stage corresponded to slower recovery or prolonged sequel
to the disease. The neuropsychiatric problems in the survivors (in about 50% of
cases) include learning and memory deficits, behavioral abnormalities, and speech
disorders. The cellular or molecular basis of the persistence of these changes is not
well understood. JEV predominantly infects children who are in a dynamic state of
brain development, and so insult on the CNS may have consequences later in life.
Since JEV infection leads to massive neuronal death, effective CNS repair processes
which restore the neuronal loss are imperative for complete recovery from JE. In
the postnatal/adult CNS, neuronal regeneration is primarily dependent on the pool
of neural stem/progenitor cells (NSPCs) and their ability to generate cells of both
neuronal and astrocyctic lineage. It is hypothesized that JEV infection and the asso-
ciated inflammation disrupt the NSPC pool in the germinal niches and their efficacy
of generating functional neurons, thereby stalling the neuronal repair. The lack of
functional CNS repair/regeneration possibly culminates in long-term neurological
consequences in JE survivors. In animal models and in vitro models of JE it has
been shown that NSPCs are permissive to infection, which leads to their growth
retardation. The pathophysiological relevance of these observations was supported

by profound decrement in actively proliferating NSPCs in the subventricular zone
(SVZ) of JEV-infected animals. Infection of the NSPCs and suppression of their pro-
liferation might be primarily responsible for dysregulated neurogenesis and develop-
ment of cognitive deficits in survivors of JE (Das and Basu 2008; Das et al. 2009).
Most of the symptoms associated with JE are also common to various other dis-
eases. Due to the close genetic similarity of flaviviruses, some or most of these clini-
cal characteristics are common to all human infections, thereby making differential
diagnosis difficult. Other than flaviviral infections, encephalitis due to viral or non-
viral reasons may also be characterized with some of these features. JE has also been
associated with other diseases. Cysticercosis is a risk factor for JE that is attributed
to the disruption of the BBB (Desai et al. 1997; Liu et al. 1957). JEV infection may
also predispose patients to Guillain-Barré syndrome in endemic areas (Ravi et al. 1994).
9.2.4 Diagnosis, Prophylaxis, and Therapy

The diagnosis of the disease is currently based on immunological detection of the
viral antigen or the detection of viral RNA or particles from patient samples. The
low viremia in blood has always been a problem in rapid diagnosis of JEV. IgM
capture ELISA had been the most widely used diagnostic method for JEV infection
detection, but the kit is expensive and not suitable for low-cost, large-scale diag-
nosis in rural conditions for its short shelf life. A rapid IgM capture ELISA called
JEV-Chex was developed having more stable reagents useful under rural conditions
(Ravi et al. 2006). A comparative study carried out between IgM capture ELISA and
nested reverse transcription-polymerase chain reaction (RT-PCR) for the diagnosis
of Japanese encephalitis from the samples of CSF and blood revealed that RT-PCR
is also useful for an early detection of JEV (Swami et al. 2008). At present, though
many advancements and modifications have been done on the ELISA methods, such
as introduction of the dipstick method (Shrivastva et al. 2008), but going by the
principle of detection, IgM capture ELISA remains the very principle in clinical and
laboratory confirmatory diagnosis of JEV, but of course a positive detection from a
gene amplification analysis reinforces the confirmation.
A sensitive quantitative assay for JEV RNA has been reportedly developed using
real-time RT-PCR. The assay was performed using LightCycler and RNA ampli-
fication kit SYBR Green I by selecting the JEV specific primer from the 3′UTR.
On comparing results obtained by real-time RT-PCR assay for JEV and infectivity
titrations, it was suggested that the real-time RT-PCR assay could have an additive
effect on the interpretation and evaluation of virus clearance, especially during the
virus removal process (Jeong et al. 2003). Again, a one step TaqMan RT-PCR using
a TaqMan probe has also been developed for detection of JEV, where it was shown
to be 10-fold more sensitive than the conventional two-step RT-PCR method (Yang
et al. 2004).
Therapy for JE is entirely supportive and currently there are no specific drugs
to target the virus. Over the years, various drugs—natural or synthetic—have been
tried out either singly or in combination with other compounds with limited or no
success. Even compounds that had shown promising results in vitro or in animal
models have failed in human trials. Interferon alpha and ribavirin are two such com-
pounds. Details about these drugs and their mode of action can be found in a review
by these authors (Dutta et al. 2011). The latest and most promising candidate for
therapy in JE is minocycline, a second generation tetracycline. Minocycline has been
found to be highly effective in preventing animal mortality following acute chal-
lenge with the virus (Mishra and Basu 2008; Mishra et al. 2009a,b) and is slated for
a randomized double blind phase II clinical trial.
Due to lack of definitive therapeutic countermeasures to combat JE, vaccination in
humans remains, to date, the most effective measure to prevent JE. Multiple vaccines
exist to control JE, but all have limitations. The formalin-inactivated vaccine against
JEV was produced from infected mouse brain-derived tissue soon after the virus
was discovered. This type of vaccine, manufactured by the Research Foundation
for Microbial Diseases of Osaka University, Japan, became commercially available
in Japan (as the Japanese Biken vaccine JE-VAX) and was produced in Korea by
the Green Cross Vaccine company. These were later licensed to be produced also
in the United States (Solomon 2008). This is the only JE vaccine recommended by
the World Health Organization (WHO), but there have been several concerns with
its side effects (Shlim and Solomon 2002). These vaccines are expensive and require
multiple doses to maintain efficacy and immunity. An inexpensive, live attenuated
vaccine (SA14-14-2) 48 was licensed by China in 1988, but WHO does not approve
it for human use because it is produced in primary hamster kidney cells. Although
the vaccine was adjudged to be safe and efficacious by the WHO’s Global Advisory
Committee on Vaccine Safety, several parameters such as safety in immunocom-
promised individuals and pregnant women, viral shedding in vaccines and implica-
tions of the shedding, and efficacy in infants younger than 1 year old, remains to be
ascertained (Dutta et al. 2011). The recently developed Vero cell-derived inactivated
JE vaccine containing the purified, inactivated JEV strain SA14-14-2 with aluminum
hydroxide as adjuvant seems to be a promising candidate and has passed the phase
III randomized controlled trial (Tauber et al. 2007). Several efforts have been and
are still being made to develop recombinant vaccines for JE, with some of them in
preclinical or various phases of clinical trial.
Even though vaccination is effective and has considerably lowered the incidence
of JE in many endemic regions, reports are available of their ineffectiveness in
some cases. A young adult man, who received four doses of JEV (Nakayama strain)
vaccination in childhood, reportedly developed acute JEV infection that was char-
acterized with acute flaccid paralysis. His deep tendon reflexes were decreased
except for the Achilles reflex. Following supportive care, one month after his dis-
charge, his muscle power level and deep tendon reflexes recovered partially (Chung
et al. 2007).
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10 Tick-Borne Encephalitis
Daniel Růžek, Bartosz Bilski, and Göran Günther
CONTENTS
10.1 Introduction................................................................................................... 211
10.2 Biological Properties..................................................................................... 212
10.3 Microevolution of TBEV............................................................................... 216
10.4 Ecology and Epidemiology............................................................................ 217
10.5 Clinical Features............................................................................................ 221
10.6 Pathology and Pathogenesis...........................................................................224
10.7 Diagnosis....................................................................................................... 228
Acknowledgements................................................................................................. 229
References............................................................................................................... 229
10.1 INTRODUCTION
Tick-borne encephalitis is recognized from 18th-century parish records in Åland
Islands in Finland. A virus, as the causative agent, was first isolated in 1937 by Zilber
and collaborators in the Far Eastern Soviet Union (Zilber 1939). In 1937–1939, the
Russian Ministry of Health organized three successive expeditions to the Far East,
with the purpose to reveal the origin of severe outbreaks of meningoencephalitis,
called “Taiga encephalitis” or “biphasic minogoencehalitis,” a disease that had been
observed in the Far East since 1914, but more frequently had occurred since 1933.
The expeditions revealed the viral origin of the disease and the tick Ixodes persulca-
tus as the main vector of the disease (Zilber 1939; Chumakov and Zeitlenok 1940).
In the European part of Russia (in Ural), the disease seems to have been known
since 1898. However, the well-documented history of TBE on the European conti-
nent starts with an outbreak in the Volkhov Front’s armies in 1942–1943. During that
time, the TBE virus of the Siberian subtype was isolated from Ixodes ricinus ticks
(Petrishcheva and Levkovich 1945). In 1943, the TBE virus of the western type was
isolated for the first time from human patients and I. ricinus ticks by Zilber and col-
laborators in Belarus (Pogodina et al. 2004).
Tick-borne encephalitis in Central Europe was first described by Austrian physi-
cian Schneider, who had studied clinical cases of TBE in the area of Neunkirchen
(Lower Austria) since 1927, but without knowing its etiology (Schneider 1931). In
1957, Moritsch and Krausler isolated the TBE virus in Neunkirchen and proved that
the so-called Schneider’s disease represented in fact TBE (Moritsch and Krausler
1957). However, the first successful isolation of the TBE virus in the central and
western part of Europe was performed in Czechoslovakia in 1948 by Gallia and
coworkers (Rampas and Gallia 1949), when local outbreaks of meningoencephalitis
211
occurred in several regions in Bohemia and Moravia (Hloucal 1949, 1960; Hloucal
and Gallia 1949; Hloucal and Rampas 1953; Krejčí 1949a). Virus isolated from
patient samples was pathogenic for white mice and was filterable through Seitz
and Chamberland filters. In the same year, Krejčí, with assistance from Gallia and
Blaškovič, isolated the virus during an outbreak of meningoencephalitis in South
Moravia (Krejčí 1949b). Gallia, Edward, and others demonstrated a close similar-
ity of the new virus with Russian spring–summer encephalitis virus and louping-ill
virus. Approximately 80% of the TBE patients recorded a tick bite before the illness,
suggesting the role of ticks as vectors of the virus. This was confirmed in 1949 by
Rampas and Gallia and subsequently by Krejčí, when the virus was successfully
isolated from different stages of ticks Ixodes ricinus collected in the endemic areas.
Shortly after the isolation of the TBE virus in Czechoslovakia, the virus was
isolated in Hungary, Poland, Bulgaria, Yugoslavia (Slovenia) (Bedjanič et al. 1955;
Vesenjak-Zmijanac et al. 1955), Austria, Romania, and Germany, but also in Finland,
Sweden, Northern China, and Japan.
There are several synonyms known for TBE, based on historical descriptions of
TBE in different countries. These include “Taiga encephalitis,” “Kumlinge disease,”
“Central European encephalitis,” “Czechoslovak encephalitis,” “Russian spring–
summer encephalitis,” “Far East Russian encephalitis,” “Biphasic milk fever,”
“Biundulating meningoencephalitis,” etc.
TBE after drinking raw goat milk was first reported in the Leningrad region in
Russia in 1948 (Smorodintsev et al. 1953). This so-called biphasic miningoencepha-
litis was characterized with a milder clinical course and, unlike the classical form of
TBE, a family character of the disease. Initially, this illness was considered to be a
different disease from TBE. The same disease occurring after drinking raw milk was
observed in the Moscow region in 1951, and was called milk fever (Drozdov 1959).
In the same year, a large outbreak of milk-borne TBE was reported in Slovakia,
when at least 660 people become infected, and 271 of those were hospitalized.
During this outbreak, extensive scientific investigation under supervision by Raška,
Bárdoš, and Blaškovič was performed, and the etiology and mode of transmission
(i.e., by goat milk) was successfully revealed (Blaškovič 1954). Transmission of TBE
by milk of infected dairy animals or by milk products (yogurt, cheese) was experi-
mentally confirmed by Grešíková (Grešíková 1957, 1958). Historically, most of the
pioneer research work on TBE has been done in Russia and the Czech Republic
(Czechoslovakia).
10.2 BIOLOGICAL PROPERTIES

TBEV is the most medically important member of the tick-borne serocomplex group
of the genus Flavivirus, family Flaviviridae. The TBE serocomplex includes Langat
virus, louping-ill virus, Negishi virus, Kyasanur Forest disease virus, Omsk hemor-
rhagic fever virus, Powassan virus, and others. TBE virus is subdivided into three
antigenic subtypes corresponding to three genotypes: European (previously Central
European encephalitis; with Neudoerfl strain as prototype), Far Eastern (previously
Russian spring–summer encephalitis; prototype strain Sofjin), and Siberian (previ-
ously Western Siberian encephalitis; prototype strains Zausaev and Vasilchenko)
Tick-Borne Encephalitis 213
(Ecker et al. 1999). However, based on antigenic properties, the European TBEV
strains are more closely related rather to Louping ill virus than to the Far Eastern and
Siberian strains (Hubálek et al. 1995).
Recently, a new taxonomic scheme based on the comparison of the complete cod-
ing sequences of all recognized tick-borne flavivirus species has been proposed. This
suggests the assignment of TBEV and Louping ill virus to a unique species (TBEV)
including four viral types (i.e., Western Tick-borne encephalitis virus, Eastern Tick-
borne encephalitis virus, Turkish sheep Tick-borne encephalitis virus, and Louping
ill Tick-borne encephalitis virus) (Grard et al. 2007). However, this classification has
not been approved by the International Committee for Taxomony of Viruses yet, and
it is not widely accepted since it combines viruses with different biological charac-
teristics into one species.
More recently, a comparison of higher number of TBEV isolates from Siberia
resulted in identification of two separate groups of TBEV strains, which meet the
criteria to be classified as new genotypes. These new genotypes are phylogeneti-
cally different from the three previously established TBEV genotypes. One of the
new possible genotypes is represented by only one isolate, 178–79, originated from
the Irkutsk region, Russia. The second new genotype is tentatively named as “group
866” and is represented by 10 isolates (Zlobin et al. 2001; Tkachev et al. 2011). In
summary, the most up-to-date taxonomical study indicates TBEV species divided
into six subtypes/genotypes. The differences of biological properties of the members
of the newly described genotypes in comparison with strains from the previously
established genotypes are currently under investigation.
The TBEV virions are spherical, lipid-enveloped particles, approximately
50–60 nm in diameter (Slávik et al. 1967). The genome consists of a linear posi-
tive single-stranded RNA molecule, which is deposited in a capsid formed by C
(capsid) protein. Two virus proteins are integrated in the viral envelope, namely E
(envelope; Mr 55,000) and M (membrane; Mr 8000) proteins. Viral RNA consists
of one open reading frame (ORF), which is flanked by untranslated (noncoding)
regions (UTRs). The 5′UTR contains a type 1 cap (m7GpppAmG), followed by a
conserved stem-loop structure. The 3′UTR is not polyadenylated and is character-
ized by extensive length and sequential heterogeneity (Wallner et al. 1995). This part
of the viral genome can be divided into two parts: a proximal (localized behind the
“stop” codon of the open reading frame) and a distal (“core,” the 3′ terminus itself).
The distal part of this region (approximately 340 nt) is highly conserved, while the
proximal part is a noticeably variable segment with common deletions and insertions
(Proutski et al. 1997; Gritsun et al. 1997). The untranslated regions form secondary
stem-loop structures that probably serve as cis-acting elements for genome replica-
tion, translation and/or packaging (Gritsun et al. 1997; Proutski et al. 1997a,b). The
ORF encodes one large polyprotein, which is co- and post-translationally cleaved by
viral and cellular proteases into three structural proteins (C, prM, and E) and seven
nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).
C protein is a relatively small basic protein with lower sequence homology
between different flaviviruses (Lindenbach and Rice 2003). The carboxyl terminus
of C protein serves as an internal signal sequence leading the structural protein prM
into the membrane of endoplasmic reticulum (ER). The viral protease NS2B-NS3
cleaves this signal sequence, releasing the N-terminus of prM protein (Kofler et al.
2002). prM protein is a glycosylated precursor of the membrane protein M. The prM
protein shows a chaperon-like activity during the envelope protein E folding (Lorenz
2002; Lorenz et al. 2002).
The E protein is the site of the major viral antigens and the main target for neutral-
izing antibodies (although antibodies directed by prM/M and NS1 also provide some
protection). Moreover, it is responsible for specific binding to a cellular receptor and
penetration of the virus into the host cell. It is supposed to be a main determinant of
the virulence (Gritsun et al. 1995). Three-dimensional structure of the protein E was
studied at the resolution of 2.0 Å by x-ray crystallography (Rey et al. 1995).
The protein forms two monomers anchored in the membrane by their distal parts
at physiological pH. After virus uptake by receptor-mediated endocytosis into the
host cell, acidic pH in endosomes triggers irreversible changes in the E protein struc-
ture including its re-arrangement to trimeric forms and this leads to the initiation
of the fusion process between the viral and endosomal membrane (Holzmann et al.
1995). Conserved histidines in the E protein function as molecular switches and, by
their protonation at acidic pH, control the fusion process (Fritz et al. 2008).
Each E protein monomer is composed of three domains (I–III). Domain I is
located in the central part of the protein. It is formed by eight antiparallel beta sheets,
contains the N-terminus of the protein, two disulfide bridges, and an N-glycosylation
site.
Domain II is formed by two long loops that extend out of domain I and form a
finger-like structure. The domain contains a number of beta sheets and three disul-
fide bridges (Rey et al. 1995; Heinz 2003). Part of the domain responsible for the
fusion of the viral envelope with the membrane of the endosome is called fusion
peptide (Heinz and Allison 2003).
The domain III has a typical fold of an IgC molecule (Heinz 2003). It contains a
beta barrel composed of seven antiparallel beta sheets. It is supposed that the lateral
part of the domain III is responsible for binding to a specific cellular receptor (Rey
et al. 1995).
Among the most conserved parts of the E protein, there are 12 cysteine residues
forming six disulfide bridges with conserved localization in comparison with all
known flaviviruses (Nowak and Wengler 1987).
NS1 is a glycoprotein containing two or three potential glycosylation sites (Lee
et al. 1989) with not exactly defined functions. It exists in dimeric forms localized
freely in the cytoplasm or associated with membranes. This protein is also secreted
into the extracellular space particularly as a pentamer or hexamer and occasion-
ally as a decamer or dodecamer (Crooks et al. 1994). This so-called soluble antigen
induces protective immune response in the host (Gould et al. 1986).
NS2A is a small, hydrophobic protein with undefined function. It is supposed
that it plays a role in the forming of a replication complex (Lindenbach and Rice
2003). A small membrane-associated protein NS2B serves as a crucial cofactor for
protease activity of the NS3 protein (Yamschikov and Compans 1995). The central
hydrophilic domain of the NS2B protein possibly interacts with the NS3 protein and
it is flanked by hydrophobic regions probably anchored in the membrane (Chambers
et al. 1993).
NS3 is the second largest viral protein. It contains conserved regions important
for the function of the NS3 as a serine protease, helicase, and RNA triphosphatase
(reviewed by Lindenbach and Rice 2003).
NS4A and NS4B are small, hydrophobic proteins. NS4A is probably part of the
replication complex (Uchil and Satchidanandam 2003), while the function of NS4B
is not known.
NS5 is the largest and highly conserved viral protein serving as a viral RNA-
dependent RNA polymerase (Steffens et al. 1999). Its C-terminus shares a sequence
homology with RNA-dependent RNA polymerases of other (+)RNA viruses
(Lindenbach and Rice 2003). Apart from their main function as RNA dependent
RNA polymerase the TBEV NS5 protein is able to interfere with type I IFN JAK-
STAT signaling (Werme et al. 2008).
The infection of the host cell with TBEV (Figure 10.1) begins with the binding
of the virus to a cell receptor, which has not been sufficiently identified until now.
Kopecký et al. (1999) identified two polypeptides of 35 and 18 kD as putative ver-
tebrate receptors for TBEV using viroblot technique with anti-idiotypic monoclo-
nal antibodies directed against antibodies that neutralize the infectivity of TBEV.
However, the anti-idiotypic monoclonal antibodies did not bind effectively to tick
cells indicating that different receptors are used by vertebrate and invertebrate cells
for the binding of TBE virus (Kopecký et al. 1999). It remains unclear whether TBEV
uses single or multiple receptors on susceptible cells. Apparently, just the ability to
use multiple receptors can be responsible for the very wide host range of flaviviruses,
which replicate in arthropods and in a broad range of vertebrates. After binding to
the receptor, virus is internalized by the process of endocytosis. As already men-
tioned above, the acidification within the endosomal vesicle triggers conformational
changes of the E proteins leading to rearrangement of the dimers to trimeric forms
2
9
3
10
8
4
7
5
FIGURE 10.1 Replication machinery of TBEV in mammalian host cell. (1) Interaction of
TBEV with host cell receptor. (2) Receptor mediated endocytosis of TBEV. (3) Fusion of viral
and host cell membrane. (4) Release of the viral genome into the cytoplasm. (5, 6) Replication
of the viral genome. (7) Transcription. (8) Viral particles assembly occurs in the endoplasmic
reticulum. (9) Maturation of viral particles in the Golgi complex. (10) Release of mature virus.
and subsequent fusion of the viral envelope with the membrane of the vesicle. The
viral nucleocapsid is then released into the cytoplasm and viral RNA is uncoated.
The positive-stranded RNA is used for translation, and negative-stranded RNA that
serves as a template for the RNA is synthesized. The polyprotein is cleaved by viral
and cellular proteases into individual viral proteins. The surface structural proteins
prM and E are translocated into lumen of the ER and their amino termini are liber-
ated through proteolytic cleavage by the host signalase. The newly synthesized RNA
is packaged by protein C into nucleocapsids on the cytoplasmic site of ER. Viral
envelope is acquired by budding of the nucleocapsid into ER. The immature non-
infectious virions containing proteins prM and E in heterodimeric association are
transported into Golgi complex, where prM is cleaved, and the E protein is reorga-
nized to the form of fusion-competent homodimers. These mature virions are finally
released from the host cell by fusion of the transport vesicle membrane with plasma
membrane (Mandl 2005).
TBEV infection is associated with dramatic morphological changes occurring in
the infected cells. These include formation of smooth membrane structures, prolif-
eration of endoplasmic reticulum, and accumulation and convolution of membranes.
The infected cells often die by apoptosis or necrosis (Růžek et al. 2009).
The TBEV maturation process in tick cells seems, however, to be very differ-
ent from the cells of vertebrates. In cell lines derived from the tick Rhipicephalus
appendiculatus infected with TBEV, nucleocapsids occur in cytoplasm and the enve-
lope is acquired by budding onto the cytoplasmic membrane or into cell vacuoles
(Šenigl et al. 2006).
10.3 MICROEVOLUTION OF TBEV

It has been recognized since the 1960s that TBEV isolated from field-collected ticks
contains a heterogeneous population of variants that produce a range of plaque sizes,
express different levels of temperature-sensitivity, and neuroinvasiveness (Mayer
and Kožuch 1969). Tick-adapted variants of TBEV were shown to exhibit small-
plaque phenotype and slower replication in mammal cells and decreased neuroin-
vasiveness in laboratory mice (Bakhvalova et al. 2011). In nature, such low-virulent
virus variants could emerge after several generations spent in ticks after transovarial
and transstadial transmission and direct transfer from one tick to another during co-
feeding on the same host.
The relatively rapid phenotype change of TBEV was first mentioned when the
TBEV passaged in ticks Hyalomma plumbeum noticeably decreased the virulence
for laboratory mouse and subsequent passaging through mouse brains led to restor-
ing of the former phenotype (Dzhivanyan et al. 1988). Similarly, passaging of TBEV
through salivary glands of ticks Ixodes ricinus led to reduction of neuroinvasiveness
(Labuda et al. 1994). Analogously, the attenuated phenotype of the passaged virus
was not stable; when the attenuated virus was passaged through mouse brains, the
virulent phenotype was restored and after additional passages, the virus was more
virulent than the parental strain (Kaluzová et al. 1994).
A Siberian strain isolated from I. persulcatus and passaged in mouse brain was
subsequently passaged in H. marginatum by artificial inoculation and then again
through mice. The tick-adapted virus exhibited small-plaque phenotype and slower
replication in pig embryo kidney cells, higher yield in ticks, and decreased neuroin-
vasiveness in mice. A total of six amino acid substitutions distinguishing genomes of
the variants were identified and two of them located in the E protein are supposed to
be responsible for the phenotypic differences (Romanova et al. 2007).
In another study, an attenuated temperature-sensitive TBEV strain (263), isolated
from field ticks I. ricinus, was either serially subcultured 5 times in mice or at 40°C
in PS cells, producing two independent strains, 263-m5 and 263-TR with identi-
cal genomes; both strains exhibited increased plaque size, neuroinvasiveness, and
temperature-resistance. Sequencing revealed two unique amino acid substitutions,
one mapping close to the catalytic site of the viral protease NS2B-NS3 (Růžek et al.
2008b).
The selection, during serial passage, of preexisting quasi-species was postulated
to be the explanation for the rapid shift of virus phenotypic characteristics. All the
results lead to the suggestion that TBE virus exists as a heterogeneous population
that contains virus variants most adapted to reproduction in either ticks or mam-
mals. Host switch results in a change in the ratio of these variants in the population
(Romanova et al. 2007). In other words, virulent and attenuated viruses may coex-
ist as quasi-species in the same TBEV population and rapid conversion of neuro-
virulence during virus tick/mammal adaptation is mediated by selection from the
quasi-species population rather than random mutagenesis during virus passage in the
laboratory (Růžek et al. 2008b).
10.4 ECOLOGY AND EPIDEMIOLOGY

In Europe, the main vector as well as reservoir of TBEV is the tick Ixodes ricinus
(Ixodidae) (Rampas and Gallia 1949), a dominant hard tick across the European
continent (Figure 10.2). I. trianguliceps, I. hexagonus, and I. arboricola are con-
sidered as amplifying vector ticks of TBEV (Grešíková 1972; Křivanec et al. 1988).
Domestic Free-living
animals Artiodactyla
and Carnivora
Man
Small vertebrates
FIGURE 10.2 Life cycle of ixodid tick and transmission cycle of TBEV.
Infected secondary vector ticks, such as Haemaphysalis inermis and Dermacentor

reticulatus exhibit lower transmission rates (Grešíková and Kaluzová 1997). The
virus was also isolated from H. inermis in the Czech Republic (Grešíková and Nosek
1966), and H. spinigera and H. turturis were shown to be able to transmit TBEV
under laboratory conditions (Nosek et al. 1967). Kožuch and Nosek (1971) confirmed
D. marginatus and D. reticulatus as possible TBEV vectors, though the infection
and transmission rates were lower than in vector species of the genera Ixodes and
Haemaphysalis (Nosek et al. 1967).
Far Eastern and Siberian subtypes are transmitted predominantly by I. persul-
catus. This tick comprises 80%–97% of all tick species in the Urals, Siberia, and
the Far East region of Russia. Dermacentor pictus, Dermacentor silvarum, and
Hyalomma concinna have also been associated with local TBE outbreaks in some
areas of Siberia and the Far East, where I. persulcatus is not the predominant species
(Zlobin and Gorin 1996). However, TBEV has been isolated from 15 other tick spe-
cies in Russia and also sporadically from other parasitic invertebrates (e.g., fly, flea,
lice; reviewed by Gritsun et al. 2003b).
The life cycle of I. ricinus consists of three stages: six-legged larva molds to
eight-legged nymph, which develops to similar but larger imago (adult). Each stage
takes approximately 1 year to develop to the next stage. However, the life cycle can
take from 2 to 6 years, depending on various factors, including climatic conditions,
availability of suitable hosts, etc. Each developmental stage prefers different verte-
brate hosts for feeding. Larvae feed on small rodents, but also insectivores or birds.
Nymphs prefer larger animals such as squirrels, hedgehogs, hares, and birds. Adults
feed on deer, foxes, goats, sheep, cows, dogs, etc. The fertilized engorged female
lays 500–5000 eggs and dies. The virus infects ticks chronically for the duration of
their life. It is transmitted from transstadially from one developmental stage to the
next and transovarially from infected female to eggs. The frequency of transovarial
transmission seems to be very low, but under certain conditions it can be sufficient to
ensure the continuity of virus populations. Sexual transmission from infected male to
uninfected female has been also suggested and verified experimentally (Chunikhin
et al. 1983). TBE virus can be transmitted to man or other hosts by all the tick stages,
i.e., larvae, nymphs, as well as adult ticks. The seasonal activity of I. ricinus has
two peaks: April–May and September–October. Comparison of the tick population
curves and the morbidity rate in humans shows that there is a difference of approxi-
mately 14 days between the peaks of the two curves. The gap between the peak of
tick activity and the highest morbidity rate in humans corresponds to the incubation
period of the disease, which is between 4 and 14 days. The activity of I. persulcatus
has only one peak and lasts from the end of April to the beginning of June. During
July only some sporadic cases can be seen (Süss 2003).
A tick can be infected with TBEV following feeding on viremic animal hosts or
following co-feeding of infected and uninfected ticks on the same host (nonviremic
transmission) (Labuda et al. 1993, 1997). Virus crossing from tick esophagus to sub-
esophageal ganglion represents the main route of tick infection. Epidermal cells are
the primary site of virus replication in the tick body. Immunofluorescence detection
of TBEV antigen in sections of virophoric female ticks feeding for 36 hours shows
clear infection of epidermal cells and salivary glands (Nosek et al. 1972). In the
period that precedes molting, the virus multiplies in the tick and invades almost all
the tick’s organs (Benda 1958).
Virus transmission from the infected tick to the host is quite fast. Theoretically, the
virus can be transmitted by saliva during the first minutes of feeding. Experimentally,
it was demonstrated that TBEV was transmitted from infected Haemaphysalis iner-
mis ticks to lab mice within the first 3 hours of feeding (Grešíková and Nosek 1966).
A number of pharmacologically active compounds is secreted in tick saliva. These
modify the microenvironment in the site of feeding and control the hemostatic,
inflammatory, and immune responses in the vertebrate host in order to facilitate
blood feeding. Such bioactive saliva molecules include immunoglobulin-binding
proteins, histamine-binding proteins, interferon regulators, and inhibitors of natural
killer cells and complement. The action of the bioactive saliva molecules can facili-
tate TBEV transmission to the vertebrate host (Nuttall and Labuda 2004).
Hosts of TBEV include various rodents (Clethrionomys, Apodemus, Mus,
Microtus, Micromys, Pitymys, Arvicola, Glis, Sciurus, and Citellus) and insectivores
(Sorex, Talpa, Erinaceus), but also reptiles, birds, bats, carnivores (Meles, Vulpes,
Mustela), and large vertebrates. Rodents, insectivores, bats, and probably some birds
and carnivores serve as reservoirs of TBEV in nature, i.e., these animals develop
viremia for a long period without becoming clinically ill and thus can serve as a
source of TBEV for the virus transmission to uninfected ticks. Small rodents and
insectivores exhibit unapparent infection after experimental TBEV inoculation
with long-lasting viremia (Kožuch et al. 1967; Achazi et al. 2011). The virus can
be detected in various organs for long time periods (Achazi et al. 2011; Knap et al.
2012). Experimentally, the transmission of TBEV from ticks to rodents and insec-
tivores, as well as from these hosts to ticks, was demonstrated. The virus has been
successfully isolated from various rodents and insectivores captured in the field
(e.g., Apodemus flavicollis, Clethrionomys glareolus, Erinaceus roumanicus, etc.;
Kožuch et al. 1967; Weidmann et al. 2006). This all clearly demonstrates the crucial
role of rodents as well as insectivores in TBEV circulation in nature. Moreover,
these animals serve as a bridge for TBEV transmission by co-feeding of infected and
uninfected ticks. The same has been verified experimentally even on immune indi-
viduals (Labuda et al. 1997). The importance of co-feeding in the virus maintenance
in nature is not clearly determined, since it requires common feeding of larvae and
nymphs on the same host, which is not, however, a frequent event.
Lizards are frequently infested with ticks in the field. Experimentally inoculated
lizards Lacerta viridis with TBEV develop viremia lasting up to 7 days and antibody
response (Grešíková and Albrecht 1959; Sekeyová et al. 1970).
The role of birds in the circulation of TBEV is not fully clear (Ernek et al. 1968), but
the virus was isolated from several species, especially from water birds (Grešíková 1972).
Experimentally infected wild ducks (Anas platyrhynchos) develop chronic infection
with viremia detectable from 4 to 36 weeks postinoculation (Ernek et al. 1969). On the
other hand, viremia was not detected in the inoculated great tit (Parus major), blackbird
(Turdus merulla), common buzzard (Buteo buteo), common kestrel (Falco tinnunculus),
common pheasant (Phasianus colchichus) (Grešíková 1972).
High and long-lasting viremia (7–23 days p.i.) was observed in bats (Myotis myo-
tis, Barbastella barbastella, Plecotus auritus) inoculated with TBEV (Kolman et
al. 1960; Nosek et al. 1961). TBEV persisted in bats during their hibernation and
posthibernation viremia was observed (Nosek et al. 1961).
Indicator hosts have only brief viremia with low virus production and are not
able to transmit the virus to vectors. Dogs or roe deer represent examples of indi-
cator hosts. Dogs develop low and short viremia (Grešíková et al. 1972) and sero-
convert upon infection but they are not capable to further spread the virus (Pfeffer
and Dobler 2011). Low and short viremia and seroconversion was also observed in
roe deer (Capreolus capreolus) after experimental inoculation with TBEV or after
experimental transmission of TBEV from infected ticks (Nosek et al. 1967). Dairy
animals (goats, sheep, cows) develop viremia after the infection, the virus can pass
from the blood of the livestock into the mammary gland and is present in milk
(Grešíková 1957, 1958; Grešíková and Řeháček 1959), but their role in TBEV circu-
lation in nature is not known.
Humans are accidental and dead-end hosts of TBEV, i.e., they can develop a dis-
ease with viremia, but they do not participate in TBEV circulation in nature. In most
cases, people are infected by a bite of an infected tick. Less frequent cases of TBE
occur after drinking infected unboiled milk or eating unpasteurized milk products. In
the Czech Republic, retrospective analysis revealed that, between 1997 and 2008, 64
cases of TBE were recorded in patients who reported consumption of unpasteurized
goats’ and dairy milk or unpasteurized sheep’s milk cheese (0.9% of the total 7288
number of TBE cases). The majority of cases involved goats’ milk (36 patients, i.e.,
56.3%) and sheep’s milk cheese (21 patients, i.e., 32.8%). Dairy milk-borne infec-
tion was responsible for 7 TBE cases (10.9%). Thirty-three cases (51.6%) occurred in
family outbreaks following purchase of cheese or milk from animal breeders (Kříž
et al. 2009). In Slovakia, 33 TBE cases after drinking raw milk were reported dur-
ing 5 years (about 9% all cases) (Labuda et al. 2002). In Poland, 119 unpasteurized
milk samples from 63 cows, 29 goats, 27 sheep from 8 farms in eastern Poland were
analyzed (Cisak et al. 2010). The most common TBEV occurrence was in the milk of
sheep (22.2%), milk of goats (20.7%), and cows (11.1%) (as determined by the RT-PCR
method). By the ELISA method, the highest prevalence of anti-TBEV antibodies was
found in the milk of sheep (14.8%), milk of cows in 3.2%, and none in milk of goats.
Sporadic laboratory-based infections caused by inhaling infected aerosol or
by accidental needle-stick injury were reported before the availability of effective
TBEV vaccine (Gallia et al. 1949; Molnár and Fornosi 1952; Bodemann et al. 1977;
Avšič-Županc et al. 1995).
Based on the biology of ticks as the vectors of TBEV, TBE has two main epide-
miological characteristics: seasonal character of the disease and territorial distribu-
tion. The case distribution according to age, sex, and occupation is determined by
contact with the source of infection, particularly in forested areas. TBEV is endemic
in areas extending from Central and Eastern Europe to Siberia and parts of Asia.
More and more TBEV endemic areas are being detected, especially in Asia (Lu et
al. 2008). In Russia, the highest TBE incidence is reported in Western Siberia and
Ural. In the European part of Russia, especially the northern part and Crimea exhibit
high TBE incidence. The countries and regions in Central and Eastern Europe with a
high risk of exposure to TBEV in 2011 are the following: Latvia, Lithuania, Estonia,
Belarus, Sweden (eastern coast, Gotland, Oland, and endemic foci in northern part),
Finland (Åland Islands, and west coast), Poland (Mazury, Podlasie, Opole, Lublin,
Warsaw, and Gdańsk Regions, single endemic areas in central part of Poland), Czech
Republic (whole country but especially South Bohemia, Plzen Region, Moravia),
Slovakia (especially between Bratislava and Banska Bystrica), Germany (south part
and endemic areas in Sachsen and in the northern Germany), Austria (area between
Vienna and Klagenfurt and between Vienna and Linz), Hungary (west part and scat-
tered endemic areas in eastern part of this country), Slovenia, Croatia (northern
part), Albania (southern part), and Norway (southern coast). Small, single endemic
areas occur in Switzerland, France, and Italy. No TBE cases were reported, e.g., in
Great Britain, Ireland, Iceland, the Netherlands, Luxemburg, Spain, and Portugal.
TBEV represents a potential risk for people traveling to endemic countries for leisure
activities in nature (Rendi-Wagner 2004; Reusken et al. 2011; Chaudhuri and Růžek
2012). During the past decades, an increase in TBE incidence has been reported in
most European countries as well as in Russia. This might be caused by several fac-
tors, which include ecological (climatic changes), agricultural, but also social factors
(changes in leisure activities) (Korenberg 2009). In relation to the climatic changes,
a shift of the upper limit of the geographical habitats of ticks to higher altitudes has
been observed. Previously, a limit of occurrence of ticks was at 700–750 m above
sea level and ticks were not able to finish their life cycle at higher altitudes. It was
revealed recently that ticks shifted to the altitudes up to 1000 m above sea level
(Danielová et al. 2008). Because these mountain zones are often used for leisure
and outdoor activities, the risk of TBEV infection in these areas increased consider-
ably (Daniel et al. 2003). Socioeconomic factors may exert a powerful effect on the
frequency of population contact with TBEV, which leads to an explosive increase
in general morbidity. But attributing the increasing incidence of TBE cases to
“the collapse of communism” in Eastern Europe as repeatedly published by some
authors (Randolph 2004, 2008) is too simplistic and has a poor epidemiological basis
(Korenberg 2009).
The most exposed groups to TBEV are forestry workers and farmers. For exam-
ple, in serological studies in Poland, seropositivity was found in about 20%–30% of
farmers and forestry workers (Cisak et al. 1998). Children and youth include 25% of
sick persons in Poland.
10.5 CLINICAL FEATURES

It is assumed that the majority of TBEV infections are asymptomatic. Although the
proportion of these cases is difficult to determine, serological surveys suggest 70%–
95% of the infections occurring asymptomatically (Kaiser 2008; Shapoval 1976,
1977; Pogodina et al. 1979).
In case of symptomatic infection, the incubation period is usually 7–14 days, but
it may vary from 2 to 28 days. Two-thirds of patients with TBE caused by European
TBEV strains have a characteristic biphasic course (Kaiser 1999; Günther et al.
1997a; Holzmann 2003). The first, viremic phase is generally sudden and is charac-
terized with nonspecific influenza-like symptoms lasting 2–4 days, with moderate
fever (sometimes with chills), fatigue, malaise, headache, myalgia, gastrointestinal
symptoms, leukocytopenia, thrombocytopenia (Lotrič-Furlan 1995), and sometimes
with elevated liver enzymes. This is followed by a symptom-free interval of about

1 week (range 1–33 days) before the second phase develops. The second phase pre
sents as meningitis (50% of all adult patients), meningoencephalitis (40%), or as
meningoencephalomyelitis (10%) (Bogovič et al. 2010). During the second phase,
an elevated white blood cell count can be seen, but rarely exceeds 15 × 109/L. The
C-reactive protein concentration and erythrocyte sedimentation is usually normal
but may be elevated in some cases. In CSF, moderately raised protein levels and
lymphocytic pleocytosis is seen (only during the early phases of the disease poly-
morphonuclear cells may predominate in CSF) (Bogovič et al. 2010).
The meningeal form is usually manifested by high fever, headache, nausea, vom-
iting, and vertigo. Encephalitis is characterized by restlessness, tremor of extremi-
ties, fasciculations of the tongue, concentration disturbances, and cognitive function
disturbances. Other symptoms include global latent paresis of the limbs, hyper
esthesia, impaired micturition, and increased physiology reflexes. Disturbance of
consciousness ranges from somnolence to stupor or very rarely to coma (Fališevac
and Balus 1981; Bogovič et al. 2010). Seizures are rare.
The meningoencephalomyelitis and meningoencephalomyeloradiculitis represent
the most severe form of TBE. In these forms, flaccid paralyses of the limbs are pres-
ent, but also the paralyses of pharyngeal or respiratory muscles can develop. The
upper extremities are affected more frequently than the lower ones (Bogovič et al.
2010).
Far Eastern TBEV strains cause more frequently a disease with a monophasic
course; the biphasic form occurs in only 3%–8% of the patients (Votiakov et al.
2002). Focal encephalitic symptoms appear in 31%–64% of the patients, meningeal
form is seen in about 26%, febrile form in 14%–16% (Votiakov et al. 2002). In about
0.5% of cases, the infection progresses into a chronic form.
The Siberian subtype is associated with focal encephalitis in 5%, meningeal in
about 47%, and febrile form in 40% of the cases. Biphasic course is seen in about
20% of patients (Votiakov et al. 2002). About 1.7% of patients with acute illness
develop a chronic progressive form of TBE (Popponikova 2008). The chronic form
of TBE is not seen in Europe; only two cases have been reported with some con
troversy (Mickiene et al. 2002). However, in Russia, several cases are well docu-
mented, although they vary in the clinical presentation, and the time of onset of
the individual neurological symptoms (Bogovič et al. 2010). Clinical manifestations
include Kozhevnikov’s epilepsy (epilepsia partialis continua), progresive neuritis,
lateral sclerosis, progressive muscle atrophy, and a Parkinson’s-like disease (Bogovič
et al. 2010).
The fatality rate in adult patients in Europe is less than 1%. Far Eastern TBEV
subtype is associated with more severe disease with case fatality rate up to 40%.
The disease caused by Siberian TBEV strains has a case fatality rate of 2%–3%
(Lindquist and Valapathi 2008; Bogovič et al. 2011; Mickiene et al. 2002). Severe
courses of TBE infection with higher mortality and long-lasting sequelae often
affecting the quality of life are in correlation with increased age (>60 years of age)
(Haglund and Günther 2003).
A postencephalitic syndrome has been identified in 35%–58% of patients after
acute TBE. The patients have permanent sequelae; the most commonly reported
problems include cognitive or neuropsychiatric complaints, balance disorders, head-

ache, dysphasia, hearing defects, and spinal paralysis (Růžek et al. 2011).
TBE in children is generally milder, although severe illness may occur and
even lead to permanent impairment of the quality of life due to neuropsychological
sequelae (Kunze et al. 2004). Encephalomyelitic manifestation of TBE is very rare in
children. The paralytic form usually heals without sequelae. The prognosis of TBE
in children is usually favorable (Falk and Lazarini 1981; Messner 1981).
TBE following alimentary infection is about 50% monophasic. In this form, the
symptoms include fever, intracranial hypertension, sharp headache, nausea, vom-
iting, weakness, loss of appetite, dizziness, drowsiness, gastrointestinal problems,
epistaxis, pharyngitis, laryngitis, and photophobia. After 2–7 days, these symptoms
disappear and the patients recover (Kubánka and Pór 1954). The biphasic form of
alimentary TBE is more severe, but benign. The first phase takes about 7 days and is
characterized with the same symptoms as the monophasic form, but in addition with
very high fever, blurred vision, and diplopia. The second phase appears after approx-
imately 8 days of remission and is characterized with meningeal irritation and/or
encephalitis. Hypersomnia and diplopia are typical signs. In mild cases, the second
phase lasts 3–4 days; in more serious cases, 14–21 days (Henner 1954; Hympán
1954; Dudáš et al. 1954; Černáček et al. 1954).
Treatment is symptomatic only, with strict bed rest. Maintenance of water and
electrolyte balance is important. Analgesics, vitamins, and antipyretics are admin-
istered. Corticoids have a favorable effect during the acute stage of TBE (intrave-
nously 5–10 mg/kg/day) (Dunyewicz et al. 1981), but the use of corticosteroids may
be questioned, as it has not been shown to be useful in prospective studies (Mickiene
et al. 2002). Administration of hyperimmunoglobulin is not recommended due to
concerns about antibody-dependent enhancement of the infection (Kaiser 1999;
Waldvogel et al. 1996; Jones et al. 2007).
Besides preventive measures, such as wearing appropriate clothing or checking
the skin for attached ticks after returning from activities in nature, TBE can be suc-
cessfully prevented by active immunization (Kunz 2003). In Russia, several inacti-
vated vaccines are available (vaccine produced by the Institute of Poliomyelitis and
Viral Encephalitis [IPVE] in Moscow and EnceVir produced by Virion, Tomsk). The
IPVE vaccine is applied for adults and children of three years and more. The basic
vaccination scheme consists of two doses within 5–7 months with revaccination after
1 year and a booster every 3 years. The EnceVir vaccine is administered to adults
(>18 years of age) with the same vaccination scheme. In case of rapid immuniza-
tion, the first two doses are administered within 1–2 months (Donoso Mantke et al.
2011). In Europe, two inactivated vaccines are available (FSME-Immun by Baxter
Bioscience, Austria; Encepur by Novartis Vaccines and Diagnostics, Germany). The
vaccination scheme is analogous for both vaccines with three doses given on 0, 1–3
months, 5–12 months after the second (FSME-Immun), or 9–12 months (Encepur).
First booster vaccination should be after 3 years, and the next booster vaccina-
tions every 5 years (or every 3 years for people older than 50 years [Encepur] or 60
years [FSME-Immun]) (Rendi-Wagner et al. 2004; Hainz et al. 2005). Both vac-
cines are comparable in terms of efficacy and tolerability. FSME-Immun has shown
a protective effect of >95% after three doses (Heinz 2007). TBE cases are observed
despite immunization with both available vaccines (Anderson et al. 2010; Stiasny
2009). Persons older than 50 years show a significantly lower antibody response
(Weinberger et al. 2010), with a higher frequency of low responders and vaccine fail-
ures. Therefore, people older than 50–60 years of age may be recommended three
doses as basic immunization, day 0, 1 month, and 3 months. The next booster dose
should be administrated as early as possible before the next season. Rapid schedules
with an interval shortened to 2 weeks between the first and second dose should be
avoided in people older than 60 years.
Vaccination is especially recommended for forestry workers, farmers, hunters, people
who are in contact with forests (collectors of mushrooms, etc.), and tourists and children
staying in those places. Most common adverse reactions after immunization are itching,
local swelling, pain, rash, fever, or moderate elevated body temperature (especially in
children), nausea, malaise, pain of the skeletal system and muscles, headache, and vomit-
ing. Contraindications for immunization include hypersensitivity (allergy) to chicken pro-
tein, formalin, neomycin, gentamicin, and/or acute reaction to previous immunization.
There are no data available concerning the influence of TBEV vaccines on pregnancy.
Immunization of pregnant women and children <12 months of age is not recommended.
Other contraindications include acute infections.
10.6 PATHOLOGY AND PATHOGENESIS

After the tick bite, the virus replicates in subcutaneous tissues. Various cell types
in the skin including keratinocytes become infected by the virus. Langerhans cells
(LCs) are probably the first distinct dendritic cells subset that present antigens of
TBEV to T cells and activate their differentiation to T helper type 1 (Th1), Th2,
and cytotoxic T lymphocyte (CTL) effector cells. However, LCs are likely to serve
also as a vehicle for the transport of the virus to draining lymph nodes (Chambers
and Diamond 2003). The lymph nodes play an important part in the pathogenesis
of TBE, although virus replication is not accompanied by virus-specific histologi-
cal changes and no marked destruction of infected lymph node cells is observed
(Málková and Filip 1968). Within the lymph nodes, macrophages represent the tar-
get cells for TBEV infection. The interaction between TBEV and macrophages is
crucial, and the inability of the virus to multiply in macrophages is in association
with the inability to induce an infection of the individual (Ahantarig et al. 2009;
Plekhova et al. 2011). Massive viral multiplication in the nodes leads to the spread
of the virus into the bloodstream and induction of viremia (Málková and Fraňková
1959; Málková and Kolman 1964). Temporary leukopenia in the white blood picture
is observed. A significant decrease is recorded in all cellular elements. In regional
lymph nodes, a significant decrease in lymphocytes appears (Málková et al. 1961).
TBEV can be isolated from blood leukocytes during the first days of infection, which
indicates virus replication in immunocompetent blood cells (Leonova et al. 1996).
The virus is probably cleared when protecting antibodies appear in serum and the
encephalitic symptoms are probably due to an intense inflammatory reaction in the
brain.
During the primary viremic phase, several extraneural tissues are infected (espe-
cially spleen, liver, and bone marrow cells). The release of the virus from these cells
induces secondary viremia, which continues for a couple of days. During the sec-
ondary viremia, the virus crosses the blood–brain barrier and reaches the CNS. The
mode of virus crossing the blood–brain barrier remains unknown. Four potential
routes were postulated: (i) by neuronal route after infection of peripheral nerves or
by olfactory neurons, (ii) by so-called Trojan horse mechanism when TBEV-infected
immune cells migrate into the CNS, (iii) by infection of vascular endothelial cells
of brain capillaries, and (iv) by diffusion of virus between the capillary endothelial
cells under the conditions of increased blood–brain barrier permeability (Růžek et
al. 2010) (Figure 10.3). Experiments with mice infected with TBEV showed that
the breakdown of the blood–brain barrier occurs at later stages of the neuroinfec-
tion when a high virus load is present in the brain; therefore, the breakdown of the
Inflammation
Clearance of virus cytokines
immmunopathology
CD4+
CNS T cells
CD8+
T cells
Neurons Microglia
apoptosis immune
necrosis response
BBB
Leukocyte trafficking
cytokine/chemokine
mediated Crossing the
BBB
Cytokine/ Replication
Infection Trojan horse chemokine
of olfactory in the BBB
mechanism mediated BBB cells
neurons disruption
Hematogenous spread
Secondary viremia
IgM/LgG Replication in peripheral

tissues
Primary viremia
Type I IFN Replication in lymph

Inflammatory cytokines nodes
Migration
Skin: primary infection

Tick saliva
Host modulates
immune responses
TBEV - infected tick
FIGURE 10.3 Schematic drawing of the steps during TBEV infection in the mammalian
host. BBB, blood–brain barrier; CNS, central nervous system.
blood–brain barrier is not necessary for TBEV entry into the brain and more likely is
in association with dramatic up-regulation of proinflammatory cytokine/chemokine
expression in the infected brain (Růžek et al. 2011).
In most cases, the morphological picture of TBE in the CNS has the characteris-
tics of nodular polioencephalomyelitis with meningeal involvement. The histopatho-
logical picture involves (i) damage to the nervous parenchyma with cell necrosis
and neuronophagy, (ii) perivascular inflammatory reaction with serous exudation,
(iii) spongiform focal necrosis in the form of facultative lesions and secondary dam-
age or anoxic vassal lesions (Jellinger 1981).
Viral antigens can be demonstrated in perikarya and processes of Purkinje cells
and large neurons of dentate nucleus, inferior olives, anterior horns, neurons of other
brainstem nuclei, isocortex, and basal ganglia (Gelpi et al. 2005).
The infected neurons are damaged—there is necrosis and neurolysis. The degra-
dation products are phagocyted by microglia cells (neurophagy). Astroglial swelling
and proliferation and increase of glycogen content take place (Jelliger 1981).
Interestingly, there is a poor topographical correlation between inflammatory
changes and distribution of viral antigen in the brain (Gelpi et al. 2005). The inflam-
matory response has two phases. The first phase is characterized by an unspecific
resorptive inflammatory reaction of granulocytes and macrophages, while the sec-
ond phase represents specific, defensive inflammatory reaction in which elements of
the lymphomonocytic system and macrophages are predominant (Jelliger 1981). In
general, the inflammatory response includes nodular and flaky tissue infiltrates of
histiocytes and rod-shaped microglia, perivascular cell cuffs, meningeal infiltrates,
and less frequent infiltrates in cerebral nerve roots, spinal nerve roots, and spinal
ganglia. The perivascular cuffs consist predominantly of T-lymphocytes, histiocytes,
plasma cells, and macrophages (Jelliger 1981). B-cells are only rarely found (Gelpi
et al. 2005).
In most cases, the spinal gray matter is infected with preference for the anterior
horns and the cervical region. There is a disseminated infection in the brainstem.
The cerebellum is mostly affected; massive infection is seen in the dentate nucleus,
cortex, and white matter. In the diencephalon, thalamus nuclei are highly infected.
The putamen and caudate nucleus are severely affected, whereas the pallidum is
only slightly affected. Widespread dissemination of the nodules is seen throughout
the cortex of telencephalon, with frontal accumulation, in insula of Reil, claustrum,
basal rhinencephalic gray matter and amygdala, in the subcortical white matter, and
less in the deep cerebral white matter and fiber systems. The hippocampus is usually
uninfected (Jelliger 1981).
TBEV induces a strong intrathecal immune activation, as demonstrated by a mas-
sive up-regulation of proinflammatory cytokines and other inflammatory mediators.
Patients with TBE react with stronger immune activation than patients with other
forms of encephalitis (enterovirus, HSV2) as indicated by CSF neopterin response
(Günther et al. 1996). The immune activation in brain involves production of various
inflammatory cytokines including IFN-γ, IL-2, IL5, IL-6, and IL-10. The impor-
tance of IL-10 during TBE has been demonstrated; low levels of IL-10 in CSF aggra-
vate the course of TBE (Günther et al. 2011) (Figure 10.4). Although IL-6 production
is generally associated with a protective immune response, it may also contribute to
Encephalitis with high production

Encephalitis with low production Up–regulation
Down–regulation
IL-10
CD4+ CD4+
Th 2 Th 1 TNF
IFN
IL-10 IL-6
IL-10
IL-10
Macrophage
B cell
MHC II
Astrocytes
Glial cells IL-6 lgM
lgG
Neopterin
Endothelial cells
FIGURE 10.4 Interaction of intrathecal inflammatory mediatos in TBE with different clini-
cal course. Proposed pathogenesis resulting in severe encephalitic or mild disease. (From
Günther, G. 1997. Tick-borne encephalitis—on pathogenesis and prognosis. Dissertation
Thesis, Karolinska Institutet, Stockholm. With permission.)
disease progression. Interestingly, an inverse correlation between raised IL-6 levels

and IgG titers was observed in TBE patients.
It has been hypothesized that the immunological mechanisms activated in the
brain can contribute to the nerve destruction during TBE, i.e., the host immune
response can act as a double-edged sword during TBE. Experiments with mice
demonstrated that mice immunosuppressed by cyclophosphamide or sublethal
X-irradiation exhibited prolonged survival after TBEV inoculation when compared
with untreated individuals (Semenov et al. 1981; Vince and Grčević 1981). Adoptive
transfer of sensitized splenocytes to immunosuppressed mice significantly decreased
the mean survival time of the infected animals (Semenov et al. 1981). The important
role of CD8+ T-cells in immunopathology during TBE has been revealed (Růžek
et al. 2009). Based on these results, it seems that the outcome of TBE represents a
result of certain “immunological conflict” between the infection control mediated by
immunity and damage to the host mediated by immunopathology (Růžek et al. 2010;
Rouse and Sehrawat 2010).
IgM and IgG antibodies against TBEV antigens are found in serum as well as in
the CSF. TBEV-IgM antibodies are detectable at an early stage of the infection when
the first symptoms appear. The level of IgM in CSF starts to increase between day 0
and day 6 after onset of encephalitic symptoms and reaches its peak approximately
2 weeks later, but variations were found in individual patients. Although in most
cases IgM antibodies are not detectable after 6–7 weeks, they may persist for several
months. In contrast, the IgG level increases only moderately at the beginning and
during the whole encephalitic phase. These TBEV-specific IgG antibodies confer a
long-term or even life-long immunity (Růžek et al. 2011). In CSF, IgG antibodies are
detectable as long as 11–13 months (Günther et al. 1996).
The course and outcome of TBE can be influenced by several factors. These include
virulence of the particular TBEV strain, and dose of the virus, but also age, sex, immune
status, and genetic background of the host. Interestingly, a functional toll-like receptor 3
gene may be a risk factor for TBEV infection (Kindberg et al. 2011). A deletion within
the chemokine receptor CCR5 (CCR5Δ32), which plays an important role in leukocyte
transmigration across the blood–brain barrier, is significantly more frequent in patients
with TBE than in TBE-naive patients with aseptic meningitis (Kindberg et al. 2008).
Moreover, the severity and outcome of TBE is associated with variability in the 2′–5′-
oligoadenylate synthetase gene cluster (family members are interferon-induced antiviral
proteins) (Barkhash et al. 2010), and the rs2287886 single nucleotide polymorphism
located in the promoter region of the human CD209 gene (Barkhash et al. 2012). This
gene encodes dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN), a C-type
lectin pathogen-recognition receptor expressed on the surface of dendritic cells and some
types of macrophages (Barkhash et al. 2012). Taken together, polymorphism in various
genes may largely influence the sensitivity of the host to the infection and determine the
severity of the disease.
10.7 DIAGNOSIS
In the later stage of the infection, specific antibodies are formed. They cut off vire-
mia and at the beginning of the second phase, no infective virus can be found in
the blood. Therefore, the most important laboratory techniques for the diagnosis of
TBE are not those of virus isolation but serological ones. Antibodies occur as early
as the beginning of the second phase, which represents the actual disease. If specific
antibodies are found, it is necessary to exclude that these are old antibodies caused
by a previous TBE infection or by TBEV vaccination (Hofmann 1981). This is ruled
out by intrathecally detecting the produced antibodies that indicate actual TBEV
infection. IgM activity can be demonstrated in 96% of patients, a median of 3 days,
after onset of encephalitis, later, serum from all patients are positive (Günther et al.
1997b). Maximum IgG activity can be detected in serum after 6 weeks and decreases
thereafter, but persists for many years (>30 years). IgG should be analyzed in paired
sera. Intrathecal antibodies are seen in 97% of patients after a median of 9 days
(Günther et al. 1997b) and the analysis may be of value in certain cases.
It is recommended to test paired sera with 1 or 2 weeks’ interval using ELISA or by
classical methods, which include virus neutralization, inhibition of hemagglutination,
and complement fixation tests. Diagnosis is established when at least a fourfold rise of
antibodies is demonstrated. However, if the patient is hospitalized at the peak of the
second phase or even later, further increase of the already high antibody titer cannot be
often observed. In this case, the analysis of IgM antibodies is crucial; the presence of
specific IgM antibodies indicates recent infection (Hofmann 1981). Generally, ELISA
is the method of choice due to its simple performance. New diagnostic ELISA kits are
available, which have very high specificity and sensitivity (Holzmann 2003). Isolation of
TBEV or detection of viral RNA by RT-PCR in serum or CSF have large limitations and
is rarely used in clinical practice (Saksida et al. 2005; Bogovič et al. 2011).
Antibodies against TBEV can be also assayed directly from CSF. Most patients
suffering from acute TBE have IgG antibodies in CSF, but IgM antibodies are
present only in about 80% of the cases. Sometimes, it is not clear if these antibodies
are actually produced in the brain or if they penetrate to the CSF though the broken
blood–brain barrier (Hofmann 1981).
In the postmortem material, the virus can be demonstrated in brain tissue by
immunofluorescence, RT-PCR, or by cultivation techniques (Hofmann 1981).
Tissue destruction in the CNS is rare. Abnormalities on MRI are seen in up to
18% in TBE-Eu with lesions confined to the thalamus, cerebellum, brainstem, and
nucleus caudatus (Lorenzl et al. 1996; Marjelund et al. 2004). Electroencephalogram
is abnormal in 77% (Kaiser 1999). Both MRI and EEG abnormalities are unspecific,
not diagnostic, and no direct correlation to the prognosis has been shown.
ACKNOWLEDGEMENTS
The authors acknowledge financial support by the Czech Science Foundation proj-
ect P302/10/P438 and P502/11/2116 and grant Z60220518 from the Ministry of
Education, Youth, and Sports of the Czech Republic. We thank Patrik Kilian for the
preparation of the figures.
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11 St. Louis Encephalitis
Luis Adrian Diaz, Lorena I. Spinsanti,
and Marta S. Contigiani
CONTENTS
11.1 Introduction................................................................................................. 239
11.2 Taxonomic Classification.............................................................................240
11.3 Virus Structure............................................................................................240
11.4 Genome Organization and Protein Functions............................................. 241
11.5 Replication Cycle......................................................................................... 242
11.6 Pathology and Pathogenesis......................................................................... 242
11.7 Clinical Presentations..................................................................................244
11.8 Diagnosis.....................................................................................................246
11.9 Ecology........................................................................................................ 249
11.10 Epidemiology............................................................................................... 252
11.11 Conclusion................................................................................................... 254
References............................................................................................................... 254
11.1 INTRODUCTION
St. Louis encephalitis virus (SLEV) was isolated for the first time during a human
encephalitis outbreak in St. Louis, Missouri United States of America (USA) in
1933 (Lumsdem 1958). The viral isolation was carried out from a brain sample of a
death patient. The outbreak took place during an exceptionally hot and dry summer.
More than 1000 cases were reported; most of them localized near open storm drains,
rain drainage and sewage channels, which worked as Culex mosquitoes breading
sites (Reisen 2003). Further ecological studies carried out during a SLEV human
encephalitis outbreak in Yakima Valley (Washington, USA) (1941–1942) incrimi-
nated peridomestics bird species as hosts and Culex mosquitoes as vectors (Hammon
et al. 1945). Understanding the ecological and epidemiological behavior of SLEV
and the development of new diagnostic techniques allowed a global vision regarding
the public health importance of SLEV in the USA.
In the past decades the concern about SLEV for the public health has decreased
in the USA. However, in our days, the epidemiological pattern of SLEV is chang-
ing. Since 2002, a reemergence scenario for this pathogen was observed in South
America. Human encephalitis cases were reported in Argentina and Brazil
239
TABLE 11.1
Taxonomic Classification of St. Louis Encephalitis Virus
Family Genus Serocomplex Species
Flaviviridae Flavivirus Japanese encephalitis Cacipacoré virus
Japanese encephalitis virus
Koutango virus
Murray Valley virus
St. Louis encephalitis virus
Usutu virus
West Nile virus
Yaounde virus
(López et al. 2011; Mondini et al. 2007; Rocco et al. 2005; Spinsanti et al. 2003,
2008).
11.2 TAXONOMIC CLASSIFICATION

SLEV belongs to the genus Flavivirus, Flaviviridae family (Table 11.1). The virus
prototype of the genus is Yellow Fever. It is composed of 53 different viral species,
most of them being pathogenic for humans and arthropod-borne viruses (ICTV 2009).
According to their serologic relatedness, flaviviruses were divided into antigenic com-
plexes (Heinz and Roehrig 1990). Based on its antigenic properties, SLEV belongs to
the Japanese encephalitis serocomplex. This group includes other viruses with medical
and veterinary concerns (West Nile (WNV), Japanese encephalitis (JEV), and Murray
Valley (MVEV), among others) (Burke and Monath 2001) (Table 11.1).
11.3 VIRUS STRUCTURE

The Flavivirus virion is a small icosahedral enveloped particle of 40 to 60 nm diame
ter, which includes a 30-nm core (Mukhopadhyay et al. 2003). Structural analysis of
mature Flavivirus virions revealed that the virus possesses an icosahedral envelope
organization and a spherical nucleocapsid core (Kuhn et al. 2002) (Figure 11.1a).
Mature virions contain three structural proteins; the capsid protein C, membrane
protein M, and the envelope protein E. Multiple copies of the C protein (11 kDa)
encapsulate the RNA genome to form the viral nucleocapsid (Jones et al. 2003). The
nucleocapsid is surrounded by a host-cell-derived lipid bilayer, in which copies of M
and E are anchored (Mukhopadhyay et al. 2003) (Figure 11.1a). The M protein is a
small (approximately 8 kDa) proteolytic fragment of its precursor form prM. The E
protein is approximately 50 kDa and is the main protein present in the virion enve-
lope. The E protein is in charge of recognizing the host cell receptors and is the main
target of neutralizing antibodies (Lindenbach and Rice 2001; Nybakken et al. 2006).
Certain mutations in the E protein can cause the virion’s lack of virulence (Beasley
et al. 2004; Engel et al. 2010; Gritsun et al. 1995).
St. Louis Encephalitis 241
(a) (b) 5ŃCR
Structural
proteins
C (Capside)
Prm–M (Membrane)
E (Envelope)
E E NS1 (Cofactor replication viral)

NS2A (Modulate antiviral response of host, viral
replicase complex)
E NS2B (Cofactor NS2B-NS3 complex) Open
E M
reading
M NS3 (Helicase, protease) frame
Non-structural
Nucleocapside proteins
NS4a Interferon signaling
RNA viral replication
NS4b
Envelope
NS5
(Methyltransferase activity,
RNA-dependent RNA polymerase)
3ŃCR
FIGURE 11.1 St. Louis encephalitis virion and genome structure.
11.4 GENOME ORGANIZATION AND PROTEIN FUNCTIONS

The SLEV genome consists of a single-stranded positive-sense RNA fragment of
approximately 10,963 nt (nucleotides). The viral genome works as a single RNA
messenger molecule, which possess an open reading frame (ORF) (Chambers et al.
1990a) (Figure 11.1b). The ORF encodes a 3429-aa polyprotein. The ORF is flanked
by noncoding regions (NCR). The 5′NCR (125 nt) presents an m7G5′ppp5′cap. The
3′NCR is 551 nt long and lacks the polyA tail (Brinton 2002; Diaz et al. 2010). These
NCR form RNA secondary structures, which could intervene directing the processes
of amplification, translation, and packaging of genomes (Hahn et al. 1987; Proutski
et al. 1997).
NS1 is a homodimer that participates in the replication process of viral RNA; it is
one of the first viral proteins being secreted and induces a strong humoral immune
response (Mackenzie et al. 1996; Schlesinger et al. 1986). NS2A is a small hydro-
phobic protein (22 kDa) thought to intervene in the RNA templates recovery associ-
ated with viral polymerase (Mackenzie et al. 1998), to module the host’s antiviral
response and cleavage of the NS1-NS2A junction after translation, and to play a
functional role in the viral replicase complex (Leung et al. 2008). NS2B is a small
membrane-associated protein, and acts as a cofactor necessary for the NS2B-NS3
complex serine-protease activity, which cleaves the viral poly-protein at the NS2A/
NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 junctions (Bera et al. 2007; Jan
et al. 1995; Shiryaev et al. 2007). NS3 is a big cytoplasmic protein (70 kDa) that
intervenes in several enzymatic activities (protease, helicase) involved in the poly-
protein processing and viral RNA replication (Chambers et al. 1990b; Li et al. 1999;
Luo et al. 2008). Interestingly, NS3 also appears to be involved in the virus assembly
through mechanisms that are independent from the enzymatic functions outlined
above (Patkar and Kuhn 2008). NS4A and NS4B are small hydrophobic membrane-
associated proteins of 16 and 27 kDa, respectively. Based on its sub-cellular local-
ization, both proteins may intervene in the viral RNA replication (Mackenzie et al.
1998; Westaway et al. 1997). NS5 is the biggest (103 kDa) and more conservative
protein throughout the Flavivirus genus (Davidson 2009). It has a methyltransferase
activity on its N-terminal region and a RNA-dependent RNA polymerase (RdRp)
activity on the C-terminal motifs (Mukhopadhyay et al. 2003; Liu et al. 2010).
11.5 REPLICATION CYCLE

The virus enters into the cell through a mechanism known as receptor-mediated
endocytosis, where the viral receptor for recognition and cell adsorption is protein
E (Hung et al. 1999). Once the endocytic vesicle is formed, the endosome acid pH
generates structural alterations in protein E, producing endosome-virion mem-
brane’s fusion and releasing the nucleocapsid to the cytoplasm (Stiasny et al. 2002);
although direct fusion of the viral envelope with the cellular membrane has been
observed too (Hase et al. 1989). After the viral uncoating process, the replication
starts. Viral RNA replication occurs in perinuclear spots and implies the synthesis
of a single complementary negative strand that works as a template for the positive
strand molecule’s synthesis (Lindenbach and Rice 2001). The genomic viral RNA is
used directly as a messenger and is completely translated from its 5′ end to produce a
big precursor poly-protein that later is cleaved to generate each individual viral pro-
tein (Clyde et al. 2006). The new viral particle’s assembly occurs in the endoplasmic
reticulum (ER), where immature particles are generated (containing prM) and then
transported to the exterior through the exocytic pathway. Evidence was found that
the acid pH in the trans Golgi network (TGN) produces conformational changes in
the prM-E complex that are necessary for the particle’s maturation. Once the cleav-
age of the complex is produced by the action of cellular furins, the mature particles
are released through exocytosis (Lindenbach and Rice 2001).

After the virus inoculation, it replicates in local tissue and regional lymph nodes.
Afterward, the virus is carried away through the lymph pathway and is poured to the
bloodstream through the thoracic duct, generating a secondary viraemia that trans-
ports it to the extra-neural tissues where also occurs viral replication and release
to circulation (Malkova 1960). The viraemia level is modulated by the clearance
rate of macrophages and finishes with the humoral response appearance, usually
one week after infection. In most of the cases, the viraemia curve presents a short-
duration peak (2 to 3 days) followed by a quick dropping. The extra-neural tissues
with greater replication are connective tissue, skeletal, cardiac and smooth muscle,
lymphoreticular tissue, and endocrine and exocrine glands. In newborn hamsters

infected with SLEV and Rocío virus, the pancreas and heart were the organs most
severely affected (Harrison et al. 1980). Experimental research on mice infected
with neurotropics Flavivirus has demonstrated the link among viraemia level, brain
infection development, and appearance of multiple sites with viral antigens in neu-
ral tissue (Albretch 1998), supporting the concept of a hematogenous release to the
central nervous system (CNS) (Johnson 1982). The mechanism used by Flavivirus
to pass through the blood-brain barrier during the natural infection remains unclear.
The viruses ability for replicating in endothelial vascular cells suggests they may
pass through the brain capillaries. However, only a few times were viral antigens
found in vivo on endothelial cells of brain capillaries, and also it was observed that
Flavivirus do not replicate at high titers in brain endothelial cells in vitro either
(Dropulic and Masters 1990). Some experimental studies suggest that CNS invasion
occurs through the olfactory epithelium. The olfactory tract has been recognized
as an alternative pathway to the CNS and an important release mode after nasal
spray exposures. Intranasal inoculation with Flavivirus in murine animal models
can result in lethal encephalitis, probably due to the direct infection of olfactory
neurons and the posterior release in the brain, while peripheral inoculation with the
same virus does not produce CNS invasion. Nevertheless, in experimental models
using mice and hamsters, olfactory neurons were the access gateway to CNS after
infection (Monath et al. 1983). On the other hand, pathologic studies on Japanese
encephalitis cases indicate a widespread involvement of the brain stem, deep nuclei,
and cortex, more consistent with a hematogenous infection.
The infection’s starting point and development are influenced by viral and
host specific factors. The intracerebral (i.c.) or intranasal (i.n.) infection with high
viral doses predisposes to fatal encephalitis. The viral strains can differ in neuro-
invasiveness, neuro-virulence or both. Among the factors involved in pathogenesis,
the most important are age, gender, genetic susceptibility, preexistent infections, or
immunity to heterologous agents. Newborn animals are more susceptible to lethal
encephalitis than adult animals. Animals inoculated peripherally are susceptible in
the first 3 to 4 weeks of life when they develop resistance, but can remain suscep-
tible to lethal encephalitis if they are i.c. inoculated. Immature neurons are more
sensible to infection (Ogata et al. 1991). In SLEV induced encephalitis, susceptibility
in humans increases with advanced age. The mechanisms underlying this increase
are unknown. Physiological factors leading to temporal immunosuppression may be
responsible for a greater susceptibility. In this way, mice exposed to cold or stress
and inoculated with WNV developed a bigger viral replication and mortality rate
(Ben-Nathan and Feuerstein 1990). Sexually mature female mice showed a bigger
resistance to Flavivirus infection (Andersen and Hanson 1974), while in humans this
has not been demonstrated yet. Genetic markers play a central role in the Flavivirus
infection pathogenesis (Sangster et al. 1998). A genetic resistance to Flavivirus infec-
tion has been observed in nonimmune inbred mice strains (Mashimo et al. 2008).
The recovery from flaviviral encephalitis depends on early intrathecal antibod-
ies synthesis and viral clearance by macrophages. The inflammatory response in
CNS-related infections consists of helper-inducer T cells and, to a lesser degree, B
lymphocytes, infiltrating from the blood to the perivascular space and parenchyma.
Macrophages and activated microglial cells in the perivascular space and paren-
chyma, respectively, are responsible for viral clearance. The outcome is determined
by the comparative rates of viral spread and neuronal infection, migration of inflam-
matory cells into the CNS, and the rapidity of the antibody response. Interferon
production is elicited in the brain of human SLE patients, but its role in limiting the
virus spread in the CNS is unclear.

The clinical spectrum of a SLEV infection can comprise from an unspecific
febrile syndrome, alike to influenza, to a disease affecting the CNS. The clini-
cal features of meningitis and encephalitis due to SLEV are not specific, and
other etiologies (bacterial, fungal, other viral, toxic, cerebrovascular), must be
considered.
Clinical manifestations of SLEV infection can be grouped in three syndromes
(Table 11.2): Encephalitis (including meningoencephalitis and encephalomyelitis),
meningitis, and febrile cephalea.
The incubation period can last from 4 to 21 days. The beginning of the symptoms
are characterized by general discomfort, fever, chills, cephalea, anorexia, nausea,
myalgia, and sore throat or cough, followed after 1–4 days by the neurological or
meningeal signs. Less than 1% of SLEV infections are clinically apparent (Tsai et al.
1987). The disease’s severity increases with age, with people over 60 years old the
TABLE 11.2
Definitions of Clinical Syndromes Caused by St. Louis Encephalitis Virus
I. Encephalitis (including meningoencephalitis and encephalomyelitis)
A. Acute febrile illness (oral temperature ≥37.8°C (≥100°F)
B. One or more signs in either of the following categories:
1. Altered level of consciousness (confusion, disorientation, delirium, lethargy, stupor, coma)
2. Objective signs of neurologic dysfunction (convulsion, cranial nerve palsy, dysarthria,
rigidity, paresis, paralysis, abnormal reflexes, tremor, etc.)
II. Aseptic meningitisa
A. Acute febrile illness
B. Sign(s) of meningeal irritation (stiff neck with or without positive Kernig’s or Brudzinski’s sign)
C. No objective signs of neurologic dysfunction
III. Febrile headachea
A. Acute febrile illness
B. Headache (may also have other systemic symptoms, such as nausea or vomiting)
C. No signs of meningeal irritation or neurologic dysfunction
Source: From Brinker, K.R.M. and Monath, T.P., The acute disease, in Monath, T.P. (ed.), St. Louis
Encephalitis. Washington, DC: American Public Health Association, 1980, pp. 503–534.
a Cerebrospinal fluid pleocytosis present in patients with encephalitis and aseptic meningitis; it may also
be found in patients with the syndrome of febrile headache.

ones having more frequent cases of encephalitis. Being elderly contributes to virus
neuropathogenesis, brain damage, and severity in the clinical manifestation (Burke
and Monath 2001).
Mortality rate increases with age (over 75 years) (Reisen 2003). Underlying dis-
eases such as diabetes, hypertension, chronic alcoholism and arteriosclerosis predis-
pose to severe infection accompanied by a tragic ending (Brinker and Monath 1980).
From clinical data obtained during the first SLEV outbreak in Cordoba (Argentina)
stood out, in the preludes, cephalea, somnolence, and some degree of temporo-spa-
tial disorientation in the first 72 h after the symptoms appeared (Spinsanti et al.
2008). Bradypsychia and temporo-spatial disorientation were the prevailing find-
ing. In some patients the symptoms progressed with a significant depression of the
sensory system, and some of them needed assisted mechanical ventilation. There
were observed tremors in the face, hands, and feet with myoclonia episodes. Other
patients presented holocranial cephalea accompanied by photophobia, some with
mixed aphasia and others with episodes of tonic clonic seizures. The encephalitis
frequency (including meningoencephalitis) varied from 80% of cases in persons
under 20 years to 95% in those over 60 years. Figure 11.2 shows the age distribution
for the most common clinical syndromes observed with SLEV infection.
Symptoms can spontaneously resolve during any stage of the disease with com-
plete recovery. The acute disease can be followed by the “convalescent fatigue syn-
drome” in <50% of the patients, characterized by asthenia, irritability, tremors,
somnolence, depression, memory loss, and cephalea, and can last up to three years.
Approximately 20% of these patients can present symptoms that persist for long peri-
ods, such as speech and sensory-motor alterations and tremors. Elderly and severity
of the acute disease seem to predispose to these sequels (Finley and Riggs 1980).
The cerebrospinal fluid (CSF) is usually under normal pressure and contains from
ten to hundreds of mononuclear cells (≤500), mainly lymphocytes. However, at the
beginning of the disease predominantly polymorphonuclear leucocytes can be pres-
ent (Luby 1994). Protein concentration can be slightly high, while glucose levels
Encephalitis
Meningitis
Percent of cases with each syndrome
100% Febrile headache

90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
0–19 20–39 40–59 >60
Age group (years)
FIGURE 11.2 Frequency of clinical symptoms notified in St. Louis encephalitis cases.
(Reproduced from Spinsanti et al. 2008.)
FIGURE 11.3 Magnetic resonance imaging (MRI) from a patient suffering from SLEV
encephalitis. The cranial axial T2-weighted MRI shows hyperintense abnormalities in the
substantia nigra with major compromise on the right side (arrows).
remain normal. In peripheral blood there is mainly a polymorphonuclear leucocy-

tosis. The interval observed between the beginning of the disease and death has
been consistent in all the studies. Approximately 50% of the fatal cases die during
the first week of the disease’s appearance and 80% during the following two weeks.
It is difficult to assign mortality directly to SLEV or to secondary complications or
underlying diseases. In general, death due to direct viral damage on CNS occurs
without secondary complications appearing during the first 2 weeks, and death late
is probably due to nonrelated causes (Brinker and Monath 1980).
The electroencephalogram shows a diffuse and generalized diminution and an
amorphous and generalized delta wave activity (Brinker et al. 1979). Computed
tomography scans may be normal, but magnetic resonance images have disclosed
T2-weighted hyper-intense abnormalities in the substantia nigra, consistent with
edema (Cerna 1999) (Figure 11.3).
11.8 DIAGNOSIS
Table 11.3 shows laboratory criteria for SLEV infection diagnosis. In the human
infection with SLEV, patient’s age, season of the year, place of residence and exposi-
tion, and information about similar cases occurring in the community are important
epidemiologic data in the differential diagnostic compared with other infections. It is
essential to discard other agents such as bacteria, mycobacteria, spirochetes, fungal
and viral infections like herpes, enterovirus (that are spread also during summer),
TABLE 11.3
Laboratory Criteria for SLEV neurological Infection Diagnosisa
Suspicious case: Is every person presenting
• Compatible clinical symptoms (febrile illness, temperature >38°C, with neurological signs, aseptic
meningitis, encephalitis, etc.).
• Symptoms onset during a known period of Flavivirus spreading.
Probable case: Probable illness that satisfies the anterior criteria and at least one of the following:
• Serum or CSF IgM without seroconversion by NT in serum paired samples.
• IgM and IgG in a single serum or CSF sample.
Confirmed case: Febrile illness associated to neurological manifestations and at least one of the
following laboratory results:
• Viral isolation or antigen or viral genome demonstration in tissue, blood, CSF or other organic
fluids.
• Specific IgM in CSF and/or serum and sero-conversion by NT technique in serum or CSF paired
samples.
a The following diagnosis criteria were adapted from Moore et al. 1993.
and other arboviruses (WNV, Rocio Virus, or Eastern, Western, and Venezuelan
Equine Encephalitis Viruses). In elder people it is possible to mistake a SLEV infec-
tion with a cerebrovascular accident (Burke and Monath 2001).
Viral isolation from serum or CSF is very difficult due to the shortness of the
viraemia period; viraemia can precede the symptoms for a few days. SLEV has been
frequently recovered in fatal cases from brain and also from spleen, liver, lung, and
kidney (Calisher and Poland 1980). The specific molecular diagnosis has a high sen-
sibility and specificity degree to detect SLEV when it is compared with traditional
techniques such as cellular culture plates assay and enzyme immunoassay with anti-
gen capture (Howe et al. 1992; Kramer et al. 2002). Several authors have developed
different RT-PCR methods able to detect a high number of SLEV strains (Kramer
et al. 2002; Lanciotti and Kerst 2001; Chiles et al. 2004). Recently, Re et al. (2008)
have developed a SLEV specific RT-nested PCR more sensitive than RT-PCR, allow-
ing a low detection limit (7 PFU-plaque forming unit). This method was able to
amplify the genome of SLEV strains of different geographic origins and also to
detect viral RNA from mosquito homogenates and from a cell culture infected with
two SLEV strains recently isolated in Argentina (Diaz et al. 2006, 2012).
The definitive diagnosis in humans depends on the serology almost exclusively.
The presumptive diagnosis of recent infection is based on the detection of immuno-
globulin M (IgM) antibodies by the IgM capture technique enzyme-linked immu-
nosorbent assay (MAC-ELISA) in CSF and/or serum (Martin et al. 2000). Serum
antibodies type IgM specific appear in the first 4 days after the beginning of the dis-
ease, with a peak at 7–14 days and decline to extinguish generally at 60 days (Burke
and Monath 2001). The rapidity of this test is of great utility on the early diagnosis
and epidemiological surveillance. However, the persistence of IgM antibodies has
been detected in some patients. Spinsanti et al. (2011) have detected specific SLEV
IgM antibodies in serum until more than a year after the beginning of the symptoms,
indicating that the presence of IgM is not systematically associated with recent infec-
tions. On the other hand this test uses a conjugated monoclonal antibody (6B6C-1)
that detects antibodies against different Flavivirus species (Monath et al. 1984).
Specific diagnosis usually relies on serological tests on appropriately timed acute
and convalescent samples. The IgG-type antibodies can be detected by ELISA tests,
Hemagglutination Inhibition (HI) and Neutralization (PRNT); the IgG-type anti-
bodies titers increase from the week following to the beginning of the symptoms.
The HI test detects mainly group-reactive antigens and is useful for screening stud-
ies due to its sensibility, but it has the disadvantage of being low-specific especially
in geographic areas where more than one flavivirus is circulating. The PRNT is the
most specific test, “gold standard” for arbovirus. Neutralizing antibodies (NTAbs)
appear in first place and in higher number than HI antibodies; they reach a maxi-
mum titer at 7–21 days after the disease’s onset and persist usually during the whole
life. The PRNT is used to confirm results derived from serological surveys realized
through HI and MAC-ELISA (Calisher and Poland 1980) (Figure 11.4).
Certain flaviviruses seem to share more antigens than others; antibodies against
Flavivirus group members closely related are more difficult to differentiate with
low-specificity techniques such as HI and MAC-ELISA (Martin et al. 2004). One
of these groups is that integrated by SLEV, JEV WNV, and MVEV (Table 11.1).
Other flaviviruses that share extensively with SLEV are Rocío virus, Ilheus virus,
and DENV (Calisher and Poland 1980).
Spinsanti et al. (2011) provide data about the patterns of response of the subclasses
IgG1, IgG2, IgG3, and IgG4 produced during a SLEV natural infection (acute and
convalescent phases) and in humoral long-term immunity. They had demonstrated
the persistence of the four IgG isotypes for more than one year in patients infected by
SLEV. However, IgG1 isotype was present at the highest titers, with a peak between
Sera/CSF
(after 4-day onset)
IgM detection
(MAC-ELISA)
(+) (–)
PRNT
Paired serum samples If patient has compatible diseases
(test against Flavivirus with SLE encephalitis, ask for a
circulating in the area) second sample and repeat test.
FIGURE 11.4 Algorithm for St. Louis encephalitis serological diagnosis.

day 8 and 30 after onset of the disease, coincident with the highest titer of NTAbs.
IgG1 antibodies have been described as the main subclass in infections by WNV, as
well as those with the highest neutralizing activity, which could be responsible for
viral clearance (Hoffmeister et al. 2011).
11.9 ECOLOGY
SLEV presents an exclusive distribution in the American continent, where it is
widely extended (Figure 11.5). In the mid-1980s, the demographic variation between
the different SLEV strains through molecular studies of genetic and biological vari-
ability was demonstrated.
Based on the envelope’s gen complete sequence, SLEV strains have been classi-
fied in 7 genotypes (I-VII), being genotypes I and II widely distributed in the USA
and the others in Central and South American countries (Kramer and Chandler 2001)
(Figure 11.5). Recent studies of SLEV molecular diversity characterization indi-
cate a new genotype’s presence (genotype VIII) exclusive of the Amazonia region
I
II
I II
I I
II V
II II
II
IV
VI
VIII
V
V
II
III VII
II V
FIGURE 11.5 Geographic distribution of St. Louis encephalitis virus genotypes.

(Brazil) (Rodriguez et al. 2010). The phylogenetic analysis indicated that different
viral strain isolations had created a monophyletic group in which most of the strains
are grouped according to the geographic origin (Auguste et al. 2009; Kramer and
Chandler 2001). In a recent phylogeographic study it has been postulated that SLEV
would originate in South America and migrate to the northern countries, resulting
in a limited interchange (Auguste et al. 2009). Recently, it was detected the presence
of genotype V (abundant in South America) in the state of Florida (Ottendorfer et
al. 2009), while genotype I was found in mosquitoes collected in Argentina (Diaz et
al. 2012). This evidence would indicate an effective introduction of SLEV strains in
regions between North and South America, probably through bird migration, as it
was proposed for WNV by Diaz et al. (2008a).
In the US, SLEV has been one of the main causes of arbovirus encephalitis epi-
demics until the introduction of WNV in 1999. Most of the clinical cases have been
reported by the states of Texas, Florida, southeastern states, and the Ohio River’s
basin (Reisen 2003). By the contrary, urban epidemics of encephalitis due to SLEV
in other American countries are rare, focal, or of small magnitude or remain unde-
tected. This low incidence in Central and South America may be due to an inad-
equate case notification system, laboratory diagnosis deficiencies, attenuated viral
strains spreading, and/or enzootic cycles involving mosquitoes that do not often feed
on humans (Spence 1980).
SLEV is maintained in nature by transmission between different Culex spp. mos-
quito species and passeriform and columbiform birds (Figure 11.6). The members
in this transmission network vary according to geographic localization and time
of the year. In the US, SLEV ecology is well characterized, while for the rest of
the American continent it remains practically unknown. Excepting Argentina where
research has been done, moving forward SLEV ecological characterization (Diaz
2006, 2008b, 2009, 2012; Flores et al. 2010).
In the eastern and western regions of the US, SLEV transmission networks are
separated by epidemiological differences based on the virus transmission’s ecological
determinants (Reisen 2003). In the eastern states, main vectors belong to the Culex
pipiens complex (Culex pipiens pipiens and Culex pipiens quinquefasciatus) and its
main hosts are house sparrows (Passer domesticus). These peridomestic mosqui-
toes vectors develop frequently in rich organic material water such as sewers and
peridomestic water reservoirs. These mosquitoes are spread in urban and suburban
ambient densely populated, especially where sanitary conditions are deficient. In the
western regions of the USA, the main vector mosquito is Culex tarsalis. This specie
reproduces in flooded and irrigated soils, and in industrial or urban residual water
(Mitchell 1980). Humans are frequently exposed in rural areas, often determined by
recreational and working activities. Periurban and wild birds act as hosts, mainly those
abundant in agricultural areas close to water sources such as house sparrows (Passer
domesticus) and house finches (Carpodacus mexicanus) (McLean and Bowen 1980).
When all the conditions are favorable for viral transmission (quick amplification,
with a progressive increment in number of infected individuals, infective vectors and
hosts), humans and other mammals can be infected accidentally (dead-end host).
At this point, other environmental and behavioral factors that determine human
exposition to mosquitoes acquire epidemiologic relevance. Humans and domestic
Hosts
Passeriformes
Columbiformes
Birds
St. Louis encephalitis
virus
Dead end host

Human and
other mammals
Vectors
FIGURE 11.6 Transmission cycle of St. Louis encephalitis virus.
mammals are excluded from the basic transmission cycle because the viraemia titers
are insufficient to infect vector mosquitoes (Monath 1980).
The “overwinter” mechanism by which the virus survives during winter, is
unknown; however, studies suggest that the virus could persist locally in verte-
brate hosts (birds, bats) with viraemia resurgence in springtime, contributing to the
local transmission’s re-initiation (Reisen et al. 2001). Other data suggest overwinter
through vertical transmission in mosquitoes of the Culex pipiens complex or the
overwinter hibernantion in adult mosquitoes (Flores et al. 2010; Monath and Tsai
1987).
In Central and South America, SLEV was isolated from humans in Argentina,
Brazil, Panama, and Trinidad; from birds in Brazil, Haiti, Jamaica, Mexico,
Panama, and Trinidad; and from arthropods in Argentina, Brazil, Ecuador, French
Guyana, Guatemala, Jamaica, Mexico, Panama, and Trinidad (Diaz et al. 2006;
Monath 1980; Rocco et al. 2007; Sabattini et al. 1998). In these regions, the virus
has been isolated from 11 different mosquito’s genera, including Culex nigripalpus
and Culex quinquefasciatus (Spence 1980). Strong evidence support birds as hosts.
Several viral strains have been isolated from 27 bird species (cormorants, egrets,
pigeons, thrushes, celestines) in six different countries; the mocking birds (Mimidae
Family) could be implicated in transmission in Jamaica, and certain formicaridos
(Formicarius canalis, Myrmotherula axillaris, Myrmotherula hauxwelli) in forest
regions of Brazil. Although, wild mammals (micro-rodents, sloths) and domestic

mammals have been found infected, the evidence supporting their role as hosts are
scarce (Spence 1980).
In Argentina the SLEV transmission cycles have been partially clarified. Even
when antibodies against SLEV have been detected in domestic mammals such as
horses, cows, sheep, and goats, they would only have relevance as dead-end hosts
(Monath et al. 1985; Sabattini et al. 1998).
Serological studies have detected the presence of birds from families Furnaridae,
Columbidae, Tyranidae, Fringilidae, Icteridae, Ardeide, and Phytotomidae infected
with SLEV (Sabattini et al. 1998). Experimental inoculation studies had demon-
strated that Eared Dove (Zenaida auriculata) and Picui Ground-dove (Columbina
picui) develop viraemia sufficiently high to infect Culex quinquefasciatus mosqui-
toes (Diaz 2009; Diaz et al. 2008b). On the contrary, House Sparrows in Argentina,
differently from the USA, do not play a significant role in transmission and mainte-
nance of SLEV (Diaz 2009).
Regarding the vector, SLEV has been isolated from Culex quinquefasciatus and
Culex spp. in Santa Fe province (Argentina) (Mitchell et al. 1985). In Cordoba City,
several mosquitoes’ species (Aedes aeygpti, Aedes albifasciatus, Aedes scapularis,
Anopheles albitarsis, Culex apicinus, Culex interfor, Culex quinquefasciatus, and
Psorophora sp.) have been detected infected by SLEV during enzootic periods (Diaz
et al. 2012). Vector competence studies carried out in Culex quinquefasciatus col-
lected in Esperanza (Santa Fe) and Cordoba showed this species’ ability to infect and
transmit SLEV strain 78V-6507 (Diaz 2009; Mitchell et al. 1980).
Activity patterns present characteristics of a temporal seasonal nature and spatial
heterogeneous nature, coincident with the focal nature of the transmission cycles that
arbovirus have in other regions such as the USA. The greater viral activity periods
are concentrated in the summer season (February and March), starting the spreading
in November and December (spring). According to recent studies by Diaz (2009),
in Cordoba would be a primary transmission network formed by Culex quinque-
fasciatus and Culex interfor mosquitoes, acting as avian host Eared Dove (Zenaida
auriculata) and Picui Ground-dove (Columbina picui), who can also be part of the
transmission network in epidemic periods.
11.10 EPIDEMIOLOGY
SLEV epidemiological transmission patterns reflect the interactions between humans
and the virus, its reservoirs, and vector mosquitoes (Day 2001; Reisen 2003). The
sporadic appearance of cases in the USA has been documented since 1933. From
1964 through 2010, an average of 102 cases were reported annually (range 2–1967)
(CDC 2011). In USA western states, SLEV has an endemic transmission pattern.
Epidemics are limited due to the high immunity level in populations with long resi-
dence in the area, setting aside to a less sensitive part of the adult population; in
consequence, cases occur frequently in children and young adults (Reisen 2003).
Infections occur mainly in rural areas associated to the vector’s habitat. In the
central-eastern region, average outbreaks occur sporadically, followed by periods,
sometimes long, without viral transmission’s evidence. The intermittent occurrence
of outbreaks has been associated with climatic factors, such as temperate winter,
rainy spring, and hot and dry summer (Monath 1980). High temperatures are favor-
able to the virus replication in mosquito, while rains below normal levels allow the
formation of small pools in drainage systems, leading to big mosquito populations
of the complex Culex pipiens. Sometimes small warning outbreaks occurred fol-
lowed by big outbreaks in the same place the next year, probably as a result of an
elevated viral amplification in the second year. The virus intermittent activity pro-
duces that the human population may be immunologically susceptible, allowing the
virus’ introduction and outbreak occurrence. Epidemics have occurred frequently
in urban areas or their periphery and were restricted to areas where environmental
factors are associated to an increased reproduction or exposition of mosquitoes. In
general, these areas are of low socioeconomical level, with precarious housing, or
with sewage effluents (Luby 1979; Monath 1980; Tsai 1991). On the contrary, during
the 1933 outbreak, the highest case rate was observed in the population with greater
economical resources and low density housing, but with open drains, pools, creeks,
and open spaces (Froeschle and Reeves 1965).
Culex pipiens and Culex quinquefasciatus are highly domestic mosquito species
that prefer the interior of houses or their surroundings. A higher infection risk has
been observed in houses without mosquito nets or air conditioning, with more cases
in women, probably due to the latter’s exposition to the peridomestic vector (Marfin et
al. 1993; Monath 1980; Tsai et al. 1988). Nevertheless, in the epidemics that occurred
in Florida in 1990, people with outdoor occupations had the highest risk of infection
(Meehan et al. 2000). In the west of the USA, where the virus is transmitted by Culex
tarsalis, rural work is a risk factor and the case rate is higher in men (Reisen and Chiles
1997). A serological survey performed during the Florida epidemic in the 1990s indi-
cated that infection rates were highest in people with outdoor occupations. In that case,
health warnings, closing recreational parks such as Walt Disney World at night, and the
use of personal protection seemed to reduce infection rates (Meehan et al. 2000).
Since 2002, SLEV is experiencing a reemergence as a human encephalitis etio-
logical agent in the south cone of South America, producing small outbreaks and
epidemics in Argentina and Brazil (López et al. 2011; Mondini et al. 2007; Rocco
et al. 2005; Spinsanti et al. 2008). Serological studies carried out in Argentina indi-
cate a wide distribution and endemicity of SLEV in temperate and subtropical areas
(central and northern Argentina) (seroprevalence from 10% to 68%) (Sabattini et al.
1998; Spinsanti et al. 2000, 2002). In two populations from Cordoba (Argentina), the
risk of infection are associated with the presence of garbage dumps near dwellings,
the practice of outdoor activities at night, and the place of residence (Spinsanti et
al. 2007). The practice of outdoor activities at night increased the chance of infec-
tion, probably due to the nocturnal habits of the Culex mosquitoes. During summer–
autumn of 2005, in this city, SLEV encephalitis epidemic occurred in humans, and
it was the first one in South America (Spinsanti et al. 2008). There were 47 reported
cases, of which most were hospitalized. The cases predominantly occurred among
people 60 years and older. Nine deaths were reported. In this study, a correlation
between age and disease severity was observed. Advanced age is associated with a
greater severity of disease caused by several flavivirus (WNV, JEV) (Brinker and
Monath 1980; Johnson 2002).
In the following years, there were only isolated cases registered, whereas between
2010 and 2011, there was an increase in notified cases with neurological compromise
in several provinces of Argentina (Fabbri et al. 2011; Vergara Cid et al. 2011; Seijo
et al. 2011).
Serological evidence of viral activity exists in other countries; some of them do
not have records of isolations, such as Uruguay, Colombia, Venezuela, El Salvador,
and Caribbean Islands. Many of the serological studies had been carried out with
low specific techniques; in consequence, those results are uncertain because there
may be serological cross reactions with other flaviviruses spread in the region as
DENV (Monath 1980).
11.11 CONCLUSION
SLEV is an emerging flavivirus in the south cone of the American continent, in
particular in Argentina and Brazil, where advances are being made for its eco-
epidemiological characterization. Most of the bordering countries (Bolivia, Paraguay,
Uruguay, Chile, Peru, Ecuador, and Colombia) do not have research programs of
regional relevance focused in this viral infection.
The SLEV epidemiology is driven for climatic, entomological, viral, and host fac-
tors that form a complex network of interactions not completely understood.
From the explained above it is very important that the design and adjustment of coop-
erative research programs move forward in this viral infection study; sharing previous
experiences with countries with more episodes and counseling to public health systems
about prevention policies and detection of early febrile and neurological symptoms.
SLEV is a neglected diseases; however, knowing and understanding its eco-
epidemiology are of concern. Other sympatric flavivirus cause similar symptoms in
human population making diagnosis more difficult.
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12 Powassan Virus
Laura D. Kramer, Alan P. Dupuis II,
and Norma P. Tavakoli
CONTENTS
12.1 Introduction................................................................................................... 261
12.2 Classification and Distribution...................................................................... 262
12.2.1 Canada...............................................................................................264
12.2.2 United States...................................................................................... 265
12.2.3 Mexico............................................................................................... 265
12.2.4 Russia................................................................................................. 265
12.3 Biological Properties (Virus Structure, Genome Organization,
Protein Functions, Replication Cycle)...........................................................266
12.4 Clinical Presentation..................................................................................... 267
12.5 Diagnosis....................................................................................................... 270
12.6 Pathology and Pathogenesis........................................................................... 273
12.7 Ecology and Epidemiology............................................................................ 275
12.7.1 Transmission Cycle............................................................................ 276
12.7.2 Arthropod Hosts................................................................................ 276
12.7.2.1 Ixodes cookei...................................................................... 276
12.7.2.2 Ixodes scapularis................................................................ 277
12.7.2.3 Dermacentor andersoni...................................................... 277
12.7.2.4 Other Species...................................................................... 278
12.7.3 Disease Incidence.............................................................................. 278
12.8 Prognosis and Treatment............................................................................... 279
12.9 Conclusion.....................................................................................................280
Acknowledgement..................................................................................................280
References...............................................................................................................280
12.1 INTRODUCTION
Powassan virus (POWV; family Flaviviridae, genus Flavivirus) is a member of
the mammalian tick-borne virus group (Grard et al. 2007). The virus appears to be
widely distributed in its enzootic hosts in North America and Far East Asia (Mandl
et al. 1993). Remarkable disease is rare (Hoang Johnson et al. 2010), but encephalitis
in humans may be associated with significant neurologic sequelae. The first case was
identified in 1958 in Powassan, Ontario, Canada (McLean and Donahue 1959). The
virus appears to be increasing in prevalence in the United States, possibly as a con-
sequence of improved diagnostics leading to increased detection, but equally likely
as a consequence of the proliferation of vector tick populations or increased contact
261
between infected ticks and humans due to lifestyle changes. An increased incidence
of the closely related TBEV has also been noted in Europe, more likely due to socio-
economic factors than climate warming (Godfrey and Randolph 2011). POWV is
comprised of two lineages, lineage I (POWV) and II (Deer tick virus; DTV), with
distinct transmission cycles (Ebel et al. 2001). It has been speculated that DTV may
lead to milder cases (Ebel et al. 1999); however, at least two recent cases of DTV that
were fatal (Tavakoli et al. 2009) (and unpublished data) demonstrate this virus has
the potential to be virulent. This chapter will address the biology, epidemiology and
ecology, pathology, and diagnostics of these two viruses.
12.2 CLASSIFICATION AND DISTRIBUTION

Flaviviruses (family Flaviviridae, genus Flavivirus) fall into distinct ecologic groups,
i.e., mosquito-borne (Culex or Aedes), tick-borne, those with no known vector, and
agents that infect arthropods only (Gaunt et al. 2001). The genus was originally
divided serologically into groups (Porterfield 1980; Calisher et al. 1989), with the
tick-borne flaviviruses grouping together. Molecular genetic analyses have upheld
the original antigenic and ecologic groupings. Further intensive analyses have indi-
cated that the tick-borne flaviviruses can be divided into mammalian, seabird, and
Kadam tick-borne flavivirus groups (Grard et al. 2007) (Figure 12.1). The mamma-
lian tick-borne virus group was originally referred to as the tick-borne encephalitis
serocomplex. It was suggested that ticks that feed on mammals and sea birds may
constitute the evolutionary bridge among these three groups (Grard et al. 2007).
Tick-borne viruses share a common ancestor with the other flaviviruses (Thiel et al.
2005), and while closely related overall to each other, they are genetically divergent
(Figure 12.2), widespread (Gritsun et al. 2003), and epidemiologically important
(Demina et al. 2010; Gritsun et al. 2003). Extensive phylogenetic analysis suggested
that acquisition of tick-borne transmission is a derived trait within the flaviviruses
(Cook and Holmes 2006). Powassan virus is the most genetically divergent member
of the TBEV antigenic complex (Calisher et al. 1989). The virus exists as two lin-
eages, POWV and DTV (Ebel et al. 2001; Kuno et al. 2001), in which the E protein
nucleotide and amino acid sequences differ by 14.6% and 4%, respectively (Beasley
et al. 2001), and the nucleotide sequence of the entire genome of DTV differs by
16% from POWV (Kuno et al. 2001). This was interpreted as indicating that they
represent distinct genetic subtypes (“genotypes”) of the same virus type. The genetic
distance between these two viruses suggests they diverged and evolved indepen-
dently into two distinct ecological niches from a single origin (Telford et al. 1997).
DTV and POWV coexist, and DTV has been found throughout the historical range
of POWV (Kuno et al. 2001). Viruses from both lineages have been responsible for
human illness (Kuno et al. 2001; Tavakoli et al. 2009). POWV and DTV appear to
be genetically stable over time suggesting evolutionary constraint (Pesko et al. 2010;
Kuno et al. 2001) consistent with other arboviruses. The viruses are also conserved
over distance, as demonstrated by POWV isolated from a human case in Primorsky
krai, Russia, in 2006, which was 99.8% similar to the LB strain that was isolated
in Canada in 1958 (Leonova et al. 1991). In this chapter, unless otherwise stated,
POWV includes both lineage I and II.
Powassan Virus 263
DENV1 DENV3
DENV2 JEV MVEV KRV
SLEV WNV
DENV4 KUNV
0.1
YFV
YFV17D
CFAV
YOKV
97
RBV
98
KADV
GGYV
MMLV 71
MODV SREV TYUV DTV

MEAV KSIVRFV
APOIV POWV
KFDV
AHFV
LGTV
SSEV OHFV UVE
TBTBEVOHF
LI/LIN
LIV
TBEVEU EV S FE
b
TSEV GGEV
0.1
AHFV: Alkhurma hemorrhagic fever virus MODV: Modoc virus

APOIV: Apoi virus MLV: Montana myotis leukoencephalitis virus
CFAV: Cell fusing agent virus MVEV: Murray valley encephalitis virus
DENV1–4: Dengue virus types 1–4 OHFV: Omsk hemorrhagic fever virus
DTV: Deer tick virus POWV: Powassan virus
GGEV: Greek goat encephalitis virus RBV: Rio bravo virus
GGYV: Gadget gully virus RFV: Royal farm virus
JEV: Japanese encephalitis virus SLEV: St. Louis encephalitis virus
KADV: Kadam virus SREV: Saumarez reef virus
KFDV: Kyasanur forest disease virus SSEV: Spanish sheep encephalomyelitis virus
KRV: Kamiti river virus TBEV: Tick-borne encephalitis virus
KSIV: Karshi virus TSEV: Turkish sheep encephalitis virus
KUNV: Kunjin virus TYUV: Tyulenly virus
LGTV: Langat virus WNV: West Nile virus
LIV: Louping ill virus YFV: Yellow fever virus
MEAV: Meaban virus YOKV: Yokose virus
FIGURE 12.1 Phylogenetic analysis based on complete polyprotein sequences. Phylogenetic

reconstruction was performed using the maximum likelihood method. All branchings were
supported by quartet puzzling frequencies at 99% or 100% except at the forks where a value
is indicated. The tick-borne flavivirus group is enclosed in a circle with a solid line, the
mosquito-borne flavivirus group in a circle with a dashed line, and the no-known vector flavi-
virus group in a circle with an intermittent dashed line. To improve the legibility of the tree,
the distal part of the TBFV branch is presented with a 3.5× magnification. (From Grard, G.
et al., Virology, 361, 80, 2007. With permission.)
CFAV: Cell fusing agent

FEV-TBEV: Far eastern subtype
LIV GGYV: Gadgets Gulley virus
KADV: Kadam virus
OHFV KSIV: Karshi virus
W-TBEV
FE-TBEV KFDV: Kyasanur forest disease virus
SSEV TSEV LGTV: Langat virus
POWV LIV: Louping ill virus
Mammalian lineage KSIV OHFV: Omsk hemorrhagic fever virus
KADV
RFV POWV: Powassan virus
Origin Seabird KFDV RFV: Royal farm virus
lineage SSEV: Spanish sheep encephalomyelitis
CFAV
TSEV: Turkish sheep encephalomyelitis
LGTV W-TBEV: Western subtype
GGYV
FIGURE 12.2 Evolution/dispersal of mammalian tick-borne viruses. (From Gritsun, T. S.

et al., Adv. Virus Res., 61, 317, 2003. With permission.)
POWV is the only tick-borne flavivirus detected in the Nearctic zoogeographic

region including mainly Canada and the United States (Clarke 1964), and is distrib-
uted rather widely within this region. Serological evidence of the virus in wild mam-
mals has been detected from the Pacific to the Atlantic coast, in British Columbia,
Alberta, Ontario, Nova Scotia, and in southeastern Siberia. Details follow describing
hosts in diverse locations.
12.2.1 Canada
POWV has been isolated from ticks (Ixodes cookei, Ix. marxi) (Artsob et al. 1984,
1989; McLean et al. 1964, 1966, 1967; McLean and Larke 1963) and small mam-
mals [woodchucks (Marmota monax), red squirrel (Tamiasciurus hudsonicus),
striped skunk (Mephitis mephitis)] in Ontario (McLean et al. 1964; McLean and
Larke 1963; Artsob et al. 1986). Serologic evidence from hemagglutination inhi-
bition (HI), complement fixation (CF), and/or neutralization tests (NT) have been
obtained from snowshoe hares (Lepus americanus) in Nova Scotia, from snow-
shoe hares and Richardson’s ground squirrels (Urocitellus richardsonii) in Alberta
(Zarnke and Yuill 1981a,b; Hoff Yuill et al. 1970), from chipmunks (Tamias stria-
tus), Columbian ground squirrels (Ur. columbianus), golden-mantled ground squir-
rels (Callospermophilus lateralis), and marmots (Marmota sp.) in British Columbia
(McLean et al. 1968, 1970), and from long-tailed ground squirrels (Ur. undulates)
in the Yukon Territory (McLean et al. 1972). Human cases have been detected in
Quebec as well as Ontario (Mahdy et al. 1979, 1982; McLean and Donahue 1959;
McLean et al. 1960; Partington et al. 1980; Rossier et al. 1974).
Powassan Virus 265
US cases 2001–2011
NY 12, MN 8,
WI 6, ME 4,
VT 1, MI 1,
VA 1
POWV isolates
Serologic evidence of POWV transmission
FIGURE 12.3 Map of North America indicating locations of viral isolates, serological evi-
dence of virus activity, and human cases from 2001 to 2011.
12.2.2 United States
POWV is distributed primarily throughout the northeastern and midwestern states.
Virus has been isolated from ticks, humans, and/or small mammals in New York
(Centers for Disease Control and Prevention 1972, 1975; Deibel et al. 1979; Embil et al.
1983; Hinten et al. 2008; Tavakoli et al. 2009; Srihongse et al. 1980; Whitney et al. 1968;
Whitney 1963, 1965), Connecticut and Massachusetts (Telford et al. 1997; Main et al.
1979), West Virginia (Artsob 1989), Colorado (Thomas et al. 1960), California (Johnson
1987), Minnesota (Minnesota Department of Health 2011), Wisconsin (Brackney et al.
2008, 2010; Ebel et al. 2000), and South Dakota (Keirans and Clifford 1983). Additional
serologic evidence has been detected in Maine, Vermont, Pennsylvania, and Michigan
(Artsob 1989; Centers for Disease Control and Prevention 2001; Hoang Johnson et al.
2010; Main et al. 1979; Telford et al. 1997; Keirans and Clifford 1983) (Figure 12.3).
12.2.3 Mexico
Reeves et al. found HI antibodies to POWV in sera collected from humans, rodents,
and chickens in Hermosillo, Mexico (Sonora State) (Reeves et al. 1962). Reported
seropositivity rates from this study were 4% in humans, 6% in chickens, and 11% in
rodents. Confirmatory plaque reduction neutralization tests (PRNTs) were not per-
formed, so the significance of these results may be diminished. HI tests, especially
for flavivirus infections, are not specific (Srihongse et al. 1980).
12.2.4 Russia
POWV was first isolated in Russia in 1972 from a pool of Haemaphysalis longicor-
nis (neumanni) ticks (L’Vov et al. 1974) and apparently co-circulates with TBEV
in Russia in the same locations (Leonova et al. 2009). Since the initial isolation,
the virus has been found throughout the Far Eastern Russia region of Primorsky
krai. Fourteen cases have been reported through 1987 (Leonova et al. 1987). POWV
isolates have been reported from humans, ticks, small mammals, and mosquitoes
(L’Vov et al. 1974; Leonova et al. 1987, 1991; Tkachenko et al. 1976; Kislenko et al.
1982). The identification of POWV-positive mosquitoes is interesting, but of ques-
tionable significance without accompanying vector competence experiments.
12.3 BIOLOGICAL PROPERTIES (VIRUS STRUCTURE,

GENOME ORGANIZATION, PROTEIN
FUNCTIONS, REPLICATION CYCLE)
Structurally, the POWV virion is similar to other members of the Flavivirus genus
(Mandl 2005). The virion is enveloped, with polyhedral nucleocapsid symmetry.
Virus particles are spherical, ~50 nm in diameter with a single envelope protein (E)
and a membrane protein (M) on the surface surrounding a lipid bilayer derived from
the host cell. The nucleocapsid core consists of single-stranded RNA complexed
with multiple copies of the capsid protein. The ~11-kb nucleotide viral genome com-
prises 3415 amino acids in a single open reading frame encoding three structural
proteins, capsid, prM/M, and E, and seven nonstructural proteins, NS1, 2a, 2b, 3, 4,
(a)
≈ 10.8 kb 3´
5´ CAP (I)
7mGpppAm
ORF OH
5´ NCR 3´ NCR
5´ CS RCS2 CS2 CS1 3´SL
Mosquito-borne flaviviruses
5´ CS R3 R3 PR
Tick-borne flaviviruses An
3´SL
(b) ≈ 3400 aa
Structural genes Nonstructural genes
?
NH3 PROT HEL MTase RdRP COOH
C prM E NS1 NS2A 2B NS3 4A NS4B NS5
PROT
pr M 2Aα cleaved NS3
FIGURE 12.4 Flavivirus genome structure and expression. (a) Genome structure and RNA
elements. NCR, noncoding region. Models of functionally important secondary and tertiary
structures within the 5ʹ and 3ʹ NCR and the coding region are shown with predicted hairpin
loops indicated. (b) Polyprotein processing and cleavage products. Structural proteins are C,
prM, E, while nonstructural (NS) proteins are NS1, NS2A and 2B, NS3, NS4A and 4B, NS5.
Cleavage sites for host signalase (♦), the viral serine protease (↓), furin or related protease (▾), or
unknown proteases (?) are indicated. (From Lindenbach, B. D. et al., Flaviviridae: the viruses
and their replication, in Fields Virology, eds. D. M. Knipe and P. M. Howley, Lippincott-
Raven Publishers, Philadelphia, 2007, 1101. With permission.)
Powassan Virus 267
4a, 5. Untranslated regions of 100 and ~500 nt, respectively, are found at the 5ʹ and 3ʹ
ends, respectively. A type I cap m7 GpppAmpN2 exists at the 5ʹ end. While the flavivi-
rus genome generally lacks a 3ʹ polyadenylated tail (Westaway et al. 1985; Lindenbach
et al. 2007), tick-borne viruses are an exception, as a poly-A structure is found in
some strains. Cleavage of the polyprotein into individual proteins occurs co- and post-
translationally through cellular and viral proteases. The differences in genome struc-
ture and expression between tick-borne and mosquito-borne flaviviruses are illustrated
in Figure 12.4 (Lindenbach et al. 2007). The majority of differences are found in the 5ʹ
and 3ʹ noncoding regions. The replication cycle of flaviviruses and TBEV specifically
have been well described (Mandl 2005) and will not be discussed further here.

POWV is transmitted to humans by tick bite. DTV has been demonstrated to be
transmitted to mice within 15 min of attachment by infected nymphal stage ticks
(Ebel and Kramer 2004), although it is possible that transmission to humans may take
longer. The first reported case of POW encephalitis occurred in Ontario, Canada, in
1958 (McLean and Donahue 1959). From 1958 to 1998, 27 cases of POWV disease
were reported in North America, and from 1999 to 2005, an additional nine cases
were reported in the United States (Hinten et al. 2008) and one in Canada (Ford-
Jones et al. 2002). The first case of POW encephalitis was reported in Russia in
1973 and 14 additional cases were reported between 1974 and 1989 (Leonova et al.
1991). In 2007 alone, six cases were reported in New York State (Centers for Disease
Control and Prevention 2009; Tavakoli et al. 2009), and from 2008 to June 2011,
eight cases were reported in Minnesota; two of the cases were reported in 2011 with
one fatality (Minnesota Department of Health 2011) (Table 12.1 and Figure 12.3),
five cases reported in New York, four cases in Wisconsin (2010), and one case in
Virginia (2009) (U.S. Geological Survey 2011). The number of POW cases reported
to the CDC strongly suggests an increase in incidence of disease in recent years
(Hinten et al. 2008; Ebel 2010). The clinical symptoms of some of the more recent
reported cases in North America are listed in Table 12.1.
The incubation period of POWV ranges from 8 to 34 days (Smith et al. 1974;
Rossier et al. 1974). The clinical syndromes most often associated with the infection
are encephalitis, meningoencephalitis, and aseptic meningitis. A review of the earli-
est reported cases of POW encephalitis indicated that the disease is characterized by
a sudden onset of fever with a temperature of up to 40°C and convulsions (Smith et
al. 1974; Rossier et al. 1974). A prodrome of nonspecific syndromes lasting 1–3 days
may include sore throat, drowsiness, generalized malaise, nausea, headache, myal-
gia, and disorientation (Smith et al. 1974). Patients showed signs of encephalitis with
vomiting, respiratory distress, prolonged fever, stupor, and possible convulsions.
Encephalopathy is common and in these cases includes such symptoms as gener-
alized weakness, ataxia, tremor, and ocular symptoms (Table 12.1) (Hinten et al.
2008). In some cases, a fine macular erythematous rash has been reported (Wilson
et al. 1979; Partington et al. 1980; Tavakoli et al. 2009; Rossier et al. 1974; Embil et
al. 1983; Smith et al. 1974; Kolski et al. 1998). Muscle weakness or rigidity and some
degree of paralysis have been observed in some patients (Table 12.1) (Hinten et al.
TABLE 12.1
Patient Symptoms and Outcomes in a Number of the Most Recent Reported
Cases of POW Encephalitis
Age Sex State Symptoms Outcome Reference
Child, age N/R Ontario, Fever, generalized Discharged with (Kolski et al.
N/R Canada seizures, altered level of normal outcome 1998)
consciousness
64 M Ontario, Drowsiness, mild right Died of massive (Gholam et al.
Canada facial weakness, clumsy pulmonary 1999)
movement, right side embolism
weakness, decreased
consciousness,
respiratory distress
70 M ME Muscle weakness, Discharged to (Centers for
somnolence, anorexia, rehabilitation Disease Control
fever, leukocytosis, facility and Prevention
anemia, left sided 2001)
hemiplegia, confusion
25 M ME Fever, headache, vomiting, Discharged to (Centers for
somnolence, confusion, rehabilitation Disease Control
inability to walk, bilateral facility and Prevention
hand twitching, bilateral 2001)
weakness in upper
extremities, lip smacking
66 M VT Somnolence, severe Discharged home (Centers for
headache, confusion, with cognitive Disease Control
bilateral leg weakness, difficulties and Prevention
slow speech, short term 2001)
memory loss
53 F ME Loss of balance, vomiting, Persistent (Lessell and
diarrhea, fever, ataxia, ophthalmoplegia Collins 2003)
diplopia, bilateral lateral
gaze palsy, dysarthria,
agitation, muscle
weakness, altered mental
status, ophthalmoplegia
74 F ME Headache, fever, myalgia, Outpatient (Hinten et al.
confusion, inability to rehabilitation 2008)
speak or walk, combative,
tremulous
69 M WI Abdominal pain, vomiting, No apparent (Hinten et al.
fever, chills, lethargy, neurologic 2008)
denied neurologic symptoms
symptoms
Powassan Virus 269
TABLE 12.1 (Continued)

Patient Symptoms and Outcomes in a Number of the Most Recent Reported
Cases of POW Encephalitis
Age Sex State Symptoms Outcome Reference
60 F MI Proximal muscle weakness Discharged to (Hinten et al.
in 4 extremities, diplopia, rehabilitation 2008)
fever, paralysis, facility
respiratory failure
83 M NY Fever, headache, altered Discharged to (Hinten et al.
mental status, stiff neck, nursing home 2008)
generalized muscle
weakness
91 F NY Fever, headache, altered Discharged to (Hinten et al.
mental status, stiff neck, nursing home 2008)
generalized muscle pain
and weakness
62 M NY Fatigue, fever, bilateral Fatal encephalitis (Tavakoli et al.
maculopapular palmar 2009)
rash, diplopia, dysarthria,
weakness in right arm
and leg
Child age N/R MN Severe neurological Required ongoing (ProMED Mail
N/R symptoms care 2009)
Adult age N/R MN Severe neurological Required ongoing (ProMED Mail
N/R symptoms care 2009)
60s M MN N/R Recovering (ProMED Mail
2011)
60s F MN N/R Fatal (ProMED Mail
2011)
Note: N/R, not reported.
2008; Gholam et al. 1999; Tavakoli et al. 2009; Lessell and Collins 2003; Goldfield
et al. 1973; Rossier et al. 1974; Jackson 1989; Partington et al. 1980). In one case,
temporal lobe involvement including olfactory hallucinations and the localization
of electroencephalogram (EEG) irregularity initially complicated the diagnosis as
such clinical evidence is typical of herpes simplex encephalitis (Embil et al. 1983).
Although definite focal features have been observed in some cases, specific temporal
lobe involvement has not been reported as a common feature of POW encephalitis
which is likely generalized throughout the brain (Embil et al. 1983). In the most
severe cases patients become comatose and a fatality rate of approximately 10% has
been reported (Artsob 1989; Ebel 2010).
The symptoms reported for POWV infections in Russia between 1974 and 1989
included fever, headache, nausea, vomiting, fatigue, drowsiness, neurological and
meningeal symptoms, paralysis, seizures, cerebellar ataxia, and spastic hemiparesis
(Leonova et al. 1991). Only one fatality was reported, which may be attributed to
a dual infection with TBEV (Leonova et al. 1991). In Russia, POWV co-circulates
with TBEV, and therefore the incidence of encephalitis cases caused by POWV may
be masked by those caused by TBEV. In addition, coinfections with both viruses
have been shown to occur (Leonova et al. 1991). It should be noted that the Russian
strains of POWV isolated between 1972 and 2006 are genetically highly homolo-
gous to strains belonging to lineage I POWV (Leonova et al. 2009). Dual infec-
tions with other tick-borne pathogens have also been noted in the United States, e.g.,
Anaplasma phaghocytophilum (Hoang Johnson et al. 2010).
Symptoms of POWV infection can be difficult to differentiate from those caused
by other arboviruses. Furthermore, asymptomatic infections occur as evidenced by
seroprevalence of up to 3% of the population in certain northern Ontario communi-
ties (McLean et al. 1962).
12.5 DIAGNOSIS
POWV is endemic in North America, and although the disease is rare, it should be
part of the differential diagnosis when arboviral encephalitis is suspected. This is
especially important in areas where tick activity has been reported. Obtaining an
in-depth patient history is a first step toward performing a diagnosis. Information
regarding travel history, contact with animals, vaccinations, and possible tick bites
will help in determining the type of diagnostic tests that should be performed. In
many cases patients do not recall being bitten by a tick. This is because ticks, in par-
ticular nymphal ticks, are small (1.5 mm in diameter) and are therefore difficult to see.
Some of the tests that are performed on an encephalitic patient include magnetic
resonance imaging (MRI), computed tomography (CT) scan, and EEG. Results of
MRI, in general, show changes consistent with microvascular ischemia or demy-
elinating disease in the parietal lobe in one case, temporal lobe in a second (Centers
for Disease Control and Prevention 2001), and superior cerebellum in a third case
(Tavakoli et al. 2009). CT scans are not as sensitive and therefore have not been
as informative as MRI scans in detecting neurologic abnormalities (Partington
et al. 1980; Lessell and Collins 2003). EEG reveals generalized slowing and dif-
fuse encephalitis (Centers for Disease Control and Prevention 2001; Hinten et al.
2008). Blood and CSF also should be collected. CSF glucose is generally normal as
expected for a viral infection. CSF protein is normal or mildly elevated (Hinten et
al. 2008; Lessell and Collins 2003; Embil et al. 1983; Jackson 1989). The initial CSF
cell count may be normal or will show a lymphocytic pleocytosis cell predominance
which on repeat examination in most cases will reveal lymphocytic pleocytosis of
less than 500/mm3 (Hinten et al. 2008; Lessell and Collins 2003; Embil et al. 1983;
Wilson et al. 1979; Jackson 1989). During the early phase of the infection, viral RNA
can be detected in CSF using molecular methods, more commonly, reverse tran-
scription polymerase chain reaction (RT-PCR). In the course of the viremic phase,
there is a window of opportunity during which the virus is present in the central
nervous system, and in this period, it is possible to detect the viral nucleic acid by
RT-PCR. A reasonable strategy would be to perform a flavivirus group-specific PCR
to narrow the range of possible etiologic agents (Whitby et al. 1993; Fulop et al.
Powassan Virus 271
1993; Kuno 1998; Scaramozzino et al. 2001; Maher-Sturgess et al. 2008), and if
that is reactive, then virus-specific PCR primers can be used in the second stage for
virus identification (Scaramozzino et al. 2001; Tavakoli et al. 2009; Lanciotti 2003).
In addition, PCR products can be sequenced. A modification of this method is to
use group-specific PCR followed by restriction enzyme analysis (Gaunt and Gould
2005). An advantage of using group-specific PCR is that lower specimen volume is
used and costs associated with PCR reactions are lower. Using this methodology, the
group-specific PCR primers should be designed for sensitive detection of all viruses
within the group and the virus-specific PCR primers should be designed for specific
virus identification. Flavivirus group-specific PCR primers have in general targeted
the NS5 gene or the 3ʹ noncoding region as these regions possess a high degree of
sequence conservation (Fulop et al. 1993; Kuno 1998; Scaramozzino et al. 2001;
Lanciotti 2003; Maher-Sturgess et al. 2008).
An RT-PCR electrospray ionization mass spectrometry (RT-PCR/ESI-MS)
method based on analysis of base composition of PCR amplicons has been reported
(Grant-Klein et al. 2010). This assay couples an 8 primer broad-range flavivirus assay
using RT-PCR to ESI-MS to detect pan-flavivirus, pan-dengue virus and WNV gene
targets (Grant-Klein et al. 2010). The method is a high-throughput assay that can
detect mosquito and tick-borne flaviviruses, including POWV, for diagnostic and
epidemiologic surveillance. A mass-spectrometer, specialized software, and trained
staff are required to perform this procedure.
RT-PCR is no longer useful once the immune system has cleared the virus. At this
stage, serology is a more effective method for diagnosis. The principle diagnostic
method is the detection of POWV-specific IgM and neutralizing antibodies in CSF or
serum. IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA)
and indirect IgG ELISA are performed to detect encephalitis caused by flaviviruses
including WNV (Johnson et al. 2000; Martin et al. 2000). Positive ELISA results
are in general confirmed by PRNT in biosafety level three containment facilities
(Lindsey et al. 1976; Beaty et al. 1995). Confirmation is required because the possi-
bility of cross-reaction with other flaviviruses is fairly high. Indirect fluorescent anti-
body tests may also be used for detection of flaviviruses. However, IFA tests are less
sensitive than ELISA and they are not suitable for high throughput testing (Gubler et
al. 2000). In general, serologic confirmation consists of the following: (1) a fourfold
or greater change in POWV-specific neutralizing antibody in the same specimen
or later specimen or (2) detection of POWV-specific IgM in CSF or (3) detection of
POWV-specific IgM in a serum specimen and POWV-specific neutralizing antibody
in the same specimen or later specimen.
In recent years, fluorescent microsphere immunoassays (MIA) have been devel-
oped that measure antibodies induced by flavivirus infections (Wong et al. 2003;
Johnson et al. 2005). Multiplex MIAs can simultaneously measure antibodies to sev-
eral antigens at the same time and can therefore save time, reagents, and patient sam-
ple. An MIA is based on the conjugation of an antigen to a fluorescent microsphere.
When patient serum is added to the suspension of microspheres, any antibody pres-
ent in the serum that recognizes the antigen will bind. Washing will remove any
nonspecifically bound molecules. The addition of a secondary reporter anti-human
immunoglobulin antibody will allow the detection of the specific antibody-antigen
complex. Measurement of the fluorescence extrinsic to the beads and fluorescence

of the fluorophore conjugated to the secondary antibody will enable the identifi-
cation of the antibody present in the patient sample. The measuring instrument is
a simplified flow cytometer with lasers that identify and measure the fluorescence
associated with the reaction. Since flavivirus proteins have a high degree of amino
acid sequence similarity, significant cross-reaction is observed in many ELISA tests.
For example, an MIA assay that was developed to detect antibodies to the E-protein
of WNV also detects antibodies to E-proteins of related flaviviruses including St.
Louis encephalitis, Japanese encephalitis, and dengue viruses (Wong et al. 2004).
However, specificity can be added to the assay by multiplexing with additional anti-
gens. For example, adding recombinant NS5 antigens can allow antibodies from
WNV to be distinguished from SLE or dengue infection, and JE or yellow fever
vaccination (Wong et al. 2003). An MIA assay to detect total antibodies (IgG, IgA,
and IgM) to recombinant WNV E protein, recombinant NS5 and recombinant DTV
E protein has been successfully used to detect cases of POWV in the state of New
York (Hinten et al. 2008; Wong et al. 2003, 2004).
The results of antibody tests could well be negative during the early phase of
infection as antibody production begins to increase to a detectable level. Therefore,
serological tests should be repeated after 1–2 weeks. Serological tests are not geno-
type specific and therefore it is not possible to determine whether an infection is
caused by POWV (lineage I) or DTV (lineage II). Furthermore, as previously dis-
cussed, flavivirus cross-reactivity can confound a diagnosis unless additional speci-
ficity is added to the testing strategy.
Historically, mouse inoculations have been used for isolation of arboviruses
including POWV. When the first case of POW encephalitis was being investigated in
1958, portions of the brain were inoculated into newborn mice; those inoculated with
suspensions of basal ganglia and cortex developed signs of acute encephalitis 5 days
after inoculation (McLean and Donahue 1959). The second, third, and fourth suck-
ling mouse brain passage also induced encephalitis (McLean and Donahue 1959).
Subsequent to inoculation, virus can be typed using neutralization tests. Intracranial
inoculation of suckling mice is a sensitive method of virus isolation, but its use has
become uncommon, as most diagnostic laboratories no longer use mice for diagnos-
tic purposes.
An alternative method that is no longer routinely used for diagnosis of POWV
is cell culture. This method, however, remains in use in a limited number of spe-
cialized laboratories for diagnostic and research purposes. POWV produces cyto-
pathic effects and/or plaques in several vertebrate cell lines including pig kidney
embryo cells (PEK) (Leonova et al. 2009), rhesus monkey kidney cells (LLC-MK2)
(Abdelwahab et al. 1964; Stim 1969), baby hamster kidney cells (BHK) (Karabatsos
and Buckley 1967; Brackney et al. 2008), and African green monkey kidney cells
(VERO) (Stim 1969), among others. POWV has been isolated from patient brain tis-
sue (McLean and Donahue 1959; Centers for Disease Control and Prevention 1975;
Deibel et al. 1979) and blood (Leonova et al. 2009).
The only laboratories that currently provide diagnostic testing for POWV are
State Health laboratories and the CDC. Commercial laboratories do not at present
offer serologic testing.
Powassan Virus 273

The first reported case of POW encephalitis occurred in 1958 and was fatal (McLean
and Donahue 1959). An autopsy was performed and macroscopically the brain and
meninges appeared normal. However, histological preparation and staining of various
areas of the central nervous system including the cortex, basal ganglia, pons, and medulla
showed an inflammatory process with characteristic perivascular infiltration and focal
parenchymatous infiltration (McLean and Donahue 1959). The amount of perivascular
infiltration varied and in general the inflammatory cells were composed of lymphocytes
and monocytes and were confined to the perivascular space. The focal infiltrations were
situated in the parenchyma, separated from the blood vessels and were varied in size.
The main cells in these foci were macrophages or microglia and occasionally polymor-
phonuclear cells. In addition, a limited number of degenerating nerve cells with satellite
inflammatory cells were also observed (McLean and Donahue 1959).
Neuropathological examination of a patient who died of a massive pulmonary
embolism following POW encephalitis in 1997 revealed mild diffuse swelling of
the cerebral hemispheres with diffuse meningeal congestion (Gholam et al. 1999).
Histologic examination of the meninges and Virchow-Robin spaces showed severe
chronic inflammatory infiltration in these areas and focal areas of infiltration into the
brain parenchyma. Tissue necrosis was observed in the most involved areas: medio-
temporal lobes, ventral midbrain, and basal ganglia (Gholam et al. 1999). Diffuse
reactive astrocytic gliosis and microglial activation were observed throughout the
gray matter. Occasionally brain stem neurons with an intranuclear eosinophilic
inclusion consistent with a viral inclusion were also observed (Gholam et al. 1999).
It should be noted that when the virus from this case was subsequently isolated and
typed it was found to belong to the POWV lineage II/DTV (Kuno et al. 2001).
Histological findings at autopsy have also been reported for a third case of POW
encephalitis (specifically, lineage II/DTV genotype) in which the patient presented
with meningoencephalitis and succumbed to the infection (Figure 12.5) (Tavakoli
et al. 2009). In this case multinodular mononuclear infiltrates and areas of necro-
sis were observed throughout the brain (Figure 12.5a and b). The predominant
cells in the parenchyma were CD8+ cytotoxic T cells (Figure 12.5c), while those in the
leptomeninges and perivascular spaces were CD4+ helper T cells. Extensive neuronal
loss was observed in the substantia nigra (Figure 12.5d) and gray matter regions and
to a lesser extent white matter tracts showed microglia-macrophage infiltration (Figure
12.5e). Virus antigen was detected by immunohistochemical staining in neuronal cell
bodies, dendrites, and axons as well as within the few surviving Purkinje cells (Tavakoli
et al. 2009). Perivascular and parenchymal infiltration of T cells, neuronal loss, and focal
necrosis appear to be characteristics of POW encephalitis and may account for the sig-
nificant neurological impairment observed in a substantial number of patients.
In animal models (monkey, rabbit, horse), POWV infection results in widespread
encephalitis which is characterized by lymphoid perivascular cuffing, a lymphocytic
meningitis, and choroiditis (Gritsun et al. 2003; Little et al. 1985). In experimentally
infected horses, neuronophagia and zones of parenchymal necrosis were observed
throughout the white and gray matter with the most severe lesions in the medulla
and mesencephalon (Little et al. 1985). It has been postulated that the intense
(a) (b) (e)
(c) (d)
FIGURE 12.5 Histological findings of fatal case of POW encephalitis attributed to Lineage
II, DTV. (a) Microglial nodules and lymphocytic infiltrates in the pons are visible in basal
pontine nuclei (arrowheads). There is less involvement of descending fiber tracts (arrow) and
pontocerebellar fibers. (b) In pontine basal nuclei, confluent foci of parenchymal necrosis is
evident. (c) Upon CD8+ immunostaining of the basal pontis, a cytotoxic T-cell infiltrate with
close association with surviving neurons (arrows) is observed. (d) Significant neuronal loss
is apparent in the substantia nigra such that surviving neurons are rare (arrows). (inset) An
eosinophilic dying neuron and remaining neuromelanin pigment are seen encased in macro-
phages or free in the parenchyma (arrowheads). (e) Phosphoglucomutase immunostaining of
lumbar spinal cord shows prominent infiltration by microglia-macrophages and in the ante-
rior horn and focal microglial nodules in the lateral corticospinal tract (arrow) and posterior
column (arrowhead). Paraffin sections in panels a, b, and d were stained with hematoxylin
and eosin. (From Tavakoli, N. P. et al., N. Engl. J. Med., 360, 2099, 2009. With permission.)
inflammatory response and destruction of nervous tissue elements relates to anti-

body and sensitized lymphocyte interaction with POWV in the brain (Little et al.
1985).
A number of studies examining the pathogenesis of POWV in diverse animals
have been published. Suckling mice demonstrate impaired movement and died by 5
days following intracranial (i.c.) inoculation of DTV (Telford et al. 1997). The LD50
for an adult CD mouse following subcutaneous injection of a 20% brain suspen-
sion was approximately 104.8/mL, and death occurred within 14 days. In contrast,
colonized adult P. leucopus, a natural host, survived similar infection without dem-
onstrating any morbidity or mortality (Telford et al. 1997). A significantly lower
concentration of POWV (104 adult mouse LD50) was measured in SM brain fol-
lowing infection compared with Central European TBEV (108 adult mouse LD50)
(Telford et al. 1997). Ninety percent of larval ticks acquired POWV from mice that
had been intraperitoneally inoculated with 105 PFU. Engorged larvae contained
approximately 10 PFU. Transstadial transmission resulted in approximately 20%
of nymphs infected following feeding as larvae on viremic mice. Titer increased
Powassan Virus 275
approximately 100-fold during molting. Nymphal deer ticks efficiently transmitted

POWV to naive mice after as few as 15 minutes of attachment (Ebel and Kramer
2004). Inflammatory degenerative changes were observed in the nerve tissue of white
mice following intraperitoneal inoculation of 107.5 LD50/ml Russian strain of POWV.
The mice developed convulsions by day 4–5 and often died suddenly. Virus was
seen by EM in neurons, dendrites, glial cells, and intracellular spaces (Isachkova et
al. 1979). Multilayered proliferation of membranes were observed in the cytoplasm
of brain cells, similar to what has been observed with other neurotropic flaviruses
(Whitney et al. 1972; Isachkova et al. 1979). Intraperitoneal injection of Vero cell-
derived DTV or POW virus into females, 5–6 weeks old, NIH Swiss mice yielded
comparable LD50 values of 102.7 and 102.3 PFU, respectively. However, onset of ill-
ness was slightly delayed in DTV-infected mice (day 9 or 10 postinfection compared
with day 7 or 8 in POWV-infected mice) (Telford et al. 1997). These results indicate
virus strains from the two POWV lineages are equally pathogenic in mice.
When Macaca rhesus primates were infected by the i.c. route with Canadian and
Russian strains of POWV, similar patterns of excitation and loss of movement were
observed as with mice. The monkeys remained viremic from 3–21 days postinfection
(pi) with a maximum titer of 105–106 LD50 3–9 dpi. There were no differences in patho-
genesis between the two viruses (Frolova et al. 1985). POWV replicates to lower levels
in wild rodents in Russia when compared with TBEV (Leonova et al. 1987), suggesting
decreased virulence. Yet in Far Eastern Russia, there appear to be more cases with mild
symptoms compared with North America; this could be a consequence of strain differ-
ences or reflect differing ecological conditions (Gritsun et al. 2003).
Gray squirrels (Sciurus carolinensis) inoculated subcutaneously (sc) with 104 whole
mouse (WM)icLD50 POWV demonstrated prolonged but low level viremia, sug-
gesting this species may be a natural reservoir of the virus (Timoney 1971). POWV
produced inapparent infection in chicks inoculated sc with 1–100 WMicLD50, and
viremia peaked d 3–4 (McLean et al. 1960). The infection threshold for adult female
Dermacentor andersoni feeding on viremic rabbits was approximately 102.5/ml
WMicLD50 (Chernesky 1969). Following feeding by experimentally infected D. ander-
soni, hamsters were found to circulate ~104 WMicLD50 POWV (Chernesky 1969), well
above the threshold of infection.
There is a low public health risk of POWV transmission through raw goat’s milk
(Woodall and Roz 1977). Following intramuscular inoculation of 103 suckling mouse
(SM)icLD50 virus, goats produced undetectable viremia, similar to TBEV which pro-
duces a low level viremia, but approximately 105 SMicLD50/ml virus was detected in the
milk 7–15 dpi. This was sufficient to infect kids, although no morbidity was observed.
In contrast, transmission through unpasteurized milk appears to be unlikely in cows
(Drozdov 1959).
12.7 ECOLOGY AND EPIDEMIOLOGY

Recent studies have focused on the virologic properties of POWV (Brackney et al.
2009; Brackney et al. 2010; Davis et al. 2005; Ebel et al. 2001; Pesko et al. 2010;
Leonova et al. 2009; Kuno et al. 2001). The ecologic and epidemiologic-based inves-
tigations have been lacking since the POWV chapter published in The Arboviruses
(Artsob 1989) except for the reporting of human clinical cases (Hicar et al. 2011;
Minnesota Department of Health 2011; Tavakoli et al. 2009; Centers for Disease
Control and Prevention 2009; Hinten et al. 2008; Ford-Jones et al. 2002; Centers for
Disease Control and Prevention 2001) and the identification and characterization of
Lineage II, DTV (Telford et al. 1997; Ebel et al. 1999, 2000; Brackney et al. 2008).
Intensive field studies are required to determine if the apparent increase in human
cases is due to the emergence of DTV in the black-legged tick (Ixodes scapularis)
population (majority of recent POWV cases are in areas with high Ix. scapularis
populations), or the result of improved diagnostic capabilities. It is unclear if pro-
totype POWV is still extant within the United States or if it has been displaced by
DTV.
12.7.1 Transmission Cycle
Protoype POWV and DTV lineage strains are maintained in nature in a transmis-
sion cycle involving ixodid ticks and small mammals. A systemic viremia within the
vertebrate host may not be necessary for efficient transmission. Co-feeding or non-
viremic transmission, proposed and demonstrated in the laboratory with TBEV by
Labuda et al. (1993), may be sufficient to maintain POWV in nature. This has been
modeled for POWV for long-term maintenance in natural foci (Nonaka et al. 2010).
Another mode of virus transmission, transovarial transmission (female to larvae
via the egg), has been documented in the laboratory with Ix. ricinus, Dermacentor
reticulatus, H. longicornis (neumanni), and other ticks and various strains of TBEV
(Naumov et al. 1980; Danielova et al. 2002).
The ecological difference between POWV lineages appears to be the result of the
invertebrate host responsible for transmission. Furthermore, as evidenced by mos-
quito isolates in Russia, transmission may involve nontraditional vectors (Tkachenko
et al. 1976; Kislenko et al. 1982), although more studies are needed to ascertain the
validity and/or importance of this alternative cycle.
12.7.2 Arthropod Hosts
12.7.2.1 Ixodes cookei
Ixodes cookei (woodchuck tick) has been incriminated as the principal vector of pro-
totype POWV, especially in the northeast United States and eastern Canada (Artsob
et al. 1986; McLean et al. 1960, 1962, 1964a,b, 1966, 1967; Whitney and Jamnback
1965). This tick is distributed from South Dakota to Texas northeasterly through the
United States and eastern Canada. At least 22 virus isolates have been acquired from
Ix. cookei ticks and a number of other isolates from the vertebrate hosts that these
ticks feed upon (Artsob 1989; Karabatsos 1985; Ebel 2010). Somewhat surprising is
the fact that experimental studies to assess vector competence of this species have
not been conducted or have not been published, possibly a consequence of the dif-
ficulty of collecting sufficient numbers of individuals to conduct such studies.
Ix. cookei behaviorally resemble the nidicolus argasids (soft ticks) rather than
other ixodids. This species is restricted to the burrows of its preferred hosts, wood-
chucks and other rodents, mustelids, raccoons, foxes, coyotes, etc. (Farkas and
Powassan Virus 277
Surgeoner 1990; Main et al. 1979; Ko 1972; Kollars and Oliver 2003) and may com-
plete its life cycle on a single host or family group. Ticks are not routinely collected
by the flagging method suggesting that dispersal of ticks is via movement of the host
from burrow to burrow during male-female encounters in early spring and young of
the year dispersals in late summer (Ko 1972).
Human encounters with Ix. cookei are infrequent as compared with other tick
species (Cohen et al. 2010; Anderson and Magnarelli 1980; Campbell and Bowles
1994; Hall et al. 1991; NYS, unpublished data) possibly explaining the relatively low
number of human cases of POW encephalitis, especially where Ix. scapularis abun-
dance is low or nonexistent.
12.7.2.2 Ixodes scapularis

With the exception of a few historical isolates (fox brain from WV and D. ander-
soni ticks in CO), DTV has been isolated primarily from the Lyme disease vector,
Ix. scapularis (black-legged tick or deer tick). Ix. scapularis is a three host tick.
Each stage—larva, nymph, and adult female—will take a bloodmeal from a sep-
arate host (Barbour and Fish 1993). Larvae predominantly feed on white-footed
mice (Peromyscus leucopus), although they have been recorded feeding on numer-
ous other species (Keirans et al. 1996). Nymphs are more indiscriminate feeders,
acquiring their bloodmeal from a wider host range including small mammals, birds,
reptiles, and humans (Keirans et al. 1996; Smith et al. 1996; Cohen et al. 2010;
Apperson et al. 1990, 1993; Levine et al. 1997). Adult females feed on larger mam-
mals, particularly deer and livestock (Keirans et al. 1996; Smith et al. 1996; Cohen
et al. 2010). The majority of tick-derived DTV isolates have been from the adult
stage of Ix. scapularis (Brackney et al. 2008, 2010; Telford et al. 1997; Ebel et al.
1999, 2000; unpublished data from NYS).
In the laboratory, Ix. scapularis has been shown to be a competent vector of
POWV. Costero and Grayson reported infection rates of 10%, 40%, and 57% for
larvae, nymphs, and females, respectively, after feeding on viremic hosts (Costero
and Grayson 1996). Transstadial (larva to nymph, nymph to adult) and transovarial
transmission were also reported in this study. Another study demonstrated the trans-
mission of Lineage II/DTV, by infected nymphal Ix. scapularis in as little as 15
minutes after feeding on naive P. leucopus (Ebel and Kramer 2004), suggesting that
unlike other tick-borne diseases, such as Lyme, ehrlichiosis, and babesiosis, there
is no grace period for removal of an attached tick to prevent POWV infection. The
authors of this study also reported transstadial transmission.
Due to the indiscriminate feeding behavior and experimentally derived vector
competence of Ix. scapularis, it is hypothesized that this species may have provided
the bridge for POWV to escape from the focal, enzootic cycle, thus becoming a
potential (re)emerging pathogen.
12.7.2.3 Dermacentor andersoni

As stated earlier, the first recorded POWV isolate, although unidentified until after
POWV and described in the literature by McLean and Donahue (1959), was isolated
from a pool of D. andersoni (Rocky Mountain wood tick) collected along North
Cache la Poudre River, Colorado in 1952 (Thomas et al. 1960). D. andersoni larvae
and nymphs primarily feed upon small mammals, including mice, chipmunks, and
ground squirrels. Adults will feed on larger mammals including wild and domes-
tic ungulates, rabbits, and porcupines (Scott and Brown 2011; Wilkinson 1984;
Burgdorfer 1969; Burgdorfer 1975). D. andersoni is the principal vector of Colorado
tick fever virus (Emmons 1988). POWV development in this species of tick follow-
ing feeding on infected rabbits indicated virus multiplication in various tick organs
leading to peroral transmission through salivary secretion of infected nymphs and
adults, and transstadial transfer of virus, but no transovarial transmission (Chernesky
1969; Artsob 1989).
12.7.2.4 Other Species

POWV has been isolated from at least two other tick species in North America, Ix.
marxi (McLean and Larke 1963) and Ix. spinipalpis (Keirans and Clifford 1983). Ix.
marxi could be an important enzootic vector of POWV transmission particularly
in arboreal squirrels of Canada as hypothesized by Main (Main 1977) based on
virus isolation and serologic results from field investigations by McLean et al. (1963,
1964a,b, 1966, 1967). Ix. spinipalpis ticks are distributed from the Rocky Mountains
westward with populations extending into the Badlands region of the Dakotas
(Gregson 1956; Dolan et al. 1997). It is primarily nidicoulous and feeds on Neotoma
rodent species (Dolan et al. 1997). Maupin et al. (1994) did not collect Ix. spinipalpis
with standard dragging procedures, and therefore this species is likely an enzootic
vector of POWV with little chance of infecting humans.
There is compelling evidence that the expansion of POWV into an Ix. scapularis
driven cycle is responsible for the apparent increase in human cases. 1. Numerous
POWV virus isolates (Lineage II DTV) have been obtained from Ix. scapularis,
2. DTV has been isolated from at least 2 recent fatal human infections, 3. Ix. scapu-
laris ticks are competent vectors of POWV, 4. Ix. scapularis ticks are able to trans-
mit virus in as little as 15 minutes, 5. Experiments have determined transstadial and
transovarial transmission of POWV in Ix. scapularis, and 6. The majority of human
cases occur during peak larval and nymphal Ix. scapularis activity and in areas
where Ix. scapularis populations are abundant.
12.7.3 Disease Incidence
Since 1999, the majority (80%) of reported human POWV cases had onset dates
between April–September. Another 15% had onset dates in October and November
(Hinten et al. 2008; U.S. Geological Survey 2011). This is not surprising since most
Lyme disease cases also occur during the summer months although vectored solely
by Ix. scapularis, especially in the northeast and upper midwestern United States
(Hinten et al. 2008), and McLean demonstrated POWV activity in the field dur-
ing these months (McLean et al. 1964a,b, 1966, 1967; McLean and Larke 1963).
Of interest, 2 cases in 2009, one each in NY (January) and VA (December), had
onset dates in winter months (U.S. Geological Survey 2011). An additional case from
Ontario had onset in December 1979 (Mahdy et al. 1982; Partington et al. 1980).
Of the first 19 POW cases reported only 2 were in individuals greater than 55
years old (57 and 82). Sixteen of the cases were in children less than 15 years old and
Powassan Virus 279
a single case was in a 19 year old (Artsob 1989). Between 1999 and 2005, Hinten
et al. reported only two POWV cases out of nine in individuals less than 55 years
old (25 and 53). The remaining seven individuals were 60, 66, 69, 70, 74, 83, and
91 years old (Hinten et al. 2008). Of cases reported by NYS, in 2007–09, five cases
were individuals between the ages of 74 and 84, while three cases were in children
younger than 10 years old and another was in a young man aged 24 years (U.S.
Geological Survey 2011; NYS, unpublished data). Of the 37 cases for which we have
information, 13 were females and 24 were males (Artsob 1989; Hinten et al. 2008;
NYS unpublished data).
12.8 PROGNOSIS AND TREATMENT

With a fatality rate of 10%, POW encephalitis is a serious disease (Artsob 1989).
Furthermore, even among patients who survive, the long term affects can be severe.
In up to 50% of cases, due to significant neurologic sequelae, prolonged inpatient
rehabilitation is required subsequent to acute care hospitalization (Table 12.1)
(Wilson et al. 1979; Mahdy et al. 1982; Hinten et al. 2008). In many patients, symp-
toms such as hemiplegia, quadriplegia, ophthalmoplegia, severe headaches, weak-
ness, and memory loss can persist for months (Hinten et al. 2008; Wilson et al. 1979;
Goldfield et al. 1973; Lessell and Collins 2003; Partington et al. 1980).
There are no vaccines or specific therapies available for prevention and treatment
of POW encephalitis. However, prevention strategies including vector and vertebrate
control can help reduce the incidence considerably. Avoidance of tick bites is the
best strategy and can include reducing human contact with small and medium-sized
mammals (woodchucks, mice, squirrels) in order to reduce the risk of exposure to
infected ticks, avoiding bushy areas, wearing light colored clothing that fully cov-
ers the arms and legs, and using insect repellant. In addition, people should check
themselves and their pets for ticks and if they detect any, they should remove them
as soon as possible. Environmental controls such as removing rodent nests will also
reduce the risk of contact with ticks.
It is doubtful that a substantial amount of effort and resources will be allocated to
vaccine development as the numbers of POW encephalitis cases are too few to justify
such an undertaking. However, there is a vaccine against TBEV, which demonstrates
broad cross-protection against three subtypes of TBEV strains and a somewhat
reduced but still protective neutralization capacity against more distantly related
viruses, such as OHFV (Orlinger et al. 2011).
Although there is no specific therapy for the treatment of POW encephalitis,
supportive therapy is used to treat some of the symptoms. Mechanical ventilation
can be used to protect the airway and reduce the affects of apnea. As seizures are
a common symptom of POW encephalitis in children (McLean and Donahue 1959;
Rossier et al. 1974; Embil et al. 1983; Smith et al. 1974; Kolski et al. 1998), anticon-
vulsant therapy can be beneficial. In addition, it is important to control the tempera-
ture and nutritional status of the patient. The initiation of early physical and speech
therapy to maintain various functions such as joint mobility and speech is key in
minimizing the long term affects of the infection (Rossier et al. 1974; Hinten et al.
2008).
Six out of nine cases reported in the United States between 1999 and 2005 had
significant neurological sequelae and required prolonged inpatient rehabilitation
(Hinten et al. 2008). None of the nine cases were fatal during the acute phase of
disease. However, as mentioned previously, death has been reported in a signifi-
cant number of cases (Table 12.1) (McLean and Donahue 1959; Centers for Disease
Control and Prevention 1975; Gholam et al. 1999; Tavakoli et al. 2009). In some
cases death was due to acute encephalitis and occurred within days of symptom
onset (McLean and Donahue 1959; Centers for Disease Control and Prevention 1975;
Tavakoli et al. 2009), while in others it was due to sequelae directly related to disease
and occurred many months later (Joshua 1979; Mahd et al. 1982; Artsob and Spence
1981; Artsob 1989).
12.9 CONCLUSION
POWV and DTV are emerging tick-borne pathogens. Neurologic sequelae follow-
ing infection are common and require medical care. The lack of awareness of this
tick-borne disease and the need for specialized laboratory tests to confirm diagnosis
suggest the frequency of POW infections may be greater than previously suspected.
Increased human surveillance following the introduction of West Nile virus into
North America and improved diagnostic methods have demonstrated increased preva-
lence of POWV activity in the northeastern United States (New York, Massachusetts,
Maine, Vermont, Connecticut) and north central United States (Michigan, Minnesota,
Wisconsin). The number of human cases has nearly doubled since 1998. The population
at risk is increasing as homes are built on the edges of wooded tracts of land that are
occupied by tick hosts, bringing humans and potential POWV hosts and vectors in close
proximity. As the northeastern range of I. scapularis continues to expand northward
and westward, so does the likelihood of contact with new populations. Research on the
ecology of this virus elucidating vertebrate hosts and environmental factors supporting
efficient virus transmission should be conducted.
ACKNOWLEDGEMENT
We thank Betsy Kauffman and Mary Franke for their invaluable help with this
chapter.
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13 Neurological Dengue
Aravinthan Varatharaj
CONTENTS
13.1 Introduction................................................................................................. 290
13.2 Virology....................................................................................................... 291
13.3 Historical Aspects........................................................................................ 291
13.3.1 Origins............................................................................................ 291
13.3.2 Discoveries..................................................................................... 292
13.3.3 The Emergence of DHF................................................................. 292
13.4 Epidemiology............................................................................................... 293
13.4.1 Global Patterns............................................................................... 293
13.4.2 South-East Asia.............................................................................. 293
13.4.3 Americas........................................................................................ 294
13.4.4 Africa............................................................................................. 294
13.4.5 Non-Endemic Areas and International Travel............................... 294
13.4.6 Economic Burden of Dengue......................................................... 294
13.5 Vectors......................................................................................................... 294
13.5.1 Insect Vectors................................................................................. 294
13.5.2 Life Cycle....................................................................................... 295
13.5.3 Other Methods of Transmission..................................................... 295
13.6 Pathogenesis................................................................................................. 295
13.6.1 Initial Events.................................................................................. 295
13.6.2 Humoral Immune Response........................................................... 296
13.6.3 Cell-Mediated Response................................................................ 296
13.6.4 Cytokine Storm.............................................................................. 296
13.6.5 Viral-Host Interplay....................................................................... 297
13.7 Clinical Spectrum of Dengue Infection...................................................... 297
13.7.1 Asymptomatic Infection................................................................. 298
13.7.2 Dengue Fever.................................................................................. 298
13.7.3 Dengue Hemorrhagic Fever........................................................... 298
13.7.4 Dengue Shock Syndrome............................................................... 299
13.7.5 Unusual Manifestations.................................................................. 299
13.7.5.1 Hepatitis........................................................................... 299
13.7.5.2 Myocarditis...................................................................... 299
13.7.6 Neurological Manifestations.......................................................... 299
13.8 Dengue Encephalopathy..............................................................................300
13.8.1 Hepatic Encephalopathy................................................................. 301
13.8.2 Cerebral Edema.............................................................................. 301
13.8.3 Intracranial Hemorrhage................................................................302
289
13.8.4 Deranged Electrolytes.................................................................... 303

13.8.5 Cerebral Hypoperfusion................................................................. 303
13.9 Dengue Encephalitis.................................................................................... 303
13.9.1 Literature Review........................................................................... 303
13.9.2 Clinical Features............................................................................304
13.9.3 Laboratory Features....................................................................... 305
13.9.4 Radiological Features.....................................................................306
13.9.5 Dengue Encephalitis in Perspective...............................................307
13.9.6 Neuropathogenesis.........................................................................308
13.10 Dengue Transverse Myelitis........................................................................308
13.11 Peripheral Nervous System Manifestations.................................................309
13.11.1 Guillain-Barré Syndrome...............................................................309
13.11.2 Mononeuropathy............................................................................ 310
13.11.3 Myositis.......................................................................................... 310
13.12 Laboratory Diagnosis of Dengue Infection................................................. 311
13.12.1 General Investigations.................................................................... 311
13.12.2 Confirming Dengue Infection........................................................ 311
13.12.3 Detection of Virus.......................................................................... 312
13.12.4 Detection of Host Immune Response............................................. 312
13.13 Management................................................................................................ 313
13.13.1 Dengue Fever.................................................................................. 313
13.13.2 Dengue Hemorrhagic Fever and Shock.......................................... 314
13.13.3 Dengue Encephalopathy................................................................. 314
13.13.4 Dengue Encephalitis....................................................................... 316
13.13.5 Future Treatments.......................................................................... 316
13.14 Vaccination.................................................................................................. 317
13.15 Vector Control............................................................................................. 318
13.16 Conclusion................................................................................................... 318
References............................................................................................................... 319
13.1 INTRODUCTION
Dengue is a viral disease which poses a significant and increasing problem to global
health. The World Health Organization (WHO) estimates that 2.5 billion people,
40% of the world’s population, are at risk of dengue infection (WHO 2009). Fifty
million infections occur each year, with an increasing incidence, and result in 24,000
deaths. The magnitude of the dengue problem demands action.
To act against the dengue virus, we must understand it. An increasing knowledge
of viral interaction with the human body is revealing how dengue has the capacity
to cause disease. The familiar form of dengue infection presents as an acute febrile
illness, but it is better to think of a spectrum of clinical manifestations, where the
features of infection range from asymptomatic carriage to a severe hemorrhagic dis-
order with multisystem involvement. The factors that determine where along the
Neurological Dengue 291
spectrum an infected individual will manifest illness are complex, but key toward
developing an effective management strategy.
The public health implications are clear, but why is dengue of interest to neuro-
scientists? The broad spectrum of dengue infection does not spare the nervous sys-
tem, peripheral or central. Neurological manifestations are well-reported, but poorly
understood. For decades it has been a matter of debate as to how these manifesta-
tions are mediated, and whether dengue virus has the potential to directly infect
the nervous system. An increasing body of evidence now places us in a position to
develop an answer, and in doing so, advances our understanding of the clinical spec-
trum of dengue infection.
13.2 VIROLOGY
Dengue is a single-stranded positive-sense ribonucleic acid (RNA) virus of the
Flavivirus genus, which includes among others yellow fever, West Nile, and Japanese
encephalitis viruses. The flaviviruses are part of the (non-taxonomic) descriptive
group of “viral hemorrhagic fevers,” which includes Ebola, Marburg, and Lassa fever.
The RNA genome of the dengue virus is protected by an icosahedral (20-sided) protein
capsid and lipid outer envelope, forming a complete virion with a diameter of around
50 nm. There are four viral serotypes, named DEN-1 to DEN-4, that are genetically
distinct although sharing a common phylogeny. Numerous strains of each serotype
have also been discovered. The complete RNA genome has been sequenced for all
four serotypes, and runs to a length of around 11,000 bases (Henchal and Putnak
2009). For comparison, the human genome contains 2.85 billion bases (IHGSC
2001). Contained within this “simple” genome are merely 10 individual genes; the
human genome has 25,000. Three structural genes code for the capsid protein (C),
the membrane-associated protein (M), and the envelope protein (E). The remain-
ing seven nonstructural genes—numbered NS1, NS2A, NS2B, NS3, NS4A, NS4B,
NS5—encode proteins with various roles in replication and infection. Together,
these ten genes and their protein products are the causative agents of the dengue
problem. Existing, as genes do, only to make more copies of themselves, one has to
concede that they have developed a tremendously successful survival strategy.
13.3 HISTORICAL ASPECTS

13.3.1 Origins
I venture to submit the following propositions, which possibly may not be deemed very
extravagant:—that dengue is a disease not yet thoroughly understood.
Charles C. Godding
Staff surgeon aboard HMS Agamemnon, 1890
It is likely that dengue has plagued humanity since antiquity, although the form in
which it has done so has undergone considerable evolution in parallel with our own.
Phylogenetic analyses suggest that dengue existed several millennia ago as a dis-
ease of forest-dwelling nonhuman primates (the “sylvatic cycle”) (Wang et al. 2000).
The first major step in the dengue-human evolutionary relationship occurred when
human civilization shifted from a hunter-gatherer society to one based in urban
aggregations, in inadvertent concert with insect vectors of the genus Aedes. A few
thousand years ago in Asia, these populations reached a critical mass of over 10,000
human individuals, which allowed dengue to make the leap into a form able to main-
tain itself in an endemic-epidemic human transmission cycle. Around this time,
chroniclers of the Chinese dynasties began to make observations regarding a febrile
illness with muscle aches that was associated with proximity to water and spread by
insects. The causative agent was likely DEN-2, and the illness likely dengue fever
(DF). Similar shifts occurred independently across the world, and the sylvatic cycle
progenitor fell into the niche of endemic-epidemic cycling in geographically isolated
human population centers, leading to the four distinct serotypes identified today.
13.3.2 Discoveries
For centuries, dengue cycled endemic-epidemic in urban population centers, causing
a self-limiting febrile illness, killing few, and attracting only occasional attention
from the medical men of the time. Benjamin Rush, Pennsylvanian physician and
25th signatory of the Declaration of Independence, gave his now infamous descrip-
tion of “breakbone fever” in 1780. The Spanish-American War of 1898 meant that
U.S. troops were stationed in dengue-endemic areas, and led to Ashburn and Craig’s
paper proving that the cause of dengue is an “ultramicroscopic agent” that is present
in the blood (Ashburn and Craig 1907). In 1919, Cleland proved that the vector was
Aedes aegypti (Cleland and Bradley 1919). Virus, insect, and man existed in balance.
All this changed in the 20th century. Population upheaval in the aftermath of the
World War II allowed unprecedented spread of the virus to pandemic levels. Rapid
urbanization coupled with inadequate public sanitation, especially in the popula-
tion centers of South-East Asia, provided ample breeding ground for Aedes aegypti,
catalyzing a string of epidemics. Air travel exposed the virus to naive populations,
ripe for infection. Something else changed too; dengue began to kill. The emergence
of dengue hemorrhagic fever (DHF) heralded the second major step in the dengue-
human relationship.
13.3.3 The Emergence of DHF

DHF was first described in the Philippines in 1953 and soon spread throughout the
region. China, the Maldives, India, Sri Lanka; one by one, DHF became endemic.
During the 1960s and 1970s, DHF progressed through South-East Asia, setting up
a pattern in each country which began with a few sporadic cases, a sudden outbreak
in the rainy season, and then a stable cycle of yearly surges and bigger 3–5 yearly
epidemics. For reasons that are now being understood, individuals in an area with an
endemic serotype are vulnerable to secondary infection by another serotype, which
results in a more fulminant disease and an increased likelihood of progression to
DHF. Dengue-naive populations in areas infested with Aedes aegypti are highly vul-
nerable; for example, when DEN-1 was introduced to Cuba in 1977, over 44% of the
population were infected, although the majority with mild disease only (Halstead
2007). Five years later, the 1981 DEN-2 epidemic was markedly more severe, with
hundreds of thousands of cases of DF and around 10,000 cases of DHF. In this pat-
tern, the worldwide spread of dengue has continued into the new millennium.
13.4 EPIDEMIOLOGY
13.4.1 Global Patterns
Today, dengue virus is endemic to human populations in over one hundred coun-
tries, and approximately 2.5 billion individuals, 40% of the global population, are
at risk (WHO 2009). Tropical and subtropical areas are worst affected (shown in
Figure 13.1). It is estimated that 50 million infections and 24,000 fatalities occur
worldwide every year. The incidence has increased by a factor of 30 in the last 50
years and continues to climb. At the 2005 World Health Assembly in Geneva, WHO
member states declared that the dengue phenomenon “may constitute a public health
emergency of international concern” (Documentation of the World Health Assembly
2005).
13.4.2 South-East Asia
South-East Asia bears the brunt of the dengue problem. Subdividing by the Köppen
climate classification, the tropical rainforest (e.g., Indonesia, Philippines) and tropical
monsoon (e.g., Sri Lanka, Thailand) areas are worst affected, with large populations
of Aedes aegypti and multiple circulating viral serotypes. In these areas, dengue has
an established foothold in the cities and is now spreading to rural areas. Meanwhile,
the tropical savannah areas such Bangladesh and parts of India are experiencing
epidemics of increasing range and frequency, and in the last decade, dengue has also
begun to encroach on the temperate areas such as Nepal and Bhutan.
FIGURE 13.1 Global distribution of dengue. The Tropics of Cancer and Capricorn are
marked. The southern United States, Mexico, Australian Queensland, and the Pacific Islands
are also variably affected.
13.4.3 Americas
In the Americas, dengue is endemic throughout large parts of South and Central
America. From 2001 to 2007, there were nearly 4.5 million reported cases, occur-
ring in regular outbreaks (WHO 2009). Brazil is particularly affected, with over 2.7
million cases during that period. DEN-1, DEN-2, and DEN-3 are in common circula-
tion, although DEN-4 is also present, especially in the Andean countries. In a large
study conducted between 2000 and 2007, enlisting 20,880 patients from Western
South America, Forshey et al. (2010) reported that dengue was the most common
cause of acute febrile illness in that region, responsible for 26% of cases.
13.4.4 Africa
Dengue in Africa is poorly understood, due to a lack of reliable surveillance and
a greater focus on malaria and HIV. Nevertheless, dengue is clearly present on the
continent, and what data there is suggests that outbreaks are occurring with increas-
ing frequency (WHO 2009). East Africa is worst affected, with circulating DEN-1,
DEN-2, and DEN-3. The WHO has called for attention to the dengue problem in
Africa, especially the particular issue of dengue in majority HIV-positive popula-
tions and the potential implications for transmission.
13.4.5 Non-Endemic Areas and International Travel

Dengue does not commonly occur in Europe, North America, or Russia, but is
an important differential diagnosis in the febrile traveler returning from the trop-
ics. Analyzing global surveillance data of 17,353 patients, Freedman et al. (2006)
showed that dengue is the most common identifiable cause of systemic febrile illness
in travelers returning from South East Asia (58% of cases with identified causes), the
Caribbean (52%), South America (31%), or South Central Asia (27%), even higher
than malaria.
13.4.6 Economic Burden of Dengue

The economic impact of dengue in endemic areas is vast. Suaya et al. (2009) con-
ducted a landmark study looking at the costs associated with dengue in 1695 patients
in eight American and Asian countries. The average cost per patient (in international
dollars) was I$514 for cases managed in the community and I$1394 for inpatients.
The total cost for the study cohort was I$587 million, a very conservative estimate
given the degree of under-reporting.
13.5 VECTORS
13.5.1 Insect Vectors
The primary vector of the dengue virus is the mosquito Aedes aegypti, and hence
dengue may be classed as an “arbovirus”—an “arthopod-borne virus.” Aedes is found
in abundance from latitudes 35°N to 35°S, reflecting locations in which winters are
above 10°C, allowing the insect to survive year-round. Summer invasions have been
recorded up to 45°N, but the mosquitoes perish in winter. Low temperatures also
ensure that high-altitude areas within the Tropics are relatively spared.
Other vectors of the genus Aedes have also been implicated in dengue outbreaks.
The 2001 Hawaiian outbreak was spread by Aedes albopictus, a less efficient forest-
living vector which resulted in a slowly-spreading epidemic (Halstead 2007). The
transport of Aedes albopictus from its Asian homeland was likely due to the global
trade in tires, inadvertently containing viable eggs. Aedes polynesiensis and scutel-
laris have also been implicated.
13.5.2 Life Cycle

The female of Aedes aegypti lays its eggs in stagnant water, which exists in nature
but is provided in abundance wherever there is human habitation—discarded bottles,
buckets, and tires are perfect locations. There is evidence that dengue virus can pass
from a female mosquito to its eggs (transovarial spread) (Arunchalam et al. 2008),
and this may be a useful strategy to ensure viral persistence out of season. The
eggs are resistant to drying and may remain in a quiescent state for several months.
Following a period of rainfall, the eggs hatch into larvae; after a week, the larvae
pupate, and 2 days later, the mature mosquitoes emerge. The insect has a predilection
for cool and dark areas, causing it to live indoors, in close contact with humans. The
females feed on human blood, which provides a nutritious diet for egg production,
and bite mainly during the day. To take up the virus, the mosquito must bite during
the period of peak viremia, the start of which usually precedes symptoms. As the
blood meal is digested the virus infects the gut and within 8–12 days undergoes mas-
sive replication and systemic spread, penetrating the salivary glands. Further feeding
then transmits the virus to humans. Aedes aegypti itself lacks the ability to clear
the virus and remains infected for the duration of its life, which may be a month or
more. Individual insects have a relatively narrow range, usually confined to around
the house in which they hatched. Humans serve to amplify the virus and transport
it to fresh pastures.
13.5.3 Other Methods of Transmission

Spread between humans through infected blood transfusions and organ transplants
is possible, and vertical transmission from human mother to fetus has also been
reported (Chye et al. 1997). However, these represent a rare minority of cases.
13.6 PATHOGENESIS
13.6.1 Initial Events
After the bite, dengue virus passes into the blood and infects and replicates within
cells of the immune system, particularly macrophages and their precursor mono-
cytes. Lymphocytes, mast cells, dendritic cells, endothelial cells—and many oth-
ers—may also be targets for infection. Epidermal dendritic cells (Langerhans cells)
around the bite area may well be the first targets, and infection down-regulates their
production of the major histocompatibility complex (MHC) and induces apoptosis,
compromising antigen presentation and delaying an effective immune response.
After an incubation period of 7–10 days, large numbers of mature virions are released
into the circulation, resulting in viremia and the variable development of symptoms
(and infectivity). Blood-borne virus infiltrates organs (especially the spleen) and
begins replication in tissue macrophages. It is likely that it is the immune response
to viremia which determines the progression and clinical manifestations of dengue
infection. It has long been observed that secondary dengue infections lead to a more
severe phenotype than the primary infection, and much work has focused on aber-
rant immune responses in secondary dengue.
13.6.2 Humoral Immune Response

Viral proteins are immunogenic and elicit a neutralizing antibody response,
especially the envelope proteins which serve as highly visible epitopes. Many
antibodies are cross-reactive, however, and bind to endothelial cells and plate-
lets resulting in their apoptosis (Lin et al. 2001, 2002). Also, antibodies (via the
classical pathway) and viral proteins including NS1 (Avirutnan et al. 2006) trig-
ger complement activation, which may contribute to vascular permeability and
coagulopathy.
Antibody-dependant enhancement (ADE) occurs in secondary dengue infection
when immunoglobulins persisting from the primary infection bind to circulating
dengue virus (Dejnirattisai et al. 2010). Present at concentrations inadequate for
effective viral neutralization, the immunoglobins form complexes with virions, bind
to immune cells, and in doing so enhance the uptake of effective virions by their tar-
gets. ADE effectively subverts the immune response to the cause of viral replication.
As IgG can cross the placenta, ADE may also contribute to the severity of dengue
infection in infants born to dengue-exposed mothers.
13.6.3 Cell-Mediated Response
Presentation of dengue antigens initiates a clonal expansion of CD8+ (cytotoxic) and
CD4+ (helper) T-lymphocytes. Following resolution a significant number remain and
render lifelong immunity to that serotype. However, the serotypes are sufficiently
heterogenous in their antigenicity that infection with one does not confer immu-
nity to the others. Preferential activation of memory T-lymphocytes from the pri-
mary infection at the expense of generating a new and more specific cell-mediated
response (a concept dubbed “original antigenic sin”) delivers a sub-optimal response
which may be at least partly responsible for the increased severity of secondary
infections (Martina et al. 2009).
13.6.4 Cytokine Storm
Large amounts of inflammatory mediators produced by infected and activated
immune cells create a “cytokine storm” that contributes to vascular endothelial
breakdown (King et al. 2000). Both Th1 and Th2 responses are elicited. The Th1
response produces IFN-γ and IL-2 and is biased toward cell-mediated immunity,
whereas the Th2 response produces IL-4, IL-5, IL-10, and TGF-β, and is biased
toward antibody synthesis. There is evidence that the Th2 response is less effective
and associated with more severe disease (Mustafa et al. 2001).
13.6.5 Viral-Host Interplay

The clinical outcome of dengue infection reflects the interplay of viral and host
factors. On the one hand, it may be that certain viral strains are simply more
virulent, and indeed, genotypic variation between strains has been implicated in
differential pathogenesis (Leitmeyer et al. 1999). Numerous host factors are also
at play, however. As previously mentioned, aberrant immune responses likely
play a large part in mediating severe dengue infection, and these may be a pre-
dominant feature of the secondary response. Host genetic factors may also be
involved, and numerous HLA polymorphisms associated with varied suscepti-
bility have been identified (Loke et al. 2001). Females (Kabra et al. 1999) and
infants (Guzman et al. 2002) are generally more susceptible to severe disease,
as are individuals with preexisting chronic disease such as asthma (Guzman et
al. 1992).
13.7 CLINICAL SPECTRUM OF DENGUE INFECTION

Dengue is one disease entity with different clinical presentations and often with unpre-
dictable clinical evolution and outcome. (WHO 2009)
For reasons that are now being understood, the clinical manifestations of dengue
infection are highly variable. The outcome of the virus-host interaction may affect
wide range of organ systems, with varying severity, and potential for systemic fail-
ure. The traditional WHO classification into distinct disease entities is widely used
and is shown in Table 13.1. However, in the face of practical difficulties in applying
this classification to patients who may straddle categories, a new classification has
been produced which divides cases into severe dengue and nonsevere dengue (with
or without warning signs).
TABLE 13.1
Traditional WHO Classification
Asymptomatic or subclinical
Dengue fever (DF)
Dengue hemorrhagic fever (DHF)
Dengue shock syndrome (DSS)
“Unusual manifestations”
13.7.1 Asymptomatic Infection
In some cases, detectable symptoms may fail to develop at all, resulting in an asymp-
tomatic carrier state. During viremia, the patient is still infectious, however, and
asymptomatic carriers likely play a significant role in disseminating the virus.
13.7.2 Dengue Fever
DF classically presents with a rapid onset of fever, malaise, headache, and retro-
orbital pain, with severe myalgia and arthralgia (“breakbone fever”). Erythema of
the face, neck, and chest is typical. A generalized maculopapular rash erupts 3 to 5
days after the onset of fever in 50%–82% of patients and is characteristically speck-
led with petechiae and larger islands of spared skin (Pincus et al. 2008). In infants
and younger children the presentation is nonspecific, with prominent coryza, and
also diarrhea, rash, seizures (usually febrile convulsions), vomiting, and abdominal
pain. In a minority of individuals with DF there may be some signs of a mild hemor-
rhagic tendency (“DF with unusual bleeding”).
DF may result from either primary or secondary dengue infection. The onset
of symptoms is usually 7–10 days after the bite, although the incubation period
can be as short as three or as long as fifteen days. As a rule, febrile illness in the
traveler more than 2 weeks after return from the tropics is unlikely to be dengue.
The fever lasts for 2–7 days and is in the majority of cases followed by a complete
recovery.
13.7.3 Dengue Hemorrhagic Fever

DHF usually results from secondary infection, although may occur after primary
infection in infants. DHF is largely indistinguishable from DF in the initial stages;
after a few days, however, vascular leak and derangement of clotting become mani-
fest. There is easy bruising, bleeding from injection sites, and widespread petechiae.
The classic bedside investigation is the tourniquet test; a blood pressure cuff around
the upper arm is inflated to halfway between systolic and diastolic pressure for 5
min, and the test is considered positive if more than 30 petechiae per square inch
are observed on the underlying skin. There may also be gastrointestinal bleeding
(especially if with existing peptic ulceration), epistaxis, hematuria, and menorrha-
gia. Vascular leak results in hemoconcentration, serous effusions (mostly pleural and
peritoneal), and hypoproteinemia. The liver is often enlarged and tender, and acute
liver failure may occur.
Fever persists for 2–7 days, after which the disease may take one of two courses.
Most patients experience sweating and some circulatory disturbance during defer-
vescence, and in less severe cases this is followed by spontaneous resolution. In
more severe cases, however, the disease progresses to a state of critical vascular
leak and circulatory collapse, known as dengue shock syndrome (DSS). Thus, the
critical window for modifying the course of disease is at and just before the time of
defervescence. Worsening thrombocytopenia and elevation of the hematocrit herald
impending DSS.
13.7.4 Dengue Shock Syndrome

DSS is both hypovolemic, due to blood loss, and distributive, due to vascular leak.
Classical signs of shock are present; tachycardia, hypotension, and cool, clammy, poorly
perfused peripheries. Complications include electrolyte and blood glucose abnormali-
ties, metabolic acidosis, hemorrhage (including intracranial hemorrhage), disseminated
intravascular coagulation (DIC), and large serous effusions. Eventually, there is multi-
organ failure. Untreated, death usually occurs within 12–24 h from the onset of shock.
13.7.5 Unusual Manifestations
13.7.5.1 Hepatitis
Dengue virus and antigens have been isolated from the liver (Nogueira et al. 1988;
Miagostovich et al. 1997). Some degree of hepatic involvement accompanies most
dengue infection, probably reflecting a predilection for viral infiltration into liver
macrophages (Kupffer cells) and hepatocytes. Direct liver tropism is not the only
mechanism, however, and dengue hepatitis is likely to be multifactorial, and may
involve liver hemorrhage and ischemic injury. Patients with preexisting hepatic
impairment are likely to be at greater risk. Use of paracetamol to control fever may
also contribute. The usual presentation of dengue hepatitis is as a rise in serum
transaminases (alanine transaminase [ALT]; aspartate transaminase [AST]) which
reflects the severity of infection, and there may be some right-upper quadrant pain.
In a retrospective study of DHF and liver failure, Kuo et al. (1992) found that the
transaminitis in dengue is usually AST-predominant. Progression to frank liver fail-
ure is uncommon but may occur together with hepatic encephalopathy. There is evi-
dence that DEN-3 and DEN-4 serotypes may have a greater predisposition to liver
involvement (Dengue Bulletin 24, 2000).
13.7.5.2 Myocarditis
Acute myocarditis with impaired left ventricular function has been reported (Wali
et al. 1998), and may add a cardiogenic element to DSS and increase the likelihood
of fluid overload with volume resuscitation. Cardiac rhythm abnormalities may also
occur. It is unclear whether these manifestations are due to dengue viral invasion of
cardiomyocytes or part of a systemic inflammatory process.
13.7.6 Neurological Manifestations
Neurological manifestations of dengue infection have been recognized for some time,
and are receiving increased attention in light of the continued spread of dengue. The lat-
est WHO guidance specifically mentions neurological manifestations (encephalopathy
and encephalitis), recommending that although these may occur in the absence of classi-
cal features, they should be considered markers of “severe dengue” (WHO 2009). Fever
and exposure in an endemic area should be sufficient to raise suspicion.
Numerous neurological manifestations have been reported. They may be classified
as primarily central (inside the meninges) or peripheral (outside the meninges), and
are outlined in Table 13.2. These will be examined in detail in the following sections.
TABLE 13.2
Neurological Manifestations of Dengue Infection
“Central” Encephalopathy (Section 13.8)
Encephalitis (Section 13.9)
Transverse myelitis (Section 13.10)
“Peripheral” Guillain-Barré syndrome (Section 13.11.1)
Mononeuropathy (Section 13.11.2)
Myositis (Section 13.11.3)
13.8 DENGUE ENCEPHALOPATHY

Encephalopathy, a clinical picture of reduced consciousness with variable other features
of central nervous system dysfunction, is the most frequently reported neurological com-
plication of dengue infection. The incidence is variably reported as 0.5% (Cam et al.
2001) to 6.2% (Hendarto and Hadinegoro 1992) of DHF cases. There is evidence that
DEN-2 and DEN-3 are most often implicated (Cam et al. 2001; Solomon et al. 2000).
Encephalopathy is often caused by infections, metabolic derangements, alcohol
or drugs. Many extra-cranial infections result in encephalopathy, not through central
nervous system penetration, but as a result of a systemic inflammatory response. In
contrast, encephalitis is a histological diagnosis of inflammation of the brain paren-
chyma, commonly due to viral infection. Organisms with the capacity to infect neu-
rons are described as neurotropic, and those causing disease are neurovirulent. The
typical clinical features of encephalitis are fever (if infective), reduced conscious-
ness, headache, seizures, and focal neurological signs. Thus, encephalitis is one of
the causes of encephalopathy, albeit not the most common.
Although dengue encephalopathy has been recognized since the 1970s, there has
been much controversy as to the underlying mechanism; in particular, whether the den-
gue virus is neurotropic and therefore has the potential to penetrate the blood–brain
barrier (BBB) and cause encephalitis. For this reason, Solomon and Barrett (2003) have
suggested the term “cerebral dengue,” by analogy to “cerebral malaria,” the term implies
brain involvement without specifying a pathological mechanism. The traditional view
has been that dengue is nonneurotropic and that dengue encephalopathy is mediated
indirectly by the systemic effects of severe dengue infection. These are outlined in Table
13.3. Later research has challenged this understanding, and will be reviewed below.
TABLE 13.3
Possible Causes of Dengue Encephalopathy
Liver failure leading to hepatic encephalopathy
Vascular leak leading to cerebral edema
Bleeding tendency leading to intracranial hemorrhage
Hyponatremia
Direct viral infiltration leading to encephalitis
13.8.1 Hepatic Encephalopathy
In a study of dengue encephalopathy in Vietnam, 24% of cases were attributable to
hepatic encephalopathy (Solomon et al. 2000). Dengue virus has a recognized tro-
pism for liver macrophages (Kupffer cells) and hepatocytes, and hepatic involvement is
common in severe dengue infection. Although this is usually manifest only as a slight
increase in transaminases, reflecting a degree of hepatocellular injury, there may be
progression to more severe forms of liver failure (Lawn et al. 2003). Mohan et al. (2000)
estimate that jaundice occurs in 12–62% of patients with DSS, representing impaired
hepatic function in the excretion of bilirubin. As the liver fails and excretory function
is further impaired, neurotoxic products of cellular metabolism, including ammonia
(which should undergo hepatic detoxification to urea), are retained in concentrations
that result in complex and deleterious effects on cerebral function. The clinical con-
sequences range from neuropsychiatric disturbances to coma. A classic clinical sign
is asterixis, a flapping tremor of the outstretched hands that results from dysfunction
of central motor areas controlling posture. In a study of 191 Thai children with vari-
ous dengue grades, Wiwanitkit (2007) found that 35% had liver dysfunction, and 8%
frank hepatic encephalopathy, while in Malaysia, Lum et al. (1996) found that hepatic
encephalopathy occurred in 20% of children with DHF/DSS.
13.8.2 Cerebral Edema
An abnormal accumulation of water in the brain parenchyma may occur in a wide
range of pathological states, reflecting the multifaceted control of intracranial
homeostasis in normal physiology. Vasogenic edema occurs when there is disruption
of tight junctions between endothelial cells that comprise the BBB, allowing uncon-
trolled fluid shift; whereas cytotoxic edema results from cellular injury and loss of
intracellular contents.
Cerebral edema compromises brain function. First, extracellular water interrupts
delicately maintained concentrations of ions and neurotransmitters. Second, the
Monro-Kellie doctrine states that, since the cranium describes a fixed volume, the
brain, blood, and cerebrospinal fluid (CSF) contained within it must maintain a state
of volume equilibrium. Thus, the cerebral perfusion pressure, intracranial pressure,
and volume of brain tissue are related, and if one is elevated there must be a com-
pensation in the others. As parenchymal volume rises, cerebral perfusion is rapidly
compromised, leading to neuronal death. The final consequence of cerebral edema is
brain herniation, as the increased intracranial pressure is relieved by the evacuation
of brain tissue through the foramen magnum, with fatal results.
Cerebral edema has been shown to occur widely in dengue encephalopathy, both
at postmortem and on brain imaging (see Table 13.4). The pathophysiology is likely
multifactorial. In part it may be a continuation of the widespread endothelial disruption
and vascular leak that occurs in severe dengue, leading to vasogenic cerebral edema.
Whether the effect on cerebral microvasculature reflects dengue infection of endothelial
cells or a systemic cytokine storm remains to be seen. In part it may also occur second-
ary to hyponatremia, leading to fluid shift. Finally, if dengue is indeed neurotropic,
cerebral edema may occur due to cytotoxic effects on neurons, as it may do in other
TABLE 13.4
Cerebral Edema in Dengue
Study Patients Cases with Cerebral Edema
Postmortem Diagnosis
Nimmannitya et al. 1987 10 (DHF) 3
Janssen et al. 1998 1 (DF) 1
Chimelli et al. 1990 5 (DHF) 3
Radiological Diagnosis
Lum et al. 1996 6 (DHF) 3 (on CT)
Kankirawatana et al. 2000 8 (DHF) 2 (on CT)
Cam et al. 2001 27 (DHF) 12 (on MRI)
Wasay et al. 2008 6 (DHF) 3 (on CT)
viral encephalitides. Interestingly, Solomon et al. (2000) identified a case of dengue

encephalopathy with associated cerebral edema on CT in which no diagnostic features
of DF or DHF were apparent—this was likely edema secondary to dengue encephalitis.
13.8.3 Intracranial Hemorrhage
The hemorrhagic diathesis and vasculopathy of severe dengue infection predisposes
to intracranial hemorrhage which may result in encephalopathy. This has been dem-
onstrated in several postmortem and neuroimaging studies (see Table 13.5). The
TABLE 13.5
Intracranial Hemorrhage in Dengue
Patients with
Study Encephalopathy Cases with Intracranial Hemorrhage
Postmortem Diagnosis
Burke 1968 12 (DHF) 2 intracerebral
1 subarachnoid
1 subdural
Nimmannitya et al. 1987 10 (DHF) 6 intracerebral
Janssen et al. 1998 1 (DF) 1 brainstem
Radiological Diagnosis
Patey et al. 1993 1 (DHF) 1 subarachnoid (on CT and MRI)
Cam et al. 2001 27 (DHF) 1 unspecified (on MRI)
De Souza et al. 2005 1 (DSS) 1 brainstem (on CT and MRI)
Kumar et al. 2007 1 (DHF) 1 basal ganglia and intracerebral (on CT)
Kumar et al. 2009 5 (DHF) 3 basal ganglia (on CT)
2 intracerebral and subdural (on CT)
distinction between postmortem and radiological diagnoses is important, as clini-

cally significant microscopic and petechial brain hemorrhage may occur without
obvious correlation on brain imaging; especially as much of the literature has uti-
lized computed tomography (CT), which provides suboptimal images of the posterior
fossa and could potentially miss small but significant hemorrhages in the brainstem.
13.8.4 Deranged Electrolytes
Several studies have reported an association between dengue infection and hypona-
tremia (Mekmullica 2005), with a recent study of 150 patients by Lumpaopong et
al. (2010) showing that 61% of DF and 72% of DHF cases are mildly hyponatremic.
Again, this is likely multifactorial, but is probably a result of intravascular volume
depletion in severe dengue infection leading to pituitary release of anti-diuretic hor-
mone (ADH) and plasma dilution. It is worth noting that viral encephalitis is a rec-
ognized cause of the syndrome of inappropriate ADH secretion (siADH).
Severe hyponatremia is generally taken as a serum sodium concentration below
125 mmol/L, the level below which cerebral edema occurs. When developing rap-
idly, a hyponatremic encephalopathy results. A more moderate and insidious decline
in serum sodium is better-tolerated, and chronic hyponatremia is common and often
asymptomatic. It appears that the hyponatremia in dengue infection tends to be fairly
mild, and is unlikely to be the sole cause of encephalopathy.
13.8.5 Cerebral Hypoperfusion
Profound hypotension in DSS may result in hypoxic-ischemic encephalopathy, and
Limonta et al. (2007) have demonstrated cell apoptoses in the brain tissue of fatal
cases of DSS. Below a mean arterial pressure of approximately 50 mmHg, there is a
failure of cerebral autoregulation and blood flow to the brain is compromised. There
is a spectrum of presentation related to the severity of the ischemic insult and reflect-
ing the selective vulnerability of various cell types. Neurons have a high metabolic
activity and are highly vulnerable; following a period of global cerebral ischemia
cellular dysfunction and death ensues, resulting in encephalopathy. The spatial dis-
tribution of ischemia and infarction is typically in the “watershed area,” found in the
border zone between the anterior and the middle cerebral arteries where perfusion
has little physiological reserve. Widespread infarction is largely irreversible and car-
ries a poor prognosis.
13.9 DENGUE ENCEPHALITIS

13.9.1 Literature Review
When you have eliminated the impossible, whatever remains, however improbable,
must be the truth. Sherlock Holmes, The Sign of the Four (A. Conan Doyle 1988).
Dengue virus was previously believed to be nonneurotropic. It was thought that den-
gue encephalopathy must be a result of the systemic disruption that occurs in severe
infection. Individual cases in which the encephalopathy was attributable to one or
TABLE 13.6
Isolating Dengue Encephalitis
Key Studies
Study Location Patients Exclusion Criteria
Kankirawatana et al. 2000 Bangkok, Thailand 8 All studies excluded:
Solomon et al. 2000 Ho Chi Minh City, Vietnam 9 Hepatic encephalopathy
Misra et al. 2006 Lucknow, India 11 Intracranial hemorrhage
Kularatne et al. 2008 Peradeniya, Sri Lanka 6 Electrolyte derangement
Cerebral hypoperfusion
more of the causes outlined above (hepatic encephalopathy, cerebral edema, intra-
cranial hemorrhage, deranged electrolytes, or cerebral hypoperfusion) lent credence
to this view. However, with the increasing prevalence of dengue encephalopathy
there has been a growing body of literature which identifies a subset of patients in
which one of these causes cannot be identified. A parallel strand of evidence has
emerged from the discovery of dengue virus and anti-dengue immunoglobulins in
the CSF of these patients. Together, these findings strongly suggest that dengue virus
is indeed neurotropic; it is capable of infection and replication within the central
nervous system, and that therefore there exists a separate clinical entity which is cor-
rectly called dengue encephalitis.
Many of the early studies in this area were unable to adequately exclude other
causes of encephalopathy and hence it is difficult to draw conclusions from them
regarding encephalitis per se. Much of the literature is limited to case reports, which
although instructive, are of limited statistical value. However, four studies with
exhaustive exclusion criteria have provided the preliminary evidence. Table 13.6 out-
lines the key clinical studies supporting the hypothesis of dengue viral neurotropism.
As discussed previously, cerebral edema is compatible with true viral encephalitis so
is not included as an exclusion criterion.
13.9.2 Clinical Features
The core clinical features of dengue encephalitis are fever, reduced consciousness,
headache, and seizures (Varatharaj 2010)—the core features of any viral encephali-
tis. These are listed in Table 13.7. Numerous other features have been associated with
dengue encephalitis and are outlined in Table 13.8.
The presence of classical features of dengue infection such as rash and arthralgia
is variable. In two studies, 50% (Soares 2006) and 78% (Solomon 2000) of cases
of dengue encephalitis did not have typical features of dengue infection. Thus, the
absence of these features should not bar the consideration of dengue encephalitis as
the diagnosis in a suitable patient.
Dengue encephalitis is more often a consequence of secondary than primary den-
gue infection (Varatharaj 2010). This likely reflects the role of the immune response
(or lack thereof) in determining disease phenotype. In the key studies identified
TABLE 13.7
Core Clinical Features of Presumptive Dengue Encephalitis
Feature Percentage of Cases
Fever 100
Reduced consciousness 100
Headache 65
Seizures 47
Source: Adapted from Varatharaj, A., 2010, Neurol. India 58(4), 585–91.
TABLE 13.8
Other Clinical Features Associated with Dengue Encephalitis
Feature Study Comments
Abnormal posturing Solomon et al. 2000 Bilateral hippocampal
hyperintensities on MRI.
Amnesia Yeo et al. 2005
Epilepsia partialis continua Verma et al. 2010 Possibility of focal involvement of
primary motor area.
Extensor plantars Solomon et al. 2000
Facial nerve palsy Kankirawatana et al. 2000
Verma et al. 2010
Frontal release signs Solomon et al. 2000 Frontal lobe involvement.
Tetraparesis Misra et al. 2006 Brainstem or spinal cord
involvement.
Meningism Solomon et al. 2000 Likely co-existent
Kankirawatana et al. 2000 meningo-encephalitis.
Kularatne et al. 2008
above, the mean time of onset of neurological symptoms ranged from three to seven
days from the start of fever. Consensus suggests that the clinical course of den-
gue encephalitis is relatively self-limiting, and that with intensive care most patients
make a full recovery in days-weeks, with little or no residual deficits. The Lucknow
patients were atypical in this regard, as out of 11, 3 died and 3 were left with residual
deficits (Misra 2006). It is difficult to determine what interplay of viral, host, or
medical factors were at play, but it would not be surprising to discover that different
strains possess varying properties of neurovirulence, nor to find that certain hosts
are more vulnerable. Clearly, this will be an important avenue of future research.
13.9.3 Laboratory Features

CSF lymphocytosis may occur and suggests a meningoencephalitic process.
Reported frequencies range from 12.5% (Kankirawatana 2000) to 73% (Misra
2006). Although dengue virus or antibody is reliably isolated from the serum, evi-
dence of dengue in the CSF (either antigen or antibody) is found in only a minority
of patients, 17% in the four key studies. In another study dengue antibody was found
in the CSF of only 2 out of 7 (29%) patients with dengue encephalitis (Soares 2006).
Is the presence of dengue virus and/or antibody in the CSF evidence of encephali-
tis? It could be argued that disruption of cerebral vascular endothelium allows serum
contents to passively leak into the CSF, without active viral CNS invasion. This
hypothesis is unsatisfying for three reasons. First, these patients with evidence of
dengue in the CSF have an encephalopathy which cannot otherwise be explained,
and it is parsimonious to conclude that viral neurotropism is the explanation. Second,
virus may be present in the CSF while simultaneously absent in the serum, a find-
ing which does not correspond with the suggestion of passive viral leak during the
viremic phase (Domingues 2008). Third, histological studies have confirmed the
presence of viral components in brain tissue, directly supporting the hypothesis of
neurotropism (Miagostovich 1997; Ramos 2008).
It then remains to be explained why only a minority of patients with dengue
encephalitis has detectable evidence of virus in the CSF. For comparison, detection
of virus in the CSF by PCR has a sensitivity of >95% for herpes simplex encephalitis
(Cinque 1996). Perhaps the apparent low sensitivity is due to a low CSF viral load,
because even though PCR has a sensitivity of 93%–100% for serum dengue virus,
this level of reliability requires a viral load of at least 100 genome copies (Lanciotti
1992). Kao et al. (2005) have commented that this problem applies to dengue anti-
body detection in the CSF. An additional problem is that the temporal fluctuations
in CSF viral load and antibody titer are not known, and hence it is difficult to time
sample collection to maximize sensitivity. These problems will need to be addressed
by future studies.
13.9.4 Radiological Features
Computed tomography (CT), magnetic resonance imaging (MRI), and other brain
imaging modalities aid in the diagnosis of viral encephalitis. MRI provides more
information than CT by delivering greater definition of brain parenchyma and
improved views of the posterior fossa. Imaging helps with the exclusion of differ-
ential diagnoses and may also identify signs suggestive of viral encephalitis, such
as cerebral edema, white matter changes, and localized necrosis. The addition of
a gadolinium-based contrast agent identifies areas of BBB breakdown. Focal and
asymmetrical abnormalities are suggestive of encephalitis over encephalopathy, the
latter tending to produce more global and symmetrical changes.
Many viral encephalitides display a tropism for particular brain structures, which
results in typical imaging patterns. The predilection of herpes simplex virus for the
temporal lobes or that of Japanese encephalitis virus for the basal ganglia, is well-
established. Is there is a similar tropism for dengue encephalitis, and correspond-
ing features on brain imaging? A summary of reported brain imaging findings in
dengue encephalitis is shown in Table 13.9. No stereotypical pattern of involvement
has yet emerged, although the focal nature of abnormalities supports the diagnosis
of encephalitis.
TABLE 13.9
Brain Imaging Findings in Dengue Encephalitis
Study Imaging Findings Comments
Cam et al. 2001 MRI Focal “encephalitis-like” changes Authors did not specify
location
Yeo et al. 2005 MRI Bilateral hippocampal hyperintensity Patient had retrograde
amnesia
Misra et al. 2006 MRI Largely normal, one showed
hyperintensity in globus pallidus
Muzaffar et al. 2006 MRI Temporal lobe hyperintensity
Kamble et al. 2007 CT Thalamic hyperintensity JE serology negative
Wasay et al. 2008 CT/MRI Cerebral edema
Focal changes in temporal, occipital,
frontal lobes, and pons and upper
spinal cord.
13.9.5 Dengue Encephalitis in Perspective

Sufficient evidence has now emerged to support the hypothesis of dengue viral neuro
tropism and establish the existence of dengue encephalitis as a distinct clinical entity. A
case definition is outlined in Table 13.10. As several authors have pointed out, the vali-
dation and adoption of a uniform case definition will greatly aid the further recognition
and study of this condition (Varatharaj 2011; Soares and Puccioni-Sohler 2011).
What is the impact of dengue encephalitis? Rather than taking patients with dengue
and narrowing down to those with encephalitis, several studies have taken patients
with encephalitis and filtered out those with dengue (see Table 13.11). Just as the cli-
nician’s thought-process works from presentation to etiology rather than vice-versa,
these studies provide a very interesting picture of the real impact of dengue encephali-
tis. The results show that dengue is not just “on the list” of causes of viral encephalitis;
in endemic areas it is an important agent and needs to be actively considered.
TABLE 13.10
Case Definition for Dengue Encephalitis
Dengue Virus or IgM in Serum
Core Features Not Explained by Corroborating Evidence

Fever Acute liver failure CSF lymphocytic pleocytosis
Reduced consciousness Shock Dengue virus or IgM in CSF
Headache Electrolyte derangement Suggestive brain imaging
Seizures Intracranial hemorrhage
TABLE 13.11
Dengue as a Cause of Encephalitis in Endemic Areas
Frequency of
Study Location Patients Dengue (%)
Kankirawatana 2000 Thailand Children with suspected viral encephalitis 18
Solomon et al. 2000 Vietnam Children with suspected CNS infection 4.2
Horm Srey et al. 2002 Cambodia Children and adults with suspected encephalitis 5
Van Tan et al. 2010 Vietnam Children with suspected viral encephalitis 4.6
Soares et al. 2011 Brazil Adults with suspected viral encephalitis 47
13.9.6 Neuropathogenesis
How does dengue defeat the BBB, and how does it damage neurons? Clearly, not all
dengue infections result in encephalitis, so some interplay of host and viral factors is
likely at work. Understanding these factors will be key in future efforts for preven-
tion, detection, and treatment.
In general, viral entry to the central nervous system occurs via transmission
through nerve axons or by spread across the BBB during viremia. There is no evi-
dence to suggest that the former occurs with dengue, although retrograde spread
through the olfactory nerve has been shown to occur with the related flaviviruses St.
Louis encephalitis virus (Monath 1983) and Japanese encephalitis virus (Yamada
2009). In contrast, there is evidence to suggest that dengue may be able to weaken the
BBB. Chaturvedi et al. (1991) showed that dengue virus causes BBB breakdown via
a histamine-dependent pathway. This is notable given that mast cells are targets of
dengue infection and are vulnerable to antibody-dependent enhancement in second-
ary infection (Brown et al. 2006). Mast cell infection results in the release of various
vasoactive and immunologically active cytokines (King et al. 2002; St John et al.
2011), and it is known that products of mast cell degranulation, including histamine,
result in BBB breakdown (Abbott 2000). Thus, it seems reasonable to postulate that
a defective immune response in secondary dengue infection coupled with the ability
of infected mast cells to open the BBB may a play a role in dengue neurovirulence.
Little is known about the specific effects of dengue on neurons. In general, neu-
rotropic viruses cause neuronal cell death either by direct cytopathic effects or by
inducing a fatal immune response against infected cells. Amaral et al. (2011) have
shown that dengue virus (DEN-3) injected directly into the brains of mice results in
behavioral changes, seizures, and death (an encephalitis-like syndrome). Viral load
within the brain increased with time, suggesting active infection and replication,
and a CNS inflammatory response was stimulated, resulting in inflammatory cell
infiltration and the release of cytokines.
13.10 DENGUE TRANSVERSE MYELITIS

Transverse myelitis is an inflammatory condition of the spinal cord. Presentation
depends on the level of involvement but typically there is initial flaccid paraparesis
TABLE 13.12
Dengue Transverse Myelitis
Author Time to Onset of Paraparesis Recovery Period
Solomon et al. 2000 4–5 days Improvement after 7–15 days with some
residual symptoms
Leao et al. 2002 12 days (although urinary Improvement after 35 days, full recovery
retention developed after after 6 months
2 days)
Chanthamat and 6 days Improvement after 10 days, full recovery
Sathirapanya 2010 after 1 year
which evolves into spasticity, reflecting interruption of descending corticospinal

fibers. Sphincters are often involved, and a sensory level is characteristic. The cause
is typically either demyelinating disease, such as multiple sclerosis, neuromyelitis
optica or acute disseminated encephalomyelitis; systemic inflammatory disease such
as lupus or sarcoidosis; or related to infections such as with herpesviruses, flavivi-
ruses, and bacteria such as Borrelia burgdorferi. In infectious transverse myelitis,
cord involvement typically occurs contemporaneously with the febrile phase of ill-
ness, and there is usually a CSF reaction with cells and protein.
Transverse myelitis occurring in the acute phase of dengue infection has been
recorded in the literature (see Table 13.12). In these cases the onset of paraparesis
coincided with the febrile phase of illness, so it is reasonable to suppose the etiology
is para-infectious rather than postinfectious. However, it remains to be seen whether
the underlying mechanism is direct viral infiltration or an immune phenomenon.
13.11 PERIPHERAL NERVOUS SYSTEM MANIFESTATIONS

13.11.1 Guillain-Barré Syndrome
The term “Guillain-Barré syndrome” describes the clinical picture of an acute
inflammatory demyelinating polyradiculoneuropathy, with variable motor, sensory,
and autonomic involvement. The typical presentation is with ascending symmetrical
paralysis and areflexia, which may progress to respiratory weakness and failure.
The majority of cases are preceded by an infection, often Campylobacter jejuni,
although numerous antecedent infections have been associated. The etiology is
thought to be molecular mimicry leading to immunological cross-reactivity against
nerve components.
Several authors have reported cases of Guillain-Barré syndrome associated with
dengue infection (Chew et al. 1998; Esack et al. 1999; Leão et al. 2002; Santo et
al. 2004; Sulekha et al. 2004; Kumar et al. 2005; Soares et al. 2006; Chen and Lee
2007; Soares et al. 2008; Gupta et al. 2009; Chanthamat and Sathirapanya 2010).
In all cases typical features of DF/DHF were initially present, followed by a delay
ranging from 7 to 30 days before the onset of paralysis. CSF analysis and nerve
conduction studies have yielded results consistent with classical Guillain-Barré,
namely cytoalbuminemic dissociation (high protein and low cells) with demyelin-
ation and/or denervation. Intravenous immunoglobulin has been used with good
outcomes.
13.11.2 Mononeuropathy
Post-infectious mononeuropathies have also been reported in association with den-
gue. Case reports describe involvement of phrenic (Chien et al. 2008; Ansari et al.
2010), long thoracic (Chappuis et al. 2004), optic (Sanjay et al. 2008), facial (Patey
et al. 1993), and ulnar and peroneal (Kaplan and Lindgren 1945) nerves. As with
postinfectious Guillain-Barré syndrome, there is a delay of days or weeks between
the initial infection and the development of neurological symptoms, reflecting the
presumed immunological etiology. Immunosuppressive therapies have been used in
a range of dengue mononeuropathies with variable success.
13.11.3 Myositis
Myalgia has long been recognized as a characteristic feature of dengue infection,
even more so than with other viral illnesses. Muscle biopsies in uncomplicated den-
gue have shown inflammatory cell infiltrates, with rare myonecrosis (Malheiros
1993). In some cases, however, the disease may progress to frank myositis with
variable degrees of muscle breakdown, weakness, and elevation of serum creatine
kinase (CK). These patients have tender muscles, flaccid weakness, and typically
no evidence of CNS involvement. CSF analysis and neuro-imaging are normal, and
evidence of dengue infection is obtained from the serum but not the CSF. Muscle
biopsies in these cases shown dense inflammatory cell infiltrates (Kalita et al. 2005;
Paliwal et al. 2011). Interestingly, Paliwal et al. (2011) have identified two distinct
presentations of dengue myositis, which likely represent points on a spectrum of
underlying immune responses. In primary infections, myositis featuring predomi-
nantly lower limb weakness developed 3–15 days after dengue symptoms, and was
associated with a moderate rise in CK. These patients gradually recovered over
weeks, with a good outcome. In secondary infections, however, patients experienced
a delay of several weeks before the onset of a rapidly progressive myositis, with gen-
eralized skeletal muscle dysfunction and respiratory failure, massively elevated CK,
and poor outcome.
Management of dengue myositis is supportive, with an emphasis on early
respiratory support for patients at risk of ventilatory failure. Some groups have
found benefit from immunosuppression (Finsterer and Kongchan 2006), a strat-
egy that has been used with some success in HIV-associated myositis (Johnson
et al. 2003). A rhabdomyolysis-like picture may occur in conjunction with renal
failure due to glomerular deposition of myoglobin, which can be prevented by
vigorous hydration (Davis and Bourke 2004; Lim and Goh 2005; Acharya et
al. 2010). Transvere myelitis and Guillain-Barré syndrome should be excluded
as other causes of a dengue-associated weakness, as these may have different
therapeutic options.
Thus, muscle involvement in dengue infection runs along a spectrum from myal-
gia and benign self-limiting myositis to fulminant myositis. The pathology is unclear
and may variably reflect direct viral infiltration of myocytes (Salgado et al. 2010)
or an immune-mediated insult. The increased severity associated with secondary
infections is in keeping with other manifestations and does suggest a degree of
immunopathogenesis.
13.12 LABORATORY DIAGNOSIS OF DENGUE INFECTION

13.12.1 General Investigations
In uncomplicated DF, routine blood tests are usually normal. In DHF/DSS,
however, the full blood count invariably shows thrombocytopenia and elevated
hematocrit. The white cell count (WCC) is variable, and there may be a reactive
lymphocytosis; however, a neutropenia and fall in total WCC at the end of the
febrile phase is almost universal. Clotting analysis shows a coagulopathy with an
increase in APTT (activated partial thromboplastin time) and PT (prothrombin
time). Plasma albumin is low due to vascular leak, and liver enzymes may be
deranged in the presence or absence of apparent hepatic failure. Additionally,
in DSS metabolic acidosis, hyponatremia, and deranged renal function may be
seen.
13.12.2 Confirming Dengue Infection

It is a general principle in the laboratory diagnosis of viral infections that one
may aim to detect either the virus or the host immune response (Table 13.13).
Which strategy to pursue is a decision that is based on timing. The initial febrile
stage of infection corresponds with the viremic phase, so it is sensible to aim to
detect the virus. In an immunocompetent individual, after about 5 days, there is
an immune response that abolishes detectable viremia and defervesence ensues.
Hence, in these patients, it is more suitable to aim to detect the host immune
response.
TABLE 13.13
Laboratory Methods of Confirming Dengue Infection
Detection of Virus
Viral culture
PCR amplification of viral RNA
Immunochemistry for viral antigens
Detection of Host Immune Response

MAC-ELISA for anti-dengue IgM
Hemagglutination-inhibition (HI) test
13.12.3 Detection of Virus
Detection of the virus has traditionally been achieved by viral culture, and this
remains the gold-standard test, though it is time-consuming and costly. Specimens
must be collected before defervescence or soon after, as the humoral response
interferes with culture. Excessive heat may inactivate the virus, and specimens
must be transported chilled. The sample is then inoculated into larval or adult
mosquitoes of the genus Toxorhynchites. After a few days, in which almost all
tissues of the inoculated mosquito become infiltrated by virus, a tissue smear is
prepared from the head of the mosquito and examined by immunofluorescence.
Alternatively, samples may be inoculated into mosquito cell culture (typically the
C6/36 clone from Aedes albopictus) with comparable results, and this method is
now widely used.
The second option is detection of viral RNA by PCR (polymerase chain reac-
tion) assay. PCR is quicker, more widely available, can detect viremia regardless of
disease phase, and can be used to differentiate serotypes based on distinct genetic
sequences. Viral RNA is extracted and purified from the sample and the desired
sequences are rapidly amplified using a combination of specific primers. The ampli-
fied product which results is separated and identified by agarose gel electrophore-
sis. A well-validated PCR assay developed by Lanciotti et al. (1992) demonstrates
sensitivities of 94% (DEN-1), 93% (DEN-2), and 100% (DEN-3 and -4). However,
the disadvantage of PCR is that it is susceptible to sample contamination and false-
positives, which may result from nonspecific primer binding or binding to conserved
sequences of other flaviviruses.
The third option now emerging is detection of viral antigens by immunochem-
istry. An interesting candidate currently being investigated is the NS1 antigen. In a
recent multicenter trial with 1385 patients, one commercially available assay kit had
a reported sensitivity and specificity of 64% and 100%, respectively (Guzman et al.
2010). In a smaller trial, another group achieved a sensitivity of 89% (Dussart et al.
2006). This test has the advantage of being easier and quicker than culture, cheaper
than PCR, and can be used in the acute phase unlike serology. The poor sensitivity
is a problem, however, and further work is needed.
13.12.4 Detection of Host Immune Response

Detection of the host immune response (serology) has numerous advantages.
Specimen collection is not time-critical, as antibodies persist for several weeks, and
there is little risk of inactivation by heat. Serological assays are also fairly simple to
use, and widely available as self-contained kits. The commonly used technique is
MAC-ELISA (IgM antibody-capture enzyme-linked immunosorbent assay) which
measures “dengue-specific” IgM. The presence of IgM antibodies indicates recent
infection (within 24 weeks), and it is possible to confirm acute infection by demon-
strating rising antibody titers in paired acute and convalescent samples. The main
disadvantage of serological tests is that they have a poor sensitivity in the acute
phase of infection, before the development of an immune response. There also have
a limited specificity as there is an element of cross-reactivity with antibodies against
other flaviviruses, especially in areas where dengue and Japanese encephalitis co-
circulate. In an evaluation of one commonly-used proprietary MAC-ELISA kit, the
initial sensitivity was 69% rising to 90% on convalescent testing, while specificity
was 80% (Singh et al. 2006).
IgG antibody-capture ELISA (GAC-ELISA) is also available. Low titers of IgG
become detectable after the first week and continue to rise for months. These likely
persist for life, though eventually at concentrations which may become undetectable.
By paired ELISA, the ratio of IgM to IgG can be calculated and this allows the dif-
ferentiation of primary (predominantly IgM) from secondary (predominantly IgG)
infections.
The hemagglutination-inhibition test has been largely superseded by newer meth-
ods. The serum sample is added to a fixed dose of dengue antigens. When red blood
cells are added the antigens normally cause hemagglutination. In the presence of
anti-dengue antibodies this hemagglutination is inhibited to a degree which is quan-
tifiable and corresponds to antibody titer.
13.13 MANAGEMENT
13.13.1 Dengue Fever
No specific treatment exists for dengue infection. Treatment is supportive and aimed
toward the early detection and management of complications. In DF, control of fever
may be achieved with cautious use of paracetamol. Aspirin is best avoided due to
the antiplatelet effect and the risk of precipitating Reye’s syndrome. Adequate nutri-
tion should be ensured and, if necessary, oral rehydration therapy considered. Close
monitoring for signs of conversion to DHF/DSS is essential, especially around the
period of defervescence. It must be remembered that in the early stage it is difficult
to accurately predict which patients will go on to develop severe infection; regu-
lar monitoring is the only sure strategy. In practice, this means that after an initial
assessment (see Table 13.14) most cases with nonsevere infection and no warning
TABLE 13.14
Initial Assessment
History Examination Investigation
Time of onset of fever Hydration Full blood count
Oral intake Hemodynamic stability Urea and electrolytes
Warning signs Respiratory distress, pleural effusion Baseline hematocrit
Diarrhea Abdominal tenderness, hepatomegaly, Confirmation of dengue infection
Urine output ascites (not usually necessary in
Dengue in family or Rash, bleeding uncomplicated cases)
neighborhood Tourniquet test
Travel history Neurological manifestations
Medical history
signs may be managed as outpatients, with daily examination and monitoring of

platelet count and hematocrit.
13.13.2 Dengue Hemorrhagic Fever and Shock

Management of DHF rests upon the careful replacement of intravascular fluid and
electrolyte losses. Early fluid resuscitation avoids complications. In mild cases fluid
replacement may be oral; if so oral rehydration solutions as used for diarrhea are
ideal. Again, close monitoring is required, especially during defervescence. Signs of
hemodynamic compromise or fall in platelet count and elevation of hematocrit are
indications for parenteral therapy. Isotonic solutions of crystalloid may be infused at
a rate of 5–7ml/kg/hr, adjusted according to response. Fluid replacement should be
carefully titrated to hemodynamic status and urine output (aiming for above 0.5ml/
kg/hr), to avoid the risk of overload. In patients with warning signs, further labora-
tory tests including blood glucose, renal function, liver function, and clotting should
also be monitored.
DSS is a medical emergency and the aim should be to urgently expand plasma
volume. Crystalloid or colloid should be given in a bolus of 10–20 ml/kg in order
to support effective circulation and organ perfusion. According to response the rate
of infusion may be gradually reduced to a maintenance regime. Again, strict fluid
balance is required to optimize intravascular filling and avoid overload. If there are
signs of bleeding or a falling hematocrit then urgent tranfusion should be arranged
along with, if appropriate, specific interventions to arrest the bleeding point (e.g.,
urgent endoscopy in the case of upper gastrointestinal tract hemorrhage). Ideally the
patient should be managed in an intensive care setting, with invasive monitoring of
physiological parameters.
13.13.3 Dengue Encephalopathy
As has been discussed previously, the term “dengue encephalopathy” covers a range
of mechanisms which may cause cerebral insult. The etiological distinction is of crit-
ical importance if effective management is to be delivered, and hence these patients
require a detailed diagnostic work-up (see Table 13.15).
If a nonencephalitic etiology is established, it may be managed according to
established protocols. All are best handled in an intensive care or high dependency
unit.
Current strategies to manage hepatic encephalopathy focus on control of the
raised serum ammonia levels which result from impaired hepatic detoxification
(Bernal et al. 2010). Lactulose is commonly given for this reason as it reduces the
number of ammonia-forming gut bacteria, as well as acidifying the contents so as
to convert NH −4 to NH3 and potentiate the excretion of ammonia. Antibiotics such as
neomycin may also be given with the same purpose of reducing gut bacterial load.
If these methods fail, serum ammonia concentrations may be reduced directly by
hemofiltration. There is some evidence that therapeutic hypothermia slows cellular
metabolism and production of ammonia, though this strategy is not yet widely used
TABLE 13.15
Investigation of Dengue Encephalopathy
Test Findings
Hemodynamic monitoring Prolonged hypotension suggests hypoxic-ischemic encephalopathy
Electrolytes Low sodium suggests hyponatremic encephalopathy
Raised creatinine suggests acute renal failure and uremic encephalopathy
Liver function tests Raised bilirubin, transaminases, and prothrombin time suggest acute liver
Clotting tests failure and hepatic encephalopathy
Lumbar puncture Lymphocytosis and evidence of dengue in CSF suggests encephalitis
Red cells and xanthrochromia suggests hemorrhage
Brain imaging Intracranial hemorrhage
Cerebral edema
Focal changes suggestive of encephalitis
EEG Generalized changes of encephalopathy (e.g., slowing)
Focal changes suggestive of encephalitis (e.g., periodic lateralized
epileptiform discharges)
Seizure activity
(Vaquero et al. 2005). Ultimately, however, the only effective treatment for severe
hepatic encephalopathy is liver transplantation.
Cerebral edema requires the careful control of several physiological param-
eters, and it may be necessary to surgically implant an intracranial pressure
(ICP) monitoring device to better guide the fine adjustment of these variables.
Management can then be instigated in a step-wise fashion. Nursing in a head-
up position is a starting point; this aims to reduce ICP, however, and care must
be taken not to compromise cerebral perfusion. As hypoxia and hypercapnia
both result in cerebral vasodilation, it is important to ensure good oxygenation
and carbon dioxide clearance. If necessary, this may be achieved by therapeutic
hyperventilation. Careful fluid balance is needed to maintain cerebral perfusion
pressure without exacerbating fluid overload. Electrolyte levels should be cor-
rected to maintain serum osmolarity, as hypo-osmolarity worsens cerebral edema.
If these holding measures fail then mannitol, an osmotically active diuretic, may
be used to draw water from the brain parenchyma into the intravascular space.
Hypertonic saline solutions may be used in a similar way. In more desperate
situations it may be possible to induce a therapeutic coma using drugs such as
phenobarbital, which suppress cerebral metabolism and reduce ICP. Finally, in
the case of catastrophic brain swelling and impending herniation the only option
is decompressive craniectomy.
General medical management of intracranial hemorrhage is largely support-
ive. Cerebral autoregulation is impaired in the acute phase so adjustments to blood
pressure (which is often high) should be avoided for fear of compromising perfu-
sion. As to specific data on the management of intracranial hemorrhage in dengue,
there is little available evidence. It is unclear to what extent dengue hemorrhagic
encephalopathy reflects damage to cerebral vascular endothelium, bleeding di

athesis, or exacerbation of preexisting cerebrovascular disease. However, as the
bleeding diathesis likely plays at least some role, it would seem reasonable to
correct the clotting abnormalities, and some groups have used platelet transfusion
for this purpose (Kumar et al. 2009). In some cases it may be appropriate to use
fresh frozen plasma (FFP), prothrombin complex concentrate (PCC), or even acti-
vated factor VII (fVIIa). A large hemorrhage may cause a significant mass effect,
especially in the confined space of the posterior fossa, and if there are signs of
raised intracranial pressure it may be necessary to perform a decompressive cra-
niectomy or urgent CSF shunting.
Hyponatremic encephalopathy is managed by correcting the serum sodium.
In dengue the patient is likely to be intravascularly volume deplete so this may be
achieved by the judicious use of intravenous 0.9% saline. Too-rapid (more than
8mmol/L/day) correction of the serum sodium should be avoided, as this carries a
risk of precipitating central pontine myelinolysis.
Hypoxic–ischemic encephalopathy carries a poor prognosis. The initial priority
of management is to stabilize hemodynamic status and secure cerebral perfusion.
As fever and seizure activity both increase brain metabolic activity, these should be
suppressed. There is great interest in the application of therapeutic hypothermia for
reducing brain metabolism, and early results have been promising.
13.13.4 Dengue Encephalitis
General management of viral encephalitis requires close support in an intensive care
setting, with meticulous attention to the airway, to oxygenation, hydration, and nutri-
tion (Solomon et al. 2007). Seizure activity should be suppressed with anti-epileptic
drugs, and intracranial pressure should be carefully monitored and controlled. Until
bacterial infection and HSV encephalitis have been positively excluded by analysis
of the CSF, empirical treatment with a third-generation cephalosporin and acyclovir
should be continued. As an inhibitor of viral DNA polymerase, however, acyclovir
has no effect on the RNA replication of dengue virus. With no specific antiviral yet
available, management of a confirmed case of dengue encephalitis must focus on
intensive organ support. If the illness is indeed self-limiting, as has been suggested,
then this approach may buy enough time to allow recovery.
13.13.5 Future Treatments

There is currently no clinically available antiviral effective against dengue, though
an increasing understanding of the molecular basis of dengue pathogenesis has
provided an array of potential drug targets. A combination of high-throughput
screening with intelligent drug design has yielded various agents which have
shown promise for future use (see Table 13.16). All are currently in laboratory
stages of development and much further work is needed before large-scale clinical
trials can be conducted. From a CNS standpoint, one must also consider the ques-
tion of drug delivery across the BBB if a viable treatment for dengue encephalitis
is to be developed.
TABLE 13.16
Potential Dengue Antivirals
Agent Putative Targets Studies
Amantadine Viral entry (in vitro) Koff et al. 1980
Zosteric acid Viral entry (in vitro) Rees et al. 2008
Ribavirin RNA synthesis (in vitro) Koff et al. 1982
(in mice) Koff et al. 1983
Interferon RNA synthesis (in vitro) Diamond et al. 2000
(in monkeys) Ajariyakhajorn et al. 2005
Triaryl pyrazoline RNA synthesis (in vitro) Puig-Basagoiti et al. 2006
Morpholino oligomers RNA synthesis (in mice) Stein et al. 2006
Geneticin RNA synthesis (in vitro) Zhang et al. 2009
Translation
Protease inhibitors Viral protease (in vitro) Tomlinson et al. 2009
13.14 VACCINATION
There is currently no effective vaccine against dengue. However, recent decades
have seen great leaps forward in vaccine development, and promising candidates are
now nearing clinical reality.
As our current understanding of dengue pathogenesis suggests that the primary
protective host response is the initiation of specific neutralizing antibodies, the ideal
dengue vaccine should generate high and long-lasting antibody titers to cover all
four viral serotypes (tetravalence). Given the suspected role of antibody-dependent
enhancement in severe dengue infection, full tetravalency is crucial to avoid the
theoretical risk that an incomplete dengue vaccine may predispose toward enhanced
infection by an omitted serotype, especially as all four serotypes are now in wide-
spread co-circulation. This requirement has proved to be a significant challenge.
TABLE 13.17
Dengue Vaccine Candidates
Vaccine Type Comments
ChimeriVax Chimeric live attenuated virus on yellow Safe, immunogenic, but requires extended
fever backbone multiple-dose course
DEN4Δ30 Live virus attenuated by gene deletion Effective monovalent vaccine, tetravalent
formulation currently in trials
DENVax Chimeric live attenuated dengue virus
DEN-80E Recombinant envelope protein Less interference between serotypes
D1ME-VR-P DNA vaccine of envelope and membrane
genes
Source: Adapted from Webster, D. P. et al., 2009, Lancet Infect. Dis. 9, 678–87; Coller, B. G., and
Clements, D. E., 2011, Curr. Opin. Immunol. 23, 1–8.
Creating a tetravalent vaccine which generates a balanced immune response requires

overcoming interference between the separate components. Promising candidate
vaccines currently in development are shown in Table 13.17.
13.15 VECTOR CONTROL

An integrated strategy for the management of dengue will necessarily require con-
trol of the insect vectors. The highly resistant eggs of Aedes aegypti mean that it is
insufficient to attempt eradication of the mosquitoes, their larvae, and pupae alone,
as following rain the population will be regenerated from quiescent eggs. Thus, a
many-pronged approach is required, involving the eradication of adult and larval
forms with insecticides coupled with the clearing out and elimination of suitable
nesting locations. Clearly, this process is related to economic development and
increasing standards of public health and sanitation. The World Health Organization
has developed detailed guidelines for Integrated Vector Management (IVM), which
promote a multidisciplinary strategy operating at a range of levels from individual to
government (Table 13.18).
13.16 CONCLUSION
Dengue infections are common worldwide and represent a significant burden to
global health. The individual clinical manifestations of infection encompass a broad
spectrum of disease states, and represent the outcome of complex human-viral inter-
actions. The virus has the capacity to disrupt all major organ systems with results
that range from benign to fatal. Neurological manifestations of dengue infection
have long been appreciated as part of this spectrum. Although relatively infrequent,
the vast scale of the dengue problem means that the absolute incidence is signifi-
cant. Whereas these presentations were previously thought to be indirectly medi-
ated, decades of research now suggest that the dengue virus is truly neurovirulent,
and that dengue encephalitis is a significant entity in endemic areas. An increasing
appreciation of the neurological manifestations of dengue infection will advance our
understanding of dengue viral-human interactions in general. Some future research
questions are outlined in Table 13.18. In particular, understanding the interplay of
host and viral factors that determine the clinical outcome of infection in any given
TABLE 13.18
Future Research in Neurological Dengue
What is the temporal pattern of CSF viral load and antibody titer?
What is the sensitivity of PCR and ELISA in CSF?
What are imaging features that characterise dengue encephalitis?
What is the role of mast cells in CNS penetration?
What viral and host factors influence dengue neurotropism?
What is the efficacy of novel antiviral agents in the treatment of dengue encephalitis?
What is the mortality, morbidity, and economic impact of neurological dengue?
individual will be an important step forward that will aid the search for an effective
management strategy. The worldwide public health and economic ramifications of
the dengue problem are vast, and have driven efforts to achieve a sustainable solu-
tion. Aggressive vector control, widespread deployment of an effective vaccine, and
development of specific antiviral agents will all help to reduce the burden of this
pernicious disease. The next decades will show if these efforts are successful in halt-
ing the continued spread of the dengue virus.
ACKNOWLEDGEMENT
I thank A. J. Phillips (University of Cambridge) for assistance with obtaining references.
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14 Influenza Virus and
CNS Infections
Jun Zeng, Gefei Wang, and Kang-Sheng Li
CONTENTS
14.1 Introduction................................................................................................... 325
14.2 Biological Properties of Influenza Virus....................................................... 326
14.2.1 Virus Structure.................................................................................. 326
14.2.2 Genome Organization........................................................................ 326
14.2.2.1 Protein Functions................................................................ 327
14.3 Epidemiology................................................................................................. 328
14.4.1 Febrile Seizures................................................................................. 329
14.4.2 Reye’s Syndrome............................................................................... 330
14.4.3 Encephalitis Lethargica..................................................................... 330
14.4.4 Acute Necrotizing Encephalopathy................................................... 330
14.5 Diagnosis....................................................................................................... 331
14.6 Pathology and Pathogenesis........................................................................... 332
14.7 Prognosis and Treatment............................................................................... 333
References............................................................................................................... 335
14.1 INTRODUCTION
Influenza, commonly referred to as the flu, is an infectious disease caused by influ-
enza virus, a group of single-stranded minus-sense RNA viruses, which affects
birds and mammals. There are three types of influenza virus, influenza A, B, and
C. Influenza virus A or B causes the flu syndrome, including chills, fever, headache,
and sore throat and muscle pains. Although it is often confused with other influenza-
like illnesses, especially the common cold, influenza is a more severe disease than
the common cold. In adults, complications may follow the primary viral infection of
the respiratory tract, such as bronchitis and pneumonia (Sessa et al. 2001). In chil-
dren less than 5 years of age, the most common infective complication is acute otitis
media (Tsolia et al. 2006). Influenza C infection is usually asymptomatic.
Since the time of the Spanish flu during 1917–1919, influenza has been recog-
nized as a virus that might cause neurological complications (Hayase and Tobita
1997; Ravenholt and Foege 1982). Influenza virus has been observed as the cause of
325
central nervous system (CNS) dysfunction. Influenza virus is associated with various
CNS lesions that have poor prognosis, including influenza-associated encephalitis/
encephalopathy (IAE), Reye’s syndrome, and acute necrotizing encephalopathy
(ANE; Wang et al. 2010). Influenza A infection was a common cause of febrile sei-
zure admissions (Chiu et al. 2001), and encephalitis/encephalopathy (Morishima et
al. 2002).
14.2 BIOLOGICAL PROPERTIES OF INFLUENZA VIRUS

14.2.1 Virus Structure
The influenza virus is about 80–120 nm in diameter; the virion is enveloped and can
be either spherical or filamentous in form. The envelope of the virus is a lipid bilayer
membrane which originates from the virus-producing cell and which contains
prominent projections formed by hemagglutinin (HA) and neuraminidase (NA), as
well as the M2 protein. The lipid layer covers the matrix formed by the M1 protein.
14.2.2 Genome Organization
Influenza A and B virus genomes consist of 8 separate segments covered by the
nucleocapsid protein. Together, these build the ribonucleoprotein (RNP), and each
segment codes for a functionally important protein (Figure 14.1). Influenza C virus
harbors only 7 genome segments, and its surface carries only one glycoprotein. Type
A viruses are divided into subtypes based on differences of two surface proteins
called hemagglutinin (HA) and neuraminidase (NA). There are 16 different HA sub-
types and 9 NA subtypes.
1 PB2 Transcriptase: cap binding
2 PB1 Transcriptase: elongation
3 PA Transcriptase: protease activity?
4 HA Hemagglutinin
5 NP Nucleoprotein: RNA binding – transport of vRNA
6 NA Neuraminidase: release of virus

Matrix protein 1: major component of virion
7 M1/M2
Matrix protein 2: integral membrane protein–ion channel
Nonstructural protein 1: RNA transport, translation, splicing
8 NS1/NS2 Nonstructural protein 2: function not known
FIGURE 14.1 The relative sizes of the eight influenza segments as well as the genes that are
specified by each. (From PatentLens (2011). The influenza genome comprises eight segments.
In Figure 1: The Eight RNA Segments of the Influenza Genome, vol. 2011, patentlens. With
permission.)
Influenza Virus and CNS Infections 327
14.2.2.1 Protein Functions

14.2.2.1.1 Hemagglutinin (HA)
Hemagglutinin (HA) is a glycoprotein that binds the virus to the cell being infected.
The HA molecule is a combination of three homogeneous proteins which are bound
together to form an elongated cylindrical shape. HA is also the main viral target
of protective humoral immunity by neutralizing antibody. The binding affinity of
HA to the sialic acid residues partly accounts for the host specificity of the various
influenza A virus subtypes. Human viruses preferentially bind to sialic acid linked to
galactose by α-2,6 linkages that are the main type found on the epithelial cells of the
human respiratory tract, while avian viruses tend to bind to α-2,3 linkages that are
found on duck intestinal epithelium (Couceiro et al. 1993; Ito et al. 1998). The speci-
ficity for different receptors has been one of the interpretations for the species barrier
between human and avian influenza viruses. The presence of both α-2,6 and α-2,3
linkages on epithelium of the pig trachea is the reason why pigs may serve as the
“mixing vessel” for the genesis of new viral subtypes through co-infection (Ito et al.
1998; Liu et al. 2009). Chickens may have a similar role, since their lung and intes-
tinal epithelia have both types of linkages (Gambaryan et al. 2002). In the human
respiratory epithelium in nasal mucosa, pharynx, trachea, and bronchi express high
amounts of α-2,6 linkage, whereas the α-2,3 linkage is the major receptor on type II
pneumocytes cells and nonciliated cuboidal bronchiolar cells at the junction between
respiratory bronchiole and alveolus (Shinya et al. 2006), thereby allowing human
infection by avian influenza viruses (Ge and Wang 2011; Suzuki 2011). A mutation
that changes just one amino acid in hemagglutinin can alter the antigenic properties
significantly. Thus, the barrier to interspecies infection can be overcome easily.
14.2.2.1.2 Neuraminidase
Neuraminidase (NA) is an enzyme that helps the virus to breach cell walls. NA is
also known as sialidase, since it breaks the linkages between sialic acid and cellular
glycoproteins and glycolipids found in cell walls. There are 9 NA antigenic subtypes.
NA forms mushroom-like projections on the surface of the influenza virus. The top
consists of four identical proteins with a roughly spherical shape.
14.2.2.1.3 M2
M2 is an ion channel crucial for the pH-dependent dissociation of matrix proteins
from the nucleocapsid during viral uncoating and pH changes across the trans-Golgi
network during maturation of hemagglutinin molecules. M2 is the target of the ada-
mantanes (amantadine and rimantadine).
14.2.2.1.4 PB1
PB1 gene encodes protein, termed PB1-F2, a mitochondrial protein that causes cel-
lular apoptosis may be related to it permeabilizes the mitochondrial membranes by
forming an apoptotic pore with lipids (Chanturiya et al. 2004; Gibbs et al. 2003).
The hemagglutinin and PB2 proteins appear to be important in determining host
specificity and virulence.
The active RNA-RNA polymerase is responsible for replication and transcription.

The polymerase contains the PB2, PB1, and PA proteins. PB1 has an endonuclease
activity and holds active site (Poch et al. 1989), whereas PB2 is responsible for cap
binding. The NS1 and NS2 proteins have a regulatory function to promote the syn-
thesis of viral components in the infected cell.
14.3 EPIDEMIOLOGY
Thousands of deaths attributable to influenza infections occur annually in the United
States. According to the Centers for Disease Control and Prevention (CDC) report,
during 1976–2007, estimates of annual influenza-associated deaths from respiratory
and circulatory causes ranged from 3349 in 1986–1987 to 48,614 in 2003–2004. The
annual rate of influenza-associated death overall ranged from 1.4 to 16.7 deaths per
100,000 persons in the United States (CDC 2010).
The various subtypes of influenza present with new combinations of the surface
glycoproteins HA (H1–H15) and NA (N1–N9). Historically, these antigenic shifts
have resulted in pandemics every 10–40 years (Webster et al. 1992). Epidemics on
a smaller scale occur yearly or every few years and are caused by minor changes in
antigenicity of influenza virus (antigenic drift) by amino acid changes in the surface
antigen (HA and NA) due to point mutations of the genome.
The cases of febrile seizures have accumulated in Asia, especially in Japan
(Waruiru and Appleton 2004). The incidence of influenza-associated encephalopa-
thy has been reported much higher in Asia than in Europe and the America (Bhat et
al. 2005; Morishima et al. 2002; Okabe et al. 2000; Togashi et al. 2004). Based on
these reports, a genetic background might be involved in the pathogenesis of these
diseases.

The spectrum of influenza may range from subclinical illness to severe respiratory
tract infection that affects multiple organs with abrupt onset of fever. The incuba-
tion period is one day to a week. The illness, with fever and cough, sore throat, and
headache may last for 1–5 days. Children often present with nonspecific symptoms
such as vomiting, diarrhea in addition to high fever, cough, and rhinorrhea. Pediatric
influenza may provoke febrile convulsions, and give rise to otitis media and pneu-
monia (Peltola et al. 2003). Symptoms in neonates are lethargy, anorexia, apnea, and
interstitial pneumonia (Yuen et al. 1998). Viral and bacterial pneumonia are frequent
complications, whereas myositis, renal failure, myocarditis, and CNS symptoms
develop more rarely. The certain risk groups, such as patients with chronic heart and
lung disease, the elderly, transplant recipients, smokers, children with underlying
medical conditions, and pregnant women are more common to show these compli-
cations of influenza infection. The most common causes of death are respiratory
complications or cardiovascular diseases.
CNS involvement during influenza infection contains plenty of syndromes, more
often described in children than in adults (Studahl 2003). The major clinical entities
are encephalitis or encephalopathy. In etiological studies of encephalitis, influenza
A and/or B have been identified in up to 8.5% of adult patients with positive viro-
logical findings and in up to 10% of pediatric cases (Koskiniemi et al. 2001). Reye’s
syndrome and ANE are special forms of encephalopathies with high mortality and
sequelae. Rare conditions are myelitis caused by influenza virus (Salonen et al.
1997), and even more seldom, autoimmune diseases elicited by influenza such as
Guillain-Barré’s syndrome (Tam et al. 2007).
Encephalitis and encephalopathy are not always distinguishable from each other,
and there is probably a continuum and/or an overlap between these clinical symptoms,
including the more severe condition acute nectrotizing encephalopathy. Although the
clinical entity of influenza-associated encephalopathy has not gained universal rec-
ognition, it has been reported frequently as a complication of influenza in Japanese
children (Sugaya 2002). Influenza A is most frequently reported, especially H3N2
and H1N1, although influenza B is associated with encephalitis/encephalopathy as
well. A national survey, conducted in Japan during 1998–1999, reported that 148
out of 202 cases were diagnosed as influenza-associated encephalitis/encephalopathy
on the basis of virologic analysis. According to their report, 87.8% (130/148) was
type A influenza and 11.5% (17/148) was type B (Morishima et al. 2002). The onset
of neurological symptoms is usually within a few days to a week after the first signs
of influenza infection. Fever, decreased consciousness, and seizures are common
symptoms, and among the less common are focal neurological signs such as pare-
sis, cranial nerve palsies, and choreoathetosis. Encephalitis/encephalopathy is more
common in children, although adult cases are described (Hakoda and Nakatani
2000; Kurita et al. 2001). Reports of influenza virus-associated encephalitis/
encephalopathy with high fatality rate, especially among children, have increased
in Japan with estimates of about 200 patients during 1998–1999 and 100 during
1999–2000 (Kasai et al. 2000). The mortality is approximately 30%, and 80.6%
reported cases were children younger than 4 years of age. The risk of neurological
sequelae is high (Sugaya 2002).
14.4.1 Febrile Seizures

Febrile seizures occur in approximately 20% of hospitalized infants and adolescents
with influenza. In Hong Kong, it has been reported that 19.9% (54/272) and 18.8%
(27/144) of children aged 6 months to 5 years hospitalized with influenza A infection
in 1997 and 1998, respectively, had one or more febrile seizures (Chiu et al. 2001).
There are two types of febrile seizures, simple febrile seizure and complex febrile
seizure. The simple one is typical and characterized by single seizures that manifest
as generalized tonic–clonic or clonic convulsions lasting no longer than 15 min and
does not recur in 24 hours. The complex febrile seizure is characterized by longer
duration, with recurrence or focus on only part of the body (Toovey 2008).
The risk of febrile seizure is associated with many factors, including age, gender, and
family history. Febrile seizures occur more frequently in boys than in girls. A genetic
predisposition to febrile seizure may also exist (Kwong et al. 2006). Influenza A
viruses have been implicatively involve in the pathogenesis of febrile seizures (Chiu
et al. 2001; Hara et al. 2007; Stricker and Sennhauser 2004), although their action
has yet to be fully determined. Indirect involvement via the initiation of fever appears
most likely, because fever is an established trigger event for febrile seizure (Toovey
2008), but direct neurological effects cannot be discounted, since some influenza
A virus subtypes are probably neurotropic viruses. Few studies have demonstrated
the presence of viral antigens in the CSF or CNS tissue (Schlesinger et al. 1998;
Steininger et al. 2003). Furthermore, high levels of cytokines in patients who devel-
oped febrile seizures in influenza A virus infection have been reported (Ichiyama
et al. 2008). Febrile seizure appears to resolve without neurological sequelae (Kolfen
et al. 1998). Neurological complications of influenza infection may be partly related
to the exaggerated cytokine response.
14.4.2 Reye’s Syndrome
Reye’s syndrome is a potentially fatal disease that causes numerous detrimental
effects to many organs, especially the brain and the liver. The syndrome is character-
ized by a rapidly progressive noninflammatory encephalopathy and hepatic failure,
largely affecting children and adolescents. Signs include vomiting, disorientation,
loss of consciousness, and seizures. Signs of hepatomegaly and cerebral edema are
often present (Gosalakkal and Kamoji 2008). The disorder commonly occurs dur-
ing recovery from a viral infection, although it can also develop 3 to 5 days after the
onset of the viral illness. Influenza viruses, especially influenza B virus infections,
may precede Reye’s syndrome (Studahl 2003). Reye’s syndrome is often misdiag-
nosed as encephalitis, meningitis, diabetes, drug overdose, poisoning, sudden infant
death syndrome, or psychiatric illness. Because manifestations of Reye’s syndrome
are not unique to Reye’s syndrome but also are seen in other conditions and given
that no test is specific for Reye’s syndrome, the diagnosis must be one of exclusion.
Early recognition and treatment are essential to prevent death and to optimize the
likelihood of recovery without neurological impairment. The serious symptoms of
Reye’s syndrome appear to result from damage to cellular mitochondria.
14.4.3 Encephalitis Lethargica
Encephalitis lethargica was a devastating, mysterious, epidemic disease that killed
as many as 500,000 people in early part of the twentieth century (Ravenholt and
Foege 1982). Encephalitis lethargic could occur at any stage of life, but the inci-
dence was greatest in those between ages 10 and 30 years. Encephalitis lethar-
gic is characterized by high fever, headache, delayed physical, sleep inversion, and
lethargy (Dale et al. 2004). In acute cases, patients may enter a coma-like state
(Vilensky et al. 2006). The mechanism of causing encephalitis lethargica is not
known for certain. Dale et al. (2004) suggested that the disease is mediated by
the poststreptococcal immune response, and autoimmune origin with IgG against
human basal ganglia antigens.
14.4.4 Acute Necrotizing Encephalopathy

ANE is characterized by multifocal symmetric brain lesions involving the bilateral
thalami, putamen, and brainstem tegmentum. ANE is a rare disease first described
in Japan by Mizuguchi in 1995 (Mizuguchi et al. 1995). The disease affects young
children of both genders. ANE manifests as acute encephalopathy following 2–4 days
of fever and minor symptoms of respiratory tract infection. The clinical course of ANE
is rapidly progressive, including constitutional symptoms of emesis, cough, and diar-
rhea in combination with neurological dysfunction such as rapid consciousness and
seizures. The hallmark of this encephalopathy consists of multifocal, symmetric brain
lesions affecting the bilateral thalami and/or cerebellar medulla (Lyon et al. 2010;
Ormitti et al. 2010; Weitkamp et al. 2004; Yadav et al. 2010). The prognosis is usually
poor and associated with severe neurological sequelae in survivors (Mizuguchi 1997).
14.5 DIAGNOSIS
Typically, neurological examinations are performed on the patient who presents
with signs and symptoms of encephalitis; several types of examination may aid in
the diagnosis, such as a lumbar puncture may be performed to assess for evidence
of infection in the CNS and help to exclude other potential causes of symptoms
like meningitis. To ascertain the CNS infection, neuroimaging with MRI/CT can
be available. Neuroimaging findings on CT or MRI may be normal initially, but
pathological changes can develop after a few days of neurological symptoms. The
pathogenesis of brain damage induced by influenza infection was quite variable. The
results from MRI were divided into five categories: normal, diffuse involvement of
the cerebral cortex, diffuse brain edema, symmetrical involvement of the thalamus,
and postinfectious focal encephalitis (Kimura et al. 1998). MRI is particularly useful
for detecting metabolic derangements in the brain, although electroencephalogram
(EEG) is usually nonspecific with pathological changes, it can reflect brain function.
High voltage amplitude slow waves and the occurrence of theta oscillation have been
shown consistent with encephalitis/encephalopathy (Cisse et al. 2010; Fukumoto et
al. 2007; Okumura et al. 2005). Whereas the brain MRI, CT, and EEG are nonspe-
cific tests for the CNS infection, it may also help to rule out other causes and narrow
down the diagnosis.
In influenza virus-associated encephalopathy or encephalitis, CSF analyses will
often reveal a lack of pleocytosis or merely a discrete elevation of mononuclear leuko-
cytes. Protein and glucose content are usually normal, although a slightly increased
protein level may be present.
Determinations of white blood cell counts and serum C-reactive protein level may
be helpful in the detection of bacterial coinfections because these values are low
in patients with uncomplicated influenza. Leukopenia observed in association with
influenza infection should not prompt any further evaluation, because influenza is
known to cause lymphopenia. It has been shown that influenza B was more clearly
associated with leukopenia than was influenza A (Peltola et al. 2003).
The diagnosis of influenza infection is based on viral isolation, the viral antigen
test or RT-PCR. It has been reported that influenza A (Fujimoto et al. 1998) and
influenza B (McCullers et al. 1999) may directly cause CNS infections by PCR ana
lyses of CSF. Peltola et al. identified 11 children with encephalitis, encephalopathy, or
status epilepticus associated with influenza infection (Peltola et al. 2003). Morishima
et al. (2002) reported a high incidence of influenza associated encephalitis and
encephalopathy in Japan, whereas they consider that direct invasion of the CNS by
influenza virus is unlikely in most of their cases. Since it can be hard to find defini-
tive evidence of influenza, brain biopsy is rarely performed to sample the infected
brain tissue.

The pathogenic mechanisms of the various neurological syndromes during influ-
enza infection in humans are largely unknown. In etiology, human encephalitis/
encephalopathies that are associated with or connected with influenza virus
infection are composed of two groups. The first group is necrotic encephalopathy of
the cerebrum, and consists of ANE of childhood (Campistol et al. 1998; Huang et al.
2004; Khan et al. 2011), hemorrhagic shock and encephalopathy (Levin et al. 1983),
and Reye’s syndrome (Gosalakkal and Kamoji 2008). The pathogenesis of ANE is
unknown but necropsy findings in fatal cases of ANE reveal diffuse cerebral edema
and perivascular hemorrhage in the bilateral thalami and putamen (Mizuguchi et al.
2002). In these encephalopathies, the breakdown of the blood–brain barrier (BBB)
was suggested to be the direct cause of the brain lesions (Hayase and Tobita 1997;
Takahashi et al. 2000), but the mechanism leading to the breakdown is unknown.
Encephalitis lethargica (van Toorn and Schoeman 2009) and postencephalitic
Parkinson’s disease (Hayase and Tobita 1997) constitute the second group, and the
CNS lesion common to them is nonpurulent encephalitis of the brain stem. There has
been no clear-cut explanation of the predilection of encephalitis for the brain stem.
Influenza replicates in the respiratory tract and is seldom isolated from the brain
(Frankova et al. 1977). However, detection of influenza virus (Okabe et al. 2000)
or viral RNA in the CSF (de Jong et al. 2005; Fujimoto et al. 1998; Gu et al. 2007)
has indicated penetration of virus into the CNS. Although most viral infections
are thought to spread to the brain hematogenously, this route has been doubted in
influenza encephalitis/encephalopathy since viremia is sparsely reported in humans,
where it has been found only during the incubation period and initial stage of dis-
ease (Xu et al. 1998). The neuronal pathway, shown in animal models via the olfac-
tory and trigeminal nerve system (Park et al. 2002; Reinacher et al. 1983), may be
favored by the free nerve endings near the influenza-infected epithelial cells in the
upper respiratory tract (Mori and Kimura 2001). Different routes of virus entry to
the brain, studied experimentally in mice, are linked to infection of different cells
and regions of the CNS (Park et al. 2002) and might result in diverse clinical pictures
and neuroimaging findings. Wang et al. reported that human H1N1 and avian H5N1
influenza viruses were replicative and productive in mouse microglia and astrocyte
(Wang et al. 2008). Influenza infection in ependymal cells has been shown in an
autopsy study of immunosuppressed patients (Frankova et al. 1977). The hypothesis
of a vascular endothelial infection is supported by studies on mice. In the study,
influenza strains replicate primarily in ependymal cells in the CNS (Ito et al. 1999).
However, a limited direct invasion of neurons is not excluded. Virus-antigen positiv-
ity has been shown in Purkinje cells in the cerebellum and in several neurons in the
pons of a 2-year-old girl who died from influenza encephalopathy (Takahashi et al.
2000).
Influenza virus RNA has been detected in CSF and brain tissues of patients who
develop acute encephalitis/encephalopathy (Fujimoto et al. 1998). Influenza virus
antigens have also been detected in glial cells and neurocytes in mouse models of
encephalitis (Gao et al. 1999; Shinya et al. 1998). Besides, proinflammatory cyto-
kines are reportedly increased in the CSF and plasma of patients with influenza
encephalopathy and encephalitis (Ito et al. 1999; Shinya et al. 1998). These observa-
tions suggest the involvement of direct viral damage and immunopathological injury
in influenza-associated encephalopathy and encephalitis.
Another hypothesis has suggested that cytokine release from virus-stimulated
glial cells may be responsible for a neurotoxic effect on the brain (Wang et al. 2008)
and a rapid breakdown of the BBB (Yokota et al. 2000). Autopsy studies of patients
with CNS complications associated with influenza are generally scarce. Fatal cases
with brain pathology have shown congestion and hyperemia of the brain without
inflammatory cell infiltration, and in rare cases demyelination (Studahl 2003).
However, other researchers considered that the clinical significance of the pres-
ence of influenza virus genome in the CSF is questionable (Lee et al. 2010; Steininger
et al. 2003). Reports found an increased permeability of the BBB in the only influ-
enza virus-positive patient, indicating passive diffusion of viral genome from the
periphery into the CSF (Fujimoto et al. 1998; Morishima et al. 2002; Steininger et
al. 2003). According to the national survey conducted in Japan, it has been suggested
that direct invasion by influenza virus and inflammation is unlikely to be the cause
of encephalopathy, and despite the occurrence of brain edema, no influenza antigen
has been detected in the brains of patients with influenza-associated encephalopathy
(Morishima et al. 2002).
Cytokines such as IL-6 and TNF-alpha were markedly elevated in cerebrospinal
fluid and in serum of patients with influenza-associated encephalopathy and enceph-
alitis (Ichiyama et al. 2003, 2004; Kawada et al. 2003). The leakage of plasma pro-
tein was found in the brain of the patient who died with a rapid and fulminant course
suggestive of damage of vascular endothelial cells, which is presumably caused by
highly activated cytokines (Togashi et al. 2004).
Proinflammatory cytokines, such as IL-6 and TNF-a, can induce apoptosis,
which may give rise to aggravated encephalopathy. In addition, chemokines includ-
ing CCL2/MCP1, CXCL8/IL-8, and CXCL10/IP-10 were greatly increased both in
CSF and serum (Mizuguchi et al. 2007). These chemokines induce injury of vas-
cular endothelium, glial cells, and neurons, cause vascular lesions and breakdown
of the BBB, and thereby induce brain edema and damage, CNS disorders, and/or
systemic symptoms (Nakai et al. 2003; Wang et al. 2010).
14.7 PROGNOSIS AND TREATMENT

The prognosis of encephalitis/encephalopathy depends mainly on age and presence
of MRI or CT pathology. Compared with patients with normal MRI or CT, patients
with pathological MRI or CT are significantly younger and have more severe sequelae
or fatal disease. Neuroimaging studies in influenza encephalitis/encephalopathy sug-
gest that the majority of those who recover or have mild sequelae are the patients
with nonpathological MRI or CT (Ormitti et al. 2010; Studahl 2003). However,
severe sequelae such as choreoathetosis, altered personality, spastic quadriparesis,

and persistent vegetative state may occur also in cases with normal imaging find-
ings (Hakoda and Nakatani 2000; Ryan et al. 1999). A diffuse severe brain edema is
associated with severe brain damage or death (Yokota et al. 2000).
For most viral infections such as influenza, there are no specific medications to
treat the disease. In mild cases patients are generally instructed to eat well, drink
plenty of fluids, and to rest. In more serious cases patients may be hospitalized.
Methylprednisolone pulse therapy and large doses of IgG reduced the mortality from
influenza-associated encephalopathy (Weitkamp et al. 2004). If the patient experi-
ences seizures, anti-seizure medication may be prescribed. Anti-inflammatory drugs
and high dosage corticosteroid therapy may be useful to reduce brain edema (Aiba
et al. 2001).
Vaccines are the principal defense against influenza, but because it takes time
to produce an antigenically appropriate and immunogenic product and deliver it to
entire populations, antiviral drugs will be a principal countermeasure to reduce the
impact of a new pandemic (Monto 2006). The current armamentarium of licensed
anti-influenza medications include influenza M2 ion channel blockers (adamanta-
dine and rimantadine), and NA inhibitors (oseltamivir and zanamivir). Nucleoside
analogues that interfere with influenza virus RNA polymerase function (ribavirin
and viramidine) were used to treat severe infections in a very limited extent (Beigel
and Bray 2008).
The main advantages of neuraminidase inhibitors, compared with amantadine and
rimantadine, are fewer adverse effects, activity against both influenza A and B, and
rare resistance (Gubareva et al. 2000). In field trials, the greatest benefit from anti-
influenza drugs is gained if therapy is started early, within 48 h after onset of symp-
toms (Bridges et al. 2002). Antiviral therapy for influenza generally should be based on
detection of the virus. The primary care level cannot be based on data from hospital-
ized patients. In addition to treatment, antiviral drugs can be used prophylactically in
specific situations when vaccination is not possible or effective (Bridges et al. 2002).

Avian influenza is very common, but crossing the species barrier to mammalian is
a rare event. However, recent decade incidences of direct passage of highly patho-
genic influenza A virus strains of the H5N1 and H1N1 subtypes from birds and pigs
to humans have become a major public concern. Although presence of virus in the
human brain has a wide discrepancy, certain avian influenza virus strains can invade
the brain of experimental animals (Reinacher et al. 1983). These avian influenza
subtypes have the propensity to invade the brain along cranial nerves to target brain-
stem and diencephalic nuclei following intranasal instillation in mice and ferrets
(Maines et al. 2005; Zitzow et al. 2002). Unfortunately, the etiology and pathogen-
esis of influenza-associated CNS infections remain largely unknown. Although the
pathogenesis remains unclear, the rapidity of the onset of symptoms and the negative
presentation of viral antigen in brain tissue suggest that a cytokine-mediated process
may be the mechanism rather than direct invasion of brain parenchyma and resulting
direct cytopathic effect.
Since no specific diagnosis for influenza-associated nervous system dysfunctions

such as encephalitis/encephalopathy, febrile seizures, Reye’s syndrome, encephalitis
lethargic, and ANE, the importance of imaging techniques (MRI/CT) should be
advocated to aid clinicians to formulate a presumptive diagnosis.
So far, rare report influenza spread to the brain in humans and any associa-
tion between influenza and neuropsychiatric disorders remains to be established.
However, because neurotropic variants of influenza can rapidly be selected in other
mammalians such as mice and ferrets, this virus may be a threat imposed by nature
also to the human brain. A fight against this terror should therefore be a concern for
clinicians, virologists, and neuroscientists.
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15 Human Paramyxoviruses
and Infections of the
Central Nervous System
Michael R. Wilson, Martin Ludlow,
and W. Paul Duprex
CONTENTS
15.1 Introduction................................................................................................... 342
15.2 Paramyxoviruses: The Historical Perspective............................................... 343
15.3 Paramyxoviruses: The Biological Properties................................................ 347
15.3.1 Virus Structure.................................................................................. 347
15.3.2 Genome Organization........................................................................ 347
15.3.3 Protein Functions...............................................................................348
15.3.4 Replication and Transcription............................................................ 351
15.3.5 Replication Cycle............................................................................... 351
15.4 Measles Virus................................................................................................ 351
15.4.1 Clinical Presentation......................................................................... 351
15.4.1.1 Acute Postinfectious Measles Encephalomyelitis............... 352
15.4.1.2 Measles Inclusion Body Encephalitis................................. 352
15.4.1.3 Subacute Sclerosing Panencephalitis.................................. 352
15.4.2 Diagnosis........................................................................................... 353
15.4.3 Pathology and Pathogenesis............................................................... 353
15.4.4 Epidemiology..................................................................................... 356
15.4.5 Prognosis and Treatment................................................................... 356
15.5 Mumps Virus................................................................................................. 356
15.5.2 Diagnosis........................................................................................... 357
15.5.4 Epidemiology..................................................................................... 358
15.6 Henipaviruses................................................................................................ 359
15.6.2 Diagnosis...........................................................................................360
15.6.3 Pathology and Pathogenesis...............................................................360
15.6.4 Epidemiology..................................................................................... 361
341
15.7 Other Paramyxoviruses Not Traditionally Associated with CNS

Manifestations............................................................................................... 362
References............................................................................................................... 363
15.1 INTRODUCTION
Paramyxoviruses represent a diverse family of human and animal pathogens which,
to a greater or lesser extent, have a propensity to infect the central nervous system
(CNS).
Common pathological themes unite paramyxovirus infections and many of the
viruses are capable of spreading to the CNS where acute encephalitis and reacti-
vation following long-term persistence are prominent clinical features. Indeed, the
prototypic morbillivirus measles virus (MV) provides a paradigm for the long-term
persistent RNA virus infection in humans. Sub-acute sclerosing panencephalitis
(SSPE) is an invariably fatal rare sequela of measles occurring months to years after
the initial infection. Likewise the recently identified, and highly pathogenic, bio-
safety level 4 (BSL-4) agent Nipah virus (NiV) has been shown to reactivate in
the CNS of a number of patients months after the acute infection. Here we seek to
compare and contrast the symptoms presented when these human paramyxoviruses
infect the CNS underpinning this by highlighting their common molecular biologi-
cal and virological properties (Table 15.1).
Although outside the scope of this chapter, much has been learned from natural
and experimental infections using animal models of paramyxovirus diseases (von
Messling et al. 2003). Such approaches are all the more important given the zoo-
notic potential of some of these viruses. In fact, only two of the four neurotropic
paramyxoviruses we discuss in detail, MV and mumps virus (MuV), are exclusively
human pathogens. Zoonotic infections by hitherto unrecognized animal viruses are
typically associated with higher levels of pathogenicity than is observed following
infection with viruses which only circulate in a single species as co-evolution of the
virus and host tends to diminish pathogenicity over time. Thus the 40–75% mortality
rates observed in NiV and Hendra virus (HeV) infections are in striking contrast to
the 0.01–0.03% mortality rates observed for MuV and MV in the developed world.
It is tempting to speculate that much higher levels of mortality were observed when
MV and MuV initially jumped species from their animal reservoirs into humans.
The high levels of morbidity and mortality resulting from NiV and HeV infections in
the 21st century may reflect what occurred several thousand years ago when human
populations reached the size and density necessary to sustain endemic MV and MuV
transmission. Given the current interest in emerging and re-emerging pathogens, the
fact that MuV recently infected thousands of college students in the United Kingdom
and the United States and with the regular importation of MV into Europe and the
United States from the developing world, it is timely to compare, contrast, and review
these neurotropic paramyxoviruses.
Given the similarities in virion structure, genome organization, replication, tran-
scription, and how paramyxovirus proteins generally function within the cell, it is
logical to address the biological aspects for MV, MuV, NiV, and HeV together. The
Human Paramyxoviruses and Infections of the Central Nervous System 343
pertinent historical perspectives and key aspects of paramyxovirus biology will be

covered first to set the scene and allow the reader to appreciate the unifying molec-
ular biological features of this family of viruses. However, there are tremendous
differences among the four viruses in terms of clinical presentation, pathogenesis,
epidemiology, and neurotropism, and therefore these aspects will be treated sepa-
rately. For those who wish to gain a greater understanding of the molecular biologi-
cal properties of the paramyxoviruses we refer the reader to the recently published
book, The Biology of Paramyxoviruses (Samal 2011), which covers many of the
viruses in greater detail as well as the relevant chapters in Fields Virology (Carbone
and Rubin 2007; Eaton et al. 2007; Griffin 2007).
15.2 PARAMYXOVIRUSES: THE HISTORICAL PERSPECTIVE

As the prototypic morbillivirus, and probably the most well-known paramyxovirus,
more is understood about the molecular and cell biology of MV than any of the other
viruses we will discuss. Following the eradication of smallpox, MV is probably the
most transmissible human virus having an R0 between 12 and 18 (Moss and Griffin
2006). The virus causes a systemic disease primarily of the immune system and
is associated with high levels of morbidity and mortality in the developing world,
largely as a consequence of the profound immune suppression induced in infected
individuals (Avota et al. 2010). The first written account of MV infection dates from
the 9th century and is ascribed to the Arabian physician Abu Becr, also known as
Rhazes of Baghdad, who dated the first description of measles to the 6th century
(Hirsch 1883). Phylogenetic analysis indicates that MV probably evolved from the
ancestor of the closely related rinderpest virus (RPV) which predominately infects
cattle (Furuse et al. 2010). However, the strict tropism displayed by current circu-
lating wild-type MV strains for humans and some species of monkey is indicative
of the typical evolution of a virus following zoonotic infection whereby the virus
becomes ever more adapted to the new host. It is assumed that MV only became
permanently established in human populations after the development of cities in
India and the Middle East of sufficient size to fulfill the requirement of 250,000
to 500,000 people necessary to maintain endemic MV transmission (Keeling and
Grenfell 1997). Consequently MV has been termed a disease of civilization due to
the requirement for a sufficiently large population of naive susceptible hosts to main-
tain an endemic infection.
The first scientific investigation into measles occurred in 1757 when the Scottish
physician Francis Home transmitted the disease to uninfected individuals by inject-
ing blood from individuals who were beginning to display the characteristic rash
produced (Plotkin 1967). This crude approach provided the first demonstration
that measles is caused by an infectious agent. Characterization of MV and vaccine
development was facilitated by the introduction of sterile mammalian cell culture
techniques in the mid-20th century, the use of which enabled Enders and Peebles
to isolate MV from a child named David Edmonston in 1954 (Enders and Peebles
1954). The subsequent generation of a closely related collection of live attenu-
ated measles virus vaccines which are used to this day can be traced back to this
discovery.
344
TABLE 15.1
Virological and Clinical Features of Neurovirulent Human Paramyxoviruses
Measles Virus Mumps Virus Nipah and Hendra Viruses
Molecular Biology
Genome length 15,894 nucleotides 15,384 nucleotides 18,246 and 18,234 nucleotides
Transcription units 6 7 6
Proteins N-P/V/C-M-F0-H-L (2 nonstructural) N-V/W/P-M-SH-F0-HN-L N-P/V/W/C-M-F0-G-L (3 nonstructural)
(3 nonstructural)
Pathogenesis
Cellular receptora CD150 and Nectin-4 Unidentified sialylated EphrinB2 (NiV and HeV) and ephrinB3
glycoproteins and/or (NiV)
glycolipids
Target cells Alveolar macrophages, dendritic cells, activated B and T cells, epithelial cells, Unknownb Capillary and arterial endothelial cells,
neurons, oligodendrocytes, astrocytes (rarely), and endothelial cells (rarely) smooth muscle, and neurons
Systems targeted Immune (circulating lymphocytes and lymphoid tissues), respiratory and CNS Endocrine (e.g., salivary, Circulatory and CNS
thyroid, pancreas, testes,
and ovaries) and CNS
Natural host range Humanc Humanc Bat, human, pig, dog, cat, and horse
Mortality rate 0.03%d 0.03d 40%–75%
Delayed or
CNS Manifestations APME MIBE SSPE Meningoencephalitis Acute Encephalitis Recurrent
Disease onset after 1 to 4 weeks 1 to 6 months 5 to 10 years 0 to 2 weeks 0 to 8 days 9 days to 2 years
primary infection
Neurologic signs and Fever, headache, Malaise, seizures (e.g., Behavioral and intellectual Fever, meningismus, focal Fever, meningismus, Fever (less
symptoms irritability, focal epilepsia partialis impairment progressing to neurologic deficits, brainstem signs, common),
neurologic continua), cortical ataxic-myoclonic malaise, seizures, and malaise, and seizures headache, focal
deficits, malaise, deficits progressing to dementia papilledema (rarely) neurologic
and seizures coma deficits, malaise,
and seizures
MRI findings Numerous, Focal T2-signal Diffuse subcortical white Frequently normal; diffuse Diffuse, punctate Confluent gray
ill-defined hyperintensities (few matter T2-signal T2-signal hyperintensities; (2–7 mm) T2-signal and white matter
T2-signal reports) hyperintensities hydrocephalus (very hyperintensities T2-signal
hyperintensities rarely) (microinfarcts) hyperintensities
predominantly at
the gray-white
junction, uniform
gadolinium
enhancement
Diagnosis CSF pleocytosis CSF normal or with mild EEG with periodic spike CSF pleocytosis and CSF pleocytosis and CSF pleocytosis,
and elevated pleocytosis and elevated and wave complexes, elevated protein, elevated protein, IgG HeV- or
protein, MRI and protein, MRI, history or MRI, elevated CSF MuV-specific antibodies HeV- or NiV-specific NiV-specific
history or recent recent infection and gamma globulins and antibodies antibodies,
infection or immunosuppression, oligoclonal bands, history of NiV
vaccination absence of intrathecal elevated serum and or HeV infection
MV-specific antibodies intrathecal production of
IgG MV-specific
antibodies
Treatment High-dose Supportive Combination of inosiplex Supportive Ribvavarin Supportive
corticosteroids and IFN-α (noncurative) (controversial)
Prognosis Excellent (<10% Fatal over days to weeks Fatal (95%) over months to Excellent Fatal (40–75%) over Fatal (20%) over
mortality) years days; Long-term days to weeks;
neurologic sequelae Long-term
(20–30%) neurologic
sequelae (60%)
a For clinical isolates.

b As a respiratory infection, it is presumed, although not proven, that MuV infects epithelial cells in the URT and local lymph modes. As a neurotropic infection, ependymal cells and
Human Paramyxoviruses and Infections of the Central Nervous System
neurons are infected.

c Nonhuman primates can act as incidental hosts but are mainly used in voluntary laboratory infections.
d In the developed world.
345
MuV is a member of the Rubulavirus genus in the family Paramyxoviridae. Like

MV, MuV is one of the earliest recorded diseases being described by Hippocrates
in 400 bc in his treatise “Of the Epidemics.” In 1790, Robert Hamilton provided the
first detailed clinical description of mumps, including a case of fulminant mumps
encephalitis (Hamilton 1790). The clinical manifestations were first demonstrated
to be viral in etiology in 1934 by Johnson and Goodpasture when they successfully
transferred the disease from children to monkeys (Johnson and Goodpasture 1934).
However, it took until 1968 for the first live attenuated MuV vaccine to be developed
(Hilleman et al. 1968). Before widespread vaccination, MuV was the leading cause
of virus-induced aseptic meningitis and encephalitis in the US and Europe, causing
meningitis in up to 65% of patients (Bang and Bang 1943). Indeed, meningoencepha-
litis is the most common extra-salivary gland manifestation of a MuV infection,
although other neurological complications include transverse myelitis, sensorineural
deafness, cerebellar ataxia, seizures, and cranial nerve palsies (Nussinovitch et al.
1995). Interestingly, MuV has recently reemerged in the developed world having
infected over 6000 college students in the United States in 2006 (Barskey et al.
2009). The virus was imported from the UK, and there have been discussions as to
whether or not it might be necessary to instigate a third round of vaccination to break
transmission (Rubin 2011).
Vaccination strategies have been complicated by the fact that a number of MuV
vaccines caused aseptic meningitis in vaccinees (Balraj and Miller 1995; da Cunha
et al. 2002; Dourado et al. 2000; Sugiura and Yamada 1991). This resulted in with-
drawal of the vaccines, public resistance to MuV vaccination, and, in some countries,
complete cessation of national vaccination programs for mumps (Colville et al. 1994;
Furesz and Contreras 1990; Hashimoto et al. 2009; Sasaki and Tsunoda 2009). As a
consequence of the withdrawal of mumps vaccines from Japan’s national vaccination
program, over 2 million cases of mumps are reported annually which are accompa-
nied by significant numbers of MuV-induced aseptic meningitis and deafness (Nagai
et al. 2007).
Unlike MV and MuV, the henipaviruses are relative newcomers into the para-
myxovirus arena. In fact, the Henipavirus genus was only created in 2002 after the
identification and molecular characterization of NiV and HeV. In September 1994,
21 horses and 2 handlers in the Brisbane suburb of Hendra, Queensland, developed
a severe respiratory illness that killed 14 of the horses and 1 person within 2 weeks
(Murray et al. 1995; Selvey et al. 1995). Initially, the agent was called equine mor-
billivirus, although HeV was renamed after flying foxes of the genus Pteropus and
not horses were identified as the reservoir (Chua et al. 2002; Halpin et al. 2000).
Following the initial outbreak, HeV infections have continued to occur in horses
once or twice a year, although unusually there have been at least 17 outbreaks in
2011 thus far (Ksiazek et al. 2011). Four of the seven infected people have died,
including one person who died from encephalitis a year after a likely HeV exposure
(O’Sullivan et al. 1997). NiV was first identified in 1998–1999 after an outbreak
of viral encephalitis in Malaysian pig farmers. Interestingly this unidentified zoo-
notic agent was initially thought to be Japanese encephalitis virus, illustrating how a
very similar clinical presentation presents significant challenges in the timely diag-
nosis of novel emerging viruses (Chua et al. 1999). The mortality rate was nearly
40% among the almost 300 people infected. After spreading to Singapore, the out-
break was stopped only after the slaughter of over one million swine (Ksiazek et al.
2011). NiV continues to expand its geographical range and virulence having caused
large outbreaks in India, Bangladesh, Australia, Singapore, and Malaysia. The most
recent outbreaks in India and Bangladesh have demonstrated increasing mortality
rates approaching 75% as well as multiple cases of person-to-person transmission
(Blum et al. 2009; Chadha et al. 2006; Homaira et al. 2010; Hsu et al. 2004). Due
to their incredibly wide host range (horses, cats, dogs, rabbits, laboratory rodents),
geographical-range, high mortality, transmissibility, and ease of culture in vitro, NiV
and HeV were assigned as BSL-4 agents soon after their discovery. Two key features
of these infections are their zoonotic potential and their propensity to establish per-
sistent infection. Indeed, what we observe for NiV and HeV in the 21st century might
well have been what was observed in ancient history for the BSL-2 agents, MV, and
MuV (see above). This highlights the benefit and utility in studying emerging viruses
alongside more established and closely related pathogens as the knowledge gained
for one can be leveraged into the treatment of those which continue to emerge from
unknown reservoirs.
15.3 PARAMYXOVIRUSES: THE BIOLOGICAL PROPERTIES

15.3.1 Virus Structure
Paramyxovirus virions are enveloped particles varying in shape from spherical to
filamentous to pleomorphic. Spherical viruses, such as MV and MuV, have aver-
age diameters of 200 nm and are indistinguishable by electron microscopy (EM).
Likewise, the filamentous virions of HeV and NiV are similar by EM and are mor-
phologically similar to other members of the subfamily Paramyxovirinae (Chua et al.
2000b). However, NiV virions are typically larger on average than other paramyxo-
viruses (500 nm) having a broader variation in diameter (180–1900 nm) (Goldsmith
et al. 2003). Paramyxoviruses are highly cell-associated and cell-to-cell spread may
be very important in vivo. All of the viruses have a glycoprotein fringe of spikes
which protrude from the cell-derived lipid envelope. Ultrastructurally, HeV surface
projections have a double-fringed appearance while NiV surface projections have a
single fringe (Hyatt et al. 2001). Two glycoproteins (see below) comprise the biologi-
cally active fusion complex which interacts with cell surface molecules to initiate
the infection.
15.3.2 Genome Organization
The basic unit of infectivity for all paramyxoviruses is a negative (–) sensed RNA
molecule which co-exists with an encapsidated helical ribonucleoprotein (RNP)
complex, the (–)RNP (Eaton et al. 2006; Samal 2011). These helical structures pro-
tect the genome from nucleases, increasing the stability of the virus. Normally only
a single (–)RNP is encapsidated, although interestingly measles virions can contain
more than one copy of the genome and be functionally polyploid (Rager et al. 2002).
Reverse genetics approaches have been used to split the MV genome and illustrate
that the virus can encapsidate two (–)RNPs (Takeda et al. 2006). Although para-
myxoviruses have differing numbers of transcription units (MV, NiV, and HeV have
six whereas MuV has seven), the genomic organization is similar (Table 15.1). A gene
start (GS) sequence contains the necessary signals for the initiation of transcription
and a 5ʹ untranslated region (UTR) prior to each open reading frame (ORF). The
ORF is followed by a gene-end (GE) sequence that contains a 3ʹUTR and sequences
which mediate the template-independent polyadenylation of the resulting messenger
RNA (mRNA). Transcription is mediated by a virus encoded RNA-dependent RNA
polymerase (RdRp) which is associated with the (–)RNP and must be packaged into
a virion to ensure infectivity (Samal 2011). Transcription units are separated by non-
transcribed intergenic (Ig) spacers. These Ig spacers are ignored by the RdRp during
primary and secondary transcription and are only copied during replication of the
complete genome (see below). The genome is flanked by a leader (Le) sequence at
the 5ʹ end which contains a single genomic promoter (GP) where the RdRp initiates
transcription and genome replication. A trailer (Tr) sequence is present at the 5ʹ end
of the genome. This contains a stronger anti-genomic promoter (AGP) from which
the RdRp synthesizes nascent copies of the genome which are encapsidated into
budding virions. Genome sizes vary with MuV (15,384 nt) being smaller than MV
(15,894 nt) which in turn is smaller than HeV (18,234 nt) and NiV (18,246 nt) mak-
ing these henipaviruses approximately 15% larger than others in the same family.
Notably, each genome length obeys the “rule of six” adhered to by all paramyxovi-
ruses. The helical nucleocapsid core is structured for each nucleocapsid (N) protein
to be associated with precisely six nucleotides. Despite the varied genome lengths,
most of the viral proteins are quite similar in size and the additional genome length
of NiV and HeV is due to the presence of longer 5ʹ and 3ʹUTRs. MuV has an extra
transcription unit that encodes the small hydrophobic (SH) protein which again adds
to the length of the genome. Genes are arranged linearly and, for the most part, there
is a common order for the four viruses (Table 15.1). From the 5ʹ end to the 3ʹ end six
ORFs encode the N protein, phospho- (P) protein, matrix (M) protein, fusion (F)
glycoprotein, an attachment glycoprotein, and the large (L) protein. Three types of
attachment proteins are encoded depending on the virus, a standard glycoprotein
(G), a hemagglutinin (H) glycoprotein, or a hemagglutinin-neuraminidase (HN) gly-
coprotein. The SH protein in MuV is located between the F and HN glycoproteins
and is the fifth transcription unit. The P gene encodes multiple proteins and is more
correctly termed the P/C/V gene for MV, HeV, and NiV and the V/W/P gene for MuV
(see below).
15.3.3 Protein Functions
Proteins are defined as either structural (N, P, M, F, G, H, HN, and L) or nonstructural
(C, V, SH, and W). The (–)RNP complex is composed primarily of the N protein with
the P and L proteins comprising the RdRp (see below). Depending on the virus, three
or four proteins form the virions. The type I F and the type II G/H/HN glycopro-
teins are integral membrane proteins which form the EM visible fusion spike com-
plexes. Most of the proteins are externalized, and their short cytoplasmic tails interact
with the membrane associated M protein which is the key bridge between the lipid
envelope and the (–)RNP. Although normally considered to be a nonstructural pro-

tein, the MuV SH protein may act as a viroporin embedded in the virus envelope. The
protein is not required for replication in vitro (Takeuchi et al. 1996), and it is unclear
to what degree it plays a role in pathogenesis in vivo (Malik et al. 2011).
Virulence, host range, and cell tropism are determined largely by the two attach-
ment proteins which are both required for entry of virus into target cells (Bossart
et al. 2001; Samal 2011; Tamin 2002). The G proteins of HeV and NiV possess
neither hemagglutination nor neuraminidase activities (Tamin 2002). Early stud-
ies suggested that the similar in vivo tropism made it possible that HeV and NiV
shared a common cellular receptor and that the molecule should be highly expressed
on endothelial cells. Furthermore, given the propensity of the viruses to infect a
range of species it was likely that the receptor would be highly conserved (Bossart
et al. 2002). Identification of ephrinB2 as the receptor (Bonaparte 2005; Negrete
et al. 2005) was consistent with these hypotheses as it is expressed on arterial and
capillary endothelial cells, smooth muscle of the tunica media, and neurons. The
molecule is the membrane-bound ligand for the ephrinB class of receptor tyrosine
kinases and is important for regulating axon pathfinding, neuronal cell migration,
and vasculogenesis (Poliakov et al. 2004). EphrinB3 serves as an alternate receptor
for NiV, although with lower affinity (Bossart et al. 2008; Negrete et al. 2006). The
highly lymphotropic and immune suppressive nature of MV made it likely that the
wild-type virus would bind a cellular receptor which is highly expressed on immune
cells. Identification of CD150 as the primary MV receptor (Tatsuo et al. 2000) was
consistent with primary pathogenesis as the molecule is expressed on activated T
and B lymphocytes and regulate several leukocyte functions (Romero et al. 2004;
Veillette 2006). However, MV also infects epithelial and neuronal cells which do not
express CD150. Identification of PVRL4 as an epithelial receptor for MV (Noyce et
al. 2011) has helped to explain the means by which MV infects epithelial cells, but
the receptor used to infect neurons remains unclear. PVRL4 is present in adherent
junctions of epithelial cells and binding is essential for virus release and probably
transmission (Leonard et al. 2010; Shirogane et al. 2010). As an HN glycoprotein,
the MuV attachment protein binds sialic acid moieties conjugated to glycoproteins
and glycolipids (Samal 2011). Therefore it is unsurprising that the virus infects a
wide range of cell types in vitro. However, this is not the case in vivo. Although basi-
cally nothing is known about the first cells targeted by MuV, it has been suggested it
initially infects the upper respiratory tract (URT). Therefore it is probable that sialic
acid molecules attached to particular molecules are used preferentially, although this
has not been demonstrated. The HN protein has two apparently opposing activities,
adsorption to sialic acid-containing cell surface molecules (hemagglutination) and
enzymatic cleavage of sialic acid (neuraminidase activity). Recent studies have indi-
cated that both functions are located in a single sialic acid recognition site. A recent
crystal structure study of the HN glycoprotein of a closely related paramyxovirus,
Newcastle disease virus, indicates that amino acid position 466 may be at or near
this active site (Yuan et al. 2011). Paramyxovirus F glycoproteins are generated as
an inactive precursor (F0) which is cleaved by cellular proteases into a disulfide-
linked F1 and F2 complex. The ubiquitous intracellular protease furin activates the
MV F0 glycoprotein in the Golgi or post-Golgi apparatus (Navaratnarajah et al.
2011; Watanabe et al. 1995). This is the same site where the MuV F0 glycoprotein is
cleaved into F1 and F2, although the specific protease has not formally been identi-
fied. Henipavirus F0 glycoproteins are activated in a unique manner by endocyto-
sis and cleavage following initial surface expression (Vogt et al. 2005). Cathepsin
I cleaves the HeV F glycoprotein whereas cathepsin L cleaves NiV F glycoprotein
(Diederich et al. 2009).
When observed by EM the M protein appears as a shell of electron dense material
just below the surface of virions. Recent image reconstruction of MV particles has
shown that the M protein forms helices coating the helical (–)RNP rather than coat-
ing the inner leaflet of the membrane, as previously thought (Liljeroos et al. 2011).
Although not proven, this structural organization may well extend to other para-
myxoviruses. The M protein provides structure to the virion by interacting with the
cytoplasmic tail of the F and H/HN/G glycoproteins, the (–)RNP complex and the
inner leaflet of the lipid bilayer (Eaton et al. 2006; Samal 2011). Above and beyond
its structural role within the virion, the MV M protein also acts as a repressor of
transcription and viral RNA synthesis (Suryanarayana et al. 1994).
The second transcription unit of the four human neurotropic paramyxoviruses is
particular for a number of reasons. First, for MV, NiV, and HeV the primary tran-
script encodes the structural P protein whereas for MuV it encodes the nonstructural
V protein. Additional coding capacity for all of the viruses is obtained by a novel
co-transcriptional process in which one or more nontemplated G nucleotides are
inserted at a conserved editing site in the gene, resulting in an altered mRNA in
which there is a shift in the ORF. This leads to the generation of V proteins for
MV, HeV, and MuV or the P protein for MuV. Second, an additional nonstructural
protein is encoded in an overlapping reading frame which is accessed when the ribo-
some fails to initiate at the first AUG codon and scans to the subsequent start codon.
Therefore it is formally correct to refer to the second gene of MV, NiV, and MuV as
the P/C/V transcription unit and for MuV it is the V/W/P.
Regardless of how they are generated during transcription the nonstructural V
proteins have similar functions in abrogating the antiviral type I interferon (IFN)
response by interfering with dsRNA and IFN signaling, reviewed by Gerlier and
Valentin (2009). The amino terminus of V, and consequently P, binds to signal trans-
ducers and activators of transcription (STAT) 1 proteins which inhibits IFN signal-
ing. Unlike MuV, which inhibits STAT signaling by eliminating STAT 1 and STAT
3, henipaviruses sequester STAT proteins in high molecular weight complexes,
preventing their dimerization and translocation into the nucleus. This activity is
shared to varying degrees by the V, W, and P proteins. All three proteins also inhibit
phosphorylation of tyrosine residues in STAT (Rodriguez and Horvath 2004; Samal
2011). The accessory W protein also blocks activation of IFN-regulatory factor 3
(IRF-3)-responsive promoters in response to intracellular dsRNA signaling and sig-
naling through Toll-like receptor 3. The carboxyl terminus of the V protein is used to
inhibit dsRNA by inhibiting oligomerization the helicase encoded by the melanoma
differentiation-associated gene 5. This, in turn, restricts signaling and inhibits IFN
production (Childs et al. 2009).
15.3.4 Replication and Transcription

An essential component of every virion is the RdRp which is comprised of the L
and P proteins. RNA initiation, elongation and termination, mRNA capping, and
methylation along with polyadenylation functions all reside with the L protein which
acts both as a replicase and as a transcriptase (see above). Primary transcription is
carried out by the incoming RdRp to produce a set of mRNAs which accumulate in
the infected cell and are translated in the cytoplasm. When sufficient levels of virus
proteins accumulate, some replication commences to produce (+)RNPs which, in
turn, act as templates for the generation of (–)RNPs. These are used in secondary
transcription and are packaged into nascent virions (see below). All RNA synthesis
takes place in the cytoplasm, and intracytoplasmic inclusions which stain positive for
the N, P, and L proteins are common. It has been suggested but not proven that these
are the sites of transcription and replication.
15.3.5 Replication Cycle
Paramyxoviruses enter the cell by fusion at the plasma membrane following bind-
ing to cell surface receptors. Deposition of the (–)RNP into the cytoplasm initiates
primary transcription, and the RdRp accesses the template at the single promoter in
the 3ʹ end of the genome. At each Ig junction, there is the possibility that the RdRp
will detach from the (–)RNP. This start-stop mechanism leads to the generation of
a transcription gradient in which more mRNA transcripts encoding the N protein
are present in the cell compared with proteins encoded from the downstream tran-
scription units. This provides an elegant means to regulate gene expression. When
sufficient levels of the N, P, and L proteins are produced, replication of the negative-
sensed genome ensues (see above). Synthesis of the M protein and F0 and G, H, or
HN glycoproteins occurs in the cytoplasm with H and F0 being trafficked to the
cell surface through the endoplasmic reticulum and Golgi apparatus. MV budding
occurs from lipid rafts at the plasma membrane (Manie et al. 2000). Neuraminidase
activity of the MuV HN glycoprotein facilitates release from the infected cell by
cleaving sialic acids moieties from the glycoproteins and glycolipids. This activity
is not required for MV, NiV, and NiV, although CD150 is down-regulated from the
surface of MV-infected cells (Erlenhoefer et al. 2001).
15.4 MEASLES VIRUS

15.4.1 Clinical Presentation
Symptoms first become apparent during the viremic phase of the disease. The devel-
opment of a cough, fever, and conjunctivitis typically precedes the appearance of
the hallmark maculopapular rash which spreads from the trunk to the extremities.
Although MV is not highly neurovirulent, three distinct neurological complications
can occur following a natural MV infection.
15.4.1.1 Acute Postinfectious Measles Encephalomyelitis

Acute postinfectious measles encephalomyelitis (APME) or acute demyelinating
encephalomyelitis (ADEM) usually presents within 1 to 3 weeks after rash onset
and is characterized by malaise, recrudescence of fever, seizures, and a mortality
rate less than 10% (Norrby and Kristensson 1997). CSF may be normal, although a
mononuclear pleocytosis and a moderate elevation in protein levels is typically pres-
ent. Intracranial pressure can be mildly elevated. Antibodies to MV and increased
intrathecal production of IgG are not found (Johnson et al. 1984). MRI demonstrates
diffuse, ill-defined white matter disease most prominently at the gray-white junction
as evidenced by T2-signal hyperintensities along with uniform gadolinium contrast
enhancement.
15.4.1.2 Measles Inclusion Body Encephalitis

Measles inclusion body encephalitis (MIBE) is characterized by progressive neu-
rological deterioration, seizures (commonly epilepsia partialis continua), and coma
over days to weeks in individuals who are unable to mount a CD4+ or CD8+ T-cell
mediated immune response (Mustafa et al. 1993; Wolinsky et al. 1977). This may be
due to a congenital immunodeficiency or a result of the immunosuppressive effects
of infections such as HIV-1 (Budka et al. 1996). Clinical symptoms become apparent
one to six months after primary infection (Kaplan et al. 1992). Unlike patients with
subacute sclerosing panencephalitis (SSPE) (see below), levels of MV antibodies do
not increase. MRI and pathological findings are also similar to what are observed for
SSPE patients, except that inflammatory changes are absent.
15.4.1.3 Subacute Sclerosing Panencephalitis

The term SSPE was first used on the recommendation of Greenfield to describe
a sporadic encephalitis occurring in Europe in the middle of the 20th century
(Greenfield 1950) although the first pathological description of an “inclusion body
encephalitis” was made by Dawson in 1933. SSPE occurs on average after a 5- to
10-year incubation period in the presence of elevated MV-specific antibody titers in
both the serum and CSF (Bouteille et al. 1965; Connolly et al. 1967). Although adults
can be affected (Prashanth et al. 2006), SSPE largely presents in immunocompetent
children whose primary MV infection occurred before age two and is one of the few
causes of progressive ataxic-myoclonic chronic dementia in children. Children typi-
cally present with intellectual decline along with psychiatric and behavioral prob-
lems (stage I) followed months to years later by the development of myoclonic jerks,
ataxia, and seizures (stage II). The electroencephalography (EEG) in SSPE demon-
strates characteristic periodic complexes that are stereotyped, bilaterally synchro-
nous and symmetrical 100–1000 mV, 1–3 Hz waves lasting 1–3 seconds separated by
a 2–20 second interval (Praveen-kumar et al. 2007). Coma ensues (stage III) months
to years later. Stage IV of SSPE is characterized by akinetic mutism. Pseudobulbar
affect and startle myoclonus are common. Death due to infection or vasomotor
collapse typically occurs 2–3 years after disease onset. Although more fulminant
courses have been reported as have spontaneous remissions in approximately 5% of
cases (Freeman 1969; Gutierrez et al. 2010; Risk and Haddad 1979). MRI demon-
strates T2 hyperintense lesions that progress from the subcortical white matter to the
periventricular regions along with progressive cerebral atrophy (Anlar et al. 1996;
Murata et al. 1987). There is little or no pleocytosis in the CSF, although total protein
concentration is elevated. The gamma globulin fraction in particular is elevated with
the presence of IgG oligoclonal bands. Neuro-ophthalmologic complications are also
common and include retinal pigmentation, optic atrophy, chorioretinitis, optic neuri-
tis and papilledema (Robb and Watters 1970). SSPE is viewed as the paradigm of a
persistent RNA virus infection in the human.
15.4.2 Diagnosis
Prior to molecular diagnostic approaches, observing the presence of the pathogno-
monic prodromal Koplik spots around the buccal mucosa was the primary means of
making the diagnosis of measles. Currently, serological assays to detect MV-specific
IgG and IgM antibodies are typically used. APME is primarily a clinical diagnosis
(see above). A presumptive diagnosis of SSPE can be made by a combination of the
clinical presentation in a young patient, the presence of the characteristic periodic
complexes on EEG, elevated gamma globulins and the presence of oligoclonal bands
in the CSF, characteristic MRI findings and elevated measles antibody titers in the
serum and CSF. Similarly, MIBE is primarily a clinical diagnosis, although for all
three neurological complications of MV infection, neuropathology provides the ulti-
mate confirmation of the diagnosis.
15.4.3 Pathology and Pathogenesis

MV is highly contagious, spreads by the aerosol route and infects alveolar macro-
phages and dendritic cells in the lower respiratory tract (Lemon et al. 2011). The virus
spreads rapidly to bronchial associated lymphoid tissues and then local lymph nodes,
where the infection is amplified. Viremia is primarily mediated by MV-infected
lymphocytes and dendritic cells (de Swart et al. 2007), and this enables spread to
many organs throughout the body (Esolen et al. 1993). Although some of the target
organs such as the spleen or tonsils are primarily lymphoid in origin, numerous other
organs and tissues are infected (e.g., kidney, lung, liver, and skin). Nebulization of
recombinant MVs expressing enhanced green fluorescent protein (EGFP) into the
macaque, the only animal model which recapitulates the natural, systemic infection,
has facilitated the identification of the primary target cells (Lemon et al. 2011).
The three neurological complications of measles display differing pathology.
Examination of fatal APME cases reveals perivascular inflammation and demyelin-
ation with a corresponding loss of myelin basic protein (Johnson et al. 1984). This
is presumed to be due to an autoimmune response as virus has not been isolated or
detected in brain tissue obtained from APME patients who succumbed to the infec-
tion (Gendelman et al. 1984). In contrast to APME, examination of brain tissues
obtained from MIBE patients shows evidence of gliosis and both intranuclear and
intracytoplasmic viral inclusion bodies but little evidence of inflammation (Aicardi
et al. 1977). Therefore MIBE appears to share many common features with SSPE
(see below) but has a more rapid disease progression due to the absence of a viable
cell mediated immune response. SSPE is characterized by inflammatory infiltrates
in both the gray and white matter, hence the term “panencephalitis.” Diffuse demye
lination, astroglial sclerosis, and viral antigen contained in inclusion bodies in neu-
rons and oligodendrocytes are also observed (Herndon and Rubinstein 1968).
The route of entry used by MV to infect the CNS is unknown, but it may involve
passage through the blood-brain-barrier in infected leukocytes or direct infection of
cerebral endothelial cells at the blood-brain-barrier (Kirk et al. 1991). Examination
of the anatomical distribution of MV antigen in the CNS of SSPE patients has shown
that MV is present in the frontal cortex, basal ganglia, cerebellum, thalamus, medulla,
and parietal cortex (McQuaid et al. 1998). EM analysis of brain tissues obtained from
SSPE patients shows cell-to-cell fusion, albeit in rare instances. Virus budding is typi-
cally absent from infected cells (Iwasaki and Koprowski 1974). In vitro studies have
shown that laboratory-adapted MV can spread between primary hippocampal neurons
in the absence of CD46 (Lawrence et al. 2000). Examination of SSPE brain tissue
sections by immunohistochemistry shows that CD150 cannot be detected on neurons
or oligodendrocytes (McQuaid and Cosby 2002) and that CD46 cannot be detected
in MV infected brain lesions (McQuaid et al. 1997). These observations have given
credence to the idea that MV may spread within the CNS through localized fusion
events at lateral cell-to-cell contacts, thus negating the need for a specific virus recep-
tor (Allen et al. 1996). The interconnectivity between neurons is readily visible when
indirect immunocytochemistry is used to detect viral antigen (Figure 15.1a). It remains
to be determined if the recently identified wild-type MV receptor PVRL4 (Noyce et al.
2011) has a role in the cell-to-cell spread of MV within the CNS.
MV was first isolated from SSPE brain tissue by co-cultivation of explants with
immortalized cells in vitro (Chen et al. 1969). The lack of virus budding in vivo
was mirrored in vitro and consequently contributed to the development of cell lines
persistently infected with “SSPE” viruses. Advances in molecular biology resulted
in PCR amplification, cloning, and sequencing of MV genes directly from SSPE
brain tissue, removing the need to generate persistently infected cell lines where
the effect of the host cell environment on the virus could be a complicating factor.
Sequence analysis showed that the N, P, and L genes are relatively well conserved
in comparison to wild-type progenitor strains (Jin et al. 2002). However, the M, F,
and H genes contain many alterations from the wild-type sequence and the M gene
is particularly prone to mutations (Hall et al. 1979). The M gene can be disrupted
in two main ways, first by biased hypermutations due to the action of the cellular
dsRNA-dependent unwinding enzyme (Cattaneo et al. 1988). This results in frequent
U to C substitutions, some of which prevent the generation of a functional M protein.
Second, single point mutations or deletions in the P GE sequence are common (Ayata
et al. 2002). This leads to read through transcription at the P-M Ig junction by the
RdRp which generates bicistronic P-M mRNAs in which the second ORF cannot be
accessed by the ribosome; this in turn decreases the levels of M protein in the cell
(Cattaneo et al. 1987). Analysis of the F gene sequences has consistently shown the
presence of single nucleotide substitutions or deletions which produce premature
stop codons leading to the production of glycoproteins with truncated cytoplasmic
(a) (b)
(c) (d)
FIGURE 15.1 (See color insert.) (a) Detection of the N protein of MV in a 7-μm section
obtained from an SSPE case by indirect immunofluorescence (green). Nuclei from uninfected
neurons are counterstained with propidium iodide (red). Interconnecting neuronal processes
(arrows) and cell bodies (asterisk) are indicated. (b) T2-weighted MRI sequence of a patient
with MuV encephalitis. T2-signal hyperintensities are indicated (arrows). (Copyright ©
MedReviews®, LLC. Adapted and reprinted with permission of MedReviews, LLC. Cooper
A.D. et al. Mumps encephalitis: return with a vengeance. Rev Neurol Dis. 2007; 4:100–102.
Reviews in Neurological Diseases is a copyrighted publication of MedReviews, LLC. All
rights reserved.) (c) T2-weighted MRI sequence of a patient with acute NiV encephalitis.
Selected punctate T2-signal hyperintensities are indicated (arrows). (d) T2-weighted MRI
sequence of a patient with relapsed NiV encephalitis. Selected confluent T2-signal hyperin-
tensities are indicated (arrows). (Adapted and reprinted from Goh, K. J. et al., 2000, N. Engl.
J. Med. 342, 1229–1235.)
tails (Schmid et al. 1992). Although the presence of truncated F glycoproteins hin-
ders efficient virus budding from the cell surface, cell-to-cell fusion functions are not
affected (Cathomen et al. 1998). Collectively these mutations keep the virus highly
cell-associated which in turn probably facilitates transneuronal spread and keeps the
virus “hidden” from the immune system.
Although small animal models have been used to examine acute encephalitis and
persistence of MV in the CNS, these require the use of either rodent-brain adapted
strains or transgenic animals which express a MV receptor and typically are interferon
incompetent (Duprex et al. 1999; Schubert et al. 2006). The disadvantage of these mod-
els is that the virus is injected via the intracerebral route and there is currently no model
in which the virus reaches the CNS from the periphery. Unpublished studies from
our group identified wild-type MV in the brain of a macaque shortly after infection,
although these observations remain to be corroborated in additional animals. Whether
this was a unique finding remains to be seen (Duprex et al., unpublished).
15.4.4 Epidemiology
MV only naturally infects humans and some species of monkey and is one of the
most infectious human pathogens in current circulation. This has enabled the disrup-
tion of endemic MV transmission in many parts of the developed world, although the
virus continues to cause significant levels of morbidity and mortality in the develop-
ing world, with 164,000 deaths being attributed to measles in 2008 (Bellini and Rota
2011). Importation of the virus from the developed world is common and is especially
problematic in countries which have low levels of vaccination. Currently there are
large outbreaks of measles in France where 14,000 people have been infected and
six have died (2011). APME is the most common neurological complication resulting
from natural MV infection occurring in approximately 1:1000 cases (Miller 1964)
with MIBE cases only observed as a complication of MV infection of immunodeficient
individuals. A re-evaluation of the incidence of SSPE in recent years revealed approxi-
mately 4 to 11 cases per 100,000 cases of measles (Campbell et al. 2007), and a recent
analysis of neurological complications resulting from measles outbreaks in Papua New
Guinea has reported rates as high a 1 in 10,000 children (Manning et al. 2011).
15.4.5 Prognosis and Treatment

Live attenuated MV vaccines were developed in the 1960s from a clinical isolate
obtained from a patient, David Edmonston (Enders and Peebles 1954). APME is
typically treated with high dose corticosteroids together with supportive care,
although the benefit of steroids or other means of immunosuppression like plasma
exchange or intravenous immunoglobulins have not been proven in prospective trials
(Tenembaum et al. 2007). In addition to the 10% mortality rate, survivors of APME
can be left with neurological sequelae including behavioral disorders, mental retar-
dation and epilepsy (Johnson et al. 1984). As discussed above, MIBE and SSPE
are almost invariably fatal, and treatment is largely supportive. Clinical trials have
evaluated drug candidates for SSPE. The best results have been seen with different
combinations of weekly intrathecal INF-alpha and daily oral isoprinosine either as
monotherapy or in combination resulting in slowed disease progression in approxi-
mately one-third of patients as compared with the 5% spontaneous remission rate
seen with historical controls (Dyken et al. 1982; Gascon 2003).
15.5 MUMPS VIRUS

The classic clinical presentation of MuV infection is parotitis, which is typically
preceded by a short prodromal phase of low-grade fever, anorexia, malaise, and
headache. One third of cases are asymptomatic. Between 15% and 30% of infected
adult men develop orchitis, and oophoritis can occur in women. Pancreatitis and
deafness are other potential complications. One of the largest studies examining the
neurological involvement of MuV by both clinical and laboratory criteria reported
an epidemic which took place from 1941 to 1942 (Bang and Bang 1943). Evidence
of neurological involvement, as defined by symptoms consistent with aseptic men-
ingitis and/or evidence of inflammation in the CSF (lymphocytic predominance),
was found in 65% of patients. In this study, lumbar puncture was routine for all
patients at the time of presentation whether they had neurological symptoms or
not. In other studies, there was no temporal association between when the parotitis
presents, if it occurs at all, and the development of meningitis (Russell and Donald
1958).
An MuV infection can lead to encephalitis in 0.1%–0.2% of cases (Bjorvatn and
Wolontis 1973; Cohen et al. 1992). Encephalitis complications include paresis (ocu-
lar, facial, hemiplegia, monoplegia) as well as seizures, ataxia, convulsions, coma,
mental disturbance, and transverse myelitis (Cohen et al. 1992; Nussinovitch et al.
1992; Venketasubramanian 1997). Some cases of MuV, encephalitis can be quite
severe, with a 1.5% mortality rate (Cooper et al. 2007; Kumar and Kuruvilla 2009).
MRI demonstrates diffuse T2 signal hyperintensities (Figure 15.1b). Whether these
more severe presentations are due to host or specific characteristics of the virus
strain remains unclear. A 1983 study of 41 children with MuV encephalitis collected
over a 12-year period found that a third of patients were ataxic, one quarter devel-
oped seizures, 20% had psychiatric complications, 20% had a depressed level of
consciousness, and 13% suffered from vertigo (Koskiniemi et al. 1983). At long-term
follow-up, a minority of patients was still ataxic and/or had psychiatric disturbances.
The male to female ratio was 4:1. One third of patients had salivary gland swelling,
which preceded the encephalitis by 1 week.
In 1967, Johnson and Johnson reported an experimental model of mumps-induced
hydrocephalus in a hamster (Johnson et al. 1967). Since that time, a number of human
cases of both acute and late onset, mumps-induced hydrocephalus in people have
been reported (Aydemir et al. 2009; Bray 1972; Cinalli et al. 2004; Lahat et al. 1993;
Ogata et al. 1992; Timmons and Johnson 1970). Transient sensorineural hearing loss
occurs at a rate of 4% in adult men, although permanent deafness is much less com-
mon (Bitnun et al. 1986; Everberg 1957; Vuori et al. 1962).
15.5.2 Diagnosis
Clinical diagnosis of mumps is based on the presence of painful, unilateral, or bilat-
eral (70%) parotitis. However, since not all mumps cases involve the parotid gland
and because there are other infectious (e.g., influenza A virus, parainfluenza virus,
Coxsackie virus, and lymphocytic choriomeningitis virus, Staphylococcus aureus),
and noninfectious (e.g., Sjögren’s syndrome, sarcoidosis, tumor, salivary gland
obstruction, etc.) causes of parotitis. Laboratory diagnosis is based on isolation of
virus, for example from urine, detection of viral nucleic acid or serological confir-
mation via detection of IgM mumps antibodies with enzyme linked immunosorbent
assay (ELISA).

MuV is restricted to humans, and transmission is via direct contact, droplet spread,
or contaminated fomites. The primary cells targeted have not been identified, and
the mechanism or route by which MuV enters the CNS is also unknown. Although
the virus does not use a single receptor and the HN glycoprotein binds sialic acid
moieties, the in vivo distribution of virus in the salivary glands, CNS, testes, ovaries,
breasts, pancreas, thyroid gland, myocardium, and the joints suggests that there must
be some predilection for particular cell types (Samal 2011).
Much of what is known about the neuropathogenesis of MuV has been gleaned from
small animal models including rats, Syrian hamsters, and mice which are infected
directly via the intracerebral route. There is some evidence for peripheral spread of
MuV from the periphery to the CNS in a marmoset animal model (Saika et al. 2002).
As discussed above, a hamster model of CNS MuV infection first demonstrated evi-
dence of obstructive hydrocephalus caused by both wild-type and vaccine strains
(Ennis et al. 1969; Herndon et al. 1974; Johnson and Johnson 1968; Johnson et al. 1967;
Takano et al. 1993). These and subsequent studies demonstrated the presence of viral
particles and intracytoplasmic viral-like inclusions in ependymal cells during the acute
phase of infection and enlarged and distorted ependymal cells lining the ventricles
during the chronic phase (Takano et al. 1993; Takano et al. 1999; Wolinsky et al. 1974).
Follow-up studies in humans confirmed the presence of ependymal cells in CSF
in six out of six patients with acute MuV infection, lending weight to the notion that
MuV causes an ependymitis (Herndon et al. 1974). While not all human cases of
MuV-induced hydrocephalus fit this model, these data suggest that hydrocephalus is
the result of accumulation of virus-infected cell debris in the aqueduct of Sylvius,
blocking CSF egress. There is evidence that neuroadapted strains in particular are
able to spread beyond the ependyma and choroid plexus and infect neuronal elements
resulting in cortical microhemorrhages, perivascular inflammatory infiltrates, and
limited neuronal necrosis. There is no evidence of endothelial cell infection (Johnson
and Johnson 1968; Wolinsky et al. 1974).
15.5.4 Epidemiology
The institution of national vaccination programs led to a dramatic decline in mumps
cases with a concordant drop in MuV meningitis and encephalitis (Modlin et al. 1975). In
1967, MuV accounted for 35.9% of encephalitis cases in the United States but only 12.5%
in 1972 when national vaccine coverage had reached 40% of children. Overall, U.S. cases
have declined from >100/100,000 to <1/100,000 (McNabb et al. 2007). Recently the
virus has reemerged in the US and UK (see above) and there have been discussions as to
whether it might be necessary to administer three doses of the MMR vaccine. However,
there is no evidence that waning immunity plays a part in reinfections (Rubin et al. 2012).

The mainstay of MuV treatment remains preventative using live attenuated vaccines
which were developed in the 1960s. Despite the success of MuV vaccination, the
neurotropism of MuV has complicated these efforts, as multiple cases of MuV vaccine-
induced aseptic meningitis and encephalitis have resulted in widespread fear and even
cessation of national vaccine programs (Arruda and Kondageski 2001; da Silveira et
al. 2002; Furesz and Contreras 1990; Odisseev and Gacheva 1994). This highlights a
key difficulty with regard to vaccine safety in that the vaccines which had to be with-
drawn had passed the monkey neurovirulence (MNVT) test, which is the primary
method by which the neurovirulence of candidate mumps vaccines is assessed. Given
that the MNVT assay is not wholly reliable, this has prompted research into other
animal models such as the (Rubin et al. 2000). Promising results have been obtained,
although it is not clear as yet whether or not such rat neurovirulence tests (RNVT)
will readily replace the MNVT which is integral in the vaccine licensing process.
There are no proven anti-viral strategies for MuV. Fortunately, as discussed above, the
prognosis for most neurological complications of MuV is excellent.
15.6 HENIPAVIRUSES
While HeV causes encephalitis in horses and humans, severe pulmonary symptoms
are more common. After a 7–10 day incubation period, people develop an influenza-
like illness characterized by fever, myalgia, headache, lethargy, sore throat, nausea,
and vomiting with some patients recovering over a few weeks and others progressing
to respiratory failure and/or fatal encephalitis.
NiV causes respiratory symptoms in up to 25% of patients, but the more promi-
nent feature is the severe acute encephalitis that typically develops within a week
of infection. Signs and symptoms include reduced levels of consciousness and signs
consistent with brainstem involvement including segmental myoclonus, areflexia,
hypertension, and tachycardia. MRI demonstrates many small (2–7 mm) T2 hyperin-
tensities in the subcortical and deep white matter likely representing microinfarctions
as a consequence of the severe vasculitis (Figure 15.1c). Laboratory abnormalities
include thrombocytopenia, leukopenia, and transaminitis as well as pleocytosis and
elevated protein levels in the CSF (Goh et al. 2000). Death typically ensues within
ten days after illness onset likely secondary to brainstem involvement and respira-
tory failure (Goh et al. 2000). Twelve survivors from the initial outbreak in Malaysia
and Singapore (7.5% of survivors) had recurrent encephalitis by 24 months while ten
people (3.4%) who had either a nonencephalitic or asymptomatic primary infection,
developed late-onset acute encephalitis. The mean interval between the first neu-
rological episode and the time of initial infection was 8.4 months. The onset of the
relapsed or late-onset encephalitis was usually acute and clinical features included
fever, headache, seizures, and focal neurological signs. MRI in these cases demon-
strates patchy areas of cortically based T2-weighted hyperintensities that are much
more confluent than the T2-weighted hyperintensities seen in acute NiV encephali-
tis (Figure 15.1d). Eighteen percent of relapsed/late-onset patients died and 61% of
late/relapsed patients had residual neurological deficits versus 22% who survived
after acute Nipah encephalitis (Chong 2003; Tan et al. 2002; Wong et al. 2001).
Interestingly, a single patient presented with fatal HeV encephalitis more than a year
after a self-limited episode of meningitis in retrospect attributed to HeV. The neuro-

pathology was similar to the relapsed NiV encephalitis cases (O’Sullivan et al. 1997).
15.6.2 Diagnosis
RT-PCR can be used to detect henipavirus nucleic acids in nasopharyngeal secre-
tions, urine, and internal organs including lung and brain (Goh et al. 2000; Harcourt
et al. 2005). Given that NiV and HeV are classified as BSL-4 viruses, molecular-
based assays represent the optimal means to make a rapid and sensitive diagnosis
without having to culture a virus. Serologic diagnosis is made by detection of IgM
antibody in serum using an ELISA. All patients have IgG antibodies by 17–18 days
after infection. Since seroconversion occurs 7–15 days postinfection (10–15 days
in the CSF), and patients die on average 9 days after infection, seroconversion is
not required in some case study definitions (Goh et al. 2000; Ramasundrum 2000).
There is no difference in outcome between patients with or without a positive serol-
ogy, although there is a poorer prognosis for patients in whom virus was isolated
from the CSF (Chua et al. 2000b).

Henipaviruses primarily infect neurons and endothelial cells of blood and lym-
phatic vessels, submandibular and bronchiolar lymph nodes, tonsil, and spleen.
Both viruses induce a systemic vasculitis with microthromboses resulting in ische
mic damage, most prominently in the CNS (Chua et al. 2000a; Wong et al. 2002a).
Multifocal, necrotic plaques in the brain parenchyma likely correspond to the micro-
infarcts visualized on MRI. The primary receptor for both viruses is ephrinB2 which
explains the majority, although not all, of the tropism in vivo.
NiV is present in cerebral vascular endothelial cells as well as the CNS of both
symptomatic and asymptomatic animals. Unusual syncytial multinucleated endo-
thelial cells are seen in both NiV and HeV infections (O’Sullivan et al. 1997). Viral
antigens and RNP containing intracytoplasmic inclusion bodies are detectable
in the brain by immunohistochemistry (Hooper et al. 2001; Wong et al. 2002b).
Interestingly, no mature viral particles are observed (Goldsmith et al. 2003; Hyatt
et al. 2001). It is not clear whether the henipaviruses enter the CNS via the cranial
nerves and/or whether free or cell-associated virus is able to cross the blood brain
barrier from the bloodstream via endothelial cells.
The delayed or relapsed encephalitis seen in NiV-infected patients presumably
develops through direct neuronal infection (Chua et al. 2000b). While NiV cannot
be cultured from autopsy material, the pathology is remarkable for encephalitis with
immunolocalization for NiV antigen in neurons primarily but also in glial and epen-
dymal cells without evidence of demyelination. The recurrent and delayed encepha-
litis cases do not demonstrate the vasculitis and the associated necrotic plaques seen
in the acute encephalitis cases. Given the gray matter predominance of the pathol-
ogy, the presence of viral antigen and the much delayed onset of recurrent illness,
the etiology is very likely secondary to direct viral attack rather than a postinfectious
demyelination syndrome like APME. Similarities in pathology and disease course
between the single delayed case of HeV encephalitis and the numerous NiV cases
have spurred comparisons to SSPE (Ksiazek et al. 2011; Wong et al. 2001). However,
hypermutation of particular genes or “signature mutations” in, for example, the
cytoplasmic tails of the F and G glycoproteins or the GE sequences have not been
identified.
15.6.4 Epidemiology
The broad geographical range of the henipaviruses is in large part a result of the
widespread distribution of the flying fox (order Chiroptera, genus Pteropus in the
family Pteropodidae) which serves as the animal reservoir (Chua et al. 2002; Halpin
et al. 2000; Yob et al. 2001). Its habitat extends from the western Indian Ocean to
Southeast Asia and from Australia and the southwest Pacific islands (Olson et al.
2002; Reynes et al. 2005). Moreover, the virus naturally infects six species from five
mammalian orders (pigs, horses, cats, dogs, and humans) and can be used to infect
guinea pigs and hamsters experimentally (Eaton et al. 2006; Hooper et al. 2001;
Williamson et al. 2001; Wong et al. 2003). Viral transmission typically occurs as a
result of contact with pigs or horses, although recent outbreaks in Bangladesh and
India were probably initiated by direct transmission from bats (Chadha et al. 2006;
Luby et al. 2006).
While only one HeV strain has been reported, two distinct strains of NiV are
currently circulating. The more recent strain reemerged from 2001 to 2004 across
a wide area of central and western Bangladesh with the case fatality rate increas-
ing from 38.5% to 75% (Blum et al. 2009; Chadha et al. 2006; Homaira et al. 2010;
Hsu et al. 2004). Human to human transmission was not documented in the initial
Malaysian outbreak (Mounts et al. 2001) but is thought to have occurred in subse-
quent outbreaks in 2001, 2003, and 2007 in Bangladesh (Blum et al. 2009; Homaira
et al. 2010; Hsu et al. 2004) and India (Chadha et al. 2006).

While there are no approved treatments for henipavirus infection, there have been
some exciting recent advances. The only medication tested in humans remains riba-
virin due to its broad-spectrum of antiviral activity. An open-label trial of ribavirin
in 140 NiV-infected patients was conducted in 2001 (Chong et al. 2001). Compared
with 54 patients who either refused ribavirin or for whom ribavirin was not available,
ribavirin appeared to have a marginal but positive impact on mortality. The relative
risk reduction was 36% with 32% mortality in the treatment group compared with
54% in the control group. However, an alternative analysis of these data from the
same outbreak and subsequent animal studies has not borne out with these findings
(Georges-Courbot et al. 2006; Goh et al. 2000).
While the efficacy of ribavirin is controversial, a recent study demonstrated the
ability of a peripherally administered recombinant human neutralizing monoclonal
antibody to completely prevent disease in African green monkeys 24 h postintrathe-
cal infusion of HeV (Bossart et al. 2011). Monkeys given the MAb 72 h post-infection
suffered some neurological sequelae, but they all survived as opposed to the 100%
mortality in the untreated group. Virus-to-cell fusion can be inhibited using peptides
that may be exploitable as a potential therapy (Rey et al. 2010). Recombinant subunit
vaccine formulation protects against lethal NiV challenge in cats (McEachern et al.
2008).
15.7 OTHER PARAMYXOVIRUSES NOT TRADITIONALLY

ASSOCIATED WITH CNS MANIFESTATIONS
Although it primarily causes a pediatric respiratory disease difficult to distinguish
from the phenotype caused by human respiratory syncytial virus (HRSV), human
metapneumovirus (HMPV) has recently been associated with a number of viral
encephalitis cases in children, including two fatalities (Arnold et al. 2009; Hata et al.
2007; Kaida et al. 2006; Schildgen et al. 2005). HRSV has also been associated with
encephalitis in young children (Glaser et al. 2003; Hanna et al. 2003; Hirayama et al.
1999; Kawashima et al. 2009; Kho et al. 2004; Millichap and Wainwright 2009; Ng
et al. 2001). Lastly, CNS manifestations have also been reported in a number of chil-
dren with human parainfluenza virus type 3 (HPIV3) infections (Glaser et al. 2003;
McCarthy et al. 1990). Clearly, these are unusual cases, although they illustrate the
unusual propensity paramyxoviruses have for the CNS.

Much can be learned by comparing and contrasting the clinical manifestations
caused by viruses which are united in their molecular biology and virology but
divided, for example, by their levels of pathogenicity and the degree of mortality
they cause. Such an analysis provokes questions such as:
1. Is CNS entry the key determinant of neurovirulence?

2. How do viral proteins, which specifically target intrinsic barriers in the host
cell or modulate the efficacy of the adaptive immune response, enhance
disease?
3. What makes one virus marginally epitheliotropic/endotheliotropic, highly
lymphotropic, and quite neurotropic (MV) and another highly specific for
the circulatory and central nervous systems (NiV)?
4. Why do some viruses readily infect a wide range of species (NiV and HeV)
whereas others are exquisitely human specific (MV, MuV, HRSV, HMPV,
and hPIV3)?
5. How can this knowledge help in the development of novel targeted thera-
peutics and the treatment and prevention of neurotropic paramyxoviruses in
particular and neurotropic viruses in general?
Advances in molecular virology which permit the recovery of recombinant para-

myxoviruses will help to answer such questions but only if the systems are gener-
ated from clinical and not laboratory-adapted isolates. Growth of these viruses in
disease-relevant cell lines for example B cells (MV) or human salivary cells (MuV)
is also vital as it must always be remembered that paramyxoviruses are products of

the cells in which they are grown. Only when these two strands complement one
another and optimal animal models are used, it will be possible to recapitulate many
aspects of the disease and shed light on viral neuropathogenesis. Generation of chi-
meric viruses expressing the envelope glycoproteins of one virus and the internal
proteins of another will be useful in dissecting the contribution virus receptors have
in tropism. This is critically important as it will help to increase our understanding
of the barriers to cross species infection which is highly relevant given that MV has
been targeted for eradication by the World Health Organization. Given the fact that
RNA viruses readily evolve to fill an empty niche, much thought needs to be given to
cessation of MV vaccination campaigns as other morbilliviruses show much higher
levels of neurotropism.
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16 Rabies Virus
Neurovirulence
Claire L. Jeffries, Ashley C. Banyard,
Derek M. Healy, Daniel L. Horton,
Nicholas Johnson, and Anthony R. Fooks
CONTENTS
16.1 Introduction................................................................................................... 373
16.1.1 Rabies................................................................................................ 373
16.1.2 Clinical Course.................................................................................. 374
16.1.3 Lyssaviruses: Classification, Morphology, and Replication.............. 375
16.2 Pathogenesis................................................................................................... 378
16.2.1 Exposure............................................................................................ 378
16.2.2 Transport of Rabies Virus from the Bite Site to the CNS................. 380
16.2.3 From the CNS to Onward Transmission............................................ 381
16.3 Pathology....................................................................................................... 381
16.3.1 Cell Damage and Death..................................................................... 382
16.3.2 Functional Impairment...................................................................... 382
16.4 Immune Response......................................................................................... 383
16.4.1 Immunity to Infection........................................................................ 384
16.4.2 Therapy.............................................................................................. 384
16.5 Studying the Neuroinvasiveness of Rabies: Experimental Models of
Infection and Vaccine Development.............................................................. 385
16.6 Future Outlook............................................................................................... 386
16.7 Key Points...................................................................................................... 387
Acknowledgements................................................................................................. 387
References............................................................................................................... 387
16.1 INTRODUCTION
16.1.1 Rabies
Rabies is an infectious encephalitic disease of mammals, caused by viruses of the
genus Lyssavirus (Family Rhabdoviridae). Exposure to rabies virus, in the absence
of timely medical intervention, almost invariably results in a fatal outcome. The
term “rabies” comes from the Latin word rabere, which means “to rage or rave” and
refers to the clinical disease progression seen following infection. This condition has
been known, and feared, by people across the globe for thousands of years (Neville
373
2004). It is only within the last few centuries, however, that progress has been made
in understanding the disease, and in the development of successful prevention and
treatment strategies (Baer 2007). In the 1880s, Louis Pasteur developed the first suc-
cessful postexposure prophylaxis (PEP) (Pasteur 1885). From this early innovation,
scientific and technological advances during subsequent decades have provided the
safe and efficacious vaccines available today (World Health Organization [WHO]
2005). Despite the existence of these tools, rabies continues to kill thousands of
people every year, mostly in countries where people live on less than US$1 each day.
The disease occurs in more than 75% of the world’s countries, causing innumerable
animal deaths and an estimated 55,000 human deaths per year, the majority of which
are children younger than 15 years (WHO 2010). It is likely that the predicted annual
figure for human rabies fatalities is a gross under-estimate due to the lack of infra-
structure and reporting systems in developing countries (Fooks 2007).
Viral neurovirulence (the capacity to infect the nervous system) is a feature
of rabies virus infection, and all lyssaviruses are known to be highly neurotropic
(Schnell et al. 2010). The ability of the virus to spread throughout the nervous sys-
tem of the host (neuroinvasiveness) and into the brain is a major contributing factor
to the pathogenesis observed following rabies virus infection (Dietzschold et al.
2008).
16.1.2 Clinical Course
Once an individual has been exposed to rabies virus, it is essential that medical
attention is rapidly sought, as it is at this first crucial stage where effective PEP can
alter the outcome of infection and save lives. Following infection, there is a variable
incubation period before the onset of clinical symptoms. This period generally lasts
between 20 and 90 days in humans but may vary considerably, in some rare cases
lasting several years (Jackson 2007a; Johnson et al. 2008a). Following incubation,
the infected individual will begin to exhibit non-specific clinical symptoms, which
may include but are not limited to malaise, weakness, loss of appetite, paresthesia,
and fever (Hunter et al. 2010; Rupprecht et al. 2002). The disease then rapidly pro-
gresses to manifest as an acute central nervous system (CNS) disorder that produces
a broad spectrum of clinical symptoms (Nicholson 1994). There are two clinical
outcomes: furious and paralytic (Schnell et al. 2010). A large proportion (approxi-
mately 80%) of human cases demonstrate symptoms associated with the furious
form including aggression, hyperexcitability, muscular spasms, and hydrophobia
(Dacheux et al. 2008; Schnell et al. 2010). However, the remainder of cases gener-
ally follow a different clinical course typified by paralytic symptoms such as incoor-
dination, lethargy, and flaccid muscle weakness that progress to paralysis and death
(Hunter et al. 2010; Solomon et al. 2005). The two forms (furious and paralytic) are
not distinct or mutually exclusive and many infections develop with clinical features
from both. The factors that influence the differences in disease presentation are not
fully understood (Hemachudha et al. 2003). Obtaining a differential diagnosis of
rabies infection can often be problematic due to variations in length of incubation
period and clinical presentation. Several of the symptoms that can be observed dur-
ing rabies infection also resemble those observed in other neurological disorders.
Rabies Virus Neurovirulence 375
For example, the clinical symptoms observed in human paralytic cases can often
be confused with Guillain-Barré syndrome, but the two diseases can be differenti-
ated when there is sphincter involvement, particularly urinary incontinence, as this
only occurs during rabies infection (Asbury and Cornblath 1990; Jackson 2007a;
Solomon et al. 2005).
Regardless of differences in disease presentation, the ultimate outcome for
infected individuals is almost always death. However, there have now been several
documented cases whereby clinically affected humans have survived following the
rapid administration of PEP and/or intensive therapies, although, all but a few of these
patients suffered substantial neurological sequelae (Willoughby 2009; Willoughby
et al. 2005). There have also been a number of experimental studies in which clini-
cally affected animals have been observed to recover and survive infection, without
intervention, although recovery was accompanied by neurological sequelae (Jackson
et al. 1989; Vos et al. 2004). A few controversial reports have also been published
describing infected animals, with productive infections, showing no clinical signs,
suggesting a carrier state (East et al. 2001; Veeraraghavan et al. 1970). These atypi-
cal outcomes can be difficult to investigate due to limitations in the diagnosis of
rabies in a live animal (Jackson 2007b). It is currently unclear why these outcomes
of infection occur, and further research is required to understand such instances.
However, it is clear that the relative virulence of the infecting virus, viral load, site
of wound, and immunological status of the infected host must all be critical to the
outcome following exposure (Vos et al. 2004; Willoughby et al. 2005).
16.1.3 Lyssaviruses: Classification, Morphology, and Replication

The genus Lyssavirus belongs within the Order Mononegavirales, Family Rhabdoviridae.
There are 12 established species within the Lyssavirus genus (ICTV 2011) and two
recently identified viruses, namely Bokeloh bat lyssavirus (Freuling et al. 2010) and
Ikoma lyssavirus (Marston et al. 2012) that are awaiting classification (Table 16.1).
All lyssaviruses contain single-stranded, negative-sense RNA genomes with envel-
oped, bullet-shaped virions measuring approximately 75 × 180 nm (Warrell and
Warrell 2004). Virions derive their envelope from the plasma membrane of the cell
that they infect. The lyssavirus genomes are approximately 12,000 bp in length
and contain genes that encode five proteins: the nucleoprotein (N); the phospho-
protein (P); the matrix protein (M); the glycoprotein (G), and the large polymerase
protein (L) (Dietzschold et al. 2008). Of these proteins, the N protein encapsidates
the negative-sense RNA genome within the virion forming a tight association with
the RNA, which, along with P and L, forms the minimal replicative unit for these
viruses: the ribonucleoprotein complex (RNP). This RNP structure is responsible
for all elements of viral transcription and replication to generate viral proteins and
nascent genomes, respectively (Schnell et al. 2010). The M protein plays a structural
role between the RNP and the virion envelope (Schnell et al. 2010). The G protein
is found throughout the virion envelope and is responsible for cell attachment and
entry mechanisms (Rupprecht et al. 2002). This G protein is also the major antigenic
determinant and is the major target for neutralizing antibodies following infection
(Dietzschold et al. 2008).
TABLE 16.1
Classification of the Lyssaviruses
Human
Geographical Species from Deaths
Species Phylogroup Range which Isolated Reported
Rabies virus (RABV) 1 Worldwide Wide range of Approx.
mammals 55,000
p.a.
Lagos bat virus (LBV) 2 Africa Fruit bats, dogs, Unknown
and cats
Mokola virus (MOKV) 2 Africa Shrews, cats, dogs, 2
rodents, and
humans
Duvenhage virus (DUVV) 1 Southern Insectivorous bats 3
Africa and humans
European bat lyssavirus 1 1 Europe Insectivorous bats, 2
(EBLV-1) sheep, stone
martens, and
humans
European bat lyssavirus 2 1 Europe Insectivorous bats 2
(EBLV-2) and humans
Australian bat lyssavirus 1 Australia Fruit and 2
(ABLV) insectivorous
bats and humans
Aravan virus (ARAV) 1 Kyrgyzstan Insectivorous bat Unknown
Khujand virus (KHUV) 1 Tajikistan Insectivorous bat Unknown
Irkut virus (IRKV) 1 Eastern Insectivorous bat Unknown
Siberia
West Caucasian bat virus 3 Caucasus Insectivorous bat Unknown
(WCBV)
Shimoni bat virus (SHIBV) 2 Africa Insectivorous bat Unknown
Bokeloh bat lyssavirus (BBLV) 1? Germany Insectivorous bat Unknown
Ikoma lyssavirus (IKOV) 3? Tanzania African civet Unknown
Source: Freuling, C. et al., 2010, Discovery of a Lyssavirus in a Natterer’s bat (Myotis nattereri) from
Germany with unusual antigenic and molecular characteristics, in Rabies in the Americas (RITA)
XXI, Guadalajara, Mexico; Healy, D. M., 2011, Comparative Pathology of Lyssaviruses in a
Murine Model, School of Medicine, Dentistry and Biomedical Sciences, Centre for Infection
and Immunity, Queen’s University, Belfast, p. 261; ICTV, 2011, ICTV Master Species List 2011
v2, in Viruses, I.C.o.T.o. (Ed.), Virology Division, International Union of Microbiological
Societies; Johnson, N. et al., 2006b, Emerg. Infect. Dis. 12, 1142–1144; Kuzmin, I. V. et al.,
2010, Virus Res. 149, 197–210; Marston, D.A. et al., 2012, Emerg. Infect. Dis. 18, 664–667;
Mensink, M. and Schaftenaar, W., 1998, When bad things happen to bats: the occurrence of a
Lyssavirus in a closed population of Egyptian frugivorous bats (Rousettus aegyptiacus) at
Rotterdam Zoo, European Association of Zoo and Wildlife Veterinarians (EAZWV) Second
Scientific Meeting, pp. 147–151; Ronsholt, L. et al., 1998, Vet. Rec. 142, 519–520.
Lyssaviruses must gain entry into a host cell in order for replication to take
place. The replication cycle of these viruses involves four main activities: uncoat-
ing, transcription, replication, and assembly (Figure 16.1) (Rupprecht et al. 2002).
Initially, the viral G protein facilitates entry into the host cell by interacting with,
and attaching to, cell surface receptors (Figure 16.1.1) (Lafon 2005). This leads to
cell entry, either through direct fusion, whereby the viral envelope fuses with the
host cell membrane, releasing the RNP into the cytoplasm of the cell or via recep-
tor mediated endocytosis (RME), when the cell engulfs the viral particles through
coated pits and uncoated vesicles on its surface (Wunner 2007). Following RME,
virions are released from endosomal vesicles within the host cell, a process known as
uncoating (Figure 16.1.2) (Schnell et al. 2010). Release is dependent on a reduction in
endosomal pH that causes a conformational change in the viral G protein, resulting
in membrane fusion and release of the RNP into the cell cytoplasm (Figure 16.1.3)
(Gaudin et al. 1993).
Once in the cytoplasm of the infected cell, the virus genome initiates its replica-
tive cycle commencing with the RNP acting as a transcriptase complex, capable of
generating messenger RNA (mRNA) from the negative-sense RNA genome (Figure
16.1.4) (Schnell et al. 2010). These viral mRNAs are capped and polyadenylated by
the host cellular machinery, and translated on free ribosomes within the cell (Figure
16.1.5) (Banerjee and Barik 1992). The exception to this is the G protein, which is
moved to the rough endoplasmic reticulum (RER) and through the Golgi for transla-
tion and transport to the cell membrane (Figure 16.1.6) (Wunner 2007). A transcrip-
tional gradient is generated where the transcriptase can only initiate the generation
Rabies viruses
Attachment Budding
1 9
Cytoplasm Assembly
Uncoating 3
Genome
2 3´ 5´ Nascent
RNP RNP
[pH] Transcription
4
3´ 5´
Endosome AAA mRNAs AAA 5´ 3´
5
Translation M Genome 8
6
3´ 5´
P N L
Replication
7
Antigenome
RER 5´ 3´
Nucleus
G
Golgi
FIGURE 16.1 The intracellular life cycle of lyssaviruses following the infection of a host
cell. All abbreviations are detailed in the text.
of transcripts from the 3ʹ terminus of the genome, at the genome promoter (Banerjee
and Barik 1992). As a result, 3ʹ proximal genes are produced in abundance while
those genes distal to the genome promoter are consecutively generated to lower levels
(Schnell et al. 2010). At some point following infection, the RNP complex switches
its function from that of a transcriptase complex, to a replicase complex, whereupon
it subsequently generates full-length positive-sense RNA replicative intermediates
(Figure 16.1.7) (Banerjee and Barik 1992). These positive-sense RNAs then act as
the template for the generation of nascent negative-sense RNA genomes (Figure
16.1.8) (Banerjee and Barik 1992). Late on in the cycle, it is thought that interactions
between nascent RNPs and the M protein enable movement of virus to the plasma
membrane. It is at this site where interaction with G brings about assembly of a new
virion, which then buds from the cell (Figure 16.1.9) (Schnell et al. 2010). The prog-
eny virions are then capable of adhering to and infecting new host cells and the cycle
is initiated once more.
16.2 PATHOGENESIS
The pathogenesis of rabies virus within a host follows a sequential pattern. This
process is instigated by the entry of viral particles into the host, and is followed
by the replication of the virus within the peripheral tissues, centripetal spread
along the peripheral nerves to the CNS, dissemination within the spinal cord
and brain, and centrifugal spread to the salivary glands and other organs (Figure
16.2) (Dietzschold et al. 2008). The duration of each step and pathology produced
in disease progression are influenced by both host and viral factors, which are
detailed below.
16.2.1 Exposure
Lyssaviruses are unable to gain entry through intact skin, which acts as an effective
defensive barrier. Virions must therefore enter where this barrier is broken, e.g., a
wound or through mucous membranes, i.e., the eyes, nose, mouth. The mechanisms
of exposure and viral entry into the host are often categorized into either “bite” or
“nonbite” incidents. The vast majority of transmission occurs as a result of the bite of
an infected animal, with transmission being facilitated by the presence of virus in the
saliva (Figure 16.2.1) (Warrell and Warrell 2004). In contrast, “nonbite” exposures
are far less common, but can occur through (i) the contamination of a preexisting
open wound, (ii) scratches received from an infected host, (iii) inhalation of aerosol-
ized virus, or (iv) through the transplantation of virus-infected organs, e.g., corneal
transplants (Jackson 2007a; Johnson et al. 2006a). There are also reports of human-
to-human transmission, which have included transplacental transmission, where
virus was detected in both mother and baby postmortem (Sipahioglu and Alpaut
1985), and two cases where transmission is thought to have occurred between mother
and child, one through a bite and the other through repeated oral contact (Fekadu et
al. 1996). If transmission occurs, but a clear history of an exposure to rabies virus
is not known or recalled (e.g., an unnoticed bite or scratch received from a bat), it
is known as cryptic rabies. This inconspicuous transmission is a cause of human
4 Virus enters brain and undergoes extensive

3 Virus travels from PNS to replication leading to neuronal dysfunction
spinal cord and brain
2 Virus enters the PNS via 5b

neuromuscular junction
Virus enters
peripheral
nerves of skin
and hair 4
follicles
Virus in purkinje cells of
cerebellum (× 40 mag.)
5b 5a
3
2 5c
5c Virus spreads from brain to

infect many tissues and organs
1 Virus enters muscle tissue of

host through bite wound Virus replication in salivary
5a
glands and excretion in saliva
FIGURE 16.2 The sequential pattern of rabies pathogenesis occurring within a dog, follow-
ing a bite wound (virions not to scale). PNS = peripheral nervous system.
fatalities in regions where effective medical intervention could have been sought had
the individual been aware of their exposure (Messenger et al. 2002).
The lyssaviruses are maintained in a variety of reservoir hosts, which vary with
viral species, strain, and location (Rupprecht et al. 2002). Rabies can follow urban
(within domestic animal populations) or sylvatic (within wildlife species) transmission
cycles. While the disease circulates within the reservoir host population, infected indi-
viduals come into contact with other species, allowing for spill-over events to occur.
Transmission to humans occurs in this way (Banyard and Fooks 2011). All but two of
the lyssavirus species have been isolated from members of the Order Chiroptera (bat
species) (Table 16.1). However, members of the Family Canidae, particularly dogs, are
the most important species as a source of human infection (Rupprecht et al. 2002).
Not all exposures to rabies virus through bite wounds received from rabid ani-
mals result in infection, clinical disease, and death (Cleaveland et al. 2002). It is
thought that one of the major factors affecting the risk of developing rabies follow-
ing a dog bite exposure is the location of the bite (Knobel et al. 2005). Estimates
suggest that if no PEP is provided, the risk of developing rabies following a rabid
dog bite is approximately 50%, depending on the severity and location of the wound
received, with the risk increasing for head wounds and decreasing for bites sustained
on extremities (Baltazard and Ghodssi 1954; Solomon et al. 2005). This should not
preclude potentially exposed individuals from seeking immediate medical attention
as the risk of developing rabies can be reduced through administration of rapid,
effective PEP (Hampson et al. 2008; Jackson 2007a).
16.2.2 Transport of Rabies Virus from the Bite Site to the CNS

For lyssaviruses to infect and successfully replicate, they must come into contact
with a susceptible host cell. Virus initially enters the peripheral nerves at the inocu-
lation site (Figure 16.2.2), then travels toward the CNS along peripheral motoneu-
rons, replicating within neuronal cell bodies, before entering the spinal cord and
brain (Figure 16.2.3) (Ugolini 2008).
The ability of the virus to enter a specific cell type is dictated by the viral G
protein, which binds to specific receptors present on permissive cells (Lafon 2005).
The viral G protein amino acid sequence is key to receptor utilization and patho-
genesis, to the extent that a single amino acid substitution at position 333 in the
G protein reduces neurovirulence dramatically, resulting in nonpathogenic variants
(Dietzschold et al. 1983; Jackson 1991; Seif et al. 1985). The first substitution in G,
combined with a second at position 330, prevents the virus from entering the nervous
system (Coulon et al. 1998). A further substitution occurring at position 194 has also
been identified (Faber et al. 2005), that is considered to be important for the develop-
ment of safe live-attenuated vaccines because it prevented reversion to the natural
virulent phenotype (Faber et al. 2009). Three specific host cell receptors have been
implicated in virus entry. The nicotinic acetylcholine receptor (nAChR), present at
neuromuscular junctions, and responsible for interneuronal communication (Lafon
2005); the neural cell adhesion molecule (NCAM) CD56, present at nerve termini
and postsynaptic membranes, deep within neuromuscular junctions (Covault and
Sanes 1986), and the low-affinity neurotrophin receptor (p75NTR), a nerve growth
factor receptor (Dechant and Barde 2002) although the latter may not be necessary
for lyssavirus–host cell interaction (Tuffereau et al. 2007). In vitro, lyssaviruses are
able to replicate successfully in numerous different cell types (Reagan and Wunner
1985), and several cells that are susceptible to infection do not express the three
currently implicated receptors, suggesting that further receptor molecules that are
yet to be identified may enable virus entry (Tuffereau et al. 2007). Variations in the
ability of viral strains to infect neuronal or nonneuronal cells have been observed
(Thoulouze et al. 1997). Highly neurovirulent strains appear to have a narrow cell
tropism, only infecting nervous tissue, whereas less-pathogenic attenuated strains
have been shown to infect a range of cells, including neuronal and nonneuronal types
(Thoulouze et al. 1997). It has also been suggested that differences in the structure,
quantity, and availability of rabies receptors between different hosts may play a role
in the variable susceptibility and pathogenesis observed between species. For exam-
ple, it has been observed that there is a large quantity of nAChRs present in the mus-
cles of red foxes (a highly susceptible species), compared with the relatively reduced
quantity of these receptors in opossums (a highly resistant species) (Baer et al. 1990).
Once the virus has entered the peripheral nervous system, infection progresses
by centripetal migration of virions from the periphery to the CNS. Migration occurs
by retrograde axoplasmic flow. The virus has been reported to migrate in this way
at a rate of 50–100 mm per day (Tsiang et al. 1991). It has been suggested that this
movement is enabled through virus phosphoprotein interacting with actin- and
microtubule-based motility networks within the nerves (i.e., dynein light chain 8)
(Jacob et al. 2000; Raux et al. 2000). However, when deletions occur in the virus
binding region for these networks, there appear to be only minor effects on virus
motility. This suggests that other, as yet unidentified, interactions could be signifi-
cant (Mebatsion 2001; Rasalingam et al. 2005). Once the virus reaches the dorsal
root ganglia the virions undergo replication, before entering the neurons of the spinal
cord, where further replication occurs. Upon entering the spinal cord, there is rapid
centripetal dissemination, followed by extensive replication of the virus within the
brain (Figure 16.2.4) (Jackson 2007b; Johnson et al. 2008b).
16.2.3 From the CNS to Onward Transmission

Following infection of the brain, there is centrifugal spread of the virions to other
tissues and organs of the body via the peripheral nerves (Figure 16.2.5). Onward
transmission is enabled at this stage as virus particles migrate along the nerves to
the salivary glands, then replicate extensively within these glands, resulting in viral
shedding in the saliva (Figure 16.2.5a) (Jackson 2007b). It is important to note that
the virus always enters and replicates within the CNS before moving to the salivary
glands. However, the pathology occurring within the CNS may not be very extensive
by the time this movement has occurred, and viral shedding begins. Therefore, an
infected host may be infectious before clinical signs are sufficient to indicate a spe-
cific diagnosis of rabies (Warrell and Warrell 2004). Neurons within the eyes, hair
follicles (Figure 16.2.5b), and thoracic and abdominal organs (Figure 16.2.5c) have
been shown to be infected at this centrifugal stage of disease progression. In addition,
infection of nonneural tissue has been observed, including salivary gland acini, epi-
thelial, and muscle cells (Jackson 2007b). The results of several studies suggest that
viral spread at this stage is highly extensive. In one study, of two EBLV-2-positive
bats, viral RNA was detected in brain, salivary glands, heart, tongue, stomach, lung,
thyroid gland, intestine, liver, kidney, and bladder (Johnson et al. 2006b). Despite
this broad tissue distribution, it is likely that the virus remains within highly inner-
vated areas of these organs although spread into non-neuronal tissue, particularly in
late stage disease, needs to be further investigated.
16.3 PATHOLOGY
Despite causing severe clinical deterioration, the pathological changes caused by
rabies virus infection are predominantly noncytopathic in nature (Jackson 2007b).
Even at a late stage in infection, where there is extensive infection of the host, the
macroscopic structural changes observed, if present at all, can be mild and non-
specific (Dupont and Earle 1965). Changes seen may include inflammation of the
spinal cord and brain, resulting in encephalomyelitis (Love and Wiley 2002) and
ganglioneuritis in nerve centres (Banyard and Fooks 2011). Mild cerebral edema and
congestion of some blood vessels has been observed, along with occasional focal
changes in the parenchyma, perhaps related to prolonged clinical course (Rossiter
and Jackson 2007; Rubin et al. 1970).
The histopathological changes are also mild and mostly related to inflamma-
tory processes. Some degree of inflammatory cell infiltration is normally present,
principally involving lymphocytes and monocytes, accompanied by some plasma
cells. However, in cases with intense inflammation, the infiltrate is predominantly

composed of neutrophils (Perl and Good 1991). In the majority of rabies cases, peri-
vascular cuffing can be seen whereby mononuclear inflammatory cells form accu-
mulations around vessels, mostly in the gray matter of the spinal cord and brain
(Hicks et al. 2009). Excessive proliferation of the neuroglial supportive tissue (glio-
sis), along with accumulations of activated microglia (Babes’ nodules) can also be
seen in many infections (Rossiter and Jackson 2007). These pathological alterations
are not unique for rabies and the occurrence, density, and distribution can be highly
variable between the differing forms, and individual cases of disease (Rossiter
and Jackson 2007). There have also been fatal cases of rabies where virtually no
inflammatory changes were observed (Hicks et al. 2009). One change that is often
observed, which distinguishes rabies infection from other viral encephalitides, is the
intracytoplasmic inclusions named “Negri bodies.” These are thought to be large
aggregations of viral N and P proteins and have been implicated as possible special-
ized sites for viral transcription and replication (Lahaye et al. 2009). These markers
of infection have been observed in many different regions of the CNS and their pres-
ence and distribution can be influenced by a range of host and virus factors including
virus strain, host species, and the clinical phase of disease.
16.3.1 Cell Damage and Death

Within the affected neuronal tissue, in addition to abnormal accumulations of con-
stituent cells and aberrant inclusions inside living cells, irreversible injury, degen-
eration, and cell death can occur. The damaged or dying cells can be highlighted
in many cases by neuronophagia, whereby a microscopic pattern of accumulated
phagocytic cells (microglia or macrophages) can be observed around the afflicted
neurons (Rossiter and Jackson 2007). As with the other pathological changes, there
is a high degree of variation in the extent of this cellular destruction. The neuroin-
vasiveness and replication of the virus is dependent upon the availability of intact
neuronal cells. Therefore, in most naturally occurring infections, with high neu-
roinvasiveness, there tends to be minimal cell death compared with the less neu-
roinvasive fixed laboratory strains, which produce a greater extent of damage and
degeneration (Dietzschold et al. 2008). It has been hypothesized that apoptotic cell
death of neuronal tissue is a potential pathogenic mechanism of rabies virus (Jackson
and Rossiter 1997). However, several studies have suggested that pathogenic strains
of rabies virus do not induce apoptosis (Yan et al. 2001), while attenuated strains
are proapoptotic (Morimoto et al. 1999), so the role of this process in rabies patho-
genesis is currently unclear. It has also been proposed that degeneration of neuronal
processes, with relative preservation of neuronal cell bodies, may be a major cause of
neuronal dysfunction. This is due to the disruption of cytoskeletal integrity that has
been observed in infections with pathogenic rabies strains (Li et al. 2005).
16.3.2 Functional Impairment

The dysfunction within the CNS of rabies virus infected individuals has been
attributed to a number of causes. The results of several studies have suggested that
functional impairment could partly be due to reduced neurotransmission capabili-

ties of infected neurons. Decreased gene expression leading to inhibition of host cell
protein synthesis has been reported (Fu et al. 1993). This has included a reduction in
the binding ability of acetylcholine receptors (Tsiang 1982), and interference in the
release and binding of neurotransmitters such as γ-amino-n-butyric acid (GABA)
(Ladogana et al. 1994) and serotonin (Bouzamondo et al. 1993; Ceccaldi et al. 1993).
Serotonin is of particular interest in rabies pathogenesis due to its possible involve-
ment in the development of certain clinical signs characteristic of rabies (Jackson
2007b). In the healthy animal, this neurotransmitter is widely distributed in the
brain, and is involved in the control of sleep, pain perception, and certain behaviors
(Julius 1991). Another possible effect of rabies virus infection, which would lead to
impairment and loss of neuronal function, is failure of the cells to produce effective
action potentials through interference with ion channel function (Iwata et al. 1999,
2000). Additional studies are required to fully elucidate the direct causal mecha-
nisms of these functional changes during infection (i.e., whether they are as a result
of the direct interaction of viral particles or due to the stress or immune responses
of the host).
16.4 IMMUNE RESPONSE

The first defense of virally infected cells is the innate immune response, which trig-
gers the secretion of interferons (IFNs) (Schnell et al. 2010). Rabies virus phospho-
protein is thought to interfere with, and subvert, the innate immune response through
the inhibition of a number of signaling molecules that up-regulate IFNs (Brzozka
et al. 2005) and would normally be activated following recognition of viral infection
(Schnell et al. 2010). However, in a murine model of rabies virus infection, the level
of IFN-α, β, and γ transcripts, as well as some chemokine transcripts, were shown
to be up-regulated in the brain (Johnson et al. 2008b). Despite the induction of these
inflammatory and antiviral transcripts, the mice developed severe disease and it
was hypothesized that the failure of innate immunity to prevent disease may be as
a result of the virus replicating at such rapidity within the CNS that it overwhelms
the host before the inflammatory and antiviral mechanisms can act (Johnson et al.
2008b).
The principal correlate of protection against rabies virus is neutralizing antibody
(Hooper et al. 1998). However, during natural infection, the development of a neu-
tralizing antibody response is rare until after the virus has reached the CNS, and
disease has developed (Johnson et al. 2010a; Noah et al. 1998). The reason for this
ineffective antibody response is not fully understood, but could be due to a number of
virus and host factors. During the early stages of infection, there tends to be limited
viral replication allowing the virus to bypass normal immune surveillance. The loca-
tions at which the majority of viral replication occurs—the dorsal root ganglia, the
spinal cord, and the brain—are thought to be immunoprivileged sites in that they are
not under the same level of immunosurveillance as other parts of the body (Johnson
et al. 2010a). It has been reported that pathogenic strains of rabies virus replicate at
a lower level while in the peripheral nervous system than attenuated variants (Faber
et al. 2009), and that this reduction in replicative rate results in production of less
viral antigen (Morimoto et al. 1999), and preservation of neuronal structures (Faber
et al. 2009). Conversely, the attenuated viral strains, with high replication rates and
large amounts of glycoprotein expressed are highly immunogenic, tending to induce
strong adaptive immune responses, which allow infection to be cleared (Faber et al.
2009).
Once the virus has reached the brain, it is much more difficult for circulating anti-
bodies to reach and neutralize infecting viral particles, as a result of the blood-brain
barrier (BBB). Under normal conditions, the BBB restricts access of pro-inflamma
tory cytokines, chemokines, and immune cells in order to protect neuronal cells
from damage caused by inflammation (Phares et al. 2006; Ruzek et al. 2011). When
infection occurs with a pathogenic rabies virus, the permeability of the BBB does
not increase (Faber et al. 2009; Ruzek et al. 2011; Schnell et al. 2010), whereas infec-
tion with an attenuated rabies virus leads to increased BBB permeability, allowing
the immune response to clear the virus (Faber et al. 2009). T lymphocytes, which
are capable of crossing the BBB and could therefore still clear the virally infected
cells from the brain during a pathogenic infection, are unable to achieve this due to
induced apoptosis of T cells as a result of the up-regulation of FasL in infected neu-
rons (Baloul et al. 2004).
16.4.1 Immunity to Infection
Vaccination is highly effective at preventing disease when given preexposure or post
exposure as it generates an adaptive immune response and triggers the production
of neutralizing antibody. Preexposure vaccination with inactivated tissue culture-
derived vaccine is recommended for veterinarians, those with occupational exposure
to the virus, and travelers to rabies endemic areas. Current WHO recommendations
are for administration of vaccine intramuscularly at days 0, 7, and 28. IgM is detect-
able within 4 days and IgG appears by day 7 (Johnson et al. 2010a). Postexposure
vaccination is more intensive, with a series of inoculations (up to 4) given in protocols
recommended by the WHO depending on the category of exposure. In the highest
exposure risk category (category III), the initial postexposure vaccination should be
accompanied by an injection of rabies immunoglobulin (RIG), which aims to neu-
tralize any virus present at the site of introduction, in the lag period before the active
immune system has responded to vaccination (WHO 2010).
16.4.2 Therapy
Currently, thorough wound cleansing, postexposure vaccination, and administration
of RIG are the only recommended interventions for rabies virus infection in humans.
However, these procedures are not effective once symptoms develop and there is no
antiviral therapy that directly inhibits rabies virus replication (Jackson et al. 2003).
Palliative measures remain the principal option for care. Therapeutic coma has been
proposed as a potential therapy after its success in saving the life of a teenager who
developed rabies after being bitten by a bat in the United States (Willoughby et al.
2005). Several attempts to repeat this have unfortunately been unsuccessful (Hunter
et al. 2010); however, there have been two further documented successes and a recent
report of a human case in the United States where the patient successfully recovered
(Pro-MED-mail 2011; Willoughby 2009).
16.5 STUDYING THE NEUROINVASIVENESS OF RABIES:

EXPERIMENTAL MODELS OF INFECTION
AND VACCINE DEVELOPMENT
A wide range of experimental models have been used to study rabies virus and to gen-
erate effective rabies virus vaccines. Louis Pasteur originally used rabbits to passage
rabies virus, using the desiccated spinal cords removed from infected animals as the
first vaccine. Mice were then developed as a simpler alternative although approaches
to vaccine production using any animal material are now discouraged due to adverse
immune reactions. The susceptibility of mice to infection has led to their use as
a method of diagnosis and the use of inbred and knockout mouse strains is com-
mon to investigate rabies virus pathogenesis (Wang et al. 2005). Table 16.2 provides
an overview of the animal models that have been used to study aspects of rabies
virus biology. These models are divided into those studies that have used animals
to assess susceptibility to infection or those that have investigated mechanisms of
rabies pathogenesis. Alternatively, animals have provided models to assess vaccine
TABLE 16.2
Selected Examples of Animal Models Used to Investigate
Rabies Pathogenesis
Animal Reference Comment
Mouse (Wang et al. 2005) Measure the up-regulation of innate immune transcripts
using microarray analysis prepared from brain tissue
Rat (Gillet et al. 1986) Stereotactic inoculation enabled demonstration of
axonal transport of RABV in rat brain
Dog (Cho and Lawson 1989) Demonstrate vaccine efficacy in an animal model
Fox (Vos et al. 2001) Efficacy studies of new rabies vaccines using a variety
of inoculation routes
Ferret (Niezgoda et al. 1997) Demonstrate susceptibility of mustelids to rabies virus
Bat (Johnson et al. 2008c) Demonstrate susceptibility and experimentally
(Daubenton’s bats) investigate routes of transmission in Daubenton’s bats
(Myotis daubentonii)
(Freuling et al. 2009) Demonstrate susceptibility and experimentally
(Serotine bats) investigate routes of transmission in serotine bats
(Eptesicus serotinus)
(Jackson et al. 2008) Demonstrate susceptibility and seroconversion in the
(Big brown bat) North American big brown bat (Eptesicus fuscus)
Syrian hamster (Hanlon et al. 2001) Used for protection studies for new rabies biologicals.
Guinea pig (Hronovsky and Benda Demonstration of aerosol transmission of rabies virus
1969)
Sheep (Hanlon et al. 2001) Susceptibility of sheep to infection
efficacy, which requires challenge with live virus. Rodents, including mice, rats, and
guinea pigs have been used due to the ease of availability, and the relatively lower
costs required for maintaining sufficient numbers to conduct statistically significant
experiments. Larger mammals have been used where a specific need has been iden-
tified, such as determining the efficacy of an oral vaccine targeted at a particular
species. One example of this is the determination of bait-acceptance and immuno-
genicity of oral rabies vaccine in the raccoon dog (Nyctereutes procyonoides) (Cliquet
et al. 2006). On occasion, primates have been used as a surrogate for the effectiveness
of experimental treatments in humans, for example, the use of monkeys to assess the
ability of an attenuated strain of rabies to modify the course of lethal infection (Warrell
et al. 1987). Another distinctive group of experimental animals are bats. These natural
host species can help to answer key questions on the susceptibility and persistence of the
lyssaviruses within bat populations (Fooks et al. 2009; Franka et al. 2006; Freuling et al.
2009; Johnson et al. 2008c).
In contrast, the neurotropic nature of rabies virus has led to the application of
the virus to study neuronal pathways as a transneuronal tracer (Ugolini 1995). This
use of the virus as a marker for neuronal networks has provided important informa-
tion about viral pathogenesis. Specifically, the receptor locations, mechanisms, and
neural pathways the virus uses to migrate from the periphery, to the CNS, have been
studied in rodent and nonhuman primate models (Ugolini 2008).
16.6 FUTURE OUTLOOK

Rabies should be considered beside the major communicable disease threats to health.
The high number of preventable injuries and deaths does not discriminate on age or
gender, however, rabies should be considered as an important pediatric disease. It
is evident that the global elimination of rabies requires an interdisciplinary control
strategy. A realistic goal focused on the control of rabies in domestic dogs would
result in a concomitant reduction of human mortality and would have a demonstrable
impact on childhood mortality meeting one of the Millennium Development Goals
to reduce child mortality.
The viruses that cause rabies belong to a large and complex group, and the contin-
ued study of these lyssaviruses, along with the effects they have on the broad range
of host species they are able to infect, will enable us to further our understanding of
the ways in which we can stop avoidable deaths from occurring. Current knowledge
of these viruses has improved our understanding of rabies virus dynamics in the host.
The use of lyssaviruses as transneuronal tracers in future studies will allow fur-
ther aspects of rabies neurovirulence and pathogenesis to be investigated and under-
stood (Ugolini 2008). Further study and modeling of the virus-host interactions of
both rabies and other neurotropic viral infections will help to clarify the subversion
and avoidance mechanisms the virus uses to circumnavigate the hosts defenses. The
continued use of technologies such as live-cell imaging in the future will enable
the dynamics and spatiotemporal relationships in these interactions to be further
analyzed (Chevalier et al. 2010). The development of safe, efficacious and affordable
tools for preexposure and postexposure prophylaxis with simpler, single immuniza-
tion regimens could lead to the elimination of rabies from areas of economic and
political instability (Faber et al. 2009). Live-attenuated vaccines are also potential
future candidates for treatment in the early stages of clinical human rabies as they
are capable of inducing effective immune responses to clear virulent rabies virus
from the CNS (Faber et al. 2009).
16.7 KEY POINTS

Lyssaviruses are neurovirulent, neurotropic viruses. They must enter a host and gain
entry into a susceptible host cell in order for viral replication to take place and for a
viable infection to be established. The factors that affect the ability of a virus popu-
lation to infect the nervous system are numerous but include the neuroinvasiveness
of the infecting viral strain and the susceptibility of the host species (i.e., the age,
immune status and response produced upon infection). For a rabies virus population
to be successful, it must be able to migrate from the site of infection, to the CNS,
where efficient replication can occur, before moving to other organs of the body, par-
ticularly the salivary glands, to enable onward transmission to occur. Lyssaviruses
are essentially noncytopathic, causing relatively minimal structural pathology, and
appear to have evolved to preserve the integrity of the nervous system and perhaps
maintain efficient viral spread. However, this does not prevent dysfunction of the
nervous system occurring, which can be advantageous to the viral population where
behavioral changes occur that improve transmission potential. The lyssaviruses have
also evolved mechanisms to avoid, and subvert, the host immune response which
could otherwise interfere with and potentially clear the infection.
ACKNOWLEDGEMENTS
This work was partially supported by Defra ROAME SV3500 and by funding from
the European Commission Seventh Framework Programme under ANTIGONE
(project number 278976).
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17 Rubella Virus Infections
Jennifer M. Best, Susan Reef, and
Liliane Grangeot- Keros
CONTENTS
17.1 Introduction................................................................................................... 396
17.2 Biological Properties of the Virus................................................................. 396
17.2.1 Classification and Virus Structure..................................................... 396
17.2.2 Replication Cycle............................................................................... 399
17.3 Epidemiology.................................................................................................400
17.4 Postnatally Acquired Infection......................................................................400
17.4.1 Clinical Presentation.........................................................................400
17.4.2 Complications....................................................................................403
17.4.2.1 Joint Symptoms...................................................................403
17.4.2.2 Postinfectious Encephalitis.................................................403
17.4.2.3 Other Complications...........................................................403
17.4.2.4 Risks of Rubella Infection in Pregnancy............................404
17.4.3 Immune Responses............................................................................404
17.4.4 Laboratory Diagnosis........................................................................405
17.4.5 Pathogenesis.......................................................................................405
17.5 Congenital Rubella........................................................................................406
17.5.1 Clinical Presentation.........................................................................406
17.5.2 Transient Abnormalities....................................................................408
17.5.3 Permanent Defects.............................................................................409
17.5.3.1 Cardiac Defects...................................................................409
17.5.3.2 Eye Defects.........................................................................409
17.5.3.3 Hearing Defects.................................................................. 411
17.5.3.4 CNS Abnormalities............................................................. 411
17.5.4 Late-Onset Disease............................................................................ 411
17.5.5 Delayed Manifestations..................................................................... 411
17.5.6 Clinical Diagnosis, Treatment, and Prognosis.................................. 412
17.5.6.1 Clinical Diagnosis of Congenital Defects........................... 412
17.5.7.1 Histological Studies............................................................ 416
17.5.7.2 Other Studies of the Eye..................................................... 416
17.5.7.3 Studies of the Brains from CRS Fetuses/Infants................ 416
17.5.7.4 Delayed CNS Disorders...................................................... 417
17.5.7.5 Possible Mechanisms of Fetal Damage.............................. 417
395
17.6 Prevention...................................................................................................... 419

17.6.1 Rubella Vaccines............................................................................... 419
17.6.1.1 Immune Responses............................................................. 420
17.6.1.2 Contraindications................................................................ 420
17.6.2 Vaccination Programs........................................................................ 421
References............................................................................................................... 422
17.1 INTRODUCTION
Rubella, also known as German measles, is generally a mild disease and received lit-
tle attention until 1941 when Norman McAlister Gregg, an Australian ophthalmolo-
gist, demonstrated an association with congenital defects. He showed that congenital
cataracts, cardiac defects, and deafness might result from maternal infection in early
pregnancy. This constellation of defects later became known as congenital rubella
syndrome (CRS). The frequency of defects was underestimated until the 1960s when
there were extensive epidemics in Europe and the United States (Cooper 1975). The
epidemic in the United States resulted in 12.5 million cases of rubella, approxi-
mately 2000 of postinfectious encephalitis, and >20,000 cases of CRS. Both post
natal and congenital infection may produce neurological symptoms, but these are
rare in postnatal infection; however, the rate of reported postnatal rubella encepha-
litis has varied by geographical location with an increased rate seen in the Asia
Pacific region. As no antiviral drugs are available to treat rubella or CRS, prevention
of disease is a priority. Rubella vaccines were licensed in 1969 and 1970, allowing
vaccination programs to be initiated in the United States, Europe, and Australia,
with the aim of preventing infection in pregnancy and thereby congenital rubella.
By 2010, 67% of the Member States of the World Health Organization included a
rubella-containing vaccine in their national immunization programs (WHO 2011).
17.2 BIOLOGICAL PROPERTIES OF THE VIRUS

17.2.1 Classification and Virus Structure
Electron microscopy shows that rubella virus (RV) is a pleomorphic virus, about
70 nm in diameter (Figure 17.1). RV is classified as a non-arthropod-borne Togavirus
and is the only member of the Rubivirus genus. It is a single-stranded (SS) RNA
virus with a lipoprotein envelope. RV genome structure and method of replication
are similar to viruses of the Alphavirus genus of the Togaviridae, but RV antigens
do not cross-react with other togaviruses. The SS RNA is contained in a protein
capsid (C, 32 kDa), surrounded by the lipoprotein envelope containing two envelope
proteins, E1 (58 kDa) and E2 (42–47 kDa). These envelope proteins induce the major
immune responses. RV is a fragile virus and is easily destroyed by heat and extremes
of pH.
There are no major antigenic differences between RV isolates, but at least 13
genotypes have been described. These are divided into two phylogenetic groups,
Rubella Virus Infections 397
(a)
(b)
FIGURE 17.1 (a) Rubella virus polymorphism in the Golgi complex showing dense spheri-
cal particles (arrowheads) budding from Golgi membranes in infected Vero cells at 16 h post
infection. (b) Viral particles (white arrows) with an annular-like morphology (dense periph-
ery and less dense center) are seen budding from membranes. (Reprinted from Virology,
312, Risco, C., Carrascosa, J. L., and Frey, T. K., Structural maturation of rubella virus in
the Golgi complex, 261–269, Copyright 2003, with permission from C. Risco and Elsevier.)
clades 1 and 2, which differ by 8%–10% at the nucleotide level (Abernathy et al.
2011; WHO 2007). Identification of genotypes is very important for tracking the
spread of infection and characterizing the virus during elimination (see Prevention).
The genome of RV is a positive sense SS RNA, usually 9762 nucleotides in length,
5ʹ capped and 3ʹ polyadenylated. This RNA is infectious and serves as a messenger
RNA during infection. The RV genome has a high G + C content (approximately
70%), which initially made sequencing difficult. The genome consists of a 40-nt 5ʹ
untranslated region, a 5ʹ proximal 6351-nt open reading frame (ORF) coding for the
nonstructural proteins (p150 and p90), a 3ʹ proximal 3192-nt ORF coding for the
structural proteins, and a 59-nt 3ʹ untranslated region (Zhou et al. 2007). The order
of genes is 5ʹ-p150-p90-C-E2-E1-3ʹ (Figure 17.2).
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 kb
40S Genomic RNA
Nonstructural ORF Structural ORF
7-methyl M X P H R C E2 E1 poly[A]n
Translation
p200
NH2 COOH
Replication
Proteolytic cleavage
p150 p90
24S Subgenomic RNA
C E2 E1
Translation
p110 precursor
NH2 COOH
C E2 E1
Capsid E2 E1
292/300a 282aa 481aa
Phosphorylation Glycosylation
Capsid 34.5 kDa E2a 47 kDa E1 58 kDa

E2b 42kDa
Key
Hydrophilic region of C with RNA binding activity
Putative signal peptide sequences of E2 and E1
Putative transmembrane sequences of E2 and E1
Potential glycosylation sites
Potential phosphorylation sites

17.2.2 Replication Cycle
RV can infect a variety of cell lines. Virus replication takes place in the cytoplasm
and is similar to the replication cycle of the alphaviruses (Best et al. 2009). Virus
production reaches a peak at 24–48 h postinfection, and no effect on total cell RNA
or protein synthesis has been noted.
The myelin oligodendrocyte glycoprotein (MOG) has recently been identified as
a cell receptor for RV, although the use of other receptors has not been excluded
(Cong et al. 2011). MOG is found mainly in the central nervous system (CNS), with
lower levels in lymphoid and other tissues. The E1 protein binds to this receptor, RV
enters the cytoplasm via the endocytic pathway, uncoating of the viral RNA occurs
in the endosome, and viral RNA is released into the cytoplasm, where reproduction
occurs.
Both 40S and 24S RNA are found in RV-infected cells. The 5ʹ 6351 nt nonstruc-
tural ORF of the 40S genomic RNA is translated to produce a 2116-amino acid
polyprotein (p200; Figure 17.2). This polyprotein is cleaved by a host cell signal
peptidase to give 2 nonstructural proteins, p150 and p90. The 24S subgenomic RNA
produced from the 3ʹ structural ORF is translated to produce a 110-kDa polyprotein,
which is cleaved by a host cell signalase to produce the three structural proteins (C,
E2, and E1; Figure 17.2). These are transported to the Golgi complex where glyco-
sylation and assembly of virus particles occurs (Risco et al. 2003). Virus particles
are released by budding from the plasma membrane and intracellular membranes
(Figure 17.1). The cytopathic effect induced by RV in some cell cultures is due to
caspase-dependent apoptosis (Cooray et al. 2003).
The biological properties of RV have been reviewed in more detail by Chen and
Icenogle (2007).
FIGURE 17.2 Schematic diagram of the replication, translation, and processing of RV

nonstructural proteins and structural proteins. (Best, J. M., Cooray, S., and Banatvala, J. E.:
Rubella, in Topley & Wilson’s Microbiology and Microbial Infections. 2010. Copyright
Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.) The RV genome
consists of two long nonoverlapping ORFs: the 5′ORF encodes the nonstructural proteins,
and the 3′ORF encodes the structural proteins. The translation RV RNA produces the p200
precursor, which is cleaved to produce the p150 and p90 structural proteins. This initiates the
synthesis of the full length negative strand RNA. The negative strand acts as a template for
the synthesis of the full length positive strand RNA for new viral progeny and the 24S sub-
genomic RNA. The 24S subgenomic RNA is translated into the p110 polyprotein precursor,
which is proteolytically cleaved and posttranslationally modified to produce the structural
proteins C, E2, and E1. Within the 5′ nonstructural ORF are putative amino acid sequence
motifs for methyltransferase (M), RNA-dependent RNA-polymerase (R), helicase (H), and
papain-like cysteine protease (P) activity. The X motif indicates a region of unknown func-
tion, which has homology to alphaviruses, hepatitis E virus, and coronaviruses.
17.3 EPIDEMIOLOGY
RV is transmitted by aerosol via the respiratory route. Close contact is usually
required for transmission to occur (e.g., within the family or at work), and it is there-
fore less infectious than measles, varicella, and influenza.
Before the introduction of rubella vaccination programs, rubella was a worldwide
disease with epidemics occurring approximately every 5–9 years in the spring. In
temperate climates RV was most frequently acquired in childhood. Serological stud-
ies showed that 50% of 9- to 11-year-old children had evidence of past infection,
but 15%–20% of women of child-bearing age remained susceptible to rubella and
therefore at risk of acquiring rubella when pregnant. In developing countries, there
is considerable variation in the age of acquisition of rubella. Cutts et al. (1997) found
that the proportion of susceptible women was 15%–20%, the same as in industrial-
ized countries, with a higher rate of susceptibility in rural areas than in cities. It was
estimated that 112,000 infants worldwide were born with CRS in 2008. (http://www
.gavialliance.org/support/nvs/rubella/) (Cutts et al. 1997). The incidence of CRS was
estimated as 0.8–4.0/100 live births during epidemics and 0.1–0.2/1000 live births
during endemic periods. This burden of CRS justifies efforts to eradicate rubella (see
Prevention).
Since the introduction of rubella vaccination programs (see Prevention) rubella
has been eliminated from such countries as Sweden, Finland, and the WHO Region
of the Americas.
Rubella incidence has decreased significantly in many other countries that intro-
duced rubella vaccine through wide age-range campaigns or routine programs as
used in the European region (Muscat et al. 2012; Zimmerman et al. 2011).
17.4 POSTNATALLY ACQUIRED INFECTION

Rubella is usually a mild disease in children, but may be more severe in adults, who
may experience a prodrome with malaise and low-grade fever. Lymphadenopathy
usually develops before the rash and may persist for 10–14 days after the rash has
disappeared. The cervical, postauricular, and suboccipital lymph nodes are most fre-
quently affected. The characteristic maculopapular rash appears after an incubation
period of approximately 14 days (range 12–23 days) (Figures 17.3 and 17.4). A dis-
crete rash appears first on the face and spreads quickly to the trunk and limbs. The
rash may persist for 1–3 days or may be fleeting; lesions may coalesce. Cough, sore
throat, conjunctivitis, and headache may also occur in adults. Occasionally, rubella
presents with a more severe fever and constitutional symptoms similar to measles.
Virus is excreted from about 7 days before the onset of rash and for 7–10 days there-
after, but patients are only infectious for about 10 days. Joint symptoms may occur,
as described below (see Complications and Pathogenesis).
Laboratory diagnosis is required to confirm a diagnosis of rubella (see below),
as clinical diagnosis is unreliable. Subclinical infection is common, and other virus
infections present with similar symptoms. Measles, enteroviruses, and human herpes
Days after exposure

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Clinical features
Rash 37º
35º
Fever 37º
Arthralgia
Lymphadenopathy
Pharynx
Virus isolation
Blood
Stool
Urine
Neut. & HAI antibody

Serology
SRH antibody
Specific IgG (EIA)
Specific IgM (EIA)
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
FIGURE 17.3 Relation between clinical and virological features of postnatally acquired
rubella. (Best, J. M., Cooray, S., and Banatvala, J. E.: Rubella, in Topley & Wilson’s
Microbiology and Microbial Infections. 2010. Copyright Wiley-VCH Verlag GmbH & Co.
KGaA. Reproduced with permission.)
FIGURE 17.4 (See color insert.) Macular–papular rash in postnatally acquired rubella
infection. (Reproduced from Dr. D. Wallach and Editions De Boeck/Estem. With permission.)
402
TABLE 17.1
Differential Diagnosis of Postnatal Rubella in Different Geographical Regions
Geographical Distribution
North Central South
Virus Infection Africa Asia Australia Europe America America America Pacific Key Features
Rubella + + + + + + + +
Parvovirus B19 + + + + + + + + Erythema infectiosum
Human herpes + + + + + + + + Exanthem subitum. Predominantly <2 years.
viruses 6 and 7
Measles + + + + + + + + Prodrome with cough, conjunctivitis, coryza
Enteroviruses + + + + + + + + Echovirus 9, coxsackie A9 most frequent.
Dengue + + + − − + + + Joint and back pain, hemorrhagic
complications in children.
West Nile fever + + − + + − − − Joint pains
Chickungunya + + − − − − − − Joint pains
Ross River − − + − − − − + Joint pains
Sindbis + + + + − − − − Joint pains
Source: Reprinted from The Lancet, 363, J. E. Banatvala and D. W. G. Brown, Rubella, pp. 1127–1137, Copyright (2004), with permission from J. E. Banatvala and
Elsevier.
virus 6 and 7 may present with a similar rash, and parvovirus B19 and some arbo
viruses (e.g., Ross River, Dengue, Chikungunya) may present with both rash and
joint symptoms (Table 17.1).
17.4.2 Complications
17.4.2.1 Joint Symptoms
Joint symptoms are the most common complication of rubella. They may be
observed in ≤70% postpubertal females but are less common in prepubertal females
and males. These usually last for 3–4 days but occasionally persist for up to 1 month.
Symptoms vary from a transient stiffness of joints to arthritis with swelling, pain,
and limitation of movement. The joints most commonly affected are the fingers,
wrists, ankles, and knees. Joint symptoms are also observed in postpubertal females
after rubella vaccination (see below).
17.4.2.2 Postinfectious Encephalitis

Usually, it develops abruptly within a week of onset of rash and with recovery within
7–30 days. It may also occur in cases without rash. The prognosis is usually good
for survivors, but death rates reported vary from 0% to 30%. It has been reported
to occur in approximately 1 in 6000 cases. However, the incidence of encephalitis
was 1 in 1600 cases of rubella in a Japanese outbreak in 1987 (Moriuchi et al. 1990),
and recently in the South Pacific islands of Tonga and Samoa, the incidence was
estimated to be between 1 in 500 and 1 in 1000 cases (A. Ruben, personal commu-
nication). These cases occurred in children. Two of the eight cases of encephalitis
in Samoa died. In 2011, a rubella outbreak occurred in Tunisia, resulting in many
cases of encephalitis among children and adolescents, which are currently being
evaluated.
The most frequent symptoms of postinfectious encephalitis include headache,
vomiting, generalized convulsions, stiff neck, retroauricular lymphadenopathy, and
lethargy, often without fever (Dwyer et al. 1992). Nystagmus, diminished superficial
skin reflexes, and variable deep tendon reflexes may be detected on examination.
Those patients who die usually develop coma with normal fundi but fixed and dilated
pupils. Death occurs within 6–8 days of onset of neural symptoms. CSF is clear,
with an average cell count of 50/mm3, comprising mostly lymphocytes. CSF protein
levels are not usually elevated (reviewed by Chantler et al. 2001). EEG shows diffuse
or localized abnormalities, which may persist for a year or more. EEG and MRI are
probably more useful than CT scanning (Dwyer et al. 1992). Relapsing encephalitis
and polyradiculoneuritis are rare (Chang et al. 1997).
17.4.2.3 Other Complications

Other complications include bleeding disorders due to thrombocytopenia, which
occurs in about 1:3000 cases. Rare complications are Guillain-Barré syndrome,
bone marrow aplasia, autoimmune hemolytic anemia, hemophagocytic syndrome,
optic neuritis, and Fuchs heterochromic uveitis.
There is no specific or proven treatment for rubella. For persons infected with
rubella, symptomatic treatment may be warranted for different manifestations such
as arthralgias, myalgias, and fever.
17.4.2.4 Risks of Rubella Infection in Pregnancy

When rubella infection occurs in the first 10 weeks of pregnancy, RV will cross the
placenta and cause a generalized and persistent infection of the fetus in about 90%
of cases. After 10 weeks of gestation, the risks decline, with those infected at 13–16
weeks having an approximate 50% risk and only rare cases of deafness reported
after infection at 17–18 weeks (reviewed by Best 2007). Infection prior to concep-
tion is not a risk to the fetus (Enders et al. 1988). Spontaneous abortion may occur
in up to 20% of cases when infection occurs in the first 8 weeks of pregnancy. There
is no treatment available to prevent transmission to the fetus, and termination of
pregnancy (TOP) is usually offered when infection occurs in the first trimester. In
those for whom TOP is not acceptable, normal human immunoglobulin or rubella
hyperimmune globulin (if available) administered soon after exposure might reduce
the amount of viremia and damage, although normal human immunoglobulin does
not seem to reduce the incidence of fetal infection (Peckham 1974).
Following asymptomatic rubella reinfection in early pregnancy, RV may be trans-
mitted to the fetus in about 10% of cases, and the risk of fetal damage is probably less
than 5% (Best et al. 1989; Health Protection Agency 2011). The risk may be higher
following symptomatic reinfection, but such cases are rare.
17.4.3 Immune Responses
Viremia is terminated by the development of antibodies. Serum antibodies may
be detected by hemagglutination inhibition (HAI) 1–2 days after onset of rash, but
antibodies are not detected by enzyme immunoassay (EIA) until 6–7 days (Figure
17.3). IgG antibodies usually persist for life but may sometimes decline to undetect-
able levels in older persons. Rubella IgM antibodies develop during the 5 days after
onset of rash. These antibodies decline fairly quickly and are usually undetectable
by 8 weeks postrash onset, although their detection depends on the sensitivity of the
assay used. Antibodies may also be detected in oral fluid, urine, and nasopharyngeal
secretions (WHO 2008). IgG, IgA, and IgM antibodies to the E1, E2, and C struc-
tural proteins have been detected.
The detection of rubella antibodies at levels >10 IU/mL is usually considered to
provide evidence of immunity (Skendzel 1996; WHO 2008).
The cell-mediated immune response is also required to control infection and
appears to persist for life. A mixed Th1/Th2 response is seen with serum interferon γ
during acute rubella. An increase in serum interleukin 10 (IL-10) levels has been
detected during the first 4 days of illness. Lymphoproliferative responses develop a
few days after onset of rash and persist at low levels for many years; the strongest
responses are against the E1 protein. MHC class II restricted CD4+ T helper and
CD8+ cytotoxic T lymphocytes can be detected shortly after the antibody response
and several antigenic domains recognized by these cells have been identified within
the E1, E2, and C proteins (reviewed by WHO 2008).
17.4.4 Laboratory Diagnosis

A laboratory diagnosis is usually made by the detection of rubella IgM in serum
(Thomas et al. 1992; Best et al. 2009). Oral fluid may be used instead of serum,
but results should be confirmed with assays done on serum when testing a pregnant
woman (Banatvala and Brown 2004; Manikkavasagan et al. 2010). EIA is the pre-
ferred assay for detection of both rubella IgG and IgM because it is sensitive and can
be readily automated. Other assays, such as radial hemolysis and latex agglutination
have been widely used in the past for the detection of rubella IgG and for screening
purposes.
Laboratory diagnosis is particularly important in pregnant women with a rash or
who have had contact with a rash illness, even if antibodies have been detected in
the past or there is a history of immunization. Blood should be obtained as soon as
possible after onset of rash, and serum tested for both rubella IgG and IgM. Rising
IgG antibodies (using the same assay in one laboratory) and a positive IgM result
confirm rubella infection. A positive IgM result alone should be confirmed by testing
a second serum. The detection of rubella IgM in a woman without a rash or history
of contact with a rash illness should be interpreted with caution (Thomas et al. 1992;
Health Protection Agency 2011). Sometimes, the measurement of rubella-specific
IgG avidity may help exclude or confirm recent primary infections (Best 2007;
Thomas and Morgan-Capner 1991). Pregnant women who have had contact with a
rubella-like illness and who are susceptible to rubella or are of unknown immune
status, should be tested for rubella IgG and IgM as soon as possible. If shown to
be susceptible, such women should be retested for up to 4 weeks after contact and
offered a rubella-containing vaccine after delivery.
Antenatal screening programs for rubella antibody are available in some countries to
identify women who are susceptible to rubella and should be offered immunization (see
Prevention).
Laboratory diagnosis of rubella encephalitis is based either on the detection of
rubella-specific IgM or the detection of viral RNA by reverse transcription poly-
merase chain reaction (RT-PCR) in cerebrospinal fluid (CSF). Viral isolation from
CSF may be used for genotyping studies (Figueiredo et al. 2011). Intrathecal synthe-
sis of rubella-specific IgG can also be investigated by calculation of the Goldmann-
Witmer coefficient as follows: (CSF rubella-specific IgG/CSF total IgG)/(serum
rubella-specific IgG/serum total IgG), but this procedure is not often used (Jacobi
et al. 2007).
17.4.5 Pathogenesis
Humans are the only known host for rubella, although the virus can infect cell cul-
tures derived from other animals. RV is spread via the respiratory route. Patients
excrete the virus for ≤7 days before onset and 7–10 days after onset of rash (Figure
17.3). High titers may be excreted for about 10 days, when patients are infectious.
Virus may also be detected for a shorter time in stools and urine. Virus particles
transmitted by aerosol infect cells in the upper respiratory tract and spread to lym-
phoid tissue of the nasopharynx and upper respiratory tract. Replication of the virus
in regional lymph nodes leads to lymphadenopathy. A viremia occurs for about

7 days before onset of rash, leading to systemic infection. Many tissues become
infected, including the placenta in pregnant women. Mononuclear cells are prob-
ably involved in the spread of virus, but extracellular virus can also be detected in
serum. Rubella induces a mild and transient immunosuppression, less pronounced
than that observed with measles. A fall in total leukocytes, T cells, and neutrophils
and a depression of lymphocyte responsiveness to mitogens and antigens are seen
(reviewed in WHO 2008).
The mechanisms by which the rash and joint symptoms are induced by rubella
are not fully understood. RV has been isolated from skin biopsies taken from skin
with rash and skin from patients with subclinical rubella (Heggie 1978). Immune
mechanisms may be involved. Administration of pooled human immunoglobulin
has been shown to prevent rash, but not viremia. RV and rubella IgG antibodies
can be detected in the synovial fluid and circulating immune complexes have been
detected in serum, suggesting that immune complexes could be formed and these
might cause joint symptoms. Hormonal factors may also play a role (reviewed by
Best et al. 2005). Although RV has been isolated from synovial fluid from occasional
patients with chronic inflammatory joint disease, there is no convincing evidence
that rubella or rubella vaccination is associated with this condition. The pathogenesis
of the encephalitis is also not understood. RV has been detected in brain tissue and
RV has been detected in CSF by isolation and RT-PCR. Rubella-specific IgG and
IgM antibodies can be detected in CSF. However, limited pathological data have not
shown demyelination or inflammatory damage, which suggests that the encephalitis
is not immune mediated.
17.5 CONGENITAL RUBELLA

The clinical features of congenital rubella infection (CRI) are variable, ranging from
a wide spectrum to no anomalies. Severe anomalies are observed when fetal infec-
tion has occurred during the first trimester. Following infection between 13 and 16
weeks of gestation, hearing defects and retinopathy are the only defects that may be
seen in up to 35% of infants, while after infection at 17–20 weeks, the risk of deaf-
ness is about 6% (Best et al. 2009). Symptoms may be present at birth or during the
early months of life, but a few, such as hearing impairment and eye defects, are fre-
quently delayed (see Delayed Manifestations). In addition, late-onset disorders such
as type 1 diabetes mellitus may develop in the second decade of life.
There is no treatment available to prevent congenital rubella infection. When
rubella is acquired in early pregnancy, almost every organ may be affected, but eye
defects, cardiac defects, and hearing defects predominate. Congenital anomalies
apparent at birth have been categorized as transient or permanent (Table 17.2). Some
clinical features are used in the case definition used by WHO and for surveillance of
CRI (Tables 17.2 and 17.3).
TABLE 17.2
Clinical Features of CRS
Early Transient Permanent Features, Use in
Features Some Recognized Late Surveillancea
Ocular defects
Cataracts (unilateral/bilateral) + A
Glaucoma + A
Pigmentary retinopathy + A
Microphthalmia +
Iris hypoplasia +
Cloudy cornea +
Auditory defects
Sensorineural deafness + A
(unilateral/ bilateral)
Cardiovascular defects
Persistent ductus arteriosus + A
Pulmonary artery stenosis + A
Ventricular septal defect + A
Myocarditis +
Central nervous system
Microcephaly + B
Pyschomotor retardation +
Meningoencephalitis + B
Behavioral disorders
Speech disorders
Intrauterine growth retardation +
Thrombocytopenia, with purpura + B
Hepatitis/ hepatosplenomegaly + B
Bone “lesions” + B
Pneumonitis +
Lymphadenopathy +
Diabetes mellitus +
Thyroid disorders +
Progressive rubella panencephalitis +
Source: Reprinted from The Lancet, 363, J. E. Banatvala and D. W. G. Brown, Rubella, pp. 1127–1137,
Copyright (2004), with permission from J. E. Banatvala and Elsevier.
a For surveillance, a clinically confirmed case is defined as one in which two complications from group
A or group B or one from group A or one from group B are present (WHO 1999).
TABLE 17.3
WHO Case Definition for CRS
Suspected Case
Any infant less than one year of age in whom a health worker suspects CRS. A health worker should
suspect CRS when an infant aged 0–11 months presents with heart disease and/or suspicion of
deafness and/or one or more of the following eye signs: white pupil (cataract), diminished vision,
pendular movement of the eyes (nystagmus), squint, smaller eyeball (microphthalmus) or larger
eye-ball (congenital glaucoma), or when an infant’s mother has a history of suspected or confirmed
rubella during pregnancy, even when the infant shows no signs of CRS.
Clinically Confirmed CRS Case

An infant in whom a qualified physician detects at least two of the complications in section A below or
one from section A and one from section B:
A. Cataracts, congenital glaucoma, congenital heart disease, hearing impairment, pigmentary
retinopathy.
B. Purpura, splenomegaly, microcephaly, mental retardation, meningoencephalitis, radiolucent bone
disease, jaundice with onset within 24 hours after birth.
Laboratory-Confirmed CRS Case

An infant with clinically confirmed CRS who has a positive blood test for rubella-specific IgM. Where
special laboratory resources are available the detection of RV in specimens from the pharynx or urine
of an infant with suspected CRS provides laboratory confirmation of CRS.
Congenital Rubella Infection (CRI)

If a mother has suspected or confirmed rubella in pregnancy her infant should have a rubella-specific
IgM test. An infant who does not have clinical signs of CRS but who has a positive rubella-specific
IgM test is classified as having congenital rubella infection (CRI).
Source: Reproduced from the World Health Organization (2003). WHO standards for surveillance of
selected vaccine-preventable diseases: Geneva, WHO/V&B/03.01. Available at: http://www
.who.int/immunization/documents/WHO_VB_03.01/en/index.html. With permission.
17.5.2 Transient Abnormalities
These are seen in the first few weeks of infancy and usually occur with permanent
defects, such as those of the eye and heart. This combination of features reflects
extensive infection and is associated with high perinatal mortality. Transient abnor-
malities include thrombocytopenic purpura (TCP) (Figure 17.5a), hepatosplenomeg-
aly (Figure 17.5b), hemolytic anemia, and cloudy cornea (Table 17.2). Babies are
often small-for-dates, with about 90% below the 50th growth percentile and 60%
below the 10th percentile. TCP is associated with a reduced number of megakaryo-
cytes in the bone marrow. A rare complication of TCP is intracranial hemorrhage.
Radiolucencies of the long bones (Figure 17.5c) are seen in about 20% of infants and
usually resolve within the first 1–2 months.
The CNS is involved in about 25% of infants who present with congenital abnor-
malities at birth. The most common manifestation is meningoencephalitis. Infants
(a) (b) (c)
(d) (e)
FIGURE 17.5 (See color insert.) Congenital anomalies in congenital rubella syndrome
(CRS). (a) Case of CRS with maculo-papular rash and purpura; (b) case of CRS with hepa-
tosplenomegaly; (c) case of CRS with radiolucencies of long bones; (d) case of CRS with
cardiac dilatation; (e) cataracts (opacity of the lens). (Kindly provided by Dr. C. A. Bouhanna
and Dr. J. C. Janaud, Hôpital intercommunal, Créteil, France, with permission from Dr. D.
Wallach and Editions De Boeck/Estem.)
may be irritable or lethargic with a full fontanelle and consistent CSF changes. Some
of those infants with severe meningoencephalitis will subsequently progress well
neurologically, whereas others may be severely retarded and have ataxia, spastic
diplegia, or communication problems.
17.5.3 Permanent Defects
17.5.3.1 Cardiac Defects
Cardiac defects are present in approximately 50% of infants whose mothers had rubella
in the first 2 months of gestation (Reef et al. 2000). Patent ductus arteriosus and branch
pulmonary artery stenosis are the most common cardiac defects and are responsible for
much of the perinatal mortality seen with CRS (Oster et al. 2010). Less frequent abnor-
malities are ventricular septal defect, tetralogy of Fallot, aortic stenosis, transposition of
the great vessels, and tricuspid atresia. With available treatment, including surgery, most
cardiac defects can be corrected. A neonatal myocarditis may also occur in infants with
these defects. Figure 17.5d shows an X-ray of cardiac dilation.
17.5.3.2 Eye Defects

The typical cataracts and pigmentary retinopathy seen in CRS were described by
Gregg in 1941. The cataracts may consist of a central dense pearly white opacity
(Figure 17.5e) or may be total with a more uniform density throughout the lens.
Bilateral cataracts are found in about 50% of affected infants; they are usually pres-
ent at birth but may not be visible until several weeks later. Cataracts, which are often
accompanied by microphthalmia (Figure 17.6a,b), are a useful marker for surveillance
of CRS (Bloom et al. 2005; WHO 1999; Vijayalakshmi et al. 2007). Retinopathy is
found in about 50% of affected infants. Hyperpigmented and hypopigmented areas
of the retina give it a “salt and pepper” appearance, which can be a useful diagnostic
indicator of CRS; however, because it does not cause any visual defects, it may not
be suspected. Retinopathy is due to a defect in pigmentation and usually involves the
macular areas. Glaucoma is less frequently observed than cataract. Other symptoms
(a)
(b)
FIGURE 17.6 (a) Coronal view of fetal face at 19 weeks gestation by ultrasound.
Microphthalmia and hyperechogenic lens (arrow). (b) Coronal view of fetal face at 19
weeks of gestation showing the ophthalmic asymmetry and microphthalmia. (Cordier, A.
G., Vauloup-Fellous, C., Grangeot-Keros, L., Pinet, C., Benachi, A., Ayoubi, J. M., Picone,
O.: Pitfalls in the diagnosis of congenital rubella syndrome in the first trimester of preg-
nancy. Prenat Diagn. 2012. 32(5), 496–7. Copyright Wiley-VCH Verlag GmbH & Co. KGaA.
Reproduced with permission.)
are pupil rigidity, cloudy cornea, corneal opacity, microcornea, iris hypoplasia, optic
atrophy, anophthalmos, chronic uveitis, corneal hydrops, choroidal neovasculariza-
tion, and keratoconus (Arnold et al. 1994; Vijayalakshmi et al. 2007). Some of these
ocular abnormalities may occur later in life (see below).
17.5.3.3 Hearing Defects

Hearing defects range from mild to severe. Sensorineural deafness occurs in ≥70%
of infants with CRI, either in combination with other defects or it may be the only
congenital defect, especially when maternal infection occurred after the first tri-
mester. Deafness may be bilateral or unilateral with no characteristic audiometric
pattern. The impact of congenital rubella deafness has been insufficiently appreci-
ated in the past and continues to be difficult to assess in developing countries. In the
early 1970s, before the widespread use of rubella immunization, a study in London
estimated that 15% of all cases of sensorineural deafness were the result of CRI
(Peckham et al. 1979); in many cases, rubella had not been diagnosed during preg-
nancy. Improvements in audiological methods and routine testing now help to detect
hearing loss in early infancy.
17.5.3.4 CNS Abnormalities

It is estimated that as many as 80% of CRS patients exhibit neurological disorders.
Lethargy, irritability, and motor disabilities are apparent in infancy (Frey 1997).
Microcephaly may be present at birth or develop subsequently (Chang et al. 1996).
As development progresses, infants affected by CRS may show mental retardation,
motor disabilities, abnormal posture and movements, and seizures. The mental retar-
dation may be associated with undetected hearing impairment.
17.5.4 Late-Onset Disease

This syndrome, consisting of chronic rubella-like rash, pneumonitis, failure to thrive,
and persistent diarrhea, may develop between 3 and 12 months of age in infants
with CRI. Mortality is high, but infants may recover if treated with corticosteroids.
Hypogammaglobulinemia has also been reported. Hanshaw et al. (1985) suggested
that this syndrome may be induced by circulating immune complexes.
17.5.5 Delayed Manifestations
Some defects may not be apparent for months or years and occur in >20% of children with
CRS (Sever et al. 1985; Cooper and Alford 2006). This may be due to failure to recognize
such defects as deafness, but sensorineural deafness and some CNS and ocular anomalies
may develop later and progress in severity. In the eye, cataracts may develop after birth
and resorption of the cataractous lens has also been reported. Glaucoma may develop
between 3 and 22 years of age, and corneal hydrops, keratic precipitates, keratocornus,
and spontaneous lens absorption have also been reported. Retinopathy has been associ-
ated with subretinal neovascularization leading to visual disturbance (Arnold et al. 1994).
Vascular changes due to renal artery and aortic stenosis may also lead to hypertension.
Some CNS manifestations such as mental retardation and other behavioral dis-
orders (autism, schizophrenia-like disease) may be delayed, and can be progressive.
As many as 7% of CRS children may develop an autistic disorder (Chess et al. 1978;
Hwang and Chen 2010). Some CRS patients develop schizophrenia-like disease
(Lane et al. 1996; Lim et al. 1995). Exceptionally, progressive rubella encephalitis
(PRP) may develop in children between the ages of 10 and 20 years. The main neu-
rological features of PRP are dementia, cerebellar ataxia, and seizures. The course
of this disease is slow and progressive, and death invariably occurs within a period
of 1–10 years. Fortunately, PRP is a rare manifestation of CRS disorders (fewer than
50 cases have been described worldwide) (reviewed by Frey 1997). It is somewhat
similar to subacute sclerosing panencephalitis due to chronic infection of the CNS
with measles virus.
The most frequent endocrine abnormality is insulin-dependent diabetes mellitus
(IDDM; juvenile-onset, type 1), which is seen in about 20% of adult CRS patients
(Ginsberg-Fellner et al. 1985). About 5% of CRS patients develop thyroid dysfunc-
tion, including hypothyroidism, hyperthyroidism, and thyroiditis, whereas 20%–
40% have thyroid autoantibodies. In addition, growth hormone deficiency, poor
growth, and early cessation of growth have been reported.
17.5.6 Clinical Diagnosis, Treatment, and Prognosis

Infants with CRS should be evaluated and treated by specialists for the different
clinical manifestations associated with CRS. Infants with cardiac anomalies, hear-
ing and visual impairments, as well as psychomotor difficulties, should be treated
as soon as possible after birth but, as some of these defects may be progressive,
repeated testing and continuing assessment of the therapeutic approach is required.
17.5.6.1 Clinical Diagnosis of Congenital Defects

17.5.6.1.1 Prenatal Diagnosis Using Imaging Techniques
Prenatal ultrasound descriptions of rubella congenital defects are relatively rare
(Degani 2006). Recently, the use of ultrasound was assessed on a few cases of CRI
(Migliucci et al. 2011): intrauterine growth retardation (IUGR), polyhydramnios,
cardiomegaly, defects of atrial septum, hepatosplenomegaly, ascites, echogenic bowel,
and placentomegaly were observed. However, as expected, ultrasound examination of
cases with isolated deafness was normal. Microcephaly can also be observed. Ocular
disorders are quite common following first-trimester infection, but only microphthal-
mia and cataract are detectable on antenatal ultrasound scans (Figure 17.6a,b). Two
clinical cases recently reported in France highlighted the limits and value of prenatal
ultrasound examination: in the first case, a pregnant woman had had rubella testing
at 7 weeks of gestation and was considered immune. When routine ultrasound scan
was performed at 16 weeks, cerebral ventricle enlargement was observed without any
other defect. At 17 weeks, ventricular enlargement was confirmed together with intra-
hepatic calcifications and hyperechogenic bowel. At 19 weeks, femur length was at
the 10th percentile. Because of these ultrasound anomalies, prenatal diagnosis of con-
genital CMV, parvovirus B19, and toxoplasma infections were performed, but were
all negative. Retrospective investigation retrieved a history of rash illness diagnosed
as an allergy at 4 weeks of gestation. Considering the ultrasound anomalies and the

history of rash, fetal blood was collected at 20 weeks. In this sample, interferon was
detected at a high level (75 UI/mL, normal <2 UI/mL) together with rubella-specific
IgM antibody, confirming the diagnosis of CRI (Furet et al. 2010). This case shows
that ultrasound scans may help diagnose CRI in settings in which rubella cases are
sporadic and very often misdiagnosed. In the second case, primary rubella infection
with rash was described at around 5 weeks. The ultrasound examination at 16 weeks
was normal, but RV RNA was detected by RT-nested PCR analysis of amniotic fluid
obtained at 18 weeks, indicating CRI. The ultrasound scan performed at 19 weeks
revealed IUGR, together with asymmetric bilateral microphthalmia (below the 5th
percentile), opacity of the right lens, and an isolated hyperechogenic mitral focus.
This latter case shows that a normal ultrasound scan does not exclude CRS, as abnor-
malities may be detected later in pregnancy and consequently points out the need of
iterative ultrasound scans to check for fetal defects (Cordier et al. 2012). Although
prenatal ultrasound results are nonspecific, they can help diagnose CRI and, in some
cases, may be used as prognostic markers of the severity of CRS.
Computed tomography (CT) and fetal magnetic resonance imaging (MRI) can
show abnormalities in the fetal brain of patients with CRI. The early detection of some
brain abnormalities, such as microcephaly and cortical anomalies, may influence the
prognosis of fetal infection. Upon examination of children with CRS, Yoshimura
et al. (1996) observed a close relationship between ventricular dilatation and CNS
sequelae (mental retardation, microcephaly, cerebral palsy) (Yoshimura et al. 1996).
In this study, nine CRS patients were examined. Five patients without dilatation of the
lateral ventricles had no sequelae except deafness, whereas among the 4 with dilata-
tion, 3 had mental retardation, cerebral palsy, or microcephaly. By contrast, abnormal
intensity areas in the white matter found by MRI were not strictly correlated with
CNS sequelae. In the same way, Chang et al. (1996) obtained cranial ultrasound scans
for 5 CRS infants whose ages ranged from 1 day to 2 months (Chang et al. 1996).
Only 2 of these infants were small for their gestational age, and none of them were
microcephalic at birth. Deafness and ocular lesions were found in 4 patients, and
congenital heart disease was found in 3. All had abnormal ultrasound scans: linear-
shaped hyperechogenicity over the basal ganglia or periventricular punctate hyper-
echogenicity or subependymal cysts were observed. Follow-up ultrasonograms for 2
of the patients showed progressively enlarging hyperechogenic lesions. Calcification
was found in both patients examined by means of CT. All patients became micro-
cephalic with profound global developmental delay. Numazaki and Fujikawa (2003)
described CT results in a CRS infant at 4 days of age (Numazaki and Fujikawa 2003).
They noted several cortical low-density areas and calcifications of the periventricular
area and basal ganglia. MRI performed at 4 weeks of age showed almost similar
findings. At 2 months of age, the patient showed severe bilateral hearing loss. At 12
months of age, she had mild mental retardation and developmental delay.
17.5.6.1.2 Postnatal Clinical Diagnosis

The WHO has produced a case definition for CRS as shown in Table 17.3. The clini-
cal diagnosis of congenital rubella should be confirmed by laboratory tests whenever
possible.
17.5.6.1.3 Prognosis
The long-term prognosis for those infants with cardiac defects or profound deafness
combined with cataracts and brain damage is extremely poor. Surviving children
with severe CRS may have multiple congenital abnormalities and therefore require
continuous and specialized medical care and education, and rehabilitation. However,
many of those who were born in Australia in the early 1940s, when examined at 25
years of age, had developed far better than might have been expected, being of aver-
age intelligence, employed, and some were married and had normal children (Menser
et al. 1967). Those born as a result of the 1964/1965 epidemic in the United States
apparently had more problems, when studied in their twenties. One third required
institutional care for severe handicaps, one third were still living with their parents,
and a third were leading normal lives (Cooper and Alford 2006). The difference is
probably due to the survival of more severely affected infants in the United States,
due to improved medical care at that time. More recent studies of CRS patients have
reported diabetes, thyroid disorders, early menopause, and osteoporosis at a higher
prevalence than in the general population (Munroe 1999; Forrest et al. 2002).
17.5.6.1.4 Laboratory Diagnosis

Infants with CRI produce rubella IgG and IgM at birth, and high titers of RV may be
excreted (Figure 17.7). The IgG response persists, but the IgM response declines and
is seldom detectable after 18 months of age. The most convenient method for diag-
nosis is the detection of rubella IgM, by M-antibody capture EIA, in serum or oral
fluid ideally taken before 3 months of age (Corcoran and Hardie 2005; Eckstein et
al. 1996; Best and Enders 2007). A diagnosis can also be made by demonstrating the
Maternal Birth Infant IgG
infection
Maternal IgG
Infant IgM
Infant
lymphoproliferative
response
1st 2nd 3rd 2 4 6 1 2 3 4

Trimesters Months Years
Age of Child
FIGURE 17.7 Serological markers for the diagnosis of congenital rubella. (Reproduced
from Best and O’Shea, Diagnostic Procedures for Viral, Rickettsial and Chlamydial
Infections, 7th edition, 1995. With permission from the American Public Health Association.)
TABLE 17.4
Laboratory Techniques for the Diagnosis of Congenital Rubella
Infection
Prenatal
Established Methods Diagnosis Diagnosis in Infancy
Rubella-specific IgM in serum Yes Birth–1 year
Rubella-specific IgM in oral fluid No Birth–1 year
Rubella-specific IgG No 7 month–1 year
RV isolation in cell culture Yesa Birth–1 year
RT-PCR (RV RNA detection) Yes Birth–1 year
Source: Reprinted from Rubella Viruses. Perspectives in Medical Virology Volume 15,
J. M. Best and G. Enders, Laboratory diagnosis of rubella and congenital
rubella, pp. 39–77, Copyright (2007), with permission from Elsevier.
a Too slow to be useful.
persistence of rubella IgG in sera taken between 6 and 12 months of age (Table 17.4),
when both IgM and IgG tests should be used. However, it is not possible to make a
serological diagnosis after immunization with a rubella-containing vaccine.
A diagnosis can also be made by detection of RV in nasopharyngeal secretions
(NPS), urine, oral fluid, lens aspirates, CSF, and EDTA-blood collected in the first
3 months (Table 17.4). As virus isolation is demanding and time consuming, RV is
usually detected by RT-PCR (Bosma et al. 1995; Feng et al. 2011). RT-nested PCR has
been used, but is currently being replaced by real-time PCR. It should be remembered
that infants with CRS excrete high titers of RV, which is readily transmitted. Therefore
isolation precautions should be employed and contact with pregnant women avoided.
Prenatal diagnosis of CRS is possible using amniotic fluid and/or fetal blood (Best
2007; Best and Enders 2007). Laboratory diagnosis has been reviewed in more detail
by Best and Enders (2007) and Best et al. (2009).

The pathogenesis of CRS is not fully understood, as much of the work on CRI was
done in the 1960s following extensive epidemics of rubella and before the introduc-
tion of vaccination. Recent studies have relied on work in cell cultures, as access to
cases of CRS is limited due to the success of vaccination programs (see Prevention),
and no reliable animal model has been established.
CRS is a progressive disease, due to the persistence of RV, which may continue
to cause damage in later life. Structural defects result from defective organogenesis,
tissue destruction, and scarring. When maternal infection occurs in the first trimes-
ter of pregnancy, at the time of organogenesis, every organ may be involved and RV
can be isolated from all such organs (Rawls 1968; Thompson and Tobin 1970). RV is
able to persist intracellularly in the presence of neutralizing antibodies. When infec-
tion occurs after the first trimester, RV may cross the placenta, but fetal immune
responses, which will have by then developed, will terminate the infection and a
persistent infection will not usually be established. Although the organ of Corti is
susceptible to infection up to 16 weeks of gestation, other fetal damage is unlikely as
organogenesis is complete by 8 weeks.
17.5.7.1 Histological Studies

In a classical histological study of fetuses obtained at TOP following maternal
rubella in the first trimester, Töndury and Smith (1966) observed some damage in
68% of the 57 fetuses examined, but no gross external malformations were noted.
Scattered foci of damage were seen in the chorion, and damaged endothelial cells
from the blood vessels were seen to be desquamated into the blood vessels. Thus,
during maternal viremia, infected cells may be transported into the fetal circulation
to establish widespread infection of organs. In the heart, necrotic cellular damage
was observed in the myocardium and the auricular wall was unusually thin in some
specimens, which might lead to the septal defect of the heart. Obstructive lesions
may occur in the pulmonary and renal arteries due to RV-induced damage to the
intimal lining of the arteries. In the eye, fiber cells of the lens were damaged, and
later, degenerative changes were seen in the lens, suggesting active disease leading to
the characteristic cataracts observed by Gregg (1941). In the inner ear, foci of necro-
sis were noted in the epithelium of the cochlea and damage to the stria vascularis.
Complete atrophy of the organ of Corti has been reported. This damage would lead
to deafness. Sporadic foci of damage were also seen in the teeth and skeletal muscle.
No inflammatory response was observed, because fetal immune responses have not
developed in the first trimester.
17.5.7.2 Other Studies of the Eye

Ocular manifestations of CRS have been reviewed by Arnold et al. (1994) and
Vijaylakshmi et al. (2002). Almost all ocular structures may be affected in CRS,
and these authors suggested that ocular abnormalities are caused either by (1) direct
disturbance of organogenesis due to inhibition of cell replication by RV or (2) tissue
destruction and scarring caused by immune responses, which may progress through-
out pregnancy. RV enters the lens before the development of the lens capsule. It
may persist within the lens and has been isolated from cataracts from children up
to the age of 3 years (Menser et al. 1967). Using RT-PCR, Rajasundari et al. (2008)
detected RV RNA in 92% of lenses from confirmed cases of CRS aged 0–11 months.
Cataract, microphthalmos, and glaucoma are probably caused by mitotic inhibition
and slow growth of cells (Rajasundari et al. 2008). Retinopathy is reported in from
13.3% to 61% cases. Pigment deposits are seen throughout the retina and vary from
fine powdery to granular shapes. Visual acuity is not affected by these pigmentory
changes. Strabismus and nystagmus (disorders of motility) are frequent and likely to
be the result of organic lesions in the eye.
17.5.7.3 Studies of the Brains from CRS Fetuses/Infants

Töndury and Smith (1966) saw no obvious damage in brain cells, but endothelial cell
damage and small hemorrhages were seen in some cases, and the cortical width was
abnormally small in 10 affected fetuses, when compared with normal fetuses. Peters
and Davis (1966) reported areas of mineralization in the blood vessel walls in the
putamen and other nuclei of the brain. In vitro studies in human fetal brain cells have
shown that RV was found mainly in astrocytes, with oligodendrocytes and neurons
only occasionally infected (Chantler et al. 1995). Infection of astrocytes may lead
to focal areas of necrosis and the pattern of neurological deficit seen in CRS. The
restricted replication in oligodendrocytes correlates with the lack of demyelination
generally seen in CRS. The cell receptor for RV, MOG is a type I integral membrane
protein and is expressed mainly in the CNS. Cong et al. have suggested that MOG is
likely to facilitate the attachment of RV to brain cells, leading to cell damage (Cong
et al. 2011).
17.5.7.4 Delayed CNS Disorders

Using MRI, Lim et al. (1995) compared brain morphology in adult patients with
CRS who had schizophrenia-like symptoms with those of adult early-onset schizo-
phrenic patients without CRS and healthy control subjects (Lim et al. 1995). CRS
patients had smaller intracranial volumes than schizophrenic patients. In both
affected groups, patients had smaller cortical gray matter volumes than the control
group. In addition, they showed significant enlargement of the lateral ventricles.
The observations in CRS patients are consistent with a developmental lesion that
limits full brain growth, with the small intracranial volume due at least in part to
reduced cortical gray matter. It is worth noting that nucleic acid from RV and other
viruses was never detected in the brains of schizophrenia patients at autopsy (Taller
et al. 1996).
Brain lesions associated with PRP include diffuse atrophy of the brain with
ventricular dilatation (Kuroda and Matsui 1997) and alteration of the white matter
(Weil et al. 1975). Extensive neuronal loss together with inflammatory damage can
be observed.
The role of RV in the pathogenesis of PRP is not clear. RV has only been detected
once in the brain of a CRS child with PRP using cocultivation techniques (Cremer
et al. 1975). Martin et al. (1989) have suggested that virus-induced autoreactivity to
brain antigens may be an important mechanism in chronic inflammatory disorders
of the CNS. Circulating immune complexes may also play a role (see below).
17.5.7.5 Possible Mechanisms of Fetal Damage

Early in vitro studies suggested that RV may cause a retardation of cell division
(Plotkin et al. 1965) and chromosomal damage (Nusbacher et al. 1967). Plotkin and
Vaheri (1967) suggested that a specific protein found in RV-infected cells caused this
slowing down of cell division, now recognized as mitotic inhibition. Recent work
suggests that this may be due to disruption of actin filaments, which are an important
component of the cytoskeleton (Lee and Bowden 2000). Naeye and Blanc (1965)
showed that the organs of RV-infected fetuses were smaller and had fewer cells than
those of uninfected fetuses of the same age (Naeye and Blanc 1965). Thus, mitotic
inhibition during the period of organogenesis could result in congenital malforma-
tions. RV-induced fetal endothelial cell damage may cause hemorrhages in small
blood vessels, leading to tissue necrosis and further damage to organs (e.g., liver,
myocardium, organ of Corti) over a longer period.
Other suggested mechanisms of RV-induced fetal damage are RV-induced apop-

tosis of essential cells and viral interference with the cell cycle (Atreya et al. 2004;
Cooray et al. 2005; Lee and Bowden 2000; Best et al. 2005). Caspase-dependent
apoptosis is responsible for the cytopathogenicity induced by RV in cell cultures,
and this is controlled by cell survival signaling pathways and by interferon (Adamo
et al. 2008; Cooray et al. 2003, 2005; Duncan et al. 2000). Adamo et al. (2004) have
shown that RV-infected fetal fibroblasts do not undergo apoptosis, although apop-
tosis does occur in RV-infected adult fibroblasts, as the background of gene expres-
sion is antagonistic to it in fetal fibroblasts (Adamo et al. 2008). This might help to
explain the persistence of RV-infected fibroblasts in utero, although these findings
do not exclude apoptosis in other cell types. In addition, the persisting virus may be
able to disrupt normal cell growth. Thus, RV infection may interfere with the cell
cycle, since the RV NSP p90 has been shown to interact with both the retinoblastoma
protein (pRB), which is a cell cycle regulator, and the cytokinesis regulatory protein
citron-K kinase (pCK) (Atreya et al. 2004). A strong interferon response and up-
regulation of chemokines in RV-infected human embryonic fibroblasts also suggest
that RV could disrupt growth and proliferation pathways (Adamo et al. 2008).
Following widespread infection of the fetus, the foci of infection may persist for
some years, but only 1 in 103 to 1 in 105 fetal cells harbor RV (Rawls 1968). RV has
been detected in NPS, CSF, and urine for up to 23 months (Best and Enders 2007),
and RV has been isolated from lens aspirates from infants up to the age of 12 months,
from a cataract from a 3-year-old child and the CSF of children with CNS involve-
ment up to 18 months of age. Rubella antigen has been detected in the thyroid of a
5-year-old child, and RV was recovered from the brain of a 12-year-old child with
PRP. Persisting RV would then be responsible for disruption of cell growth and dif-
ferentiation, since viral proteins are known to bind to cell proteins involved in cell
division and cellular proliferation pathways (see above). RV persistence in cell lines
is more likely to be established at low multiplicities of infection, and virus repli-
cation may be limited by interferon production (Adamo et al. 2008) and defective
interfering RNAs (Derdeyn and Frey 1995). Clones of infected cells may persist
in such organs as the eye and brain for many years, as virus will not be exposed to
the immune response. Defects of cell-mediated immunity would also help the per-
sistence of RV. Defective lymphoproliferative responses may persist for 2–3 years
(O’Shea et al. 1992), and other defective cell mediated immune responses may per-
sist into the second decade of life (reviewed by Best et al. 2005). Mauracher et al.
found that patients with CRS had significantly lower levels of antibodies to the RV
E1 protein than rubella seropositive healthy controls when linearized E1 was used in
Western blots under reducing conditions, and functional affinity of the E1-specific
IgG was also significantly lower (Mauracher et al. 1993). As there was no difference
in the levels of antibodies to the E2 and capsid proteins, selective immune toler-
ance to the E1 protein was suggested, which may be due to defects in cell-mediated
immune responses.
Autoimmune mechanisms are thought to play a role in the pathogenesis of IDDM
and other endocrine disorders, but the mechanisms are not fully understood. RV
has been isolated from the pancreas of several infants who died from CRS (Sever
et al. 1985). RV infection may damage islet cells and initiate a process that leads to
IDDM in later life. Rubella antigens have also been identified in the thyroid follicles
of a patient with CRS and thyroid autoimmunity (Ziring et al. 1977). RV infection
is not cytolytic in human fetal islet cells in vitro, but a depression of immunoreac-
tive-secreted insulin has been observed (Numazaki et al. 1990). Immunoreactive
epitopes in the RV capsid have been shown to share antigenicity with β-cell pro-
tein (Karounos et al. 1993). Autoantibodies to islet cells, which predict the onset of
diabetes, have been detected in 20% of these patients. There may also be genetic
susceptibility; the HLA types of patients with IDDM are typical of those with auto-
immune disease, an increase of the haplotype HLA-A1, B8, DR3, and a decrease in
the prevalence of HLA-DR2 (Forrest et al. 2002; Sever et al. 1985). Thus, CRI may
increase the susceptibility to IDDM in these patients.
It has been suggested that circulating immune complexes (CICs) play an impor-
tant role in “late-onset disease” and PRP. Coyle et al. (1992) reported rubella
antibody-containing CICs in 21 of 63 (33%) patients with CRI (aged 5 months to 28
years), which were associated with late-onset clinical problems in 10 of the 21 posi-
tive subjects. Tardieu et al. (1980) studied eight boys with CRI, who developed severe
clinical symptoms between 3 and 6 months of age—interstitial pneumonia, diarrhea,
skins rash, hepatosplenomegaly, rapid neurological deterioration, and purpura, six of
whom died (Tardieu et al. 1980). Rubella-specific CIC were detected in 4 patients
in the acute phase of this illness. An initial disequilibrium of T and B lymphocytes
was later corrected. Verder et al. (1986) studied an infant born to a mother who
had laboratory-confirmed rubella at 13 weeks of gestation (Verder et al. 1986). The
infant appeared well at birth, but failure to thrive and interstitial pneumonia were
noted at 2 months of age. Other symptoms of late-onset disease developed including
meningoencephalitis at 5½ months, which subsequently progressed with increasing
lethargy, bulging fontanel, hydrocephalus, and hypodense areas on cerebral CT scan.
Her condition improved on treatment with prednisone and plasma exchange transfu-
sions. RV was isolated from PBL up to 8 months of age. High titers of rubella IgM,
low rubella IgG, and an abnormally high total IgM were noted, and total lymphocyte
counts were normal. Few CD8+ T cells were detected at 5 months of age, and there
was only low activity of K and NK cells. Exchange transfusions at 9 months pro-
duced clinical improvement, cessation of viremia, and normalization of cytotoxic
cell functions. These authors suggested that CIC were composed of rubella antigen
and rubella IgM antibodies and that deficient cytotoxic lymphocyte functions were
responsible for the failure to clear the CIC. CIC have also been found in the serum
and occasionally in CSF from patients with PRP. These CIC contain rubella-specific
IgG antibodies and rubella antigen (Coyle and Wolinsky 1981).
17.6 PREVENTION
17.6.1 Rubella Vaccines
The first live attenuated rubella vaccines were licensed in 1969–1970. Several vac-
cines were developed, but the RA27/3 strain is the most widely used, as it induces
the best immune response (Reef and Plotkin 2007; WHO 2008). Rubella vaccine
is generally administered in combination with measles (MR), measles and mumps
(MMR), or with the further addition of varicella (MMRV) (Centers for Disease
Control and Prevention 2008).
Rubella vaccines cause few side effects in children, but rash, low-grade fever,
irritability, lymphadenopathy, myalgia, paresthesia, and joint symptoms may occur
10–30 days after vaccination. Joint symptoms occur most frequently in postpubertal
females (≤25%). Rubella vaccine is not associated with chronic joint disease. The
first dose of MMRV may be associated with febrile seizures (Klein et al. 2010).
Symptoms are rare after a second dose. MMR vaccines containing the Urabe strain
of mumps have been associated with aseptic meningitis, but this is not found with the
Jeryl Lynn strain of mumps (Department of Health 2010a).
In 1998, Wakefield et al. suggested an association between MMR vaccine and a
“new syndrome” of autism and bowel disease, but subsequent epidemiological stud-
ies in several countries consistently failed to find evidence of a link between MMR
vaccine and autism or inflammatory bowel disease (Honda et al. 2005; Pebody et
al. 1998; Institute of Medicine 2001; WHO 2003; Department of Health 2010b). It
is now accepted that Wakefield’s paper was a small case series with no controls
and relied on parental recall and beliefs. Ten of Wakefield’s coauthors retracted the
article’s interpretation of a link with MMR vaccine (Murch et al. 2004), and the
paper was finally retracted in 2010. It was recently suggested that Wakefield’s article
linking MMR vaccine and autism was not only wrong but fraudulent (Godlee et
al. 2011). The consequences of Wakefield’s paper were disastrous since MMR vac-
cination rates in the United Kingdom decreased sharply and were as low as 80% in
2003–2004, resulting in outbreaks of measles (Jansen et al. 2003).
17.6.1.1 Immune Responses

Rubella antibodies usually develop 10–28 days after vaccination. Only about 5%
of vaccinees will fail to develop an immune response, which may be due to a con-
current infection or to maternal antibodies in infants or from blood transfusion or
immunoglobulin. Some vaccinees will have preexisting low levels of antibodies
detectable by alternative assays and may fail to develop higher concentrations of
antibody. Vaccine-induced antibodies probably persist for life in most vaccinees,
but it is advised to confirm immunity in women before pregnancy if they were vac-
cinated in childhood.
17.6.1.2 Contraindications
Pregnancy is a contraindication to vaccination with rubella-containing vaccines
(RCV) and should be avoided for at least 1 month after vaccination (Centers for
Disease Control and Prevention 2001; Department of Health 2010b). However, if a
woman who is unknowingly pregnant is vaccinated, there is no indication for abor-
tion or prenatal diagnosis, since no abnormalities compatible with CRS have been
detected in infants after vaccination in pregnancy (Centers for Disease Control and
Prevention 2001; Reef and Plotkin 2007; Castillo-Solorzano et al. 2011).
Other contraindications to vaccination with a RCV are severe immunodefi-
ciency and intensive immune suppressive therapy, congenital immune disorders,
a history of allergic reactions to components of the vaccine, and untreated active
tuberculosis (WHO 2011). If MMR or other measles-containing vaccine is used,
the contraindications to this vaccine should also be followed. Vaccination of per-

sons who have received blood transfusion or other blood products should be delayed
for at least 3 months. Administration of human anti-Rh (D) immunoglobulin is not a
contraindication to postpartum vaccination. Vaccination should also be delayed in those
who have a fever >38.5°C or other serious disease (Department of Health 2010b; WHO
2011). Neurological conditions are not normally a contraindication, unless there is cur-
rent neurological deterioration, in which case vaccination should be delayed.
17.6.2 Vaccination Programs

The aim of rubella vaccination programs is to prevent infection in pregnancy includ-
ing CRS. Rubella vaccination programs were started in some countries in the early
1970s. Countries such as the United States introduced universal vaccination, where
all children were offered vaccination to eliminate the disease. Other countries such
as the United Kingdom introduced selective vaccination of prepubertal girls and
susceptible adult women to protect the population at risk (i.e., pregnant women), but
these programs were not entirely effective (Best et al. 2009; WHO 2011). Currently,
most (92%) countries use two doses of MMR, with the first dose being administered
between 12 and 24 months (WHO 2011). As of December 2010, a total of 130 WHO
member states had introduced RCV, a 57% increase from 83 member states in 1996
(Figure 17.8). In addition, goals to eliminate rubella and CRS were established in
the WHO Region of the Americas (by 2010), the WHO European Region (by 2015),
and the WHO Western Pacific Region has established targets for accelerated rubella
control and CRS prevention by 2015.
1996
65 countries
12% of birth cohort
2010
131 countries
42% of birth cohort
FIGURE 17.8 Countries using rubella vaccine in their national immunization system.
(From WHO/IVB database and the “World Population Prospects: the 2010 Revision,” New
York, UN 193 WHO Member States. Date of Slide: 28 September 2011.)
In 2000, the first WHO rubella vaccine position paper was published, which
placed an emphasis on direct protection of women of child-bearing age (WHO
2000). In 2011, the WHO guideline was updated and supports a paradigm shift
in vaccination strategy for introduction of RCVs. The position paper recommends
countries take advantage of the measles platform of two doses of measles vaccine
to introduce MR or MMR vaccine using the strategy recommended (WHO 2011).
This experience in part is based on country and regional experiences and focuses
on the interruption of rubella transmission targeting children and adolescents. The
recommended strategy includes: an initial catch-up campaign, followed immediately
with introduction of the MR/MMR vaccine in the routine program. Vaccination of
women of child-bearing age is now considered an additional strategy, as women
were difficult to access in many settings, resulting in limited vaccine coverage,
which allowed the continuing circulation of RV. Thus, susceptible pregnant women
were at risk of exposure and subsequent rubella infection. In addition, since 2000,
all countries have added delivery of a second dose of measles vaccine for all children
either in campaigns or through the addition of a routine immunization visit. The
second measles dose provides an opportunity to use combined MR/MMR vaccines
that can reach 80% of all children, thereby effectively blocking rubella transmission
and its associated risk of CRS.
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Section II
Retroviruses
18 Human T-Lymphotropic
Virus
Motohiro Yukitake and Hideo Hara
CONTENTS
18.1 Introduction................................................................................................... 431
18.1.1 Structure and Biology of HTLV-I...................................................... 432
18.1.2 Epidemiology of HTLV-I................................................................... 433
18.1.3 Transmission...................................................................................... 434
18.1.4 Prevention.......................................................................................... 435
18.2 HAM/TSP...................................................................................................... 435
18.2.1 Pathology........................................................................................... 435
18.3 Immunopathology.......................................................................................... 437
18.3.1 T-Cell-Mediated Immune Responses in the Spinal Cord.................. 437
18.3.2 Detection of HTLV-I-Infected Cells and HTLV-I Provirus in
Spinal Cord Lesions........................................................................... 438
18.3.3 Clinical Features of HAM/TSP......................................................... 439
18.4 Risk Factors for HAM/TSP...........................................................................444
18.5 Treatment of HAM/TSP................................................................................ 445
18.5.1 Immunomodulatory Therapy for the Treatment of HAM/TSP......... 445
18.5.1.1 Interferon α and β............................................................... 445
18.5.1.2 Corticosteroid Hormone.....................................................446
18.5.1.3 Other Immunomodulatory Agents for the Treatment
of HAM/TSP.......................................................................446
18.5.1.4 Antiviral Therapy for the Treatment of HAM/TSP............446
18.5.2 Symptomatic Treatment..................................................................... 447
18.6 Other Neuromuscular Disorders Associated with HTLV-I Infection............448
18.7 HTLV-II and Neurological Diseases.............................................................448
References...............................................................................................................449
18.1 INTRODUCTION
In 1980, human T-lymphotropic virus type I (HTLV-I) was isolated from cultured
CD4+ T-lymphocytes of a cutaneous T-cell lymphoma patient (Poiesz et al. 1980),
who was later considered to have adult T-cell leukemia/lymphoma (ATL) (Poiesz
et al. 1980; Uchiyama et al. 1977; Yoshida et al. 1984). In 1985, it was reported that
59% of patients with tropical spastic paraparesis (TSP) in Martinique were HTLV-
I-seropositive (Gessain et al. 1985). Then, TSP patients in Jamaica and Colombia
431
were also shown to have anti-HTLV-I antibodies in both serum and cerebrospinal
fluid (CSF) (Rodgers-Johnson et al. 1985). Moreover, Osame et al. (1986) reported
cases of HTLV-I-seropositive chronic progressive myelopathy in the south of Japan
and named it HTLV-I-associated myelopathy (HAM). Soon after, HAM and HTLV-
I-seropositive TSP were identified as the same disease, termed HAM/TSP (Osame
1990; Roman and Osame 1988). Epidemiological studies suggested that 10 to 20
million people worldwide are infected with HTLV-I (de The and Bomford 1993),
but prevalence of HAM/TSP is only 0.1%–5% of HTLV-I-infected individuals
(Hollsberg and Hafler 1993; Kaplan et al. 1990). In addition, there are some large
endemic areas of HTLV-I infection in the south of Japan, Central and West Africa,
the Caribbean, Central and South America, and the Middle East.
18.1.1 Structure and Biology of HTLV-I

HTLV-I has been classified into a type C oncovirus group (group VI; ssRNA-RT) by
the International Committee on Taxonomy of Viruses and belonging to the family
of Retroviridae. Unlike the human immunodeficiency virus (HIV), HTLV-I causes
diseases, such as adult T-cell leukemia (ATL) and HAM/TSP, in less than 5% of
infected individuals (Yamaguchi and Takatsuki 1993). In general, HTLV-I-related
diseases (including not only HAM/TSP and ATL but also alveolitis, uveitis, poly-
myositis, and arthropathy) occur in around 10% of HTLV-I carriers. In other words,
most people infected with HTLV-I are asymptomatic throughout their lives, whereas
between 0.1% and 5% of HTLV-I carriers develop HAM/TSP (in comparison, life-
time risk of ATL ranges from 2% and 5%) (Kaplan et al. 1990).
HTLV-I is a round, enveloped virus of around 100 nm in diameter (Ohtsuki et al.
1982). The length of the HTLV-I genome has 9032 base pairs and encodes structural and
enzymatic proteins similar to those of other retroviruses. The three typical structural
and enzymatic genes, the group antigen (gag), polymerase (pol), and envelope antigen
(env) genes, are flanked by long terminal repeats (LTRs) (Seiki et al. 1983). However, the
HTLV-I genome has a unique region at the 3ʹ end called the pX region, which contains
at least four partially overlapping reading frames (ORFs) encoding regulatory proteins
such as the Tax transactivator (ORF4), the posttranscriptional regulator Rex (ORF3), and
accessory proteins (p12, p13, p30) (Matsuura et al. 2010).
The full-length mRNA encodes both gag protein (p55) and pol protein. The gag pro-
tein is cleaved by the viral protease to yield the matrix (MA, p19), capsid (CA, P24), and
nucleocapsid (NC, P15) proteins. The pol protein is synthesized by ribosomal frame-
shifting. In addition, a single-spliced mRNA encodes the env protein, and a double-
spliced mRNA encodes the Tax and Rex proteins. Some other RNA frameshifts and
transcript splicing patterns are also known. The LTRs located at the 5ʹ and 3ʹ ends of the
viral genome are divided into U3, R, and U5 regions. The U3 region contains essential
elements for control of proviral transcription, mRNA termination, and polyadenylation.
The R region contains the Rex-responsive element (Ciminale et al. 1992). The functions
of HTLV-I proteins and glycoproteins are listed in Table 18.1 (Albrecht and Lairmore
2002; Johnson et al. 2001; Lemasson et al. 2007; Satou et al. 2011).
HTLV-I has six subtypes: subtype A is also known as the cosmopolitan sub-
type sequenced from Japan (Seiki et al. 1982), which is found in many HTLV-I
Human T-Lymphotropic Virus 433
TABLE 18.1
Functions of HTLV-I Proteins and Glycoproteins
HTLV-I Proteins and Glycoproteins Functions
Envelope Proteins (Encoded by env)

Surface glycoprotein (gp46) Bind to host cell receptor
Transmembrane protein (gp21) Anchors surface glycoproteins to virus
Structural Proteins (Encoded by gag)

Matrix layer (p19) Organize viral components at the inner cell membrane
Capsid (p24) Protect viral RNA and proteins
Nucleocapsid (p15) Nucleic acid-binding protein
Functional Proteins (Encoded by pol)

Protease (p14) Cleaves polyproteins into functional components
Reverse transcriptase (p95) Converts single-stranded RNA to double-stranded DNA
Integrase Facilitates insertion of provirus into host cell DNA
Regulatory Proteins
Tax Activates transcription provirus and host genes
Transcriptional and posttranscriptional regulator
Rex Modulates transport of viral RNA
Posttranscriptional regulator of viral gene expression
HTLV-I bZIP factor Down-regulates viral transcription
p12I Role in viral replication and T-cell activation
pl3II Targets mitochondria
p30II Modulates transcription
endemic areas worldwide; subtypes B, D, and F from Central Africa; subtype C

from Melanesia, and subtype E from South and Central Africa (Proietti et al. 2005).
18.1.2 Epidemiology of HTLV-I
Although it is difficult to estimate the exact global prevalence of HTLV-I because of lim-
ited population-based reports, it is estimated that approximately 10–20 million people
are infected by HTLV-I globally (de The and Bomford 1993; de The and Kazanji 1996).
There are some geographical clusters of HTLV-I in the south of Japan, Central and West
Africa, the Caribbean, Central and South America, and the Middle East (Figure 18.1)
(Levine et al. 1988; Mueller 1991; Proietti et al. 2005; Sonoda et al. 2011).
Japan is a key endemic area of HTLV-I infection. There are some endemic areas
in the south of Japan, such as Kyusyu Island, Shikoku Island, and Okinawa Island
(Yoshida et al. 1982). Although the prevalence in the general population varies from
0% to 37% (Yoshida et al. 1982), the migration of infected Japanese individuals has
recently changed the prevalence throughout the whole of Japan. In the Caribbean
islands, the rate of HTLV-I infection is observed to be high, and the prevalence
is estimated with approximately 5% in Jamaica (Murphy et al. 1991). Africa is
Europe
Asia
North America
Japan
Caribbean Africa
islands
South America
Australia
HTLV-I endemic areas
Less than 1%
1 to 5%
More than 5%
FIGURE 18.1 Worldwide distribution of HTLV-I. There are several high-prevalence areas:
in the southern region of Japan, the Caribbean, the equatorial regions of Africa, South
America, the Middle East, and Melanesia. It should be noted that HTLV-I endemic areas do
not correspond exactly to the country boundaries.
estimated to be the origin of HTLV-I because all different primate T-lymphotropic

viruses (PTLV) have been found there. Phylogenetic studies also suggest central
Africa as the birthplace of PTLV. In Africa, although the study of the region is lim-
ited, the prevalence varies from 0.5% to around 5% (Dumas et al. 1991). In Brazil,
the highest prevalence (1.35%) was reported for the central area and the coast, with
the lowest (0.08%) in the north and south (Goncalves et al. 2010). In addition, there
are smaller endemic foci in Australia (Aboriginal populations), Iran, and Papua New
Guinea (Goncalves et al. 2010; Yoshida et al. 1982). In nonendemic areas such as
Europe and North America, HTLV-I infection is mainly observed in immigrants
from endemic areas, their offspring, and sexual contacts, as well as intravenous drug
users and sex workers. From the data of blood donors in Europe and North America,
the prevalence is quite low, for example, 0.0039% in France and 0.01%–0.03% in the
United States and Canada (Biggar et al. 2006; Murphy et al. 1991).
18.1.3 Transmission
There are several routes of HTLV-I transmission. The main route from mother to child
is through breastfeeding. The frequency of transmission through breastfeeding is esti-
mated to be between 15% and 25%, and there are several factors that influence this,
such as HTLV-I proviral load, mother-child HLA class I concordance, and the duration
of breastfeeding (Biggar et al. 2006). Mother-to-child transmission during the gestation
period also occurs, but the frequency is less than 5%. As HTLV-I is present in the genital
secretions of infected individuals, sexual transmission of HTLV-I is the other main route.
In sexual transmission, higher transmission efficiency from men to women than from
women to men has been suggested (Murphy et al. 1996). Intravenous exposure to blood is
also an important route of HTLV-I transmission. Transfusion of HTLV-I-infected cellular
blood components results in seroconversion in more than 40% of recipients (Murphy et

al. 1996). Compared with that for cellular blood component transfusion, plasma products
and cold storage of blood transfusion result in a lower rate of transmission, presumably
due to the absence and/or death of HTLV-I-infected lymphocytes (Donegan et al. 1990).
The use of contaminated needles and syringes among the drug users is another route of
HTLV-I transmission (Feigal et al. 1991).
18.1.4 Prevention
Considering the poor prognosis of ATL and HAM/TSP, prevention of HTLV-I infec-
tion is important. For HAM/TSP, patients experience long-lasting progressive physi-
cal impairments. Thus, public health interventions such as counseling and education of
high-risk individuals and populations are indispensable. Since the implementation of
a program to prevent intravenous exposure to HTLV-I in Japan in 1986, many coun-
tries, especially those with endemic areas (United States, Canada, Brazil, and several
European countries), started to implement systematic and permanent screening of all
blood donors (Osame et al. 1990). This important public health intervention has been
shown to be effective in preventing HTLV-I transmission, and, as a result, this inter-
vention succeeds in decreasing the number of new infections in the overall population.
For HTLV-I-seropositive individuals, it is advisable not to donate blood, organs, semen,
or milk (Goncalves et al. 2010). As mother-to-child transmission is the other important
problem, prenatal screening for HTLV-I should be carried out, especially in endemic
areas (Carneiro-Proietti et al. 2002). It is well recognized that neonatal infection with
HTLV-I is preventable through short-term breastfeeding for less than 3 months after
birth and/or bottle-feeding (Takahashi et al. 1991). Cesarean delivery should also be con-
sidered to minimize the risk of perinatal transmission (Goncalves et al. 2010).
Counseling and education for HTLV-I-infected individuals, as well as the general
public in endemic areas, are important not only to prevent HTLV-I infection but also
to provide psychological and social support. In addition, access to correct informa-
tion about HTLV-I infection is very important (Guiltinan et al. 1998).
18.2 HAM/TSP
18.2.1 Pathology
Macroscopically, the spinal cord shows mild to severe atrophy with thickening of the
leptomeninges. Spinal cord atrophy is symmetric and can occur throughout the entire
spinal cord, especially the thoracic cord. In general, representative pathological findings
in HAM/TSP are inflammatory infiltration and diffuse loss of myelin and axons (Figure
18.2) (Akizuki et al. 1988; Iwasaki 1990; Izumo 2010). Microscopic pathological find-
ings can be divided into two phases depending on the duration of the disease. In patients
with a relatively short clinical course, for example, a few years or shorter, infiltration of
mononuclear cells and degeneration of both myelin and axons are the main findings.
Inflammatory lesions continuously extend to the entire spinal cord but are the most
severe in the middle to lower thoracic spinal cord. Inflammation is observed in both gray
and white matter with inflammatory lymphocyte infiltration. In addition, lymphocyte
(a)
(b)
FIGURE 18.2 Pathological features of HAM/TSP patient. (a) Macroscopic findings

of thoracic spinal cord. Arrows show the symmetrical degeneration in the lateral column
(Klüver-Barrera stain). (b) Microscopic findings of thoracic spinal cord. Perivascular and
parenchymal infiltration of mononuclear cells was observed (hematoxylin–eosin stain).
These pictures were kindly provided by Dr. Izumo, Molecular Pathology, Center for Chronic
Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University,
Kagoshima, Japan.
infiltration is observed more frequently in the deeper portion of the cord than in the
surface areas, and more severe in the anteriolateral column than in the posterior column
(Izumo 2010). On the other hand, patients with a longer clinical course show less inflam-
matory change in the spinal cord, and both myelin and axon are degenerated monotoni-
cally. Tissue in the spinal cord shows gliosis with foamy cells, microglial cells, and a
small number of lymphocytes. Fibrous thickening of the vessel wall and pia mater is also
observed. Inflammatory changes and gliosis are also present in the gray matter, but neu-
ronal cells are relatively well preserved in the spinal cord. Although the tissue damage is
most severe in the thoracic cord, corticospinal damage is also observed as ascending to
the cervical spinal cord and brainstem. These types of damage are recognized as a result
of Wallerian degeneration. In the brain, similar inflammatory changes are also observed
to milder degrees (Aye et al. 2000).
The predominance of inflammation in the middle to lower thoracic cord is
explained by the assertion that an anatomical site with slow blood flow, namely, the
Longitudinal distribution Cross-sectional distribution
Blood flow Inflammatory lesions Blood flow
Inflammatory lesions
FIGURE 18.3 Distribution of inflammatory lesions and blood supply of the spinal cord.
Longitudinal and cross-sectional distribution of inflammatory lesions of HAM/TSP seemed
to be identical with slow blood flow area. (Modified from Aye, M.M. et al. 2000, Acta
Neuropathol. 100, 245–252; Izumo, S. 2010, Neuropathology 30, 480–485.)
area of endings of central and peripheral spinal arteries, may be associated with the
distribution of pathological changes (Figure 18.3) (Aye et al. 2000). Similar findings
are also observed in the brain.
18.3 IMMUNOPATHOLOGY
18.3.1 T-Cell-Mediated Immune Responses in the Spinal Cord
In patients with a relatively short clinical course, there are many inflammatory cells
including CD4+ T cells, CD8+ T cells, and macrophages in affected spinal cord
parenchyma (Umehara et al. 1993). B cells are also observed in the affected lesion
but are mainly located in perivascular spaces. Proinflammatory cytokines such as
interleukin (IL)-1β, (TNF)-α, and interferon (IFN)-γ were detected in perivascu-
lar infiltrating cells including macrophages, astrocytes, and microglia at the active
inflammatory lesions (Umehara et al. 1994). Expression of myeloid-related protein
(MRP) 14 and MRP-8, essential proteins in Ca2+-dependent functions during inflam-
mation, has been observed in infiltrating/activated macrophages and microglia (Abe
et al. 1999). Among various adhesion molecules, high expression of vascular cell
adhesion molecule 1 (VCAM-1) on the endothelium (Umehara et al. 1996), and up-
regulation of very late antigen 4 (VLA-4) and monocyte chemoattractant protein 1
(MCP-1) in the infiltrating cells, has been observed. Intracellular adhesion molecule 1
(ICAM-1) and lymphocyte function-associated antigen 1 (LFA-1) are also considered
as being related to lymphocyte infiltration (Cabre et al. 1999). The immunoreactivity for
HLA class I and up-regulation of HLA class II are found on various cells such as endo-
thelial cells, microglia, and infiltrating mononuclear cells in the lesions. On the other
hand, patients with a longer clinical course show CD8+ T-cell predominance with down-
regulation of proinflammatory cytokine expression in the affected lesions (Matsuura et
al. 2010). Although macrophages are also detectable in the affected lesions, down-regu-
lation of activated markers such as MRP-14 or MRP-8 has been observed (Umehara et al.
1994). Even in cases with a long clinical course, active inflammatory pathological change
has been reported in some cases (Iwasaki et al. 2004). Thus, the disease progression of
HAM/TSP is different among individuals. In the brain, perivascular inflammatory infil-
tration was observed in deep white matter and in the marginal area of cortex and white
matter with similar types of infiltrating cells. Taken together, these findings strongly
suggest that immune responses, especially T-cell-mediated immune responses, play a
critical role in the pathogenesis of HAM/TSP.
18.3.2 Detection of HTLV-I-Infected Cells and

HTLV-I Provirus in Spinal Cord Lesions
In general, strong HTLV-I-specific antibody response with high titer is corre-
lated with proviral load, and an increased number of HTLV-I-specific HLA class
I-restricted CD8+ cytotoxic T lymphocytes (CTLs) is one of the characteristic fea-
tures of HAM/TSP. These observations support the presence of persistent HTLV-I
proviral expression in vivo. In addition, CD4+ T cells are recognized as a predomi-
nant target of HTLV-I infection. In PBMC of patients with HAM/TSP, despite high
HTLV-I proviral loads being observed at higher levels than in asymptomatic carri-
ers, HTLV-I tax-expressing cells are hardly detected. As HTLV-I tax has functions
to activate cellular genes including inflammatory cytokines, HTLV-I tax-expressing
cells are believed to play important roles in the pathogenesis of HAM/TSP. On the
other hand, tax includes a dominant epitope recognized by HTLV-I-specific CD8+
CTLs. These findings have suggested that strong HTLV-I tax expression is necessary
to increase the number of HTLV-I-specific CD8+ CTLs. In the CSF of patients with
HAM/TSP, HTLV-I proviral load and HTLV-I tax expression were reported more
frequently than in the PBMC. It is noteworthy that HTLV-I proviral load has been
shown to have a significant association with clinical progression and with recent
onset of HAM/TSP. In the spinal cord lesions of patients with HAM/TSP, pathologi-
cal studies have revealed that HTLV-I DNA was localized to inflammatory infiltrat-
ing UCHL-1-positive cells (suggestive of T cells) but not in CD68+ cells (suggestive
of macrophages) around the perivascular areas in the affected lesions by PCR in
situ hybridization (PCR-ISH) (Matsuoka et al. 1998). The percentage of PCR-ISH-
positive T cells was estimated at about 10% of infiltrated T cells in active chronic
lesions. Another study showed that Tax mRNA expression was detected in infil-
trating CD4+ T cells in active lesions by PCR-ISH (Moritoyo et al. 1996). Among
the CD4+ T cells, CD45RO+helper/inducer T cells were reported as major HTLV-
I-harboring cells in vivo. Furthermore, quantitative PCR analysis showed that the
Peripheral blood Blood–brain barrier Central nervous system
HTLV-I-infected T cells Proliferation of infected T cells CNS resident cells

(mainly CD4+ T cell) Expansion of viral antigens
CD4+ CD4+ CD4+ CD4+

CD4+
Immune response to
CD4+ infected T cells
CD8+ CD4+
CTL Migration Bystander
CD8+ CD4+ damage
CTL
CD8+ CD8+ CD8+ CD8+ Cytokine

CD8+
CTL CTL CTL CTL CTL
Microglia
B cell
Migration Antibody to self-antigen
B cell (ex., anti-hnRNP-A1ab)?
Immunoglobulin
FIGURE 18.4 Hypothesis on the pathogenesis of HAM/TSP. Immune responses of CD8+

CTL to HTLV-I-infected T cells (mainly CD4; T cells) produce cytokines, which would dam-
age bystander neuronal cells. Autoimmunity to self-antigen on the neuronal cells (for exam-
ple, antibodies to hnRNP-A1 cross-reacted with HTLV-1-tax) is still uncertain. ab, antibody;
CNS, central nervous system; CTL, cytotoxic T lymphocytes; hnRNP-A1, heterogeneous
nuclear ribonuclear protein A1.
amount of HTLV-I DNA decreased in parallel with the number of infiltrating CD4+
T cells in the affected spinal cord. These findings suggest that infiltrating CD4+ T
cells, especially those with HTLV-I tax expression, should be a preferential viral
reservoir in the CSF. Although humoral immunity might also play a role in the devel-
opment of HAM/TSP, definitive pathological data have not been reported (Levin et
al. 1998; Yukitake et al. 2008a). Taken together, these data strongly indicate that
T-cell-mediated immune responses against HTLV-I-infected cells (mainly CD4+ T
cells) play a main role in the pathogenic mechanism of spinal cord injury in HAM/
TSP patients. Furthermore, because there is no evidence that HTLV-I infects neu-
ronal cells, neuronal cell damage is interpreted as a bystander effect (Figure 18.4).
18.3.3 Clinical Features of HAM/TSP

It should be helpful to understand the history of HAM/TSP discovery to shed light
on the clinical features of HAM/TSP. In Japan, HAM/TSP was established from dis-
ease entities such as primary lateral sclerosis and spinal spastic paraparesis (Osame
and Igata 1989), which show gait disturbance with upper motor symptoms and patho-
logical reflexes. Therefore, clinical features of HAM/TSP resemble those of such
neurodegenerative diseases to some degree.
HAM/TSP is characterized by spastic paraparesis with the presence of anti-

HTLV-I antibodies in both serum and CSF (Table 18.2) (Osame 1990). The weakness
of lower extremities is symmetric, and disease progression usually occurs slowly. In
addition, in some cases, symptoms are static after initial progression. In over 60%
of patients with HAM/TSP, weakness of the lower limbs is the first symptom. In con-
trast, weakness in the arms is rarely present. Almost all patients show spastic weakness
TABLE 18.2
Diagnostic Guidelines for HAM/TSP
I. Clinical criteria
The florid clinical picture of chronic spastic paraparesis is not always seen when the patient first
presents. A single symptom or physical sign may be the only evidence of early HAM/TSP.
A. Age and sex incidence
Mostly sporadic and adult, but sometimes familial, occasionally seen in childhood; females
predominant
B. Onset
This is usually insidious but may be sudden
C. Main neurological manifestations
1. Chronic spastic paraparesis, which usually progresses slowly, sometimes remains static
after initial progression
2. Weakness of the lower limbs more marked proximally
3. Bladder disturbance usually an early feature. Constipation usually occurs later; impotence
or decreased libido is common
4. Sensory symptoms such as tingling, pins and needles, burning, etc., are more prominent
than objective physical signs
5. Low lumbar pain with radiation to the legs is common
6. Vibration sense is usually impaired; proprioception is less often affected
7. Hyperreflexia of the lower limbs, often with clonus and Babinski sign
8. Hyperreflexia of upper limbs; positive Hoffmann and Trömner signs are common; weakness
may be absent
9. Exaggerated jaw jerk in some patients
D. Less frequent neurological findings
Cerebellar signs, optic atrophy, deafness, nystagmus, other cranial nerve deficits, hand tremor,
absent, or depressed ankle jerk. Convulsions, cognitive impairment, dementia, or impaired
consciousness are rare
E. Other neurological manifestations that may be associated with HAM/TSP:
Muscular atrophy, fasciculations (rare), polymyositis, peripheral neuropathy,
polyradiculopathy, cranial neuropathy, meningitis, encephalopathy
F. Systemic nonneurological manifestations that may be associated with HAM/TSP:
Pulmonary alveolitis, uveitis, Sjören syndrome, arthropathy, vasculitis, ichthyosis,
cryoglobulinemia, monoclonal gammopathy, adult T-cell leukemia/lymphoma
II. Laboratory diagnosis
A. Presence of HTLV-1 antibodies or antigens in blood and cerebrospinal fluid (CSF)
B. CSF may show mild lymphocyte pleocytosis
C. Lobulated lymphocyte may be present in blood and/or CSF
D. Mild to moderate increase of protein may be present in CSF
E. Viral isolation when possible from blood and/or CSF
in the lower extremities, hyperreflexia, and extensor plantar responses. Although the
strength of the arms is usually preserved, brisk deep tendon reflexes tend to develop in
the upper extremities (Nakagawa et al. 1995). Bladder dysfunction is the other common
symptom in HAM/TSP patients. Patients often experience urinary frequency, urgency,
or incontinence (Oliveira et al. 2007). Coexistence of irritative and obstructive urinary
dysfunction is characteristic of HAM/TSP, and urinary symptoms sometimes occur
before the development of weakness of the lower extremities. Urodynamic studies usu-
ally reveal an overactive bladder, and detrusor sphincter dyssynergia (DDS) is also com-
mon. Constipation, back pain, and sensory disturbance/numbness in the lower limbs are
also common symptoms. Numbness in the lower limbs is usually mild. In patients with a
longer clinical course, autonomic dysfunctions such as dyshidrosis, orthostatic hypoten-
sion, and impotence are also observed. In addition, small numbers of patients show finger
tremor, cerebellar signs, and mild cognitive impairment.
The main laboratory finding is high antibody titers against HTLV-I in both serum
and CSF (Osame 1990). Atypical lymphocytes called “flower cells” are some-
times observed in peripheral blood and CSF (Figure 18.5) (Osame and Igata 1989).
Various systemic laboratory abnormalities are also found in HAM/TSP patients.
Hypergammaglobulinemia and increased β2-microglobulin are also found in the
serum. In the CSF, pleocytosis and elevation of protein concentration are com-
mon abnormal findings. Increased neopterin concentration in CSF is also observed
(Nakagawa et al. 1995; Nomoto et al. 1991). The presence of oligoclonal IgG bands,
elevated concentrations of various cytokines such as TNF-α and IL-6, and increased
intrathecal antibody synthesis specific for HTLV-I have also been reported (Hollsberg
and Hafler 1993; Link et al. 1989; Osame et al. 1987).
FIGURE 18.5 “Flower cell” in the peripheral blood. Flower cells are atypical lymphoid
cells with lobulated nuclei. They are commonly observed in the peripheral blood of HTLV-1
infected individuals, but less common in the CSF of HAM/TP patients. (Courtesy of Dr.
Fukushima, Division of Hematology, Department of Internal Medicine, Faculty of Medicine,
Saga University, Saga, Japan.)
On neuroradiological aspects, thoracic cord atrophy without signal intensity

changes on magnetic resonance images (MRI) is accepted to be characteristic of
HAM/TSP, but its incidence is varied, ranging from 20% to 74% (Alcindor et al.
1992; Bagnato et al. 2005; Ferraz et al. 1997). In addition, reports of HAM/TSP
patients with signal intensity changes on MRI have been reported (Shakudo et al.
1999; Umehara et al. 2006; Watanabe et al. 2001). In our study, the MRI find-
ings of the spinal cord can be classified into three types: “normal” (57.9%), “atro-
phy” (34.2%), and “T2 hyperintensity” (7.9%) (Figure 18.6). Spinal cord atrophy on
(a)
(c)
(b) (d)
FIGURE 18.6 Spinal cord MR images in the atrophy, and T2 hyperintensity type of HAM/
TSP patients. Diffuse spinal cord atrophy (arrow in panel b) was observed on T2WI in the
atrophy type (a, sagittal image; b, axial image of the thoracic cord at the Th7 spine level).
Diffuse hyperintensity areas (arrows in panels c and d) were observed on T2WI (c, sagittal
image at the cervical spine; d, axial image of the cervical cord at the C7 spine level).
MRI was shown to have little value for the prediction of prognosis of disability or
responsiveness to interferon α therapy. In contrast, HAM/TSP patients showing T2
hyperintensity tend to show subacute onset and rapid progression of severe parapa-
resis of lower extremities (Yukitake et al. 2008b). Chronic progressive HAM/TPS
patients with T2 hyperintensity in the cervical cord were also reported (Umehara
et al. 2004).
A subacute progressive form of HAM/TSP, which progresses to a severe stage
within a few months, is also known (Lima et al. 2007; Nakagawa et al. 1995; Yukitake
et al. 2008b). Nakagawa et al. (1995) found 14 patients (9.2%) showing rapid pro-
gression of motor impairments within two years among 153 HAM/TSP patients. As
mentioned above, the subacute progressive form of HAM/TSP sometimes shows T2
hyperintensity on spinal MRI. Furthermore, the incidence of the subacute progres-
sive form of HAM/TSP (9.2%) is similar to that of T2 hyperintensity on spinal MRI
(7.9%). Taking these finding together, the incidence of a clinically malignant form of
HAM/TSP, which usually shows T2 hyperintensity on spinal MRI, is estimated to
be less than 10%. In laboratory findings, the subacute progressive form of HAM/TSP
tends to show increased CSF IgG levels, high CSF anti-HTLV-I antibody titers, and
increased CSF neopterin concentration (Kuroda et al. 1991; Nakagawa et al. 1995).
For the treatment of such rapid progression, high doses of methylprednisolone are
sometimes given intravenously, but the efficacy is limited. HAM/TSP patients with
suspected HTLV-I infection via blood transfusion or organ transplantation also show
relatively fast progression in some cases (Kuroda et al. 1992). Summarized clinical
features of HAM/TSP are shown in Table 18.3.
TABLE 18.3
Summarized Clinical Features of HAM/TSP
Neurological Manifestations Laboratory/MRI Findings
Main findings Spastic paraparesis Presence of HTLV-I antibody in both
Neurogenic bladder serum and CSF
Common Hyperreflexia of upper limbs Mild pleocytosis in CSF
Impaired vibration sense Elevation of protein in CSF
Sensory disturbance of lower limbs
Low lumbar pain Normal spinal MRI findings (around 60%)
Less common Hand tremor Flower cells in CSF
Cerebellar sign
Peripheral neuropathy Spinal cord atrophy on MRI (over 30%)
Rare Subacute progressive myelopathy T2 hyperintensity on spinal MRI
Dementia (less than 10%)
Leukoencephalopathy
ALS-like manifestation
Note: CSF, cerebrospinal fluid; ALS: amyotrophic lateral sclerosis.

18.4 RISK FACTORS FOR HAM/TSP

The prevalence of HAM/TSP is only 0.1%–5% in HTLV-I-infected individuals, and
the remaining individuals spend their lifetime as asymptomatic carriers. The life-
time risk of developing HAM/TSP among carriers is estimated to be 0.23% in Japan.
Therefore, HTLV-I is necessary but not sufficient to develop HAM/TSP. Although
the crucial risk factors for the development of HAM/TSP among HTLV-I carri-
ers are still unknown, several factors have been considered. Female predominance
(female/male, 2–2.3:1) has been reported. Although HAM/TSP can develop at all
ages, many HAM/TSP patients notice the neurological impairments at middle age.
HTLV-I PVL is considered as an important risk factor for the development of HAM/
TSP. Several studies have shown that HTLV-I PVL in PBMCs of HAM/TSP patients
is about five to sixteen fold higher than that of asymptomatic carriers (Hashimoto
et al. 1998; Kubota et al. 1993; Nagai et al. 1998). It has also been reported that the
prevalence of HAM/TSP increased as HTLV-I PVL exceeded 1% in PBMCs. In
CSF, HTLV-I PVL of HAM/TSP patients also increased compared with that in the
PBMCs. In particular, it is noteworthy that the ratio of HTLV-I PVL in CSF cells to
that in PBMCs was significantly associated with clinical progression of HAM/TSP
and with recent onset of HAM/TSP (Takenouchi et al. 2003). These observations
strongly suggest that increased HTLV-I PVL is a strong risk factor associated with
clinical progression of HAM/TSP. HTLV-I tax expression is also considered to be
a risk factor for HAM/TSP. The level of HTLV-I tax mRNA expression in HTLV-I-
infected cells was significantly higher in HAM/TSP patients than in asymptomatic
carriers (Yamano et al. 2002), and this finding correlated with the HTLV-I provi-
ral load, Tax-specific CD8+ T-cell frequency, and disease severity. It has also been
shown that persistent HTLV-I gene expression in vivo was necessary for the main-
tenance of HTLV-I PVL (Asquith et al. 2007). In the view of genomic integrations,
it was reported that transcriptionally active genomic regions in HTLV-I determine
the rate of HTLV-I proviral expression (Meekings et al. 2008). It has been suspected
that the genomic integration of HTLV-I to certain regions may induce high HTLV-I
tax expression, and subsequent tax-induced proliferation that defines HTLV-I PVL.
Thus, both HTLV-I tax expression and HTLV-I PVL may drive the expansion of
HTLV-I-specific CTLs (Bangham et al. 2009).
Although HTLV-I tax expression is thought to be a risk factor, HTLV-I tax expres-
sion in PBMCs of HAM/TSP is less frequent. Recently, HBZ has also been consid-
ered as not only part of the pathogenesis of HAM/TSP but also having a correlation
with disease severity, with much higher expression than HTLV-I tax (Saito et al.
2009; Satou et al. 2011). It has been reported that HTLV-I HBZ mRNA load in
PBMCs was significantly correlated with HTLV-I PVL, neopterin concentrations in
CSF, and motor disability in HAM/TSP patients (Saito et al. 2009).
Although HTLV-I PVL is an important risk factor for HAM/TSP, HTLV-I PVL
observed in both HAM/TSP patients and asymptomatic carriers is varied and over-
lapping (Nagai et al. 1998). Interestingly, HTLV-I PVL in asymptomatic carriers
with HAM/TSP in their families was higher than in asymptomatic carriers without
HAM/TSP in their families. On the other hand, it has been thought that the sequence
of HTLV-I varies little other than in terms of the tax gene within or between hosts.
These data suggest that genetic factors in hosts cause variation in HTLV-I PVL among
HTLV-I-infected individuals. In human leukocyte antigen (HLA) class I, HLA-A*02
and Cw*08 were found to be independently associated with a lower risk of develop-
ing HAM/TSP. The association between these two class I alleles and low HTLV-I PVL
was observed in an asymptomatic carrier group (Jeffery et al. 2000; Vine et al. 2002).
On the other hand, HLA-B*5401 was associated with higher HTLV-I PVL and an
increased risk of developing HAM/TSP (Vine et al. 2002). These results suggest that
HLA class I-restricted immune responses influence HTLV-I PVL. In HLA class I, HLA-
DRB1*0101 increased the risk of HAM/TSP (Jeffery et al. 2000). Interestingly, these
associations between HLA genotypes and susceptibility to HAM/TSP sometimes show
different results among ethnic groups. Other genetic factors have also been reported.
Polymorphisms of TNF-α, stromal-cell-derived factor 1, and IL-15 were shown to influ-
ence the outcome of HTLV-I infection (Vine et al. 2002). Polymorphism in the IL-10
promoter was also reported to affect both HTLV-I PVL and risk of developing HAM/
TSP (Sabouri et al. 2004). On the viral side, an association between HTLV-I tax gene
sequence variation and the risk of HAM/TSP was reported. This previous study dem-
onstrated that the HTLV-I tax subgroup A was more frequently observed in HAM/TSP
than in asymptomatic carriers (Furukawa et al. 2000).
18.5 TREATMENT OF HAM/TSP

Although numerous therapeutic trials have been reported, there is no definitive treat-
ment for HAM/TSP (Izumo et al. 1996; Nagai and Osame 2002; Nakagawa et al.
1996; Nakamura 2009). On the basis of the pathogenesis of HAM/TSP as mentioned
above, two main strategies should be considered: immunomodulatory therapy and
antiviral therapy. The strategies of immunomodulatory therapies for the suppression
of chronic inflammatory status with immune activation can be characterized as fol-
lows: suppression of immune activation, especially that caused by HTLV-I-infected
cells, inhibition of the transmigration of HTLV-I-infected cells into the spinal cord,
and reduction of chronic inflammation in the spinal cord via the down-regulation of
proinflammatory cytokines and/or adhesion molecule expression. On the other hand,
the therapies focusing on antiviral effects are divided mainly into three parts: sup-
pression of HTLV-I expression and/or replication, inhibition of the proliferation of
HTLV-I-infected cells, and elimination of HTLV-I-infected cells.
18.5.1 Immunomodulatory Therapy for the Treatment of HAM/TSP

18.5.1.1 Interferon α and β
IFN-α and IFN-β are classified as type I IFNs and have various biological func-
tions including immunomodulation and antiviral effects. Among various therapeu-
tic agents for HAM/TSP, IFN-α is the only agent to have been proven as effective
in multiple, randomized, double-blind, and controlled trials (Izumo et al. 1996).
Therapeutic effects were observed in a dose-dependent manner, and significant
improvements of motor and urinary dysfunctions were observed in about 70% of
HAM/TSP patients treated with 3.0 MU of IFN-α. Therapeutic benefits continued
for 4 weeks after the trial without serious adverse effects. Significant decrease of
spontaneous PBL proliferation in vitro was observed. HTLV-I proviral loads in the
peripheral blood were significantly decreased in combination with the reduction
of memory T cells among CD8high+ T cells. These observations suggested that the
reduction of HTLV-I proviral loads or HTLV-I tax mRNA expression in the periph-
eral blood occurred under IFN-α therapy for HAM/TSP. These findings probably
show that one of the immune mechanisms of IFN-α therapy for HAM/TSP is a cor-
rection of Th1/Th2 imbalance, which is thought to deviate toward Th1 in HAM/TSP.
IFN-β has also been reported for the treatment of HAM/TSP (Oh et al. 2005). As
well as the improvements of motor dysfunctions, reductions of HTLV-I tax mRNA
load and the frequency of HTLV-I-specific CD8+ T cells were observed in the periph-
eral blood. Significant decrease of spontaneous PBL proliferation in vitro was also
observed. Interestingly, HTLV-I proviral loads in the peripheral blood remained
unchanged.
Although the effects of HTLV-I proviral loads were different, both IFN-α and
IFN-β have been thought to have efficacy for the treatment of HAM/TSP via immuno
modulatory mechanisms such as correction of Th1/Th2 imbalance.
18.5.1.2 Corticosteroid Hormone

Corticosteroid has been shown to be an efficient agent for various inflammatory
and autoimmune diseases. As HAM/TSP is a chronic inflammatory disease in spi-
nal cord showing immunopathological features, corticosteroid has been one of the
most popular agents for the treatment of HAM/TSP. Although high doses of meth-
ylprednisolone are sometimes given intravenously, oral prednisolone (PSL) has been
the most popular treatment for HAM/TSP. Short-term efficacy of PSL treatment
for HAM/TSP has been reported (Nakagawa et al. 1996). In this study, 107 of 131
HAM/TSP patients (81.7%) showed improvement of motor functions. On the other
hand, data showing nonefficient or transient benefit of PSL treatment for HAM/TSP
have also been reported. In addition, the long-term efficacy of PSL treatment for
HAM/TSP is unclear because of several adverse effects such as opportunistic infec-
tions, gastric ulcer, glucose intolerance, hypertension, and osteoporosis. Although
the efficacy of PSL treatment for HAM/TSP seems to be limited, it has to be men-
tioned that HTLV-I proviral loads in PBMCs of HAM/TSP patients were signifi-
cantly decreased in PSL treatment over 5 years.
18.5.1.3 Other Immunomodulatory Agents for the Treatment of HAM/TSP

Blood purification (plasmapheresis and lymphocytapheresis), oral administration of
pentoxifylline, intravenous heparin administration, high-dose intravenous gamma-
globulin administration, intermittent high-dose oral administration of vitamin C,
intravenous administration of fosfomycin followed by oral administration of eryth-
romycin, and fermented milk drink were applied in attempts to treat HAM/TSP
(Nakagawa et al. 1996; Nakamura et al. 2009). In general, these agents had less
adverse effects, but the efficacy for HAM/TSP tended to be limited and transient.
18.5.1.4 Antiviral Therapy for the Treatment of HAM/TSP

As mentioned above, IFN-α and IFN-β are considered as having both antiviral
effects and immunomodulatory effects.
Some nucleoside reverse-transcriptase inhibitors, such as zidovudine, lamivudine,

tenofovir, abacavir, zalcitabine, and stavudine, have successfully inhibited HTLV-I
replication (Hill et al. 2003). Treatment with zidovudine, a thymidine analogue, at
a high dose in 10 HAM/TSP patients for 24 weeks showed clinical benefits in some
patients without referring to the changes of HTLV-I proviral loads. In a lamivudine
therapeutic trial on 5 HAM/TSP patients, significant reduction of HTLV-I proviral
loads in the PBMCs was observed in all 5 patients, but clinical improvements were
observed in only 1 patient. However, a clinical trial with combination therapy by
zidovudine and lamivudine in a randomized, double-blind, placebo-controlled study
showed no significant results in terms of both clinical features and laboratory find-
ings (Taylor et al. 2006).
The use of anti-Tac, an antibody to the IL-2 receptor (IL-2R), was also attempted
for the treatment of HAM/TSP patients. Productions of IL-2 and IL-2R by HTLV-I
tax transactivation in HTLV-I-infected cells are thought to dysregulate cellular gene
expression by HTLV-I tax, and this dysregulation is believed to initiate a process of
T-cell activation and proliferation by autocrine or paracrine loop. Thus, IL-2 and/or
IL-2R blockage might decrease HTLV-I-infected cells through apoptosis of HTLV-
I-infected cells by IL-2 deprivation. Treatment for HAM/TSP patients using dacli-
zumab, humanized anti-Tac antibody, showed mild improvement of motor disability
in three of nine patients without serious adverse effects (Lehky et al. 1998). In terms
of anti-HTLV-I effects, selective down-regulation of activated T cells expressing
IL-2R receptor in the PBMCs and a decrease of spontaneous proliferation of PBMCs
were observed. In addition, HTLV-I proviral load in the PBMCs was markedly
reduced after the treatment. These results suggest that humanized anti-Tac antibody
has the efficacy to reduce IL-2R-presenting HTLV-I-infected cells in the peripheral
blood of HAM/TSP patients.
Anti-CC chemokine receptor 4 (CCR4) antibodies, which is another humanized
antibody, might also be considered for use in the treatment of HAM/TSP in future.
Anti-CCR4 antibody has a strong antibody-dependent cellular cytotoxic effect, and
favorable results in a phase I study of anti-CCR4 antibody focused on the treat-
ment of relapsed CCR4-positive ATL and peripheral T-cell lymphoma have been
presented (Yamamoto et al. 2010).
Some favorable anti-HTLV-I results for histone deacetylase enzyme inhibitor and
prosultiamine have also been reported in the treatment of HAM/TSP (Nakamura
2009; Nakamura et al. 2009).
18.5.2 Symptomatic Treatment
Symptomatic treatment is still an important arm of HAM/TSP therapy because many
of these therapies are tolerable in the long term (Araujo and Silva 2006; Goncalves et
al. 2010). Antispastic drugs are used to reduce spasticity, but the efficacy is usually
limited. Rehabilitation programs are also useful for HAM/TSP patients. In progres-
sive cases of HAM/TSP, patients often use a cane and/or a wheelchair. For neuro-
logical bladder, the best bladder management is intermittent cleaning catheterization
associated with an anticholinergic drug and an antispastic muscle agent. Because
urinary tract infections, such as cystitis and pyelonephritis, are common in HAM/
TSP patients, antibiotic agents are used when the active infections are observed.
Renal and ureteral lithiasis, vesicoureteral reflux, and chronic renal failure some-
times coexist with HAM/TSP. Analgesics are also used for the treatment of pain and
dysesthesia associated with myelopathy, elevated muscle tones, and joint contrac-
tures. Constipation is a very common bowel dysfunction. Not only the use of laxative
products, but also adequate and timely food and fluid intake, should be considered.
18.6 OTHER NEUROMUSCULAR DISORDERS

ASSOCIATED WITH HTLV-I INFECTION
It is also known that people with HTLV-I infection sometimes develop not only
HAM/TSP and ATL but also other nonneuromuscular or neuromuscular disorders.
The same as for HAM/TSP, the pathogenic association between these disorders and
HTLV-I infection is still unclear.
In nonneuromuscular disorders, uveitis, alveolitis, arthritis, dermatitis, opportu-
nistic infections, Sjögren syndrome, and SLE have been reported as possibly associ-
ated with HTLV-I infection (Goncalves et al. 2010; Verdonck et al. 2007).
In neuromuscular disorders, the following disorders have been discussed (Araujo
and Silva 2006; Goncalves et al. 2010; Verdonck et al. 2007). Inflammatory muscle
disorders in HTLV-I-infected individuals with and without myelopathy have been
reported. Pathological studies have demonstrated that HTLV-I proviral DNA and
HTLV-I tax expression were observed in the infiltrating cells but not in muscle fibers
in patients with HTLV-I-seropositive polymyositis (Higuchi et al. 1995). In inclu-
sion body myositis (IBM), which is also common in elderly people, it has also been
observed that HTLV-I proviral DNA and HTLV-I tax mRNA were present in the
infiltrating cells (Matsuura et al. 2008). Although peripheral neuropathies sometimes
coexist in HAM/TSP, those in HTLV-I-infected individuals without myelopathy have
also been reported (Kiwaki et al. 2003). The incidence of peripheral neuropathies
in HTLV-I-seropositive individuals is reportedly higher (8.6%) than that in HTLV-
I-seronegative individuals (2.6%). Other neurological impairments such as amyo-
trophic lateral sclerosis-like syndrome, cognitive deficits, depression, dysautonomia,
leukoencephalopathy, cerebellar ataxia, ophthalmological diseases, and meningitis
are reported in HTLV-I-seropositive individuals (Araujo and Silva 2006; Goncalves
et al. 2010; Verdonck et al. 2007). It is important that we consider the difficulty in
proving a true association between HTLV-I infection and these neurological mani-
festations. Unlike for HAM/TSP, we have limited data to prove strong associations
between HTLV-I infection and these neurological manifestations.
18.7 HTLV-II AND NEUROLOGICAL DISEASES

HTLV-II is closely related to HTLV-I with approximately 70% genomic homology.
HTLV-II is endemic in Native Americans and epidemic in injecting drug abusers
worldwide. The mode of transmission of HTLV-II is similar to that of HTLV-I.
Breastfeeding, sexual transmission, contaminated blood products, and intravenous
drug abuse are established modes of transmission. Clinical features of HTLV-II-
associated neurological diseases are also similar to those of HAM/TSP (Araujo
and Hall 2004). Although HTLV-II-associated diseases have been reported less
frequently than HAM/TSP, the main neurological feature is chronic progressive
myelopathy resembling HAM/TSP, but with much lower frequency. Spinal cord atro-
phy has been the most common abnormal feature on spinal MRI. Abnormal T2 high
intensities with or without cord swelling have also been reported on spinal MRI. On
head MRI, high-intensity signals in the periventricular and subcortical white matter
on T2-weighted images are frequent abnormal features. In contrast with HTLV-I,
the role of HTLV-II in the development of neurological disorders has been much less
clear. In addition, concomitant HIV infection makes its difficult to prove the exact
association between HTLV-II and such neurological diseases.
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19 Human
Immunodeficiency Virus
Neuropathogenesis
Ritu Mishra and Sunit K. Singh
CONTENTS
19.1 Introduction................................................................................................. 457
19.2 Brain Cells: As HIV Reservoir and Executor of the Neuroinflammation..... 458
19.3 Role of HIV Proteins in Neuropathogenesis............................................... 461
19.4 Cytokines: Additive Role in Neuropathogenesis......................................... 462
19.5 Neuronal Damage........................................................................................ 463
19.5.1 Excitotoxicity and Oxidative Stress............................................... 463
19.5.2 Impairment of Neurogenesis..........................................................466
19.6 HIV-Associated Neurological Disorders.....................................................466
19.7 General Symptoms and Classification of Neuro-AIDS............................... 467
19.7.1 Asymptomatic Neurocognitive Impairment...................................468
19.7.2 Mild Neurocognitive Disorder.......................................................468
19.7.3 HIV-Associated Dementia.............................................................468
19.8 Diagnosis of HIV-Associated Neurological Disorders (HAND)................469
19.9 HIV-Associated Neuropathologies and Association of
Opportunistic Infections.............................................................................. 470
19.10 Treatment: CNS Complications and HAART............................................. 471
References............................................................................................................... 473
19.1 INTRODUCTION
Inflammation encompassing the brain tissues as a result of virus infection is known
as viral encephalitis. There are many viruses responsible for viral encephalitis, and it
is one of the emerging health issues worldwide (Shoji et al. 2002). To combat against
viral encephalitis, a better understanding of events that occur within the central ner-
vous system (CNS) after viral exposure is needed. Viral infections immensely activate
the host immune responses at periphery and in CNS, results in neuroinflammation and
acts as a key process in the viral neuropathogenesis. Human immunodeficiency virus
1 (HIV-1) is well studied (Wang, Rumbaugh, and Nath 2006) among other viruses
responsible for encephalitis or dementia. Within the retrovirus family, HIV belongs
to subgroup known as lentiviruses (Worlein et al. 2005) and responsible for acquired
457
immunodeficiency syndrome (AIDS). HIV has two copies of single-stranded RNA

in its genome. The transcript produced from the viral promoter is approximately 10
kb long. This transcript contains seven structured subdomains throughout its length,
namely long terminal repeats (LTR), transactivating region (TAR), rev responsive ele-
ment (RRE), psi elements (PE), a TTTTTT slippery site (SLIP), cis-acting repressive
sequences (CRS), and inhibitory/instability RNA sequences (INS). The ends of each
strand of HIV RNA contain an RNA sequence called LTR. LTR regions are reported
to have promoter sequences. The HIV genome encodes nine open reading frames
(ORFs), namely group specific antigen (gag), polymerase (pol), envelope (env), trans-
activator of transcription (tat), regulator of virion protein expression (rev), negative
effector (nef), viral infectivity factor (vif), viral protein R (vpr), and viral protein U
(vpu) (Johri et al. 2011). Broadly, these genes can be categorized into two categories,
structural and nonstructural genes. The gag, pol, and env genes are structural genes in
HIV genome, which contain information to make the structural proteins for new virus
particles. Gag, Pol, and Env are synthesized as polyproteins, which subsequently get
proteolyzed into individual proteins (Frankel and Young 1998). Nonstructural genes
can be further classified as accessory and regulatory proteins. Accessory proteins
include Vif, Vpr, Vpu, and Nef, which are unique to primate lentiviruses (Johri et al.
2011). Whereas, HIV Rev and Tat proteins are designated as HIV regulatory proteins.
HIV is known to infect cells of the host’s immune system, causing persistent and
chronic infection (Narayan and Clements 1989). HIV is neurovirulent and manifests
neurological complications (Williams et al. 2008). More than 65 million people glob-
ally have been infected with the HIV since its discovery in the early 1980s (Power
et al. 2009). Neurological disorders caused by HIV infection covers both the central
and the peripheral nervous systems (McArthur et al. 2005). Neurological complica-
tions relevant to CNS have been reported in more than 50% of untreated HIV patients
(Clifford 2008). In addition to dementia symptoms, AIDS patients show some com-
mon viremia-related symptoms such as fever, skin rash, oral ulcers, and lymphade-
nopathy after 2–3 weeks of primary HIV infection (Pope and Haase 2003). However,
HIV can enter into nervous suystem at any stage of infection and can have adverse
effects on overall quality of life (Power et al. 2009). The neuropathology of primary
HIV-1-associated CNS disorders, characterized as HIV-1-associated neurocognitive
disorders (HANDs) (Antinori et al. 2007) and that can be distinguished from second-
ary processes such as opportunistic infections or malignancies.
The development of HIV-associated dementia (HAD) or AIDS dementia complex
(ADC) is a unique syndrome and is characterized by clinical manifestations such
as neurocognitive impairment (forgetfulness, poor concentration and comprehen-
sion, slowed mental processing), accompanied by emotional disturbances (agitation,
apa thy), and followed by motor dysfunctions (tremor, bradykinesia, ataxia, and spas-
ticity) (Power et al. 2009).
19.2 BRAIN CELLS: AS HIV RESERVOIR AND EXECUTOR

OF THE NEUROINFLAMMATION
HIV is known to enter the brain soon after infection (Figure 19.1). The appearance
of meningitis (Carne et al. 1985; Hoffmann et al. 2001) and intrathecal synthesis
Human Immunodeficiency Virus Neuropathogenesis 459
Blood
2
3
4 1
Basement 2
Brain
membrane
Pr
od
uc
tiv
e te d
Restric
?
Productive
?
Astrocyte T cell Olygodendrocyte
Microglia Monocyte Perivascular macrophage
HIV-1 virion Neuron Microvascular endothelial cell
FIGURE 19.1 (See color insert.) HIV-1 neuroinvasion. (1) According to the “Trojan horse”
hypothesis, the entry of HIV-1 into the brain takes place by the migration of infected mono-
cytes, which differentiate into perivascular macrophage. (2) The passage of infected CD4+
T cells can be another source of infection in the brain. Other probable causes of CNS infec-
tion might be (3) the direct entrance of the virus or (4) entrance of HIV-1 by transcytosis of
brain microvascular endothelial cells. Once the virus is in the brain, it productively infects
macrophages and microglia. Astrocyte infection is known to be restricted. The infection of
oligodendrocytes, especially neurons, is questionable. (Reproduced from Ghafouri, M. et al.,
2006, Retrovirology 3, 28. With permission.)
of HIV-1 antibody (Carter et al. 1988) are among the evidence of HIV infection in
brain. HIV-DNA has been detected in the postmortem brain samples of patients
who died with HIV infection (Davis et al. 1992). Many groups have revealed a
condition of immune activation in the brain, with enhanced levels of cytokines
and other proinflammatory factors. Astrocytes and microglia have been primarily
reported to be productively infected by HIV (An et al. 1999). The increased expres-
sion of adhesion molecules (Seilhean et al. 1997), an enhanced astrocyte activation
(Geiger et al. 2006), and loss of astrocytic functions have been reported to attribute
to HIV-associated neuropathology (Saito et al. 1994; Tornatore et al. 1994; Vallat
et al. 1998).
The most accepted mechanism of HIV entry into the brain is the “Trojan horse”
mechanism. The mechanism postulates the role of HIV-infected monocytes/
macrophages crossing the blood–brain barrier (BBB) and disseminating the virus
(Meltzer and Gendelman 1992; Vazeux et al. 1987). Many studies have found the
evidence of HIV infection in astrocytes (Tornatore et al. 1994; Wiley et al. 1986).
It is not well understood whether neuroectodermal cells, particularly neurons, get
infected by HIV. There are few reports stating HIV-1 infection in cerebral endo-
thelial cells (An et al. 1999; Tornatore et al. 1994). Endothelial cell loss can take
place with apoptosis through the HIV proteins secreted out extracellularly from
the HIV infected cells (Acheampong et al. 2005). The microglial cells and mono-
cytes can be called as a long term reservoir and source of transmission of HIV
infection in the CNS. HIV RNA has been detected in a variety of cell types such
as macrophage/microglial cells and multinucleated giant cells (Stoler et al. 1986).
Microglial physiology is the main focus for a cascade of events, which can lead to
neuronal dysfunction and death. Several molecular mediators of neuronal injury in
HAD originate from microglia (Garden 2002). HIV-1 infection in the CNS is cen-
tered around viral replication in cells of glial and macrophage lineage (Gendelman
et al. 1994b), which has been found to be correlated with development of dementia.
Microglial nodules develop much before the onset of AIDS or HIV induced viral
encephalitis (HIVE) and are known as the hallmark of neuro-AIDS (Kibayashi et
al. 1996).
The detailed role of macrophages/microglia in the pathogenesis of HIV-1-
associated neurocognitive impairment has been reviewed extensively elsewhere
(Yadav and Collman 2009). In general, HIV-1 infection and immunopositivity is
restricted to the perivascular compartment, as shown by widespread staining of the
parenchymal microglia (Morris et al. 1999). The privileged areas of CNS act as a
sanctuary site for the persistence of HIV-1, which again turns into a challenging task
from a treatment point of view.
HIV infection and the complexity of disease progression focus on the cytopathic
effects of the infection first in CD4+ T cells, and then later, in the cells of macro-
phage lineage (Pantaleo et al. 1993; Rosenberg and Fauci 1991). The rapid loss in the
number of the CD4+ T-cells in peripheral blood is the reflection of highly productive
infection of HIV in CD4+ T-cells. Such productive HIV infection leads to a loss of
cell-mediated immune (CMI) responses and suppression of Th1 cytokines, such as
Interleukin-2 (IL-2) and Interferon-gamma (IFN-γ) (Dalgleish 1995). This lays the
foundation for the development of state of immunodeficiency in the host and mani-
fests itself in two ways: first, development of selected tumors and turning the host
favorable for various types of opportunistic pathogens (Liu et al. 1999); second, sup-
port the progressive virus replication in the brain (Gendelman et al. 1994a).
Infiltration of infected macrophages into the brain is accompanied by massive
cytokine/chemokine induction (Della Chiara et al. 2010). Pathological changes of
HIV induced neuroinflammation include perivascular accumulations of mono
nuclear cells (Bell 1998; Gendelman et al. 1994b). The mechanism of the neuro-
logical complications in HIV-infected individuals is not well understood. How does
HIV enter into the CNS early during infection and remain slow/silent for such a long
period? It is not well understood whether heightened neurological complications in
final AIDS are due to virus reactivation or due to a renewed phase of viral neuroinva-
sion. However, many groups support the notion that the virus replicates continuously
in the CNS at low levels (Williams et al. 2008).
19.3 ROLE OF HIV PROTEINS IN NEUROPATHOGENESIS

The HIV-1-encoded small nuclear transcriptional activator protein, Tat (trans-activator
of transcription), is the first protein to be expressed in the virus life cycle (Li et al.
2009). Tat is a virally encoded protein that can be released by HIV-1-infected cells
and can activate HIV-infected and/or uninfected cells to release potentially neuro-
toxic substances. Tat has been detected in the extracellular space and sera of infected
individuals (Hudson et al. 2000; Wiley et al. 1996) and may directly interact with
surrounding neurons (Cheng et al. 1998; Tardieu et al. 1992). Anti-tat antibodies
have been reported in the brains of patients with HIV encephalitis (Del Valle et al.
2000). The source of Tat proteins in the CNS is still confusing—whether the Tat
protein is released by infected cells of the brain or transported across the BBB from
peripheral viral sources (Banks et al. 2005).
Tat can contribute to neuropathogenesis through various mechanisms. It can stim-
ulate proinflammatory responses through production of several cytokines includ-
ing Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha (TNF-α), Interleukin-6
(IL-6), and transforming growth factor beta (TGF-β) (Zauli et al. 1992). Tat induces
TNF-α production, activation of different G proteins, and followed by Ca2+ mobili-
zation and activation of the protein kinase C pathway, which might result in down-
stream activation of NF-kB (Contreras et al. 2005). In addition, Tat stimulates the
p38 MAPK and the JAK/STAT pathways, which activate Interferon gamma-induced
protein 10 (IP-10) production. Tat induces host-soluble factors such as IFN-γ, which,
in turn, induces the chemokines as IP-10 production by macrophages (Dhillon et al.
2008). These effects of Tat and other HIV proteins on macrophage/microglia sug-
gest that viral proteins are important contributors to the activation events leading to
HIV-induced neuroinflammation.
Tat exerts a toxic effect on neurons via cytokines, chemokines, and nitric oxide
(NO) released by microglia (Thomas et al. 2009), which in turn leads to the apoptosis.
Astrocytes, neurons, and endothelial cells are equally prone to Tat-induced
inflammatory responses, which can further augment the infiltration of monocytes
into the brain (Pu et al. 2003). Tat is known to significantly contribute in the disrup-
tion of the BBB by altering the distribution of endothelial cell-tight junction proteins
such as claudin 1, claudin 5, ZO-1, and ZO-2, which ultimately leads to enhanced
transmigration of monocytes and lymphocytes (Andras et al. 2003; Toborek et al.
2003) in the brain.
Another HIV-1 protein, gp120, can also be directly neurotoxic by inducing neu-
ronal apoptosis, mainly through interactions with the chemokine receptor CXCR4.
Recombinant gp120 from the R5 strains of HIV-1 is known to induce apoptosis in a
human neuronal cell line. These receptors are expressed on neurons, suggesting the
role of CCR5 in neuronal activation and damage (Xu et al. 2004). gp120 disrupts
calcium homeostasis in neurons, which triggers mitochondrial membrane disrup-
tion and activation of caspases and endonucleases via the intrinsic pathway leading
to apoptosis (Mattson et al. 2005). The extrinsic pathway, which involves the up-
regulation of the death receptor Fas, has also been demonstrated to be a mechanism
for gp120-induced neuronal apoptosis (Thomas et al. 2009).
Vpr is a 96-amino acid HIV-1-encoded virion-incorporated protein and essen-

tial for HIV-1 replication in macrophages (Subbramanian et al. 1998). Vpr has been
recently reported in sustantial amounts in both the basal ganglia and the frontal cor-
tex of HIVE patients. It was mainly found in the resident macrophages and neurons
(Wheeler et al. 2006). Soluble HIV-1 Vpr protein is reported in the serum of HIV-
infected patients with neurological disorders (Levy et al. 1994). In the mouse model,
the expression of HIV-1 Vpr in brain monocytoid cells has been implicated in neu-
ronal injury and other motor dysfunctions (Jones et al. 2007). Exogenous treatment
of soluble Vpr also perturbs the neuronal membrane potentials, leading to apoptosis
(Patel et al. 2000). Vpr directly exert cytotoxic effects on neurons by activating the
glia, which results into the release of neurotoxic substances. Vpr can also alter the
expression of various important cytokines and inflammatory proteins in infected as
well as uninfected cells (Mukerjee et al. 2011). Vpr is known to be taken up by neu-
rons and leads to the deregulation of calcium homeostasis. Vpr can also activate the
oxidative stress pathway involving mitochondrial dysfunction (Mukerjee et al. 2011).
Neuronal injuries by various viral proteins, including Tat, Nef, Vpr, and the Env
proteins gp120 have been explained by multiple theories. HIV envelope proteins,
such as gp120 as well as Tat, have been reported to mimic of many chemokines;
therefore, HIV-induced neuronal injury can be mediated by chemokine receptor sig-
naling. Various experiments showed that blocking of chemokine receptor signaling
can prevent HIV/gp120-induced neuronal apoptosis (Meucci et al. 2000; Zheng et al.
1999), and Stromal cell-derived factor-1 (SDF-1) directly exerts neurotoxic effects
through the stimulation of chemokine receptors (Kaul and Lipton 1999). Various
reports demonstrate the different mechanisms of neuroinflammation through vari-
ous HIV proteins and differential clinical picture by different HIV clades. Models of
HIV infection, including simian and feline immunodeficiency virus infection, have
shown the role of individual viral strains and the specific viral proteins in contribut-
ing to neuroinflammation (van Marle and Power 2005). Differences in genetic sus-
ceptibility at the individual or population level might play an important role because
clinically evident disease affects only a subset of HIV-infected patients, although
HIV-1 neuroinvasion and neurotropism occur in most of the HIV-infected patients
(van Marle and Power 2005). HIV-associated neurological disorders (HANDs) have
also been reported among patients having higher CD4+ T-cell levels, which were
earlier believed to show up among HIV patients having CD4+ T-cell counts below
200 cells/μL of blood (Power et al. 2009).
19.4 CYTOKINES: ADDITIVE ROLE IN NEUROPATHOGENESIS

Microglia and macrophages within the brain release both α- and β-chemokines in
response to HIV infection of the CNS (Lindl et al. 2010). These chemokines and cyto-
kines can bind to neurons because neurons express chemokine receptors (Rottman
et al. 1997). This bystander way of HIV-induced inflammatory response might play
a critical role in HIV infection in the brain. There is experimental evidence showing
the elevated levels of the α-chemokines, CXCL10/IP-10 and CXCL12/SDF-1α, in the
brains and CSF of HAD patients (Cinque et al. 2005). α-chemokines are expressed
in many types of CNS cells constitutively and can have both neuroprotective and
neurotoxic effects (Khan et al. 2008). It can also bind CXCR chemokine receptors
and thereby increases intracellular calcium in G protein-dependent signaling. During
HIV infection, the major chemoattractants, i.e., CCL3, CCL4, CCL5, CCL2, and
CX3CL1, have been implicated in increased trafficking of monocytes into the brain.
Brain autopsy samples of patients having HAD symptoms died with HIV infection
(diagnosed with HAD) have been reported to have infected monocytes (Kanmogne
et al. 2007; Mukhtar and Pomerantz 2000).
In the peripheral circulation, chronic immune activation takes place due to immune
responses against HIV, and extracellularly secreted HIV proteins such as gp120 and
Tat. Immune activation results into the activation of monocytes, which acquire inva-
sive phenotype by induced expression CD16 and CD163. These activated monocytes
have enhanced migratory capacity and cross through the blood brain barrier, whose
integrity is comprised by HIV proteins and other pro-inflammatory mediators gen-
erated by activated cells (“push” mechanism). In the CNS, monocytes differentiate
into macrophages and release infectious viruses, which in turn infect other cells
through the CD4/CCR5 receptor complex. The infected cells release viral proteins
(i.e., gp120 & Tat), express cytokines, chemokines and other proinflammatory fac-
tors, which activate neighbouring uninfected and/or infected cells in bystander fash-
ion. Chemokines such as monocyte chemoattractant protein-1 (MCP-1) and SDF-1α
further recruit monocytes into the CNS (“pull” mechanism). Some of these factors
also activate the brain microvascular endothelial cells (BMVECs), which results into
induced expression of adhesion molecules such as intercellular adhesion molecule-1
(ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on BMVECs, which
helps in the entry of inflammatory cells in brain. These two mechanisms, “push” and
“pull,” to contribute to monocytes/macrophage accumulation and activation inside
CNS (Yadav and Collman 2009). HIV is believed to cross the BBB via the transport
of infected CD4+ T cells and/or monocytes. However, free HIV particles can also
enter the CNS through transcytosis of the BBB, which is not believed to be a major
route.
19.5 NEURONAL DAMAGE

19.5.1 Excitotoxicity and Oxidative Stress
Despite the lack of data supporting widespread productive neuronal infection, it is
clear that neuronal loss occurs in the brain of HIV-infected persons (Adle-Biassette
et al. 1995; Dunfee et al. 2006; Gray et al. 2000). Neuronal damage can happen by
various direct and indirect pathways, induced by viral infection or after exposure
to viral toxic proteins. Both virus and glial cell-derived proteins may contribute to
neuronal damage (Nath et al. 1999) (Figure 19.2). Infected macrophages and micro
glial cells are believed to contribute to pathological changes in the neurons through
the secretion of viral (gp120, Tat, and Nef) and cellular (cytokines, chemokines,
and nitric oxide) products (Kolson and Pomerantz 1996; Lipton and Gendelman
1995). Viral proteins, particularly gp120, gp41, tat, nef, and cytokines, prostaglan-
dins, proteases, arachidonic acid, or quinolinic acid metabolites produced by acti-
vated brain resident cells are attributed to neuroinflammation (Everall et al. 1993).
Blood
Released proteins
1
Brain
gp120 3
2
A Tat
Vpr 6
1 6
Quinolinic acid
2 Secreted factors 3
Arachidonic acid 5
B Nitric oxide
4 IL-1
PAF 6
TNF
Neuronal death
apoptosis
6
Dementia
FIGURE 19.2 (See color insert.) Mechanism of neuropathogenesis. Two components of

this mechanism are (A) the direct effect of the HIV-1 infection, including HIV-1 proteins, and
(B) the indirect consequence of infection comprising the secretion of cytokines and neuro
toxins. The infected macrophages and microglia participate actively in the neurodegeneration
by (1) shedding viral proteins and (2) releasing a significant amount of cytokines and neu-
rotoxins into the CNS. (3) Tat and TNF-α contribute to the disruption of the BBB, which in
turn become more permeable to infected monocytes and cytokines present in the periphery.
The secreted proinflammatory cytokines activates (4) microglia and (5) astrocytes, which
in turn secrete neurotoxins; moreover, the alteration of astrocytes function results in an
increase in the level of neurotoxicity in the brain. (6) Multifactorial neuronal injury: neuro
toxins released from several sources, as the direct and indirect consequences of HIV-1 infec-
tion lead to neuronal injury. (Reproduced from Ghafouri, M. et al., 2006, Retrovirology 3,
28. With permission.)
In particular, HIV Tat protein is known to be transported along neural pathways and
is well described as the cause of neurotoxic damage at remote sites (Bruce-Keller
et al. 2003). These reports emphasized that each of these toxins can damage nerve
cells, but severity increases manifold for neuronal apoptosis in a combination of
both cellular and viral factors (Xu et al. 2004). Many in vitro studies also confirmed
the glutamate-mediated excitotoxicity as a factor of neuroinflammation. Glutamate,
an excitatory neurotransmitter (Fonnum 1984), is known to be involved in HIV-
induced neurotoxicity (Jiang et al. 2001; Zhao et al. 2004). Many studies demon-
strated that astrocytes normally take up glutamate, keeping extracellular glutamate
concentration low in the brain, and thus astrocytes display their neuroprotective role
(Fonnum 1984). In astrocytes, the glutamate gets converted into glutamine via mito-
chondrial glutamine synthetase and transported to neurons for synthesis of GABA
(γ-amino butyric acid), a neurotransmitter (Porcheray et al. 2006). This is known as
the glutamine–glutamate cycle between astrocytes and neurons, which establishes
the brain nitrogen homeostasis. This action is inhibited in HIV infection, probably
due to cellular inflammatory mediators and viral proteins. An increase in extracellu-
lar glutamate concentration contributes to neuronal death through hyperactivation of
N-methyl-d-aspartate receptors (NMDAR), a mechanism addressed as excitotoxic-
ity. Excitatory amino acid transporters (EAAT), are mainly expressed on glial cells,
which ensures the clearance of extracellular glutamate (Gegelashvili and Schousboe
1997). EAAT-1 and EAAT-2 genes are reported to be expressed on glia (Chaudhry
et al. 1995; Danbolt et al. 1992; Lehre et al. 1995) and provide in vivo protection
against glutamate toxicity (Rothstein 1996). Astrocytes play a key role as a neuro-
protector against glutamate stress in the course of HIV infection. In a study, the 6-h
exposure of either HIV-1 gp120 or Tat to astrocytes resulted in a 60% reduction in
glutamate uptake by astrocytes and reduction in EAAT-2 expression but not EAAT-1
(Fallarino et al. 2003; Kort 1998). Considerable loss in EAAT expression in peri-
neuronal microglia in HIV infection has been reported in cases of HIVE (Vallat-
Decouvelaere et al. 2003).
Tat activates the neuronal excitatory NMDAR, leading to excitotoxicity and con-
sequent apoptosis of neurons (Li et al. 2008; Song et al. 2003; Kaul et al. 2001). In
vivo studies showed that production of super oxide anion and nitric oxide is sig-
nificantly increased in demented, compared with AIDS patients without dementia
(Boven et al. 1999).
Tat has been shown to depolarize neurons through direct interaction with neuro-
nal membranes and may act as substrate to increase the aggregation of neural cul-
tures (Nath 2002). A minor exposure ranging in nanomolar concentrations of gp120
can hyperactivate the NMDAR by binding through the glycine-binding sites of the
NMDAR (Fontana et al. 1997). This is another mechanism by which HIV/gp120 or
Tat may exert an adverse effect on neuronal cells. Another HIV-1 protein Vpr is also
now shown to affect cultured hippocampal neurons through formation of a cation-
permeable channel (Piller et al. 1998).
Hyperactivation of the NMDAR provokes intracellular signals leading to apop-
tosis or necrosis of neuronal cells (Bonfoco et al. 1995). In the case of severe excito-
toxic insults, the cells die early through the process of necrosis/apoptosis (Bonfoco et
al. 1995). Molecular mechanism imparting neuronal apoptosis due to excitotoxicity
is diversified involving Ca2+ overload, p38 MAPK activation, release of cytochrome
c from mitochondria, activation of caspases, free radical formation, lipid peroxida-
tion, and chromatin condensation (Budd et al. 2000; Ghatan et al. 2000).
Oxidative stress alters the cellular lipid metabolism, producing harmful mole-
cules, such as ceramide, sphingomyelin, hydroxynonenal, etc., which are observed in
patients with HAND (Sacktor et al. 2004). Detection of oxidized proteins in the CSF
also confirms the role of oxidative stress in HAND (Turchan et al. 2003). Tat and Vpr
are reported to increase oxidative stress in neurons due to mitochondrial dysfunction.
These reports suggest that oxidative stress is an important mode of neuronal death
and subsequent neurodegeneration (Lindl et al. 2010). Neuroprotective potentials of
many antioxidants have been demonstrated in vitro, which establishes that oxidative
stress is an important factor in neurodegeneration (Turchan et al. 2003).
19.5.2 Impairment of Neurogenesis
Adult neurogenesis (ANG) was initially thought to occur only in rodents. Recent
findings show that ANG also takes place in humans and other primates (Eriksson
et al. 1998). ANG is important for maintaining the homeostatic state of CNS and
is involved in learning, memory, olfaction, and anxiety-related behaviors (Revest
et al. 2009). ANG has been reported to get perturbed in HAND (Rodriguez et al.
2008; Taupin 2009). Astrocytes provide trophic support to both mature and imma-
ture neurons, but this support gets impaired in cases of HIV infection in the brain
and that restricts the proliferation and migration of Neural Progenitor Cells (NPCs)
(Eriksson et al. 1998). Maturation and differentiation of NPCs are dependent on
cell cycle regulation (Herrup and Yang 2007). Disruption at the level of cell cycle
proteins such as the transcription factor, E2F1, and its regulator, the retinoblastoma
gene, are reported to be dysregulated in patients having HAND (Hoglinger et al.
2007). Another cell cycle protein doublecortin (dcx) (microtubule protein) expressed
in immature neurons was shown to be disrupted in HAND (Herrup and Yang 2007).
Dysregulated expression of E2F1 disrupts the dcx, which ultimately leads to the
disruption in ANG. Disruption of ANG and its molecular regulation trims down
the plasticity of the CNS, which leads to devastating consequences in brain regions
assaulted by HIV-induced toxicity (Karl et al. 2005).
19.6 HIV-ASSOCIATED NEUROLOGICAL DISORDERS

In 1983, Snider et al. reported for the first time that the underlying pathogen in AIDS
likely affect the CNS. In 1982, the first descriptive report appeared explaining about
the damage to the nervous system due to AIDS (Scaravilli et al. 2007). However,
Snider et al. (1983) are credited with the first systematic description of the CNS
complications of AIDS in a case series of 50 patients. The existence of neurocog-
nitive impairment directly attributable to HIV was not well understood until 1987
(Woods et al. 2009). It is a relatively recent event revealing involvement of the CNS
in AIDS (Snider et al. 1983; Vivithanaporn et al. 2010). In the last three decades,
numerous experimental studies contributed to a better understanding of the HIV
neuropathogenesis.
The neurological complications in AIDS patients are mostly due to HIV infec-
tion in the CNS, and it is also confirmed by viral detection within brain tissues
(Shaw et al. 1985), together with the observation of multinucleated giant cells
(MGC). These cells are considered unique to AIDS and considered as a hallmark of
the syndrome (Budka 1986). The neuropathological abnormalities are collectively
called HIV-associated neurological disorders (HAND). Although the severe form of
HAND is typically ascertained during the late stages of infection. It was later found
that moderate neuropsychological, neurophysiological, and neuroimaging abnor-
malities can occur well before the advanced stages of AIDS (Heaton et al. 1995).
This suggests that HIV neuropathogenesis is a gradual process and HIV-induced
neuroinflammation may begin at any time in the course of infection and can act as a
trigger for the development of HAND.
19.7 GENERAL SYMPTOMS AND

CLASSIFICATION OF NEURO-AIDS
HAND is a broad term given to describe the cumulative anomalies observed and
best correlated with the altered CNS structure and function in HIV-infected indi-
viduals. Based on the stage of infection and AIDS development, the severity of these
disorders varies. Among AIDS patients, about half are symptomatic for neurological
disorders and a few of them fit in the research classification of dementia (Hogan and
Wilkins 2011). Variability can be attributed to disease stage, treatment, and the tech-
niques and diagnostic tools used to detect the impairment (Hogan and Wilkins 2011).
Patients should fulfill certain criteria to be considered as a subject of HAND. The
affected person should demonstrate an acquired deficiency in at least two neurocog-
nitive domains and show impairment in work or activities of daily living (ADLs),
and an abnormality in motor abilities or specified neuropsychiatric functions (e.g.,
motivation, liability, and social behavior) (Woods et al. 2009). However, Grant and
Atkinson elaborated the definitions of the American Academy of Neurology (AAN)
in 1995 and included an additional diagnosis of “subsyndromic neurocognitive
impairment.” The neuro-AIDS patients falling in this category demonstrate mild
neurocognitive deficits that do not interfere with ADLs (Woods et al. 2009).
HAND defines three categories of disorders according to the extent and severity
of dysfunctions (Ghafouri et al. 2006; Valcour et al. 2011) (Table 19.1):
1. Asymptomatic neurocognitive impairment (ANI)

2. Mild neurocognitive disorder (MND)
3. HIV-associated dementia (HAD)
TABLE 19.1
Research Criteria for HIV-Associated Neurocognitive Disorders
Diagnosis Entity Cognitive Performance Functional Performance
Normal cognition Normal Normal
Asymptomatic Acquired impairment in at least Does not impact daily functions
neurocognitive impairment two cognitive domains (<1 SD)
Mild neurocognitive Acquired impairment in at least Interferes with daily function to at
disorder two cognitive domains (<1 SD) least a mild degree (eg, work
ineficiency, reduced mental acuity)
HIV-associated dementia Acquired impairment in at least 2 Marked impact on daily functions
domains, typically in multiple
domains with at least 2 domains
with severe impairment (<2 SD)
Source: Reproduced from Valcour, V., Sithinamsuwan, P., Letendre, S., and Ances, B. (2011). Curr HIV/
AIDS Rep 8(1), 54–61. With permission.
19.7.1 Asymptomatic Neurocognitive Impairment

ANI does not affect everyday functioning among patients. It was added as a diagnosis
for the initial and mild cases of HAND, also referred to as subsyndromic neurocog-
nitive impairment (Grant et al. 1995). This class of neurological impairments repre-
sents over 50% of diagnosed cases, including around 21%–30% of the asymptomatic
HIV-infected individuals (Robertson et al. 2007). These abnormalities, while mild,
are demonstrable, neurocognitive impairment.
It is very important to preidentify those at risk for more significant cognitive as
well as functional deficits. This can be regarded as a window for foreseeing the pat-
terns of individual susceptibility for neurological impairments and make judicial
decisions for further treatment before severe cognitive deficits start affecting the
everyday functioning with detrimental consequences (Woods et al. 2009). In other
words, detection and treatment of patients at this earliest stage of HAND actually
provide the best chance to prevent further progression of disease.
19.7.2 Mild Neurocognitive Disorder

MND subjects show an advanced and severe form of impairment, which involve dif-
ficulties in pursuing daily activities. About 20%–40% of HAND cases are encoun-
tered with MND, which comprises 5%–20% of the HIV population overall (Woods
et al. 2009; Grant et al. 1999).
This category of subjects should meet at least two of the following criteria:
1. Decline in more than two instrumental activities of daily living (ADLs)

(e.g., financial management).
2. Reduced cognitive abilities, followed by significant reduction in job respon-
sibilities and unemployment.
3. Decline in vocational skills, e.g., frequent errors, decreased productivity,
and difficulty in achieving prior levels of skill and performance.
4. Self- or proxy-report of increased problems in areas of day-to-day life activ-
ities (this criterion should exclude self-report of current depression because
depression itself is a kind of biased self-report).
5. Inabilities of subject for everyday functioning (e.g., medication management).
19.7.3 HIV-Associated Dementia
HIV dementia is a result of multifactorial events, and it is difficult to name any single
cellular or viral factor alone as a causative factor. There are many gaps in under-
standing the exact correlation between the cognitive disorders and the different HIV-
induced changes. HAD represents the most severe form of HAND. It includes all the
terms and criteria listed for MND in terms of its functional impact, but all symptoms
are given in their more severe form and chronic disabilities found in AIDS patients.
It also demonstrates a decline in two or more of the cognitive domains accompanied
with marked decline in ADL. In the early 1990s, HAD was thought to be faced by
an extremely wide range (6%–30%) of HIV-infected individuals after onset of AIDS
(Maj et al. 1994; McArthur et al. 1993).
The use of combined antiretroviral therapy (cART) improved the circumstances

by lowering the incidences of HAD, but approximately 4%–7% of persons with
AIDS still experience HAD (Grant et al. 1999). Recent assessments suggest that
only 1%–2% of HIV persons actually meet the criteria for HAD (Grant et al. 2005).
Other neurocognitive impairments such as severe movement abnormalities, i.e.,
chorea, myoclonus, dyskinesia, and dystonia, are not very common in HIV infec-
tion but can be observed in individuals with HAD (Mirsattari et al. 1999). However,
slowed movement (bradykinesia) and slowed information processing (bradyphre-
nia) are the commonly observed features among neuro-AIDS patients (Woods et al.
2009).
Learning and memory dysfunctions are known to be highly prevalent in HIV
infection and reported in almost 40%–60% of AIDS patients (Rippeth et al. 2004).
The level of impairment ranges from mild to moderate but may increase from mod-
erate to severe among patients in advanced stages of AIDS (Reger et al. 2002). The
severity of AIDS affects the attention and memory of patients. During early stages
of HIV infection, basic attention/concentration skills appear relatively unaffected
(Reger et al. 2002). Neuroimaging studies have suggested that increased attention
load increases the frontoparietal activation, leading to impairment in attention and
working memory recollection (Chang et al. 2004). Visuoperception/visuospatial
functioning are very important aspects of cognitive health. These are reported to
be generally unaffected in HIV infection, as the occipital and parietal cortices are
generally not affected very badly in neuro-AIDS. Cognitive and visual impairments,
language impairments, and particularly speech difficulties are also frequently chal-
lenged cognitive features among HIV-infected children (Wolters et al. 1997), com-
pared to adults. Language and speech irregularities appear more frequently in HIV
patients with CNS opportunistic infection (e.g., in progressive multifocal leukoen-
cephalopathy). Thorough investigations of spatial cognition are needed to charac-
terize the nature and origin of various neurological impairments in HIV-infected
patients.
19.8 DIAGNOSIS OF HIV-ASSOCIATED
NEUROLOGICAL DISORDERS (HAND)
Dementia is initiated through substantial immune deficiency caused by HIV and
consequent systemic opportunistic infections and complications of AIDS. HIV-
associated dementia is characterized by some clinical features such as cerebral
and basal ganglia atrophy and diffuse periventricular white matter hyperintensi-
ties, which can be visualized through magnetic resonance imaging (Dal Pan et
al. 1992; Simpson and Tagliati 1994). Diminished levels of neuronal metabolite
N-acetyl-aspartate levels are also used to diagnose dementia by magnetic reso-
nance spectroscopy (Sacktor et al. 2005). Elevated levels of choline are used as
an indicator of inflammation (Ernst et al. 2003). Serological investigations show
high protein and IgG levels with an accompanying pleocytosis in the cerebro-
spinal fluid of 66% of HAD patients. HIV is known to preferentially infect the
basal ganglia and deep white matter, thereby displaying the cardinal features of
a “subcortical dementia.” This makes HAD not readily detected by the routine
mental status examination such as Folstein Mini, unless the patient is critically
demented (Skinner et al. 2009). Nevertheless, background correction caused by
substance abuse is essential to be taken into consideration, helping the exclusion
of residual neuropsychiatric deficits, especially evident with long-standing crack/
cocaine use. These neuroimaging techniques as well as cerebrospinal fluid analy-
ses are sufficient to exclude other factors causing similar images in the brain.
However, paradoxes exist, and deterioration in neurological status after initiation
of highly active antiretroviral therapy (HAART) has been reported and is termed
as neurological immune reconstitution inflammatory syndrome (neuro-IRIS) and
requires careful analysis of disease history and neuroimaging as well (Power et
al. 2009).
19.9 HIV-ASSOCIATED NEUROPATHOLOGIES AND

ASSOCIATION OF OPPORTUNISTIC INFECTIONS
Autopsy and other histological studies suggest the neuropathological changes in
more than 90% of HIV/AIDS patients (Johnson 1998). The changes in brain tissues
of HIV-1-infected patients are very evident, and some of them have been found to be
unique to HIV infection. The pathobiology of primary HIV-related neurological dis-
orders are generally defined by neuroinflammation and neuronal injury (Jones and
Power 2006). There are multiple histopathological hallmarks of HIV-1-associated
neuropathologies. Significant infiltration and accumulation of macrophages in brain
tissues, the appearance of microglial nodules, and specially multinucleated giant
cells (MGCs) become prominent in the central white matter and deep gray matter
in HIV dementia (Budka 1986). These MGCs are actually virus-induced fusion of
microglia and/or macrophages. The neuropathological hallmarks such as appear-
ance of MGCs, perivascular cuffs comprised of macrophages and lymphocytes, and
diffuse white matter pallor are now confirmed with magnetic resonance imaging and
spectroscopy findings (Sharer 1992). Viral antigens particularly viral proteins have
been detected and well correlated with the state of HIVE in the CNS. Enhanced
astrogliosis, indicative of astrocyte activation and damage as well as the loss of spe-
cific neuron subpopulations, particularly those in hippocampus and basal ganglia,
are very frequently seen in cognition and motor dysfunction. The loss is also evident
at synaptic connections and myelin pallor accompanied with the loss of myelin and
oligodendrocytes surrounding neuronal axons (Gendelman et al. 1994b; Lawrence
and Major 2002). Neuronal apoptosis along with other forms of cell deaths, autoph-
agy, and necrosis can likely contribute to reduced neural cell survival and health
(Jones and Power 2006).
Three main types of changes have been reported in brain tissue of patients with
HAD (Scaravilli et al. 2007). First, the presence of disseminated foci or more dif-
fuse lesions including myelin loss, reactive astrocytosis, and microglial activation
displayed as microglial nodules, macrophages, and accumulation of MGCs during
high viral load. Second, diffuse myelin pallor, particularly the severe form in the
centrum semiovale, is frequently associated with HIV leukoencephalopathy. In
the case of extreme HIV-induced pathology, these kinds of lesions are regarded
as very common and may overlap in one third of cases (Budka et al. 1991). In
addition to these characterized lesions, axonal damage has also been reported by
immunocytochemistry studies, which reveal the extremely frequent deposition of
the beta-amyloid protein precursor in neurons of AIDS patients (Giometto et al.
1997).
In other cases of noninfectious neurodegenerative disorders, focal CNS involve-
ment is more prominent in the early stages of the disease (Seeley et al. 2009),
whereas in HIV induced neurodegeneration shows a broader CNS impact; mainly
focused on the deep gray matter structures and subcortical regions. This explains
the clinical manifestations that revolve around cognitive, motor, and behavioral dys-
functions. Other frequent CNS associated changes include apathy and depression in
HAD (Hoare et al. 2010; Sharer 1992; Warriner et al. 2010).
When HIV directly infects the cells of CNS, it causes HAD, which falls under
the category of progressive subcortical dementia. However, HIV infection of the
peripheral nervous system results in a painful sensory neuropathy termed as distal
sensory polyneuropathy. These sensory neuropathies were shown to be exacerbated
by several antiretroviral drugs (Power et al. 2009).
HIV-induced neurological manifestations can also be categorized in two major
groups. The first group includes the neurological syndromes that are directly caused
by HIV-1 infection and more frequently encountered in AIDS patients. The second
group of neurological syndromes includes opportunistic infections and consequent
neurological perturbations. HIV infection exacerbates the opportunistic infections
in the brain, and enhances the coexistence of morbid illnesses (Valcour et al. 2011).
Secondary infections make the treatment of HAND a harder goal to achieve despite
the availability of HAART. The CNS environment is reported to be more discordant
than in the lymphoid system, and it is evident that the CNS can harbor virus, very
different from virus in plasma (Valcour et al. 2011).
Opportunistic infections take place as a consequence of HIV-induced immuno-
suppression. Opportunistic infection in the central and peripheral nervous system
consists of toxoplasmic encephalitis, cryptococcal meningitis, progressive multifocal
leukoencephalopathy (PML), primary CNS lymphoma, CNS tuberculosis, cytomeg-
alovirus encephalitis and radiculitis, or multidermatomal herpes zoster (Mamidi et
al. 2002; Roullet 1999).
19.10 TREATMENT: CNS COMPLICATIONS AND HAART

Currently, highly active antiretroviral therapy (HAART) is the most effective treat-
ment for controlling HIV replication in patients and thereby avoiding the early onset
of the AIDS. Therefore, HAART also seems promising in controlling HAND. The
usual HAART regimen combines three or more different drugs such as two nucle-
oside reverse transcriptase inhibitors (NRTIs) and a protease inhibitor (PI), two
NRTIs and a non-nucleoside reverse transcriptase inhibitor (NNRTI) or other such
combinations. Treatment with either two nucleoside analogue reverse transcriptase
inhibitors and the nonnucleoside analogue reverse transcriptase inhibitor, nevirapine,
has been shown to improve neuropsychological performance in HIV/AIDS patients

(Price et al. 1999). Improvement in neuropsychological performance has also been
reported with two nucleoside analogue reverse transcriptase inhibitors and a prote-
ase inhibitor (Langford et al. 2006; Sacktor et al. 1999; von Giesen et al. 2002).
HAART has shown convincing benefit in turning back deteriorative HIV-
associated dementia (Anthony and Bell 2008; Boisse et al. 2008; Brew et al.
2009), but poor drug penetrance into the CNS still restrains the efficacy of
HAART (Langford et al. 2006). The therapy became ineffective in those cases,
where patients were exhibiting a plateau or “burnt-out” phase of HIV-associated
dementia. Even the subjects receiving HAART showed continued neuropsychi-
atric disease progression (Brew et al. 2007). Other than HAART, there are sev-
eral drugs reported to be neuroprotective and have been employed for treating
HIV-associated dementia (McArthur et al. 2005). Amantidine has shown protec-
tive effect in HAD patients, especially in improving motor disabilities. Matrix
metalloproteinase (MMP) inhibitors have also shown promising results against
HIV-induced neurotoxicity (Zhang et al. 2003). Minocycline, a broad-spectrum
antibiotic, has been found to be useful in suppressing microglia/macrophage
activation and can control the severity of neuroinflammation (Zink et al. 2005).
Valproic acid, a well-known antidepressant, has also been used in treating demen-
tia (Schifitto et al. 2006).
According to WHO and the Joint United Nations Programme on HIV/AIDS
(2004), HAART therapy has been significantly helpful in increasing the life expec-
tancy of AIDS patients. Surprisingly, neuroinflammation still persists even after the
use of HAART in AIDS patients. Age has been consistently identified as a risk factor
for cognitive impairment in HIV-infected patients (Valcour et al. 2011).
Successive reports after the introduction of HAART has shown two most peculiar
effects: first, being effective in suppressing HIV replication, which in turn reduces
both the morbidity and neurobehavioral disorders. Second, it modifies the patho-
logical pattern of neuro-AIDS HAART is successful in suppressing HIV replica-
tion (Pakker et al. 1997) and improving cellular immunity (Autran et al. 1997) and
provides a wide range of protection against opportunistic infections (Jellinger et al.
2000; Maschke et al. 2000; Chiappini et al. 2007). A decrease in immune activa-
tion has also been observed compared with the pre-HAART era. HAART therapy
suppresses the level of HIV RNA in plasma but has shown high levels of microglial/
macrophage activation in the basal ganglia and hippocampus region (Anthony et al.
2005). The prevalence of severe forms of dementia can be minimized by the use of
HAART, but the occurrence of HIV dementia is increasing with the increased chro-
nicity of HIV-1 infection and prolonged survival of HIV-infected individuals carry-
ing on HAART therapy (Dore et al. 2003; McArthur et al. 2003; Neuenburg et al.
2002). About 25% of patients under treatment show intolerance to the drugs, which
manifests as nausea, anorexia, skin rash, hepatitis, and neuropsychiatric disorders.
The appearance of dementia mostly starts during the advanced stages of AIDS.
These observations suggest that CNS does not get complete therapeutic effects of
HAART compared to the effects at periphery.
In conclusion, the introduction of HAART has dramatically modified the course

and prognosis of HIV infection and is helpful in increasing the life expectancy
of patients. Therefore, it is crucial to understand the multidimensional course of
HAND, which differs from normal pathological manifestations of AIDS and thus
need different approaches to deal with HIV induced neuroinflammation and neuro-
degeneration effectively.
ACKNOWLEDGEMENT
Authors thankfully acknowledge the financial support provided by “Indo-Swiss
Grant” DST/INT/SWISS/P-44/2012, through Dept. of Science and Technology,
Govt. of India, New Delhi.
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(a) (b)
(c)
(d) 8
HCoV-OC43
6
TCID50/ml
4
2
0
0 24 48 72 96 5 10 15 20 25
MAP1b Hours p.i. Days p.i.
FIGURE 5.4 The main target of HCoV-OC43 infection is the neuron in mouse and human cell
cultures. (a) Mixed primary cultures from the murine CNS. (b) Cocultures of human neurons
and astrocytes obtained from differentiated human NT2 cell line using a protocol, which gives
rise to a mixture of neurons and astrocytes. In both type of cultures, HCoV-OC43 primarily
targets the neuron for infection leading to axonal beading (white arrows in a and b). The +viral
S protein is in green in infected neurons and red represents the glial fibrillary acidic protein
(GFAP) in activated astrocytes. The blue signal is the nucleus detected by the DNA-specific dye
DAPI. (c) NT2-N cells (95% pure human neuronal culture) infected by HCoV-OC43. The viral
S protein is in red in infected neurons and green represents the microtubule associated protein
1b (MAP1b) expression in differentiated neurons. (d) HCoV-OC43 can establish a long term
infection of the NT2-N cells for up to 25 days postinfection even though cell death occurred in
a portion of the NT2-N cells after acute infection.
(a) (b)
(c) (d)
FIGURE 15.1 (a) Detection of the N protein of MV in a 7-μm section obtained from an SSPE
case by indirect immunofluorescence (green). Nuclei from uninfected neurons are counter-
stained with propidium iodide (red). Interconnecting neuronal processes (arrows) and cell bod-
ies (asterisk) are indicated. (b) T2-weighted MRI sequence of a patient with MuV encephalitis.
T2-signal hyperintensities are indicated (arrows). (Copyright © MedReviews®, LLC. Adapted
and reprinted with permission of MedReviews, LLC. Cooper A.D. et al. Mumps encephalitis:
return with a vengeance. Rev Neurol Dis. 2007; 4:100–102. Reviews in Neurological Diseases
is a copyrighted publication of MedReviews, LLC. All rights reserved.) (c) T2-weighted MRI
sequence of a patient with acute NiV encephalitis. Selected punctate T2-signal hyperintensi-
ties are indicated (arrows). (d) T2-weighted MRI sequence of a patient with relapsed NiV
encephalitis. Selected confluent T2-signal hyperintensities are indicated (arrows). (Adapted
and reprinted from Goh, K. J. et al., 2000, N. Engl. J. Med. 342, 1229–1235.)
FIGURE 17.4 Macular–papular rash in postnatally acquired rubella infection. (Reproduced

from Dr. D. Wallach and Editions De Boeck/Estem. With permission.)
(a) (b) (c)
(d) (e)
FIGURE 17.5 Congenital anomalies in congenital rubella syndrome (CRS). (a) Case of CRS
with maculo-papular rash and purpura; (b) case of CRS with hepatosplenomegaly; (c) case of
CRS with radiolucencies of long bones; (d) case of CRS with cardiac dilatation; (e) cataracts
(opacity of the lens). (Kindly provided by Dr. C. A. Bouhanna and Dr. J. C. Janaud, Hôpital inter-
communal, Créteil, France, with permission from Dr. D. Wallach and Editions De Boeck/Estem.)
1
Blood
2
3
4 1
Basement 2
Brain
membrane
Pr
od
uc
tiv ted
e Restric
?
Productive
?
Astrocyte T cell Olygodendrocyte
Microglia Monocyte Perivascular macrophage
HIV-1 virion Neuron Microvascular endothelial cell
FIGURE 19.1 HIV-1 neuroinvasion. (1) According to the “Trojan horse” hypothesis, the
entry of HIV-1 into the brain takes place by the migration of infected monocytes, which
differentiate into perivascular macrophage. (2) The passage of infected CD4+ T cells can be
another source of infection in the brain. Other probable causes of CNS infection might be
(3) the direct entrance of the virus or (4) entrance of HIV-1 by transcytosis of brain micro-
vascular endothelial cells. Once the virus is in the brain, it productively infects macrophages
and microglia. Astrocyte infection is known to be restricted. The infection of oligoden-
drocytes, especially neurons, is questionable. (Reproduced from Ghafouri, M. et al., 2006,
Retrovirology 3, 28. With permission.)
Blood
Released proteins
Brain
gp120 3
2
A Tat
Vpr 6
1 6
Quinolinic acid
2 3
Secreted factors
Arachidonic acid 5
B Nitric oxide
4 IL-1
PAF 6
TNF
Neuronal death
apoptosis
6
Dementia
FIGURE 19.2 Mechanism of neuropathogenesis. Two components of this mechanism are

(A) the direct effect of the HIV-1 infection, including HIV-1 proteins, and (B) the indirect
consequence of infection comprising the secretion of cytokines and neurotoxins. The infected
macrophages and microglia participate actively in the neurodegeneration by (1) shedding
viral proteins and (2) releasing a significant amount of cytokines and neurotoxins into the
CNS. (3) Tat and TNF-α contribute to the disruption of the BBB, which in turn become
more permeable to infected monocytes and cytokines present in the periphery. The secreted
proinflammatory cytokines activates (4) microglia and (5) astrocytes, which in turn secrete
neurotoxins; moreover, the alteration of astrocytes function results in an increase in the level
of neurotoxicity in the brain. (6) Multifactorial neuronal injury: neurotoxins released from
several sources, as the direct and indirect consequences of HIV-1 infection lead to neuronal
injury. (Reproduced from Ghafouri, M. et al., 2006, Retrovirology 3, 28. With permission.)
BIOLOGICAL SCIENCES
Neuroviral Infections
Neurovirology is an interdisciplinary field representing a melding of virology, clinical

neuroscience, molecular pathogenesis, diagnostic virology, molecular biology, and
immunology. Neuroviral Infections: RNA Viruses and Retroviruses presents an up-to-date
overview on general principles of infections and major neuroviral infections caused by RNA
viruses and retroviruses. It is designed for virologists, specialists in infectious diseases, teachers
of virology, and postgraduate students of medicine, virology, neuroscience, and immunology.
Rooted firmly in basic principles, this book guides readers through 19 chapters, each dedicated
to a major RNA virus, retrovirus, and virus family. Each chapter details the organization
of the viral genome and its pattern of gene expression, explains molecular mechanisms
of pathogenesis, and examines interactive host dynamics—encompassing principles,
transmission cycle, and vector species.
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Sunit K. Singh and Daniel Růžek

Boca Raton London New York

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© 2013 by Taylor & Francis Group, LLC

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International Standard Book Number-13: 978-1-4665-6723-8 (eBook - PDF)

and the CRC Press Web site at

Section I RNA Viruses

Chapter 1 Alphavirus Neurovirulence................................................................... 3

Chapter 2 Neurological Chikungunya: Lessons from Recent Epidemics,

Chapter 3 Arenaviruses and Neurovirology........................................................ 39

Chapter 5 Human Coronaviruses: Respiratory Pathogens

Chapter 6 Nonpolio Enteroviruses, Polioviruses, and Human

Chapter 7 Neurovirulence of the West Nile Virus............................................. 149

Chapter 8 Murray Valley Encephalitis Virus.................................................... 167

Chapter 9 Japanese Encephalitis Virus and Human CNS Infection.................. 193

Chapter 10 Tick-Borne Encephalitis.................................................................... 211

Chapter 11 St. Louis Encephalitis....................................................................... 239

Chapter 12 Powassan Virus................................................................................. 261

Chapter 13 Neurological Dengue......................................................................... 289

Chapter 14 Influenza Virus and CNS Infections................................................. 325

Chapter 15 Human Paramyxoviruses and Infections of the

Chapter 16 Rabies Virus Neurovirulence............................................................ 373

Chapter 17 Rubella Virus Infections................................................................... 395

Chapter 18 Human T-Lymphotropic Virus.......................................................... 431

Chapter 19 Human Immunodeficiency Virus Neuropathogenesis...................... 457

Dr. Daniel Růžek is a research scientist at the Institute of

Cristian Baicus Marta S. Contigiani

Ashley C. Banyard Vlasta Danielová

Luis Adrián Diaz and

W. Paul Duprex Liliane Grangeot-Keros

Daniel L. Horton Laura D. Kramer

Hélène Jacomy Shiril Kumar

Mathieu Meessen-Pinard Duksha Ramful

Norma P. Tavakoli Michael R. Wilson

many alphaviruses causes fatal disease in experimental models, whereas subcutane-

1.3 INTERFERENCE WITH ANTIVIRAL TRANSCRIPTION

1.4 CHANGES IN CELLULAR TROPISM

1.5 HOST-ANTIVIRAL RESPONSE

indicating alterations of the neurological disease in the immunocompromised host.

1.7 INNATE IMMUNE RESPONSE

1.8 ADAPTIVE IMMUNE RESPONSE

antibody-independent Th1-biased response characterized by CD4+ T-cell production

1.9 HOST RESPONSE AND VACCINE DEVELOPMENT

nonformalin-fixed virus utilizing this platform. Following systemic challenge with

2.1 INTRODUCTION: CHIKUNGUNYA, A PARADIGM SHIFT

peripheral neuropathies, and deaths among neonates (mother-to-child infection), and

2.2 NEUROLOGICAL CHIKUNGUNYA IN HUMAN NEONATES

Chikungunya alphavirus neurological involvements (human and animal models)

Perivascular white Meningoencephalitis

Neuronal necrosis Encephalitis

FIGURE 2.1 Chikungunya neuropathology. CHIKV is not classically considered as a true

2.3 NEUROLOGICAL MANIFESTATIONS IN PEDIATRIC PATIENTS

2.4 NEUROLOGICAL CHIKUNGUNYA IN ADULTS

2.5 NEUROLOGICAL CHIKUNGUNYA: ROUTES TO

TABLE 2.2 (Continued)

* Italicized, NHP model; boldface, human studies.

2.6 NEUROINFECTION BY “OLD WORLD” SINDBIS VIRUS

2.1 INTRODUCTION: CHIKUNGUNYA, A PARADIGM SHIFT

2.5 NEUROLOGICAL CHIKUNGUNYA: ROUTES TO

2.7 NEUROINFECTION BY NEW WORLD

5.6 HUMAN CORONAVIRUSES: EPIDEMIOLOGY

5.7.1 Human Coronaviruses Other than SARS-CoV: Possible

5.7.2 SARS-CoV: Possible Association with

5.8 POSSIBLE MECHANISMS OF HUMAN CORONAVIRUS-

(Bechill et al. 2008; Chan et al. 2006) in different cell types, HCoV-OC43 induces