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Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013

 April 2013

On the Cover Transmission of Hepatitis E Virus

from Rabbits to Cynomolgus
Egon Schiele (1890–1918) Macaques............................................. 559
Self-Portrait with Physalis P. Liu et al.
(1912)
Cross-species transmission suggests that
Oil and opaque color on wood rabbits may be a new source of human infection.
(32.2 cm × 39.8 cm)
Leopold Museum, Vienna
Description and Nomenclature of
www.leopoldmuseum.org
Neisseria meningitidis
About the Cover p. 694
Capsule Locus..................................... 566
O.B. Harrison et al.
Perspective To meet the need for consistency, revised
Discrepancies in Data Reporting nomenclature is proposed for serogroups and
for Rabies, Africa................................. 529 capsule biosynthesis genes.
L.H. Nel
Synchronized, shared, or unified data reporting Detection of Spliced mRNA from Human
is necessary and feasible.
Bocavirus 1 in Clinical Samples from
Children with Respiratory
Research Tract Infections.................................... 574
Circovirus in Tissues of Dogs with A. Christensen et al.
Vasculitis and Hemorrhage................ 534 HBoV1 mRNA could be used instead of HBoV1
L. Li et al. DNA to diagnose these infections.
Previously found in only 1 other kind of p. 538
mammal—pigs—circovirus likely is associated Predicting Hotspots of Influenza
with vascular disease in dogs. Reassortment...................................... 581
T.L. Fuller et al.
Cost-effectiveness of Novel System Reassortment is most likely to occur in eastern
of Mosquito Surveillance and Control, China, central China, or the Nile Delta in Egypt.
Brazil..................................................... 542
K.M. Pepin et al. Effect of 10-Valent Pneumococcal
Cost-effectiveness of this system is comparable Vaccine on Childhood Pneumonia,
p. 569
with that of traditional vector control methods. Brazil..................................................... 589
E.T. Afonso et al.
Vaccination reduced hospitalization rates within
1 year.
Serotype IV and Invasive
Group B Streptococcus Occult Hepatitis B Virus Infection in
Disease in Neonates, Chacma Baboons, South Africa......... 598
Minnesota, 2000–2010......... 551 C. Dickens et al.
P. Ferrieri et al. Whether baboons were infected with HBV by
Serotype predominance has shifted, and drug humans or vice versa is unclear.
resistance is emerging.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013

 April 2013
Risk Factors for Influenza
among Acute Care Hospital 648 Response to Rabies Epidemic,
Workers during 2009 Bali, Indonesia, 2008–2009
Pandemic, Toronto, Ontario, A. Putra et al.
Canada.................................. 606 p. 644
S.P. Kuster et al. 652 Genetic Relatedness of Dengue
Influenza was associated with household Viruses in Key West, Florida,
exposure, aerosol-generating procedures, 2009–2010
and lower adherence to hand hygiene
J.L. Jordan et al.
recommendations.

Deaths Associated with Influenza 655 Control of Foot-and-Mouth

Pandemic of 1918–19, Japan.............. 616 Disease during 2010–2011
S. Chandra Epidemic, South Korea
J. Park et al.
The proportion of the population lost in Japan
was similar to that lost in other Asian countries.
Photo Quiz
Methicillin-Resistant Staphylococcus 660 P. Kumar and F. A. Murphy
aureus Colonization of the Groin and
Risk for Clinical Infection among Another Dimension
HIV-infected Adults.............................. 623 664 Myth Dispelled
P. Peters et al. A. Possner
Treatment would likely reduce clinical infections.

Letters
Dispatches
630 Feline Origin of Rotavirus Strain, 665 Novel Serotype of Bluetongue
Tunisia, 2008 Virus, Western North America
M. Ben Hadj Fredj et al.
666 Hepatitis E Virus Genotype
635 Tick-borne Encephalitis Virus in 3 Strains in Domestic Pigs,
Horses, Austria, 2011 Cameroon
J.O. Rushton et al.
668 Novel Respiratory Syncytial
638 Hepatitis Virus in Long-Fingered Virus Subtype ON1 among
Bats, Myanmar Children, Cape Town,
B. He et al. South Africa
p. 660
641 Hand, Foot, and Mouth Disease 670 Henipaviruses and Fruit Bats,
Caused by Coxsackievirus A6, Papua New Guinea
Thailand, 2012
J. Puenpa et al. 672 High Incidence of Japanese
Encephalitis, Southern China
644 Early Introduction and Delayed
Dissemination of Pandemic 673 Novel Hantavirus in Field Vole,
Influenza, Gabon United Kingdom
S. Lekana-Douki et al.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013

 686 Hepatitis E Virus and Porcine-
Derived Heparin
April 2013
676 Hand, Foot, and Mouth Disease 689 Human Enterovirus Genotype
Outbreak and Coxsackievirus C104, China
A6, Northern Spain, 2011
691 Monkey Bites among US
678 Rabies Update for Latin Military Members, Afghanistan,
America and the Caribbean 2011

680 Serosurvey of Dogs for Books and Media

Human, Livestock, and Wildlife
Pathogens, Uganda 693 The Foundations of Virology:
Discoverers and Discoveries,
682 Iatrogenic Creutzfeldt-Jakob Inventors and Inventions,
Disease from Commercial Developers and Technologies
Cadaveric Human Growth S. Bloom
Hormone
p. 683 About the Cover
684 West Nile Virus Infection in 694 Pale horse, pale rider done
Belgian Traveler Returning from taken my lover away
Greece P. Potter
Etymologia
686 Powassan Virus Encephalitis, 692 Syncytium
Minnesota, USA

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4 April 2013

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 Discrepancies in Data Reporting
for Rabies, Africa
Louis H. Nel

Human rabies is an ancient disease but in modern and responsible data reporting. Analyses of examples
times has primarily been associated with dog rabies–en- from Africa indicate that the above aspects are seriously
demic countries of Asia and Africa. From an African per- compromised.
spective, the inevitable and tragic consequences of rabies
require serious reflection of the factors that continue to drive Factors Leading to Complacency and Neglect
its neglect. Established as a major disease only after mul-
of Rabies
tiple introductions during the colonial era, rabies continues
to spread into new reservoirs and territories in Africa. How-
Rabies virus, a classical zoonotic pathogen, has an
ever, analysis of reported data identified major discrepan- extensive host range and can probably infect all terrestrial
cies that are indicators of poor surveillance, reporting, and mammals. Although vampire bat rabies has a major effect
cooperation among national, international, and global au- with regard to livestock losses in Latin America (1), rabies
thorities. Ultimately, the absence of reliable and sustained is generally not associated with agricultural animals be-
data compromises the priority given to the control of rabies. cause the main terrestrial reservoirs are domestic dog pop-
Appropriate actions and changes, in accordance to the One ulations of the developing world and wildlife carnivores
Health philosophy and including aspects such as synchro- elsewhere. Thus, rabies is often handled unconnectedly by
nized, shared, and unified global rabies data reporting, will health and veterinary authorities, and there is regular confu-
not only be necessary, but also should be feasible. sion as to who is responsible for controlling this disease. It
is also likely that public demand for effective control mea-

R abies, despite its high case-fatality rate and

preventability (through efficacious preexposure and
postexposure prophylaxis), has in recent years progressively
sures would have been far greater if rabies had been a major
disease of economically vital animals with a corresponding
effect on livelihood. For Africa, a case in point is rinder-
become established as a neglected disease, and most human pest, a viral disease for which there had been, figuratively
cases are associated with dog rabies endemic to countries in speaking, as many eradication campaigns as pandemics.
Africa and Asia. However, there are numerous other serious These campaigns were typically pan-African and driven by
infectious diseases that, like rabies, are underreported and considerable international support and cooperation (2).
linked with poverty in the developing world. How then The history of rabies in Africa is not well recorded, but
should these diseases be prioritized? it is well accepted that the disease must have been present
This report presents considerations that influence in northern Africa for hundreds of years, particularly as
the priority status of rabies, as well as issues that could an urban disease of dogs and also associated with cycles
differentiate rabies and should play a role in establishing in the Middle East. Rabies became epizootic in many
the relative role of this disease in Africa and other parts countries of sub-Saharan Africa only during the nineteenth
of the developing world. It also discusses 1 key area that and twentieth centuries; in this region, the disease became
needs to be addressed before a bona fide demonstration of well-established in dogs and involved wildlife species over
rabies incidence and progress toward effective dog rabies large areas (3).
control will be feasible. This area is the need for true Today, no regions of or countries in mainland Africa
cooperation and synergy between global organizations and are known to be free of rabies (4). In addition, Africa harbors
national authorities with respect to responsibilities related several rabies-related viruses. Historically, the isolation and
to effective and thorough surveillance with synchronized epidemiologic analyses of these viruses largely correlated
with specific surveillance studies or diagnostic competencies
Author affiliation: University of Pretoria, Pretoria, South Africa (5). This scenario is also true for the most recent discoveries
of 2 novel African lyssaviruses (6,7), and all indications are
DOI: http://dx.doi.org/10.3201eid1904.120185

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 529
PERSPECTIVE

that other as yet unknown lyssaviruses remain to be found. in children <15 years of age. For this reason, WHO has
Without more comprehensive and routine surveillance deemed rabies a reportable disease. Although WHO
and laboratory-based diagnosis, the epidemiology and the recognizes rabies as a reportable disease, many countries
potential role of these viruses or emergence of these viruses (e.g., India) (20) do not.
remain speculative. Unfortunately, a lack of consistent and To attempt an assessment of the incidence of this
sustained or routine laboratory-based diagnosis for rabies disease globally, WHO has collected rabies data since 1959
(and no capacity to distinguish rabies-related viruses) may and in the late 1990s created and administered Rabnet, a
well be the status quo in many countries in Africa. rabies-dedicated Web platform, to which countries have
In humans, rabies often develops with a wide variety of been requested to submit annual rabies statistics (21).
nonspecific clinical symptoms, and symptoms believed to be These figures were published under various categories,
typical of rabies (e.g., foaming at the mouth, hydrophobia, including human cases, animal cases, presence or
and extreme aggressiveness) are frequently not observed. absence of rabies, national rabies vaccine production and
Approximately 30% of human rabies cases develop in the importation, and rabies vaccine administration. In addition,
paralytic or dumb form (8), and the overlap of symptoms there were several subsections, which enabled specification
with those of other infections often leads to misdiagnosis of certain criteria. For instance, under animal rabies, one
(9–12). Apart from misdiagnosis, rabies exposures are could choose total number of dog cases and further specify
often ignored or deemed as minimal in dog rabies– whether one would want to observe dog rabies positive,
endemic areas of the world. Because dogs are common dog rabies negative, or both. Figures obtained from Rabnet
as companion animals in most cultures, the exposure risk were frequently used in publications and in country reports.
is actually greater than for many other zoonotic diseases. Despite having been a worthwhile undertaking, the Rabnet
However, postexposure prophylaxis is unlikely to be sought website has been closed indefinitely (until further notice)
after lick-associated exposures to dogs. Some cultures are since late 2011, given the realization of incorrect reporting
known to believe that the lick from a dog is useful for and to avoid subsequent misrepresentation.
wound treatment (13). In conjunction with this belief, some The World Organisation for Animal Health (OIE) also
cultures believe that the aggressive and uncharacteristic regards rabies as a reportable disease, and OIE statistics for
behavior of persons or animals with symptoms of rabies such diseases are published on the World Animal Health
is caused by sorcery or demon possession. Far too often Information Database (22). The structure of this database
rabies patients end up at tribal or traditional healers whose includes 1) immediate notifications and follow-up reports
treatments include exorcism, administration of toxic herbs, submitted by member countries in response to exceptional
and other such undesirable interventions (14,15). disease events occurring in these countries and follow-
Rabies is one of the oldest recognized diseases in human up reports about these events; 2) reports every 6 months
history, and there is anecdotal evidence of its presence in describing OIE-listed disease situations in each country;
Mesopotamia and elsewhere in the Mediterranean basin and 3) annual reports providing further background
since antiquity (16). Because this evidence has been known information about animal health, and laboratory and
for many years, an unfortunate consequence has been a loss vaccine-production facilities. Thus, information for
of newsworthiness, which has compromised awareness and specific diseases is available on the website, and one can
priority in public and professional practice. A contrasting compare multiple countries with each another. Reports are
example could be witnessed with the newly emerged submitted biannually by each country, and a final report is
influenza A(H1N1)pdm09 virus, which caused widespread issued at the end of each year. The information for rabies
panic and has received much attention over the 3 years since gives detailed monthly case reports of rabies in animals
its emergence. In the 17 months from April 2009 through for every month of each year and, in some cases, for each
August 2010, a total of 18,000 deaths caused by swine- district or province of the country.
origin influenza (mostly associated with other primary risk The Southern and Eastern African Rabies Group
factors) were recorded worldwide (17). In contrast, rabies (SEARG), founded in 1992, focuses on control of dog
is conservatively estimated to cause ≈50,000 deaths per rabies in countries in Africa. Official meetings are held
year, mostly in children, and is not associated with any approximately every 2 years, at which representatives
other health risk factors (18). from member countries gather and present data regarding
rabies in their country in standardized country reports. The
Global and Regional Structures and information from these reports is published on an open-
Reporting of Rabies Cases access website (www.searg.info). The country reports
The World Health Organization (WHO) has reported focus on human and animal rabies (domestic and wildlife)
that rabies has the highest case-fatality rate of all infectious and request information regarding vaccine purchases and/
diseases of humans (19), and most human exposures occur or production and vaccination strategies.

530 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Discrepancies in Data Reporting for Rabies, Africa

Discrepancies and Deficiencies in likely receive rabies data from medical health authorities.
Rabies Reporting In contrast, the animal disease focus of OIE suggests that
Lack of reporting on rabies data by most developing veterinary services will submit rabies data to this body.
countries is disconcerting. Examination of data on WHO Inconsistencies described in this report suggest a need for
and OIE web sites showed that information was frequently improved collaborative effort and effective communication
missing for an entire year or more in several countries. Data between all relevant authorities with regard to diseases
from selected countries from the southern African region for that simultaneously effect human and animal health. The
which SEARG, WHO, and OIE data reports were available imminent neglect of any zoonosis of which the main
are shown in Figure 1. Data submitted to the 3 authorities reservoir host is not an economically vital animal species
varied considerably in all examples. The only data from is predictable unless addressed by unconditional execution
any of the countries that showed some correlation was that and instruction of the One Health paradigm on global,
of Swaziland, where data submitted to SEARG and WHO regional, and country levels.
were the same, but data submitted to OIE were different.
In addition, when the ratio of human to animal rabies cases Conclusions
was analyzed, a vast range of ratios was typically found Rabies remains endemic throughout Africa, and for
for data for 2010 (Figure 2). Data for individual countries all the reasons discussed, loses visibility in Africa because
ranged from high ratios of human rabies cases to animal it typically oscillates disconnectedly between authorities
rabies cases to (the more rational) low ratios of human concerned with either human or animal health. Poor
cases to animal cases. Some countries reported only human epidemiologic surveillance and inconsistent reporting,
rabies cases (clinical diagnoses for most countries in including that to responsible global authoritative bodies,
Africa). Such inconsistent data can be considered a further has created a lack of rabies awareness and appreciation
indicator of poor surveillance practices. of its effect on humans in Africa. The absence of reliable
Inconsistencies in reporting of rabies epidemiologic and sustained rabies data compromises the priority that
information to WHO and OIE did not apply only to the control of rabies should be given, considering that a
developing countries. Although inconsistencies were not lack of laboratory-based proof of disease incidence will
as great as those observed in developing countries, which innately counteract attempts to justify (on national levels
was likely caused by the fact that rabies cases are less or to global funding agencies) the need for extensive and
frequent because of adequate control measures, they were expensive rabies elimination programs.
still evident from the industrialized world. Also, for various OIE has recently released its Fifth Strategic Plan,
countries, data for specific years were submitted only to which includes the One Health approach and is committed
OIE, only to WHO, or to neither organization. to improved cooperation with human–animal–environment
Discrepancies for rabies epidemiologic data between interfaces (23). Despite this fact, the forms required by each
various authorities could be interpreted as symptomatic of of the organizations to be completed (e.g., for rabies) are
a larger problem, which should be addressed on a global different. This difference creates additional work for the
scale. One likely reason for the lack of consistency of authority responsible for submission of data and can lead
rabies data is the different focus areas of WHO and OIE. to inconsistencies. If submission forms are standardized,
WHO focuses mainly on human disease, and will most then the same form can be sent to the various organizations.

Figure 1. Number of rabies cases

in animals reported in 2007 from
countries in Africa classified as
developing countries. Data were
obtained from Southern and Eastern
African Rabies Group reports (black
bars), the World Health Organization
(Rabnet) (21) (white bars), and the
World Organisation for Animal Health
World Animal Health Information
Database (gray bars).

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 531
PERSPECTIVE

Figure 2. Ratio of human to animal cases of rabies reported in 2010 from Southern and Eastern African Rabies Group countries. Ratios
are indicated above the bars. CAR, Central African Republic.

Another alternative would be to have 1 body to which to disease in animal and human populations in Africa. The
report epidemiologic data to, and from this body the major success of such a venture is certain to be conditional to the
organizations can use and publish the appropriate data. synchronized cooperative support of OIE, WHO, and other
This uniformity will prevent submission of inconsistent global partners. In this regard, it is encouraging that during
data but will require true collaboration between medical a high-level technical meeting in Mexico at the end of 2011
and veterinary sectors (the One Health approach). Because (26), the Food and Agricultural Organization of the United
of the need for consistent and transparent data, appropriate Nations together with OIE and WHO have affirmed their
actions and changes, in accordance to the One Health commitment to alignment and honing of their respective
philosophy, will be necessary and feasible. coordination mechanisms to defend against emerging
On a continental level, the One Health approach has diseases at the animal–human–ecosystems interfaces.
already been shown to be beneficial toward rabies control in
at least 1 part of the developing world, when implemented Acknowledgments
by the Pan American Health Organization in Latin America I thank Terence Scott for data analysis and technical
(24). In contrast, there is no pan-African approach to rabies assistance, and Terence Scott and Jacques Barrat for administering
control, although small regional efforts present hope. The the SEARG website.
rabies control program in Kwa-Zulu Natal in South Africa
This study was supported by the National Research
(25), which is supported by the Bill and Melinda Gates
Foundation of South Africa.
Foundation, celebrated a year free of human rabies on June
24, 2011. That occasion constituted the first time in 20 years Dr Nel is a professor of virology and head of the lyssavirus
that Kwa-Zulu Natal has not recorded a single human death research program at the University of Pretoria. His research
caused by rabies in a year. However, this is a small victory interests are rabies and rabies-related viruses and the epidemiology
in the face of the continent-wide challenge. Although dog and control of zoonotic diseases, such as rabies.
rabies is rapidly decreasing in Kwa-Zulu Natal, it is still
present, and until eliminated from dogs, the likelihood of
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nants in diagnosis with special reference to paralytic form. J Neurol dx.doi.org/10.1590/S0102-311X2007000900013
Neurosurg Psychiatry. 2010;81:812–20. http://dx.doi.org/10.1136/ 25. Nel LH, Le Roux K, Atlas R. Rabies control program in South Af-
jnnp.2009.185504 rica. Microbe. 2009;4:61–5.
12. Buyuk Y, Uzun I, Aybar Y, Kurnaz G, Ozaras R. Rabies in Tur- 26. Food and Agriculture Organization of the United Nations. Animal
key: three human cases illustrating the importance of suspecting Production and Health Division; 2012 [cited 2012 Jan 20]. http://
exposure. Wilderness Environ Med. 2007;18:214–7. http://dx.doi. www.fao.org/ag/againfo/home/en/news_archive/2011_Tripartite_
org/10.1580/06-WEME-BR-054R.1 against_Diseases.html.
13. Hatfield G. Encyclopedia of folk medicine: old world and new world
traditions. Santa Barbara (CA): ABC-CLIO; 2004. Address for correspondence: Louis H. Nel, Department of Microbiology
14. Mahawar MM, Jaroli DP. Traditional knowledge on zootherapeutic
and Plant Pathology, New Agricultural Bldg, Office 9-13, University of
uses by the Saharia tribe of Rajasthan, India. J Ethnobiol Ethnomed.
2007;3:25. http://dx.doi.org/10.1186/1746-4269-3-25 Pretoria, Pretoria 0001, South Africa; email: louis.nel@up.ac.za

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Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 533
RESEARCH

Circovirus in Tissues of Dogs with

Vasculitis and Hemorrhage
Linlin Li, Sabrina McGraw, Kevin Zhu, Christian M. Leutenegger, Stanley L. Marks, Steven Kubiski,
Patricia Gaffney, Florante N. Dela Cruz Jr, Chunlin Wang, Eric Delwart, and Patricia A. Pesavento

We characterized the complete genome of a novel a spectrum of swine diseases called porcine circovirus–
dog circovirus (DogCV) from the liver of a dog with severe associated diseases that have been described in pigs world-
hemorrhagic gastroenteritis, vasculitis, and granulomatous wide. PCV2 infection causes severe economic losses be-
lymphadenitis. DogCV was detected by PCR in fecal sam- cause of increased mortality and reduced production, mak-
ples from 19/168 (11.3%) dogs with diarrhea and 14/204 ing it one of the most economically important viruses in the
(6.9%) healthy dogs and in blood from 19/409 (3.3%) of
global swine industry. Among lesions that have been attrib-
dogs with thrombocytopenia and neutropenia, fever of un-
known origin, or past tick bite. Co-infection with other canine
uted to PCV2 infection are pneumonia, enteritis, lymph-
pathogens was detected for 13/19 (68%) DogCV-positive adenitis, vasculitis, nephritis, and reproductive disease (4).
dogs with diarrhea. DogCV capsid proteins from different In cases for which PCV2 is considered causative, immu-
dogs varied by up to 8%. In situ hybridization and trans- nohistochemical and in situ hybridization (ISH) analyses
mission electron microscopy detected DogCV in the lymph demonstrate large amounts of PCV2 antigen or nucleic ac-
nodes and spleens of 4 dogs with vascular compromise and ids in the cytoplasm of macrophages and dendritic cells in
histiocytic inflammation. The detection of a circovirus in tis- the depleted follicles in lymphoid tissues (4,5). Naturally
sues of dogs expands the known tropism of these viruses to occurring porcine circovirus–associated diseases is often
a second mammalian host. Our results indicate that circo- accelerated or exacerbated by concurrent viral or bacterial
virus, alone or in co-infection with other pathogens, might infections, and secondary infections often occur as a result
contribute to illness and death in dogs.
of immunosuppression (6).
Random nucleic acid amplification with or without

C ircoviruses are nonenveloped, spherical viruses with a

single-stranded circular DNA genome of ≈2 kb; they
group as a genus within the family Circoviridae, together
prior enrichment for viral particle–associated nucleic acids
(7,8), followed by deep sequencing and in silico similarity
searches for sequences related to those of known viruses,
with the proposed genus Cyclovirus and the phylogeneti- have been highly productive in the field of animal virus
cally more distinct genus Gyrovirus (1). Most of the known discovery (9–11). We used this technique to identify virus
species in the genus Circovirus infect birds and cause signs sequences in affected tissues from companion animals with
including malformations and necrosis of the integument, diseases of unknown cause. We identified a canine circovi-
lymphoid depletion, and immunosuppression (2). rus in the liver of a dog that had necrotizing vasculitis and
Before 2012, the only circoviruses reported to infect granulomatous lymphadenitis, both of which are described
mammals were the 2 closely related porcine circoviruses in PCV2-infected pigs (4). We named this virus dog circo-
(PCVs) (3). PCV2 is the primary pathogen associated with virus (DogCV) rather than canine circovirus to avoid con-
Author affiliations: Blood Systems Research Institute, San Fran-
fusion with the CaCV notation used for canary circovirus
cisco, California, USA (L. Li, E. Delwart); University of California,
(12,13), canine calicivirus (14,15), and Capsicum chlo-
San Francisco (L. Li, E. Delwart); University of California School of
rosis virus (16). A closely related variant of DogCV was
Veterinary Medicine, Davis, California, USA (S. McGraw, K. Zhu,
sequenced independently in canine serum samples and
S.L. Marks, S. Kubiski, P. Gaffney, F.N. Dela Cruz Jr, P.A. Pesaven-
was published recently (17); however, no disease associa-
to); IDEXX Laboratories, West Sacramento, California, USA (C.M.
tion was described with the virus. To determine whether
Leutenegger); and Stanford Genome Technology Center, Stanford,
DogCV could be associated with canine vascular disease,
California, USA (C. Wang)
we identified additional dogs with vascular and granuloma-
tous lesions and examined the distribution of DogCV by
DOI: http://dx.doi.org/10.3201/eid1904.121390 ISH analysis.

534 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Circovirus in Dogs

Materials and Methods assembled sequences and singlets >100 bp were compared
with the GenBank nonredundant nucleotide and protein
Animal Sample Collection databases (www.ncbi.nlm.nih.gov/genbank) by using
A 1-year-old castrated male dog that had been ken- BLASTn and BLASTx, respectively (http://blast.ncbi.
neled for 3 weeks was brought to the University of Cali- nlm.nih.gov/Blast.cgi). Potential viral sequences with sig-
fornia, Davis (UC Davis), Veterinary Medical Teaching nificant hits (E-value <0.001) to known virus sequences
Hospital for evaluation of progressive vomiting and diar- were identified.
rhea with hematochezia. Despite initial supportive therapy
at the referring veterinarian, clinical signs worsened; at UC Genome Sequencing and Analyses
Davis, the dog was treated for hypovolemic shock. Because PCR and Sanger sequencing were used to confirm the
of suspected disseminated intravascular coagulation and a presence of virus genome sequences assembled from deep
poor overall prognosis, the owner elected to have the dog sequencing reads. Inverse PCR was then used to amplify
euthanized and granted permission for routine necropsy, the genome of target circoviruses with primers based on
which was performed at the Anatomic Pathology Service of the sequences obtained by deep sequencing. Virus genome
the UC Davis School of Veterinary Medicine. The clinical sequences obtained were deposited in GenBank (accession
and postmortem workups for infectious causes in this case nos. KC241982–KC241984). Putative open reading frames
included negative test results for infectious causes of enter- (ORFs) with coding capacity >100 aa were predicted by
ic disease, such as canine parvovirus, canine enteric coro- Vector NTI Advance 11 (Invitrogen, Carlsbad, CA, USA).
navirus, Salmonella spp., canine distemper virus, Campy- The stem-loop structure was predicted by using Mfold (21).
lobacter spp., Clostridium perfringens enterotoxin A gene,
Cryptosporidium spp., and Giardia spp. Histologic results Phylogenetic Analysis
showed extensive fibrinoid vascular necrosis, thrombosis, Phylogenetic analyses based on aligned amino acid se-
and hemorrhage throughout the gastrointestinal tract and quences from full-length replicate (Rep) proteins were gen-
kidneys, as well as granulomatous lymphadentitis of the erated by using the neighbor-joining method in MEGA4
mesenteric lymph nodes. Special stains of histologic speci- (22), using amino acid p-distances with 1,000 bootstrap
mens revealed no detectable bacteria or other infectious replicates. Other tree-building methods, including maxi-
agents. Liver tissue samples were collected, stored in whirl- mum parsimony and maximum likelihood, were used to
pack bags, and frozen at −80°C until further processing. confirm the topology of the neighbor-joining tree.

Sample Preparation and Nucleic Acid Extraction Prevalence Study of DogCV in Sample Cohorts
A liver tissue sample (≈25 mg) were immersed in 1 mL Real-time PCR using 2 primers and a conventional hy-
cold Hank’s balanced saline solution and disrupted with a drolysis probe with a 5-primer 6-FAM and 3-primer TAM-
tissue homogenizer for 30 sec on ice. The resulting homog- RA label and TaqMan Universal PCR Master Mix (Applied
enates were placed on dry ice for 5 min and then thawed Biosystems, Foster City, CA, USA) was used to detect
at room temperature (18). Freezing and thawing were re- DogCV in DNA extracts from 3 dog sample cohorts: 1) fe-
peated twice. Samples were clarified by centrifugation at cal samples from 204 healthy dogs; 2) fecal samples from
10,000 × g for 3 min; the supernatants were then filtered 168 dogs with diarrhea; and 3) blood samples from 480 dogs
and underwent nuclease treatment as described (19). Viral with thrombocytopenia and neutropenia, fever of unknown
nucleic acids were extracted by using the QIAamp Viral origin, or past tick bite. Primer pairs and probes for canine
RNA Mini Kit (QIAGEN, Valencia, CA, USA) and stored circovirus are given in the Table. Total nucleic acid was ex-
at −80°C. tracted by using the Corbett X-Tractor Gene platform (QIA-
GEN). Real-time PCR was conducted by using the real-time
Library Preparation and Sequencing PCR instrument LightCycler 480 (Roche, Indianapolis, IN,
Viral nucleic acid libraries were prepared as described USA) under these conditions: 50°C for 2 min, then 95°C for
(19). The library of single-stranded DNA fragments was 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C
sequenced by using the Genome Sequencer FLX Instru- for 1 min. Synthetic DNA fragments (≈150 bp) of the cor-
ment (Roche, Indianapolis, IN, USA). responding regions were used to produce a standard curve
and an analytical sensitivity of 10 molecules.
Sequence Data Analysis
The pyrosequencing reads were sorted and trimmed ISH Analysis
as described (19). Trimmed reads from each sample were A fourth sample cohort consisted of tissue samples
assembled de novo by using the MIRA assembly program from 21 necropsy cases of dogs whose clinical signs or
(20), with a criterion of >95% identity over 35 bp. The microscopic lesions matched the sentinel animal (i.e.,

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 535
RESEARCH

Table. Oligonucleotide primer pairs and probe for DogCV sequences*

Region and amplicon Primers Sequences, 5′3′
Replicate gene, 66 bases DogCV-forward CTTGCGAGAGCTGCTCCTTATAT
DogCV-reverse CTCCACTTCCGTCTTCCAGTTC
DogCV-probe TCCGGAGATGACCACGCCCC
Capsid gene, 68 bases DogCV2-forward CTGTTGTGAAACTGAAAGAGACGAA
DogCV2-reverse TGACGTAGGTCTCCGGATACG
DogCV2 -probe AGCCTTGCCGCTGTCGCGTC
*DogCV, dog circovirus.

hemorrhagic diarrhea, vasculitis, and/or granulomatous Electron Microscopy

disease); these samples were selected from the tissue ar- Selected pieces of formalin-fixed lymph node tissue
chives of Anatomic Pathology at the UC Davis Veterinary from the sentinel dog were post fixed in 2.0% glutaralde-
Medical Teaching Hospital. Control tissues were obtained hyde and then routinely processed and embedded in ep-
from 5 dogs whose cause of death was unrelated to vascular oxy resin (Eponate12 kit; Ted Pella, Inc., Redding, CA,
disease. Tissue sections chosen for analysis were, in part, USA). Selected thick sections were stained with toluidine
case dependent because the presence of vasculitis or in- blue, as described (26). Ultrathin sections from se-
flammation among these cases was not limited to a sin- lect areas of the lymph node were examined by using a
gle tissue type. Spleen, lymph node, jejunum, and ileum Zeiss (Göttingen, Germany) 906E transmission electron
were examined in all cases; other examined tissues were microscope.
kidney, brain, adrenal gland, pancreas, duodenum, heart,
and lung. Tissues from the sentinel dog were included in Metagenomic Identification of Canine Circovirus
this analysis. Of ≈10,000 squesnce reads, 5 contigs and singlets
Tissue sections were mounted on 3-aminopropyl- composed of 52 sequence reads from the liver tissue had
triethoxylane–coated slides (Fisher Scientific, Pittsburgh, significant similarity to the Rep protein of circoviruses (E-
PA, USA). A 25-nt oligomer (CTCAGACAGAGACAC- value <1–10). Two bocavirus sequences were also detected.
CGTTGCTATG) complementary to a segment of ORF2 Because circoviruses have a circular genome, the full viral
(capsid) was 3′-end labeled by the addition of a single genome was then amplified by inverse nested PCR and the
digoxigenin-II-dideoxy undine (Eurofins MWG Operon, amplicon sequenced by primer walking. The assembled ge-
Huntsville, AL, USA). A manual capillary-action work nome was named DogCV strain UCD1 (DogCV-UCD1).
station (Fisher Scientific) was used to perform colorimet-
ric DNA in situ hybridization. Tissue sections were depa- Results
raffinized and digested by incubation at 37°C for 10 min
in 0.25% pepsin in 1× Tris-buffered saline (pH 2.0); pep- Genome Analysis and Phylogenetic Relationship
sin activity was stopped by a 5-min incubation at 105°C. The complete circular genome of DogCV-UCD1 was
Nucleic acid was denatured by 5-min incubation at 105°C 2,063 nt (GenBank accession no. KC241982). Analysis
in 100% formamide. Tissue sections were then incubated of the genome sequence showed characteristics typical of
at 37°C in 10 µmol of the digoxigenin-labeled probe in circoviruses, including an ambisense organization with 2
hybridization buffer (22.5% deionized formamide, 7.5% major inversely arranged ORFs encoding the putative rep-
chondroitin sulfate, 5× saline sodium citrate, 0.25% block- lication-associated (Rep, 303 aa), and capsid (Cap, 270
ing reagent, and 50 mmol phosphate buffer). Sections were aa) proteins. A characteristic stem-loop structure with a
incubated in an antidigoxigenin antibody solution (500:1 conserved nonanucleotide motif (5′-TAGTATTAC-3′,
dilution) containing 2.5 mL buffer 1 with 5 μL of antidi- similar to the consensus of bird and pig circoviruses) was
goxigenin Fab fragments conjugated with alkaline phos- also found in the 5′-intergenic region (135 nt, between
phatase (750 U/mL) (Roche). Sections were then washed the start codons of the 2 major ORFs). The 3′-intergenic
and developed according to manufacturer instructions region of DogCV-UCD1 between the stop codons of the 2
before counterstain with 1% fast-green FCF for 5 min. major ORFs was 203 nt (Figure 1, panel A) (27).
Slides were mounted with ImmunoHistoMount (Immuno- The complete genome of another DogCV strain
bioscience, Mukilteo, WA, USA) and coverslipped with (DogCV-UCD2, GenBank accession no. KC241984)
SHUR/Mount (Triangle Biomedical Sciences, Durham, was amplified and sequenced from a fecal sample from a
NC, USA). No background hybridization was seen when shelter-housed dog that had vomiting and diarrhea. This
replicate tissue sections were incubated with an unrelated strain shared 95% overall genome nucleotide identity with
digoxigenin-labeled probe with similar guanine-cytosine DogCV-UCD1, and both strains showed 96%–97% nucle-
content or when matched tissue from unaffected dogs were otide identity to the recently reported canine circovirus iso-
incubated with the DogCV-specific probe (23–25). late 214 from blood (17).

536 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Circovirus in Dogs

The putative Rep proteins of DogCV-UCD1 showed 6.9%; p>0.05 by χ2 test). Of the 19 dogs with diarrhea who
42%–54% amino acid identity to the Rep proteins of por- had DogCV detected in fecal samples, 13 (68%) were co-
cine and avian circoviruses, with the closest identity to infected with >1 other pathogens, including canine enteric
PCV1. The DogCV-UCD1 capsid showed <30% amino coronavirus, Cryptosporidium spp., C. perfringens α toxin,
acid identity to known circovirus capsids. Sequence align- Giardia spp., Salmonella spp., Campylobacter jejuni, and
ment of the putative Rep protein of DogCV-UCD1 with Campylobacter coli (tested by PCR).
those of known species in the genus Circovirus identified The prevalence of DogCV in blood samples from
several highly conserved amino acid motifs, including the cohort of dogs with thrombocytopenia and neutrope-
WWDGY, DDFYGW, and DRYP. Motifs associated with nia, fever of unknown origin, or past tick bite was 3.3%
rolling circle replication (FTLNN, TPHLQG, and CSK) (16/480), similar to that reported for canine serum samples
and the dNTP binding (GXGKS) were also identified. The (2.9%, 6/205) (17). The partial Rep and/or Cap protein re-
N terminal region of the Cap protein was highly basic and gions (≈350 bp) were amplified from 11/16 samples. All
arginine-rich, as is typical for circoviruses. A phylogenetic showed >96% nucleotide identity, except 1 amplicon,
analysis of the complete Rep protein of DogCV strains which had <90% nucleotide distance to DogCV-UCD1
and all known circoviruses was performed, with chicken and -UCD2. We sequenced the complete genome of this
anemia virus (genus Gyrovirus) as the outgroup (Figure 1, virus (DogCV-UCD3; GenBank accession no. KC241983);
panel B). The phylogenetic tree showed that DogCV strains it showed 91%–92% amino acid identity of the complete
grouped with PCV1 and PCV2, forming a distinct clade Rep and Cap proteins to DogCV-UCD1 and -UCD2 and
of mammalian circovirus, whereas circoviruses affecting to the published canine circovirus (CaCV-1 strain NY214;
birds clustered separately. GenBank accession no. JQ821392) (17).

Prevalence Study ISH Analysis and Pathologic Findings in

DogCV was detected by real-time PCR in fecal samples Positive Cases
from 14/204 healthy dogs and 19/168 dogs with diarrhea; To establish tissue distribution and investigate whether
the difference in prevalence was not significant (11.3% vs. DogCV contributes to canine disease, we developed and

Figure 1. A) Genome organization of dog circovirus (DogCV) and porcine circovirus 2 (PCV2). B) Phylogenetic analysis of DogCV strains
(UCD1–3, isolated from tissue, feces, and blood respectively) based on the amino acid sequence of the replicate (Rep) protein. GenBank
accession numbers for circoviruses used in the analysis: Finch circovirus (FiCV), DQ845075; Starling circovirus (StCV), DQ172906; Raven
circovirus (RaCV), DQ146997; canary circovirus (CaCV), AJ301633); Columbid circovirus (CoCV), AF252610; Gull circovirus (GuCV),
DQ845074; beak and feather disease virus (BFDV), AF071878; Cygnus olor circovirus (SwCV), EU056310; Goose circovirus (GoCV),
AJ304456; Duck circovirus (DuCV), DQ100076; Porcine circovirus 1 (PCV1), AY660574; PCV2, AY424401; canine CV, JQ821392; Barbel
CV, GU799606; Cyclovirus (CyCV) NG13, GQ404856; Silurus glanis circovirus (Catfish CV), JQ011378; CyCV TN25, GQ404857; CyCV
PK5034, GQ404845; CyCV PK5006, GQ404844; CyCV NG Chicken8, HQ738643); and chicken anemia virus (CAV), M55918.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 537
RESEARCH

validated an ISH oligomeric probe and examined the sen- dog 3 was restricted to the ventral surface of the brain along
tinel dog and dogs from 21 suspected, retrospective cases the basilar artery overlying the medulla; dog 4 had bicavitary
that included >2 of these 3 signs: vasculitis, hemorrhage, or hemorrhage. The common histologic lesion in all dogs was
granulomatous disease. A wide spectrum of affected tissues fibrinonecrotizing vasculitis, although the distribution of af-
was represented in this group; matching tissues were also fected vessels and the amount of associated hemorrhage var-
examined from 5 control dogs in which these signs were not ied. In dog 1, segments of inflamed or necrotic vessels were
present. Samples from the sentinel dog (dog 1) and 3 other seen in the intestine (multiple segments), urinary bladder,
dogs (dogs 2–4) were positive for DogCV by ISH analysis. liver, spleen, and lungs. In dog 2, vasculitis was limited to
All other tissue samples from control dogs were negative by the intestine and spleen, and in dog 3, vasculitis was in kid-
ISH analysis. Clinical signs, gross and histologic findings, neys, intestine (Figure 2, panel G), heart, liver, spleen, and
and distribution of virus DNA as determined by ISH were meninges. In dog 4, only a few vessels were affected, at the
used to examine a possible causal role for DogCV. corticomedullary junction in the kidneys. For all dogs, his-
Dog 1 was a male beagle who had acute onset of tiocytic drainage or granulomatous lymphadenitis were seen
vomiting and hemorrhagic diarrhea. Dog 2 was a 5-year- in Peyer’s patches (Figure 2, panels C, E) and in >1 lymph
old, female, spayed Boston terrier who had vomiting and node. In addition, in dogs 2 and 3, multiple lymph nodes
diarrhea. Dog 3 was a 1-year-old, female, spayed boxer were severely necrotic. All dogs had microscopic lesions in
with a 5-day history of lameness and progressive tetrapa- the kidneys, but intensity and character varied widely. Dogs
resis. Dog 4 was a 2-year-old Greyhound found dead with 1 and 2 had tubular necrosis with little inflammation, dog 3
bicavitary hemorrhage; blood smear and PCR showed this had a severe granulomatous interstitial nephritis, and dog 4
dog was co-infected with Babesia conradae. had multifocal hemorrhage and minimal lymphocytic, plas-
Gross examination revealed consistent lesions among macytic nephritis. Dogs 1 and 3 had multifocal pancreatitis
these 4 dogs, including lymphadenopathy and hemorrhage. and adrenalitis. No multinucleate giant cells, common in
In dogs 1 and 2, the hemorrhage was associated with the gas- pigs infected with PCV2, were seen.
trointestinal tract (Figure 2, panels A, C); dog 2 had addition- By ISH analysis, abundant cytoplasmic viral nucleic
al multifocal to coalescing regions of hemorrhage in the kid- acid was detected in macrophages within germinal centers
neys (Figure 2, panel B). Gross evidence of hemorrhage in and subcapsular and medullary sinuses of the mesenteric

Figure 2. Organ tissues from sentinel dog (dog 1) and 2 other dogs (dogs 2 and 3) that were positive by in situ hybridization (ISH) analysis
for dog circovirus (DogCV). A) Gross view of the gastrointestinal system from dog 1. Multifocal to coalescing hemorrhages are shown in
the stomach and intestinal serosa. B) Gross view of the kidney from dog 2. Segmental regions of hemorrhage and necrosis (infarction)
can be seen within the cortex and radiating from the medullary papilla to the capsule. C) Hemolyxin and eosin (H&E) stain of the ileum
from dog 1. Peyer’s patches are moderately depleted, and multifocal regions of hemorrhage are present within the muscular intestinal
wall. D) ISH of the ileum from dog 1. Peyer’s patches circumferentially contain abundant DogCV DNA. E) H&E stain of a Peyer’s patch
in the ileum from dog 1, showing moderate depletion of lymphocytes and an increased population of macrophages within the germinal
center and the peripheral base of the follicle. F) ISH of a Peyer’s patch in the ileum from dog 1. DogCV DNA is rich within the cytoplasm
of abundant cells at the periphery of the follicle, and individual cells are scattered within the germinal center, lymphatic channels, and
submucosa. The positive cells have the morphologic appearance of macrophages. G) H&E stain of the jejunum from dog 3, showing
segmental, circumferential, fibrinoid necrosis of the artery. H) ISH of a mesenteric lymph node from dog 2. Macrophages in the medullary
sinus and lymphatic cords contain abundant DogCV DNA.

538 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Circovirus in Dogs

lymph nodes from all 4 dogs (Figure 2, panel H), mandibu- fecal and plasma samples and tissue distribution in infected
lar lymph nodes from 2 dogs, ileal Peyer’s patches from all animals, and detected paracrystalline arrays in inclusion
4 dogs (Figures 2, panels D, F), ellipsoids (terminal arteri- bodies in macrophages. Real-time PCR analysis showed a
oles) of the spleen for 1 dog, and germinal centers in splenic prevalence of 11.3% and 6.9% in fecal samples from dogs
white pulp for 2 dogs. Although the reticular network was with diarrhea and healthy dogs, respectively. DogCV DNA
abundantly positive in dog 1 (Figure 2, panel H), the com- was also found in 3.3% of blood samples from dogs with
mon pattern was localization to the centers of lymphoid or thrombocytopenia and neutropenia, fever of unknown ori-
Peyer’s patch follicles (Figure 2, panels D, F), corresponding gin, and past tick bite, which is approximately the same
to the morphology and distribution of dendritic cells. Rare percentage as previously reported (17).
nuclei of elongate cells (presumed endothelium) lining small ISH analysis of the sentinel dog and 21 additional dogs
vessels of the adrenal cortex or intestinal lamina propria su- selected retrospectively from past necropsies detected viral
prajacent to the Peyer’s patches were positive in 2 dogs. No nucleic acid in 4 dogs, including the sentinel dog, and the
nucleic acid was detected by ISH analysis in other tissues, histopathologic features and distribution of virus in tissue
regardless of the character or intensity of the inflammation. samples from these dogs were evaluated. The organs af-
None of the 5 control dogs showed DogCV DNA by ISH. fected varied even in this small set of animals, but all dogs
had necrotizing vasculitis and hemorrhage, and all but 1 had
Transmission Electron Microscopy Analysis lymphadenitis and granulomatous disease. Because the test-
Ultrastructural analysis of the mesenteric lymph node ed retrospective animals were chosen to match the sentinel
from dog 1 revealed macrophages laden with large num- case, our sampling is biased, and the spectrum of diseases
bers of intracytoplasmic inclusions (Figure 3, panel A). associated with this virus might be broader than we detected.
Inclusions were round, oblong, or irregular; were <0.5 Among dogs positive by ISH, disease signs varied, and clini-
µm; and often clustered (up to 25 per cell) within the cyto- cal, gross, and microscopic features in some of the disease
plasm, (Figure 3, panel B). Most inclusions were granular syndromes were similar to those associated with PCV2 in-
and electron-dense and had a distinct periphery but were fection (4,5). In particular, porcine dermatitis and nephropa-
nondelineated by membrane. Some inclusions contained thy syndrome shares many of the histologic features seen in
paracrystalline arrays of icosahedral virions that were 9–11 DogCV-positive dogs (28,29), and PCV2 has been reported
µm in diameter (Figure 3, panel C). to cause necrotizing lymphadenitis, vasculitis, or neurologic
disease (4,30,31). Virus distribution, as assessed by ISH
Discussion analysis, is also similar between DogCV and PCV2.
We identified a novel circovirus, DogCV, in the liver Viral DNA was consistently detected in the cytoplasm
of a dog that had necrotizing vasculitis and granulomatous of macrophages and monocytes in lymphoid tissues of in-
lymphadenitis. We characterized the genome of multiple fected dogs. Virus distribution in pigs infected with PCV2
DogCV strains, determined DogCV prevalence in dog is most consistently within lymphoid tissue, with sporadic

Figure 3. Lymph node from sentinel dog from which dog circovirus was identified. A) Toluidine blue stain shows multiple macrophages
within the medullary sinus contain vacuoles and discrete, oblong to round, variably stained cytoplasmic bodies (arrows). B) A single
macrophage adjacent to a lymphocyte (upper left) and partial profiles of other cells. Intracytoplasmic inclusion bodies are distributed
throughout the macrophage cytoplasm, along with mitochondria and vacuoles. Scale bar indicates 2 µm. C) Intracytoplasmic inclusion
bodies contain granular content and sometimes paracrystalline to herringbone arrays of 10–11 nm diameter viral-like particles. Scale
bar indicates 100 nm.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 539
RESEARCH

reports of virus in other tissues (4,23,32,33). For example, Dr Li is a staff scientist at the Blood Systems Research In-
in pigs with dermatitis and nephropathy syndrome, granulo- stitute and Department of Laboratory Medicine, University of
matous inflammation of the kidneys is commonly reported; California, San Francisco, USA. Her research interests are viral
however, virus is detected in renal tissue in only a few cases discovery and viral infectious diseases.
by any method. In our study, DogCV DNA was detected in
lymphoid tissues, including Peyer’s patches, even for dogs
3 and 4, where no clinical or histologic enteric lesions were References
detectable. DogCV DNA was also found in small, end-capil- 1. Li L, Kapoor A, Slikas B, Bamidele OS, Wang C, Shaukat S, et al.
lary endothelial channels of the intestinal lamina propria and Multiple diverse circoviruses infect farm animals and are commonly
the adrenal cortex; however, confirmation of these cells as found in human and chimpanzee feces. J Virol. 2010;84:1674–82.
endothelial will require further investigation. http://dx.doi.org/10.1128/JVI.02109-09
2. Todd D. Avian circovirus diseases: lessons for the study of PMWS.
Two distinct histologic features of PCV2 infection Vet Microbiol. 2004;98:169–74. http://dx.doi.org/10.1016/j.
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ruses, porcine circoviruses 1 and 2. Virus Res. 2012;164:4–9. http://
however, macrophages within the lymph node contained dx.doi.org/10.1016/j.virusres.2011.09.013
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were found in both the sinus and medullary cords of the 9. http://dx.doi.org/10.1016/j.virusres.2011.10.007
5. Opriessnig T, Meng XJ, Halbur PG. Porcine circovirus type
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that have been described in PCV2 infected tissues (35,36). strategies. J Vet Diagn Invest. 2007;19:591–615. http://dx.doi.
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6. Opriessnig T, Halbur PG. Concurrent infections are important
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Among the dogs with diarrhea in our study, most (68%) of Natl Acad Sci U S A. 2001;98:11609–14. http://dx.doi.org/10.1073/
those positive for DogCV had co-infection with >1 enteric pnas.211424698
pathogens. Also, for the small set of cases in which we iden- 8. Breitbart M, Salamon P, Andresen B, Mahaffy JM, Segall AM,
Mead D, et al. Genomic analysis of uncultured marine viral com-
tified virus in situ, 1 dog was co-infected with a canine boca- munities. Proc Natl Acad Sci U S A. 2002;99:14250–5. http://dx.doi.
virus and 1 with Babesia conradae. The role of co-infection org/10.1073/pnas.202488399
in the pathogenesis of disease in these cases is unclear. 9. Bexfield N, Kellam P. Metagenomics and the molecular identi-
In summary, DogCV should be considered in cases fication of novel viruses. Vet J. 2011;190:191–8. http://dx.doi.
org/10.1016/j.tvjl.2010.10.014
of unexplained vasculitis in dogs, although further stud- 10. Delwart E. Animal virus discovery: improving animal health,
ies will be required to ascertain whether and when DogCV understanding zoonoses, and opportunities for vaccine develop-
causes disease. DogCV also could be a complicating ment. Curr Opin Virol. 2012;2:344–52. http://dx.doi.org/10.1016/j.
factor in other canine infectious diseases, as is the case with coviro.2012.02.012
11. Tang P, Chiu C. Metagenomics for the discovery of novel human vi-
PCV2, which is most dangerous in pigs co-infected with ruses. Future Microbiol. 2010;5:177–89. http://dx.doi.org/10.2217/
other pathogens. Future research into the contribution of fmb.09.120
DogCV to disease should carefully consider these potential 12. Phenix KV, Weston JH, Ypelaar I, Lavazza A, Smyth JA, Todd D,
viral and host factors. et al. Nucleotide sequence analysis of a novel circovirus of canaries
and its relationship to other members of the genus Circovirus of the
family Circoviridae. J Gen Virol. 2001;82:2805–9.
Acknowledgments 13. Stewart ME, Perry R, Raidal SR. Identification of a nov-
el circovirus in Australian ravens (Corvus coronoides) with
We thank Scott Fish and the California Animal Health Food
feather disease. Avian Pathol. 2006;35:86–92. http://dx.doi.
Safety Laboratory in Davis for electron microscopy processing org/10.1080/03079450600597345
and expertise. 14. Roerink F, Hashimoto M, Tohya Y, Mochizuki M. Organization of
the canine calicivirus genome from the RNA polymerase gene to the
This work was supported by the Blood Systems Research poly(A) tail. J Gen Virol. 1999;80:929–35.
Institute and National Institutes of Health grant R01 HL083254 15. Matsuura Y, Tohya Y, Nakamura K, Shimojima M, Roerink F, Mo-
(to E.D.). P.A.P. is supported by the Bernice Barbour Foundation chizuki M, et al. Complete nucleotide sequence, genome organiza-
tion and phylogenic analysis of the canine calicivirus. Virus Genes.
and the UC Davis, Center for Companion Animal Health. 2002;25:67–73. http://dx.doi.org/10.1023/A:1020174225622

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16. Knierim D, Blawid R, Maiss E. The complete nucleotide sequence of 27. Delwart E, Li L. Rapidly expanding genetic diversity and host range
a capsicum chlorosis virus isolate from Lycopersicum esculentum in of the Circoviridae viral family and other Rep encoding small cir-
Thailand. Arch Virol. 2006;151:1761–82. http://dx.doi.org/10.1007/ cular ssDNA genomes. Virus Res. 2012;164:114–21. http://dx.doi.
s00705-006-0749-4 org/10.1016/j.virusres.2011.11.021
17. Kapoor A, Dubovi EJ, Henriquez-Rivera JA, Lipkin WI. Com- 28. Allan GM, Ellis JA. Porcine circoviruses: a review. J Vet Diagn
plete genome sequence of the first canine circovirus. J Virol. Invest. 2000;12:3–14. http://dx.doi.org/10.1177/10406387000
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2011;6:e28879. http://dx.doi.org/10.1371/journal.pone.0028879 with naturally acquired porcine circovirus type 2 associated dis-
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Metagenomic analyses of viruses in stool samples from children vetmic.2003.10.006
with acute flaccid paralysis. J Virol. 2009;83:4642–51. http://dx.doi. 31. Opriessnig T, Langohr I. Current state of knowledge on porcine
org/10.1128/JVI.02301-08 circovirus type 2–associated lesions. Vet Pathol. 2012; Epub ahead
20. Chevreux B, Pfisterer T, Drescher B, Driesel AJ, Müller WE, Wetter T, of print.
et al. Using the miraEST assembler for reliable and automated mRNA 32. Szeredi L, Dan A, Solymosi N, Csagola A, Tuboly T. Asso-
transcript assembly and SNP detection in sequenced ESTs. Genome ciation of porcine circovirus type 2 with vascular lesions in por-
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org/10.1093/nar/gkg595 spohl J, Segales J, et al. Porcine circovirus type 2-associated cer-
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ware for evolutionary analysis of DNA and protein sequences. Brief (PMWS)–affected pigs. Vet Pathol. 2007;44:621–34. http://dx.doi.
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23. Sarli G, Mandrioli L, Panarese S, Brunetti B, Segales J, Dominguez 34. Segalés J, Domingo M. Postweaning multisystemic wasting syn-
J, et al. Characterization of interstitial nephritis in pigs with naturally drome (PMWS) in pigs. A review. Vet Q. 2002;24:109–24. http://
occurring postweaning multisystemic wasting syndrome. Vet Pathol. dx.doi.org/10.1080/01652176.2002.9695132
2008;45:12–8. http://dx.doi.org/10.1354/vp.45-1-12 35. Stevenson GW, Kiupel M, Mittal SK, Kanitz CL. Ultrastructure of
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25. Langohr IM, Stevenson GW, Nelson EA, Lenz SD, Hogenesch H, multisystemic wasting syndrome. Vet Pathol. 2009;46:729–35.
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porcine circovirus type 2 serogroup B. Vet Pathol. 2010;47:140–7.
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26. Woc-Colburn AM, Garner MM, Bradway D, West G, D’Agostino
J, Trupkiewicz J, et al. Fatal coxiellosis in Swainson’s Blue Moun- California, Davis, Vet Med: PMI, 4206 VM3A, 1 Shields Ave, Davis, CA
tain rainbow lorikeets (Trichoglossus haematodus moluccanus). Vet 95616, USA; email: papesavento@ucdavis.edu
Pathol. 2008;45:247–54. http://dx.doi.org/10.1354/vp.45-2-247

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RESEARCH

Cost-effectiveness of Novel System

of Mosquito Surveillance
and Control, Brazil
Kim M. Pepin, Cecilia Marques-Toledo, Luciano Scherer, Maira M. Morais,
Brett Ellis, and Alvaro E. Eiras

Of all countries in the Western Hemisphere, Brazil has medical and nonmedical costs and indirect costs from loss
the highest economic losses caused by dengue fever. We of work (7). This high economic cost of the disease occurs
evaluated the cost-effectiveness of a novel system of vector even after Brazil spent $1 billion annually on the dengue
surveillance and control, Monitoramento Inteligente da Den- vector control program. Cost-effective methods of vector
gue (Intelligent Dengue Monitoring System [MID]), which control are needed to decrease the huge economic effects
was implemented in 21 cities in Minas Gerais, Brazil. Traps
of this disease in Brazil.
for adult female mosquitoes were spaced at 300-m intervals
throughout each city. In cities that used MID, vector con-
The most accurate method of assessing dengue risk by
trol was conducted specifically at high-risk sites (indicated vector surveillance is one that specifically counts dengue
through daily updates by MID). In control cities, vector con- vectors that are actively in search of a blood meal: adult
trol proceeded according to guidelines of the Brazilian gov- female Aedes aegypti and occasionally Ae. albopictus mos-
ernment. We estimated that MID prevented 27,191 cases of quitoes. Traditional methods of vector monitoring in Bra-
dengue fever and saved an average of $227 (median $58) zil, which include surveys of larvae and pupae (8,9) and
per case prevented, which saved approximately $364,517 in capture of adult mosquitoes by aspiration (10), are less
direct costs (health care and vector control) and $7,138,940 specific and labor-intensive. Surveys of larvae target both
in lost wages (societal effect) annually. MID was more effec- vector sexes and can only predict the number of mosquitoes
tive in cities with stronger economies and more cost-effec- that will survive to adulthood, rather than directly measure
tive in cities with higher levels of mosquito infestation.
adults. Capturing adults by aspiration does not specifically
target female mosquitoes, is labor-intensive, and requires

D engue viruses cause ≈50 million infections annually

worldwide, and ≈1% of these infections require hos-
pitalization because of dengue hemorrhagic fever (1). Bra-
access to premises.
Fixed-position traps designed to capture gravid mos-
quitoes (e.g., MosquiTRAPs) (Ecovec SA, Belo Horizonte,
zil accounts for ≈75% of all dengue cases in the Western Brazil) have been developed to reduce personnel costs and
Hemisphere (2), and during 2000–2005, Brazil reported directly measure adult female mosquito abundance in Brazil
more cases than any other country in the world (3). Since (11,12). MosquiTRAPs have been implemented in the form
the reemergence of dengue in Brazil in 1982, there has of a large-scale mosquito surveillance system, Monitora-
been an epidemiologic shift to hyperendemicity (4,5) and mento Inteligente da Dengue (Intelligent Dengue Monitoring
more severe disease (5,6). Moreover, of all countries in System [MID]; Ecovec SA), which is used to count mosqui-
the Western Hemisphere, Brazil has the highest economic toes in real time. MID involves weekly monitoring of Mos-
losses caused by dengue ($1.35 billion) annually for direct quiTRAP (placed in a 300 m × 300 m grid format) counts and
trapped-mosquito infection status with automated database
Author affiliations: National Institutes of Health, Bethesda, Maryland,
updating (in situ mosquito data entry by cell phones directly
USA (K.M. Pepin); Colorado State University, Fort Collins, Colorado,
to a Web-based database). The mosquito data are managed
USA (K.M. Pepin); Ecovec SA, Belo Horizonte, Brazil (C. Marques-
by a spin-off company (Ecovec SA), which provides daily
Toledo, L. Scherer); Universidade Federal de Minas Gerais, Belo
updates to control personnel so they can specifically target
Horizonte (M.M. Morais, A.E. Eiras); and Duke–National University
highly infested areas. Preliminary results from 3 cities (Tres
of Singapore Graduate Medical School, Singapore (B. Ellis)
Lagoas in Mato Grosso do Sul State, and Presidente Epita-
DOI: http://dx.doi.org/10.3201/eid1904.120117 cio and Bastos in Sao Paulo State) during 1 season of MID

542 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Novel System of Mosquito Surveillance and Control

implementation showed that this system is effective in de- highest dengue incidence in the state were chosen by the
creasing dengue cases (13). However, an estimate of cost- Minas Gerais State Department of Health to receive MID.
effectiveness for more cities over a longer period is needed These cities were Aguas Formosas, Araguari, Bom Despa-
for deciding whether MID should be maintained. cho, Caratinga, Conselheiro Lafaiete, Coronel Fabriciano,
We evaluated the cost-effectiveness of supplement- Curvelo, Governador Valadares, Ipatinga, Itabira, Joao
ing vector control methods with MID in 21 cities in Minas Monlevade, Lavras, Malacacheta, Manhuaçu, Padre Para-
Gerais State, Brazil, after use during 2 dengue seasons. iso, Paracatu, Pirapora, Ponte Nova, Sete Lagoas, Teofilo
We also identified factors that affected efficacy and cost- Otoni, and Visconde do Rio Branco. The only difference
effectiveness of MID. We reported direct savings for in vector-control activities between cities that used MID
health care costs and vector control activities separately and those that did not use MID was that vector control in
from indirect savings for lost wages so that results are MID cities targeted sites that MID identified as highly in-
relevant to public health budgets and societal concerns. fested with gravid adult mosquitoes. Details of the struc-
ture and function of MID and control efforts are shown
Methods in online Technical Appendix 1 (wwwnc.cdc.gov/EID/
article/19/4/12-0117-Techapp1.pdf).
Case Data
Monthly dengue cases during January 2007–June 2011 Data Analysis
were obtained from each municipality in Minas Gerais, Dengue incidence was strongly seasonal, and outbreak
Brazil, by using Sinan Net (Information System for Notifi- probability varied substantially between cities (Figure 2,
cation of Grievances), a publicly available database of the panel A), which did not follow any common statistical
Health Ministry of Brazil. Dengue cases were expressed as probability distribution in the exponential family. Thus,
incidence per 100,000 inhabitants on the basis of the Bra- we adopted a nonparametric approach to data analysis. On
zilian Institute of Geography and Statistics (Rio de Janeiro, the basis of potential differences in dengue transmission
Brazil) 2010 population census. caused by population size (14) and demographics (15), the
21 treatment cities were divided into 5 groups by popula-
MID Mosquito Surveillance System tion size: 18,000–21,000, 35,000–60,000, 70,000–90,000,
MID was implemented in 21 cities in Minas Gerais 100,000–140,000 and 150,000–300,000 for comparison
during April 2009–June 2011. These cities are dispersed with control cities (Figure 2, panel B). Cities within Minas
throughout the state in areas that included a range of popu- Gerais that did not implement MID were referred to as
lation sizes and incidences (Figure 1). Cities that had the control cities. There were 147 control cities that could be

Figure 1. Spatial distribution of 21 cities tested with Monitoramento Inteligente da Dengue (Intelligent Dengue Monitoring System [MID]),
Minas Gerais, Brazil, 2009–2011. A). Size of city centroids (n = 218) (circles) is proportional to population size. B) Size of city centroids (n
= 147) (circles) is proportional to total dengue fever incidence during 2007–2011. Gray circles indicate cities that never implemented MID,
and black circles indicate cities that implemented MID during mid-2009–June 2011. Areas of higher and lower total incidence are positively
clustered with each other (Moran’s I, p<0.0001). Cities that implemented MID and those that had not implemented MID are distributed
throughout areas of high and low incidence. Only cities with populations >15,000 are shown. Incidence data were not available for all cities.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 543
RESEARCH

Figure 2. Changes in incidence of dengue fever in 21 cities that implemented Monitoramento Inteligente da Dengue (Intelligent Dengue
Monitoring System [MID]), Minas Gerais, Brazil, mid-January 2007–June 2011. A) Annual incidence in 21 cities that implemented MID
(bars outlined in black) and 147 cities that had not implemented MID (bars outlined in gray). Horizontal lines in boxplots indicate medians of
1,000 medians. Whiskers indicate ± 2.7 SD. Circles indicate points that fall outside ± 2.7 SD. B) Distribution of population sizes in cities that
implemented MID. C) Time that MID was implemented in each city. D) Median relative increase (RI) in incidence for cities that implemented
MID versus cities that had not implemented MID. RI was calculated as the sum of monthly incidence after MID was implemented divided by
the sum of monthly incidence before MID was implemented for the same number of months. For cities that implemented MID, the median is
a single value for the 21 cities. For cities that had not implemented MID, 21 cities with the same distribution of population sizes as MID cities
were selected at random 1,000 times and their median relative differences during the same set of time frames were calculated. Horizontal
line in the boxplot indicates median of 1,000 medians. Whiskers indicate ± 2.7 SD. Circles indicate points that fall outside ± 2.7 SD.

grouped into a distribution of population sizes of treat- of the 1,000 sets of control cities and the set of treatment cit-
ment cities. ies and calculated the difference (d = RIcontrol – RIMID). Under
To compare this large sample size in a case–control for- the null hypothesis that MID had no effect at decreasing RI,
mat to only 21 MID cities, we generated 1,000 random sets the median of the 1,000 differences would be 0. We tested
of 21 control cities with the same population distribution as this hypothesis using a sign test. The alternative hypothesis
the MID cities. Next, we calculated the relative difference in was that the median of the 1,000 differences would be sig-
incidence (RI) for the same period before and after the start nificantly >0 if MID decreased the RI of treatment cities.
of surveillance for each treatment city (i.e., incidence for x We identified factors that affected the effectiveness
time before MID/incidence for x time after MID). Likewise, and cost-effectiveness of MID by using a generalized lin-
for each set of the control cities, we calculated RI using the ear model (γ distribution, log link) with either RI or US
distribution of time frames in each group of treatment cities dollars/prevented case as response variables. Factors con-
(Figure 2, panel C) matched to the corresponding group in sidered were population size (PS); distance to 3 large popu-
control cities. Lastly, we calculated the median RI for each lations (D3L); distance to 3 high-incidence populations; a

544 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Novel System of Mosquito Surveillance and Control

ranking system for the effectiveness of using MID (PED); a maximum on the number of possible new cases (K). In the
measure of average mosquito infestation during the dengue main text, we assumed that K was equal to 30% of the pop-
season in 2011 (IMFA); population density; income per ulation in city i, which has been observed (16). However,
capita; and an index between 0 and 1 that included employ- we also considered higher and lower values of K (5%, 10%,
ment, income, education, and health, all with equal weight. 20%, and 50%) (Figure 3).
Distances were the sum of Euclidian distance to 3 cities
with population size (D3L) or density in the 90th percen- Cost Data
tile. We fit each variable individually and fit all possible All costs were in US dollars. Costs per dengue case
linear combinations of the 8 variables. were taken from the report of Sheppard et al. (7). They calcu-
We chose between competing models by using delta lated direct and indirect costs for ambulatory ($69 and $317,
Akaike Information Criterion (ΔAIC  =  AIC of intercept respectively) and hospitalized ($428 and $460, respectively)
only model – AIC of target model). A higher ΔAIC indi- case-patients. We considered dengue fever case-patients to
cates a better model of the data. When comparing nested be ambulatory and dengue hemorrhagic fever case-patients,
models that differed by only 1 factor, 2 AIC points is con- dengue shock syndrome case-patients, and case-patients
sidered a significant difference (α = 0.05). Statistics for all who died to be hospitalized. We did not distinguish deaths
single-variable models, model selection results, and fits of (0.045% of case-patients) from severe cases (0.38% of case-
the best multivariable and full models are shown in online patients) because we could not obtain the age distribution of
Technical Appendix 2 in Tables 1, 2, and 3 (wwwnc.cdc. deaths and gross domestic product estimates from each city.
gov/EID/article/19/4/12-0117-Techapp2.xlsx). Indirect costs assumed an average of 4.5 days of lost work
We estimated the number of cases prevented by MID for ambulatory case-patients and 14 days for hospitalized
by predicting the number of cases that would have occurred case-patients (7). Estimates of indirect costs per case were
in the absence of MID and taking the difference between adjusted to account for case-patients who did not miss work
those and the number of observed cases (i.e., cases pre- by using the age distribution of case-patients in Brazil (7).
vented/year = predicted cases in the absence of MID [E] – Total costs for MID in the 21 cities were measured di-
observed annual cases [O]). We calculated E by using a lo- rectly by Ecovec SA (Table). MID costs in individual cities
gistic model according to the equation Ei = diOi(1 – Oi/Ki), varied from $25,566 to $163,944 (online Technical Appen-
where K is the maximum number of possible cases in city dix 2 Table 4). Cost-effectiveness per city was calculated
i. The logic is that the number of cases prevented depends as the measured cost of MID in a given city divided by its
on the estimated growth coefficient (d = RIcontrol – RIMID) number of cases prevented, as estimated from the model.
and the observed cases (O) but is capped by a theoretical In Minas Gerais, vector control activities are conducted

Table. Total costs of MID in 21 cities, Brazil, 2007–2011*

Product or service Cost in US dollars (%)
Royalties to UFMG and FAPEMIG
MID† 29,918.96 (2.0)
MI-Virus† 38,894.65 (2.6)
Consumables (licensing)
MosquiTRAP† 77,533.50 (5.2)
Sticky card 112,776.00 (7.5)
AtrAedes† 131,572.00 (8.8)
Web software 21,000.00 (1.4)
Mobile software 96,041.00 (6.4)
Services
MI-Vírus kit and analyses 58,485.00 (3.9)
MID 61,303.96 (4.1)
Shipping and freight
MI-Virus and traps 11,056.45 (0.7)
Stationary and materials 668.40 (0.0)
Technical and supervision (employees and taxes)
Technical support at Ecovec SA, 12 h/d 115,499.00 (7.7)
Technical support at cities visited 80,208.00 (5.4)
Full-time biologist 372,520.54 (24.9)
Technical visits on site 19,200.00 (1.3)
Taxes 269,270.66 (18.0)
Total 1,495,948.13 (100.0)
*MID, Monitoramento Inteligente da Dengue (Intelligent Dengue Monitoring System); UFMG, Universidade Federal de Minas Gerais; FAPEMIG,
Fundação de Amparo a Pesquisa do Estado de Minas Gerais; MI-Virus, Intelligent Virus Monitoring System; MosquiTRAP, fixed-position trap designed to
capture gravid mosquitoes; AtrAedes, synthetic ovipostion attractant.
†Manufactured by Ecovec SA (Belo Horizonte, Brazil).

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 545
RESEARCH

Figure 3. Effectiveness of Monitoramento Inteligente da Dengue (Intelligent Dengue Monitoring System [MID]), Minais Gerais, Brazil,
mid-2009–mid 2011. Predicted number of dengue fever cases prevented per year during the time of MID are plotted against the annual
incidence of dengue fever in each city during the same time. K is a percentage value of the population size in a city. Error bars indicate 2
SE. A) 29,533 cases were prevented when K = 50%. B) 24,263 cases were prevented when K = 20%. C) 16,578 cases were prevented
when K = 10%. D) 9,219 cases were prevented when K = 5%. Shaded symbols distinguish population size classes as follows: black circles
indicate 18,000–21,000; gray circles indicate 35,000–60,000; white circles indicate 70,000–90,000; triangles indicate 100,000–140,000;
squares indicate 150,000–300,000.

according to guidelines of the National Program for Den- medical and nonmedical direct costs, as well as vector
gue Control (17) and the state department of health in Minas control and MID. Indirect costs comprised costs for lost
Gerais. Government resources are apportioned to cities on wages and MID costs in treatment cities. Costs for MID
the basis of their population size and history of dengue inci- cities were calculated from the number of observed cases
dence. Thus, we assumed that the per capita cost of control (divided into ambulatory and hospitalized case-patients).
was similar in each treatment city. To estimate the cost of Similarly, the estimated costs of dengue in the absence of
mosquito control activities in each city, we took the per capi- MID were divided into ambulatory and hospitalized case-
ta cost ($1.11) from a study in Sao Paulo, Brazil, in 2005 (18) patients by multiplying the sum of the number of observed
and multiplied this cost by the population size in each city. cases plus the number of prevented cases by the propor-
The previous study measured 3 components of dengue con- tion of observed cases in persons who were ambulatory or
trol costs: vector control activities (larval survey, insecticide hospitalized. The dollars saved annually were calculated
spraying); laboratory activities (entomology and serologic by subtracting the cost of dengue in MID cities from the
analysis); and public education and database maintenance. predicted cost of dengue if MID were not implemented.
Labor comprised ≈60% of costs, and materials needed for Underreporting was not accounted for because we had no
conducting the work comprised 31% of costs (18). city-specific data to inform estimates. Costs were not dis-
For each treatment city, we calculated the direct, in- counted because we considered all cases to be nonfatal and
direct, and total costs of dengue. Direct costs comprised our study period was only 2.5 years.

546 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Novel System of Mosquito Surveillance and Control

Results
The annual incidence of dengue in control cities varied
more widely than in treatment cities, and the median annual
incidence in treatment cities was generally higher (Figure
2, panel A). However, there was a trend of decreased dif-
ference in incidence between annual incidence in treatment
cities relative to control cities during the years (2010–2011)
in which MID was used during the peak dengue season
(January–May or June) (Figure 2, panel A). This trend was
confirmed by the finding that the RI before and after the
time frame of MID was 2.7× higher (a decrease from 4.0 in
control cities to 1.3 in treatment cities; 68%), in control cit-
ies relative to those that used MID (z = −31.59, p<0.0001)
(Figure 2, panel D). The RI for treatment cities for each
population group is shown in Figure 4.
The most parsimonious generalized linear model of
RI in MID cities included PS and IDFM (online Technical
Appendix 2 Table 2). PS and IDFM showed a negative cor-
relation with RI, although the correlation of PS was mar-
ginally not significant (p = 0.083 for PS and p = 0.0023 for Figure 4. Mean relative difference in incidence (RI) of dengue
fever cases for treatment cities grouped by population size
IDFM) (online Technical Appendix 2 Table 3). This finding using Monitoramento Inteligente da Dengue (Intelligent Dengue
indicates that MID effectiveness was higher in cities with Monitoring System), Minas Gerais, Brazil, mid-2009–mid 2011.
stronger economies and that there is a trend of higher ef- Horizontal line indicates mean RI for the 1,000 median RI of
fectiveness in larger populations. In contrast, the most par- control city sets. Error bars indicate 2 SD. Error bars for the largest
simonious model of cost-effectiveness included IMFA and population size group are too small to be shown. The black dot is
an outlier that was excluded from the general linear model results.
D3L (online Technical Appendix 2 Table 2). IMFA and
D3L showed a negative correlation with cost-effectiveness
(p = 0.0086 and p = 0.032, respectively) (online Technical variability in clinical disease, high underreporting rates,
Appendix 2 Table 3). Thus, cost-effectiveness was higher and lack of studies that directly measure the efficacy of
in cities with higher mosquito infestation levels and cities controls. Consequently, only a few studies have demon-
that were farther from cities with large populations. strated the cost-effectiveness of vector control activities
Under the assumption that dengue could affect 30% of a (19–21). In one of these studies, targeted source reduc-
population, we estimated that the number of cases prevented tion was more effective than nontargeted vector control,
by MID annually in the largest cities (>130,000 inhabitants) reducing vector abundance by 52%–82% depending on
was 2,300–3,900 (Figure 5). In the smallest cities (<40,000 the country (21). Another study found that targeted vector
inhabitants), these estimates decreased to 143–182, and the control reduced the dengue case load by 53% (20). Our es-
total number in all 21 cities was 27,191. However, these timate of a 68% reduction in incidence caused by targeted
numbers depend on the assumed number of potentially sus- control efforts by MID was higher than that in the study
ceptible persons (Figure 3). The average cost-effectiveness by Suaya et al. (20). One reason may be geographic differ-
was $227/case prevented, which was driven mainly by a ences in the effects of source reduction methods (the previ-
few larger values (Figure 6, panel A). The median value ous study was conducted in Cambodia). Alternatively, our
was $58, indicating that the average value was higher than higher estimate may be caused by implementation of MID
the cost-effectiveness value in most cities. The number of at a fine spatial scale over a broader area, which produced
cases prevented translated to net total savings of $8,999,406 higher intervention efficacy.
annually. Savings in health care and vector control costs The trend of increased effectiveness in larger popula-
was $364,517, and savings in lost wages was $7,138,940 tions might not be significant in the multivariable model
(Figure 6, panel B; online Technical Appendix 2 Table 4). (which includes IDFM) because PS and IDFM showed a
positive correlation (r = 0.53). The single-variable model
Discussion results suggest that MID may be more effective in larger
Accurate estimates of dengue incidence and its eco- populations (online Technical Appendix 2 Table 1). The
nomic effects are more limited (16,19) than are estimates fact that MID was more cost-effective in cities with higher
of other infectious diseases that pose similarly serious pub- mosquito infestation levels emphasizes the power of tar-
lic health threats. This finding is caused mainly by high geting vector control practices to areas in which gravid

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 547
RESEARCH

MID activities and does not evaluate control activities. It

is likely that the quality with which cities conduct pre-
scribed control practices varies, which could also explain
the lack of relationship between PED and RI. Further-
more, PED is assessed by a yes or no checklist for MID
activities, rather than by an evaluation of the quality of
each activity. If only minor activities constitute most of
the variation in PED, then little meaningful variation in
MID quality between cities might be observed. Regard-
less, the lack of relationship between PED and efficacy
of MID suggests that an additional method for assessing
MID quality, perhaps through collaboration with city con-
trol personnel, might be useful for maintaining, standard-
izing, and improving quality.
One caveat to our method of estimating cost-effective-
ness is that it was necessary to estimate the number of sus-
ceptible hosts in the absence of MID (we used 30%) to pre-
Figure 5. Effectiveness of Monitoramento Inteligente da Dengue dict the number of cases that would be prevented. Although
(Intelligent Dengue Monitoring System [MID]), Minas Gerais, 30% is not unreasonable based on previous studies (16),
Brazil, mid-2009–mid-2011. Predicted number of dengue fever the maximum incidence observed in a given city in Minas
cases prevented per year during the time of MID are plotted
Gerais during 2007–2010 was only 8.8%. This discrep-
against the annual incidence of dengue fever cases in each city
during the same time. A total of 27,191 cases were prevented. ancy was partly caused by underreporting, which was not
Cases prevented/year = predicted cases in the absence of MID (E) accounted for in our study. Nevertheless, we also provided
– observed annual cases (O), where Ei =  diOi(1 – Oi/Ki), d is the predictions for lower values of K to understand how it could
difference between median relative difference (RI) in incidence in affect our estimates. When fewer hosts are susceptible, the
control cities (mean 1,000 datasets) minus the RI in each treatment
number of cases prevented is also lower, which decreases the
city, and K is 30% of the population size in city i. Error bars indicate
2 SE of the number of predicted cases that were prevented (points cost-effectiveness of MID. Thus, previous large outbreaks
without bars are shown because the SEs are smaller than the size with the same serotype and vaccination programs would be
of the point). Shaded symbols distinguish population size classes as expected to decrease the cost-effectiveness of MID. Another
follows: black circles indicate 18,000–21,000; gray circles indicate caveat to our assumption is that K varies by city because
35,000–60,000; white circles indicate 70,000–90,000; triangles
of historical disease patterns and other factors. Collection
indicate 100,000–140,000; squares indicate 150,000–300,000.
of longitudinal serologic data would be useful for more ac-
curate, city-specific predictions of K. Last, our study used
mosquitoes are most abundant. A possible reason for high- previously estimated costs for control activities, health care,
er cost-effectiveness in cities that were farther from large and lost wages. These costs were per capita estimates that we
cities could be that proximity to larger cities may enable a assumed could be extrapolated to each city equally. The ac-
higher proportion of cases that were contracted elsewhere curacy of our results could be improved through microcost-
(i.e., during travel or commuting to large metropolitan ing analyses within each city.
areas) (22,23). Although MID showed an average cost-effectiveness
PED, a measure of MID quality, was not correlated value of $227 (median $58) per case prevented in Minas
with effectiveness or cost-effectiveness of MID. This re- Gerais, the average value increased to $616 in 6 moder-
sult suggests that variation in the force of infection be- ately sized cities (population 73,000–117,000) that did not
tween cities overwhelmed differences in PED, the current show any savings in direct costs (online Technical Appen-
measure of PED is inaccurate, or both. The relationship dix 2 Table 4). Three of the cities saved on indirect costs
between mosquito infestation and human incidence is and total costs, but the 3 other cities (Joao Monlevade,
highly variable in space and time (24–26). Studies of den- Itabira, and Conselheiro Lafeite) had a net loss of up to
gue virus serotype circulation in Brazil have found domi- $81,042 in direct costs and $66,246 in indirect costs be-
nance of a single serotype during any given year, and dif- cause of incorporating MID into their budgets. These 3
ferent genotypes within the serotypic groups have caused cities had relatively low annual dengue incidence in the
large, severe outbreaks because of reduced population 2 years before MID implementation (12, 18, and 72 cases
immunity (5,27–29). Thus, variations between cities in per 100,000 population relative to a range of 104–2,014
novel genotype dynamics might have affected variation cases in the other 18 cities except for Paracatu, which had
in RI because of PED. In addition, PED pertains only to 4 cases). Thus, in general, cities with annual incidences of

548 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Novel System of Mosquito Surveillance and Control

Figure 6. Cost-effectiveness of and savings from Monitoramento Inteligente da Dengue (Intelligent Dengue Monitoring System [MID]),
Minas Gerais, Brazil, mid-2009–mid-2011. A) For cost-effectiveness, the number of US dollars (USD$) spent per dengue fever case
prevented is plotted against the annual incidence of dengue fever cases during MID for the city. Each point represents cost-effectiveness
for a city. Points are coded by population size classes. Horizontal line indicates average cost-effectiveness ($227) per case prevented.
B) Savings for each cost component from the benefits of MID. Direct savings include only health care, nonmedical direct savings, and
vector control savings. Indirect savings include only savings in the work force. Total savings include direct and indirect savings. Negative
values indicate dollars lost because of implementing MID. Vertical line indicates 0. Shaded symbols distinguish population size classes as
follows: black circles indicate 18,000–21,000; gray circles indicate 35,000–60,000; white circles indicate 70,000–90,000; triangles indicate
100,000–140,000; squares indicate 150,000–300,000.

>72 cases per year were more likely to have higher MID K.M.P. was supported by the Research and Policy for In-
cost-effectiveness. fectious Disease Dynamics Program of the Science and Technol-
Furthermore, cities in which MID was implemented ogy Directorate, US Department of Homeland Security; and the
had historically high dengue incidences relative to control Fogarty International Center, National Institutes of Health. A.E.E.
cities (mean ± SD 2007 and 2008 were 549.1 ± 592 in MID was supported by Conselho Nacional de Desenvolvimento Cientí-
cities and 240.4 ± 567.6 in control cities). Thus, average fico e Tecnológico (PRONEX-Dengue), Fundação de Amparo à
estimates of cost-effectiveness may be high in cities in Pesquisa do Estado de Minas Gerais, Departamento de Ciência e
which MID was implemented. However, factors determin- Tecnologia–Ministério da Saúde, and Instituto Nacional de Ciên-
ing incidence patterns in a given city, such as population cia e Tecnologia–Dengue.
immunity, infrastructure, or human behavior, may not be
Dr Pepin is an epidemiologist at Colorado State University in
static over time because high population immunity is not
Fort Collins, Colorado. Her research interests are epidemiologic
protective against novel serotypes (or genotypes with high
modeling and statistical analyses of infectious disease data to fa-
forces of infection) and human behavior and infrastructure
cilitate decision making for public health practices.
are continually changing. A predictive model of serotype
dynamics across cities formulated on the basis of serologic
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49892010000300004 Minas Gerais, Brazil; email: alvaro@icb.ufmg.br

550 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Serotype IV and Invasive Group B
Streptococcus Disease in Neonates,
Minnesota, USA, 2000–20101
Patricia Ferrieri, Ruth Lynfield, Roberta Creti, and Aurea E. Flores

Medscape, LLC is pleased to provide online continuing medical education (CME) for this journal article, allowing clinicians
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Learning Objectives
Upon completion of this activity, participants will be able to:

• Describe the classification of infant group B Streptococcus (GBS) disease and the impact of
maternal screening on this illness
• Analyze the epidemiology of infant GBS disease in the current study
• Distinguish the most common GBS genotypes among infants infected in the current study
• Evaluate the significance of GBS genotype IV

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Authors
Disclosures: Patricia Ferrieri, MD; Ruth Lynfield, MD; Roberta Creti, PhD; and Aurea Flores PhD have disclosed no relevant
financial relationships.

Group B Streptococcus (GBS) is a major cause of inva- Isolates were characterized by capsular polysaccharide
sive disease in neonates in the United States. Surveillance serotype and surface-protein profile; types III and Ia pre-
of invasive GBS disease in Minnesota, USA, during 2000– dominated. However, because previously uncommon sero-
2010 yielded 449 isolates from 449 infants; 257 had early- type IV constituted 5/31 EO isolates in 2010, twelve type IV
onset (EO) disease (by age 6 days) and 192 late-onset (LO) isolates collected during 2000–2010 were studied further.
disease (180 at age 7–89 days, 12 at age 90–180 days). By pulsed-field gel electrophoresis, they were classified into
3 profiles; by multilocus sequence typing, representative
Author affiliations: University of Minnesota Medical School, Minne-
isolates included new sequence type 468. Resistance to
apolis, Minnesota, USA (P. Ferrieri, A.E. Flores); Minnesota Depart-
clindamycin or erythromycin was detected in 4/5 serotype
ment of Health, St. Paul, Minnesota, USA (R. Lynfield); and Istituto
This work was presented in part at the XVIII Lancefield International
1
Superiore di Sanità, Rome, Italy (R. Creti)
Symposium on Streptococci and Streptococcal Diseases,
DOI: http://dx.doi.org/10.3201/eid1904.121572 September 4–8, 2011, Palermo, Italy.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 551
RESEARCH

IV isolates. Emergence of serotype IV GBS in Minnesota 11 years, the recent emergence of invasive disease in infants
highlights the need for serotype prevalence monitoring to with serotype IV GBS, and an increase in disease caused by
detect trends that could affect prevention strategies. this serotype in 2010 compared with our previous findings
(15). We provide serotyping results of all isolates from EO
treptococcus agalactiae, or group B Streptococcus and LO disease collected during 2000–2010 and present data
S (GBS), is one of the leading causes of invasive bacterial
diseases, such as bacteremia, pneumonia, and meningitis,
from molecular characterization by pulsed-field gel electro-
phoresis (PFGE) and multilocus sequence typing (MLST) of
in newborns and infants in the first months of life in the serotype IV isolates from EO and LO disease.
United States (1,2) and in other parts of the world (3–6). In
newborns birth through 6 days of age, invasive GBS disease Materials and Methods
is designated as early-onset (EO) and late-onset (LO) in in-
fants 7 days to 3 months of age; some investigators report an Study Design
ultra-late period extending well beyond 3 months of age (3). As part of the CDC Active Bacterial Core Surveillance
During the past 20 years, prevention of EO and LO in- Program, GBS isolates from 453 infants in Minnesota with
vasive GBS disease in the United States has been an area of invasive GBS disease reported during January 1, 2000–
interest for clinicians and public health officials. In the early December 31, 2010, were submitted to the MDH Public
1990s, the overall rate of EO disease in the United States was Health Laboratory, and the case details were reviewed (7).
1.7/1,000 live-born infants (7,8) but differed from one part Infants were classified according to GBS disease onset and
of the country to another: for example, 0.56 for Minneapo- age: 257 (age birth–6 days) with EO disease, 180 (age 7–89
lis/St. Paul, 1.3 for Houston (9), and 1.3 for Maryland (10). days) with LO disease, and 12 (age 90–180 days) with de-
This high rate prompted the Centers for Disease Control and layed LO disease. Four infants with first GBS-positive
Prevention (CDC) to issue guidelines in 1996 for preventing culture at age 6–13 months were classified with ultra-LO
EO disease by screening pregnant women at 35–37 weeks’ disease (ULOD) (3) and were not included in our analyses.
gestation for GBS colonization and administering antimicro- The total numbers of live births in Minnesota by year were
bial drug prophylaxis to women at risk of transmitting the provided by MDH.
organism to the child (11). Although implementation of the
prescribed measures reduced the rate of EO disease in the Study Isolates and Culture Sites
United States to 0.47/1,000 live births by 1999–2001, prob- Of the 449 EO and LO isolates studied and analyzed,
lems remained (e.g., risk-based vs. culture-based approaches 403 were from blood, 42 from cerebrospinal fluid (CSF),
to prevention, laboratory detection of colonized mothers, use and 4 from normally sterile sites (1 bone, 1 joint, and 2
of antimicrobial drugs in women with allergies to penicillin, obtained postmortem from liver and lung). One isolate per
use of secondary prevention among infants) (12); these fac- infant was included in the analysis. Isolates received from
tors required revision of the guidelines in 2002 (7) and again MDH without patient identifiers were serotyped and stud-
in 2010 (13). However, measures designed to prevent EO ied by molecular methods at the GBS Molecular Reference
disease have had little effect on the rate of LO disease, which Laboratory (University of Minnesota Medical School Twin
remained 0.4/1,000 live births throughout the 1990s, varying Cities Campus, Minneapolis, MN, USA). Isolates were
only slightly from year to year (8,12). tested by using single-colony picks in Todd-Hewitt Broth
GBS isolates are characterized according to capsular (THB) (Bacto; Becton, Dickinson and Company, Sparks,
polysaccharide (CPS) serotype, of which 9 are recognized: MD, USA), supplemented with 2% sheep blood.
Ia, Ib, II–VIII (9, 14–16), and a recently proposed sero-
type IX (17). Results from earlier studies in various parts Reference Strains and Antiserum for Serotyping
of the United States, including Minnesota, indicated that Ia, We used GBS prototypes from our laboratory of in-
III, and V were the predominant serotypes in EO disease, ternationally recognized reference strains of serotypes Ia,
whereas serotype III and Ia were predominant in LO dis- Ib, II–VIII, and newly proposed IX; Rabinowitz 3139 was
ease (9,10,12,15). used for serotype IV (17–19). The prototype strains for sur-
Since 1995, our laboratory has collaborated with the face-expressed proteins were those used previously (18).
Minnesota Department of Health (MDH) to serotype isolates Monospecific rabbit antisera to serotypes Ia, Ib, II–VIII,
from cases of EO and LO disease in Minnesota in conjunc- proposed IX, and atypical V (serotype V genetic variant);
tion with the CDC Emerging Infections Program (7). This surface proteins C-α and C-β; group B protective surface
collaboration has enabled us to follow for almost 2 decades (BPS) protein; and the R4(Rib), R1, and R1, R4 (Alp3)
changes in serotype distribution of GBS isolates that cause species of R were produced in our laboratory against in-
invasive disease in Minnesota. Here we report on the epi- ternational reference strains (17,18,20,21). All antisera
demiology of EO and LO GBS disease in Minnesota over were tested against all serotypes to ensure no immunologic

552 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Invasive GBS Disease in Neonates

cross-reactions. In addition, molecular typing by PCR con- had alleles at 6 of 7 loci in common with >1 other member
firmed all prototype strains used for production of each of the complex. The study STs were compared with all STs
type-specific antiserum, including serotype IV. in the Streptococcus agalactiae MLST Database.

CPS and Detection of Surface-expressed Proteins Antimicrobial Drug Susceptibility Studies

Isolates grown in THB were extracted with hot hydro- All 45 isolates from 2010 were tested for susceptibility
chloric acid and typed by double-diffusion immunoprecipi- to clindamycin and erythromycin according to Clinical and
tation in agarose slides with specific antisera to serotypes Laboratory Standards Institute guidelines (www.microbi-
Ia, Ib, and II–VIII, as described (18). An isolate nonreac- olab-bg.com/CLSI.pdf) by a microdilution MIC method or
tive with any of these antisera was considered nontypeable gradient diffusion E-test. For 2000–2009, only the 7 serotype
and was regrown in supplemented THB to upregulate CPS IV isolates were tested. Isolates sensitive to clindamycin
production; the extract was retested (concentrated 3-fold) but resistant to erythromycin were further tested by using a
with antisera to Ia–VIII, proposed IX, and atypical V. Ex- double-disk diffusion D-zone test to determine whether re-
tracts from nontypeable and serotype IV isolates were also sistance to clindamycin could be induced (26).
tested with antisera to GBS surface-expressed proteins
(16,18–20). Statistical analyses of the typing results (2-sid- Results
ed p value, Fisher exact test) were done by using InStat During January 2000–December 2010, a total of 257
(GraphPad Software, Inc., San Diego, CA, USA); p values infants in Minnesota with invasive GBS disease had EO
<0.05 were considered significant. disease, with GBS isolated from cultures taken during the
first 6 days of life (Table 1). An additional 192 infants had
DNA Macrorestriction Profile Analysis LO disease, 180 with GBS-positive cultures at 7–89 days
All 12 serotype IV and 2 nontypeable isolates were and 12 at 90–180 days of age (delayed LO). Four isolates
studied by PFGE according to published methods (16,22,23) from infants with ULOD were not included in our analyses.
by using bacterial DNA digested with SmaI (Invitrogen, La Nearly 90% of all isolates were from blood, but the source
Jolla, CA, USA). The PFGE profile of an isolate was de- of the isolates differed significantly for EO versus LO dis-
termined by comparing its macrorestriction band pattern to ease: 251 EO isolates were from blood, compared with 152
those of the prototype isolates in our PFGE library (16,22), LO isolates (p<0.0001), but CSF was the culture source for
including the prototypes from 4 PFGE profile groups delin- 4 EO isolates compared with 38 LO isolates (p<0.0001).
eated among our serotype IV isolates from recent years. III and Ia were the predominant serotypes, accounting
for approximately two thirds of the 449 GBS isolates from all
MLST and Clonal Complex Assignment EO and LO invasive disease (Table 2). Nearly another third
At least 1 serotype IV isolate from each PFGE pro- consisted of serotypes V, II, and Ib. A few isolates, most-
file was studied by MLST as described (14,16,24). Also ly from infants with EO disease, were serotype IV or VII;
studied were 4 serotype IV blood isolates collected from 2 5 (1.1%) were nontypeable. All serotype IV isolates were
mothers during the peripartum period and from 2 nonpreg- identified by routine serotyping and did not require supple-
nant adults. Results from partial sequencing of 7 house- mental growth or concentration of the extracts; only a few
keeping genes were compared with data from the Strep- isolates of other serotypes required these extra approaches.
tococcus agalactiae MLST Database (http://pubmlst.org/ When the distribution of serotypes in EO versus LO
sagalactiae/) to arrive at an allelic profile and sequence type disease was compared, Ia was most commonly isolated in
(ST) for each isolate, as described (16). The clonal complex the 257 EO cases (79, 30.7%), followed by III (57, 22.2%),
(CC) assignment of each isolate was determined by using II (43, 16.7%), and V (39, 15.2%). In contrast, among
eBURST software (25) so that each CC comprised STs that the 192 LO isolates, serotypes III (95, 49.5%) and Ia (57,

Table 1. Distribution of GBS invasive disease in infants by isolate source and age of infant at time of culture, Minnesota, USA, 2000–
2010*
No. infants with GBS invasive disease†
Late onset
Culture source Early onset 7–89 90–180 >181 Total
Blood 251 142 10 3 406
Cerebrospinal fluid 4 37 1 1 43
Other sites‡ 2 1 1 0 4
All 257 180 12 4 453
*GBS, group B Streptococcus.
†No. isolates studied were 1 per infant (n = 453). Early-onset is birth–6 days. Late-onset categories (age in days at time of culture): classical (7–89);
delayed (90–180); ultra (>181; not included in analyses).
‡Other sites, normally sterile: 1 bone, 1 joint, 2 tissues (1 liver, 1 lung).

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RESEARCH

Table 2. Serotype distribution of invasive GBS isolates for

cultures collected from infants, Minnesota, USA, January 2000–
December 2010*
No. (%) patients†
CPS type Early onset Late onset Total
Ia 79 (30.7) 57 (29.7) 136 (30.3)
Ib 25 (9.7) 12 (6.2) 37 (8.2)
II 43 (16.7) 5 (2.6) 48 (10.7)
III 57 (22.2) 95 (49.5) 152 (33.9)
IV 8 (3.1) 4 (2.1) 12 (2.7)
V 39 (15.2) 18 (9.4) 57 (12.7)
VII 2 (0.8) 0 (0.0) 2 (0.4)
Nontypeable 4 (1.6) 1 (0.5) 5 (1.1)
Total 257 (100.0) 192 (100.0) 449 (100.0)
*GBS, group B Streptococcus; CPS, capsular polysaccharide serotype.
†Early-onset, patient age birth–6 days; late-onset, patient age 7–180 days.
Figure 1. Distribution of early-onset and late-onset invasive group
B Streptococcus disease in infants, by year, Minnesota, USA,
29.7%) predominated. Serotypes Ia and IV were distributed 2000–2010. Bars indicate isolates of all capsular polysaccharide
similarly among EO and LO isolates (Ia, 30.7% of EO vs. serotypes (CPS); line indicates all serotype IV isolates. A total of
29.7% of LO; IV, 3.1% of EO vs, 2.1% of LO). Serotypes 257 infants had early-onset and 192 infants late-onset disease; 12
II and III, however, had significantly different distribution infants had type IV infection.
in EO and LO disease; serotype II accounted for 16.7% of
EO versus 2.6% of LO isolates (p<0.0001), whereas sero- Emergence of resistance to clindamycin in serotype IV
type III accounted for 22.2% of EO versus 49.5% of LO isolates during 2010 led us to investigate antimicrobial drug
isolates (p<0.0001). Of the 42 isolates from CSF, serotype resistance for all 45 serotype IV isolates cultured from in-
III accounted for 50%, whether from infants with EO (2/4) fants with invasive GBS disease during that year. Antimi-
or LO (19/38) disease. The 4 isolates from ULOD were 1 crobial drug susceptibility profiles (Table 4) revealed that 14
each of serotypes Ia, Ib, II, and V (data not shown). (31.1%) of these isolates were resistant to clindamycin and
During the 11-year study period, the number of infants erythromycin; clindamycin resistance was inducible for 4 of
with EO or LO invasive disease varied from year to year; these 14 isolates. In addition, a higher percentage (>60%)
most years had more EO than LO cases (Figure 1). This of isolates of serotypes IV, V, or Ib were clindamycin resis-
variation resulted in yearly incidence that ranged from tant compared with isolates of the predominant serotypes Ia
0.21 to 0.45 (mean 0.33) per 1,000 live births for EO and (8.3%) and III (11.1%). Specifically, the percentage of sero-
from 0.14 to 0.34 (mean 0.25) per 1,000 live births for LO type IV isolates that were clindamycin resistant (4/5, 80%)
disease (Figure 2). Although serotype IV was seen only was significantly higher than for all other serotypes com-
sporadically in previous years (Figure 1), incidence of this bined (10/40, 25%; p = 0.027). In 2010, serotype IV account-
serotype increased in 2010, when it was isolated from 5 ed for 28.6% (4/14) of all isolates resistant to clindamycin.
(16.1%) of 31 infants with EO disease. These 5 cases were Because of the increase of invasive disease caused by
not clustered geographically or temporally. serotype IV GBS, we pursued molecular studies on isolates
Among the 12 infants who had serotype IV invasive
disease during 2000–2010, 11 survived (Table 3). Eight in-
fants had EO disease, all of which were diagnosed within 2
days of birth; only 4 infants, separated temporally, had LO
disease. All infants with EO disease were full term (ges-
tational age 37–42 weeks); for LO infants, all but 1 were
not full term (gestational age 23–34 weeks). Overall, the
proportion of babies born prematurely (<37 weeks) and in-
fected with any GBS serotype was 23.4% (54/231) for EO
disease, 47.0% (77/164) for LO disease, and 53.8% (7/13)
for delayed LO or ULOD disease (data not shown).
Blood was the most common culture source for sero-
type IV isolates (11/12); 1 was from a joint fluid. Isolates
from all 7 infants who had serotype IV GBS disease during
2000–2009 were susceptible to clindamycin, but 4/5 (80%) Figure 2. Incidence of early-onset and late-onset group B
isolates from infants who had EO disease in 2010 were Streptococcus disease per 1,000 live births, by year, Minnesota,
clindamycin resistant. USA, 2000–2010.

554 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Invasive GBS Disease in Neonates

Table 3. Patient characteristics of and clinical data for serotype IV GBS isolates from infants with invasive GBS disease, Minnesota,
USA, January 2000–December 2010*
Patient age at Patient Clindamycin Isolate protein Isolate PFGE
Period Disease type† diagnosis outcome Culture source susceptibility‡ profile profile
2001–2009 Late-onset 98 d Survived Joint fluid S C-α and BPS 37a
Late-onset 30 d Survived Blood S C-α and BPS 37
Late-onset 9d Died Blood S BPS 37a
Early-onset 1d Survived Blood S None 40
Late-onset 9d Survived Blood S BPS 37a
Early-onset 2d Survived Blood S BPS 37a
Early-onset 1d Survived Blood S BPS 37a
2010 Early-onset 1d Survived Blood R C-α 39a
Early-onset Newborn Survived Blood R C-α 39c
Early-onset Newborn Survived Blood R C-α 39a
Early-onset Newborn Survived Blood R C-α 39a
Early-onset Newborn Survived Blood S BPS 37a
*GBS, group B Streptococcus; PFGE, pulsed-field gel electrophoresis; C-α, C-protein α; BPS, group B protective surface protein.
†Early-onset, patient age birth–6 days; late-onset, patient age 7–180 days.
‡S, susceptible, MIC <0.25 μg/mL; R, resistant, MIC >1 μg/mL.

of this serotype. Figure 3, panel A, shows the PFGE DNA recently described (16), while ST459 was a single-locus
macrorestriction band patterns of the serotype IV prototype variant of the much older ST196. Of the 8 isolates, 1
isolates, designated 37, 38, and 39 (16), and profiles we (PFGE profile 39a, ST459) was resistant to clindamycin
classified as 39c and 39a from 2 study isolates expressing (data not shown), as were other isolates in subgroups of
only C-α protein. Profiles of 6 isolates with C-α protein and PFGE profile group 39 (Table 3). The PFGE profiles of all
BPS protein or only BPS protein appeared to be identical the serotype IV isolates were classified into 5 groups or
or very similar and were designated 37 or 37a for their re- their subgroups: 37, 39, and 40 for isolates from infants;
semblance to the group 37 prototype (Figure 3, panel B). 36, 38, and 39 for isolates from adults (Tables 3, 5).
The profile of an isolate that did not express any of the
surface proteins studied was unique and was classified as Discussion
profile 40. Overall, isolates with C-α and BPS proteins or As in the rest of the United States and other parts of
BPS protein alone were in PFGE profile group 37 or its the world (8,12,13,27,28), in Minnesota, GBS is one of the
subgroups and were susceptible to clindamycin; those with leading causes of invasive bacterial infections in infants
only C-α protein were in subgroups of PFGE group 39 and during the first year of life. Overall, the yearly incidence
were resistant to clindamycin (Table 3). rate of EO and LO disease in this state decreased modestly
To investigate further genetic relatedness among se- from 2000 to 2010; however, even with implementation of
rotype IV isolates from invasive GBS disease in Minne- control guidelines issued by CDC, some years had an in-
sota, we studied 4 isolates from infants and 4 from adults crease in incidence, as occurred for EO disease in 2010.
by using MLST (Table 5). We found that the serotype IV Our finding that GBS caused invasive disease beyond
prototype strain 3139, studied for comparison, was ST2 in the third month of life for ≈9% of infants in our study was
CC1 (data not shown). Among isolates from infants, 1 was in keeping with previous reports (3). Investigators have
ST196, 2 were ST452, and 1 was ST468, a new sequence found that premature birth was a major risk factor for LO or
type (allelic profile 5,25,4,3,2,3,1). Among isolates from ULOD GBS disease (8,13,28,29). Prematurity was likely
adults, 1 was ST196, 2 were ST291, and 1 was ST459. The a contributing factor for LO disease in infants <90 days
5 STs were grouped into 3 major CCs: ST196 and ST459 of age, in particular for those with delayed LO disease or
in CC1, ST291 in CC17, and ST452 and ST468 in CC23. ULOD. The proportion of infants with these disease types
ST468 was a single-locus variant of ST452, which we who were born preterm was >2× that for infants with EO

Table 4. Clindamycin susceptibility profiles of GBS isolates from 45 infants with invasive GBS disease, Minnesota, USA, 2010*
Antimicrobial susceptibility profile† CPS serotype
Clindamycin Erythromycin D-test Ia Ib II III IV V Total
S S Not done 7 1 0 13 1 2 24
S R Negative 4 0 0 3 0 0 7
SR R Positive 1 1 0 2 0 0 4
R R Not done 0 2 1 0 4 3 10
Total no. 12 4 1 18 5 5 45
No. (%) resistant 1 (8.3) 3 (75.0) 1 2 (11.1) 4 (80.0) 3 (60.0) 14 (31.1)
*GBS, group B Streptococcus; CPS, capsular polysaccharide serotype.
†S, susceptible, MIC <0.25 μg/mL; R, resistant, MIC >1 μg/mL; SR, inducible resistance to clindamycin indicated by positive D-zone test (double-disk
diffusion test).

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 555
RESEARCH

Figure 3. DNA macrorestriction profiles for serotype IV isolates from invasive group B Streptococcus (GBS) disease in infants, Minnesota.
Isolates were studied by SmaI digestion and pulsed-field gel electrophoresis (PFGE) analysis and were designated as expressing
C-protein α (C-α) or group B protective surface protein (BPS). Lane number is at the top and PFGE profile number at the bottom of each
lane. A) Lane 2, λ molecular size standard; lanes 3 and 4, serotype IV/C-α GBS isolates from early-onset disease; lanes 5–7, prototypes
of PFGE profile groups 39 (IV/C-α), 38 (IV/C-α), and 37 (IV/C-α and BPS); lane 8, internal standard 89-022 (Ib/C-α and C-β). B) Lane 1,
λ molecular size standard; lane 2, PFGE profile 37 prototype (IV/C-α and BPS); lanes 3–6, isolates from late-onset disease; lanes 7–9,
isolates from early-onset disease; lane 10, internal standard 89-022. Protein profile of isolates in lanes 2–4, C-α and BPS; lanes 5–8, BPS
only. The isolate in lane 9 did not express any of the proteins studied.

disease; in Minnesota, as in other parts of the United States newborns but also among older infants through the first
(30), ≈75% of infants with EO disease are born at full term. year of life, just as occurred with serotype V during the past
Our results on the distribution of serotypes from all GBS 2 decades (33,34). The sharp increase in 2010 of disease
invasive disease were consistent with studies showing that, caused by serotype IV isolates and concurrent emergence
worldwide, serotype III continues to predominate, followed of clindamycin resistance within this serotype could fore-
by serotypes Ia and V (27,31). We found that the predomi- shadow problems similar to those for serotype V; a high
nant serotypes in EO disease continued to be Ia, followed percentage of antimicrobial drug–resistant serotype V iso-
by III, with these serotypes in reverse order for LO disease, lates (14,26,35) cause disease in infants and older adults
similar to our previous findings for Minnesota (15) and find- (33,36). We found not only an increase in the percentage
ings for other parts of the country (9). However, in marked of nonpregnant women in the United States colonized with
contrast to the 1990s, when the prevalence of serotype V was serotype IV in vaginal/rectal sites (16) but also evidence of
2× that for serotype II (15), serotype II was the third most clindamycin resistance, with 3 of 8 representative serotype
prevalent serotype for EO disease in our study. IV isolates studied found to be resistant (P. Ferrieri, unpub.
Characterization of GBS isolates by serotype over time data). Because the vaginal tract is a reservoir for GBS caus-
enabled us to track the emergence and spread of serotype ing EO disease (13,37), increased colonization of this site
IV as a cause of invasive disease in Minnesota. These re- with clindamycin-resistant serotype IV GBS is of concern.
sults showed that this previously less common serotype has Careful attention must be given to the type of intrapartum
become more common, starting in the mid-1990s, when se- antimicrobial drug prophylaxis administered; GBS isolates
rotype IV was found in a few adults (2 mothers in the peri- should be assessed for inducible clindamycin resistance
partum period and 1 nonpregnant adult) (15); subsequently, when penicillin, ampicillin, or cefazolin cannot be used.
in 2001, the first case of LO disease caused by serotype IV Among the serotype IV isolates from invasive GBS
was found, and in 2010, this serotype became prominent disease, we observed association among PFGE profile,
in causing GBS disease. Isolation of this serotype in 2011 surface protein profile, and susceptibility/resistance to
from 2 infants with LO disease suggests its continued pres- clindamycin. Isolates with only BPS protein or C-α and
ence in the community (P. Ferrieri, unpub. data). BPS proteins were in PFGE group 37 and susceptible to
Our findings raise the possibility that serotype IV, al- clindamycin; those with only C-α protein were in group 39
though reported previously from a case of EO disease in or its subgroups and resistant to clindamycin. Results from
the United States (32) and in small numbers from other a study of serotype IV isolates from colonized nonpregnant
parts of the world (6), has the potential to emerge as a women living in various areas in the United States showed
notable cause of invasive GBS disease not only among a similar association between surface protein profile and

556 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Invasive GBS Disease in Neonates

Table 5. Molecular characteristics of serotype IV GBS isolates causing invasive disease in infants and adults, Minnesota*
Isolate source† PFGE profile Allelic profile‡ Sequence type§ Clonal complex¶
Mother, early-onset 38d 1,1,3,1,1,12,2 196 1
Infant, early-onset 40 1,1,3,1,1,12,2 196 1
Nonpregnant adult 39a 1,1,3,1,41,12,2 459 1
Mother, early-onset 36c 2,25,1,2,1,1,1 291 17
Nonpregnant adult 36d 2,25,1,2,1,1,1 291 17
Infant, late-onset 37 5,25,4,3,2,3,3 452 23
Infant, late-onset 37a 5,25,4,3,2,3,3 452 23
Infant, early-onset 37a 5,25,4,3,2,3,1 468 23
*GBS, group B Streptococcus; PFGE, pulsed-field gel electrophoresis.
†Early-onset, patient age birth–6 days or mother during peripartum period; late-onset, patient age 7–180 days.
‡Multilocus sequence type of 7 housekeeping genes (adhP, pheS, atr, glnA, sdhA, glcK, tkt).
§From http://pubmlst.org/sagalactiae database.
¶Determined by eBurst analysis (25).

PFGE profile (16). MLST results suggested a possible as- and epidemiologic data for this work and Dave Guse for perform-
sociation between clindamycin resistance and ST459. ing antimicrobial drug susceptibility testing.
In contrast to colonizing serotype IV isolates, which
Funding for GBS surveillance in Minnesota was provided
have limited clonal diversity (16), results from our mo-
through the CDC Emerging Infections Program.
lecular studies of invasive serotype IV isolates showed
that the 16 isolates cultured since 1995 spanned 5 PFGE Dr Ferrieri is a professor in the Departments of Laboratory
profiles, 5 STs, and 3 CCs, which suggests greater genetic Medicine/Pathology and Pediatrics, medical director of the Clini-
diversity. Among 97 colonizing serotype IV isolates from cal Microbiology Laboratory, and attending physician in pediatric
a wider area of the United States, we found 3 PFGE profile infectious diseases at the University of Minnesota Hospital, Min-
groups (37–39) and 2 STs (452 and 459) (16); in contrast, neapolis. Her research interests include GBS and pneumococcal
among just 16 invasive isolates in this study, we found the disease, animal models of infection, pathogenic mechanisms of
same PFGE profiles and STs and 2 additional PFGE pro- disease, host defense responses, innovation in diagnostic microbi-
file groups (36 and 40) and STs (196 and 468). In other ology, and clinical infectious diseases.
MLST studies of GBS isolates (4–6,14,38,39), only small
numbers of serotype IV have been included, and these have
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et al. Molecular analysis of group B protective surface protein, a sota, USA, 2003–2007. Emerg Infect Dis. 2009;15:1279–81. http://
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ferring patterns of evolutionary descent among clusters of related Address for correspondence: Patricia Ferrieri, University of Minnesota
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1530.2004 MN 55455, USA; email: ferri002@umn.edu

558 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Transmission of
Hepatitis E Virus from
Rabbits to Cynomolgus Macaques
Peng Liu,1 Qiu-Ning Bu,1 Ling Wang, Jian Han, Ren-Jie Du, Ya-Xin Lei, Yu-Qing Ouyang,
Jie Li, Yong-Hong Zhu, Feng-Min Lu, and Hui Zhuang

The recent discovery of hepatitis E virus (HEV) strains The zoonotic nature of HEV was first confirmed
in rabbits in the People’s Republic of China and the United in 1997 with the identification of HEV isolates in swine
States revealed that rabbits are another noteworthy in the United States, which were most closely related to
reservoir of HEV. However, whether HEV from rabbits can an isolate of HEV from a person in the United States,
infect humans is unclear. To study the zoonotic potential for and this isolate could experimentally infect nonhuman
and pathogenesis of rabbit HEV, we infected 2 cynomolgus
primates (5,6). Zoonotic transmission of HEV was further
macaques and 2 rabbits with an HEV strain from rabbits in
China. Typical hepatitis developed in both monkeys; they
substantiated with the demonstration of HEV infection in
exhibited elevated liver enzymes, viremia, virus shedding persons after they ate undercooked infected meat from wild
in fecal specimens, and seroconversion. Comparison of the boars and wild deer (7,8). Antibodies against HEV have
complete genome sequence of HEV passed in the macaques been detected in numerous animal species, including dogs,
with that of the inoculum showed 99.8% nucleotide identity. cats, sheep, goats, horses, cattle, bison, and rats; and HEV
Rabbit HEV RNA (positive- and negative-stranded) was strains have been genetically identified from domestic and
detectable in various tissues from the experimentally wild pigs, chickens, deer, mongooses, and rabbits (4,9).
infected rabbits, indicating that extrahepatic replication may The recent discoveries of HEV-like viruses in rats and fish
be common. Thus, HEV is transmissible from rabbits to have further broadened understanding of the host range and
cynomolgus macaques, which suggests that rabbits may be diversity of HEV (10–12).
a new source of human HEV infection.
The first strain of rabbit HEV was isolated from Rex
Rabbits on 2 rabbit farms in Gansu, People’s Republic of

H epatitis E virus (HEV) is the causative agent of acute

hepatitis E, which is endemic to many developing
countries and occurs sporadically in some industrialized
China (13). Additional studies indicated that rabbit HEV
was prevalent among various breeds of farmed rabbits
throughout much of China, and the prevalence of antibodies
countries. HEV is a small nonenveloped virus with a pos- against HEV was 57.0% in Lanzhou and 54.6% in Beijing
itive-sense single-stranded RNA genome of ≈7.2 kb; it is (13–15). Rabbit HEV has also been isolated from rabbits
currently classified as the sole member of the genus Hep- in Virginia, USA, which showed a high prevalence of
evirus, family Hepeviridae (1). Thus far, at least 4 geno- antibodies against HEV (36%) and HEV RNA (16.5%)
types, which comprise a single serotype, of HEV have been (16). Phylogenetic analyses revealed that rabbit HEV was
identified in mammals: genotypes 1 and 2 are restricted to most closely related to genotype 3 HEV, which has been
strains that infect humans, and genotypes 3 and 4 are zoo- confirmed to infect humans. Furthermore, a recent study
notic (2). More recently, a putative fifth HEV genotype was indicated that rabbit HEV is antigenically related to the
identified in wild boars in Japan (3). HEV from chickens, other known animal strains of HEV and is experimentally
which is phylogenetically distinct from HEV from mam- transmissible to swine (17). However, to our knowledge,
mals, is likely to be classified as a new genus within the no study had determined the zoonotic potential of rabbit
family Hepeviridae (4). HEV. Therefore, in this study, we endeavored to ascertain
whether rabbit HEV can cross species barriers and infect
Author affiliation: Peking University Health Science Center, Beijing,
nonhuman primates and to further clarify the pathogenesis
People’s Republic of China
and replication of rabbit HEV in its natural host.

DOI: http://dx.doi.org/10/3201/eid1904.120827 1
These authors contributed equally to this article.

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RESEARCH

Materials and Methods (2 rabbits per group) and inoculated intravenously with
either 1 mL of PBS (negative control) or 1 mL of rabbit
Virus Inocula HEV inoculum. Serum and fecal specimens were collected
The rabbit HEV strain (CHN-BJ-R14) used in this weekly after inoculation. Serum samples were tested for
study was originally recovered from the feces of a farmed ALT activity and HEV RNA. Fecal specimens were also
Rex Rabbit in the suburbs of Beijing in 2011. The fecal assayed for HEV RNA. If serum and fecal specimens
sample was diluted in phosphate-buffered saline (PBS) became simultaneously positive for HEV RNA, a complete
(pH 7.4) containing 1% bovine serum albumin to make necropsy was performed of each rabbit. Bile and various
a 10% (wt/vol) suspension. The clarified suspension was different types of tissues and organs, including liver,
subsequently filtered through 0.45-μm and 0.22-μm filters. kidney, small intestine, spleen, stomach, heart, brain,
Titers of the rabbit HEV inoculum were determined by a bladder, and lung, were collected and stored at -80°C.
semiquantitative nested reverse transcription PCR (RT- To prevent cross-contamination during necropsy, we used
nPCR) (18), and the titer was 104 genome equivalents (GE) individually wrapped, sterile disposable materials and new
per milliliter (mL). sterile scalpel blades for each sample.
Approximately 100 mg of each tissue and organ was
Animals homogenized in 1 mL of sterile PBS (pH 7.4) to make
Two juvenile male cynomolgus monkeys (Macaca 10% (wt/vol) suspensions and clarified by centrifugation
fascicularis), weighing 2.0–2.5 kg, designated as Cy1 and at 4,500 g for 10 min at 4°C. Thereafter, 100 µL
Cy2, were obtained from the Beijing Xierxing Institute of of the clarified supernatants was used for total viral
Biologic Resources (Beijing, China) for the cross-species RNA extraction, and positive-stranded and negative-
infection study. For the rabbit infection study, four 7-week- stranded HEV RNA were detected by RT-nPCR as
old specific-pathogen free (SPF) New Zealand white rabbits, described below.
weighing 750–1,000 g, were obtained from the Department
of Laboratory Animal Science of Peking University Health Determination of ALT Levels
Science Center. Preinoculation serum and feces specimens All serum samples were tested immediately for
were collected once a week for 3 weeks, and all animals ALT levels with a Hitachi Automatic Clinical Analyzer
were tested for alanine aminotransferase (ALT) to establish 7180 (Hitachi High-Technologies, Tokyo, Japan), by
a baseline, and were confirmed as negative for antibodies using chemical reagents purchased from Roche (Basel,
against HEV by an ELISA and negative for HEV RNA Switzerland), according to the manufacturer’s instructions.
by RT-nPCR. The animal experiments were approved by Biochemical evidence of hepatitis was recorded when the
the Committee of Laboratory Animal Welfare and Ethics, serum ALT level exceeded the baseline ALT level by >2-
Peking University Health Science Center. The regulations fold, as defined by a peak ALT value that was equal to or
of the review committee of Laboratory Animal Welfare greater than double the prechallenge values (19,20).
and Ethics and the protocol for the review on Laboratory
Animal Welfare and Ethics, Peking University Health ELISA to Detect Antibodies against HEV
Science Center, were followed. The serum specimens collected from monkeys were
tested for IgM and IgG against HEV by using an ELISA
Experimental Inoculation of Nonhuman Primates based on the virus E2 protein (amino acids 394–606 of
To determine whether rabbit HEV strains are HEV open reading frame [ORF] 2) (20), according to the
transmissible to nonhuman primates, we inoculated manufacturer’s instructions (Wantai, Beijing, China). The
intravenously 2 cynomolgus monkeys, housed separately, serum samples collected from rabbits were also examined
with 2 mL of the rabbit HEV inoculum. After inoculation, for antibodies by using the same assay. Signal-to-cutoff
serial serum and fecal samples were collected 2×/week for values were calculated, and values >1 were considered
16 weeks. positive. Preinoculation baseline serum specimens were
Serum samples were tested for ALT levels and for IgM used as negative controls for each monkey.
and IgG against HEV. All samples were also assayed for
HEV RNA by RT-nPCR (15). RT-nPCR to Detect Positive-stranded and
Negative-stranded HEV RNA
Experimental Infection of Rabbits RNA was extracted from 100 μL of serum, bile, tissue
To clarify the extrahepatic replication sites of HEV, suspension, or 10% fecal suspension by using TRIzol
rabbits were experimentally infected with rabbit HEV as reagent (Invitrogen, Burlington, ON, Canada), and purified
described (19). In brief, 4 SPF rabbits, which were housed RNA was resuspended in 11 μL of RNase-free water. To
in separate cages, were divided randomly into 2 groups detect positive-stranded HEV RNA, 11 μL of purified

560 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Transmission of HEV from Rabbits to Macaques

RNA was reverse transcribed at 42°C for 60 min with entire viral genome. The nested PCR was done as described
SuperScript II reverse transcription (Invitrogen) and the (21). The nucleotide sequences at the 5′ and 3′ termini of
external reverse primer P4 or S4 in a reaction mixture of the genome were determined by using a rapid amplification
20 mL. Then, nested PCRs were carried out to amplify the of cDNA ends (RACE) kit (Invitrogen), according to the
partial fragments of ORF1 (129–373 nt) and ORF2 (5,983– manufacturer’s instructions.
6,349 nt) of the HEV genome by using the 2 sets of specific
external and internal primer pairs listed in online Technical Sequence Analyses
Appendix Table 1 (wwwnc.cdc.gov/EID/article/19/4/12- The expected PCR products amplified from the
0827-Techapp1.pdf). The PCR parameters for both sets of inoculum and monkey fecal sample at 3 weeks wpi were
primers and both rounds of PCR were the same, with an purified and ligated into a pGEM-T vector (Promega). At
initial incubation at 94°C for 5 min, followed by 30 cycles least 3 positive clones for each region of the viral genome
of denaturation at 94°C for 30 s, annealing at 50°C for 30 were sequenced commercially in both directions by using
s, and extension at 72°C for 40 s, with a final incubation at an automated DNA sequencer (ABI model 3730 sequencer;
72°C for 10 min. Applied Biosystems, Foster City, CA, USA).
Tissues with detectable positive-stranded HEV RNA Nucleotide sequences were assembled and analyzed
were then assayed for negative-sense HEV RNA by RT- with the MEGA 4.0 and ALIGNX software (Vector NTI
nPCR with the same 2 sets of universal primers (online package version 9.0; Invitrogen). ORFs were identified
Technical Appendix Table 1). The extracted RNA was by using the EMBOSS software (version 5.0.0; emboss.
subjected to cDNA synthesis with the external forward sourceforge.net). The full-length genomic sequences of
primer P1 or S1. Then parental RNAs were degraded CHN-BJ-R14 and rHEV-Cy1 reported in this study have
by RNaseH, and this was followed by nested PCR. The been deposited in GenBank under accession nos. JX109834
amplification conditions for negative-stranded HEV RNA and JX121233, respectively.
detection were essentially the same as those used in the
detection of positive-sense HEV RNA. Results
The PCR protocol used in this study could detect as
few as 10 GE copies of HEV plasmid DNA. Negative and Cross-Species Transmission of Rabbit HEV
positive controls were included in each assay to exclude the to Nonhuman Primates
possibility of contamination and failure of amplification. In both of the macaques inoculated with rabbit
A recombinant plasmid containing HEV ORF1 and ORF2 HEV, hepatitis developed, as determined on the basis of
fragments at a concentration of 102 copies per mL and ALT elevation, viremia, fecal shedding of viruses, and
serum or fecal specimens or tissues from naive rabbits seroconversion (Figure). Dramatic elevations in serum
were used as positive and negative controls, respectively. ALT were observed 5 and 10 wpi for both monkeys, with a
Samples showing a band of the expected size on a 1.5% peak value of 135 U/L at 9 wpi for monkey Cy1 and 97 U/L
(w/v) agarose gel were considered positive, and the positive at 5.5 wpi for monkey Cy2.
products were directly sequenced. Before inoculation, both monkeys were seronegative
for HEV and became seropositive for antibodies against
Amplification of the Full-Length Genome HEV at 6–7 wpi. IgM against HEV was detectable from
of Rabbit HEV 7 to 12 wpi for Cy1 and from 6 to 8 wpi for Cy2. The rise
To compare the complete genome sequence of the in IgM against HEV was followed closely by a strong
HEV passed in the macaques to that of the inoculum, response of IgG against HEV for Cy1, whereas both
the fecal sample (rHEV-Cy1) of 1 monkey at 3 weeks’ responses occurred at about the same time for Cy2. The
postinoculation (wpi) and the inoculum (CHN-BJ-R14) IgG level against HEV remained markedly elevated at the
were sequenced to determine the full-length genome as end of the 16-week experiment.
reported (21). Briefly, total RNA was extracted from 120 Serum and fecal samples taken before inoculation
μL of the rabbit HEV inoculum and a 10% monkey fecal from both monkeys were negative for HEV RNA. Viremia
suspension in PBS by using the Total RNA Isolation System and fecal shedding of viruses were detected in both
(Promega, Madison, WI, USA). cDNA was synthesized monkeys after intravenous inoculation. Fecal excretion of
from 12 μL of purified RNA by using 1 μL (200 U) of rabbit HEV, indicative of replication, was first detected
Moloney murine leukemia virus reverse transcription at 1 wpi and persisted for 5–9 weeks. HEV viremia was
(Promega) and 2 μL (10 pmol/L) of OligodT primer. With first detected at 5.5 wpi for Cy1 and at 2 wpi for Cy2 and
6 sets of specific external and internal primer pairs (online lasted for 2.5–3.5 weeks. The partial sequences of the PCR
Technical Appendix Table 2), a set of nested PCRs were products from both monkeys shared 99%–100% nucleotide
performed by using the first-strand cDNA to amplify the identity with the original inoculum.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 561
RESEARCH

Figure. Cross-species trans-

mission of rabbit hepatitis E
virus (HEV) to 2 cynomolgus
macaques (Cy1 and Cy2).
Alanine aminotransferase (ALT)
levels are plotted as U/L. The
baseline ALT levels were 33
U/L and 38 U/L for Cy1 and
Cy2, respectively. The titers of
HEV IgM and IgG are plotted as
ELISA signal-to-cutoff (S/CO)
values. Presence and absence
of HEV RNA in serum or feces
are indicated by + and – signs,
respectively.

Sequence Analyses of Rabbit HEV during the inoculum (CHN-BJ-R14) revealed 18 nt mutations over
Cross-Species Transmission the entire genome, resulting in 9 nonsynonymous amino acid
To analyze mutations in the rabbit HEV genome that changes. ORF1 harbored 16 of the 18 nt mutations; 11 were
appeared during a single passage between the 2 different in the helicase domain and in the RNA-dependent RNA
host species, we sequenced rabbit HEV strains recovered polymerase domain (Table).
from the inoculum (CHN-BJ-R14) and from experimentally Nucleotide BLAST (http://blast.ncbi.nlm.nih.gov/
infected cynomolgus monkeys (rHEV-Cy1) over the entire Blast.cgi) analysis showed that CHN-BJ-R14 and rHEV-
genome. The CHN-BJ-R14 and rHEV-Cy1 isolates had the Cy1 were most closely related to genotype 3 HEV with a
same genomic length of 7,284 nt, excluding the 3′ poly (A) maximum nucleotide identity of 81%, with the exception
tail, and contained 3 ORFs—ORF1, ORF2, and ORF3— of 3 other rabbit HEV strains isolated in Gansu (13) and
which encoded proteins of 1,722 aa (nt 26–5194), 660 aa (nt Beijing (21). However, several unique features possessed
5232–7214), and 113 aa (nt 5221–5562), respectively. The 5′ only by rabbit HEVs, but not genotype 3 or other HEV
untranslated region (UTR) and 3′ UTR comprise 25 nt and strains, were observed in the 2 rabbit HEV isolates of
71 nt, respectively. Sequence analyses showed that CHN- this study. These features, discovered in a previous study
BJ-R14 and rHEV-Cy1 shared 99.8% nucleotide identity with (21), were characterized by an insertion of 31 aa in ORF1
each other. Comparison of the complete genome sequence of (929–959 aa) and a unique A residue at nt 13 (sites based
rabbit HEV passed in the macaques (rHEV-Cy1) with that of on CHN-BJ-R14) in the 5′ UTR (data not shown).

562 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Transmission of HEV from Rabbits to Macaques

Extrahepatic Replication of HEV in Experimentally food safety (22,23). The recent discovery of rabbit strains
Infected Rabbits of HEV in China (13) and the United States (16) showed
Both control rabbits remained negative for HEV RNA that farmed rabbits are another key reservoir of HEV. In
throughout the study. Viremia and fecal shedding of HEV our previous study, phylogenetic analysis of the genome
were detected in rabbits inoculated with the rabbit HEV of rabbit HEV suggested the potential for cross-species
inoculum. Both rabbits were necropsied, at 5.5 wpi and transmission of rabbit HEV (21). A recent study also
12 wpi, respectively, when ALT elevation was observed, demonstrated that rabbit HEV can cross species barriers
and HEV RNA was detected simultaneously in serum and infect SPF pigs (17). In the study described here, we
and feces. Bile and 9 different types of tissues and organs showed that under experimental conditions, rabbit HEV is
were collected and tested for positive-stranded HEV RNA. transmissible to cynomolgus macaques, which can serve
Positive-stranded HEV RNA was detected in bile and in 5 as surrogates for humans. This finding suggests that rabbit
of the tissues—liver, kidney, small intestine, spleen, and HEV may be a new source of human HEV infection.
stomach. Detection of positive-stranded HEV RNA from In both cynomolgus monkeys infected in this study
various tissues and organs did not indicate that the virus with 104 GEs of rabbit HEV, typical acute hepatitis E
was replicating in these tissues because contamination of developed. The patterns of HEV infection in cynomolgus
the tissue samples by virus circulating in the blood could monkeys infected with rabbit HEV were similar to those
not be ruled out. To further identify the replicating sites of of animals inoculated with HEV strains of genotypes 1–4,
HEV, we screened for negative-stranded RNA, which is an that is, characterized by fecal excretion of virus, followed
intermediate product during HEV replication, in all tissues by viremia and liver enzyme elevation and finally by
that were positive for the positive-stranded HEV RNA. seroconversion (24–27). Although the same viral doses
Negative-stranded RNA was also detectable in the 5 types were inoculated into both monkeys, the overall course of
of tissues. The positive products were sequenced and found disease varied somewhat, findings in accord with those
to be identical to the original inoculum. of previous studies (28). In an earlier study, cross-species
infection of pigs infected with rabbit HEV showed a delayed
Discussion onset and short duration of viremia and fecal virus shedding
Since the first animal strain of HEV, swine HEV, was and an absence of seroconversion (17), which differed from
identified from a pig in the United States in 1997 (5), the findings observed in infected monkeys of this study. The
increasing identification of HEV infection among a wide differences might suggest that pigs are less susceptible than
range of animals, including pigs, chickens, wild boar, and nonhuman primates to rabbit HEV. However, because the
deer (4), has raised public health concern for zoonoses and inocula in both the current study and in other studies (17,19)

Table. Comparison of the complete genome sequence of rabbit HEV passed in macaques with that of the inoculum*
Nucleotide Amino acid
Nucleotide position† Genomic region CHN-BJ-R14‡ rHEV-Cy1§ Position† Substitution
614 ORF1-MeT C T 197 Silent
957 ORF1-Y T C 311 Thr to Ile
1667 ORF1-PCP T C 548 Silent
1875 ORF1 T C 617 Pro to Leu
2706 ORF1-X G A 894 Asp to Gly
3553 ORF1-Hel A T 1176 Silent
3571 ORF1-Hel C T 1182 Silent
3859 ORF1-RdRp C A 1278 Silent
3889 ORF1-RdRp C T 1288 Silent
3972 ORF1-RdRp G A 1316 Glu to Gly
4215 ORF1-RdRp C T 1397 Leu to Pro
4285 ORF1-RdRp A G 1420 Silent
4414 ORF1-RdRp T C 1463 Silent
4427 ORF1-RdRp C T 1468 Tyr to His
4882 ORF1-RdRp T C 1619 Silent
5028 ORF1-RdRp T C 1668 Ala to Val
5531 ORF2 C T 100 Silent
ORF3 C T 104 Ala to Val
5713 ORF2 T A 161 Ile to Asn
*HEV, hepatitis E virus; ORF, open reading frame; Thr, Threonine; Ile, Isoleucine; Pro, proline; Leu, leucine; Asp, aspartic acid; Gly, glycine; Glu, glutamic
acid; Tyr, tyrosine; His, histidine; Ala, alanine; Val, valine; Asn, asparagine.
†Nucleotide or amino acid position according to the rabbit HEV CHN-BJ-R14 strain.
‡CHN-BJ-R14, HEV isolate recovered from the rabbit HEV inoculum in this study.
§rHEV-Cy1, HEV isolate recovered from the fecal sample of 1 monkey at 3 wpi in this study.
¶Putative domains in ORF1. MeT, methyltransferase domain; Y, Y domain; PCP, papain-like cysteine protease domain; X, X or macro domain; Hel,
helicase domain; RdRp, RNA-dependent RNA polymerase domain.

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RESEARCH

have not yet been titrated for infectivity and because HEV HEV found in this study and the other reports of extrahepatic
infections are virus dose dependent (18), additional studies manifestations of HEV infection in humans (34), clinicians
should be performed to determine the infectivity titer of should consider the possibility of HEV infection in patients
rabbit HEV and to demonstrate whether the rate of inducing with nonhepatic diseases, especially patients with acute
hepatitis increases with virus dose of infection. pancreatitis, neurologic syndromes, thrombocytopenia,
In the current study, although comparison of the full- hemolysis, and autoimmune manifestations.
length sequences of rHEV-Cy1 and CHN-BJ-R14 showed In conclusion, the successful infection of cynomolgus
99.8% nucleotide identity, 18 nt changes, resulting in 9 macaques with rabbit HEV suggests that humans might
nonsynonymous amino acid substitutions, were found in be at risk for infection with rabbit HEV. Further, rabbit
the genome of HEV. These results suggest that adaptation HEV was detectable in multiple rabbit tissues and organs,
of rabbit HEV to growth in cynomolgus monkeys may indicating extrahepatic replication may be a common
be associated with a certain number of mutations. Eleven feature of rabbit HEV. These findings raise additional
of the 16 mutations fell within ORF1, accompanied by concern for zoonotic transmission of HEV infection among
4 nonsynonymous substitutions, mapped to the helicase persons who have occupational exposure to rabbits or
region and the RNA-dependent RNA polymerase region, persons who eat undercooked rabbit meat. Future studies
which are essential for efficient replication of the genomes should be conducted to investigate rabbit HEV infection in
of HEV (29). Moreover, although most mutations are human populations and assess whether close contact with
expected to be in the third codon position, of the 16 rabbits is a risk factor for HEV infection.
substitutions in ORF1, 7 occur at the first codon position
and 3 at the second codon position. These facts may Acknowledgments
indicate that positive selection is operating in the infection We are grateful to Malcolm A. McCrae for proofreading the
of the cynomologus monkeys with the rabbit HEV revised manuscript.
inoculum. A recent study revealed that high-throughput
sequencing of isolates from bile and feces from 2 pigs This work was partially supported by the National Science
experimentally infected with human HEV of genotype 3f Foundation of China (grant no. 81271827).
shared the same full-length consensus sequence as in the Dr Peng is a PhD student at Department of Microbiology,
human sample, although a limited spectrum of mutations School of Basic Medical Sciences, Peking University. His
were observed during the interspecies transmission (30). primary research interests are the molecular epidemiology and
The genomic sequences in this study were determined by pathogenesis of HEV.
sequencing several randomly selected positive clones,
which is much less extensive than high-throughput
sequencing; consequently, additional studies will be References
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analysis of the full genome of rabbit hepatitis E virus (rbHEV) Address for correspondence: Ling Wang, Department of Microbiology,
and molecular biologic study on the possibility of cross species
School of Basic Medical Sciences, Peking University, 38 Xueyuan Road,
transmission of rbHEV. Infect Genet Evol. 2011;11:2020–5. http://
dx.doi.org/10.1016/j.meegid.2011.09.006 Haidian District, Beijing 100191, China; email: lingwang@bjmu.edu.cn

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RESEARCH

Description and Nomenclature

of Neisseria meningitidis
Capsule Locus
Odile B. Harrison, Heike Claus, Ying Jiang, Julia S. Bennett, Holly B. Bratcher, Keith A. Jolley,
Craig Corton, Rory Care, Jan T. Poolman, Wendell D. Zollinger, Carl E. Frasch, David S. Stephens,
Ian Feavers, Matthias Frosch, Julian Parkhill, Ulrich Vogel, Michael A. Quail,
Stephen D. Bentley, and Martin C.J. Maiden

Pathogenic Neisseria meningitidis isolates contain a cause invasive meningococcal disease. The polysaccharide
polysaccharide capsule that is the main virulence determi- capsule is a key virulence determinant, and for serogroups
nant for this bacterium. Thirteen capsular polysaccharides A, C, W, and Y, it forms the basis of polysaccharide con-
have been described, and nuclear magnetic resonance jugate vaccines. In one of the first reports distinguishing
spectroscopy has enabled determination of the structure of N. meningitidis, disease isolates were serologically classi-
capsular polysaccharides responsible for serogroup speci-
fied into types I–IV on the basis of agglutination reactions
ficity. Molecular mechanisms involved in N. meningitidis
capsule biosynthesis have also been identified, and genes
with immune rabbit serum (1). In 1950, the subcommittee
involved in this process and in cell surface translocation are on Neisseria of the Nomenclature Committee of the Inter-
clustered at a single chromosomal locus termed cps. The national Association of Microbiologists recommended that
use of multiple names for some of the genes involved in types I and III be combined into serogroup A; type II be-
capsule synthesis, combined with the need for rapid diagno- come serogroup B; a type II subgroup, termed type II-α,
sis of serogroups commonly associated with invasive me- become serogroup C; and type IV become serogroup D.
ningococcal disease, prompted a requirement for a consis- After the report of a fourth serogroup, Z′ (later shown to be
tent approach to the nomenclature of capsule genes. In this serogroup E), 3 new serogroups (X– Z) were identified by
report, a comprehensive description of all N. meningitidis using double agar diffusion (2,3). In 1981, three more sero-
serogroups is provided, along with a proposed nomencla- groups (H, I, K) were proposed, and a fourth (serogroup L)
ture, which was presented at the 2012 XVIIIth International
was identified in 1983 (4,5).
Pathogenic Neisseria Conference.
Nuclear magnetic resonance spectroscopy enabled
determination of the structure of capsular polysaccharides

T hirteen Neisseria meningitidis serogroups have been

described on the basis of serologic differences of the
capsule; of these 13 serogroups, 6 (A, B, C, W, X, Y)
responsible for serogroup specificity, and structures for 12
of the 13 serogroups (all but serogroup D) from N. menin-
gitidis capsular polysaccharides have been reported (6–15).
Molecular mechanisms of capsular polysaccharide synthe-
Author affiliations: University of Oxford, Oxford, UK (O.B. Harri-
sis have been elucidated; genes involved in polysaccharide
son, J.S. Bennett, H.B. Bratcher, K.A. Jolley, M.C.J. Maiden); Uni-
biosynthesis and cell surface translocation are clustered at a
versity of Würzburg, Würzburg, Germany (H. Claus, M. Frosch,
single chromosomal locus termed cps. Genes within this lo-
U. Vogel); The Sanger Institute, Cambridge, UK (Y. Jiang, C.
cus are divided into 6 regions: A–D, D′, and E (16). Genes
Corton, J. Parkhill, M.A. Quail, S.D. Bentley); National Institute
in region A encode enzymes for biosynthesis of the capsu-
for Biological Standards and Control, Potters Bar, UK (R. Care, I.
lar polysaccharide, and genes in regions B and C are im-
Feavers); Crucell, Leiden, the Netherlands (J.T. Poolman); Walter
plicated in the translocation of the high molecular weight
Reed Army Institute of Research, Silver Spring, Maryland, USA
polysaccharides to the cell surface.
(W.D. Zollinger); Frasch Biologics Consulting, Martinsburg, West
Complete nucleotide sequences of cps loci encod-
Virginia, USA (C.E. Frasch); and Emory University, Atlanta, Geor-
ing serogroups A–C, W, and Y have been elucidated.
gia, USA (D.S. Stephens)
Serogroup-specific capsule biosynthesis genes located
DOI: http://dx.doi.org/10.3201/eid1904.111799 in region A have been published for serogroup X, and

566 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 N. meningitidis Capsule Locus

nucleotide sequences for serogroups E, L, and Z have Genome Sequencing

been submitted to GenBank (accession nos. AJ576117, Genomic data for serogroups D, H, I, K, and L were
AF112478, and AJ744766, respectively) (17–19). This obtained by using the Illumina sequencing platform and de
study provides a comprehensive description of all N. novo assembly (Illumina, San Diego, CA, USA), using the
meningitidis serogroups and presents proposed revisions shuffle and Velvet Optimization scripts found within Vel-
to the nomenclature. vet 1.1.03 (25) (Table 1). By using VelvetOptimiser version
2.1.7 (http://bioinformatics.net.au/software.velvetoptimiser.
Materials and Methods shtml), optimal k-mer lengths ranging from 41 to 65 were
selected and contigs were generated. These contigs were
Strain Selection deposited in the Bacterial Isolate Genome Sequence Data-
Serogroup D, H, I, and K isolates were obtained base (BIGSDB) along with the isolate provenance and name,
from a collection maintained at the National Institute for after which contigs were scanned for genes within the cps
Biological Standards and Controls, Potters Bar, UK; the locus and tagged and alleles were assigned (26). New alleles
isolates were originally from a collection (1980s) from were manually checked for a correct start and stop codon
the People’s Republic of China Committee for Culture and aligned with known alleles before assignation. Addition-
Collection of Microorganisms. Serogroup E, W, X, and al data (e.g., multilocus sequence type [MLST]), and PorA,
Y isolates were from the Bavarian N. meningitidis car- PorB, and FetA designations were obtained.
riage collection (20). Three serogroup L isolates were
analyzed with additional serogroup H, I, and K isolates Annotation and Bioinformatic Methods
obtained from Paula Kriz (Czech Republic), who had ob- Predicted proteins were clustered into homology
tained the isolates from Fraser Ashton, who had acquired groups by using TribeMCL algorithm with a cutoff of 1e-
them from People’s Republic of China (Table 1). The se- 70 (http://doc.bioperl.org/bioperl-run/lib/Bio/Tools/Run/
quenced genomes from serogroup A isolate Z2491, sero- TribeMCL.html#General). The genes within the cps loci
group B isolate H44/76, and serogroup C isolates FAM18 that encoded proteins with the same homology group were
and 053442 were used (Table 1) (21–24). Isolates were assigned the same name, exceptions being those genes
grown on Mueller-Hinton agar supplemented with 5% found in Region A. BLASTp (http://blast.ncbi.nlm.nih.
(vol/vol) defibrinated sterile horse blood for 15 h at 37°C gov/Blast.cgi?PAGE=Proteins) searches were done by us-
in a 5% (vol/vol) CO2 atmosphere. DNA was extracted ing a reference sequence database with default settings.
by using the Wizard Genomic DNA Purification Kit Annotation was performed by using the genome view-
(Promega, Southampton, UK) according to the manufac- er Artemis (27). The cps nucleotide sequence locus for
turer’s instructions. each serogroup was stored in BIGSDB and linked with the
corresponding isolate record.
PCR and Nucleotide Sequencing of the Capsule Locus MEGA5 (http://megasoftware.net/) was used to calcu-
The genes for the N. meningitidis cps locus are lo- late overall p-distances and G+C content and to obtain the
cated between a gene encoding a putative inner mem- number of polymorphisms observed within each gene. The
brane transport protein (NMC0044 FAM18 genome cps loci were compared by using the Artemis Comparison
annotation) and a gene encoding the sodium/glutamate Tool (www.sanger.ac.uk/resources/software/act/).
symport carrier protein, gltS (NMC0069). PCR reactions
were performed for isolates 29013, α707, 29031, 29043, Results and Discussion
29046, 3608, α275, α388, α162, WUE171, WUE172, and
WUE173 (Table 1) by using the Expand Long Template General Organization
PCR System (Roche Applied Science, Burgess Hill, UK) All of the serogroups examined had comparable cps loci,
according to the manufacturer’s recommended protocol, and regions occurred in the following order D-A-C-E-D′-B,
with annealing at 55°C and extension at 68°C for 20 min. revealing a conserved gene synteny and a replication of the
Initial reactions used 1 of the following primer pairs: genomic rearrangements (Figure). Genes in regions B–D, D′
gltS (NMC0069) to tex (NMC0059) (primers gltS 5′-CC- and E were conserved, whereas genes in region A were di-
GACCAAGCCGTATTGC + ATGATACTCGAAGGC- verse; this finding is consistent with the distinct biochemical
GTGGTT-3′ and tex 5′-TGTCGAAGCCGTCCATA- composition found within each serogroup (Table 2).
ATCT + GCCCTGTCCAACAAGTTCGT-3′) and tex to The lowest GC content was in regions A and C, in-
NMC0044 (primers tex 5′-CGCCCGGTTCGTCATCC dicative of genes in these regions resulting from horizon-
+ TTGCTGCTGGTAGGCGAATCC-3′ and NMC0044 tal recombination (Table 2; Table 3, Appendix, wwwnc.
5′-CGGGCGAACACGGTAAT + TATCGTTGGTGC- cdc.gov/EID/article/19/4/11-1799-T3.htm); regions B,
GCTGGTTAT-3′). D, and E displayed GC contents of 50%, 52%, and 52%,

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 567
RESEARCH

Table 1. Description of Neisseria meningitidis isolates*

Disease GenBank
Isolate name Serogroup Origin status Strain designation accession no.
N. meningitidis Z2491 A The Gambia Invasive A:P1.7, 13–1: F1–5: ST-4 (cc4) AL157959
N. meningitidis H44/76 B Norway Invasive B:P1.7,16: F3–3: ST-32 (cc32) CP002420
N. meningitidis FAM18 C United States Invasive C:P1.5,2: F1–30: ST-11 (cc11) AM421808
N. meningitidis 053442 C PRC Invasive C:P1.7–2,14: F3–3: ST-4821 (cc4821) CP000381
N. meningitidis 29013 D PRC Unknown C:P1.22,14–6: F3–16: ST-8723 (cc213) ERR028660
N. meningitidis α707 E Germany Carrier E:P1.5–1,2–2: F4–3: ST-254 (cc254) HF562982
N. meningitidis 29031 H PRC Carrier H:P1.21,3: F4–21: ST-4959 (-) ERR028662
N. meningitidis H-ASH/87 H PRC Carrier H:P1.21,3: F4–21: ST-4959 (-) ERR036095
N. meningitidis H-ZANE/83 H PRC Carrier H:P1.21,3: F4–21: ST-4959 (-) ERR036096
N. meningitidis 29043 I PRC Carrier I:P1.22,14–6: F5–2: ST-5594 (-) ERR028663
N. meningitidis I-ZANE/87 I PRC Carrier I:P1.22,14–6: F5–2: ST-5594 (-) ERR036097
N. meningitidis 29046 K PRC Carrier K:P1.7–2,14: F5–14: ST-8724 (-) ERR028664
N. meningitidis K-ASH/87 K PRC Carrier K:P1.7–2,14: F5–14: ST-8724 (-) ERR036087
N. meningitidis WUE3608 L Unknown Carrier L:P1.18–1,3: F1–5: ST-963 HF562986
N. meningitidis L-ASHTON/87 L PRC Carrier L:P1.22,14: F1–38: ST-8902 (-) ERR036088
N. meningitidis 21033 L Dublin, Carrier L:P1.7–2,13–1:F1–5: ST-3258 (-) ERR063490
Ireland, UK
N. meningitidis α275 W Germany Carrier W:P1.18–1,3: F4–1: ST-22 (cc22) HF562987
N. meningitidis WUE171 W Germany Unknown W:P1.5–9, 10: F3–6: ST-11 (cc11) HF562992
N. meningitidis α388 X Germany Carrier X:P1.5–1,2–2: F5–1: ST-765 (cc254) HF562988
N. meningitidis α162 Y Germany Carrier Y:P1.5–2,10–1: F4–1: ST-23 (cc23) HF562989
N. meningitidis WUE172 Y Unknown Unknown Y:P1.5,2: F1–1: ST-166 (cc11) HF562992
N. meningitidis WUE173 Z Unknown Unknown Z:P1.7–1,1: F1–7: ST-4443 (-) HF562991
*PRC, People’s Republic of China; UK, United Kingdom.

respectively, similar to those found in the Neisseria ge- propose a comprehensive description of all N. meningitidis
nomes (21,22,33). Bacterial genomic GC content varies serogroups and a nomenclature for the cps locus, which
considerably among species but remains uniform within a was presented at the 2012 XVIIIth International Pathogenic
bacterial genome, such that genes acquired through hori- Neisseria Conference (Table 3, Appendix).
zontal genetic exchange will have a GC content different We propose that the capsule biosynthesis genes within
from the overall GC content found within the genome. region A should be termed cs (for capsule synthesis) fol-
lowed by a letter representing the serogroup and by a capi-
Nomenclature tal letter defining each gene according to the Demerec sys-
Reports identifying genes and proteins involved in N. tem of genetic nomenclature (34). For example, serogroup
meningitidis capsule biosynthesis, combined with the in- A capsule biosynthesis genes would be termed csaA–D (cs
creasing availability of bacterial genomes, have necessi- for capsule synthesis and a for serogroup A) (Table 3, Ap-
tated a more unified approach to the nomenclature of genes pendix). Under this proposal, the sialic acid capsule bio-
within N. meningitidis cps locus (Table 3, Appendix). The synthesis genes would be termed cssA–C (cs for capsule
capsule locus from each serogroup has been uploaded to synthesis and s for sialic acid capsule).
the BIGSDB platform, enabling sequences from each gene Serogroups B, C, W, and Y are commonly associated
within the N. meningitidis cps locus to be indexed and mul- with invasive meningococcal disease, and rapid diagnosis
tiple cps loci to be typed. However, during the process, it of the serogroup is key in monitoring the epidemiology
was found that the nomenclature of some genes within this of the disease and in developing prevention strategies.
locus posed a problem. For instance, genes within region B Thus, it is necessary to have nomenclature that identifies
were thought to encode lipidation enzymes and, thus, have the serogroup simply and quickly. The polysialyltransfer-
been known as lipA and lipB. However, within the annotat- ase genes belonging to serogroups B and C share >70%
ed genomes from N. meningitidis FAM18, MC58, Z2491, sequence identity and should be termed csb and csc, re-
and 05342 and from N. lactamica 020–06, two other dis- spectively. The equivalent gene in serogroups W and Y
tinct lipA and lipB genes have been described encoding a is also a sialyltransferase, but it also has a glycosyltrans-
lipoic acid synthetase and a lipoate-protein ligase protein, ferase function and is distinct from the serogroups B and
respectively. Furthermore, there are, in some instances, C genes. These distinctions should be reflected in the
multiple names for the same gene. nomenclature: we propose that the gene for serogroups
Continued surveillance of meningococcal disease W and Y be termed csw and csy, respectively. The O-
combined with the use of multiple names for some genes acetyltransferases should be termed cssE for serogroup
made it apparent that a consistent approach to the nomen- C and cssF for serogroups W and Y; the nomenclature
clature of capsule genes was needed. To meet that need, we for ctrG, which has been shown to have a role in surface

568 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 N. meningitidis Capsule Locus

Figure. Genetic organization of the cps locus among Neisseria meningitidis serogroups A (N. meningitidis Z2491); B (N. meningitidis
H44/76); C (N. meningitidis FAM18, 053442, and 29013); W α275 (clonal complex sequence type [ST] 22); W WUE171 (clonal complex
ST-11); Y α162 (clonal complex ST-11); Y WUE172 (ST-23); E (N. meningitidis α707); H (N. meningitidis 29031); I (N. meningitidis
29043); K (N. meningitidis 29046); L (N. meningitidis WUE3608); X (N. meningitidis α388); and Z (N. meningitidis WUE173); and a cnl N.
meningitidis isolate. Letters on left represent serogroups. Arrows depict gene orientation.

translocation of sialic capsules, should be retained (30). (UDP-GlcNAc) 2-epimerase, which converts UDP-
The remaining serogroups would also follow this scheme GlcNAc into UDP-N-acetyl-d-mannosamine (UDP-Man-
(Table 3, Appendix). Genes within region C that encode NAc); csaB is the polymerase linking ManNAc-phosphate
the capsule transport genes have been termed ctrA–D (af- monomers together with csaC, encoding an O-acetyltrans-
ter capsule transport), and this nomenclature should be ferase, which transfers acetyl groups to ManNAc (18,28).
preserved (Table 3, Appendix) (32). Genes within region The fourth gene, csaD, is predicted to be involved in either
B that are involved in capsule translocation should be des- capsule transport or in cross-linking of the capsule to the
ignated ctrE and ctrF (31). Capsule genes are accessible meningococcal cell surface. The serogroup A cps did not
through the PubMLST database (http://pubmlst.org/neis- contain insertion sequences, and genes within region A
seria). In addition to the common gene name, these loci contained some of the lowest GC content values among all
are allocated a value-free nomenclature consistent with serogroups (Table 3, Appendix).
the FAM18 genome annotation but using the prefix NEIS
instead of NMC (Table 3, Appendix). Serogroups B, C, W, and Y
The W-135 and 29E serogroup designations originated Capsular polysaccharides from serogroups B, C, W,
at the Walter Reed Army Institute of Research as a result and Y are composed of sialic acid derivatives; serogroups
of a paper published by Evans et al. (35). We propose to B and C express (α2→8)- and (α2→9)-linked sialic acid
rename these W and E because the numbers are historic and homopolymers, and alternating sequences of d-galactose
supply no useful information. or d-glucose and sialic acid are expressed by serogroups
W and Y (6,7). Region A is 5,313 bp long in serogroup B,
Region A Capsule Biosynthesis Genes 6,690 bp long in serogroup C, and averages ≈7,581 bp in
serogroups W and Y.
Serogroup A All 4 serogroups contain the conserved cssA–C genes
The serogroup A capsule is composed of repeat- for cytidine-5′-monophosphate-N-acetylneuraminic acid
ing units of O-acetylated (α1→6)-linked N-acetyl-d- synthesis; other designations for cssA–C include siaA–C,
mannosamine-1-phosphate (13). The capsule biosyn- synA–C, and neuA–C (17). These are followed by csb, csc,
thesis region is 4,365 bp long and contains 4 genes: csw, and csy genes (siaD, siaDBCWY, or synD–G), which
csaA–D (also known as mynA–D or sacA–D) (18,28). The first are the capsule polymerases and determine the functional
gene, csaA, encodes the UDP-N-acetyl-d-glucosamine and nucleotide specificity for the 4 serogroups.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 569
RESEARCH

Table 2. Genetic diversity of Neisseria meningitidis cps genes*

Region, locus Size, bp No. alleles No. polymorphisms (%) p-distance dN/dS ratio G+C content
MLST
abcZ 433 11 52 (11) 0.046 0.04 51
adk 465 7 18 (4) 0.015 0.02 52
aroE 490 11 131 (7) 0.102 0.28 56
fumC 465 12 27 (6) 0.02 0.02 57
gdh 501 8 18 (4) 0.014 0.05 52
pdhC 480 13 61 (13) 0.041 0.08 56
pgm 450 10 62 (14) 0.055 0.11 54
Region B
ctrE 2,115 13 386 (18) 0.061 0.2 51
ctrF 1,260 12 59 (5) 0.012 0.14 49
Region C
ctrA 1,179 11 175 (15) 0.032 0.18 47
ctrB 1,164 8 264 (23) 0.051 0.10 46
ctrC 726 8 177 (24) 0.06 0.12 43
ctrD 651 7 87 (13) 0.057 0.1 46
Region D
rfbA 867 13 75 (9) 0.034 0.26 53
rfbB 1,083 15 312 (28) 0.067 0.12 53
rfbC 558 14 124 (220 0.09 0.22 55
galE 1,020 15 382 (37) 0.119 0.12 50
Region E
tex 2,274 13 173 (8) 0.019 0.09 59
NMC0060 1,007 8 24 (2.4) 0.003 0.91 40
NMC0061 345 5 24 (7) 0.013 0.19 36
*Genes in region A were too diverse among serogroups for direct comparison. Region D was omitted because galE2 is truncated and rfbA, rfbB, and rfbC
were duplicates. MLST, multilocus sequence type.

Serogroups C, W, and Y contain O-acetyltransferase within the cps locus. The genomes belonging to N. menin-
genes termed cssE (oatC) and cssF (oatWY) (29). The 2 gitidis Z2491, MC58, FAM18, and 053442; N. lactamica
sequenced serogroup C and serogroup Y isolates harbored 020–06; and N. gonorrhoeae FA1090 also contained galU
functional cssE/cssF genes with an IS1301 transposase ad- genes adjacent to argH. These were highly conserved (p-
jacent to cssF in serogroup Y. Serogroup W cssF was in- distance  =  0.015) but were more distantly related to those
terrupted by the insertion sequence IS1301. Another gene, found within the cps locus (p-distance = 0.180), indicating a
ctrG, found in all 4 serogroups, encoded a protein essential different origin for cps-associated galU genes.
in enabling the correct expression of sialic acid polysac- The serogroup D isolate was found to contain sero-
charides (30). The orientation of this gene differed between group C capsule biosynthesis genes (Figure and online
serogroups such that it was in the same direction as the css Technical Appendix Figure 1, panel A, wwwnc.cdc.gov/
genes in serogroups B and C and the opposite in serogroups EID/article/19/4/11-1799-Techapp1.pdf). The cps locus
W and Y (Figure). from the prototype serogroup D isolates deposited by Gor-
Serogroups W and Y contained an additional gene in don and Murray in 1917 and Branham in 1928 also con-
region A, galU, encoding UTP-glucose-1-phosphate uridyl- tained serogroup C-specific capsule genes; however, nei-
yltransferase, which was also found in region A of serogroup ther isolate gave precipitins with antiserum to serogroup
H. BLASTp searches of the derived amino acid sequence of C, suggesting that these isolates were unencapsulated (C.
GalU from serogroups W, Y, and H revealed that this protein Frasch. pers. comm.). The presence of internal stop codons
shared an average of 49% sequence identity with GalU pro- in the ctrA and ctrE genes is consistent with this and further
teins from Streptococcus pneumoniae isolates. GalU has an confirms that serogroup D does not exist.
essential function in capsule formation among S. pneumoni-
ae isolates, catalyzing the reversible formation of UDP-Glc Serogroup E
(uridine diphosphate glucose) and inorganic pyrophosphate The serogroup E capsule consists of alternating d-ga-
from UTP (uridine 3-phosphate) and glucose 1-phosphate, lactosamine and 2-keto-3-deoxyoctulosonate (KDO) resi-
and a role in virulence has been recognized for GalU in dues (9). Region A is ≈9,613 bp long, including IS1301,
several bacterial species (36). Additional galU genes were and contains 7 genes, cseA–G (formerly 29eA–H); gene
found adjacent to the gene argH encoding argininosuccinate cseE encodes a putative 3-deoxy-phosphooctulonate
lyase in the sequenced genomes from isolates α275, 29031, synthase, cseF encodes a putative 3-deoxy-d-manno-octu-
H-ASH/87, and H-ZANE/83, and although the genes were losonate cytidylyltransferase, and cseG encodes a d-arabi-
conserved, they were not identical to the galU genes found nose 5-phosphate isomerase protein. The deduced amino

570 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 N. meningitidis Capsule Locus

acid sequence from cseE shared 83% sequence identity amino acid sequence revealed that these genes shared 97%
with a 3-deoxy-8-phosphooctulonate synthase belonging sequence identity. The longest gene, cslB, was 2,633 bp
to N. elongata subsp. glycolytica and 81% identity with and putatively encoded a capsule polymerase (Figure and
N. subflava NJ9703; cseF shared 66% sequence identity online Technical Appendix Figure 2).
with a putative 3-deoxy-d-manno-octulosonate cytidyl- The serogroup X capsular polysaccharide is composed
yltransferase belonging to multiple bacterial species, in- of (α-1→4)-linked N-acetylglucosamine 1-phosphate (15).
cluding Pseudomonas putida and Escherichia coli. The In agreement with findings in a previous study, we found
genes cseD and cseE were found to be fused and were that region A in the serogroup X isolate contained 3 genes,
predicted to form 1 protein; this prediction is in agree- csxA–C (xcbA–C) and was 4,467 bp long, including the in-
ment with the suggestion that the genes for KDO synthe- sertion sequence, IS1016, located upstream of csxA (Figure
sis were dispensable probably because of complementa- and online Technical Appendix Figure 2) (19). The deduced
tion of lipopolysaccharide–KDO synthesis elsewhere in amino acid sequence from csxA shared 40% sequence iden-
the genome (37). tity with the previously mentioned LcbA protein belonging
to N. mucosa C102, indicating that csxA encoded a puta-
Serogroups H and Z tive capsule phosphotransferase; however, the remaining 2
The biochemical structures of serogroups H and Z con- serogroup X capsule biosynthesis genes did not share sub-
tain monosaccharide glycerol-3-phosphate repeat units and stantial sequence identities with any known protein.
share similarities with teichoic acid polymers. Region A
varied from 6,182 bp in serogroup H to 7,198 bp in sero- Serogroups I and K
group Z, with the latter containing the insertion element Different structural compositions have been de-
IS1301. Four genes were present in both serogroups (Fig- scribed for these serogroups, with serogroup I consisting
ure and online Technical Appendix Figure 3, panel A). of O-acetylated alternating N-acetyl-guluronic acid and
BLASTp searches of the deduced amino acid sequences of N-acetylmannosaminuronic acid units and serogroup K
the first 2 genes, termed cshA/cszA and cshB/cszB, shared composed of O-acetylated disaccharide repeat units con-
91% sequence identity with each other and also shared 76% taining N-acetylmannosaminuronic acid (10,11). Both se-
and 63% sequence identity with the capsule biosynthesis rogroups had almost identical capsule biosynthesis genes,
genes cps2B and cps2C belonging to Actinobacillus pleu- csiA–E or cskA–E; region A was ≈9,026 bp long (Figure
ropneumoniae serovars 2, 3, 6, 7, 9, 11, and 13 (38). These and online Technical Appendix Figure 3, panel B). MLST
genes are predicted to encode a glycerol-3-phosphate cy- analysis showed that serogroup I and K isolates investi-
tidylyltransferase, and a hypothetical protein containing a gated in this study did not possess the same sequence types
LicD domain for a role in phosphorylcholine incorpora- or PorA and FetA variable regions (Table 1). Antiserum
tion into teichoic acid polymers has been suggested (38). is not commercially available to verify these serogroups;
The third genes in region A, termed cshC and cszC, each however, the immunochemical difference between sero-
shared 65% sequence identity with a putative teichoic syn- group I and K polysaccharides may reflect differences in
thase genes (cps2D and cps9D, respectively) belonging to the original methods used to purify them (10,11). In addi-
A. pleuropneumoniae serovars 2, 7, 9, and 13. Last, cshD tion, 2 nonsynonymous substitutions were observed in the
and cszD were 75% homologous to A. pleuropneumoniae capsule biosynthesis genes csiC/cskC and csiD/cskD, for
capsule synthesis genes (cps7E). which the deduced amino acid sequence encoded putative
glycosyltransferases belonging to families 1 and 2, respec-
Serogroups L and X tively. The capsule polymerases belonging to serogroups
Serogroup L capsule polysaccharides contain 2-acet- W and Y (csw and csy) are closely related; a single amino
amido-2-deoxy-d-glucosyl residues and phosphate groups. acid substitution in the N-terminal glycosyltransferase do-
Region A contained 3 genes, cslA–C (formerly lcbA–C) main of the capsule polymerase produces the glucose or
and was 4,438 bp long. BLASTp searches of the deduced galactose substrate specificity for the enzyme (39) (online
amino acid sequence from cslA predicted a capsule phos- Technical Appendix). It is therefore possible that the non-
photransferase sharing 73% sequence identity with LcbA synonymous changes observed in the glycosyltransferases,
proteins from N. mucosa C102 (GenBank accession no. csiC/cskC and csiD/cskD, may produce the serogroup I
ACRG00000000) and N. subflava NJ9703 (GenBank ac- and K capsules. Further investigation of these serogroups
cession no. ACEO00000000), whereas cslC encoded an is required.
acetyltransferase protein. A gene similar to cslA was iden- The derived amino acid sequences from csiA, B, and E/
tified between regions D′ and B among serogroups I and K cskA, B, and E shared >70% sequence identity with capsule
and a serogroup W isolate belonging to clonal complex se- biosynthesis proteins belonging to Mannheimia haemolyt-
quence type 22 (Figure); BLASTp searches of the deduced ica serotype A1. The capsule of M. haemolytica serotype

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 571
RESEARCH

A1 is composed of a disaccharide repeat of N-acetylman- Dr Harrison is a postdoctoral research assistant in the De-
nosaminuronic acid linked with N-acetylmannosamine. partment of Zoology, University of Oxford. Her research interests
include the molecular epidemiology and population structure of
Regions B and C Neisseria meningitidis.
The genes ctrE and ctrF (formerly lipA and lipB) are
required for surface expression of a properly anchored cap-
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RESEARCH

Detection of Spliced mRNA from

Human Bocavirus 1 in
Clinical Samples from Children with
Respiratory Tract Infections
Andreas Christensen, Henrik Døllner, Lars Høsøien Skanke, Sidsel Krokstad,
Nina Moe, and Svein Arne Nordbø

Human bocavirus 1 (HBoV1) is a parvovirus associ- aspirates (NPAs), and monodection of HBoV1 DNA in
ated with respiratory tract infections (RTIs) in children, but NPAs (5). In addition, RTIs in HBoV1 DNA–positive chil-
a causal relation has not yet been confirmed. To develop dren are associated with HBoV1 seroconversion (6). This
a qualitative reverse transcription PCR to detect spliced evidence supports a causal relation between HBoV1 and
mRNA from HBoV1 and to determine whether HBoV1 RTIs in children, but DNA-based PCR tests do not seem to
mRNA correlated better with RTIs than did HBoV1 DNA, we
diagnose HBoV1 infection accurately. We propose that de-
used samples from HBoV1 DNA–positive children, with and
without RTIs, to evaluate the test. A real-time reverse tran-
tection of HBoV1-specific mRNA, as a measure of actively
scription PCR, targeting 2 alternatively spliced mRNAs, was transcribing virus, may be a better method.
developed. HBoV1 mRNA was detected in nasopharyngeal The main objectives of this study were to develop a
aspirates from 33 (25%) of 133 children with RTIs but in qualitative reverse transcription PCR (RT-PCR) detecting
none of 28 controls (p<0.001). The analytical sensitivity and spliced mRNA from HBoV1 and to clarify whether HBoV1
specificity of the test were good. Our data support the hy- mRNA detection may correlate better than DNA detection
pothesis that HBoV1 may cause RTIs, and we propose that with RTIs in children. NPAs and blood samples from a
HBoV1 mRNA could be used with benefit, instead of HBoV1 group of children, with and without RTIs, who tested posi-
DNA, as a diagnostic target. tive for HBoV1 DNA were used for this purpose.

H uman bocavirus 1 (HBoV1) is a small nonenveloped Materials and Methods

virus in the Parvoviridae family. It was discovered in
human respiratory samples in 2005 (1). The virus does not Samples
grow in standard cell lines, and diagnosis has mainly been HBoV1 DNA–positive NPA samples from an ongoing
based on DNA detection with PCR. Detection of multiple project on RTIs in children 0–16 years of age were used for
viruses in HBoV1 DNA–positive airway samples from evaluation of the test (5). In particular, 161 NPA samples
children with respiratory tract infections (RTIs) has been collected at admittance from 161 children at the Depart-
a characteristic finding in many studies (2–4). In addition, ment of Pediatrics, St. Olav’s Hospital, Trondheim Univer-
many healthy children have tested positive for HBoV1 sity Hospital (Trondheim, Norway), during June 2007–June
DNA (2,5); thus whether the virus actually causes RTIs in 2010 were included. A blood sample was also available for
children or is just a bystander to other infections has been 63 of the children. All samples had been stored at −70°C.
debated. However, we have shown that the following 3 fac-
tors are associated with RTIs: HBoV1 viremia (HBoV1 Children with RTIs
DNAemia), a high HBoV1 DNA load in nasopharyngeal Of the 161 HBoV1 DNA–positive NPA samples, 133
were from children with RTIs. Median age was 17 months
Author affiliations: Trondheim University Hospital, Trondheim, Nor- (range 3 months–5 years) and 60% were boys. They were
way (A. Christensen, H. Døllner, L.H. Skanke, S. Krokstad, N. Moe, classified as having either lower (86 children) or upper
S.A. Nordbø); and Norwegian University of Science and Technol- (47 children) RTI (LRTI; URTI). LRTI was diagnosed in
ogy, Trondheim (A. Christensen, H. Døllner, N. Moe, S.A. Nordbø) the presence of dyspnea, signs of lower airway obstruc-
tion (wheezing, retractions), and/or a chest roentgenogram
DOI: http://dx.doi.org/10.3201/eid1904.121775

574 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 mRNA from Human Bocavirus

with positive results (infiltrates, atelectasis, air trapping). designed: forward 5′-CGGCGAGTGAACATCTCTG-
URTI was diagnosed when rhinitis, pharyngitis, and/or oti- GA-3′ (positions 203–223) and reverse 5′-TGCTT-
tis media was found without signs of LRTI. In addition, 3 GTCTTTCATATTCCCT-3′ (positions 2438–2418). The
children with RTIs, admitted during winter 2011–12, were estimated PCR product spanned a spliced segment from
followed up on 3 occasions, each over 2 months. positions 241 to 2236 of the complete genome for HBoV1
(GenBank accession no. NC007455), which gives a theo-
Children without RTIs (Controls) retical PCR product of 242 bp and an alternative product,
Twenty-eight HBoV1 DNA–positive NPA samples including a short segment from positions 2044 to 2164,
were collected from a group of children who were admit- yielding a product of 363 bp (Figure 1). These estimations
ted for elective surgery and who had exhibited no signs were based on in vitro studies performed by Chen et al.
or symptoms of RTI during the previous 2 weeks. The (7). The probe targeting the untranslated region upstream
children were included prospectively during the same pe- of the nuclear phosphoprotein-1 gene had the following
riod in 2007–2010. Median age was 31 months (range 15 sequence: 5′-FAM-TGTCCACCCAAGAAACGTCGTC-
months–6 years), and 70% were boys. TAA-TAMRA-3′ (positions 2295–2319). The PCR for
every sample was also run without reverse transcription
Tests for Other Respiratory Agents to test for potential unspecific reactions with viral DNA.
All NPA samples from patients and controls were also The theoretical PCR product from HBoV1 DNA would be
tested with PCRs for adenovirus, coronavirus (OC43, 229E, 2,236 bp in length, which is too long for amplification by
and NL63), enterovirus, parechovirus, human metapneu- real-time PCR under normal conditions.
movirus (HMPV), influenza A and B viruses, parainfluenza Total DNA and RNA were extracted by using Nu-
virus types 1–4, respiratory syncytial virus (RSV), rhino- cliSens easyMag extractor (bioMérieux, Marcy l’Etoile,
virus, Bordetella pertussis, Chlamydophila pneumoniae, France), and reverse transcription was carried out with Uni-
and Mycoplasma pneumoniae. The PCRs were in-house, versal RiboClone random primers (Promega, Fitchburg,
real-time assays with TaqMan probes (Roche Diagnostics, WI, USA) and M-MLV Reverse Transcriptase (Life Tech-
Basel, Switzerland) (5). The analyses were conducted as nologies Corp., Carlsbad, CA, USA) at 37°C for 60 min.
part of the daily laboratory routine and performed within followed by 94°C for 10 min. The PCR was performed for
24 hours after sample collection. The target for the HBoV1 45 cycles at 95°C for 5 s., 55°C for 10 s, and 72°C for 20 s.
DNA PCR was the nuclear phosphoprotein-1 gene. This Amplification efficiency was calculated by us-
PCR has been described (4). A semiquantitative approach ing the formula E=10(-1/S) -1, where S is the slope of the
was chosen, and a cutoff value of 106 copies/mL was used standard curve. A human DNA PCR (specific for the
to distinguish between high and low HBoV1 DNA load in γ-glutamyltransferase light chain 1 gene on chromosome
NPAs. 20) was used as amplification control (8). Nucleic acid
extract from a clinical sample positive for RSV was used
Spliced HBoV1 mRNA-PCR as cDNA control. To make sure that mRNA had not been
We developed a real-time RT-PCR on the basis of degraded during storage, we used an RT-PCR to detect hu-
TaqMan technology (Roche). The following primers were man β actin mRNA (9).

Figure 1. Schematic representation of the 2 human bocavirus 1 (HBoV1) mRNA PCR products, illustrating alternative splicing. Positions of
primers and probe are shown. The total length of the upper product is 242 bp, and the length of the lower is 363 bp (reference sequence:
GenBank accession no. NC007455).

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 575
RESEARCH

RNA stability was studied by using clinical NPA sam- Statistical Analysis
ples and nucleic acid extracts from the easyMag extractor Statistical analysis was by χ2 test for categorical vari-
(bioMérieux). Four clinical NPA samples collected within ables and Student t test for continuous variables. Multiple
the previous 2 hours were stored for 0, 1, 3, and 5 days logistic regression analysis was used to evaluate the as-
at 4°C before nucleic acid extraction and testing with the sociation between detection of HBoV1 mRNA and LRTI,
HBoV1 mRNA PCR. One NPA sample was stored at room controlling for differences in age, sex, and the presence of
temperature and tested likewise. Two other clinical NPA other viruses among case-patients and controls. We report
samples were frozen and thawed 0, 1, 2, and 4 times be- the odds ratio (OR) with 95% CIs and the corresponding p
fore extraction and testing with the HBoV1 mRNA PCR value as a measure of the strength of the association. All
(3 times was skipped to save NPA material). Furthermore, analyses were performed by using IBM SPSS Statistics
HBoV1 mRNA PCR results from 3 HBoV1-positive NPA version 19.0 (SPSS Inc., Chicago, IL, USA).
samples stored at −70°C for 3 years were compared with
nucleic acid extracts from the same samples stored under Results
equal conditions for the same period. This was done to
determine whether the stability of RNA in clinical NPA Spliced HBoV1 mRNA PCR
samples added to virus transport medium was comparable HBoV1 mRNA was detected in 33 of the 161 HBoV1
to the stability of RNA in nucleic acid extracts from the DNA–positive NPA samples (Table 1). Gel electrophore-
easyMag at this temperature. Relative changes in RNA sis showed that primarily 2 PCR products were amplified
load were measured by comparing logarithmically trans- with sizes of ≈250 bp and ≈400 bp (Figure 2). Sequence
formed cycle threshold values (Ct values) obtained from analysis showed that they were spliced products with ei-
the same experiment. ther 1 or 2 introns cut out as expected (schematically il-
Quantitative standards for the real-time HBoV1 lustrated in Figure 1). The exact product sizes were 242
mRNA PCR were made by cloning a plasmid (pCR4-TO- bp and 363 bp. Direct PCR analysis for HBoV1 mRNA
PO; Life Technologies Corp.) containing the PCR product. on the nucleic acid extracts, without initial reverse tran-
The amount of nucleic acid was measured, and serial dilu- scription, was negative for 30 of 33 NPA samples. For
tions covering a range of 7 logs were made to measure the the remaining 3 samples, however, weak signals were de-
analytical sensitivity of the HBoV1 mRNA PCR. tected. These 3 samples had very high HBoV1 DNA loads
Analytical specificity was evaluated by using cDNA (range 4 × 108 copies/mL to >1010 copies/mL), and were
from NPA samples positive for all respiratory agents in- also strongly positive by the HBoV1 mRNA PCR after
cluded in the study. cDNA from NPA samples contain- cDNA synthesis. The products of the 3 PCRs that were
ing viruses that can be reactivated in the respiratory tract done without cDNA synthesis were sequenced. Product
were also included (i.e., herpes simplex virus, cytomega- sizes were 145, 261, and 457 bp, and sequence analysis
lovirus, Epstein-Barr virus, and human herpes virus 6). showed gaps at different positions, all of them lacking
Finally, cDNA from NPA samples positive for the more splice site characteristics (data not shown).
closely related parvovirus B19 and cDNA from fecal sam- Amplification efficiency of the HBoV1 mRNA PCR
ples positive for human bocaviruses 2 and 3 (HBoV2 and was calculated on the basis of dilutions of both the nucleic
HBoV3) were tested. The primers and probe described acid extract and cDNA. It was measured to 100% in both
by Kantola et al. were used for detection of HBoV2 and cases, indicating a high efficiency of both the PCR and
HBoV3 (10). Two samples positive for each agent were cDNA synthesis (data not shown). The assays’ reportable
used, and all samples had undergone extraction within range was from 500 copies/mL (10 copies/reaction) to 1010
2–20 hours after sample collection. Sequence analysis on copies/mL.
the PCR products was performed by using the BigDye Results of the β-actin PCR performed after DNase
Terminator Cycle sequencing method and the ABI Prism treatment were positive for all samples studied. Results of
3130 Genetic Analyzer (Applied Biosystems, Foster City, the HBoV1 mRNA PCR were negative for all other respira-
CA, USA). tory agents, herpesviruses, and parvoviruses tested.

Table 1. HBoV1 mRNA PCR results in NPAs from children with and without RTIs, Norway, 2007–2010*
Sample source Total no. No. (%) HBoV1 mRNA+ No. (%) HBoV1 mRNA– p value
Children with RTIs 133 33 (25) 100 (75) p<0.001
Controls (without RTIs) 28 0 28 (100)
Children with LRTIs 86 27 (31) 59 (69) p = 0.02
Children with URTIs 47 6 (13) 41 (87)
*HBoV1, human bocavirus 1; NPAs, nasopharyngeal aspirates; RTIs, respiratory tract infections; LRTIs, lower RTIs; URTIs, upper RTIs.

576 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 mRNA from Human Bocavirus

mRNA Stability
The HBoV1 mRNA load in NPA remained stable for 5
days at 4°C. At room temperature, it was unaltered after 24
h but was reduced by 1 log after 3 days and by 1.5 log after
5 days. Freezing and thawing of the NPA samples once or
twice did not affect yield, but after 4×, it was reduced by
≈0.5 log. For 3 NPA samples that had been stored at −70°C
for 3 years, the results were equal for both the nucleic acid
extract and the original sample. HBoV1 mRNA PCR re-
sults for nucleic acid extracts were stable for weeks when
samples were stored at 4°C (samples stored for up to 8
weeks were tested; data not shown).

Performance of Spliced HBoV1 mRNA

PCR on Samples
First, we compared the rates of positive test results for
HBoV1 mRNA among children with positive results for
HBoV1 DNA, with and without RTIs. Only one fourth of
the patients and none of the controls had test results posi-
tive for HBoV1 mRNA (Table 1). More children with LRTI
(27/86 [31%]) than with URTI (6/47 [13%]) had positive
test results for HBoV1 mRNA (Table 1). After we adjusted
for age, sex, and presence of other viruses, this difference
persisted (OR 3.5, 95% CI 1.3–9.8, p = 0.02).
We previously found that 3 factors (HBoV1 DNAemia,
high HBoV1 DNA in NPAs, and monodection of HBoV1
DNA in NPAs) were each associated with RTIs in children
(5). In the present study, these factors were strongly asso-
ciated with a positive test result for HBoV1 mRNA (Table
2). The close relationship between HBoV1 DNA load and Figure 2. Agarose gel stained with ethidium bromide. Reverse
HBoV1 mRNA detection in NPAs is also illustrated in Fig- transcription PCR products from 4 patients are shown in lanes
ure 3. Of the 100 RTI patients who were negative for HBoV1 2–5 and 1-kb DNA Ladder (Life Technologies Corp., Carlsbad, CA,
mRNA, 75 were positive for >1 other respiratory viruses. USA) in lane 1. Arrows indicate 2 bands corresponding to ≈250 and
≈400 bp.
Twenty-eight (37%) of these children were infected with
the highly pathogenic RSV. Distribution of the viruses most
commonly co-detected with HBoV1 is shown in Table 3. boys (boys 2 and 3), who had been included in an HMPV
follow-up study. Their NPA samples became positive for
Follow up of 3 Children with RTIs during HBoV1 DNA 1 week after HMPV infection was diag-
Winter 2011–12 nosed. The initial samples were negative for HBoV1 DNA.
During winter 2011–12, sequentially collected samples The clinical condition was unaltered for both when HBoV1
from 3 HBoV1 DNA–positive children made it possible to DNA appeared. HBoV1 DNA loads reached moderate lev-
gain some information about changes in HBoV1 mRNA els for boy 2 and high levels for boy 3, but for both boys,
and HBoV1 DNA over time. One of these children (boy the PCRs were negative for HBoV1 mRNA in 2 consecu-
1) was a 2-year-old boy with cerebral palsy who had been tive NPA samples taken 5 days apart. Unfortunately, no
admitted with bronchiolitis. On admission, he had HBoV1 blood samples were taken from these 2 boys.
DNAemia and analysis of NPAs showed that he had 1) a
high HBoV1 DNA load, 2) monodection of HBoV1 DNA, Discussion
and 3) positive HBoV1 mRNA PCR results. He recovered We report here the development of a robust PCR for
slowly, and after 10 days a new NPA sample was taken. detection of spliced mRNA from HBoV1. We found that
The HBoV1 DNA load was still high, but the results for the HBoV1 mRNA correlated significantly better with RTIs in
HBoV1 mRNA PCR were negative. Two months later, his children than did HBoV1 DNA, indicating that this PCR
NPAs still were positive for HBoV1 DNA and negative for may diagnose HBoV1 infection more accurately than PCR
HBoV1 mRNA. The other patients were two 1.5-year-old for HBoV1 DNA.

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RESEARCH

Table 2. HBoV1 mRNA PCR results in NPAs in relation to HBoV1 DNAemia, a high HBoV1 DNA load, and monodection of HBoV1
DNA, Norway, 2007–2010*
Factor Total no. No. (%) HBoV1 mRNA+ No. (%) HBoV1 mRNA– p value
HBoV1 DNAemia, n = 63 17 13 (77) 4 (23) p<0.001
No HBoV1 DNAemia 46 5 (11) 41 (89)
HBoV1 DNA load, n = 161
 106 copies/mL 59 33 (56) 26 (44) p<0.001
<106 copies/mL 102 0 102 (100)
 108 copies/mL 18 17 (94) 1 (6) p<0.001
<108 copies/mL 143 16 (11) 127 (89)
Monodection of HBoV1 DNA, n = 161 43 14 (33) 29 (67) p = 0.022
Multiple virus detections 118 19 (16) 99 (84)
*HBoV1, human bocavirus 1; NPAs, nasopharyngeal aspirates; HBoV1 DNAemia, HBoV1 viremia.

Splicing is a process specific for mRNA synthesis, and 241 and 2236 would have been an alternative approach,
with use of a primer pair spanning an intron, spliced viral ensuring specific detection of mRNA spliced at this exact
mRNA should be specifically detected within the frame of location (Figure 1). However, because our results indicated
the familiar and robust RT-PCR. This diagnostic technique high specificity with the chosen probe, we did not develop
has been used to diagnose parvovirus in dogs and may also this approach further.
be an option for diagnosing HBoV1 infections in humans Previously, RNA molecules were believed to have
(11). Furthermore, detection of mRNA is routinely used for short half-lives because RNases may be present every-
diagnosing human papillomavirus infections and has been where and easily degrade RNA. Recent studies, however,
studied for diagnosing human herpesvirus 6 and HIV in- have suggested that mRNA may be stable when molecules
fections (12–15). However, the mRNA tests for these vi- are kept in the original biologic material (16,17). We found
ruses have been based on either specific mRNA extraction, that the mRNA content in NPA samples was stable dur-
nucleic acid sequence–based amplification technology, ing a 5-day period at 4°C. This stability suggests that NPA
or pretreatment with DNase. The advantage with our ap- samples kept in a refrigerator and processed within 1–2
proach is that no pretreatment, other than cDNA synthesis, days, which is standard in our laboratory, are safe to use for
is needed. The procedure is performed as a regular RT-PCR HBoV1 mRNA testing.
with standard equipment and will be easy to use in most In 3 samples that had strong signals in the HBoV1
routine laboratories. The analytical performance of the test mRNA test, a weak signal was detected also without prior
was good with high analytical specificity and sensitivity. cDNA synthesis, evoking the question of whether HBoV1
The probe target was chosen to detect the 2 products DNA could give false-positive reactions in the HBoV1
(Figure 1) and thereby to maximize analytical sensitivity. mRNA test. The PCR was designed so that the theoretical
A probe spanning the spliced segment between positions DNA product would be 2,236 bp—too large for amplifica-
tion to occur in a regular, real-time PCR. For this reason,
the PCR products were expected to result from recombina-
tion events. Gel and sequence analysis showed that all 3
products had different sizes, ranging from 145 to 457 bp.
Moreover, no common sequence profiles were found near
the gap junctions, which seemed to be located at random.
Homologous recombination, a concentration-dependent
process, may explain this phenomenon because it occurred
only in NPA samples with extremely high levels of HBoV1
DNA. We speculate that the PCR products might have been
subpopulations of nonviable HBoV1 mutants that appeared
when virus replication was at its highest. However, the
specificity of the test was not affected because it happened
only in patients with very high viral DNA loads and strong
HBoV1 mRNA signals.
In addition to being a diagnostic test, this method
may be used to gain information on HBoV1 transcription
in vivo. Our data confirmed previous in vitro results on
Figure 3. Distribution of human bocavirus 1 (HBoV1) DNA loads in
nasopharyngeal aspirates either positive (n = 33) or negative (n = the splicing pattern at the 5′ end of the HBoV1 genome
128) for HBoV1 mRNA. Each dot indicates 1 sample. (Figure 1) (7,18).

578 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 mRNA from Human Bocavirus

Table 3. Most commonly co-detected viruses in NPAs from children with HBoV1 DNA, distributed by presence of RTI and HBoV1
mRNA, Norway, 2007–2010*
HBoV1 DNA+ children with RTIs HBoV1 DNA+ controls
No. (%) HBoV mRNA+, No. (%) HBoV1 mRNA–, HBoV mRNA+, HBoV1 mRNA–,
Virus n = 33 n = 100† n=0 n = 28†
Respiratory syncytial virus 4 (12) 28 (28) – 0
Rhinovirus 7 (21) 25 (25) – 10 (36)
Enterovirus 5 (15) 24 (24) – 15 (54)
Adenovirus 1 (3) 20 (20) – 9 (32)
*NPAs, nasopharyngeal aspirates; HBoV1, human bocavirus 1; RTIs, respiratory tract infections; +, positive; –, negative.
†Triple and quadruple infections were common, and percentages within the columns may therefore add up to >100%.

For evaluation of the HBoV1 mRNA test, we were which were negative for HBoV1 mRNA and positive for
able to use available clinical samples from children with HBoV1 DNA could be from patients with past HBoV1
or without RTIs who had been tested for 18 respiratory infections who were still shedding viral DNA. Boy 1, who
agents and had HBoV1 DNA in NPAs (5). None of the was followed up during winter 2011–12, may illustrate
children without RTIs had detectable HBoV1 mRNA. Be- this. Results of PCR on NPA samples from this boy were
cause mRNA is a marker of active viral transcription, this positive for HBoV mRNA only for a short period (<10
finding indicates that the 28 asymptomatic children carried days), coinciding with the acute symptomatic infection,
inactive HBoV1 or HBoV1 with low activity. The strong whereas HBoV1 DNA persisted for months. An alterna-
association found between active HBoV1 transcription and tive hypothesis might be that the samples negative for
RTI in children supports the hypothesis that HBoV1 may HBoV1 mRNA and positive for HBoV1 DNA were from
cause RTIs. The hypothesis is further supported by the as- children with a latent HBoV1 infection. The findings in
sociations found between HBoV1 mRNA and the 3 fac- boys 2 and 3, who were followed up during the same win-
tors: HBoV1 DNAemia, high HBoV1 DNA load in NPAs, ter, may support this hypothesis. HBoV1 DNA in NPAs
and monodection of HBoV1 DNA—all factors strongly appeared during an ongoing HMPV infection in both
related to RTIs in children (5,6,19). In addition, the fact children, but results of PCR for HBoV1 mRNA remained
that HBoV1 mRNA was more frequently detected in chil- negative in 2 consecutive samples. The lack of detectable
dren with LRTIs than with URTIs indicates that LRTI is a HBoV1 mRNA may indicate that HBoV1 did not play a
prominent manifestation of HBoV1 infection. role in these infections. Release of latent HBoV1 DNA
Only one fourth of the HBoV1 DNA–positive chil- from cells disrupted by inflammation caused by HMPV
dren with RTIs had detectable HBoV1 mRNA. Similar may be a better explanation. More longitudinal studies,
findings were recently reported by Proenca-Modena et al. including serologic analyses, are needed to further study
(20). The absence of HBoV1 mRNA in most of the chil- these relationships.
dren with RTI may indicate that these children did not
have a clinical HBoV1-infection, despite positive test re- Acknowledgments
sults for HBoV1 DNA. Indeed, other respiratory viruses We are grateful to Anne-Gro Wesenberg Rognlien for par-
were frequently detected among the children who had a ticipating in the establishment of the clinical study.
negative HBoV1 mRNA test result; RSV accounted for This study was supported by the Liaison Committee between
one third of infections. the Central Norway Regional Health Authority and the Norwe-
The previously mentioned strong relation between gian University of Science and Technology.
HBoV1 DNA load in NPAs and HBoV1 mRNA is illus-
trated in Figure 3. It shows 2 distinct populations with little Dr Christensen is a consultant in the Section for Virology,
overlap, and good discrimination between HBoV1 mRNA– Department of Medical Microbiology, St. Olav’s Hospital, and
positive and –negative samples can be achieved with cut- a doctoral candidate at the Institute of Laboratory Medicine,
off values from 106 to 107 HBoV1 DNA copies/mL. We Children’s and Women’s Health, Norwegian University of Sci-
suggest that, for clinical purposes, HBoV1 mRNA is more ence and Technology, Trondheim. His research interests include
accurate than HBoV1 DNA in diagnosing active HBoV1 viral respiratory infections, parvoviruses, picornaviruses, and
infection, but a high HBoV1 DNA load (>107 copies/mL) paramyxoviruses.
may also be useful in diagnosis.
Previously, HBoV1 has been found to persist in References
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Med Virol. 2009;81:1276–82. http://dx.doi.org/10.1002/jmv.21496 Norway; email: a.christensen@ntnu.no

580 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Predicting Hotspots for
Influenza Virus Reassortment
Trevon L. Fuller, Marius Gilbert, Vincent Martin, Julien Cappelle, Parviez Hosseini, Kevin Y. Njabo,
Soad Abdel Aziz, Xiangming Xiao, Peter Daszak, and Thomas B. Smith

The 1957 and 1968 influenza pandemics, each of which have killed ≈1 million persons (3,4). The exchange of genes
killed ≈1 million persons, arose through reassortment events. between pairs of influenza virus subtypes increased viru-
Influenza virus in humans and domestic animals could lence in animal models, including reassortment between
reassort and cause another pandemic. To identify geographic subtypes H9N2 and H1N1, between H5N1 and H1N1, and
areas where agricultural production systems are conducive between H3N2 and H5N1 (5,6). We focus on reassortment
to reassortment, we fitted multivariate regression models between subtypes H3N2 and H5N1 because extensive data
to surveillance data on influenza A virus subtype H5N1
are available, but given sufficient data, our approach could
among poultry in China and Egypt and subtype H3N2 among
humans. We then applied the models across Asia and Egypt
be extended to other subtypes.
to predict where subtype H3N2 from humans and subtype For seasonal influenza virus A subtype H3N2,
H5N1 from birds overlap; this overlap serves as a proxy for person-to-person transmissibility and prevalence among
co-infection and in vivo reassortment. For Asia, we refined the humans are high (7). Furthermore, subtype H5N1, which
prioritization by identifying areas that also have high swine is primarily found in birds, can be highly pathogenic; the
density. Potential geographic foci of reassortment include fatality rate among humans is 60% (8). In mice, ≈8% of
the northern plains of India, coastal and central provinces reassortant viruses formed from human subtype H3N2 and
of China, the western Korean Peninsula and southwestern avian subtype H5N1 resulted in increased virulence and a
Japan in Asia, and the Nile Delta in Egypt. mortality rate of 100% (5). This finding among mice raises
the possibility that among humans reassortment events

S imultaneous infection with multiple influenza virus between subtypes H3N2 and H5N1 could generate a novel
strains can affect virus fitness components, such as vi- influenza virus that could spread rapidly, resulting in many
rus growth performance, and thus affect virus pathogenic- deaths. To prioritize areas where future reassortment is
ity, transmission, or recombination (1). In a host infected most likely to occur, we analyzed surveillance data for
with 2 closely related influenza viruses, the stains can re- subtype H5N1 among poultry in the People’s Republic
assort, exchanging gene segments to produce new strains, of China and Egypt and subtype H3N2 among humans.
some of which might have increased virulence. Virulence We chose China and Egypt because both countries have
might also trade off with transmission such that more patho- had recent outbreaks of subtype H5N1 infection among
genic viruses spread more slowly (2). However, in some poultry, human deaths from subtype H5N1 infection, and
instances, a reassortant virus can have high transmissibility extensive spatial data on cases of infection with subtype
and high pathogenicity. For example, reassortment between H5N1. This information would help decision makers
influenza viruses of humans and birds resulted in the 1957 implement policies to reduce spillover in these areas (9).
and 1968 pandemic viruses, each of which is estimated to Areas with high risk for co-occurrence of these 2 influenza
virus subtypes along with high densities of susceptible
Author affiliations: University of California, Los Angeles, California,
hosts, such as swine, quail, or turkeys, could benefit from
USA (T.L. Fuller, K.Y. Njabo, T.B. Smith); Université Libre de
enhanced monitoring and farm and market biosecurity.
Bruxelles, Brussels, Belgium (M. Gilbert); Food and Agriculture
Organization of the United Nations, Beijing, People’s Republic
Materials and Methods
of China (V. Martin); Centre de Cooperation International en
Recherche Agronomique pour le Developpement, Montpellier,
Influenza Data
France (J. Cappelle); EcoHealth Alliance, New York, New York,
USA (P. Hosseini, P. Daszak); National Laboratory for Quality
Egypt
Control on Poultry Production, Dokki, Giza, Egypt (S.A. Aziz); and
All data for Egypt were aggregated to the scale
University of Oklahoma, Oklahoma City, Oklahoma, USA (X. Xiao)
of the markaz, an administrative district that includes
DOI: http://dx.doi.org/10.3201/eid1904.120903 several villages. The dataset for subtype H5N1 infections

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RESEARCH

during 2009–2012 consisted of 453 cases among poultry

in backyard flocks, farms, and live-bird markets in 35
markazes. Screening assays are described elsewhere (10).
See online Technical Appendix Figure 1, wwwnc.cdc.gov/
EID/article/19/4/12-0903-Techapp1.pdf, for a workflow
analysis. Most (72%) positive samples came from chickens
in backyard flocks. The available geographic data on subtype
H3N2 in Egypt are limited (online Technical Appendix Table
3), so we used human population density as a surrogate for
cases of infection with subtype H3N2 because virtually all
humans (except those who have been vaccinated or infected
recently) will be susceptible to infection with subtype H3N2.

China
All data for China were aggregated to the spatial scale
of the prefecture, an administrative unit within a province
that typically contains several towns and villages. We
examined 2 independent datasets for cases of subtype H5N1
infection on the basis of outbreaks on poultry farms and
active surveillance of live-bird markets (11). The data on
cases of subtype H3N2 infection were retrieved by querying
GenBank and the EpiFlu database available from the Global
Initiative on Sharing All Influenza Data (GISAID) website
(http://platform.gisaid.org) for all occurrences of subtype Figure 1. Potential influenza reassortment areas in Egypt. Districts
H3N2 in China that included fine-scale geographic data on in red are predicted to have an above average number of cases
the prefecture in which the sample was collected (online of influenza subtype H5N1 virus in poultry and an above average
human population density, which is a proxy for subtype H3N2 virus
Technical Appendix Table 1). Data on subtype H3N2 cases
infections.
were available for 35 prefectures and comprised 632 human
cases collected over 14 years. However these data are limited
because in a typical influenza year in China, hundreds of and duck density, human population density, percentage
millions of cases might occur. Therefore, we also compared of each prefecture occupied by bodies of water, and
the GenBank and GISAID data on subtype H3N2 cases with percentage of cultivated cropland per prefecture or markaz
human population density, assuming that the density is a (online Technical Appendix Table 2). These variables were
proxy for the true number of subtype H3N2 cases. included because results of previous studies have associated
them with risk for subtype H5N1 (11,13,14). For example,
Ecologic Variables human population was included as a predictor of subtype
H5N1 because it serves as an indirect measure of intensity
Subtype H3N2 of poultry trade (15). For Egypt, we used overall poultry
For China, we predicted the probability of occurrence density because density of chickens and ducks separately
of subtype H3N2 cases by using environmental factors was not available.
hypothesized in previous studies to be major drivers of
human influenza: human population density, percentage Swine Density
urban area, precipitation, and temperature (online Technical To identify potential areas of influenza reassortment,
Appendix Table 2). For instance, we incorporated we refined the co-occurrence maps by also incorporating
population into the model because we hypothesized that swine density. We used density data from the Food and
human influenza cases would be more likely to occur in Agriculture Organization of the United Nations, which
high-density urban areas with a large number of susceptible constructed these data by extrapolating from agricultural
human hosts (12). For Egypt, we used human population censuses and livestock surveys by using regression models
density as a proxy for subtype H3N2 infections. (16). The rationale was to focus on areas where subtypes
H3N2 and H5N1 might exchange genes in livestock
Subtype H5N1 because swine support co-infections with multiple lineages
To predict occurrence of subtype H5N1 cases, we of the influenza virus, which occasionally generate novel
used the following as environmental covariates: chicken strains (17,18).

582 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Hotspots for Influenza Virus Reassortment

Statistical Models used as a surrogate for subtype H3N2 infections in humans,

were located in the Nile Valley and Delta in Lower Egypt
Egypt (Figure 1). These areas could be sites of human co-infection
We constructed a Poisson regression model in which with subtypes H3N2 and H5N1, leading to the evolution
the dependent variable represents the count of a rare of novel influenza strains. Major cities located within 10
event. The dependent variable was the number of cases km of potential reassortment hotspots are Benha, Cairo,
of subtype H5N1 in poultry per district. The independent Dumyat, El Faiyum, and Shibin el Kom, which could be
variables were poultry density, human population density, prioritized for increased surveillance to detect reassortment
percentage cropland, and percentage water per district. events and prevent spread. Poultry density per district was
The dataset included sites where poultry were negative for a highly statistically significant predictor of subtype H5N1
subtype H5N1. We identified districts predicted to have an in poultry (Table), probably because high bird densities
above average number of cases of subtype H5N1 among facilitate transmission of the virus among flocks in a
poultry and above average human population density. Such village. The percentage of cropland per district was highly
districts could be the site of double infections with subtypes correlated with poultry density (ρ = 0.72), so we included
H3N2 and H5N1 in humans and of in vivo reassortment. only the latter in the regression model. The percentage
of water per district also approached significance, which
China could be because family compounds in rural areas where
We used multivariate logistic regression to backyard flocks are raised are typically located near canals
relate occurrence of subtypes H3N2 and H5N1 to the and irrigated fields.
aforementioned ecologic variables (online Technical
Appendix Table 2). The logistic regression models were China
built by using population density, percentage urban area, Cases of subtype H3N2 in humans in China were
temperature, and precipitation as predictors of subtype H3N2 mostly concentrated along the east coast (online Technical
presence and chicken and duck density, human population Appendix Figure 2, panel A). The association between
density, percentage cropland per prefecture, and percentage subtype H3N2 and human population density was
water as predictors of subtype H5N1 presence. The datasets significant (Table). Human population density, climate, and
comprise occurrences of subtypes H5N1 or H3N2 but lack the percentage of urban areas per prefecture collectively
negative occurrences. Therefore, we selected negative sites explained ≈60% of the risk for subtype H3N2 occurrence
at random and then fitted a logistic regression model to the (R2 = 0.596, area under the curve [AUC] = 0.902). Subtype
positive and random negative occurrences. To reduce the bias H3N2 is expected to occur primarily in central, eastern, and
of random negative occurrences, we selected negative sites at southern China (online Technical Appendix Figure 2, panel
random 10,000× and calculated the average of the parameters B). In the surveillance and the outbreak datasets, statistically
of the logistic regression model over these randomizations. significant drivers of subtype H5N1 occurrences were
human population, duck density, and percentage of water.
Results The models for subtype H5N1 had moderate predictive
power (R2 surveillance = 0.604, AUC surveillance = 0.918,
Egypt R2 outbreak = 0.424, AUC outbreak = 0.848).
Areas with a high number of cases of subtype H5N1 After creating maps of the probability of occurrence of
in poultry and high human population density, which we subtypes H3N2 and H5N1, we multiplied the maps by one

Figure 2. Influenza empirical data and occurrence maps for influenza virus subtypes H3N2 and H5N1. A) Observed cases of subtypes
H3N2 and H5N1 in People’s Republic of China, according to outbreaks reported to the Chinese Ministry of Agriculture. B) Spatial model of
the probability of subtype H3N2 at the prefecture scale predicted by using logistic regression. C) Risk for subtype H5N1 according to the
outbreak dataset. See online Technical Appendix Figure 2, wwwnc.cdc.gov/EID/article/19/4/12-0903-Techapp1.pdf, for the corresponding
map for the surveillance dataset.

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RESEARCH

Table. Effect of environmental variables on occurrence of influenza virus subtypes*

Location, subtype (data source) Coefficient SE p value
China
Subtype H3N2
Intercept 5.184 0.946 8.7 × 108
Human population 9.47 × 104 3.23 × 104 4.47 × 102
Percentage urban 0.113 0.117 0.284
Precipitation 4.59 × 103 8.87 × 103 0.573
Temperature 1.24 × 102 8.27 × 103 0.195
Subtype H5N1 (surveillance dataset)
Intercept 7.93 1.66 3.01 × 105
Chicken density 6.94 × 102 0.438 0.572
Duck density 1.53 0.433 3.45 × 103
Human population 1.41 × 103 2.85 × 104 1.77 × 104
Percentage agriculture 2.18 × 104 1.52 × 104 0.218
Percentage water 7.32 × 102 1.7 × 102 2.36 × 103
Subtype H5N1 (outbreak dataset)
Intercept 9.24 1.14 3.15 × 1012
Chicken density 1.24 0.277 2.87 × 104
Duck density 0.542 0.24 0.117
Human population 1.37 × 103 2.67 × 104 1.41 × 103
Percentage agriculture 2.13 × 104 8.84 × 105 5.4 × 102
Percentage water 0.101 1.91 × 102 4.53 × 103
Egypt
Subtype H5N1
Intercept 1.83 0.516 4 × 104
Poultry density 7.86 × 104 2.31 × 104 7 × 104
Human population 3.36 × 102 6.49 × 102 0.605
Percentage water 0.752 0.41 6.64 × 102
*Boldface indicates p<0.05.

another to predict the probability of co-occurrence of the East Asia

2 subtypes (Figure 3, panel B; online Technical Appendix We applied the influenza virus subtypes H3N2 and
Figure 3, panel B). We classified an area as a potential H5N1 logistic regression models that were fitted to the data
reassortment hotspot if the probability of both subtypes from China to neighboring countries for which chicken and
occurring at the site was >50% and the density of swine was duck density data were available (19). As in the analysis for
above average. Analysis of other swine density thresholds China, we multiplied the subtype H3N2 and H5N1 models
yielded similar results. The consensus of the spatial models to predict areas of co-occurrence between the subtypes
(Figure 3, panel C; online Technical Appendix Figure 3, and overlaid swine density. To the extent that these areas
panel C) is that in China, there are 2 main geographic foci have above average swine density and a >50% chance for
of risk for reassortment of subtypes H3N2 and H5N1: 1) the co-occurrence of subtypes H3N2 and H5N1, potential
coastal provinces bordering the South China Sea and East reassortment hotspots are the northern plains of India (Uttar
China Sea (Guangdong, Jiangsu, Shanghai, and Zhejiang Pradesh), the western Korean Peninsula (Daejeon, Gyeonggi,
Provinces) and 2) central China (Hunan and Sichuan Jeollabuk Provinces of South Korea and Pyonganbuk and
Provinces). The added value of modeling areas where Pyongannam Provinces of North Korea), and southwestern
subtypes H3N2 and H5N1 co-occur versus modeling based Japan (Saga Prefecture on Kyushu Island) (Figure 4, panel
exclusively on subtype H5N1 is that the former approach B; Technical Appendix Figure 4, panel B). Major cities with
pinpoints a smaller geographic region that can be prioritized >500,000 persons near these hotspots include Kanpur, India;
for increased surveillance or farm biosafety. Mapping areas Chengdu, Sichuan, central China; Hangzhou and Shanghai,
based on the probability of subtype H5N1 occurrence alone eastern China; and Seoul, South Korea. Risk is higher in these
would prioritize additional provinces to the southwest cities because they have high densities of swine, which could
(Henan, Hebei, and Hubei) and to the north (Beijing, Hebei, be a mixing vessel for reassortment of subtypes H5N1 and
Liaoning, and Tianjin) of the 6 provinces that we identified as H3N2, and a high potential for infection with subtype H5N1
potential areas for reassortment between subtypes H3N2 and and H3N2 according to our regression models; for example,
H5N1 (Figure 2, panel C; online Technical Appendix Figure the models indicate that the ecologic suitability of Shanghai
2, panel C). Our prioritization of a smaller geographic area is 0.97 for subtype H3N2 and 0.996 for subtype H5N1.
is valuable if the resources for surveillance are insufficient Incorporating population density as a proxy for infection
to enable sampling of all of the provinces that are at risk for with subtype H3N2 results in predictions that are compatible
subtype H5N1. with the models based on swine density but also identifies 2

584 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Hotspots for Influenza Virus Reassortment

Figure 3. Potential influenza reassortment areas in People’s Republic of China determined by using the influenza virus subtype H5N1
outbreak dataset. A) Density of swine. B) Spatial model of the risk for subtype H3N2 and H5N1 co-occurrence according to the outbreak
dataset. C) Areas with a probability of subtype H5N1 and H3N2 co-occurrence >50% and above average swine density. D) Areas with a
probability of subtype H5N1 and H3N2 co-occurrence >50% and above average human population density. See online Technical Appendix
Figure 3, wwwnc.cdc.gov/EID/article/19/4/12-0903-Techapp1.pdf, for corresponding maps based on the subtype H5N1 surveillance dataset.

other megacities of >10 million persons that could be at high A caveat is that even if virus subtypes H3N2 and H5N1
risk for virus reassortment: Dhaka, Bangladesh and Delhi, were to reassort in swine, the spread of the reassortant
India (Figure 4, panel C; online Technical Appendix Figure virus among humans might require further virus adaptation
4, panel C). events; for example, mutations might be required for the
virus to replicate efficiently in humans or to be transmitted
Discussion among humans (22). Recent work has shown that as few as
The spatial models presented here predict that a 5 aa substitutions are required for aerosol spread of subtype
reassortant influenza (H3N2/H5N1) virus is most likely to H5N1 among mammals (23). With these qualifications in
originate in the coastal and central provinces of China or the mind, this analysis provides actionable recommendations
Nile Delta region of Egypt. The probability that subtypes about which areas to target for intensified farm and
H3N2 and H5N1 will co-occur in these regions is high market surveillance. Such surveillance could enable early
(Figure 1; Figure 3, panel C; online Technical Appendix detection of a reassortant influenza (H3N2/H5N1) virus,
Figure 4, panel C), which could lead to dual infection in should it arise in swine, and facilitate containment of the
mammalian hosts, such as swine or humans in China or virus before it crosses the species barrier to humans.
humans in Egypt. Co-infection could subsequently result Our finding that in China the probability of subtype
in in vivo reassortment. Although the influenza A(H1N1) H3N2 infection increases with human population density
pdm09 virus is hypothesized to have originated from is compatible with previous studies that detected a positive
Mexico (20), southern China remains a major hotspot for association between population, influenza cases, and
the generation of novel influenza viruses (21). Our spatial mortality rates (12,24). Reasons for this association could be
models are compatible with this longstanding observation that the number of susceptible human hosts increases with
insofar as we predict that the southern coastal province of population (11) or that surveillance efforts are greater in
Guangdong is a potential hotspot for the evolution of novel populous areas (25). Our results with regard to subtype H5N1
influenza viruses by reassortment. in birds are also largely consistent with those of previous

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 585
RESEARCH

Figure 4. Reassortment areas elsewhere in Asia based on the People’s Republic of China model constructed from the influenza virus
subtype H5N1 outbreak dataset. A) Probability of subtype H3N2 and H5N1 co-occurrence (according to the subtype H5N1 outbreak
dataset). B) Areas with a probability of subtype H5N1 and H3N2 co-occurrence >50% and above average swine density. C) Areas with a
probability of subtype H5N1 and H3N2 co-occurrence >50% and above average human population density. See online Technical Appendix
Figure 4, wwwnc.cdc.gov/EID/article/19/4/12-0903-Techapp1.pdf, for corresponding models based on the surveillance dataset.

studies that mapped subtype H5N1 hotspots in China and In Egypt, our results support increased surveillance of
Egypt. In China, several provinces identified as having high backyard flocks near Benha, Cairo, Dumyat, El Faiyum,
ecologic suitability for subtype H5N1 (including Shandong, Shibin el Kom, and Tanta, where suitability for subtypes
Jiangsu, and Sichuan) were also identified as subtype H5N1 H5N1 and H3N2 is predicted to be high. Control measures
hotspots in a previous study that used a different statistical could include compensation plans and vaccination of poultry
model and different predictor variables (11). In China, with a recently developed subtype H5N1 vaccine that is more
previous analyses have concluded that risk for subtype H5N1 effective than previous vaccines against strains circulating in
increases with the density of domestic ducks (26). In Egypt, Egypt (10). Reporting of poultry disease outbreaks in Lower
earlier studies identified high-intensity crop production as a Egypt is poor (31), probably because farmers fear loss of
statistically significant predictor of subtype H5N1 in poultry income if authorities cull their flocks. Indeed, birds suspected
(27). Similarly, we found that subtype H5N1 infections in to be infected with subtype H5N1 are often sold quickly at
poultry were associated with poultry density, which was a discount, resulting in virus transmission to buyers’ flocks
highly correlated with crop production. In a previous study, and families (32). If equitable compensation schemes were
models constructed from satellite images of vegetation implemented, reporting of subtype H5N1 might increase
predicted that the highest environmental suitability for and outbreaks could be contained more quickly, reducing
subtype H5N1 is along the Nile River and in the Nile Delta opportunities for subtypes H5N1 and H3N2 to co-infect
(28). Our models were constructed from different predictor humans or domestic animals and, thus, for reassortment.
variables, such as poultry density, but yielded similar results: In general, policies such as culling must have a
the highest number of subtype H5N1 cases in poultry were scientific basis because these measures have major effects
predicted to occur in districts in the Nile Delta. on the economy and animal welfare. For example, when
Efforts to contain the A(H1N1)pdm09 virus would part of a swine herd is culled to contain an outbreak, it might
have been more effective if the virus had been detected become necessary to euthanize the entire herd, including
in animal populations before it was transmitted to animals with no influenza exposure, because buyers will not
humans (29). Continuous zoonotic influenza surveillance accept them (33). Furthermore, influenza outbreaks among
is needed in China and Egypt and requires a network of livestock can trigger major global declines in meat prices,
laboratories to screen surveillance samples and requires and the nature and timing of veterinary health authorities’
financial incentives to encourage poultry producers and responses to an outbreak can affect the extent to which
sellers to report outbreaks. One strategy for early detection demand recovers after the crisis. In particular, when control
of a reassortant virus could involve increasing farm and measures such as culling are scientifically well justified and
market surveillance in the identified areas (i.e., live-bird explained to the public soon after the start of an outbreak,
markets in 6 provinces in China [Guangdong, Hunan, consumer confidence is restored more quickly (34).
Jiangsu, Shanghai, Sichuan, and Zhejiang] that have a Although our maps suggest a risk for reassortment
>50% chance of subtype H3N2 and H5N1 co-occurrence in Lower Egypt and eastern and central China, in vivo
and above average swine density). Increased monitoring reassortment of subtypes H3N2 and H5N1 has not been
could identify hotspots where subtype H5N1 is circulating, detected in humans in these areas. On the other hand,
leading to more efficient targeted vaccination of poultry, numerous infections with influenza (H3N2)v, a reassortant
and could pinpoint prefectures at high risk for a reassortant virus that contains genes from a subtype H3N2 virus
virus. In China, sanitary practices, such as cage disinfection circulating in swine and from the A(H1N1)pdm09 virus,
and manure disposal, would substantially reduce risk for have been detected in humans in North America (35,36).
subtype H5N1 in live-bird markets (30). This finding raises the question of why subtype H3N2v

586 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Hotspots for Influenza Virus Reassortment

has spread but subtype H3N2/H5N1 reassortants have not. (29,40). Although our analysis focused on the influenza
Spread of subtype H3N2v could result from the fact that the virus, our modeling framework can be generalized to
reassortant virus contains the M gene from the A(H1N1) characterize other potential emerging infectious diseases at
pdm09 virus, which increases aerosol transmission (35,37). the human–animal interface.
Our models might explain why, in contrast with subtype
H3N2v reassortants, no subtype H3N2/H5N1 reassortants Acknowledgments
have been detected in humans. For example, we predict We thank Stephen Felt, Bob Gilman, Ryan Harrigan,
that subtypes H3N2 and H5N1 occur in Hunan, China, a Christine Jessup, Brenda Larison, Tony Marfin, Makoto Ozawa,
province that has high swine density and was the geographic John Pollinger, Josh Rosenthal, Erin Toffelmier, Cuiling Xu, and
origin of subtype H5N1 viruses in clade 2.1 (38). Influenza 2 anonymous reviewers for their comments.
(H3N2/H5N1) reassortants in which the nonstructural gene
This work was supported by a National Institutes of Health/
comes from a clade 2.1 virus replicate poorly in mice (5).
National Science Foundation award, “Ecology and Evolution
Thus, subtype H3N2/H5N1 reassortants might not have
of Infectious Diseases,” from the Fogarty International Center
emerged as often as subtype H3N2v reassortants because the
3R01-TW005869. This research was conducted in the context
provinces where subtypes H3N2 and H5N1 overlap contain
of the Zoonotic Influenza Collaborative Network, led by the
a clade of subtype H5N1, whose genes reduce the fitness of
Fogarty International Center, National Institutes of Health. The
reassortant viruses. If this hypothesis is correct, if subtypes
Collaborative Network is supported by international influenza
H5N1 and H3N2 infect a pig in central China and exchange
funds from the Office of the Secretary of the Department of
genes, the hybrid virus might not replicate efficiently or
Health and Human Services.
transmit to other hosts. Furthermore, a reassortant virus
with surface proteins similar to those of subtype H3N2 Dr Fuller is a postdoctoral researcher at the Center for
viruses that have circulated in humans recently might have Tropical Research at the Institute of the Environment and
poor transmissibility because of preexisting immunity (18). Sustainability, University of California, Los Angeles. His primary
Applying our modeling framework to other zoonotic research interest is influenza in birds.
influenza subtypes, such as H3N2v, could yield insight
about geographic hotspots of reassortment and the pattern References
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588 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Effect of 10-Valent
Pneumococcal Vaccine on
Pneumonia among Children, Brazil
Eliane Terezinha Afonso, Ruth Minamisava, Ana Luiza Bierrenbach, Juan Jose Cortez Escalante,
Airlane Pereira Alencar, Carla Magda Domingues, Otaliba Libanio Morais-Neto,
Cristiana Maria Toscano, and Ana Lucia Andrade

Pneumonia is most problematic for children in develop- Brazil is composed of 5 administrative regions with
ing countries. In 2010, Brazil introduced a 10-valent pneu- different climatic and socioeconomic characteristics. In
mococcal conjugate vaccine (PCV10) to its National Immu- 2010, the estimated population of infants (children <12
nization Program. To assess the vaccine’s effectiveness for months of age) was ≈2,800,000, and the infant mortality
preventing pneumonia, we analyzed rates of hospitalization rate was 17 deaths per 1,000 live births (6,7). In Brazil, the
among children 2–24 months of age who had pneumonia
main reason for hospitalization of infants is pneumonia (6).
from all causes from January 2005 through August 2011.
We used data from the National Hospitalization Information
Vaccination with pneumococcal conjugate vaccine
System to conduct an interrupted time-series analysis for 5 (PCV) is a public health intervention to prevent pneumo-
cities in Brazil that had good data quality and high PCV10 coccal disease. PCV has been in use since 2000, when a
vaccination coverage. Of the 197,975 hospitalizations ana- 7-valent vaccine (PCV7) was licensed in the United States
lyzed, 30% were for pneumonia. Significant declines in hos- for routine use in children. In 2010, PCV7 was replaced
pitalizations for pneumonia were noted in Belo Horizonte by a 13-valent vaccine. Recently, a 10-valent pneumococ-
(28.7%), Curitiba (23.3%), and Recife (27.4%) but not in cal conjugate vaccine (PCV10) was licensed in Brazil; this
São Paulo and Porto Alegre. However, in the latter 2 cities, vaccine includes the same serotypes that are in PCV7 (4,
vaccination coverage was less than that in the former 3. 6B, 9V, 14, 18C, 19F, 23F), plus 3 more (1, 5, and 7F) (8).
Overall, 1 year after introduction of PCV10, hospitalizations In 2010, Brazil introduced PCV10 into its routine Na-
of children for pneumonia were reduced.
tional Immunization Program. Previously, no PCV had
been incorporated into the routine immunizations. The vac-
treptococcus pneumoniae infections are the leading cination was introduced in all cities from March through
S cause of bacterial pneumonia, meningitis, and sepsis
among children (1,2); in developing countries, these infec-
September 2010; 3 doses (at 2, 4, and 6 months of age) plus
1 booster (at 12–15 months of age) were recommended.
tions account for almost a half million deaths among chil- Two routine catch-up schedules were also in place: 1) two
dren <5 years of age (3). In Brazil, the largest country in doses for children 7–11 months of age plus a booster at
South America, the role of S. pneumoniae in pneumonia in 12–15 months of age, and 2) one dose for children 12–24
children is considerable (4,5). months of age. PCV10 is not given to children >24 months
Author affiliations: Federal University of Goiás, Goiania, Goiás, of age (9).
Brazil (E.T. Afonso, R. Minamisava, A.L. Bierrenbach, O.L. Morais- In Brazil, vaccination of children with PCV10 is free
Neto, C.M. Toscano, A.L. Andrade); Pontifical Catholic University through the National Unified Health System (10). By Oc-
of Goiás, Goiania (E.T. Afonso); Ministry of Health, Brasilia, Brazil tober 2011, the mean vaccination coverage rapidly reached
(J.J.C. Escalante, C.M. Domingues); University of Brasília, Brasília 80% for a full primary series for children <12 months of
(C.M. Domingues); and University of São Paulo, São Paulo, Brazil age in >5,000 municipalities (Brazilian Ministry of Health,
(A.P. Alencar) unpub. data).
Studies that assessed the effect of PCV7 found a sta-
DOI: http://dx.doi.org/10.3201/eid1904.121198 tistically significant reduction in the overall incidence of

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 589
RESEARCH

invasive pneumococcal disease and hospitalizations for hospitalized longer than anticipated. To avoid including
pneumonia among children <2 years of age shortly after duplicate records, we used a deterministic record linkage
the first year of vaccination (11–14). Our aim was to assess algorithm to find records for the same patient (17). We
the effectiveness of PCV10 for reducing hospitalizations then considered that consecutive records of the same pa-
for all-cause pneumonia. We analyzed trends in rates of tient with a 14-day interval between discharge and reentry
hospitalization for pneumonia among children soon after belonged to the same episode of disease (18).
the introduction of PCV10 in Brazil. Ethical approval was We studied 5 state capital cities in Brazil: Belo Hori-
granted by the Ethics Committee, Federal University of zonte, Curitiba, Recife, São Paulo, and Porto Alegre (Fig-
Goiás, Goiania, Brazil. ure 1). The cities were initially selected from a list of 10
cities participating in an ongoing case–control study evalu-
Methods ating the effect of PCV10 vaccination on pneumococcal
disease. The selection of cities for the case–control study
Data Sources was based on data quality and willingness of the local sur-
We conducted an interrupted time-series analysis by veillance teams to participate in the study. Of the initial 10
using individual-level secondary data from the Hospital- cities, 5 were excluded a priori from the time-series analy-
ization Information System of the National Unified Health sis; 3 cities had not reached vaccination coverage of at least
System from January 2005 through August 2011. The Hos- 75% for the first dose of vaccine (primary series) 3 months
pitalization Information System records ≈75% of all hospi- after vaccine introduction, and 2 cities were excluded be-
talizations in Brazil and 60%–80% of the hospitalizations cause of poor data quality in the initial descriptive analyses.
for the cities in the analyses (15). During the study period, The 5 chosen cities account for 50% of the population of
there were no major changes in the amount of hospital care the state capitals of the country and are located in 3 of the 5
provided by the National Unified Health System. administrative regions of the country.
Variables in the Hospitalization Information System The annual numbers of live births were obtained from
are demographics, date of admission/discharge, residential the Live Birth Information System and used to calculate
address, hospital code, and International Classification of the annual population of children 2–24 months of age. The
Diseases 10th Revision (ICD-10) codes for primary and monthly population was calculated by interpolating an ex-
secondary diagnoses. Because the Hospitalization Infor- ponential growth model to the annual data.
mation System database is mainly used for reimbursement
purposes, the likelihood that hospitalizations would be un- Definitions
derreported and that data would be missing are small (16). We identified Hospitalization Information System re-
The structure of the Hospitalization Information cords of children 2–24 months of age who were hospital-
System made it possible for 1 episode of hospitalization ized from January 2005 through July 2011 with specific
for a given patient to be recorded multiple times. Addi- ICD-10 codes: pneumonia (J12-J18), bronchiolitis (J21),
tional records might be generated when patients remain respiratory causes (J00–J99), nonrespiratory causes, and all
causes (19). We considered nosocomial pneumonia more
likely to be reported as a secondary discharge diagnosis;
therefore, only the primary diagnosis of the first record of
each episode of disease was used in all data analyses.
In a descriptive analysis, which included only the pre-
vaccination period, we obtained average annual numbers
and rates of pneumonia hospitalizations for each city and
the proportion of pneumonia out of all respiratory causes
and out of all causes of hospitalizations. Specific pneu-
mococcal pneumonia–coded cases in the Hospitalization
Information System represented only 0.06% of the pneu-
monia cases reported in the system because confirming
bacteriologic pneumonia in children is difficult; thus, we
considered use of pneumococcal pneumonia–coded cases
not appropriate in a time-series analysis.

Figure 1. Capital cities of Brazilian states, and their populations,

Vaccination Coverage
in which effectiveness of 10-valent pneumococcal vaccine was In Brazil, the National Immunization Program, estab-
studied. Population data obtained from Brazilian Census 2010. lished in 1973, led to high rates of coverage (20). PCV10

590 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Effect of PCV10 on Pneumonia among Children, Brazil

vaccination was introduced in March 2010 in all selected 2005–February 2010 and had 62 time points (monthly
cities except Porto Alegre, where it started in June 2010. data) in the final model (except for Porto Alegre, which
PCV10 coverage data for each city were obtained from had 65 time points because vaccination introduction was
the National Immunization Program vaccine coverage da- delayed for 3 months). The transition period was the time
tabase of the National Unified Health System, in which of vaccine introduction through 4 months after. The post-
number of vaccine doses and administrative vaccination vaccination period was the time after the transition period;
coverage are made available for all municipalities in the it comprised 14 time points in the analysis (3 fewer for
country. Numerator data are obtained from the number of Porto Alegre). The transition period was excluded from
doses administered in the vaccination rooms, by vaccine analysis, although it is shown in the figures. The time-se-
type, patient age, and municipality. PCV10 coverage for a ries analysis was based on a generalized linear model for
full PCV10 primary series (3 doses) was estimated as the rates of hospitalizations for pneumonia and for nonrespi-
number of third doses of PCV10 administered (numerator) ratory causes by using the negative binomial distribution
to children <12 months of age divided by the number of with a logarithmic link function and an offset equal to the
births in a population over time in each municipality (de- log of the population divided by 100,000 (21). Residual
nominator) multiplied by 100 (see online Technical Appen- analyses showed no substantial deviations from model as-
dix, wwwnc.cdc.gov/EID/article/19/4/12-1198-Techapp1. sumptions. The main outcome was rates of hospitalization
pdf, for sources of data on vaccination coverage). for all-cause pneumonia. The explanatory variables in the
We calculated the moving average of vaccine cover- model were calendar month (to control for seasonality),
age for every 3-month period. The value attributed to a giv- linear trend over time (to control for preexisting trends),
en month was the average vaccine coverage in that month and a variable equal to 1 after vaccination and 0 other-
and the coverage for the months before and after the given wise. After estimation of the models, 2 outputs were pre-
month. This calculation was done to smooth out short-term sented: 1) the percentage change in hospitalization rates,
vaccination coverage fluctuations (Figure 2). which compare the prevaccination and postvaccination
periods and their corresponding p values and 95% CIs,
Data Analyses and 2) a graph showing the predicted hospitalization rates
In the interrupted time-series analysis, 3 immuniza- for pneumonia and their 95% CIs for the postvaccination
tion periods were defined: prevaccination, transition, and period based on models fitted with data for the prevaccina-
postvaccination. The prevaccination period was January tion period. With the latter output, it is possible to visually

Figure 2. Monthly coverage for third dose of 10-valent pneumococcal vaccine achieved 11–14 months after vaccination among children
<12 months of age in 5 cities in Brazil. Dotted horizontal lines represent 100% vaccination coverage.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 591
RESEARCH

evaluate the observed and the predicted monthly hospi- all 3 doses of PCV10). Vaccination coverage varied by city.
talization rates for the postvaccination period; that is, the Belo Horizonte and Curitiba rapidly reached 100% cover-
rates that would have resulted had the changes during the age and maintained stable rates of ≈100% from September
transition period not taken place. 2010 on. Recife showed a tendency toward sustained and
Sensitivity analyses for each city compared models continuously rising coverage over the study period, eventu-
with and without removal of the months of July and August ally reaching ≈100%. Porto Alegre reached 100% coverage
2009. This comparison was an attempt to reduce potential on January 2011, followed by a gradual decrease to 85% in
bias resulting from the influenza epidemic. To compare the July 2011. São Paulo coverage increased to 90% in Novem-
estimated percentage change for hospitalizations for pneu- ber 2010, after which it continuously declined, reaching 75%
monia and nonrespiratory causes, we performed separate in July 2011.
time-series analyses. In theory, the vaccination-induced Trends in patterns of hospitalization rates for pneumo-
percentage change would be higher for hospitalizations nia, respiratory causes, and all causes are shown for each city
for pneumonia than for nonrespiratory causes, although a (Figure 3). Seasonal variations are evident for all cities. The
minority of possible pneumococcal disease codes was in- contribution of pneumonia to the total number of hospital-
cluded in nonrespiratory causes (such as meningitis). The ized patients varied widely by city but not by years. Rates of
percentage change for hospitalizations for nonrespiratory hospitalization for all causes, particularly from mid-2007 on,
causes was expected to reflect influences other than the decreased notably for Belo Horizonte and Recife; however,
PCV10 vaccination effect that might have concomitantly the observed reductions in rates of hospitalization for pneu-
affected the data series. We expected the effect of report- monia for these cities and for Curitiba seem to be restricted
ing/processing delays to still be present during the post- to the postvaccination period (from mid-2010 on).
vaccination period, therefore inducing an artificially lower Rates of hospitalization for bronchiolitis were lower
number of hospitalizations in more recent months (although than those for pneumonia in all cities except Porto Alegre
we had discarded the most recent ones from analysis). To (Figure 4). The seasonal variations of bronchiolitis and
compensate for this effect, we calculated the differences pneumonia were mostly parallel and were found for all
between the percentage changes in rates of hospitalizations cities. Hospitalization rates progressively increased in the
for pneumonia and nonrespiratory causes for each city. The more recent years for Porto Alegre, São Paulo, and pos-
equality of the percentage changes for hospitalization for sibly Curitiba.
pneumonia and nonrespiratory causes was tested by using Table 2 and Figure 5 show results derived from the
the Wald test (22). same time-series models. During the postvaccination period,
The observed trends for bronchiolitis, respiratory, and rates of hospitalization for pneumonia decreased significant-
all-cause hospitalizations are shown for comparison. The ly (p<0.001) in Belo Horizonte (−40.3%), Curitiba (-37.6%),
linkage/classification procedures were conducted by using and Recife (−49.3%). Rate reductions were borderline sig-
STATA version 12.0 (www.stata.com/), and the statistical nificant for São Paulo (-13.4%; p = 0.074) and Porto Alegre
analysis was done by using R (www.r-project.org/). (-23.5%; p = 0.052) (Table 2). Rates of hospitalization for
nonrespiratory causes also decreased in all cities, albeit at
Results a lower rate. The following differences between the per-
In the 5 cities, 197,975 hospitalizations of children 2 centage changes in hospitalization rates for pneumonia and
months to 2 years of age were identified during the study nonrespiratory causes represent our best estimate of the vac-
period; 109,155 (55.1%) were for respiratory causes, in- cination effect: Belo Horizonte (-28.7%), Curitiba (-23.3%),
cluding 59,636 (30.1%) for pneumonia. During the prevac- Recife (-27.4%), São Paulo (-1.8%), and Porto Alegre
cination period, the rates of pneumonia hospitalizations (-2.3%). During the postvaccination period, reductions in
varied substantially by city (Table 1). rates of hospitalization for pneumonia did not differ signifi-
Figure 2 shows the moving average for PCV10 cover- cantly from rates of hospitalization for nonrespiratory causes
age (percentage of children <12 months of age who received in São Paulo (p = 0.827) and Porto Alegre (p = 0.845).

Table 1. Rates of hospitalization for pneumonia among children 2 months–2 years of age, Brazil, prevaccination period (2005–2009)*
% Hospitalizations for % Hospitalizations for
No. cases, annual Rates, annual mean pneumonia/hospitalizations for pneumonia/hospitalizations for
City mean ( SD) ( SD) all respiratory causes all causes
Belo Horizonte 939 (133) 1.643 (217) 53.4 34.8
Curitiba 359 (49) 790 (114) 72.2 27.9
Recife 538 (41) 1.304 (107) 47 23.4
São Paulo 3999 (312) 1.247 (103) 61.4 36.1
Porto Alegre 292 (38) 863 (112) 25.8 15
*Pneumonia identified by International Classification of Diseases, 10th Revision, codes: J12–J18.

592 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Effect of PCV10 on Pneumonia among Children, Brazil

Figure 3. Trends in rates of hospitalization for pneumonia (black) and for all respiratory causes (light gray) and all causes (dark gray)
among children 2 months–2 years of age in 5 cities, Brazil, January 2005–August 2011. PCV10, 10-valent pneumococcal vaccine.

Figure 5 compares the observed monthly rates of recently been introduced in some countries in North America
hospitalization for pneumonia with the forecasted val- and Europe. Preliminary evaluations indicate a reduction of
ues that were modeled with use of data exclusively from invasive pneumococcal disease. In the province of Quebec,
the prevaccination period. For Belo Horizonte, Curitiba, Canada, PCV10 was introduced to the routine immunization
and Recife, the observed numbers are close to or below schedule 5 years after PCV7 was introduced. Data obtained
the lower limit of the 95% CI, particularly for the most by a sentinel laboratory surveillance network showed lower
recent months. incidence of invasive pneumococcal disease among children
vaccinated with PCV10 than with PCV7 (35.3 vs. 64.1 cas-
Discussion es/100,000 person-years) (23). In Finland, results of a recent
This study indicates that the introduction of PCV10 field trial found a marked decrease in the incidence of inva-
through the routine immunization program in Brazil has sive pneumococcal disease among children who were vac-
effectively lowered rates of hospitalization for pneumonia cinated according to a 3+1 or a 2+1 immunization schedule;
among children. Rates of hospitalization for all causes de- vaccine effectiveness reached 100% (95% CI 83%–100%)
clined in 3 of the 5 cities studied (Belo Horizonte, Curi- and 92% (95% CI 58%–100%), respectively, after 2 years
tiba, and Recife). In the other 2 cities (São Paulo and Porto (24). The Clinical Otitis Media and Pneumonia Study con-
Alegre), these rates did not decline significantly, possibly ducted at urban sites in Argentina, Colombia, and Panama
because vaccination coverage for these 2 cities in 2011 was showed that the efficacy of PCV10 for reducing community-
lower (≈80%) than it was in the other 3 cities (>90%). An- acquired pneumonia and alveolar consolidation among chil-
other possible reason is that Porto Alegre started its vac- dren was 7.3% and 23.4%, respectively (25).
cination program 3 months after the other cities, so its post- For PCV7, studies have already documented its ef-
vaccination period was shorter, leaving less time for the fect on rates of hospitalization for pneumonia among
vaccination to become effective. children (11,12,26–28). In the United States, the rates of
Comparison of our results with those of other studies hospitalization for pneumonia were reduced 39%–52%.
is not straightforward because, to our knowledge, no com- However, aside from the use of different vaccines, our
parable studies have been published (e.g., effects of PCV10 study is not directly comparable. Vaccination coverage
on rates of hospitalization for all causes). PCV10 has was generally lower in the United States, increasing from

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 593
RESEARCH

Figure 4. Trends in rates of hospit-alization for pneumonia (dark gray) and bronchiolitis (light gray) among children 2 months–2 years of
age in 5 cities, Brazil, January 2005–August 2011. PCV10, 10-valent pneumococcal vaccine.

68% to 83% during the postvaccination period, which the study period. Another explanation is the rapid increase
was much (4 years) longer. This longer time might have in coverage of the Family Health Programme. This pro-
allowed time for herd immunity to protect the nonvac- gram reached 85% of Brazilian municipalities in 2010 and
cinated population (26,28). Also, the illnesses compared greatly reduced deaths and hospitalizations of infants for
in each study were not the same; the United States study primary-care sensitive diseases like diarrhea and for lower
evaluated dehydration and diarrhea, whereas our study respiratory tract diseases (29–31).
evaluated all nonrespiratory conditions since the rotavi- Across all 5 cities, we found differences in rates of hos-
rus vaccine was introduced in 2006 to Brazil. pitalization for pneumonia before introduction of PCV10.
The introduction of rotavirus vaccination might actual- Marked regional differences had already been documented
ly be one of the best explanations for the decreasing trends (32). Possible reasons, other than differences in health care
of all-cause hospitalizations in the target age group during provision, are variations in epidemiology, demographics,
Table 2. Annual percent change (trend) and percentage change in rates of hospitalization among children 2 months–2 years of age,
Brazil, postvaccination period (January 2005–August 2011)
Hospitalizations for pneumonia Hospitalizations for nonrespiratory causes Difference in
City % Change (95% CI) p value % Change (95% CI) p value change p value
Belo Horizonte 40.30 <0.001  11.61 0.093 28.69 0.002
50.88 to 27.44) 23.48 to 2.10)
Curitiba 37.59 <0.001  14.27 0.012 23.32 0.011
49.63 to 22.68) 23.94 to 3.38)
Recife 49.32 <0.001  21.93 0.001 27.39 0.007
61.63 to 33.05) 32.18 to 10.13)
São Paulo 13.38 0.074  11.60 0.008 1.78 0.827
26.02 to 1.42) 19.31 to 3.15)
Porto Alegre 23.51 0.052  21.18 0.001 2.33 0.845
41.60 to 0.18) 31.08 to 9.86)

594 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Effect of PCV10 on Pneumonia among Children, Brazil

Figure 5. Observed (solid lines) and predicted (dashed lines) rates of hospitalization for pneumonia and 95% CIs (shaded area) among
children 2 months–2 years of age in 5 cities, Brazil, January 2005–August 2011. The 95% CIs are shown only for the 4 months after start
of vaccination. Decline represents the reduction in hospitalizations for pneumonia. PCV10, 10-valent pneumococcal vaccine.

socioeconomic status, and climate. Health care provisions monia has been shown to have high sensitivity and low
might play a progressively lesser role in explaining the dif- specificity for ascertaining pneumococcal pneumonia;
ferences in rates of hospitalization for pneumonia because thus, any bias resulting from misclassification of ICD-10
the results of National Household Sample Surveys show would be toward reduction of the observed effect of vac-
a trend toward equity in access to and use of health care cination (34).
facilities (31,33). Our results could also have been influenced by chang-
Several potential limitations of our study should be es in disease diagnosis and management over time. We
highlighted. Our data represent only the population served observed an increase in hospitalizations for bronchiolitis
by National Unified Health System in Brazil. The find- in the cities of Curitiba, Porto Alegre, and São Paulo. Al-
ings observed for the 5 capital cities cannot be consid- though this increase might represent a real increase in dis-
ered representative of the entire country. We attempted to ease incidence and/or severity, a more likely reason could
include mostly community-acquired cases of pneumonia simply be improvements in the diagnosis of bronchiolitis.
by restricting our analysis to the primary diagnosis for Thus, hospitalizations for bronchiolitis, which would oth-
hospitalization. By doing so, we hypothetically increased erwise be coded as nonspecific pneumonia or other lower
the proportion of hospitalizations for pneumococcal pneu- respiratory infections, could be increasingly coded correct-
monia out of all hospitalizations for pneumonia. Because ly in these locations. Potentially, this reduction in misclas-
only a few cases have pneumonia listed as a secondary di- sification over time would tend to increase the observed ef-
agnosis, we missed only a few community-acquired cases fect of vaccination in these cities, but no vaccination effect
of pneumonia by not including it. was observed in Porto Alegre and São Paulo. We have not
Information about the extent of coding errors in identified other major changes in diagnosis and reporting
Brazil is scarce. None of the information is specific to habits, including those motivated by knowledge of PCV
pneumonia. However, the clinical diagnosis of pneu- introduction or the fact that its effect was being assessed.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 595
RESEARCH

The study was conducted at the time of the 2009 in- no. 306096/2010-2) and of the National Institute of Science and
fluenza pandemic, and an influenza A (H1N1) vaccination Technology for Health Technology Assessment. A.L.A. received
campaign was conducted in Brazil as a time-limited in- a research grant from GlaxoSmithKline and financial support to
tervention. This campaign took place from March through attend scientific meetings from Pfizer and GlaxoSmithKline and
June 2010 and achieved high vaccination coverage among has served as an advisor for Pfizer.
children <2 years of age (Brazilian Ministry of Health, un-
Dr Afonso is a PhD student in epidemiology and public
pub. data). This age group was only slightly affected by
health at the Federal University of Goiás, Goiânia, and an associ-
the pandemic, as evidenced by the lack of a temporary in-
ate professor in the Department of Pediatrics. Her scientific inter-
crease in the rates of hospitalization for pneumonia and
ests include the epidemiology of respiratory tract infections.
rates of hospitalization for influenza (data not shown be-
cause numbers were so small). Therefore, we consider it
unlikely that the pandemic or its vaccination campaign References
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Scientific and Technological Development, Brazil (research grant INF.0b013e31817cf76f.

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 Effect of PCV10 on Pneumonia among Children, Brazil

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RESEARCH

Occult Hepatitis B Virus Infection in

Chacma Baboons, South Africa
Caroline Dickens, Michael C. Kew, Robert H. Purcell, and Anna Kramvis

During previous studies of susceptibility to hepatitis B zees (10); Barbary macaques (11); and tree shrews (12), in
virus (HBV) infection, HBV DNA was detected in 2/6 wild- addition to humans.
caught baboons. In the present study, HBV DNA was am- Baboons (Papio species) have been proposed as a pos-
plified from 15/69 wild-caught baboons. All animals were sible animal model of HBV infection. Phylogenetically,
negative for HBV surface antigen and antibody against HBV baboons are close to humans, showing ≈96% homology at
core antigen. Liver tissue from 1 baboon was immunohisto-
the DNA level, and they have an immune system similar
chemically negative for HBV surface antigen but positive for
HBV core antigen. The complete HBV genome of an isolate
to that of humans (13). Early studies involving injection
from this liver clustered with subgenotype A2. Reverse tran- of baboons with HBV-positive serum failed to detect any
scription PCR of liver RNA amplified virus precore and sur- clinical or biochemical signs of infection in these primates,
face protein genes, indicating replication of virus in baboon and initial serologic surveys failed to detect HBV surface
liver tissue. Four experimentally naive baboons were inject- antigen (HBsAg) in serum, leading to the conclusion that
ed with serum from HBV DNA–positive baboons. These 4 baboons were not susceptible to HBV infection (14). This
baboons showed transient seroconversion, and HBV DNA supposed lack of susceptibility of baboons to infection with
was amplified from serum at various times after infection. HBV, and the fact that unlike chimpanzees, baboons are
The presence of HBV DNA at relatively low levels and in the not an endangered species, intimated that baboons were
absence of serologic markers in the baboon, a nonhuman good candidates for sources of liver for xenotransplants.
primate, indicates an occult infection.
The use of xenotransplants from pigs and nonhuman pri-
mates to humans was considered to overcome the donor

H epatitis B virus (HBV) is a 3.2-kb partially double-

stranded virus belonging to the family Hepadnaviri-
dae. The outcome of infection with this virus is determined
shortage and to bridge patients with terminal hepatic failure
until a human donor organ became available (15).
To confirm that baboons were not susceptible to HBV
mainly by the immune response of the host and can be infection, Kedda et al. injected 6 wild-caught Chacma ba-
acute, chronic, or occult. HBV is divided into 9 genotypes boons (Papio ursinus orientalis) with pooled HBV-positive
(A–I); an additional genotype, J, has also been proposed serum and analyzed the baboons for 52 weeks by using sen-
(1–3). Several genotypes are further divided into subgeno- sitive molecular techniques to detect evidence of transmis-
types. In sub-Saharan Africa, subgenotypes A1 and D3 and sion (16). HBV DNA was detected by nested PCR in serum
genotype E circulate (4). and liver of 4 of the baboons <52 weeks after injection.
Hepadnaviruses can infect avian and mammalian hosts Liver function and histologic results were within reference
but have a limited host range, infecting only their natural ranges, and HBsAg was not detected in serum (16). How-
hosts and a few closely related species. Naturally occurring ever, during that study, HBV DNA was detected by using
infections have been found in several Old and New World nested PCR in serum of 2 of the 6 baboons at baseline,
nonhuman primates, such as chimpanzees (5), gorillas before injection with HBV. The presence of HBV DNA
(6), gibbons (7), orangutans (8) and woolly monkeys (9). was confirmed by retesting samples in independent labo-
HBV, whose natural host is humans, also infects chimpan- ratories. This finding raised the possibility that baboons
were naturally infected with a hepadnavirus. The aims of
Author affiliations: University of the Witwatersrand, Johannesburg, the present study were to determine the prevalence of HBV
South Africa (C. Dickens, M.C. Kew, A. Kramvis); Groote Schuur in wild baboons, molecularly characterize the virus isolated
Hospital, Cape Town, South Africa (M.C. Kew); and National Insti- from these baboons, determine whether the virus replicates
tutes of Health, Bethesda, Maryland, USA (R.H. Purcell) in the baboon liver, and demonstrate viral transmission to
DOI: http://dx.doi.org/10.3201/eid1904.121107 experimentally naive baboons.

598 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Hepatitis B Virus Infection in Chacma Baboons

Methods Southern Hybridization Slot Blot

Serum samples obtained from 49 adult and 20 juve- Baboon serum samples were blotted onto a nylon mem-
nile Chacma baboons caught in the Western Cape, Eastern brane by using a slot-blot manifold according to the protocol
Cape, and Limpopo Provinces of South Africa were stored at described by Zaaiger et al., which has a detection limit of 2.5
−70°C. Liver tissue (fresh frozen and formalin-fixed) was ob- × 107 HBV genomes/mL (17). HBV DNA was detected by
tained from 1 of the adult wild-caught baboons (hereinafter Southern blot hybridization with a 32P-labeled HBV DNA
referred to as baboon 9732), which was euthanized for medi- probe (HBV DNA in pBV325 vector, adr subtype).
cal and ethical reasons. Permission for this study was ob-
tained from the Animal Ethics Committee of the University DNA Extraction and Amplification and
of the Witwatersrand. All procedures were approved by this Phylogenetic Analysis
Committee. Baboons were provided care according to the DNA was extracted from 200 μL of baboon serum by
guidelines of the South African Medical Research Council. using the QIAamp DNA Blood Mini Kit (QIAGEN, Hilden,
Germany) according to the manufacturer’s instructions.
Serologic and Immunohistochemical Analysis DNA was also extracted from baboon liver tissue by us-
Baboon serum samples were tested for HBsAg and for ing a phenol-chloroform extraction method (18). Extracted
antibodies against HBV core antigen (anti-HBc) by using DNA was quantified by using spectrophotometric analysis
commercially available assays (Abbott Laboratories, Ab- with a NanoDrop ND-1000 Spectrophotometer (NanoDrop
bott Park, IL, USA). Alanine and aspartate aminotransfer- Technologies Inc., Wilmington, DE, USA).
ase levels were measured by using a 747 Automatic Ana- Subgenomic nested PCR amplifications of the precore/
lyzer (Hitachi, Tokyo, Japan). core (nt 1732–2045 and nt 1765–1968), core (nt 1687–
Formalin-fixed liver tissue from baboon 9732 was 2498 and nt 2267–2436), polymerase (nt 2540–2896 and
used for histologic and immunohistologic preparations. nt 2566–2858), and surface (nt 255–759 and nt 459–710)
Liver tissue was embedded in paraffin and sectioned. The regions were used to confirm the presence of HBV DNA in
sections were stained with hematoxylin and eosin for his- the serum extracts by using 40 cycles (16). The sensitivity
tologic examination or with immunoperoxidase and poly- of these amplifications is 40–400 HBV genomes/mL (19).
clonal antibodies to HBsAg and HBV core antigen (HB- The complete viral genome was amplified by using 8
cAg) to detect HBsAg and HBcAg in hepatocytes. overlapping subgenomic fragments (Figure 1). Thermocycling

Figure 1. Amplification of the hepatitis B virus (HBV) genome by using overlapping subgenomic fragments. Shown are 8 overlapping
subgenomic fragments amplified by nested PCR, These fragments were used to generate the complete HBV sequence isolated from
liver tissue of Chacma baboon 9732, South Africa. Dashed gray boxes indicate first-round PCRs, and white boxes indicate second-round
PCRs. Values along the 2-headed arrow at the top are in basepairs from the EcoRI site of the circular genome of HBV. Small arrows within
boxes indicate the direction of amplification.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 599
RESEARCH

conditions and sequences for primers 58, 409, 730, 1101, 1450, (cccDNA) intact. The cccDNA was detected by real-time
1860, 2440, and 2853 were obtained from Hu et al. (20). Ad- PCR (23) with the Power SYBR Green PCR Master Mix
ditional primers used were 1575 (5′-CCGGCAGATGAGA- (Applied Biosystems).
AGGCACAGACGG-3′), 1528 (5′-ACCTCTCTTTACGCG-
GTCTC-3′), 1552 (5′-TCTGTGCCTTCTCATCTGCC-3′), Transmission of HBV to Experimentally
1800 (5′-AGACCAATTTATGCCTACAGCCTCCTA-3′), Naive Baboons
1803 (5′-CGCAGACCAATTTATGCCTAC–3′), 1898 Transmission of HBV to experimentally naive ba-
(5′-GGCATGGACATTGACCCGTA-3′), 1921 (5′-TT- boons was performed at the National Institutes of Health
TATACGGGTCAATGTC-3′), 2800 (5′-CAGGTAGC- (Bethesda, MD, USA). Animals were housed and main-
GCCTCATTTTGTGGGTCACCATATTCT-3′), and 2898 tained at Bioqual, Inc. (Rockville, MD, USA). Housing
(5′-GAGGATTGGGAACAGAAAGATT-3′). All nucleotide and care of animals complied with all relevant guidelines
numbering refers to the position from the EcoRI site as posi- and requirements, and the animals were housed in facili-
tion 1. ties that are fully accredited by the Association for As-
Amplicons were sequenced directly by using the Big- sessment and Accreditation of Laboratory Animal Care
Dye Terminator version 3.1 Cycle Sequencing Ready Re- International. All protocols were reviewed and approved
action Kit (Applied Biosystems., Foster City, CA, USA) by the Institutional Animal Care and Use Committees of
and sequenced on an ABI3130xl Genetic Analyzer with 16 the National Institute of Allergy and Infectious Diseases
capillaries (Applied Biosystems) and the same primers that of the National Institutes of Health and Bioqual, Inc. Ex-
were used for amplification. The sequence has been depos- perimentally naïve, domestically raised baboons were ob-
ited in GenBank under accession no. JX507080. The HBV tained from a domestic breeder (Mannheimer Foundation,
genomic sequence obtained from the baboon was compared Homestead, FL, USA). Before inclusion of animals in
with corresponding sequences of HBV from GenBank as the study, serum was free of all markers of HBV repli-
described (4). cation when tested serologically and by nested PCR as
described above. A liver biopsy to detect HBV DNA was
RNA Extraction, Reverse Transcription, not performed.
and Amplification Four experimentally naive baboons were each inocu-
RNA was extracted from varying amounts of baboon lated with 500 μL of serum obtained from HBV DNA–pos-
liver tissue by using the guanidinium-acid-phenol method itive wild-caught baboons from South Africa. Each baboon
(21) and digested with RNase-free DNase I (Fermentas, was injected with serum from a single wild-caught baboon.
Waltham, MA, USA) to remove any contaminating DNA. After injection, serum was obtained from each of the 4
The RNA concentration was determined by spectropho- newly injected baboons at weekly intervals. Serum was
tometry with the NanoDrop ND-1000 Spectrophotometer. used to measure levels of alanine aminotransferase, iso-
cDNA was generated by using SuperScript III reverse tran- citrate dehydrogenase, γ-glutamyltranspeptidase, HBsAg,
scriptase (Invitrogen, Carlsbad, CA, USA) and oligo(dT)18 HBV e antigen, antibodies against HBV e antigen, antibod-
primers (Invitrogen) in accordance with the manufacturer’s ies against HBsAg, and anti-HBc.
instructions. Non–reverse transcribed negative controls DNA extracted from serum samples was used for nest-
were prepared in an identical manner except that diethy- ed PCR amplification of a region of the surface gene (nt
lopyrocarbonate–treated water (Invitrogen) was added to 459–710). Twenty-five weeks after injection, the study was
each reaction instead of reverse transcriptase. The suc- terminated and the baboons were euthanized. At necropsy,
cess of the reverse transcription reaction was confirmed liver tissue and serum was obtained from each baboon for
by PCR amplification of a portion of the glyceraldehyde- further testing to confirm that virus extracted from experi-
3-phosphate dehydrogenase gene (22). Subgenomic nested mentally naive baboons was the same as that found in the
PCR amplifications of the precore/core (nt 1732–2045 and original baboons.
nt 1765–1968) and surface (nt 255–759 and nt 459–710)
regions were also performed. Results

Detection of Covalently Closed Circular DNA Prevalence of HBV in Wild-caught Baboons

DNA was extracted from baboon liver tissue by us- in South Africa
ing the QIAamp DNA Mini Kit (QIAGEN). DNA extracts The prevalence of HBV in the baboon (P. ursinus ori-
were treated with Plasmid-Safe ATP-Dependent DNase entalis) population in South Africa was determined by ex-
(Epicenter Biotechnologies, Madison, WI, USA) to se- tracting DNA from serum of 69 wild-caught baboons and
lectively hydrolyze linear double-stranded chromosomal amplifying 4 regions of the viral genome (the precore/core,
DNA while leaving HBV covalently closed circular DNA core, polymerase, and surface regions) by nested PCR.

600 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Hepatitis B Virus Infection in Chacma Baboons

HBV DNA could not be amplified with a single-round because of the small volumes of serum available, liver tis-
PCR, indicating that HBV DNA was present at low levels sue obtained from baboon 9732 was used to further char-
in baboon serum. acterize HBV in baboons. The complete HBV genome was
Using the criterion of >3 of the 4 regions being PCR amplified by nested PCR of 8 overlapping subgenomic
positive, we found that 11 (22.4%) of 49 adult and 4 fragments (Figure 1).
(20.0%) of 20 juvenile wild-caught baboons were positive Phylogenetic analyses of the complete genome
for HBV. The overall prevalence of hepadnaviral DNA in showed that HBV from the baboon was closely related
baboons was 21.7% (15/69). Furthermore, the presence and to HBV subgenotype A2 (Figure 3). This finding was
specificity of the HBV DNA was confirmed when detected confirmed by comparison of mean ± SD nucleotide di-
directly in the serum of 5 of the 69 baboons by using South- vergence calculations compared to subgenotype A2 (1.00
ern blot analysis. Only 5 of the 15 PCR-positive serum ± 0.55) and to subgenotype A1 (4.52 ± 0.42). Similar re-
samples were positive by Southern hybridization because sults were obtained for each of the 4 open reading frames.
of the relatively lower sensitivity of this method. The baboon HBV had mutations in the basic core pro-
moter and precore regions not found in subgenotype A2.
Serologic, Liver Function, and Immunohistologic Tests These mutations included the G1809T/C1812T double
Serum samples from 4 of the 15 HBV DNA–positive mutation in the Kozak sequence preceding the precore
baboons, for which additional serum was available, were protein start codon and G1888A in the precore region.
tested and found to be negative for HBsAg and anti-HBc. G1809T/C1812T mutations are characteristic of sub-
Alanine and aspartate aminotransferase levels were within genotype A1, and G1888A is unique to subgenotype A1
reference ranges, and after high-speed centrifugation and (4,24). Translation of the 4 open reading frames showed
treatment with antibody against HBsAg, no viral particles them to be well conserved relative to the consensus se-
were observed in the serum by electron microscopy. His- quence of subgenotype A2 with the following exceptions:
tologic examination of liver tissue from 1 of these baboons T380C resulting in an rtV84A in conserved region A of
(9732) showed mild focal lobular hepatitis but no evidence the polymerase and a C76R in HBsAg; A2019G resulting
of interface hepatitis, bridging necrosis, dysplasia of he- in an E40G in the core protein; and C1470T resulting in
patocytes, cirrhosis, or hepatocellular carcinoma (Figure a P33G in the X protein and T1765C resulting in a P145S
2, panel A). Immunohistochemical staining of liver tissue in the X protein.
showed HBcAg in nuclei of some hepatocytes with a patchy
distribution (Figure 2, panel B). HBsAg and 42-nm envel- Expression of HBV RNA in Baboon Liver Tissue
oped (Dane) particles were not detected in the cytoplasm. RNA extracted from liver tissue of baboon 9732
was reverse transcribed and amplified in precore/core
Amplification, Sequencing, and Phylogenetic Analysis (nt 1765–1968) and surface (nt 459–710) open reading
of HBV Genome from Baboon Liver frames (Figure 4). Sequences of these amplicons were
The low viral loads indicated that the complete HBV identical to sequences of the DNA isolated from liver of
genome could only be amplified subgenomically and baboon 9732.

Figure 2. Liver tissue from Chacma baboon 9732, South Africa, showing lobular hepatitis. Liver tissue was obtained at necropsy, fixed
in formalin, embedded in paraffin, and sectioned. A) Hematoxylin and eosin staining, showing a focus of mild lobular hepatitis but no
evidence of interface hepatitis or bridging necrosis. Portal tracts are normal. B) Immunoperoxidase staining with polyclonal antibody
against hepatitis B core antigen. Core antigen was detected in the occasional hepatocyte nucleus. Arrows indicate selected positive nuclei.
Original magnifications ×400.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 601
RESEARCH

HBV DNA–positive baboons. Serum samples from the

experimentally naive baboons were HBV DNA negative
before injection, and baseline levels of aspartate amino-
transferase, alanine aminotransferase, and IgG were also
determined. The baboons were transiently positive for sev-
eral of these markers and were intermittently positive for
HBV DNA in serum. Results for a representative sample
(baboon 2) infected with serum from baboon 9732 are
shown in Figure 5. DNA extracted from liver tissue ob-
tained at necropsy from baboon 2 was used to amplify and
sequence a portion of the surface gene (nt 409–1101). The
sequence of virus extracted from liver tissue of baboon 2
six months after injection was identical to the HBV se-
quence of this region found in original baboon 9732.

Discussion
Detection of HBV DNA in serum samples of 2 Chacma
baboons before injection with human HBV (16) suggested
that baboons might be chronically infected with HBV. Our
objective was to determine the prevalence of HBV in wild-
caught Chacma baboons and to characterize the virus iso-
lated from these animals. The overall prevalence (21.7%)
of HBV in the baboons is similar to the HBV prevalence in
other nonhuman primates in areas to which HBV is highly
endemic, including sub-Saharan Africa (25).
The baboons were serologically negative, and the 1
baboon examined histologically had mild focal lobular hepa-
Figure 3. Dendogram of the complete hepatitis B virus (HBV)
genome isolated from Chacma baboon 9732, South Africa. Samples
representative of all 8 HBV genotypes and primate hepadnaviruses
are included. Samples are numbered according to their GenBank
accession numbers, followed by their country of origin. The
sample from baboon 9732 (boldface) clusters strongly with the
subgenotype A2 isolates (bootstrap value 100). Letters along the
right indicate genotypes. Values along branches are bootstrap
values. Scale bar indicates nucleotide substitutions per site.
Figure 4. Nested PCR amplification of a subgenomic region of cDNA
for baboon hepatitis B virus, South Africa. Reverse transcribed,
Identification of cccDNA in Baboon Liver Tissue DNase I–treated cDNA products were amplified by PCR, and
cccDNA was detected in liver tissue of baboon 9732 amplicons were resolved by electrophoresis on a 1% agarose gel
by using real-time PCR. DNA from HBV DNA–positive containing ethidium bromide. Non–reverse transcribed samples
(in which diethyl pyrocarbonate [DEPC]–treated water was added
tumorous and nontumorous human liver and plasmid DNA
instead of enzyme during reverse transcription) were included as
containing a greater than full-length HBV genome were negative controls. Top panel: nested PCR 1: 255F–759R; PCR
used as positive controls, and DNA from rat liver tissue 2: 459F–710R. Bottom panel, 550-bp region of glyceraldehyde-
was used as a negative control. HBV cccDNA–positive 3-phosphate dehydrogenase gene amplified to assess quality of
controls had melting temperatures of 82.7°C–84.0°C, and mRNA. Lanes 1 and 8, 100 mg of baboon liver tissue used for RNA
extraction; lanes 2 and 9, RNA extraction negative control; lanes
negative controls had melting temperatures <80.0°C. Sam-
3 and 10, 200 mg of baboon liver tissue used for RNA extraction;
ples from the baboon liver tissue showed similar melting lanes 4 and 11, RNA extraction negative control; lane 5, DNase I
temperatures as positive controls (82.7°C–83.1°C) indicat- treatment negative control in which DEPC-treated water was added
ing that HBV cccDNA was present. instead of RNA; lane 6, reverse transcription negative control in
which DEPC-treated water was added instead of cDNA; lanes 7
and 12, double-round nested PCR negative control containing
Transmission of HBV from Wild-caught Baboons to
best-quality water instead of cDNA; lane 13, single-round PCR
Experimentally Naive Baboons negative control containing best-quality water instead of cDNA;
Each of 4 experimentally naive, domestically raised lane 14, PCR-positive control containing DNA extracted from liver
baboons was injected with serum from 1 of 4 wild-caught tissue of baboon 9732 as template.

602 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Hepatitis B Virus Infection in Chacma Baboons

titis, and HBcAg was detected in liver tissue by immunohis-

tochemical staining. Detection of low levels of HBV DNA in
baboon liver tissue and in serum in the absence of HBsAg and
at relatively low levels of HBV DNA classifies this infection
as occult (26). Lack of any serologic markers of HBV infec-
tion further distinguishes this infection as a seronegative oc-
cult HBV infection (26). This finding is analogous to a sec-
ondary occult infection in the woodchuck (27). Furthermore,
HBV DNA was detected in juvenile and adult baboons, sug-
gesting lifelong persistence of the virus, which was success-
fully transmitted to experimentally naive baboons. Detection
of HBV DNA alone does not necessarily correspond to an
HBV infection (28). Thus, detection of cccDNA and viral
RNA in liver tissue of baboon 9732 showed that HBV was
replicating. The low levels of viral nucleic acids detected in
baboon liver are a further characteristic of occult infections
(27). This study demonstrates a naturally occurring occult
HBV infection in a nonhuman primate.
Phylogenetic analysis of the baboon HBV genome
showed that it belonged to genotype A, clustering with
subgenotype A2. This finding is an unexpected result be-
cause subgenotype A1 predominates in South Africa (24).
However, in the basal core promoter/precore region, the
baboon HBV had distinct characteristics of subgenotype
A1. Four additional mutations in the polymerase, surface,
X, and core regions of the baboon HBV strain differenti-
ated the baboon isolate from most subgenotype A2 iso-
lates. These mutations are not known to cause any major
functional or conformational changes.
Paradoxical identification of subgenotype A2 in the
baboon when subgenotype A1 predominates in Africa
might indicate that subgenotype A2 is an older strain that
previously circulated in Africa, which has been replaced by
other strains, including subgenotype A1 and genotypes D
and E (29). An analogous trend might have occurred in the
Mediterranean region where genotype D now predominates
over genotype A (30). Similarly, a change in the prevalent Figure 5. Levels of alanine aminotransferase (ALT) and hepatitis
HBV genotype in central and western Africa has been pos- B virus (HBV) serologic markers and detection of HBV DNA in
tulated to have occurred over the past 200 years, and geno- baboon 2. Serum obtained from baboon 2 (which was injected
with serum from baboon 9732), and ALT and HBV serologic
type E, originally restricted to the west coast of Africa, now
marker levels were measured at weekly intervals after injection.
spreads over a large crescent stretching from The Gambia, Serum for week 0 levels was obtained just before injection.
through Nigeria and the Democratic Republic of the Congo OD:CO indicates optical density:cutoff value ratios. An OD:CO
into Namibia and Mozambique (31). There is a paucity of >1 indicates a positive result. HBV DNA was detected by nested
sequencing data for subgenotype A2 from Africa. Only PCR amplification (255–761 in the first round and 459–710 in the
second round) of a 252-bp region of the virus surface gene by using
4 complete subgenotype A2 genomes from South Africa
DNA extracted from serum obtained from the infected baboon at
have been deposited in GenBank. Shorter subgenomic se- weekly intervals. HBsAg, HBV surface antigen; Anti-HBs, antibody
quences of subgenotype A2 from South Africa (32), Tuni- against HBV surface antigen; Anti-HBc, antibody against HBV core
sia (33), and Kenya (34) have also been deposited. More antigen; HBeAg, HBV e antigen; Anti-HBe, antibody against HBV
extensive molecular epidemiologic studies in Africa might e antigen. + indicates that the sample amplified successfully at this
time point, and – indicates no amplification of virus DNA. The week
uncover a higher circulation of subgenotype A2 in more
26 time point indicates the result of amplification by using DNA
remote regions. extracted from serum at necropsy, and the final time point indicates
Another explanation for the paradoxical finding of successful amplification of the virus product from DNA extracted
subgenotype A2 in the baboon could be that in Africa, as from liver tissue obtained from baboon 2 at necropsy.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 603
RESEARCH

shown in India, subgenotype A2 isolates are confined to M.C.K. was supported by a grant (NRF2K421) from the Na-
peripheral blood leukocytes (PBLs). In India, subgenotype tional Research Foundation, South Africa. A.K. was supported by
A1 and genotype D circulate, but actively replicating HBV a grant (00/52) from the Polio Research Foundation, South Africa.
subgenotype A2 isolates have been detected in PBLs (35). C.D. was supported by bursaries from the National Research Foun-
These subgenotype A2 isolates were confined to PBLs and dation and the Polio Research Foundation. R.H.P. was supported
were not detected in serum. Differential immune pressures by the Intramural Research Program of the National Institute of
are believed to compartmentalize HBV to different parts of Allergy and Infectious Diseases, National Institutes of Health.
the body in which different strains evolve independently
Dr Dickens is a researcher in the Department of Internal
(35). Further study will be needed to determine whether
Medicine, University of the Witwatersrand, Johannesburg, South
subgenotype A2 is compartmentalized to PBLs in Africa.
Africa. Her main research interests include molecular virology
In sub-Saharan Africa, most human HBV carriers are
and cancer.
negative for HBV e antigen, and in these persons, spread
of HBV is mainly horizontal (36). In baboons, their natural
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pnas.97.4.1661 Molecular genetic diversity of hepatitis B virus in Kenya. Intervirol-
21. Chomczynski P, Sacchi N. Single-step method of RNA isolation ogy. 2008;51:417–21. http://dx.doi.org/10.1159/000205526
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Anal Biochem. 1987;162:156–9. http://dx.doi.org/10.1016/0003- hapatra PK, et al. Genetic characterization of hepatitis B virus in
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occult hepatitis B virus infection. J Hepatol. 2008a;49:652–7. http:// June 12, 2003 [cited 2010 Feb 19]. http://news.nationalgeographic.
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Research Programme, Department of Internal Medicine, University
28. Hollinger FB, Sood G. Occult hepatitis B virus infection: a covert
operation. J Viral Hepat. 2010;17:1–15. http://dx.doi.org/10.1111/ of the Witwatersrand Medical School, 7 York Rd, Parktown, 2193,
j.1365-2893.2009.01245.x Johannesburg, South Africa; email: anna.kramvis@wits.ac.za

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RESEARCH

Risk Factors for Influenza among

Health Care Workers during 2009
Pandemic, Toronto, Ontario, Canada
Stefan P. Kuster, Brenda L. Coleman, Janet Raboud, Shelly McNeil, Gaston De Serres,
Jonathan Gubbay, Todd Hatchette, Kevin C. Katz, Mark Loeb, Donald Low, Tony Mazzulli,
Andrew Simor, and Allison J. McGeer, on behalf of the Working Adult Influenza Cohort Study Group1

Medscape, LLC is pleased to provide online continuing medical education (CME) for this journal article, allowing clinicians
the opportunity to earn CME credit.
This activity has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation
Council for Continuing Medical Education through the joint sponsorship of Medscape, LLC and Emerging Infectious Diseases.
Medscape, LLC is accredited by the ACCME to provide continuing medical education for physicians.
Medscape, LLC designates this Journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit(s)TM.
Physicians should claim only the credit commensurate with the extent of their participation in the activity.
All other clinicians completing this activity will be issued a certificate of participation. To participate in this journal CME
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Release date: March 18, 2013; Expiration date: March 18, 2014

Learning Objectives
Upon completion of this activity, participants will be able to:

• Define an influenza pandemic in this study

• Report signs and symptoms of influenza
• Determine whether the risk of influenza was higher in health care workers (HCWs) than in non-HCWs
• Report risk factors for influenza among HCWs

CME Editor
Jean Michaels Jones, Technical Writer/Editor, Emerging Infectious Diseases. Disclosure: Jean Michaels Jones has disclosed no
relevant financial relationships.

CME Author
Hien Nghiem, MD, freelance writer, Medscape, LLC. Disclosure: Hien Nghiem, MD, has disclosed no relevant financial
relationships.

Authors
Disclosures: Stefan Kuster, MD; Brenda Coleman, PhD; Janet Raboud, PhD; Shelly McNeil, MD; Jonathan Gubbay, MBBS;
Kevin Katz, MD; Mark Loeb, MD; Donald Low, MD; Andrew Simor, MD; and Allison McGeer, MD, have disclosed no relevant
financial relationships. Gaston De Serres, MD, PhD, has disclosed the following relevant financial relationships: served as an
advisor for GlaxoSmithKline; received grants for clinical research from GlaxoSmithKline and Sanofi Pasteur. Todd Hatchette, MD,
has disclosed the following relevant financial relationships: received grants for clinical research from GlaxoSmithKline. Tony
Mazzulli, MD, has disclosed the following relevant financial relationships: served as a speaker for Merck.

Author affiliations: Mount Sinai Hospital, Toronto, Ontario, Canada This prospective cohort study, performed during the
(S.P. Kuster, B.L. Coleman, D. Low, T. Mazzulli, A.J. McGeer); 2009 influenza A(H1N1) pandemic, was aimed to determine
University of Toronto, Toronto (B.L. Coleman, J. Raboud, K.C. Katz, whether adults working in acute care hospitals were at
D. Low, T. Mazzulli, A. Simor, A.J. McGeer); Dalhousie University, higher risk than other working adults for influenza and to
Halifax, Nova Scotia, Canada (S. McNeil, T. Hatchette); Laval assess risk factors for influenza among health care workers
University Québec, Québec City, Québec, Canada (G. De Serres); (HCWs). We assessed the risk for influenza among 563
Ontario Agency for Health Protection and Promotion, Toronto (J. HCWs and 169 non-HCWs using PCR to test nasal swab
Gubbay, D. Low); North York General Hospital, Toronto (K.C. Katz); samples collected during acute respiratory illness; results
McMaster University, Hamilton, Ontario Canada (M. Loeb); and for 13 (2.2%) HCWs and 7 (4.1%) non-HCWs were positive
Sunnybrook Health Sciences Centre, Toronto (A. Simor).
Additional members of the Working Adult Influenza Cohort Study
1

DOI: http://dx.doi.org//10.3201/eid1904.111812 Group are listed at the end of this article.

606 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Risk Factors for Influenza among Health Care Workers

for influenza. Influenza infection was associated with the recruitment of these control participants are included
contact with family members who had acute respiratory in the online Technical Appendix (wwwnc.cdc.gov/EID/
illnesses (adjusted odds ratio [AOR]: 6.9, 95% CI 2.2–21.8); article/19/4/11-1812-Techapp.pdf). The study was approved
performing aerosol-generating medical procedures (AOR by the Research Ethics Boards of all participating hospitals
2.0, 95% CI 1.1–3.5); and low self-reported adherence to and universities and by the human resources departments of
hand hygiene recommendations (AOR 0.9, 95% CI 0.7–
participating employers.
1.0). Contact with persons with acute respiratory illness,
rather than workplace, was associated with influenza
Upon enrollment, participants received a collection
infection. Adherence to infection control recommendations kit, an illustrated guide, and instruction from a nurse for
may prevent influenza among HCWs. mid-turbinate nasal swab sample self-collection. They also
completed a Web-based questionnaire detailing influenza
vaccination history, underlying medical conditions,

T he numerous outbreaks of influenza described in acute

care hospitals indicate that influenza transmission in
this setting is of major concern (1–3). Nonetheless, it re-
demographic data, potential work- or school-related
risk factors for respiratory virus infection, and potential
community risk factors. Blood samples were taken from
mains unclear whether health care workers (HCWs) are at consenting participants at enrollment and again in April or
higher risk for infection than are adults working in non- May of 2010.
clinical settings (non-HCWs). Vaccination recommenda- Participants were asked to complete weekly Web-
tions for HCWs are intended primarily to protect patients based diaries from enrollment until March 31, 2010,
from hospital-acquired influenza and influenza-associated detailing respiratory symptoms and acute respiratory
death (4,5). Although working in hospitals has been pro- illness (ARI) or febrile illnesses and documenting time-
posed as a risk factor for influenza (6), findings that support dependent risk factors (e.g., contact with persons with
that working in health care settings poses an occupational ARI symptoms). Per the study protocol, if any signs or
risk (7), or that performing particular activities or working symptoms suggestive of an ARI developed, participants
in specific health care disciplines are associated with an in- provided a self-collected mid-turbinate nasal swab sample
creased risk for influenza infection, are sparse. as soon as possible after onset to be tested for influenza by
Better understanding of risk factors for infection among using PCR. ARI was defined as 1) fever without another
HCWs would support decision-making regarding priorities obvious source; or 2) new symptoms, including >2 of the
for seasonal influenza vaccination, antiviral treatment or following: runny or stuffy nose, sneezing, sore or scratchy
prophylaxis programs, implementation of other measures throat, hoarseness, or cough; or 3) one local (runny/
to reduce influenza transmission in hospitals, and planning stuffy nose, sneezing, sore/scratchy throat, hoarseness, or
for pandemics. Therefore, we aimed to assess risk factors cough) and 1 systemic symptom (fever, malaise, myalgia,
for influenza among HCWs and to determine whether, headache, or fatigue).
during the first 2 waves of influenza A(H1N1)pdm09, Participants whose specimens tested positive for
HCWs working in acute care hospitals were at higher risk influenza were offered treatment in accordance with
than non-HCWs for symptomatic influenza. public health recommendations (8). All participants with
undetermined A(H1N1)pdm09 vaccine status as of March
Materials and Methods 31, 2010, were contacted again to confirm whether they
had received it and, if so, when. For logistical reasons,
Participants and Setting participants with unconfirmed 2009–2010 seasonal
The Influenza Cohort Study, initiated by the Working influenza vaccine status could not be contacted again;
Adult Influenza Cohort Study Group, a research team based instead, these participants were assumed not to have
in Toronto, Ontario, Canada, was started in May 2009. The received it. In Canada, vaccine for A(H1N1)pdm09
purpose of the study was to examine incidence, clinical became available for HCWs and patients at high risk
features, and epidemiology of infection caused by A(H1N1) for complications of influenza during calendar week 43
pdm09 among HCWs and other working adults in Canada. (starting October 25, 2009) and was available for healthy
For this analysis, participants were enrolled during May 29– adults during calendar week 47 (starting November 22).
September 27, 2009. Participants were eligible if they were
18–75 years of age and either worked >8 hours per week Definitions
in 1 of 5 acute care hospitals (HCW) or in an office-based For this study, HCW were defined as persons working
setting in Toronto (non-HCW). Non-HCWs were intended in an acute care hospital. A non-HCW was defined as
to provide a sample of working adults at low occupational a person working in an office-type environment not
risk for influenza, so as to bias the study toward the ability associated with the provision of health care. The first and
to identify an occupational risk in health care. Details of second waves of the influenza pandemic in Ontario were

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 607
RESEARCH

Table 1. Characteristics of study participants in the Influenza Cohort Study performed during 2009 pandemic, Toronto, Ontario,
Canada*
Participant values by cohort, n = 732
No. HCWs in acute care No. non-HCWs in office
Characteristic facilities, n = 563 settings, n = 169 p value
Mean age, y ( SD) 42.2 (11.3) 45.4 (10.8) 0.001
Female sex 478/563 (84.9) 133/169 (78.7) 0.06
Recipient of vaccine
A(H1N1)pdm09 vaccine† 469/554 (84.7) 66/165 (40.0) <0.001
Seasonal influenza vaccine 2009–10 226/563 (40.1) 41/169 (24.3) <0.001
Seasonal influenza vaccine 2008–09 407/552 (73.7) 76/164 (46.3) <0.001
Underlying health conditions
Asthma 55/555 (9.9) 14/167 (8.4) 0.56
Diabetes mellitus 22/555 (4.0) 5/165 (3.0) 0.58
Allergies to airborne irritants 235/509 (46.2) 60/155 (38.7) 0.10
Current smoker or smoker in household 73/551 (13.3) 27/163 (16.6) 0.28
Potential exposure conditions
Hand-to-face habits 275/557 (49.4) 76/168 (45.2) 0.35
Wearing of prescription eyeglasses 386/561 (68.8) 119/168 (70.8) 0.62
Reusable water bottle use 1/week 232/556 (41.7) 81/169 (47.9) 0.15
Public transit: 8 trips per week 196/558 (35.1) 45/168 (26.8) 0.04
Group gathering attendance 524/563 (93.1) 150/167 (89.8) 0.17
Face-to-face contacts/d, median (IQR) 10 (5, 20) 10 (5, 20) 0.94
>1 person/bedroom in household 192/551 (34.9) 51/166 (30.7) 0.33
Children in workplace 62/558 (11.1) 9/167 (5.4) 0.03
Child <5 y in household 77/563 (13.7) 10/169 (5.9) 0.006
Child <18 y in household 212/563 (37.7) 59/169 (34.9) 0.52
Child in household attends day care 77/555 (13.9) 16/164 (9.8) 0.17
*Data are no./total (%) unless otherwise specified. HCW, health care worker; A(H1N1)pdm09, pandemic influenza A(H1N1) 2009 virus; IQR, interquartile
range.
†Participants who had acquired A(H1N1)pdm09 <7 d after vaccination were considered unprotected.

defined as the periods for which the weekly proportion facial protection were performed according to infection
of respiratory specimens that were positive for A(H1N1) control recommendations (9). Symptomatic influenza
pdm09 was >5%, as reported by the Ontario Agency for infection was defined as influenza-positive PCR results for
Health Protection and Promotion. Similarly, seasonal a participant-collected mid-turbinate nasal swab sample.
influenza waves were defined as periods for which >5%
of weekly specimens tested positive for seasonal influenza. Antibody Assays and Interpretation
By this definition, the first pandemic wave occurred during Serum specimens were extracted from blood samples
calendar weeks 21–31 of 2009 (May 17–August 8); the and 1 mL aliquots frozen at -70°C. Aliquots were tested
second wave occurred during calendar weeks 39–48 by hemagglutination-inhibition (HAI) assay to determine
(September 27–December 5). Peak weeks were defined antibody titers against the A(H1N1)pdm09 strain (A/
as weeks during which positivity rates were >15% and California/07/2009-like) and the 2008–09 seasonal
comprised calendar weeks 21–27 (May 17–July 11) during A(H1N1) strain (A/Brisbane/59/07) to identify potential
wave 1 and calendar weeks 41–46 (October 11–November cross-reactivity by using a protocol adapted from World
21) during wave 2. As expected, few cases of seasonal Health Organization methods (10). Two HAI assays were
influenza were identified during the study period. performed per aliquot by using 0.5% turkey erythrocytes
Aerosol-generating medical procedures were defined and 4 hemagglutination units per 25 µL of virus. For
as any of the following: administration of nebulized therapy discordant pairs, the higher of the 2 geometric mean titers
or humidified oxygen at >40%, use of bag-valve mask, was used. Serum specimens were tested at the Queen
manual ventilation, noninvasive ventilation, open airway Elizabeth II Health Sciences Centre, Halifax, Nova Scotia,
suctioning, bronchoscopy or other upper airway endoscopy, Canada. Seroprotection was defined as having HAI antibody
tracheostomy, endotracheal intubation, cardiopulmonary titers of >40. Seroconversion was specifically defined as a
resuscitation, oscillatory ventilation, or any procedure prevaccination HAI titer of <10 and a postvaccination titer
that involved manipulation of open ventilator tubing in of >40 or a 4-fold change in titers for participants with a
a mechanically ventilated patient or sputum induction or prevaccination titer of >10 (11,12).
other deliberate induction of coughing.
Adherence to hand hygiene and facial protection Data Management and Statistical Analyses
recommendations was defined as the self-reported Data were entered online by the participants, then
proportion of situations during which hand hygiene and cleaned and manually inspected for errors and outlying

608 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Risk Factors for Influenza among Health Care Workers

Figure. Flowchart of 732 persons

enrolled in the Influenza Cohort
Study, Toronto, Ontario, Canada.

values. Differences in group proportions were assessed by infection after receipt of A(H1N1)pdm09 vaccine >7 days
the c2 or Fisher exact test, as appropriate, and differences before symptom onset. Participants who acquired A(H1N1)
in means (for normally distributed data, on the basis of the pdm09 within 7 days after vaccination were considered not
Shapiro-Wilk test for normality) and medians (for non- fully protected. To evaluate the validity of this assumption,
normally distributed data) were calculated by using Student we performed sensitivity analyses by calculating lags of 0
t test and Wilcoxon rank-sum test, respectively. days and 14 days, respectively. The same criteria were used
The analysis for the primary objective (i.e., to in the analysis of the secondary objective (i.e., to determine
determine whether the risk for laboratory-confirmed risk factors for laboratory-confirmed symptomatic
symptomatic influenza was higher in HCWs than in non- influenza among HCWs). The models with the lowest
HCWs) included all participants who were enrolled by quasi-likelihood under the independence model criterion
the start of the second wave of the 2009 H1N1 influenza were preferred.
pandemic (calendar week 39, starting September 27, 2009). Data were analyzed in SAS, version 9.1 for PC (SAS
Multivariable generalized estimating equation logistic Institute, Cary, NC, USA). We considered p values <0.05
regression analysis was used to determine adjusted odds as statistically significant.
ratios with 2-sided 95% CIs for constant and time-dependent
risk factors for symptomatic influenza infection on the basis Sample Size
of information from baseline questionnaires and weekly This study was initiated at the onset of the 2009 influenza
diaries. Model construction was performed on the basis of pandemic; because the expected incidence of infection was
the method proposed by Harrell (13) including A(H1N1) unknown, a formal sample size was not established. Details
pdm09 vaccination status and changing risk for influenza of the sample size estimate for the planned seasonal study
infection over time (community influenza activity). Our a can be found in the online Technical Appendix.
priori approaches to adjust for changing risk for influenza
infection over time were to 1) adjust for weekly percentage Results
of specimens positive for influenza reported to the Ontario
Agency for Health Protection and Promotion (continuous Study Population, Symptomatic Influenza
variable) and 2) adjust for peak weeks (defined as weeks Case-patients and Community Influenza Activity
during which >15% of specimens were positive for The first participant was enrolled in the study on May
influenza; [dichotomous variable]). Vaccine failure among 28, 2009 (calendar week 21). By October 11 (calendar week
participants was defined as acquiring A(H1N1)pdm09 41), at the start of the second wave of the pandemic, 732

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 609
RESEARCH

Table 2. Results of influenza virus testing of nasal swab specimens from 732 study participants in the Influenza Cohort Study
performed during 2009 pandemic, Toronto, Ontario, Canada*
Influenza test status
Characteristic No. negative or not ill, n = 712† No. positive, n = 20‡ p value
Mean age, y ( SD) 42.9 (11.3) 43.9 (9.3) 0.69
Female sex 595/712 (83.6) 16/20 (80.0) 0.76
Worker in acute care hospital 550/712 (77.3) 13/20 (65.0) 0.28
Recipient of vaccine
A(H1N1)pdm09 vaccine§ 533/699 (76.3) 2/20 (10.0) <0.001
Seasonal influenza vaccine 2009–10 258/712 (36.2) 9/20 (45.0) 0.42
Seasonal influenza vaccine 2008–09 470/696 (67.5) 13/20 (65.0) 0.81
Underlying health conditions
Asthma 67/702 (9.5) 2/20 (10.0) 1.00
Diabetes mellitus 26/700 (3.7) 1/20 (5.0) 0.54
Allergy to airborne irritants 284/646 (44.0) 11/18 (61.1) 0.15
Current smoker 63/704 (9.0) 3/20 (15.0) 0.42
Potential exposure conditions
Hand-to-face habits¶ 337/705 (47.8) 14/20 (70.0) 0.05
Wearing of prescription eyeglasses 493/709 (69.5) 12/20 (60.0) 0.36
Reusable water bottle use >1/ wk 307/705 (43.6) 6/20 (30.0) 0.23
Public transit 8 trips per week 236/706 (33.4) 5/20 (25.0) 0.43
Group gathering attendance >1 655/710 (92.3) 19/20 (95.0) 1.00
Face-to-face contacts/d, median (IQR) 10 (5, 20) 15 (8, 20) 0.53
Household crowding index >1# 233/697 (33.4) 10/20 (50.0) 0.12
Children in workplace 68/705 (9.7) 3/20 (15.0) 0.43
Child <5 y in household 83/712 (11.7) 4/20 (20.0) 0.28
Child <18 y in household 258/712 (36.2) 13/20 (65.0) 0.009
Child in household attends day care 87/699 (12.5) 6/20 (30.0) 0.03
*Data are no./total (%) unless otherwise specified. A(H1N1)pdm09, pandemic influenza A(H1N1) 2009 virus; IQR, interquartile range.
†Participants who did not report any illness or whose nasal swab samples tested negative for influenza.
‡All participants who tested positive were symptomatic.
§Participants who had acquired A(H1N1)pdm09 <7 d after vaccination were considered unprotected
¶Defined as biting one’s nails or cuticles or habitually putting one’s fingers in his or her mouth or nose.
#Household crowding index is defined as number of persons per household divided by the number of bedrooms.

participants were enrolled in the Influenza Cohort Study: of calendar weeks 42, 44, and 45; and 7 cases during
563 (76.9%) were HCWs who worked in 1 of 5 community calendar week 43. Thus, 16 of 19 cases occurred during
and teaching acute care hospitals in the Toronto area and weeks of peak A(H1N1)pdm09 activity. Seasonal influenza
169 (23.1%) were non-HCWs who worked in an office A(H3N2) virus was isolated in a sample from 1 participant
environment not associated with the provision of health care during calendar week 43.
(Table 1; Figure). Of the 2 cohorts, HCWs were younger
and were more likely to have been vaccinated against Risk Factors for Symptomatic Influenza Infection
seasonal and pandemic influenza, to work with children, The probability of symptomatic influenza infection
to have children <5 years of age in their households, and to did not differ between HCWs and non-HCWs (p = 0.28)
use public transportation >8 times per week. Of 422 HCWs (Table 2). Study participants who had a child <18 years of
who were vaccinated against A(H1N1)pdm09, 403 (95.5%) age living in the household (36.2% of influenza negative/
received vaccine within 2 weeks after its availability; of 61 untested participants vs. 65.0% of influenza positive
non-HCWs, 28 (45.9%) were vaccinated during the same participants; p = 0.009), a child who attended day care
time period (p<0.001). living in the same household (12.5% vs. 30.0%; p = 0.03),
A total of 334 (45.6%) study participants submitted and who were not vaccinated against A(H1N1)pdm09 >7
436 nasal swab samples. More than half (52.1%) of these days before onset of infection (76.3% vs. 10.0%; p<0.001)
samples were collected on the day of symptom onset (day were more likely to have respiratory illness with positive
1), 19.4% on day 2, 9.9% on day 3, and 12.1% on or after test results for influenza.
day 4. Among the 20 (4.6%) specimens yielding influenza, After adjusting for A(H1N1)pdm09 vaccination
12 (60.0%) were collected on day 1, four (20.0%) on history and community influenza activity, we found no
day 2, three (15.0%) on day 3, and one (5.0%) on day 4 difference in the risk for influenza infection between
of illness. Thirteen (2.2%) of 563 HCWs and 7 (4.1%) of persons working in an acute care hospital (HCWs) and
169 non-HCWs submitted samples that tested positive for other healthy adults (non-HCWs) (Table 3). Rather,
influenza. A(H1N1)pdm09 was detected in 19 (95%) of contact with a family member with an ARI in the previous
the 20 positive participants: 1 case during each of calendar week was the main risk factor for symptomatic influenza
weeks 24, 25, 31, 39, 40, and 47; two cases during each infection, irrespective of the method of adjusting for

610 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Risk Factors for Influenza among Health Care Workers

changing risk over time. In general, quasi-likelihood HCWs were: contact with a family member with ARI in
under the independence model criterion statistics were the previous week, performing or assisting with aerosol-
lower in models adjusting for weekly percentage of generating medical procedures, and lower adherence to
specimens yielding influenza than in those adjusting for hand hygiene recommendations (Table 5).
weeks of peak influenza activity (results not shown). A
sensitivity analysis calculating lags of 0 and 14 days (vs. 7 HAI Antibody Assays
days) from the time of receipt of A(H1N1)pdm09 vaccine Among the combined study population, 450 (61.5%)
did not alter these results. of 732 participants provided pre- and post-influenza season
Analyses restricted to HCWs and including potential blood samples. Among those, 3.6% had protective HAI
occupational risk factors in health care are shown in titers against A(H1N1)pdm09 at baseline. There was no
Table 4. During the study period, 49.6% of HCWs association with workplace and baseline HAI titers. Of
worked in emergency departments, medical inpatient the 142 (31.6%) participants who tested positive after
wards, intensive care units, or pediatric wards; 12.9% enrollment, 137 (96.5%) had received the A(H1N1)pdm09
were present during >1 and 9.4% performed >1 aerosol- vaccine, 2 (1.4%) submitted a nasal swab that tested positive
generating medical procedure per week. Approximately by PCR, and 3 (2.2%) did not submit a swab for testing or
one quarter (26.5%) of HCWs reported providing direct report an ARI (consistent with asymptomatic infection).
care for >1 patient per week who had ARI. The analysis Analysis of data collected during the period after
of risk factors for infection indicates that, similar to the vaccine became available for unvaccinated participants
combined study population, HCWs with symptomatic without known previous A(H1N1)pdm09 infection showed
influenza infection confirmed by positive nasal swab that 8 (16.3%) of 49 HCW and 3 (5.3%) of 57 non-HCWs
sample were more likely to have children <18 years of seroconverted or had a positive mid-turbinate nasal swab
age in their households (69.2% of HCWs who tested sample. Although persons working in an acute care hospital
positive vs. 36.9% who tested negative or were untested; were 3.1× as likely as other working adults to be infected
p = 0.02) and less likely to have been vaccinated against with influenza, the results were not significant in this small
A(H1N1)pdm09 >7 days before onset of infection unvaccinated group (95% CI 0.9–11.1). Influenza among
(15.4% vs. 86.3%; p<0.001) (Table 4). Compared with unvaccinated participants was not associated with age, sex,
other HCWs, those with symptomatic influenza infection or any of the other characteristics listed in Table 1.
were more likely to be present during aerosol-generating
medical procedures >1× per week (38.5% vs. 12.7%; p Discussion
= 0.02) and reported lower adherence to hand hygiene In this prospective cohort study conducted in Canada
recommendations (77.5% vs. 95%; p = 0.02). After during the 2009 influenza A(H1N1) pandemic, we found
adjustment for changing risks for influenza infection no association between working in an acute care hospital
over time, risk factors for influenza infection among and risk for influenza infection. Our findings are similar

Table 3. Risk factors for symptomatic influenza infection among health care workers in acute care hospitals and non–health care
workers in office settings during 2009 pandemic, Toronto, Ontario, Canada*
Risk factor OR (95% CI), adjusted† OR (95% CI), multivariable‡
Worker in acute care hospital 0.49 (0.19–1.27) 0.47 (0.17–1.32)
Age, y, per 10 y increase 1.08 (0.76–1.54) NA
Female sex 1.03 (0.30–3.56) NA
Recipient of A(H1N1)pdm09 vaccine§ 0.28 (0.03–2.28)¶ 0.34 (0.04–2.85)
Weekly percentage of specimens yielding influenza per 5% increase 1.49 (1.28–1.73)# 1.36 (1.13–1.63)
Potential exposure conditions
Hand-to-face habits** 3.09 (1.12–8.52) NA
Child <18 y in household 3.13 (1.21–8.07) NA
Contact with family member with ARI in prior wk 5.51 (1.81–16.76) 6.89 (2.17–21.84)
Contact with co-worker with ARI in prior wk 0.77 (0.10–6.16) NA
Household crowding index >1†† 1.99 (0.79–5.05) NA
Public transit >8 trips per wk 0.62 (0.22–1.76) NA
*Constant and time-dependent risk factors for symptomatic influenza infection (positive nasal swab specimen) in 732 primary contacts of the Influenza
Cohort Study followed during June 2009–April 2010, Toronto, Ontario, Canada. OR, odds ratio; NA, not applicable; A(H1N1)pdm09: pandemic influenza
A(H1N1) 2009 virus; ARI: acute respiratory illness.
†Adjusted for receipt of A(H1N1)pdm09 vaccine and weekly percentage of specimens yielding influenza.
‡Multivariable model including all variables with ORs listed below.
§Participants who had acquired A(H1N1)pdm09 <7 d after vaccination were considered unprotected.
¶Adjusted for weekly percentage of specimens yielding influenza only.
#Unadjusted.
**Defined as biting one’s nails or cuticles, habitually putting one’s fingers in his or her mouth or nose.
††Household crowding index is defined as number of persons per household divided by the number of bedrooms.

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RESEARCH

to those of Williams et al., who assessed serologically adherence to hand hygiene, which may have an effect
confirmed influenza during the 2007–08 influenza season similar to appropriate glove use, was protective. Although
in Berlin, Germany (14). They found no association collinearity of both putative risk and protective factors
between HCW status and influenza but demonstrated that may continue to make it difficult to accurately identify risk
the presence of children in the household and ownership factors for acquisition of influenza in health care settings,
of a car among participants with no children in the our data highlight the role of hand hygiene in the control of
household were risk factors, whereas receipt of seasonal influenza infection (18), and of protective equipment use
influenza vaccine was found to be protective. Similarly, by persons who perform or assist with aerosol-generating
Marshall et al. found no overall difference in influenza medical procedures.
infection rates between hospital workers who did and The mode of transmission of influenza remains a
did not have patient contact during the 2009 pandemic in matter of ongoing debate. Although most experts believe
Australia, but the authors identified exposure to children that droplet and aerosol transmission are the most
as a risk for influenza (15). common modes of spread of influenza, our finding and
The results of this cohort study also add insight into that of Marshall et al. (15), as well as the evidence from
occupational risk factors for influenza among persons who the elementary school–based study by Talaat et al. that
work in acute care hospitals. In contrast to a finding by increasing hand hygiene adherence reduces the risk for
Kawana et al. (16), neither our study nor those of Marshall infection with influenza, suggest that transmission by direct
et al. and Seto et al. detected an increased risk for influenza or indirect contact contributes substantially to influenza
among workers who had direct patient care responsibilities transmission (18). Appropriate hand hygiene practice
(17). However, Marshall et al. indicated that working in should continue to be recommended to prevent influenza
an intensive care unit of a hospital was a risk factor for transmission.
influenza, and wearing gloves while caring for patients who Pandemic influenza vaccine became available in
were on droplet precaution was protective. These findings Canada at the peak of the second wave of the pandemic.
are similar to ours in that exposure to aerosol-generating This complicated our analysis in that the risk for
medical procedures, which are most often performed in influenza infection depended on differing times of receipt
intensive care units, was a risk factor for influenza, and of influenza vaccine and on timing of the pandemic

Table 4. Characteristics of 563 health care workers in acute care hospitals during 2009 pandemic, Toronto, Ontario, Canada*
Influenza test status
Characteristic No. negative or not ill, n = 550† No. positive, n = 13‡ p value
Mean age, y ( SD) 42.2 (11.4) 42.5 (10.1) 0.91
Female sex 467/550 (84.9) 11/13 (84.6) 1.00
Occupation
Nurse 180/539 (33.4) 3/13 (23.1) 0.55
Physician, physiotherapist, respiratory therapist 103/539 (19.1) 5/13 (38.5) 0.15
Other§ 256/539 (47.5) 5/13 (38.5) 0.52
Potential exposure conditions
Received A(H1N1)pdm09 vaccine¶ 467/541 (86.3) 2/13 (15.4) <0.001
Child <18 y in household 203/550 (36.9) 9/13 (69.2) 0.02
Child attending day care in household 74/542 (13.7) 3/13 (23.1) 0.40
Cares for >1 patient with ARI per week 141/539 (26.2) 5/12 (41.7) 0.32
Working in high-risk area# 227/461 (49.2) 7/11 (63.6) 0.35
Present during aerosol-generating medical procedure >1/wk** 66/521 (12.7) 5/13 (38.5) 0.02
Performs aerosol-generating medical procedure >1/wk 49/540 (9.1) 3/13 (23.1) 0.11
Years’ experience, mean ( SD) 13.6 (11.4) 14.0 (9.3) 0.91
% adherence to hand hygiene, median (IQR) 95.0 (80.0–100) 77.5 (60.0–92.5) 0.02
Adherence to facial protection, %, median (IQR) 80 (50–99) 50 (30–75) 0.16
Hours worked per week, no. median (IQR) 40.0 (37.5–45.0) 37.5 (32.0–40.0) 0.22
*Data are no./total (%) unless otherwise specified. A(H1N1)pdm09, pandemic influenza A(H1N1) 2009 virus; ARI, acute respiratory illness; IQR,
interquartile range.
†Participants who either did not report any illness or whose nasal swab samples tested negative for influenza.
‡All participants who tested positive were symptomatic.
§The distribution of other persons working in acute care hospitals was: administrative personnel: 30.4%; patient attendant/health care aide/service
assistant: 0.4%; housekeeper/porter/central sterile supply/dispatch: 0.5%; medical imaging technologist/technician: 1.6%; pharmacist/pharmacy
technician: 2.0%; ward clerk/unit coordinator: 1.4%; psychologist/social worker: 1.6%; laboratory technologist/technician: 4.7%; nutritionist/other food
service staff: 1.1%; other: 3.4%.
¶Participants who had acquired A(H1N1)pdm09 <7 d of vaccination were considered unprotected.
#Emergency room, medical inpatient ward, intensive care unit, or pediatric ward.
**Aerosol-generating medical procedures are defined as any one of: administration of nebulized therapy or humidified oxygen at >40%, use of bag-valve
mask, manual ventilation, non-invasive ventilation, open airway suctioning, bronchoscopy or other upper airway endoscopy, tracheostomy, endotracheal
intubation, cardiopulmonary resuscitation, oscillatory ventilation, any procedure performed that involves manipulation of open ventilator tubing in a
mechanically ventilated patient, sputum induction or other deliberate induction of coughing.

612 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Risk Factors for Influenza among Health Care Workers

Table 5. Risk factors for symptomatic influenza infection in health care workers in acute care hospitals during 2009 pandemic, Toronto,
Ontario, Canada*
OR (95% CI), OR (95% CI),
Risk factor adjusted† multivariable‡
Age, y, per 10 y increase 0.99 (0.63–1.56) NA
Female sex 1.79 (0.23–14.04) NA
Potential exposure conditions
Receipt of A(H1N1)pdm09 vaccine§ 0.49 (0.07–3.67)¶ 0.40 (0.04–3.99)
Weekly specimens yielding influenza, %, per 5% increase 1.56 (1.29–1.88) # 1.43 (1.17–1.73)
Child <18 y in household 3.33 (1.00–11.05) NA
Contact with family member with ARI in prior week 7.26 (2.15–24.54) 7.86 (2.20–28.04)
Contact with co-worker with ARI in prior week 1.40 (0.16–12.40) NA
Cared for patient with ARI in prior week 1.50 (0.44–5.14) NA
Adherence to hand hygiene recommendations, per 10% increase 0.84 (0.73–0.98) 0.86 (0.74–0.99)
Adherence to facial protection recommendations, per 10% increase 0.92 (0.79–1.07) NA
No. AGMP performed or assisted during previous week, per 10 procedures increase** 2.29 (1.26–4.17) 1.95 (1.10–3.48)
*Generalized estimating equation logistic regression analysis of constant and time-varying risk factors for influenza infection in 563 health care workers in
acute care hospitals, Influenza Cohort Study, followed during June 2009–April 2010, OR, odds ratio; A(H1N1)pdm09: pandemic influenza A(H1N1) 2009
virus; NA, not applicable; ARI: acute respiratory illness; AGMP: aerosol-generating medical procedures.
†Adjusted for receipt of A(H1N1)pdm09 vaccine and weekly percentage of specimens yielding influenza.
‡Multivariable model including all variables with ORs listed below.
§Participants who had acquired A(H1N1)pdm09 <7 d after vaccination were considered unprotected.
¶Adjusted for weekly percentage of specimens yielding influenza only.
#Undadjusted.
**AGMP are defined as any one of the following: administration of nebulized therapy or humidified oxygen at >40%, use of bag-valve mask, manual
ventilation, noninvasive ventilation, open airway suctioning, bronchoscopy or other upper airway endoscopy, tracheostomy, endotracheal intubation,
cardiopulmonary resuscitation, oscillatory ventilation, any procedure performed that involves manipulation of open ventilator tubing in a mechanically
ventilated patient, sputum induction or other deliberate induction of coughing; OR for being in the same room during AGMP (>1/week) 6.63 (95% CI 2.05–
21.41); OR for participants performing AGMP (>1/week) 4.21 (1.12–15.76).

waves. We addressed these issues by using multivariable signs or symptoms of influenza infection (e.g., influenza-
generalized estimating equation logistic regression for like illness) might differ between HCWs and non-HCWs,
the analysis, which facilitated adjustment for timing of but differences might have remained. Although the self-
receipt of vaccine, and we accounted for the dynamics of collection of swab specimens occurred over 1–4 days
the pandemic waves by incorporating weekly percentages after illness onset, it is unlikely that any cases would
of laboratory specimens that tested positive for influenza have been missed because previous studies have shown
virus. We believe that our results are robust because 2 that A(H1N1)pdm09 remains readily detectable within
different approaches to adjust for changing risk over time this period (19–21). The study encompasses a selective
led to the same results. Nevertheless, whether the relative sample of persons working in a limited number of acute
percentage of positive specimens reflects the relative care hospitals and other working adults with Internet
number of influenza cases in the community remains a access in a single geographic area during the 2009
matter of debate. influenza A(H1N1) pandemic. Although we deliberately
Our study has several limitations. It has a lack selected controls likely to be at low risk for occupational
of power related to the small number of cases of exposure to influenza (e.g., not working in an occupation
symptomatic influenza during the second wave of the exposed to numerous children) in an effort not to miss an
pandemic in this population of working adults. We effect of the health care work environment, unmeasured
attempted to minimize selection bias by using broad biases in our control selection could have been present. In
inclusion and limited exclusion criteria; nevertheless, addition, our results may not be generalizable to seasonal
the possibility of having access to rapid diagnosis and influenza or to geopolitical areas where infection control
treatment during the second pandemic wave might have practices in hospitals are different.
resulted in biased enrollment of participants who had a The yield of self- or parent-collected nasal swab
higher self-perceived risk for influenza infection, and specimens has been shown to be comparable to health
perception of risk might differ between persons working care provider–collected nasopharyngeal aspirates from
in acute care hospitals and persons working in nonclinical children and adults (22–24), but whether the yield of
settings. Similarly, generalizability may be hampered self-collected nasal swabs differs between HCWs and
because participants in studies of influenza could differ non-HCWs has not been assessed. There is evidence that
from others in their attitudes toward vaccine acceptance microneutralization of antibody assays may demonstrate
and infection prevention practices. We tried to reduce the a greater sensitivity than HAI (25); as a result, we may
possibility of measurement bias in nasal swab collection have missed seroconversion by using the latter. Further
by having a broad interpretation of respiratory illness seroconversions might have been missed by the delay
because the interpretation of more detailed criteria for between the first (upon enrollment) and the second

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 613
RESEARCH

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bmj.322.7279.138

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 615
RESEARCH

Deaths Associated with Influenza

Pandemic of 1918–19, Japan
Siddharth Chandra

Current estimates of deaths from the influenza pan- Although the epidemiologic approach used by Richard et
demic of 1918–19 in Japan are based on vital records and al., which also uses death statistics reported by the Japanese
range from 257,000 to 481,000. The resulting crude death health authorities, raises the estimate to 481,000 (or 0.88%
rate range of 0.47%–0.88% is considerably lower than paral- of the population at the time) (7), even this estimate is ex-
lel and conservative worldwide estimates of 1.66%–2.77%. traordinarily lower than estimates from other parts of Asia.
Because the accuracy of vital registration records for early
As Taeuber argued in her classic book, The Population
20th century Asia is questionable, to calculate the percent-
age of the population who died from the pandemic, we used
of Japan, Japan occupies a special place in demography (8).
alternative prefecture-level population count data for Japan Worldwide it remains one of the largest economies (third
in combination with estimation methods for panel data that in 2011) and one of the most populous countries (tenth in
were not available to earlier demographers. Our population 2011). Yet, surprisingly, substantial knowledge gaps re-
loss estimates of 1.97–2.02 million are appreciably higher main with regard to the influenza pandemic of 1918–19 in
than the standing estimates, and they yield a crude rate of Japan, rendering it “a strangely neglected episode in mod-
population loss of 3.62%–3.71%. This rate resolves a major ern Japanese history” (4, p. 389). For example, a search of
puzzle about the pandemic by indicating that the experience Taeuber’s work for the term “influenza” revealed only 1
of Japan was similar to that of other parts of Asia. mention of the influenza epidemic of 1918, in the context
of speculation that it “may have led to reduced concep-

T he influenza pandemic of 1918–19 caused unprec- tions” (8, p. 233).

edented devastation (1); worldwide, it is estimated to The few scholars who have studied the influenza pan-
have taken 25–100 million lives (2,3), exceeding the com- demic in Japan have approached it from 1 of 3 broad per-
bined death toll of both world wars. One of the strangest spectives: historical, epidemiologic, or demographic. The
aspects of the currently held wisdom about the pandemic is historical approach is exemplified by the works of Palmer
the curiously low death rate attributed to Japan compared and Rice, which provide a qualitative contextualization
with other countries in Asia. Official records for Japan put of aspects of the pandemic and its management in Japan
the death toll at 257,363 persons (4), resulting in a crude in- (4,5,9,10). A second line of research is epidemiologic,
fluenza-attributable death rate of 0.47%. Patterson and Pyle within which 2 broad goals are pursued. The first goal is
(2) reported 350,000 deaths, and Johnson and Mueller (3) to produce estimates of major epidemiologic characteris-
cited a figure from Palmer and Rice (5) of 388,000 deaths. tics of the virus (11–13), and the second goal is to produce
Given Japan’s population of >54 million at the time (6), epidemiology-based estimates of mortality rates from the
the influenza-attributable mortality rates (0.64%–0.71%) pandemic (7). The demographic approach is exemplified
are remarkably low by Asian standards, although they are by Morita, Okazaki, Taeuber, and Yasukawa and Hiroo-
similar to the rates calculated for the United States, Canada, ka (8,14–16). Although these studies emphasize broader
and western Europe (0.65%, 0.61%, and ≈0.48%, respec- patterns of population growth in Japan, a few address the
tively) (3). Patterson and Pyle’s (2) conservative estimate question of death rates during the pandemic. For example,
of a global rate of 1.66% and Johnson and Mueller’s (3) Yasukawa and Hirooka (16) relied directly on official death
substantial upward revision of that percentage to 2.77% statistics, including those from the pandemic, to produce
suggest that the estimates for Japan, which are less than estimates of the population in early 20th-century Japan.
one quarter of the latter estimate, merit closer scrutiny. Unfortunately, the quantitative literature seems to have
more or less accepted the official vital statistics on disease-
Author affiliation: Michigan State University, East Lansing, specific deaths, feeding them (and therefore their inaccura-
Michigan, USA cies) into otherwise technically refined estimates of popula-
DOI: http://dx.doi.org/10.3201/eid1904.120103 tion and population growth.

616 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Deaths and Influenza Pandemic 1918–19, Japan

A common characteristic of the above studies is their The number of persons who physically resided in different
heavy reliance on official vital and health statistics of the parts of Japan (de facto A-type population) was computed
time. Such data are widely recognized by demographers by adjusting the numbers of persons with honseki status
as being plagued by the often-severe problem of under- downward to account for those who had honseki status but
reporting. Indeed, according to Johnson and Mueller, “it lived in other locations. These numbers were further ad-
is generally accepted that recorded statistics of influenza justed to account for the discrepancy between numbers of
morbidity and mortality are likely to be a significant un- registered persons who immigrated into the various prefec-
derstatement” (3, p. 108). For India, Davis estimated that tures, which always exceeded the numbers of persons who
the “amount of underregistration certainly exceeds 30 per migrated out (de facto B-type population ([27]). Over time,
cent at all times, and is probably nearer 50 per cent” (17, p. through a process of learning by doing (habituation), the
34). For Indonesia, Gooszen advised that such data “should registration data became reasonably accurate (8).
be regarded with a good deal of caution” (18, p. 32), and For this study, we used data from the quinquennial (ev-
Nitisastro opined that “for the system of registering deaths, ery 5 years) summations of 1898 and after. Given the de
the quality of the results was poor” (19, p. 101). Japan is no facto nature of the censuses of 1920 and after, we used the
exception to this pattern. According to Mosk, “we do not B-type population statistics for comparable data for 1918
have a trustworthy picture of what happened to vital rates and before (6). Because the population figures are based on
in the Tokugawa period…. The same can be said for the repeated summations of records that were repeatedly up-
Meiji period” (20, p. 658), and Taeuber’s assessment was dated, the count for a household was periodically revised
that “the critical question is the accuracy of the records of upward or downward, and hitherto unreported births and
vital events” (8, p. 50). The uncharacteristically low esti- deaths would have been more accurately captured by these
mates of deaths from influenza in Japan provide a strong revisions, even if they had not been reported in the annual
rationale for cross-checking the findings in the manner of vital registration records. Taeuber (8) provides evidence
Davis’ classic study of India (17). as follows: “Early publications of the Bureau of Statistics
We therefore used recently developed statistical meth- included a warning statement that the majority of the ad-
ods to estimate the loss of population in Japan from the ditions to the registers were the survivors of unrecorded
influenza pandemic of 1918–19. We adopted an approach births of earlier years,” and “Failures to report deaths dur-
that intentionally avoids heavy reliance on vital registration ing the earlier years are evident in the accumulations of the
data and is based instead on population count data for Japan aged in the successive reports.” This phenomenon forms
of that period. By applying data for multiple prefectures the basis for our reasoning that the quinquennial population
over time to prefecture-level population statistics, we esti- count data are more accurate than the annual vital registra-
mated population loss from the pandemic to be the differ- tion records.
ence between expected population (using the prepandemic The second data-associated issue is the change in the
trajectory) and observed population (using the postpan- regime for population enumeration that began in 1920,
demic trajectory) (17,21,22). The new estimates are appre- when Japan conducted its first census of its de facto popula-
ciably higher than the earlier estimates, bringing Japan’s tion. This census is widely regarded as having been accurate
pandemic experience in line with that of other parts of Asia and yielded a population count of 55.96 million (6). After
and resolving a major puzzle in the epidemiology of the conducting the 1920 census, Japan conducted quinquennial
1918–19 pandemic. censuses until the beginning of World War II. The problem
with the timing of the change in the system is that quinquen-
Methods nial registration count totals are available up to 1918, and
the quinquennial census counts started in 1920. Therefore,
Data-associated Issues the break point in the system of population enumeration ap-
With regard to data, 3 issues should be considered. proximately coincides with the break point (1918) for which
The first is the coverage of the population count data for population loss is to be estimated. Any statistical estima-
Japan in the late 19th and early 20th centuries, described tion of change across such a break point must satisfactorily
by Matsuda (23) and Taeuber (8). In 1871, the Imperial address discontinuity in the data collection system. Fortu-
Japanese government passed a law, the koseki-ho, which nately, earlier demographers and statisticians went to great
required registration of households and persons in Japan. lengths to splice the data across this break point, producing
A major emphasis of the registrations was legal domicile, similar estimates. Taeuber (8), for example, demonstrated
or honseki status. The first set of summations of these reg- that a backward projection of population from 1920 through
isters was made in 1898, after which they were computed 1898 produces a 1898 population estimate that is remark-
every 5 years until 1918, for a total of 5 nationwide popula- ably similar to a forward projection of population from 1871
tion counts derived directly from the registers (8,24–26). through 1898. The theme of splicing is also covered in the

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 617
RESEARCH

works of Morita, Okazaki, and Yasukawa and Hirooka (14– The third data-related issue is the unreliability of the
16). Ohbuchi (28) compared the estimates of these authors, data before 1898 (8,16); therefore, we used the 1898 popu-
all of which are within 2% of each other for 1915 and 1920, lation count as the starting point in our analysis. To main-
and concluded that the estimates of Yasukawa and Hirooka, tain balance of the dataset across the break point of 1918,
which are based on a reverse survival method, are the most we also limited our data to the censuses including and
reliable. Although the procedure used by Yasukawa and Hi- before 1935. The full dataset consists of observations for
rooka is generally robust, its adjustment for the influenza each of 47 prefectures for the population count for the years
pandemic fails because the official influenza death statistics 1898, 1903, 1908, 1913, and 1918, and the census data for
of ≈178,000 for 1918–1920 were taken at face value and 1920, 1925, 1930, and 1935, for a total of 423 observations.
incorporated into estimates of life expectancy at birth (16).
Therefore, inaccuracies in the official vital statistics of the Data Analyses
period flow directly into the estimates of Yasukawa and Hi- Although for decades scholars have been intrigued by
rooka. When selecting the data for the analysis, therefore, the subject of low mortality rates from the pandemic in
we started with the observations of Yasukawa and Hirooka Japan, the currently circulating estimates were produced
(16), who stated that by 1900, the most widely used popula- before the development and mainstreaming of panel data
tion estimates of demographers (14–16) tend to converge estimation methods. The studies described above based
and are close to the official population statistics. Next, be- population estimates on annual or quinquennial observa-
cause the official statistics are the only ones that contain tions for all of Japan and used datasets that were small
published data at the prefectural level (6) (Morita, Okazaki, in terms of numbers of observations. Given the existence
and Yasukawa and Hirooka [14–16] focus on producing of a panel of prefectural data on population for 47 pre-
Japan-wide data), we used the official statistics pertaining fectures and multiple time points straddling the pandemic
to the quinquennial population count (1918 and before) and years, more recently developed panel data methods can
census years (1920 and after). be used to estimate a standard population growth process

Table 1. Population growth models and population loss estimates for Japan, 1903–1930 data*
Model
Estimate 1 2 3 4 5 6 7 8
Includes Kanto earthquake Yes Yes Yes Yes No No No No
prefectures†
Includes Hokkaido outlier Yes Yes No No Yes Yes No No
Includes 1918 population Yes No Yes No Yes No Yes No
count data
Intercept, 00 13.6120‡ 13.5957‡ 13.6197‡ 13.6040‡ 13.5799‡ 13.5623‡ 13.5876‡ 13.5706‡
0.0524 0.0530 0.0530 0.0535 0.0534 0.0537 0.0541 0.0543
Time trend, 10 0.0103‡ 0.011‡ 0.0095‡ 0.0105‡ 0.0098‡ 0.0110‡ 0.0089‡ 0.0100‡
0.0012 0.0012 0.0009 0.0008 0.0012 0.0012 0.0009 0.0008
Flu dummy, 20 –0.0344‡ –0.0477‡ –0.0364‡ –0.0492‡ –0.0374‡ –0.0518‡ –0.0397‡ –0.0536‡
0.0055 0.0070 0.0052 0.0069 0.0055 0.0067 0.0051 0.0067
Flu dummy  time trend, 30 0.0006 –0.0004 0.0013§ 0.0003 0.0002 –0.0010 0.0009 –0.0002
0.0009 0.0010 0.0006 0.0007 0.0009 0.0010 0.0006 0.0006
No. observations 329 282 322 276 301 258 294 252
Hausman test statistic <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
p>0.9999 p>0.9999 p>0.9999 p>0.9999 p>0.9999 p>0.9999 p>0.9999 p>0.9999
Breusch-Pagan test statistic 924.89 653.01 921.41 652.71 848.22 599.17 847.85 601.55
p<0.0001 p<0.0001 p<0.0001 p<0.0001 p<0.0001 p<0.0001 p<0.0001 p<0.0001
Population change from –1.38 –1.97 –1.50 –2.02 –1.48 –2.12 –1.61 –2.17
influenza, millions
Population change from –2.53 –3.62 –2.87 –3.87 –3.15 –4.51 –3.59 –4.85
influenza, %
Population change, 1918– –0.66 –1.21 –0.89 –1.38 –0.90 –1.49 –1.13 –1.65
19, millions
Population change, 1918– –1.20 –2.19 –1.69 –2.60 –1.89 –3.13 –2.51 –3.65
19, %
Annual population growth 1.03 1.13 0.95 1.05 0.98 1.10 0.89 1.00
rate to pandemic, %
Annual population growth 1.09 1.09 1.08 1.08 1.00 1.00 0.98 0.98
rate after pandemic, %
*Italics indicate SE of the coefficient.
†Chiba, Kanagawa, Shizuoka, Tokyo.
‡p<0.01.
§p<0.05.

618 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Deaths and Influenza Pandemic 1918–19, Japan

Table 2. Population growth models and population loss estimates for Japan, 1898–1935 data*
Model
Estimate 1 2 3 4 5 6 7 8
Includes Kanto earthquake Yes Yes Yes Yes No No No No
prefectures†
Includes Hokkaido outlier Yes Yes No No Yes Yes No No
Includes 1918 population Yes No Yes No Yes No Yes No
count data
Intercept, 00 13.6053‡ 13.5971‡ 13.6137‡ 13.6059‡ 13.5755‡ 13.5673‡ 13.5839‡ 13.5762‡
0.0523 0.0526 0.0528 0.0530 0.0537 0.0539 0.0543 0.0545
Time trend, 10 0.0106‡ 0.0113‡ 0.0097‡ 0.0104‡ 0.0100‡ 0.0107‡ 0.0091‡ 0.0097‡
0.0012 0.0012 0.0009 0.0008 0.0012 0.0012 0.0008 0.0008
Flu dummy, 20 –0.0355‡ –0.0464‡ –0.0373‡ –0.0476‡ –0.0379‡ –0.0486‡ –0.0399‡ –0.0501‡
0.0053 0.0060 0.0050 0.0060 0.0054 0.0062 0.0051 0.0062
Flu dummy  time trend, 30 0.0002 –0.0005 0.0009 0.0002 –0.0002 –0.0009 0.0005 –0.0001
0.0009 0.0009 0.0006 0.0006 0.0009 0.0010 0.0006 0.0006
No. observations 423 376 414 368 387 344 378 336
Hausman test statistic <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
p>0.9999 p>0.9999 p>0.9999 p>0.9999 p>0.9999 p>0.9999 p>0.9999 p>0.9999
Breusch-Pagan test statistic 1525.90 1171.30 1535.75 1183.05 1403.42 1078.01 1422.01 1097.53
p<0.0001 p<0.0001 p<0.0001 p<0.0001 <0.0001 p<0.0001 p<0.0001 p<0.0001
Population change from –1.49 –2.02 –1.59 –2.05 –1.52 –1.98 –1.62 –2.01
Influenza, millions
Population change from –2.72 –3.71 –3.03 –3.92 –3.23 –4.23 –3.62 –4.50
Influenza, %
Population change, 1918 to –0.75 –1.25 –0.96 –1.39 –0.93 –1.37 –1.14 –1.51
1919, millions
Population change, 1918 to –1.35 –2.26 –1.81 –2.63 –1.96 –2.88 –2.53 –3.34
1919, %
Annual population growth 1.06 1.13 0.97 1.04 1.00 1.07 0.91 0.97
rate to pandemic, %
Annual population growth 1.08 1.08 1.06 1.06 0.98 0.98 0.96 0.96
rate after pandemic, %
*Italics indicate SE of the coefficient.
†Chiba, Kanagawa, Shizuoka, Tokyo.
‡p<0.01.

that explicitly builds in a break point for the influenza pan- models without these 2 time points. Second, we estimated
demic (21,22). By treating these 47 prefectures of Japan models without the 4 prefectures that were most affect-
as individual units, each with its own set of observations, ed by the devastating Kanto earthquake of 1923: Chiba,
the panel data method leverages the large amount of ad- Kanagawa, Shizuoka, and Tokyo. Third, because the 1918
ditional information available at the prefectural level to population count was reported as of December of that year
generate a more robust picture of population change and (i.e., the year of the pandemic), thereby introducing the
the effect of the influenza pandemic on that process. This possibility of contamination in the growth rate estimate
method is also flexible enough, given the large sample for the prepandemic trajectory, we estimated models with-
size, to accommodate prefecture-specific variation. In out the 1918 data. Finally, given the atypical population
this manner, the method enables estimation of prefecture- dynamic in Hokkaido, a frontier region in the early 20th
specific growth processes, each with a prefecture-specific century to which a large and prolonged wave of migra-
estimate of population loss from the pandemic, while still tion was in progress, we estimated models without data for
leveraging the entire set of observations to create an ag- Hokkaido. These sensitivity exercises yielded a total of 16
gregate estimate for Japan. This method is implemented possible permutations of the model. Additional sensitiv-
by running a regression of the logarithm of population on ity analyses involved using the alternative A-type statistics
a linear time trend while allowing for a 1-time (downward) (6) and dropping the data for 1898 (i.e., using 1903 as the
shift in that time trend during 1918–19 to capture influen- earliest year) to account for the above-mentioned habitu-
za-attributable population loss. Details of this method are ation process.
provided in the online Technical Appendix (wwwnc.cdc.
gov/EID/article/19/4/12-0103-Techapp1.pdf). Results
To examine the robustness of the estimates, we con- Tables 1 and 2 contain the parameter estimates for
ducted a variety of sensitivity analyses. First, to control for the 16 models. Without exception, the models show the
the possible inaccuracy of the 1898 data and for the effects significant negative effect of the influenza pandemic on
of outliers in time (the 1898 and 1935 data), we estimated Japan’s population (via the flu dummy described in the

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 619
RESEARCH

Discussion
For nearly a century, Japan’s experience during the
influenza pandemic of 1918–19 has been viewed as an
anomaly within the broader Asian experience. In stark
contrast with significantly higher estimates for deaths in
Asia and globally, which themselves are often conserva-
tive, the standing mortality rates for Japan, based heav-
ily on vital registration data, are <1%. There is, however,
substantial reason to believe that vital registration data for
the early 20th century in the most densely populated parts
of Asia, including British India (17), the Dutch East In-
dies (19), and Japan (8,20), are inaccurate, suggesting the
need for verification of mortality rates by using the Davis
Figure. Effect of including 1918 data on estimated population method (17), which is based on population count or cen-
of Japan. Data cover 1903–1930 and include observations for sus data. The key result of this study is that when these al-
Hokkaido and the prefectures affected by the Kanto earthquake of ternative population counts and census data are used, the
1923 (Chiba, Kanagawa, Shizuoka, and Tokyo).
experience of Japan conforms more closely to that of the
rest of Asia; in Japan, rates of population loss approach
online Technical Appendix); the calculated population 4% and an actual loss of ≈2 million. These estimates are
loss ranged from 1.38 to 2.05 million persons. The range similar to those for India (17,22). This result has implica-
of estimated population growth rates across the models is tions for the large bodies of work on the epidemiology
0.89%–1.13% per year, which is in line with the summary of the influenza pandemic of 1918–19 and, more broadly,
of estimates presented by Ohbuchi (28). In all but 1 model, the demographic history of Japan. Even adjusting for the
there is no appreciable difference in the rates of popula- possibility that a brief decline in fertility partly explains
tion growth before and after the pandemic. As predicted, the population loss estimated in this study, the number of
the inclusion of the population count data for 1918, which deaths in Japan were in all probability much higher than
already reflect some but not all deaths from the pandemic, previously believed.
pulls the prepandemic population growth trajectory down The results of this study come from using an alter-
(Figure), yielding substantially lower estimates of death native data source rather than vital registration data. Al-
and population loss than corresponding models that did not though the alternative data source is vulnerable to any in-
include those data (Tables 1, 2). For this reason, the models accuracies inherent in the population counts and censuses
that exclude the 1918 data are preferred to the models that of Japan, it nevertheless provides a way to confirm or con-
include the 1918 data. tradict prior results that were based on vital registration
Table 3 demonstrates that the models that control for data in the manner of Davis (17) and Chandra et al. (22)
other phenomena, including the Kanto earthquake of 1923, for India and Chandra (21) for Indonesia. Given the rela-
the 1898 and 1930 data, and the Hokkaido outlier, generate tively reliable nature of population count and census data
ranges of estimates that are similar to each other. Use of the in comparison with vital registration data, however, the
alternative but less preferred A-type statistics (6) greatly inaccuracies in the above analysis, in percentage terms,
increased the estimates of the number of deaths, thereby are probably smaller for population count and census data
strengthening our conclusions. than for vital registration data.
The only control that yielded distinct estimates condi- A second possible limitation of the family of models
tional on its inclusion was the 1918 data control; the ranges estimated above is the implicit assumption of constant
of estimates for models that include the 1918 data (−1.38 population growth rates for the periods before and after the
to −1.61 million and −1.49 to −1.62 million) do not over- pandemic. The analyses of Japanese demographers suggest
lap with the ranges of estimates for models that exclude some variation in birth and death rates during this period
the 1918 data (−1.97 to −2.17 million and −1.98 to −2.05 (29). Yet because we assumed stable population growth
million). Because other controls seem to have no material (derived from the differential between birth and death
effect on the results, the final models selected are the ones rates, with adjustment for migration), the models are ten-
in which the 1918 data are dropped but none of the other able in view of the findings of these demographers of fairly
controls are implemented (i.e., the models in the second stable population growth rates in Japan between 1900 and
column of Tables 1 and 2). The estimated population loss 1920 (28). The finding of population growth in the models
is therefore 1.97 or 2.02 million persons, which translates (Tables 1, 2) that lies within the range of earlier estimates
to a drop in population of 3.62% or 3.71%. is further cause for confidence in these models.

620 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Deaths and Influenza Pandemic 1918–19, Japan

Table 3. Sensitivity analysis of influenza-induced change estimates for Japan

Ranges for estimated population change from influenza (millions)
1903–1930 data 1898–1935 data
Included Excluded Included Excluded
Controls for models Low High Low High Low High Low High
Kanto earthquake prefectures* –1.38 –2.02 –1.48 –2.17 –1.49 –2.05 –1.52 –2.01
Hokkaido outlier –1.38 –2.12 –1.50 –2.17 –1.49 –2.02 –1.59 –2.05
1918 population count data –1.38 –1.61 –1.97 –2.17 –1.49 –1.62 –1.98 –2.05
*Chiba, Kanagawa, Shizuoka, Tokyo.

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622 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Methicillin-Resistant
Staphylococcus aureus Colonization
of the Groin and Risk for Clinical
Infection among HIV-infected Adults
Philip J. Peters, John T. Brooks, Sigrid K. McAllister, Brandi Limbago, H. Ken Lowery,
Gregory Fosheim, Jodie L. Guest, Rachel J. Gorwitz, Monique Bethea, Jeffrey Hageman,
Rondeen Mindley, Linda K. McDougal, and David Rimland

Data on the interaction between methicillin-resistant (4–6) and health care–associated MRSA infections have
Staphylococcus aureus (MRSA) colonization and clinical also been described among HIV-infected persons (7), al-
infection are limited. During 2007–2008, we enrolled HIV- though the underlying basis for this association is unknown.
infected adults in Atlanta, Georgia, USA, in a prospec- Proposed mechanisms include immune dysfunction (5,7,8),
tive cohort study. Nares and groin swab specimens were behavioral risk factors (9), and increased exposure to the
cultured for S. aureus at enrollment and after 6 and 12
health care system (10). The prevalence of MRSA colo-
months. MRSA colonization was detected in 13%–15% of
HIV-infected participants (n = 600, 98% male) at baseline, 6
nization among HIV-infected persons is also high (10%–
months, and 12 months. MRSA colonization was detected 17%) (11,12), compared with that in the general US popu-
in the nares only (41%), groin only (21%), and at both sites lation (0.8%–1.5%) (13,14). Colonization with S. aureus
(38%). Over a median of 2.1 years of follow-up, 29 MRSA is a risk factor for subsequent clinical infection (15,16),
clinical infections occurred in 25 participants. In multivariate and the site of colonization may also be an key risk factor
analysis, MRSA clinical infection was significantly associ- (17). For example, although the anterior nares is consid-
ated with MRSA colonization of the groin (adjusted risk ratio ered the primary reservoir of S. aureus (18), MRSA PFGE
4.8) and a history of MRSA infection (adjusted risk ratio 3.1). type USA300 might preferentially colonize the buttocks,
MRSA prevention strategies that can effectively prevent or genitals, and perineum (17), leading to more infections in
eliminate groin colonization are likely necessary to reduce these anatomical areas. Improving our understanding of the
clinical infections in this population.
interaction between MRSA colonization and clinical infec-
tion among persons with HIV is necessary so that effective

M ethicillin-resistant Staphylococcus aureus (MRSA)

infections are a substantial cause of illness and a
major public health problem (1). Although MRSA was tra-
prevention strategies can be developed for this population.

Methods
ditionally considered a health care–associated pathogen, it
has emerged worldwide as a notable cause of community- Study Design
associated skin and soft tissue infections (2). In the United Study participants were recruited from the Atlanta
States, MRSA pulsed-field gel electrophoresis (PFGE) type Veterans Affairs Medical Center (Atlanta, GA, USA) HIV
USA300 strains have caused most community-associated clinic, which provides medical care to ≈1,400 HIV-infected
MRSA infections (3). High rates of community-associated veterans and is the largest Veterans Affairs Medical Center
HIV clinic in the United States. This study was approved
by institutional review boards for Emory University and
Author affiliations: Centers for Disease Control and Prevention,
the Centers for Disease Control and Prevention (CDC)
Atlanta, Georgia, USA (P.J. Peters, J.T. Brooks, S.K. McAllister, B.
and the Veterans Affairs Research and Development
Limbago, G. Fosheim, R.J. Gorwitz, J. Hageman, L.K. McDougal);
Committee. Eligible participants were HIV-infected,
Veterans Affairs Medical Center, Atlanta (H.K. Lowery, J.L. Guest,
>18 years of age, receiving outpatient medical care at
M. Bethea, R. Mindley, D. Rimland); and Emory University School
the Atlanta Veterans Affairs Medical Center HIV clinic,
of Medicine, Atlanta (D. Rimland)
and competent to provide informed consent. All eligible
DOI: http://dx.doi.org/10.3201/eid1904.121353 participants who attended the clinic from September 2007

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 623
RESEARCH

through April 2008 were invited to participate in the study. infections for 24 months. Participants were classified
Participants completed study visits at enrollment and then with a MRSA clinical infection if a clinical infection
at 6 and 12 months. was documented in the medical record and MRSA was
At enrollment, data on patients’ demographic isolated from the culture. Participants with a MRSA
characteristics, medical history, and antimicrobial drug clinical infection completed a supplemental questionnaire
use within the past 12 months, and microbiologic data that focused on the signs and symptoms of their infection
on previous S. aureus infections were obtained from and its clinical course. We defined a skin and soft tissue
electronic medical records. Participants also completed a infection in the groin as an infection that involved the
questionnaire at enrollment and at 12 months that focused buttocks, perineum, genitals, anus, or proximal thigh.
on their living situation, self-reported history of skin
infections, personal hygiene, sexual behavior, and drug use Statistical Methods
over the past 12 months. The primary analysis compared participants in whom
a MRSA clinical infection developed with those in whom
Microbiologic Procedures a MRSA clinical infection did not develop. All analyses
At each study visit, specimens for S. aureus culture were performed by using SAS version 9.2 (SAS Institute
were collected from the anterior nares and the groin by Inc., Cary, NC, USA). The Wilcoxon rank-sum test
using sterile rayon swabs and placed in liquid Stuart’s (continuous variables) and the χ2 and Fisher exact tests
transport media (Becton Dickinson, Sparks, MD, USA). (categorical variables) were used to test for differences
Study staff collected specimens from the anterior nares, and in clinical, demographic, and behavioral variables among
participants were instructed (using a diagram of the human participants with and without MRSA clinical infection.
body) to collect specimens from the groin by swabbing in Statistical significance was indicated by a p value <0.05.
the skin folds between the thigh and genital area. Swabs By using a multivariate log-linked binomial regression
were plated on mannitol salt agar (Becton Dickinson) and model (Proc Genmod; SAS Institute Inc.) (23), adjusted
CHROMagar MRSA (Becton Dickinson) and then placed risk ratios (aRRs), and 95% CIs were calculated to identify
in 5 mL of trypticase soy broth with 6.5% sodium chloride variables associated independently with the development
(Becton Dickinson) as described (19,20). At each study of MRSA clinical infection. All statistically significant
visit, participants were classified as MRSA colonized if (p<0.05) variables in univariate analysis (unadjusted RR)
MRSA was detected from either the nares or groin culture. were included in a multivariate model (24), and variables
Participants were classified as colonized with methicillin- with p>0.2 in the adjusted model were dropped sequentially
susceptible S. aureus (MSSA) if MSSA was detected and to create a parsimonious model that was examined for
MRSA was not detected. Participants colonized with both goodness of fit after each step. We also evaluated variables
MSSA and MRSA (regardless of site) were classified as in the final parsimonious model with Kaplan-Meier
MRSA colonized. survival methods with corresponding log-rank tests and
All MRSA isolates were genotyped by PFGE with Cox proportional hazards models (Proc PHREG; SAS
SmaI (New England Biolabs, Beverly, MA, USA) as Institute Inc.) of time to MRSA clinical infection.
described (13,19,20). PFGE patterns were analyzed with
BioNumerics Software v 5.10 (Applied Maths, Austin, Results
TX, USA) and were assigned to USA pulsed-field types We enrolled 600 HIV-infected veterans, most of
by using Dice coefficients and 80% relatedness. USA500, whom (98%) were male with a median age of 52 years
Iberian, and Archaic PFGE types were grouped together (interquartile range [IQR] 45–59 years). Four hundred
as USA500/Iberian because they are closely related and forty-one (74%) participants were non-Hispanic Blacks,
difficult to separate by PFGE (21). PCR was used to screen and 315 (53%) were men who had sex with men. The
for staphylococcal cassette chromosome mec type and to median most recent CD4 cell count was 416 cells/μL
detect Panton-Valentine leukocidin genes for all isolates (IQR 250–579 cells/μL), and 474 (79%) participants
(22). USA300 was defined as an isolate with a USA300 were receiving antiretroviral therapy. MRSA colonization
PFGE pattern that was positive for Panton-Valnetine was detected in 79 (13%) of 600 participants at baseline,
leukocidin genes and contained staphylococcal cassette in 66 (13%) of 502 at 6 months, and in 62 (15%) of 426
chromosome mec type IVa. participants at 12 months (Table 1). In addition, MRSA
colonization with 2 distinct MRSA strains was detected in
Prospective Monitoring for Incident MRSA 2 participants at baseline, in 1 participant at 6 months, and
Clinical Infections in 2 participants at 12 months, resulting in a total of 81,
Electronic medical and microbiology records were 67, and 64 isolates collected at each of the 3 time points,
prospectively monitored for incident MRSA clinical respectively. MSSA was detected in 180 (30%) participants

624 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 MRSA Colonization and Clinical Infection

Table 1. Prevalence of Staphylococcus aureus colonization In univariate analysis, factors associated with an
among HIV-infected adults, Atlanta, Georgia, USA, 2007–2009* increased risk of developing MRSA clinical infection
S. aureus Participants, no. (%)
colonization At enrollment, At 6-mo visit, At 12-mo visit,
included MRSA colonization detected in the groin at
status† n = 600 n = 502 n = 426 baseline, a lower CD4 cell count, a previous history of
MRSA 79‡ (13) 66‡ (13) 62‡ (15) an abscess, a medical history of MRSA clinical infection,
MSSA 180 (30) 156 (31) 118 (28) renal insufficiency, a history of syphilis, the use of certain
No S. aureus 341 (57) 280 (56) 246 (58)
*MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. antistaphylococcal agents in the past 12 months, contact
aureus; PFGE, pulsed-field gel electrophoresis. with a prison or jail, and certain hygienic factors (Table 3,
†Obtained from nares and groin swab specimens.
‡MRSA and MSSA co-colonization was detected in 11 participants at Appendix, wwwnc.cdc.gov/EID/article/19/4/12-1353-T3.
baseline, 10 participants at 6 mo, and 9 participants at 12 mo. For analysis htm). A suppressed HIV viral load (<400 copies/mL) and
these participants were classified as MRSA colonized. In addition, MRSA
colonization with 2 distinct MRSA PFGE patterns was detected in 2 use of antiretroviral therapy were associated with a lower
participants at baseline, 1 participant at 6 mo, and 2 participants at 12 mo. risk for development of MRSA clinical infection. Of note, no
MRSA clinical infections developed in the 30 participants
at baseline, 156 (31%) at 6 months, and 118 (28%) at 12 with MRSA colonization that was detected solely in
months (Table 1). USA500/Iberian (n = 112, 53%) and the nares at baseline. In multivariate analysis, MRSA
USA300 (n = 71, 33%) were the most common colonizing colonization detected in the groin at baseline (aRR 4.8
MRSA PFGE types identified. USA100 (n = 14, 7%) and 95% CI 2.1–10.8) and a medical history of MRSA clinical
other PFGE types (n = 15, 7%) were uncommon. Among infection (aRR 3.1 95% CI 1.4–7.3) were the only 2 factors
MRSA-colonized participants, MRSA was detected solely that remained significantly associated with an increased risk
in the nares of 87 (41%) participants, in both the nares
and groin of 81 (38%), and only in the groin for 44 (21%)
participants. USA300 accounted for 20 (23%) isolates
detected solely in the nares, 29 (36%) detected in the nares
and the groin, and 22 (50%) detected solely in the groin
(Figure 1). Compared with other PFGE types, USA300
was more likely to be detected solely in the groin (RR =
1.9; 95% CI 1.2–3.3; p = 0.02). Including groin cultures
increased the overall detection of MRSA by 26% and of
USA300 by 44% compared with nasal culture alone.
Over a median of 2.1 years of follow-up, 29 MRSA
clinical infections occurred in 25 participants (2.5
infections/100 person-years). Skin and soft tissue infections
(n = 24) were the most common, followed by pneumonia (n
= 3) and bacteremia (n = 2). Three (13%) of the skin and soft
tissue infections required hospitalization, and 13 (54%) of 24
skin and soft tissue infections occurred in the groin. Of the
25 participants in whom MRSA clinical infection developed,
MRSA colonization was detected at baseline in the groin
only or groin and nares in 12 (48%) of 25 participants in
whom a MRSA clinical infection developed, compared with
37 (6%) of 575 participants in whom an infection did not
develop (p<0.0001). MRSA colonization was also detected
at a study visit (baseline, 6 months, or 12 months) preceding
clinical infection in 17 (68%) of 25 participants. Among
clinical isolates available for PFGE typing from an initial
MRSA clinical infection (n = 22), USA300 (n = 14, 64%)
was the most common and was identified in 9 (69%) of 13
skin and soft tissue infections that occurred in the groin
Figure 1. Percentage of pulsed-field gel electrophoresis (PFGE) types
(Table 2). USA500/Iberian (n = 8, 36%) was also common by anatomic site of detection in methicillin-resistant Staphylococcus
and was identified in all of the pneumonia and bacteremia aureus (MRSA)–colonized HIV-infected adults (n = 212 MRSA
infections. In patients with preceding colonization, the colonizing isolates; 3 study visits aggregated), Atlanta, Georgia,
PFGE type of the clinical isolate and preceding colonizing USA, 2007–2009. Other PFGE types: USA100 (n = 14), USA600 (n
isolate always matched (n = 17/17). = 1), USA700 (n = 4), USA800 (n = 7), USA1000 (n = 3).

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 625
RESEARCH

Table 2. Location of infection and preceding colonization status of 25 HIV-infected adults with MRSA clinical infection, Atlanta,
Georgia, USA, 2007–2009*
Infection/ Clinical infection Colonization status Colonizing PFGE
participant Infection location PFGE type preceding clinical infection type Second clinical infection
Skin and soft tissue infections in the groin –
VA1 Buttock USA300 None
VA2 Buttock USA300 Nares and groin USA300
VA3 Buttock USA300 None –
VA4 Perianal USA300 None –
VA5 Perianal USA300 Nares and groin USA300
VA6 Pubic area USA300 Nares and groin USA300
VA7 Scrotum USA300 None –
VA8 Thigh USA300 None –
VA9 Hip USA500/Iberian Nares and groin USA500/Iberian
VA10 Rectum USA500/Iberian Nares and groin USA300 and 1 mo later: USA500/Iberian
USA500/Iberian rectal infection
VA11 Buttock No specimen Groin only USA300
VA12 Buttock No specimen Nares and groin USA300
Skin and soft tissue infections outside of the groin
VA13 Axilla USA300 Nares and groin USA300
VA14 Axilla USA300 Groin only USA300
VA15 Lip USA300 None –
VA16 Lower extremity USA300 None –
VA17 Lower extremity USA300 Nares and groin USA300 and
USA100
VA18 Scalp USA300 Groin only USA300
VA19 Back USA500/Iberian Nares and groin USA500/Iberian
VA20 Scalp USA500/Iberian Nares and groin USA500/Iberian 12 mo later:
USA500/Iberian decubitus
ulcer infection
VA21 Scalp No specimen Nares and groin USA300
Invasive clinical infections
VA22 Bloodstream USA500/Iberian Nares and groin USA500/Iberian
VA23 Bloodstream USA500/Iberian Nares and groin USA500/Iberian
VA24 Lung USA500/Iberian Nares and groin USA500/Iberian 6 mo later: USA500/Iberian
pneumonia
VA25 Lung USA500/Iberian None – 2 mo later: USA500/Iberian
infection on foot
*MRSA, methicillin-resistant Staphylococcus aureus; PFGE, pulsed-field gel electrophoresis; –, not applicable because the participant was not colonized
before their clinical infection.

for development of MRSA clinical infection (online Table 3 visits, MRSA colonization was detected in 48 (13%)
3, appendix). MRSA colonization detected in the groin participants at baseline, in 52 (14%) at 6 months, and in
included participants with MRSA colonization detected 50 (13%) at 12 months. Approximately equal numbers of
only in the groin (n = 14; aRR 6.6) and participants with participants became colonized with MRSA or were no longer
MRSA colonization detected in the nares and groin (n = 35; colonized at each sequential study visit to maintain this stable
aRR 4.2). This analysis was repeated by using a multiple- colonization prevalence (Figure 2). On a percentage basis at
predictor Cox proportional hazards model to account for each sequential study visit, 21%–31% of MRSA-colonized
time to MRSA clinical infection and MRSA colonization participants were no longer colonized (without treatment)
detected in the groin at baseline (adjusted hazard ratio 5.9; and 4%–6% of previously uncolonized participants became
95% CI 2.5–13.9) and a medical history of MRSA clinical colonized with MRSA. Over 12 months, MRSA colonization
infection (aHR 4.0; 95% CI 1.6–9.6) were significant was persistent (detected at all 3 visits) in 26 (7%) participants
predictors of time to MRSA clinical infection. Among and intermittent (detected in 1 or 2 visits) in 54 (14%)
the 79 participants with MRSA colonization at baseline, participants (Figure 2). The PFGE type remained stable in
USA300 colonization was associated with a nonsignificant 23 (88%) of 26 participants with persistent colonization and
but increased risk of developing MRSA clinical infection, in 16 (89%) of 18 participants with intermittent colonization
compared with other PFGE types (RR 2.1; 95% CI 0.7–5.9). at 2 visits. Swab specimens from participants with persistent
In a separate analysis, MSSA colonization was not associated colonization (n = 78 MRSA isolates from 26 participants)
with developing a MRSA clinical infection (RR 0.8; 95% were more likely to yield heavy growth of MRSA (growth
CI 0.3–2.7). detected on direct agar plating without broth enrichment)
In a subanalysis of MRSA colonization in 383 HIV- than were isolates from participants with intermittent
infected adults from whom samples were cultured at all colonization (n = 72 MRSA isolates from 54 participants)

626 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 MRSA Colonization and Clinical Infection

the association between MRSA clinical infection and risk

factors for exposure to MRSA (e.g., contact with jails and
prisons) and risk factors related to hygiene (e.g., shaving
the groin, genital, or buttock area). These findings suggest
that MRSA colonization in the groin may also be a marker
of more frequent exposure to MRSA in the environment or
poor hygiene or an indicator of immunologic dysfunction
(i.e., impaired neutrophil function [27]) that in turn increases
a person’s susceptibility to clinical infection.
Prior studies have demonstrated that USA300 causes
most community-associated MRSA infections in the United
States, whereas USA500/Iberian clones are associated
with health care–associated MRSA infections (1). This
epidemiology, however, is changing (28), and participants
in this study had risk factors for both community-associated
and outpatient health care–associated MRSA exposures.
Figure 2. Prevalence of methicillin-resistant Staphylococcus aureus
(MRSA) recovered from nares and groin swabs of HIV-infected In this study, USA300 caused most skin and soft tissue
adults at each study visit among participants who had specimens infections and was more likely colonize the groin only. Other
cultured at all 3 visits (n = 383). Atlanta, Georgia, USA, 2007–2009. PFGE types, however, also caused both clinical infections
and groin colonization, and PFGE type (USA300 vs. other
[91% vs. 75%; p = 0.009]. PFGE type (USA300 vs. other PFGE types) was not independently associated with risk
PFGE types) was not significantly associated with persistent for MRSA clinical infection. These findings suggest that
vs. intermittent colonization (p = 0.27). the presence of MRSA colonization in the groin is more
useful clinical knowledge than identifying the PFGE type
Discussion causing colonization (which is rarely determined in clinical
MRSA clinical infections (mainly skin and soft tissue practice anyway).
infections) were common among HIV-infected adults in this The association of MRSA colonization and the
study. The prevalence of MRSA colonization was also high development of clinical infection in this study suggest that
at each study visit (13%–15%), and MRSA colonization in MRSA decolonization with topical or systemic treatment may
the groin was a risk factor for developing a MRSA clinical be an effective method to prevent clinical infections in this
infection. MRSA PFGE types USA300 and USA500/ population. A randomized clinical trial of adult hospitalized
Iberian were common causes of colonization and clinical surgical patients found that using intranasal mupirocin and
infection. USA300 more commonly caused colonization chlorhexidine gluconate soap total-body wash substantially
of the groin and clinical skin and soft-tissue infection in reduced the rate of health care–associated S. aureus infection
the groin. MRSA prevention strategies with HIV-infected by 58% in patients who were nasal carriers of S. aureus
adults that can effectively address colonization at this (29). Although several randomized controlled trials have
anatomical site are likely necessary to reduce MRSA demonstrated that MRSA colonization can be eliminated
clinical infections in this population. from the groin (30), and short-term clinical benefits of
HIV-infected persons have been found to have 6× the S. aureus decolonization have been demonstrated in the
risk for community-associated MRSA skin and soft-tissue hospitalized setting, data have not been available to support
infections than HIV-negative patients (25) and an increased decolonization as a method of preventing MRSA clinical
odds of having community-acquired S. aureus bacteremia infections in a community or outpatient setting (31). In this
(26). In this study, MRSA colonization in the groin and a study, we observed that although MRSA colonization was
medical history of MRSA clinical infection were risk factors frequent, it also fluctuated considerably over time. MRSA
for clinical infection. Because, in our study, most skin and colonization spontaneously resolved in approximately half
soft tissue infections occurred in the groin, colonization of of participants over 12 months, but new MRSA colonization
the groin may have directly precipitated clinical infection in was detected in as many previously uncolonized participants.
this anatomical area. In a previous analysis, we demonstrated A MRSA decolonization program would therefore treat a
that MRSA colonization was also associated with a medical substantial number of persons whose MRSA colonization
history of MRSA clinical infection, contact with jails and would have resolved spontaneously and would require
prisons, and correlates of risky sexual behavior (i.e., rarely ongoing screening to identify new colonization. The
or never using condoms) (20). In addition, in this analysis, substantial fluctuations in MRSA colonization status in
adjusting for MRSA colonization in the groin diminished this study suggest that strategies that emphasize hygiene

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 627
RESEARCH

and avoidance of potential MRSA exposures might be hygiene; avoiding shared personal items, such as towels
more effective at preventing MRSA clinical infections in and razors; and decolonization in certain situations
this setting than decolonization, but this hypothesis should (31). Given the frequency of MRSA colonization in
be tested in a clinical study that includes decolonization of the groin and its association with clinical infection,
MRSA from the groin as an intervention. MRSA prevention strategies (both hygienic practices
Our study had several limitations. First, our study and decolonization treatments) with HIV-infected adults
population was 98% male and our findings are not should be used to prevent or eliminate colonization at
generalizable to HIV-infected women. Second, the this anatomic site to reduce MRSA clinical infections in
MRSA epidemic in the United States continues to evolve this population.
(7), and new risk factors for MRSA infection in HIV-
infected adults may emerge that were not significant in This publication was made possible by support from the
this study. In addition, in our study, some risk factors Division of HIV/AIDS Prevention and the Division of Healthcare
for community-associated MRSA clinical infections, Quality Promotion, CDC. These data were presented in part at
such as methamphetamine use (9) and close contact the 48th Annual Infectious Disease Society of America Meeting,
with someone with a skin infection, were not associated Vancouver, British Columbia, Canada, October 21–24, 2010.
with MRSA clinical infection. These differences might Dr Peters is a medical officer in the Division of HIV/AIDS
be explained by low frequencies of certain risk factors Prevention at CDC, Atlanta, Georgia. His research interests
(i.e., methamphetamine use) in our study population include HIV-associated infections, such as those caused by
or a social desirability bias may have limited the full MRSA, influenza, and hepatitis B virus.
disclosure of drug use and sexual and hygienic behavior.
Third, we evaluated 45 variables in univariate analysis
and 16 variables in the initial multivariate model before References
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colonization in the United States, 2001–2002. J Infect Dis. 27. Pugliese A, Vidotto V, Beltramo T, Torre D. Phagocytic activity in
2006;193:172–9. http://dx.doi.org/10.1086/499632 human immunodeficiency virus type 1 infection. Clin Diagn Lab
15. Nguyen MH, Kauffman CA, Goodman RP, Squier C, Arbeit RD, Immunol. 2005;12:889–95.
Singh N, et al. Nasal carriage of and infection with Staphylococcus 28. Seybold U, Kourbatova EV, Johnson JG, Halvosa SJ, Wang YF,
aureus in HIV-infected patients. Ann Intern Med. 1999;130:221–5. King MD, et al. Emergence of community-associated methicillin-
16. von Eiff C, Becker K, Machka K, Stammer H, Peters G. Nasal carriage resistant Staphylococcus aureus USA300 genotype as a major cause
as a source of Staphylococcus aureus bacteremia. N Engl J Med. of health care-associated blood stream infections. Clin Infect Dis.
2001;344:11–6. http://dx.doi.org/10.1056/NEJM200101043440102 2006;42:647–56. http://dx.doi.org/10.1086/499815
17. Szumowski JD, Wener KM, Gold HS, Wong M, Venkataraman 29. Bode LG, Kluytmans JA, Wertheim HF, Bogaers D, Vandenbroucke-
L, Runde CA, et al. Methicillin-resistant Staphylococcus aureus Grauls CM, Roosendaal R, et al. Preventing surgical-site infections
colonization, behavioral risk factors, and skin and soft-tissue in nasal carriers of Staphylococcus aureus. N Engl J Med.
infection at an ambulatory clinic serving a large population of HIV- 2010;362:9–17. http://dx.doi.org/10.1056/NEJMoa0808939
infected men who have sex with men. Clin Infect Dis. 2009;49:118– 30. Ammerlaan HS, Kluytmans JA, Wertheim HF, Nouwen JL, Bonten
21. http://dx.doi.org/10.1086/599608 MJ. Eradication of methicillin-resistant Staphylococcus aureus
18. Wertheim HF, Melles DC, Vos MC, van LeeuwenW, van Belkum carriage: a systematic review. Clin Infect Dis. 2009;48:922–30.
A, Verbrugh HA, et al. The role of nasal carriage in Staphylococcus http://dx.doi.org/10.1086/597291
aureus infections. Lancet Infect Dis. 2005;5:751–62. http://dx.doi. 31. Gorwitz RJ, Jernigan DB, Powers JH, Jernigan JA, and Participants
org/10.1016/S1473-3099(05)70295-4 in the CDC Convened Experts’ Meeting on Management of MRSA
19. McAllister SK, Albrecht VS, Fosheim GE, Lowery HK, Peters PJ, in the Community. Strategies for clinical management of MRSA in
Gorwitz R, et al. Evaluation of the impact of direct plating, broth the community: summary of an experts’ meeting convened by the
enrichment, and specimen source on recovery and diversity of Centers for Disease Control and Prevention. March 2006 [cited
methicillin-resistant Staphylococcus aureus among HIV-infected 2011 Dec 1]. http://www.cdc.gov/mrsa/pdf/MRSA-Strategies-
outpatients. J Clin Microbiol. 2011; 49:4126–30. http://dx.doi. ExpMtgSummary-2006.pdf
org/10.1128/JCM.05323-11
20. Peters PJ, Brooks JT, Limbago B, Lowery HK, McAllister SK,
Address for correspondence: Philip J. Peters, Centers for Disease Control
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colonization in HIV-infected outpatients is common and detection is and Prevention, 1600 Clifton Rd NE, Mailstop E45, Atlanta, GA 30333,
enhanced by groin culture. Epidemiol Infect. 2010;139:1–11. USA; email: pjpeters@cdc.gov

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DISPATCHES

Feline Origin of RVA strains have been detected in humans, and they are
believed to be the result of direct interspecies transmission
Rotavirus Strain, from cats or dogs to humans, possibly in combination with
reassortment (10–13).
Tunisia, 2008 Previously, 2 genotype constellations among feline
and canine RVA strains, cat97-like and AU-1-like, were
Mouna Ben Hadj Fredj, Elisabeth Heylen, described (13). The genotype constellations were G3-P[3]-
Mark Zeller, Imene Fodha, I3-R3-C2-M3-A9-N2-T3-E3-H6 and G3-P[9]-I3-R3-C3-
Meriam Benhamida-Rebai, Marc Van Ranst, M3-A3-N3-T3-E3-H3, respectively. Recently, the complete
Jelle Matthijnssens, and Abdelhalim Trabelsi genomes of a feline strain (RVA/cat-wt/ITA/BA222/2005/
G3P[9]) and 2 feline-like human RVA strains (RVA/
In Tunisia in 2008, an unusual G6P[9] rotavirus, RVA/ human-wt/ITA/PAI58/1996/G3P[9] and RVA/human-
human-wt/TUN/17237/2008/G6P[9], rarely found in hu- wt/ITA/PAH136/1996/G3P[9]) were shown to possess a
mans, was detected in a child. To determine the origin of distinct genotype constellation, G3-P[9]-I2-R2-C2-M2-
this strain, we conducted phylogenetic analyses and found A3-N1/N2-T3/T6-E2-H3 (11), representing a tentative
a unique genotype constellation resembling rotaviruses
third feline genotype constellation (BA222-like). This
belonging to the feline BA222-like genotype constella-
tion. The strain probably resulted from direct cat-to-human
tentative third feline BA222-like genotype constellation is
transmission. an intriguing genotype mosaic, sometimes possessing Wa-
like nonstructural protein (NSP) 2 or NSP3 gene segments
and partially resembling the genotype constellation found in

G roup A rotaviruses (RVAs) are a leading cause of se-

vere acute gastroenteritis in infants and young chil-
dren. An infectious RVA virion is a triple-layered icosahe-
RVA strains from cattle and other artiodactyla (5,7,10,11).
Full-genome sequences of unusual human RVA strains
are being analyzed to detect interspecies transmission,
dral particle that contains 11 segments of double-stranded reassortment, and evolutionary relationships between
RNA (1). The outer protein layer is formed by virus capsid human and animal RVAs (10,11).
protein (VP) 4 (P antigen) and VP7 (G antigen), each of In 2008, during continuous surveillance for human
which is used for binomial nomenclature (1). At least 27 RVA in Tunisia, we identified an unusual G6P[9] strain in
G genotypes and 35 P genotypes have been identified (2). an 8-month-old hospitalized child (14). To understand the
Globally, only 6 G/P-genotype combinations are of epide- evolution and origin of this unusual strain, RVA/human-
miologic relevance to humans: G1P[8], G3P[8], G4P[8], wt/TUN/17237/2008/G6P[9] (hereafter referred to as strain
G9P[8], and G12P[8], which are typically found in com- 17237), we conducted phylogenetic analyses.
bination with a Wa-like genotype constellation (I1-R1-
C1-M1-A1-N1-T1-E1-H1), and G2P[4], which is found in The Study
combination with a DS-1-like genotype constellation (I2- The full-length genome sequence of the virus was
R2-C2-M2-A2-N2-T2-E2-H2) (3). determined as described (5). Primers used for all 11
Certain G genotypes rarely encountered in humans are segments are shown in online Technical Appendix Table 1
commonly associated with RVA strains from animals (4). (wwwnc.cdc.gov/EID/article/19/4/12-1383-Techapp1.pdf).
For example, G6 RVA strains are occasionally detected in Multiple sequence alignments and phylogenetic analyses
humans but are a common genotype in cattle (4). Complete were conducted by using MEGA version 5.05 (www.
genomes have been determined for 11 human G6 RVA megasoftware.net). Sequences were deposited in GenBank
strains: 7 G6P[14], 2 G6P[9], 1 human–animal reassortant (accession nos. JX271001–JX271011).
G6P[6], and 1 unique G6P[11] (5–9). Strain 17237 possessed the unique genotype
The P[9] genotype is commonly associated with the constellation G6-P[9]-I2-R2-C2-M2-A3-N1-T6-E2-H3.
G3 or G6 genotype and is believed to be typical for feline This constellation was compared with that of the human
and canine RVA strains (4). A few G3P[9] and G3P[3] G6P[9] strain Se584, feline/canine-like human RVA
strains (KF17, PAH136, PAI58, and 0537), and several
Author affiliations: Sahloul University Hospital, Sousse, Tunisia
animal strains (Table). Strain 17237 shared the same
(M. Ben Hadj Fredj, I. Fodha, M. Benhamida-Rebai, A. Trabelsi);
combination of genotypes with human RVA strain
University of Monastir, Monastir, Tunisia (M. Ben Hadj Fredj,
PAH136 (10) except for VP7 (strain 17237 contained G6
I. Fodha, M. Benhamida-Rebai, A. Trabelsi); and University of
instead G3). Overall, strain 17237 shared 8–10 genotypes
Leuven, Leuven, Belgium (E. Heylen, M. Zeller, M. Van Ranst, J.
with RVA strains possessing the BA222-like genotype
Matthijnssens)
constellation and 8–9 genotypes with several bovine or
DOI: http://dx.doi.org/10.3201/eid1904.121383 bovine-like RVA strains.

630 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Feline Origin of Rotavirus Strain, Tunisia

Table. Comparison of genomic constellation of group A rotavirus strain RVA/human-wt/TUN/17237/2008/G6P[9] from Tunisia with
reference strains*
Genotype
Strain constellation VP7 VP4 VP6 VP1 VP2 VP3 NSP1 NSP2 NSP3 NSP4 NSP5
RVA/human- BA222-like G6 P[9] I2 R2 C2 M2 A3 N1 T6 E2 H3
wt/TUN/17237/2008/G6P[9]
RVA/human- BA222-like G3 P[9] I2 R2 C2 M2 A3 N1 T6 E2 H3
wt/ITA/PAH136/1996/G3P[9]
RVA/cat- BA222-like G3 P[9] I2 R2 C2 M2 A3 N1 T3 E2 H3
wt/ITA/BA222/2005/G3P[9]
RVA/human- BA222-like G3 P[9] I2 R2 C2 M2 A3 N2 T6 E2 H3
wt/ITA/PAI58/1996/G3P[9]
RVA/human- BA222-like G6 P[9] I2 R2 C2 M2 A3 N2 T1 E2 H3
tc/USA/Se584/1998/G6P[9]
RVA/human- BA222-like G6 P[9] I2 R2 C2 M2 A3 N2 T3 E3 H3
wt/JAP/KF17/2009/G6P[9]
RVA/human- BA222-like G3 P[9] I2 R2 C2 M2 A3 N2 T1 E2 H3
wt/USA/0537/2002/G3P[9]
RVA/cat- BA222-like G3 P[9] I3 R3 C2 M3 A3 N1 T6 E3 H3
tc/AUS/Cat2/1984/G3P[9] /cat97-like
RVA/human- Bovine-like G6 P[14] I2 R2 C2 M2 A3 N2 T6 E2 H3
tc/ITA/PA169/1988/G6P[14]
RVA/human- Bovine-like G6 P[14] I2 R2 C2 M2 A3 N2 T6 E2 H3
wt/BEL/B10925/1997/G6P[14]
RVA/human-wt/ITA/111–05– Bovine-like G6 P[14] I2 R2 C2 M2 A3 N2 T6 E2 H3
27/2005/G6P[14]
RVA/cow-tc/FRA/RF/1982/G6P[1] Bovine G6 P[1] I2 R2 C2 M2 A3 N2 T6 E2 H3
RVA/cow- Bovine G6 P[1] I2 R2 C2 M2 A3 N2 T6 E2 H3
tc/VEN/BRV033/1990/G6P6[1]
RVA/cow- Bovine G6 P[5] I2 R2 C2 M2 A3 N2 T6 E2 H3
tc/USA/WC3/1981/G6P[5]
RVA/cow-tc/KOR/KJ19– Bovine G6 P[7] I2 R2 C2 M2 A3 N2 T6 E2 H3
2/2004/G6P[7]
RVA/rhesus- Bovine-like G8 P[1] I2 R2 C2 M2 A3 N2 T6 E2 H3
tc/USA/PTRV/1990/G8P[1]
RVA/human- Bovine-like G8 P[1] I2 R2 C2 M2 A3 N2 T6 E2 H3
tc/KEN/B12/1987/G8P[1]
RVA/human-tc/USA/DS- DS-1-like G2 P[4] I2 R2 C2 M2 A2 N2 T2 E2 H2
1/1976/G2P[4]
RVA/human-tc/JPN/AU- AU-1-like G3 P[9] I3 R3 C3 M3 A3 N3 T3 E3 H3
1/1982/G3P3[9]
RVA/human- Wa-like G1 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1
tc/USA/Wa/1974/G1P1A[8]
*Boldface indicates genotypes that are identical to group A rotavirus RVA/human-wt/TUN/17237/2008/G6P[9]. VP, virus capsid protein; NSP,
nonstructural protein.

Phylogenetic analyses showed that all 11 genome of strain 17237 clustered in the N1 genotype and was
segments of strain 17237 were most closely related to distantly related to typical human Wa-like RVA strains.
strains of either feline-like human or feline origin (Figures The VP2, NSP3, and NSP5 gene segments were closely
1, 2). Strain 17237 clustered most closely with RVA/ related to RVA/human-wt/ITA/PAI58/1996/G3P[9].
human-wt/ITA/PA43/2003/G6P[9], RVA/human-wt/ The VP3, NSP1, and NSP3 genome segments clustered
JAP/KF17/2009/G6P[9], and RVA/human-wt/BEL/ closely with RVA/human-wt/ITA/PAH136/1996/G3P[9].
B1711/2002/G6P[6] strains, all of which are suspected NSP1 and NSP5 clustered closely with RVA/human-wt/
to have at least a partial animal (bovine-like or feline- USA/0537/2002/G3P[9]. These 3 human strains (PAI58–
like) origin (6,7). The P[9] genome segment was most 96, PAH136–96, and 0537) are believed to be of feline
closely related to RVA strains RVA/human-wt/RUS/ origin and possess a BA222-like genotype constellation.
Nov10-N507/2010/G3P[9], BA222, and KF17. The VP1,
VP6, NSP2, and NSP4 genome segments of strain 17237 Conclusions
were closely related to BA222, clustering in the R2, I2, N1, The genome constellation of strain 17237 is similar
and E2 genotypes, respectively. This G3P[9] feline RVA to that of strains belonging to the tentative feline BA222-
strain BA222 is believed to have a common origin with like genotype constellation (Table). It has been speculated
animal RVA strains and RVA strains that are zoonotically that several of these BA222-like RVA strains resulted
transmissible to humans (11). The NSP2 gene segment from multiple reassortment events among RVA strains

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 631
DISPATCHES

originating from different hosts (cattle, other ruminants, Europe (Italy), North America (United States), Asia
humans, cats, dogs) (10,11). However, a recent article (Japan), and now Africa (Tunisia) (7,10). RVA strains
speculates that this genotype constellation, although with this BA222-like genotype constellation are much
reminiscent to bovine-like RVA strains, might represent a more likely to circulate in a certain host species rather
true feline genotype constellation (12). than result from distinct multiple reassortment events in
Our results support this hypothesis in 2 ways. The each of the above-mentioned countries. The second source
first source of support comes from the fact that BA222- of support comes from the fact that our phylogenetic
like RVA strains have been detected on several continents: analyses confirmed that each of the 11 gene segments

Figure 1. Phylogenetic trees of the full-length nucleotide sequences of the group A rotavirus (RVA) virus capsid protein (VP) 7, VP4,
VP6, VP1, VP2, and VP3 genes. Phylogenetic trees were constructed by using the neighbor-joining method with the Kimura 2-parameter
method. Bootstrap values (1,000 replicates) >70% are shown. Filled circles indicate strain RVA/human-wt/TUN/17237/2008/G6P[9] from
Tunisia; filled triangles indicate feline RVA strains; and open triangles indicate feline/canine-like human RVA strains. GenBank accession
numbers of the sequences of reference strains are shown in online Technical Appendix Table 2 (wwwnc.cdc.gov/EID/article/19/4/12-1383-
Techapp1.pdf). Scale bars indicate nucleotide substitutions per site.

632 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Feline Origin of Rotavirus Strain, Tunisia

of strain 17237 was more closely related to BA222-like To further support or refute this hypothesis, more
RVA strains than to bovine or bovine-like RVA strains. complete genomes must be determined from RVA strains
This finding strengthens the hypothesis that each of the from cats and dogs. Moreover, because P[9] is believed
BA222-like RVA strains did not result from individual to be typical for feline/canine RVA strains, it would be
multiple reassortment events but rather that this genotype intriguing to determine whether this strain could persist
constellation now circulates (most likely in cats) around in the human population and could become competitive
the world and might have resulted from >1 reassortment with already established P genotypes in humans. The
events in the more distant past. recently emerged human G9 RVA strain is believed to have

Figure 2. Phylogenetic trees of the full-length nucleotide sequences of the group A rotavirus (RVA) nonstructural protein (NSP) genes.
Phylogenetic trees were constructed by using the neighbor-joining method with the Kimura 2-parameter method. Bootstrap values (1,000
replicates) >70% are shown. Filled circle indicates strain RVA/human-wt/TUN/17237/2008/G6P[9] from Tunisia, filled triangles indicate the
feline RVA strains, and open triangles indicate the feline/canine-like human RVA strains. GenBank accession numbers of the sequences
of reference strains are shown in online Technical Appendix Table 2 (wwwnc.cdc.gov/EID/article/19/4/12-1383-Techapp1.pdf). Scale bars
indicate nucleotide substitutions per site.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 633
DISPATCHES

originated from pigs and to have become established in the 4. Martella V, Banyai K, Matthijnssens J, Buonavoglia C, Ciarlet M.
human population as the fifth major human RVA genotype, Zoonotic aspects of rotaviruses. Vet Microbiol. 2010;140:246–55.
http://dx.doi.org/10.1016/j.vetmic.2009.08.028
after multiple genome reassortment events with typical 5. Matthijnssens J, Potgieter CA, Ciarlet M, Parreno V, Martella V,
human Wa-like RVA strains (15). Banyai K, et al. Are human P[14] rotavirus strains the result of
The unusual G6P[9] RVA strain 17237 most likely interspecies transmissions from sheep or other ungulates that belong
resulted from direct interspecies transmission from a cat to the mammalian order Artiodactyla? J Virol. 2009;83:2917–29.
http://dx.doi.org/10.1128/JVI.02246-08
to a human. Interspecies transmission increases potential 6. Matthijnssens J, Rahman M, Van Ranst M. Two out of the 11
for spread of unusual and uncommon RVA strains. The genes of an unusual human G6P[6] rotavirus isolate are of bovine
findings of this study highlight the need for continuous origin. J Gen Virol. 2008;89:2630–5. http://dx.doi.org/10.1099/
monitoring of RVA strains and timely recognition of novel vir.0.2008/003780-0
7. Yamamoto D, Kawaguchiya M, Ghosh S, Ichikawa M, Numazaki K,
or rare genotypes. Continued surveillance of RVA strains Kobayashi N. Detection and full genomic analysis of G6P[9] human
in industrialized and developing countries, and in humans rotavirus in Japan. Virus Genes. 2011;43:215–23. http://dx.doi.
and animals, will provide more insights into interspecies org/10.1007/s11262-011-0624-6
transmission processes of RVAs. In turn, this information 8. El Sherif M, Esona MD, Wang Y, Gentsch JR, Jiang B, Glass RI, et
al. Detection of the first G6P[14] human rotavirus strain from a child
could help determine how the introduction of novel genes with diarrhea in Egypt. Infect Genet Evol. 2011;11:1436–42. http://
might affect the evolution of the RVA populations that dx.doi.org/10.1016/j.meegid.2011.05.012
infect humans. 9. Steyer A, Sagadin M, Kolenc M, Poljsak-Prijatelj M. Whole genome
sequence analysis of bovine G6P[11] rotavirus strain found in a
child with gastroenteritis. Infect Genet Evol. 2013;13:89–95. http://
Acknowledgments dx.doi.org/10.1016/j.meegid.2012.09.004
We thank all colleagues of the Laboratory of Clinical and 10. De Grazia S, Giammanco GM, Potgieter CA, Matthijnssens J,
Epidemiological Virology, Department of Microbiology and Banyai K, Platia MA, et al. Unusual assortment of segments in 2
rare human rotavirus genomes. Emerg Infect Dis. 2010;16:859–62.
Immunology, Rega Institute for Medical Research, University of http://dx.doi.org/10.3201/eid1605.091826
Leuven, Belgium, for their help and valuable advice. 11. Martella V, Potgieter AC, Lorusso E, De Grazia S, Giammanco
GM, Matthijnssens J, et al. A feline rotavirus G3P[9] carries
This study was supported by a grant from the World Health traces of multiple reassortment events and resembles rare human
Organization (GL.GLO.IVD.646.XC.04.2.999.00). M.Z. was G3P[9] rotaviruses. J Gen Virol. 2011;92:1214–21. http://dx.doi.
supported by the Institute for the Promotion of Innovation through org/10.1099/vir.0.027425-0
12. Matthijnssens J, De Grazia S, Piessens J, Heylen E, Zeller M,
Science and Technology in Flanders. J.M. was supported by a
Giammanco GM, et al. Multiple reassortment and interspecies
Fonds voor Wetenschappelijk Onderzoek postdoctoral fellowship. transmission events contribute to the diversity of feline, canine
and feline/canine-like human group A rotavirus strains. Infect
Dr Ben Hadj Fredj is a PhD student at the Faculty of Genet Evol. 2011;11:1396–406. http://dx.doi.org/10.1016/j.
Pharmacy of Monastir, Tunisia, and a member of the research meegid.2011.05.007
unit UR06SP20, Laboratory of Microbiology, Sahloul University 13. Tsugawa T, Hoshino Y. Whole genome sequence and phylogenetic
Hospital, Sousse, Tunisia. Her primary research interests focus analysis reveal human rotavirus G3P[3] strains Ro1845 and
HCR3A are examples of direct virion transmission of canine/feline
on virus typing, viral enteric pathogens, and virus epidemiology. rotaviruses to humans. Virology. 2008;380:344–53. http://dx.doi.
org/10.1016/j.virol.2008.07.041
14. Ben Hadj Fredj M, Zeller M, Fodha I, Heylen E, Chouikha A,
References Van Ranst M, et al. Molecular characterization of the NSP4 gene
of human group A rotavirus strains circulating in Tunisia from
1. Estes M, Kapikian A. Rotaviruses. In: Knipe M, Howley P, editors. 2006 to 2008. Infect Genet Evol. 2012;12:997–1004. http://dx.doi.
Fields virology. 5th ed. Philadelphia: Lippincott Williams & org/10.1016/j.meegid.2012.02.011
Wilkins; 2007. p. 1917–74. 15. Matthijnssens J, Heylen E, Zeller M, Rahman M, Lemey P, Van
2. Matthijnssens J, Ciarlet M, McDonald SM, Attoui H, Banyai Ranst M. Phylodynamic analyses of rotavirus genotypes G9 and
K, Brister JR, et al. Uniformity of rotavirus strain nomenclature G12 underscore their potential for swift global spread. Mol Biol
proposed by the Rotavirus Classification Working Group (RCWG). Evol. 2010;27:2431–6. http://dx.doi.org/10.1093/molbev/msq137
Arch Virol. 2011;156:1397–413. http://dx.doi.org/10.1007/s00705-
011-1006-z
Address for correspondence: Abdelhalim Trabelsi, Laboratory of
3. Matthijnssens J, Van Ranst M. Genotype constellation and
evolution of group A rotaviruses infecting humans. Curr Opin Virol. Microbiology, Sahloul University Hospital, 4054 Sousse, Tunisia; email:
2012;2:426–33. http://dx.doi.org/10.1016/j.coviro.2012.04.007 abdelhalim.trabelsi@gmail.com

634 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Tick-borne ruses. ELISA-positive serum samples were further investi-
gated by using virus-specific neutralization assays for the 3
Encephalitis Virus flaviviruses circulating in Austria (West Nile virus [WNV],
Usutu virus [USUV], and TBEV). Neutralization assays
in Horses, were conducted independently by 2 laboratories (9,10).
Results were analyzed by using SPSS version 17 software
Austria, 2011 (SPSS IBM, Armonk, NY, USA). Associations of sex,
age, and location with positive results were tested by using
James O. Rushton, Sylvie Lecollinet, 1-way analysis of variance and tested for significance by
Zdenek Hubálek, Petra Svobodová, Helga Lussy, using the χ2 test. Differences in age between horses positive
and Norbert Nowotny or negative for flaviviruses were determined by using the
Student t-test. A p value <0.05 was considered significant
An unexpectedly high infection rate (26.1%) of tick- for all analyses.
borne encephalitis virus (TBEV) was identified in a herd of The study comprised 113 (44.0%) mares, 139 (54.0%)
257 horses of the same breed distributed among 3 federal
stallions, and 5 (2.0%) geldings. The mean ± SD age of
states in Austria. Young age (p<0.001) and male sex (p =
0.001) were positively associated with infection.
horses was 8.1 ± 6.3 years (range 1–32 years). A total of
154 (59.9%) horses were boarded in Styria, 66 (25.7%)
in Vienna, and 37 (14.4%) in Lower Austria and kept in

T ick-borne encephalitis (TBE), which is caused by tick-

borne encephalitis virus (TBEV), is a potentially fatal
disease of the central nervous system, mainly in humans,
various types of housing. All 3 locations are considered ar-
eas to which WNV, USUV, and TBEV are endemic. The
animals were free from clinical symptoms associated with
but also in monkeys, dogs (1), and horses. Ruminants such flavivirus infections. None of the horses were vaccinated
as goats, sheep, and cattle are considered to be sporadically with WNV and TBEV vaccines (TBEV vaccines are not
infected subclinically. However, they might be the source licensed for use in horses).
of disease in humans who consume nonpasteurized milk Sixty-seven (26.1%) horses were positive for antibod-
and milk products (2). TBEV-associated central nervous ies against flaviviruses by ELISA, and all 67 were posi-
system disease in ruminants is rare (3). tive for TBEV by virus-specific neutralization tests (Table,
TBEV occurs in natural foci and is endemic to many Appendix, wwwnc.cdc.gov/EID/article/19/4/12-1450-T1.
countries in Europe and parts of central and eastern Asia htm). Positive results were distributed among 17 mares, 49
(4). The principal vectors for transmission are ticks of the stallions, and 1 gelding. The difference in results between
genus Ixodes. Although TBEV in humans has been stud- sexes was significant (p = 0.001) (Figure). The mean ± SD
ied extensively, there are only a limited number of reports ages of horses positive and negative for TBEV antibodies
on TBEV in animals (5), especially horses. Only 2 reports were 5.9 ± 4.2 and 8.9 ± 6.7 years, respectively (p<0.001)
were found in the German literature on the epidemiology of (Figure). Thirty-seven positive horses were kept in Styria,
TBEV infection in horses (6,7), and 1 case report was found 16 in Vienna, and 14 in Lower Austria. The difference in
on clinical symptoms of TBE in a mare (8). The purpose of positive results for horses at the 3 locations was not signifi-
this study was to determine the status of TBEV infection in cant. Low-level cross-reactivity for WNV and USUV was
a large population of a single horse breed in Austria. observed in 9 animals (Table).

The Study Conclusions

Serum samples from 257 horses of the same breed that The main findings of our study were a comparatively
were distributed among 3 federal states in Austria were high seropositivity rate of 26.1%, a higher prevalence of
obtained in April 2011 and screened by using a commer- TBEV-specific antibodies in younger horses, and a higher
cial ELISA (ID Screen West Nile Competition ELISA Kit; prevalence of TBEV-specific antibodies in stallions. We ex-
IDvet, Montpellier, France) for antibodies against flavivi- pected the horses to have subclinical infections with WNV
lineage 2, which was introduced recently into central Eu-
Author affiliations: University of Veterinary Medicine, Vienna, Aus-
rope (11), including Austria (12). However, it is well known
tria (J.O. Rushton, H. Lussy, N. Nowotny); Agence Nationale de
that WNV IgG ELISAs show cross-reactivity with other fla-
Sécurité Sanitaire, Maisons-Alfort, France (S. Lecollinet); Acad-
viviruses, necessitating the use of virus-specific neutraliza-
emy of Sciences of the Czech Republic, Brno, Czech Republic (Z.
tion assays for identification of an etiologic flavivirus.
Hubálek, P. Svobodová); and Sultan Qaboos University, Muscat,
The population in our study had a 2-fold higher infection
Oman (N. Nowotny)
rate than that observed in a similar study in Austria in 1999 in
DOI: http://dx.doi.org/10.3201/eid1904.121450 a population of 468 horses (6). A partial explanation for this

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 635
DISPATCHES

Figure. Percentages of horses

seropositive for tick-borne encephalitis
virus (TBEV) by age and sex, Austria,
2011. Geldings were excluded for
better illustration. The difference
between groups was significant for
young age (p<0.001) and male sex
(p = 0.001).

difference might be yearly fluctuating TBEV prevalence, as The reason for the high number of seropositive
measured by diagnosed human infections (http://zecken.at/ stallions is unclear because stallions in the study were
fsme/fsme-faelle-in-oesterreich/). In 1999, the lowest num- distributed among all 3 locations, and 33 (67.3%) of 49
ber (41) of human TBE cases was recorded in Austria (87, were boarded in boxes (individual stable compartments
79, 63, and 113 human cases were diagnosed in 2008, 2009, that limit contact with other members of the population).
2010, and 2011, respectively). A study in Germany in 2006 Mares were kept exclusively in 1 TBEV-endemic loca-
identified 2.9% of 240 horses with TBEV neutralizing anti- tion, mainly in pastures. However, stallions were more
bodies (7). A more recent update on TBEV seropositivity in frequently transferred to other regions (e.g., for mating),
other animal species showed prevalence rates of 26.5% in where they might have been infected because of poten-
cattle and 7.0% in sheep (10). This study did not detect anti- tially higher tick infestation rates. It is also possible that
bodies against TBEV in 40 horses. A study on the prevalence for unknown biological reasons males are more frequent-
of TBEV in dogs in Austria reported that 131 (24.0%) of 545 ly affected by ticks than females, as suggested by Perkins
dogs examined had antibodies against TBEV (13). et al. (15) in a study on the yellow-necked mouse (Apode-
We did not observe any influence of location on the mus flavicollis).We plan to conduct further experiments
likelihood of TBEV seropositivity. All 3 locations are within to elucidate why ticks seem to be more attracted to male
TBEV-endemic areas (http://zecken.at/fsme/verbreitungs- hosts than female hosts.
gebiete/). However, none of the seropositive horses showed We observed comparatively low antibody prevalence in
any clinical symptoms of an arbovirus infection at any time. yearlings of both sexes, which was probably caused by de-
Regarding age distribution, a higher proportion of creasing, but still protective, maternally transmitted immu-
younger horses (mean age 5.9 years) had antibodies against nity. Seropositivity peaks in both sexes at 4, 7, 11, 13, and
TBEV than older horses (e.g., none of the horses 15–18 and 19–20 years of age indicated infections and subsequent rein-
21–32 years of age had antibodies against TBEV). Older fections in certain years with higher TBEV activity (Figure).
horses were in the same pastures as young horses. This find- Our study suggests that horses are prone to TBEV in-
ing contrasts with results of epidemiologic studies in cattle, fection. However, they remain mostly asymptomatic. Thus,
in which animals ≤3 years of age showed a lower prevalence horses may be considered sentinel hosts for monitoring the
of antibodies against TBEV than did older animals (10). . spread of TBEV.
Tick exposure has always been high in the investigated This study was partially supported by European Union grants
areas, as reported in a study conducted >10 years ago, in HEALTH.2010.2.3.3-3 Project 261391 EuroWestNile (http://
which 52%–93% of horses were positive for antibodies eurowestnile.isciii.es/ewn) and FP7-261504 EDENext and is
against Borrelia afzelii by immunoblotting. Most of these catalogued by the EDENext Steering Committee as EDENext073
horses had already been infected during their first year of (www.edenext.eu).
age and were subsequently reinfected (14). Thus, older
horses in our study might have been infected at a young Dr Rushton is a second-year PhD student at the University of
age and showed a subsequent decrease in neutralizing anti- Veterinary Medicine, Vienna, Austria. His research interests are
bodies below the detection limit. equine ophthalmology and virology.

636 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Tick-borne Encephalitis Virus in Horses, Austria

References 11. Bakonyi T, Ivanics E, Erdélyi K, Ursu K, Ferenczi E, Weissenböck

H, et al. Lineage 1 and 2 strains of encephalitic West Nile virus,
1. Pfeffer M, Dobler G. Tick-borne encephalitis virus in dogs: is this an central Europe. Emerg Infect Dis. 2006;12:618–23. http://dx.doi.
issue? Parasit Vectors. 2011;4:59. http://dx.doi.org/10.1186/1756- org/10.3201/eid1204.051379
3305-4-59 12. Wodak E, Richter S, Bagó Z, Revilla-Fernández S, Weissenböck
2. Holzmann H, Aberle SW, Stiasny K, Werner P, Mischak A, Zainer B, H, Nowotny N, et al. Detection and molecular analysis of West
et al. Tick-borne encephalitis from eating goat cheese in a mountain Nile virus infections in birds of prey in the eastern part of Austria
region of Austria. Emerg Infect Dis. 2009;15:1671–3. http://dx.doi. in 2008 and 2009. Vet Microbiol. 2011;149:358–66. http://dx.doi.
org/10.3201/eid1510.090743 org/10.1016/j.vetmic.2010.12.012
3. Bagó Z, Bauder B, Kolodziejek J, Nowotny N, Weissenböck H. 13. Kirtz G, Kölbl S, Czettel B, Thalhammer JG. Tick-borne-enceph-
Tickborne encephalitis in a mouflon (Ovis ammon musimon). Vet alitis (TBE) in dogs in Austria: a serological prevalence study [in
Rec. 2002;150:218–20. http://dx.doi.org/10.1136/vr.150.7.218 German]. Kleintierpraxis. 2003;48:133–40.
4. Süss J. Tick-borne encephalitis 2010: epidemiology, risk areas, and 14. Müller I, Khanakah G, Kundi M, Stanek G. Horses and Bor-
virus strains in Europe and Asia: an overview. Ticks Tick Borne Dis. relia: immunoblot patterns with five Borrelia burgdorferi sensu
2011;2:2–15. http://dx.doi.org/10.1016/j.ttbdis.2010.10.007 lato strains and sera from horses of various stud farms in Austria
5. Hubálek Z, Rudolf I. Tick-borne viruses in Europe. Parasitol Res. and from the Spanish Riding School in Vienna. Int J Med Micro-
2012;111:9–36. http://dx.doi.org/10.1007/s00436-012-2910-1 biol. 2002;291(Suppl 33):80–7. http://dx.doi.org/10.1016/S1438-
6. Luckschander N, Kolbl S, Enzesberger O, Zipko HT, Thalhammer 4221(02)80017-0
JG. Tick borne encephalitis (TBE) in an Austrian horse population 15. Perkins SE, Cattadori IM, Tagliapietra V, Rizzoli AP, Hudson PJ.
(in German). Tierarztliche Praxis Ausgabe G. 1999;27:235–8. Empirical evidence for key hosts in persistence of a tick-borne dis-
7. Müller K, König M, Thiel HJ. Tick-borne encephalitis (TBE) with ease. Int J Parasitol. 2003;33:909–17. http://dx.doi.org/10.1016/
special emphasis on infection in horses [in German]. Dtsch Tierarztl S0020-7519(03)00128-0
Wochenschr. 2006;113:147–51.
8. Waldvogel A, Matile H, Wegmann C, Wyler R, Kunz C. Tick-borne Address for correspondence: Norbert Nowotny, Viral Zoonoses, Emerging
encephalitis in the horse [in German]. Schweiz Arch Tierheilkd.
and Vector-Borne Infections Group, Institute of Virology, University of
1981;123:227–33.
9. Vittecoq M, Lecollinet S, Jourdain E, Thomas F, Blanchon T, Arnal Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria;
A, et al. Recent circulation of West Nile and other flaviviruses in email: norbert.nowotny@vetmeduni.ac.at
southern France. Vector Borne Zoonotic Dis. 2013. In press.
10. Sikutová S, Hornok S, Hubálek Z, Dolezálková I, Juricová Z, Rudolf All material published in Emerging Infectious Diseases is in
I. Serological survey of domestic animals for tick-borne encepha- the public domain and may be used and reprinted without
litis and Bhanja viruses in northeastern Hungary. Vet Microbiol. special permission; proper citation, however, is required.
2009;135:267–71. http://dx.doi.org/10.1016/j.vetmic.2008.09.082

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 637
DISPATCHES

Hepatitis Virus Myanmar; the counties are adjacent to Yunnan Province,

People’s Republic of China. The bats covered 6 species:
in Long-Fingered Miniopterus fuliginosus (n = 640), Hipposideros armiger
(n = 8), Rhinolophus ferrumequinum (n = 176), Myotis chi-
Bats, Myanmar nensis (n = 11), Megaderma lyra (n = 6), and Hipposideros
fulvus (n = 12). All bat tissue samples were subjected to vi-
Biao He,1 Quanshui Fan,1 Fanli Yang, ral metagenomic analysis (unpublished data). The sampling
Tingsong Hu, Wei Qiu, Ye Feng, Zuosheng Li, of bats for this study was approved by the Administrative
Yingying Li, Fuqiang Zhang, Huancheng Guo, Committee on Animal Welfare of the Institute of Military
Xiaohuan Zou, and Changchun Tu Veterinary, Academy of Military Medical Sciences, China.
We used PCR to further study the prevalence of or-
During an analysis of the virome of bats from Myanmar, thohepadnavirus in the 6 bat species; the condition of the
a large number of reads were annotated to orthohepadnavi- samples made serologic assay and pathology impracticable.
ruses. We present the full genome sequence and a morpho- Viral DNA was extracted from liver tissue of each of the
logical analysis of an orthohepadnavirus circulating in bats. 853 bats by using the QIAamp DNA Mini Kit (QIAGEN,
This virus is substantially different from currently known
Hilden, Germany). To detect virus in the samples, we con-
members of the genus Orthohepadnavirus and represents
a new species.
ducted PCR by using the TaKaRa PCR Kit (TaKaRa, Da-
lian, China) with a pair of degenerate pan-orthohepadnavi-
rus primers (sequences available upon request). The PCR

T he family Hepadnaviridae comprises 2 genera (Ortho-

hepadnavirus and Avihepadnavirus), and viruses clas-
sified within these genera have a narrow host range. The
reaction was as follows: 45 cycles of denaturation at 94°C
for 30 s, annealing at 54°C for 30 s, extension at 72°C for
40 s, and a final extension at 72°C for 7 min. Positive re-
genus Orthohepadnavirus consists of pathogens that infect sults were obtained for 22 long-fingered bats (Miniopterus
mammals, and it currently contains 4 species: hepatitis B fuliginosus). Of these bats, 2.19% (7/320) were from Sedon
virus, woodchuck hepatitis virus, ground squirrel hepati- County and 4.69% (15/320) from Wutao County; the viruses
tis virus, and woolly monkey hepatitis B virus. The genus they harbored shared >98% nt identity. No other species had
Avihepadnavirus contains 2 avian species: duck hepatitis positive amplification results, indicating that M. fuliginosus
B virus and heron hepatitis B virus (1). Hepadnaviruses was the most likely species to harbor orthohepadnaviruses.
mainly infect the liver cells of their hosts and, in humans, Of the 22 positive samples, 3 were randomly selected
cause hepatitis B, cirrhosis, and hepatocellular carcinoma for full genome amplification: M086 from Sedon County
(2). Approximately 2 billion persons worldwide are infect- and 776 and M005 from Wutao County. PCR was con-
ed with hepatitis B virus (HBV), and 600,000 persons die ducted by using the PCR protocol defined above with high-
every year from the consequences of hepatitis B (3). fidelity Pfu DNA polymerase (Promega, Madison, WI,
Bats are associated with an increasing number of USA) and 4 pairs of specific primers (sequences available
emerging and reemerging viruses, many of which pose upon request). Four overlapping amplicons were obtained,
major threats to public health (4). We conducted a viral sequenced in both directions, and assembled into the full
metagenomic analysis of 6 species of bats from Myanmar. genomic sequence by using SeqMan, version 7.1.0 (DNA-
The analysis revealed a large number of viral contigs an- STAR, Madison, WI, USA). All 3 full genomes (GenBank
notated to orthohepadnavirus with <70% nt identity (B. accession nos. JX941466– JX941468) were 3,230 nt in
He, unpub. data), suggesting the presence of orthohepad- length, which is close to the size of primate hepatitis viruses
naviruses in these animals. We describe the virus by full (≈3,200 nt) but smaller than rodent hepatitis viruses (≈3,300
genomic analysis and morphologic observation. nt). We analyzed the genome structure by using Vector NTI
Advance 10 (Invitrogen, Carlsbad, CA). The results showed
The Study that the bat hepatitis viruses (BtHVs) contained the same
We purchased 853 freshly killed insectivorous bats in circular and compact genomic structure as other orthohe-
Sedon and Wutao Counties in southeastern Kachin State, padnaviruses, comprising 4 open reading frames encoding
the multifunctional Pol, preS1/preS2/S, preC/C, and X pro-
Author affiliations: Academy of Military Medical Sciences, Changc-
teins in the same direction (Figure 1, panel A).
hun, People’s Republic of China (B. He, F. Yang, Y. Feng, Y. Li, H.
Genomic sequence comparison and phylogenetic anal-
Guo, X. Zou, C. Tu); and Center for Disease Control and Preven-
ysis based on amino acids of the pol gene (2,562 bp) were
tion of Chengdu Military Region, Kunming, People’s Republic of
constructed with ClustalW version 2.0 (www.clustal.org/)
China (Q. Fan, T. Hu, W. Qiu, Z. Li, F. Zhang)

DOI: http://dx.soi.org/10.3201/eid1904.121655 1
These authors contributed equally to this article.

638 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Hepatitis Virus in Long-Fingered Bats, Myanmar

Figure 1. Predicted schematic representation of the bat hepatitis virus (BtHV) genome and its phylogenetic relationship with other
hepadnaviruses. A) Genomic structural map of BtHV. Boxes and arrows represent the open reading frames encoding the main proteins:
pol gene (2,305–1,636), preS1/S2 and S gene (2,864–833), preC/C gene (1,815–2,468) and X gene (1,378–1,812). Two 12-nt direct
repeat sequences (DR1 from 1,825 to 1,836 and DR2 from 1,594 to 1,605), the encapsidation signal e (1,848–1,903), and YMDD domain
(734–745) are also depicted in the map. B) Phylogenetic analysis of BtHVs and other hepadnaviruses based on amino acid sequences
of pol genes. Representatives of hepadnavirus species belonging to Orthohepadnavirus and Avihepadnavirus genera were used; their
GenBank accession nos. are shown in the trees. The different genotypes of human hepatitis B virus are also included. The 3 BtHV isolates
are identified by black triangles. Scale bar indicates nucleotide substitutions per site.

and MEGA5 (5). Phylogenetic tree analysis showed that Hepadnaviruses have not been grown in any avail-
previously described orthohepadnaviruses formed 2 clus- able in vitro cell system; thus, we did not attempt to iso-
ters, primate hepatitis viruses and rodent hepatitis viruses, late BtHV in cell culture. To detect the presence of virus
whereas the 3 newly identified BtHVs formed an indepen- particles, we used pooled liver tissues from the 3 bats that
dent cluster within the Orthohepadnavirus genus (Figure were randomly selected for full genome amplification. We
1, panel B). Sequence comparison showed that the full ge- homogenized the pooled tissues in SM buffer (50 mM Tris,
nomes of the BtHVs were 63.1%–65.3% and 33.9%–34.8% 10 mM MgSO4, 0.1M NaCl; pH7.5), followed by clarifica-
identical to members of the Orthohepadnavirus and Avi- tion by low-speed centrifugation to remove cell debris. We
hepadnavirus genera, respectively. Similar low identities then passed the pooled sample through a 0.22-µm syringe
were also observed separately in the 4 genes of the BtHVs filter (Millipore, Carrigtwohill, Ireland). Polyethylene
(Table). These results support the classification of the BtH- glycol 6000 was added, and the resulting precipitate was
Vs within the Orthohepadnavirus genus, being distantly sedimented at 12,000 × g in a desktop centrifuge (Eppen-
related to current species and likely to form a new species dorf, Hamburg, Germany) for 40 min at 4°C. The pellet
designated as BtHV. was resuspended and examined after negative staining in a

Table. Gene lengths and percentage identity between bat orthohepadnavirus and other hepadnaviruses*
pol gene preS1/preS2/S gene preC/C gene X gene
Virus† nt % ID aa % ID nt % ID aa % ID nt % ID aa % ID nt % ID aa % ID
BtHV776 2562 – 853 – 1200 – 399 – 654 – 217 – 435 – 144 –
HBV 2532 63 843 57 1203 63 400 59 639 65 212 66 465 61 154 49
WMHBV 2508 63 835 55 1176 64 391 60 636 65 211 63 459 66 152 50
WHV 2640 66 879 56 1281 66 426 51 678 69 225 71 426 67 141 44
ASHV 2634 67 877 53 1284 67 427 52 654 68 217 71 417 69 138 52
DHBV 2526 41 841 30 1104 43 367 30 888 42 295 22 NA – NA –
*nt, nucleotide length; % ID, percentage identity of nt and amino acid sequence between BtHV and other viruses; aa, amino acid length; BtHV, bat
hepatitis virus; –, not applicable; HBV, hepatitis B virus; WMHBV, woolly monkey HBV; WHV, woodchuck hepatitis virus; ASHV, arctic squirrel hepatitis
virus; DHBV, duck HBV; NA, not available.
†GenBank accession nos. for HBV, WMHBV, WHV, ASHV, and DHBV are D00329, AF046996, AY344076, U29144, and EU429324, respectively.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 639
DISPATCHES

(although no evidence of hepadnavirus was found in any of

the 176 R. ferrumequinum bats) or to a narrow host range of
the virus. Further study is required to determine the tropism
and prevalence of BtHVs in other bat species.

This study was supported by the National Natural Science

Foundation of China–Yunnan Province Joint Fund (U1036601)
and the National “973” Program (grant no. 2012CB722501)
to C.T.
Mr He is a doctoral candidate at Institute of Military Veteri-
nary, Academy of Military Medical Sciences. He is majoring in
animal virology, with research interests focusing on the discovery
of bat emerging viruses.

References

1. International Committee on Taxonomy of Viruses. ICTV 2011 mas-

ter species list v2 [cited 2012 Aug 20]. http://talk.ictvonline.org/
files/ictv_documents/m/msl/4090.aspx
2. Seeger C, Mason WS. Hepatitis B virus biology. Microbiol Mol Biol
Rev. 2000;64:51–68. http://dx.doi.org/10.1128/MMBR.64.1.51-
68.2000
3. World Health Organization. Hepatitis B, fact sheet no.204, July 2012
[cited 2012 Jul 16]. http://www.who.int/mediacentre/factsheets/
fs204/en/index.html
4. Calisher CH, Childs JE, Field HE, Holmes KV, Schountz T. Bats:
important reservoir hosts of emerging viruses. Clin Microbiol Rev.
2006;19:531–45. http://dx.doi.org/10.1128/CMR.00017-06
Figure 2. Electron microscopy of negative-stained orthohepadnavirus 5. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S.
particles (arrow) from a bat. Clumps of Australia antigen–like particles MEGA5: Molecular Evolutionary Genetics Analysis using maxi-
are seen. mum likelihood, evolutionary distance, and maximum parsimony
methods. Mol Biol Evol. 2011;28:2731–9. http://dx.doi.org/10.1093/
molbev/msr121
JEM-1200 EXII transmission electron microscope (JEOL, 6. Dane DS, Cameron CH, Briggs M. Virus-like particles in serum
Tokyo, Japan). Numerous spherical particles of ≈20 nm di- of patients with Australia-antigen–associated hepatitis. Lancet.
1970;295:695–8. http://dx.doi.org/10.1016/S0140-6736(70)90926-8
ameter were observed (Figure 2). The particles were mor- 7. Heermann KH, Goldmann U, Schwartz W, Seyffarth T, Baumgarten
phologically similar to the Australia antigens of HBV, the H, Gerlich WH. Large surface proteins of hepatitis B virus contain-
most abundant viral component found in HBV-infected ing the pre-s sequence. J Virol. 1984;52:396–402.
humans and animals and also known as surface protein or 8. Hu KL, Wei L, Zhu TT, Wang XZ, Zhang LB. Dietary composition,
echolocation pulses and morphological measurements of the long-fin-
S antigen (6,7). PCR amplification of DNA extracted from gered bat Miniopterus fuliginosus (Chiroptera: Vespertilioninae) [in
the virus pellet revealed the full genome of the BtHV, with Chinese]. Dongwuxue Yanjiu Dongwuxue Yanjiu. 2011;32:163–7.
the expected size of ≈3200 bp (image not shown). 9. Shirato K, Maeda K, Tsuda S, Suzuki K, Watanabe S, Shimoda H,
et al. Detection of bat coronaviruses from Miniopterus fuliginosus
in Japan. Virus Genes. 2012;44:40–4. http://dx.doi.org/10.1007/
Conclusions s11262-011-0661-1
Our observations provide strong evidence for the cir- 10. Watanabe S, Maeda K, Suzuki K, Ueda N, Iha K, Taniguchi S, et
culation of orthohepadnaviruses in at least 1 species of al. Novel betaherpesvirus in bats. Emerg Infect Dis. 2010;16:986–8.
bats, M. fuliginosus, in Myanmar. These bats have a wide http://dx.doi.org/10.3201/eid1606.091567
distribution (8), and increasing numbers of viruses, includ-
Address for correspondence: Changchun Tu, Institute of Military
ing coronaviruses and betaherpesviruses, are being isolated
Veterinary, Academy of Military Medical Sciences, 666 Liuying West
from them (9,10). Of the 6 bat species we sampled, only
Rd, Jingyue Economic Development Zone, Changchun 130122, People’s
M. fuliginosus was positive for BtHV. The prevalence of
Republic of China; email: changchun_tu@hotmail.com
BtHV-positive bats in the 2 counties from which we ob-
tained bats, was 2.2% and 4.7%, respectively, indicating
The opinions expressed by authors contributing to this
that this species is likely a natural reservoir host of BtHV. journal do not necessarily reflect the opinions of the Centers for
The lack of detection of BtHV in bats from the other 5 spe- Disease Control and Prevention or the institutions with which
cies may be due to the limited numbers of bats sampled the authors are affiliated.

640 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Hand, Foot, and cases per 100,000 population or >36.000 cases; the 2012
outbreak included 2 fatal cases of EV71 encephalitis (11).

Mouth Disease In this outbreak, 2 clinical patterns were observed, and 2

case definitions were applied. Suspected HFMD cases were
Caused by defined as painful blisters in the oropharynx and blisters on
the palms, soles, knees, elbows, and/or buttocks. Suspected
Coxsackievirus herpangina cases were defined as painful blisters in the

A6, Thailand,
mouth only, predominantly on the soft palate. Suspected
HFMD and herpangina cases were virologically confirmed

2012
if samples were positive for viral RNA by nested PCR.
During January–October 2012, a total of 847 samples
were collected from 825 patients with suspected cases.
Jiratchaya Puenpa, Thaweesak Chieochansin, Among those 825 patients, the diagnosis was HFMD for
Piyada Linsuwanon, Sumeth Korkong, 672 (81.4%) and herpangina for 153 (18.6%). Patients’
Siwanat Thongkomplew, ages ranged from 1 month to 38 years. The samples were
Preyaporn Vichaiwattana, collected from hospitalized patients and outpatients who
Apiradee Theamboonlers, and Yong Poovorawan had a clinical diagnosis of HFMD or herpangina and who
came from different parts of Thailand: Bangkok, 566 cases;
In Thailand, hand, foot, and mouth disease (HFMD) is
Khonkaen, 252 cases; Suphanburi, 4 cases; and Saraburi,
usually caused by enterovirus 71 or coxsackievirus A16.
To determine the cause of a large outbreak of HFMD in Rayong, and Chantaburi, 1 case each (Figure 1). Of the 847
Thailand during June–August 2012, we examined patient samples, 695 were rectal swabs, 73 fecal, 39 throat swabs,
specimens. Coxsackievirus A6 was the causative agent. To 20 serum, 9 vesicle fluid, 7 nasal swabs, 3 cerebrospinal
improve prevention and control, causes of HFMD should fluid, and 1 saliva. All samples, other than stool samples,
be monitored. were collected in virus transport media modified according
to recommendations by the World Health Organization (12).

C
Fecal samples were diluted 1:10 with phosphate-buffered
oxsackievirus A6 (CAV6) is 1 of 10 genotypes within
saline and centrifuged, and the supernatant was collected
the family Picornaviridae, genus Enterovirus, species
for testing. Viral RNA was extracted from 200-mL samples
Human enterovirus A. Other genotypes include coxsacki-
evirus A16 (CAV16) and enterovirus 71 (EV71). Although
CAV6 is commonly associated with hand, foot, and mouth
disease (HFMD) and herpangina (1,2), it has not been of
concern until the recent global outbreaks of HFMD (3–6).
In Thailand, the viruses predominately associated with
HFMD have been EV71 and CAV16 (7,8); to our knowledge,
CAV6 has not been implicated. In 2012, extensive outbreaks
of HFMD occurred in Thailand. To determine the pattern,
causative agents, and clinical manifestations of HFMD in
this 2012 outbreak, we analyzed specimens from patients.
This study was approved by the institutional review board
of the Faculty of Medicine, Chulalongkorn University;
the requirement for written informed consent was waived
because the samples were analyzed anonymously.

The Study
In Thailand, HFMD usually occurs during the rainy
season (June–August); average incidence during 2007–2011
was 20.2 cases per 100,000 population (9,10). In 2012, an
extensive outbreak of HFMD occurred; the incidence rate
was 3-fold higher than the average incidence rate of 58.15

Author affiliation: Chulalongkorn University, Bangkok, Thailand

Figure 1. Location of sample collection sites during outbreak of
DOI: http://dx.doi.org/10.3201/eid1904.121666 hand, foot, and mouth disease, Thailand, January–October 2012.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 641
DISPATCHES

Figure 2. Weekly number of reported

suspected cases of hand, foot, and mouth
disease and herpangina during outbreak,
Thailand, 2012. EV, enterovirus; CA6,
coxsackievirus 6; CA16, coxsackievirus
16; EV71, enterovirus 71.

by using the Viral Nucleic Acid Extraction Kit (RBC cases, 13.7% were caused by CAV6 and 1.3% by CAV16.
Bioscience, Taipei, Taiwan) according to the manufacturer’s Moreover, samples from 51.0% of patients with herpangina
instructions. cDNA was synthesized by using the ImProm- were positive for an untyped enterovirus (Table).
II Reverse Transcription System (Promega, Madison, WI, Generally, the clinical manifestations of HFMD
USA) with random hexamers as primers according to the were fever; drooling, and refusal to eat (among young
manufacturer’s recommendation (First BASE Laboratories, children); painful lesions in the mouth, especially on the
Selangor Darul Ehsan, Malaysia). soft palate (online Technical Appendix Figure 2, panel
To identify enteroviruses, we performed 3 separate A); and vesicular rashes on the palms and feet (online
PCRs. The first PCR, which could detect most enteroviruses, Technical Appendix Figure 2, panels B, C). For patients
was used to screen for panenterovirus. The 5′ untranslated affected by this outbreak, physicians from reporting sites
region of the viruses was amplified by nested PCR as reported anecdotally that they observed more severe skin
described (13). The second PCR was selective for EV71 manifestations than usual, especially on the buttocks
and CAV16; the primers and reaction conditions were and perianal area (online Technical Appendix Figure 2,
identical to those used in a previous study (7). The third panel D), knees, and elbows. Two cases with neurologic
PCR, for CAV6 detection, used primers designed to amplify involvement (convulsion, altered consciousness) were
the viral protein (VP) 1 gene by seminested PCR with CU- caused by EV71 and were treated with intravenous
EVF2632 (5′-TGTGTGATGAATCGAAACGGGGT-3′) immunoglobulin. No patients died.
and CU-EVR3288 (5′-TGCAGTGTTAGTTATTGT Direct sequencing was performed on the VP1 region
TTGGCT-3′) as first-round primers and CU-EVR3053 of 143 randomly selected CAV6-positive samples. The
(5′-GGGTAACCATCATAAAACCACTG-3′) as a reverse sequences were submitted to GenBank under accession
primer for the second round. The expected 420-bp PCR nos. JX556422–JX556564.
product was examined under UV light after being resolved The VP1 nucleotide sequences of CAV6 were aligned
in 2% agarose gel electrophoresis and subsequently stained with the reference sequences by using ClustalW in the BioEdit
with ethidium bromide. program version 7.0.9.0 (www.mbio.ncsu.edu/BioEdit/
Most samples were collected during the rainy season, bioedit.html). A phylogenetic tree was constructed with
from the end of June through early August 2012 (weeks MEGA software, version 5.0, by applying the maximum-
25–32), which accounted for 83.1% of all reported cases. likelihood method and using the Kimura 2-parameter model,
Altogether, enterovirus results were positive for 459 (68.3%) in which 1,000 replications were selected for bootstrapping
HFMD and 101 (66.0%) herpangina patients (Figure 2), (14) (online Technical Appendix Figure 3). The sequences
Of note, 93.1% of patients were <5 years of age. A of EV71 strain BrCr (accession no. U22521) and CAV16
high proportion of cases was found among children 1, 2, strain G10 (accession no. U05876) were used as outgroups
and 3 years of age and accounted for 68.4% of HFMD in the phylogenetic analysis.
cases and 64.2% of herpangina cases (online Technical The relationship between the CAV6 characterized in
Appendix Figure 1, wwwnc.cdc.gov/EID/article/19/4/12- this study and the prototype strain (Gdula) was investigated
1666-Techapp1.pdf). by phylogenetic analysis of partial VP1 sequences. All
Of the 672 HFMD cases, 221 (32.9%) were caused CAV6 clustered in the same lineage and with the reference
by CAV6, 62 (9.2%) by EV71, 62 (9.2%) by CAV16, and strain CAV6 (Gdula); nucleotide homologies among these
114 (17.0%) by untyped enteroviruses. Of the herpangina strains were 81.4%–84.7%.

642 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Coxsackievirus A6, Thailand

Table. Causative agents identified during hand, foot, and mouth References
disease outbreak, Thailand, 2012
No. (%) cases 1. Yamashita T, Ito M, Taniguchi A, Sakae K. Prevalence of
Hand, foot, and mouth coxsackievirus A5, A6, and A10 in patients with herpangina in Aichi
Virus disease Herpangina Prefecture, 2005. Jpn J Infect Dis. 2005;58:390–1.
Coxsackievirus A6 221 (32.9) 21 (13.7) 2. Mirand A, Henquell C, Archimbaud C, Ughetto S, Antona D, Bailly JL,
Coxsackievirus A16 62 (9.2) 2 (1.3) et al. Outbreak of hand, foot and mouth disease/herpangina associated
Enterovirus A71 62 (9.2) 0 with coxsackievirus A6 and A10 infections in 2010, France: a large
Panenterovirus only 114 (17.0) 78 (51.0) citywide, prospective observational study. Clin Microbiol Infect.
None detected 213 (31.7) 52 (34.0) 2012;18:E110–8. http://dx.doi.org/10.1111/j.1469-0691.2012.03789.x
Total 672 153 3. Fujimoto T, Iizuka S, Enomoto M, Abe K, Yamashita K, Hanaoka N.
Hand, foot, and mouth disease caused by coxsackievirus A6, Japan,
2011. Emerg Infect Dis. 2012;18:337–9. http://dx.doi.org/10.3201/
eid1802.111147
Conclusions 4. Centers for Disease Control and Prevention. Notes from the field:
Although the positive samples collected during severe hand, foot, and mouth disease associated with coxsackievirus
January–October 2012 were mostly from patients in A6—Alabama, Connecticut, California, and Nevada, November 2011–
Bangkok and Khonkaen, they partially represented the February 2012. MMWR Morb Mortal Wkly Rep. 2012;61:213–4.
5. Flett K, Youngster I, Huang J, McAdam A, Sandora TJ, Rennick
HFMD and herpangina cases in Thailand’s 30,000- M, et al. Hand, foot, and mouth disease caused by coxsackievirus
case outbreak. Virus prevalence in Thailand was A6. Emerg Infect Dis. 2012;18:1702–4. http://dx.doi.org/10.3201/
highest in HFMD and herpangina patients 1–3 years of eid1810.120813
age (Technical Appendix Figure 1). For this seasonal 6. Österback R, Vuorinen T, Linna M, Susi P, Hyypiä T, Waris M.
Coxsackievirus A6 and hand, foot, and mouth disease, Finland.
outbreak, the most common causative agent was CAV6. Emerg Infect Dis. 2009;15:1485–8. http://dx.doi.org/10.3201/
All CAV6 strains shared an isolated cluster and had eid1509.090438
high similarity, as shown in the phylogenetic analysis 7. Puenpa J, Theamboonlers A, Korkong S, Linsuwanon P, Thongmee
of VP1 region. Although CAV6 has been a predominant C, Chatproedprai S, et al. Molecular characterization and complete
genome analysis of human enterovirus 71 and coxsackievirus
emerging pathogen since 2012, no patients infected with A16 from children with hand, foot and mouth disease in Thailand
CAV6 died. According to the study conducted during during 2008–2011. Arch Virol. 2011;156:2007–13. http://dx.doi.
2008–2011 EV71 and CAV16 were the main pathogens org/10.1007/s00705-011-1098-5
contributing to the disease (7). However, we found a 8. Chatproedprai S, Theanboonlers A, Korkong S, Thongmee C,
Wananukul S, Poovorawan Y. Clinical and molecular characterization
different main pathogen: CAV6. For prevention and of hand-foot-and-mouth disease in Thailand, 2008–2009. Jpn J
control of future outbreaks, the causes of HFMD should Infect Dis. 2010;63:229–33.
be monitored. 9. Bureau of Epidemiology, Department of Disease Control. Hand,
foot and mouth disease [in Thai] [cited 2012 Oct 29]. http://www.
boe.moph.go.th/
Acknowledgments 10. Thongcharoen P. Hand, foot and mouth disease. Thailand: world-
We thank Petra Hirsch and Noppawat Charoensinphon for shaking outbreaks; 2012; vol 19. Bangkok (Thailand): Aksorn
reviewing the manuscript. Sampan Press; 2012. p. 136–44.
11. Bureau of Epidemiology, Department of Disease Control. Hand,
The study was supported by grants from The Higher foot and mouth disease, 506 surveillance report [in Thai] [cited
Education Research Promotion and National Research University 2012 Oct 29]. http://www.boe.moph.go.th/boedb/surdata/506wk/
y55/d71_4055.pdf
Project of Thailand Office of the Higher Education Commission 12. World Health Organization. Collecting, preserving and shipping
(HR1155A-55); the National Research Council of Thailand, specimens for the diagnosis of avian influenza A(H5N1) virus
Center of Excellence in Clinical Virology, Chulalongkorn infection. 2006:42–3 [cited 2012 Oct 29]. http://www.who.int/csr/
University; Chulalongkorn University Centenary Academic resources/publications/surveillance/Annex8.pdf
13. Kapusinszky B, Szomor KN, Farkas A, Takács M, Berencsi G.
Development Project, Integrated Innovation Academic Center; Detection of non-polio enteroviruses in Hungary 2000–2008 and
Chulalongkorn University Centenary Academic Development molecular epidemiology of enterovirus 71, coxsackievirus A16,
Project (CU56-HR01); Outstanding Professor of the Thailand and echovirus 30. Virus Genes. 2010;40:163–73. http://dx.doi.
Research Fund (DPG5480002); the RGJ PhD program org/10.1007/s11262-009-0440-4
14. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar
(PHD/0087/2554); and by generous support from the National S. MEGA5: molecular evolutionary genetics analysis using
Research Council of Thailand and King Chulalongkorn maximum likelihood, evolutionary distance, and maximum
Memorial Hospital. parsimony methods. Mol Biol Evol. 2011;28:2731–9. http://dx.doi.
org/10.1093/molbev/msr121
Ms Puenpa is a researcher who works in the Center of
Excellence in Clinical Virology, Department of Pediatrics, Address for correspondence: Yong Poovorawan, Center of Excellence
Faculty of Medicine, Chulalongkorn University. Her research in Clinical Virology, Department of Pediatrics, Faculty of Medicine,
interests focus on the molecular epidemiology of human Chulalongkorn University, Bangkok 10330, Thailand; email: yong.p@
enteroviruses. chula.ac.th

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 643
DISPATCHES

Early Gabon is a typical humid, tropical, forested country in

Central Africa, with 1,517,685 inhabitants and a surface

Introduction area of 270,000 km2. The country has a short dry season
during January–February, a long rainy season during
and Delayed March–May, a long dry season during June–September,
and a short rainy season during October–December. We
Dissemination report the results of a large surveillance study for pH1N1 in

of Pandemic
Gabon during a 2-year period, July 2009–June 2011.

Influenza, Gabon
The Study
Surveillance for influenza-like illness (ILI) was
performed during July 2009–June 2011 in the capital city
Sonia Etenna Lekana-Douki, of Gabon, Libreville, and in 3 other towns in rural Gabon
Augustin Mouinga-Ondémé, Dieudonné Nkoghe, (Franceville, Oyem, and Koulamoutou) (Figure 1). ILI was
Christian Drosten, Jan Felix Drexler, defined as fever (>38°C) and runny nose, cough, or sore
Mirdad Kazanji, and Eric M. Leroy throat. Study participants were enrolled at 3 health care
centers in Libreville and at the regional hospitals in the
Active surveillance in health care centers in Gabon
other towns; all patients who visited these health centers for
during 2009–2011 detected 72 clinical cases of pandemic
(H1N1) 2009 (pH1N1). We found that pH1N1 virus was ILI were systematically sampled. Individual oral consent
introduced in mid-2009 but spread throughout the country was obtained from patients for nasal sampling.
in 2010. Thus, Gabon was also affected by pH1N1. Epidemiologic data (name, age, sex, and travel
history during the month before onset) and clinical
data were collected for each patient. Nasal swabs were

I n April 2009, a pandemic strain of influenza A (H1N1)

(pH1N1) virus emerged in Mexico and the United
States; the World Health Organization declared a pan-
sent each week to Centre International de Recherches
Médicales de Franceville for analysis. Real-time reverse
transcription PCR (RT-PCR) was used to detect pH1N1,
demic alert on June 11, 2009 (1,2). This virus was respon- seasonal influenza A (H1N1 and H3N2), and seasonal
sible for a large outbreak with thousands of cases in the influenza B viruses (12). Specimens positive for pH1N1
Reunion Islands and in several French tropical Pacific is- virus were also tested by specific quantitative PCR for
lands during July–October 2009 (3). The circulation and the following common respiratory viruses: adenovirus,
public health effects of pH1N1 virus are largely unknown
in Africa, with the exception of South Africa and Kenya,
which were heavily affected by disease outbreaks during
2009 and 2010 (4–6). Other pH1N1 cases were reported
in several countries of North, West, and East Africa and in
Madagascar (7,8).
In the humid tropical forest of Central Africa, a
study demonstrated the circulation of influenza virus in
Cameroon during 2007–2008 (9); another reported cases of
pH1N1 in Cameroon in 2009 (10). A sentinel surveillance
program for influenza in Kinshasa, Democratic Republic
of the Congo, during 2009–2011 reported several cases of
pH1N1 (11).

Author affiliations: Centre International de Recherches Médicales

de Franceville, Franceville, Gabon (S.E. Lekana-Douki, A. Mouinga-
Ondémé, D. Nkoghe, E.M. Leroy); Ministère de la Santé Publique,
Libreville, Gabon (D. Nkoghe); Bonn Medical Centre Institute of
Virology, Bonn, Germany (C. Drosten, J.F. Drexler); Institut Pasteur
Figure 1. Towns in the influenza sentinel network in Gabon. Libreville
de Bangui, Bangui, Central African Republic (M. Kazanji); and was chosen as a typical urban community; Franceville, in the
Institut de Recherche pour le Développement, Montpellier, France southeast, represents a savannah/forested rural region of 100,000
(E.M. Leroy) inhabitants; and Oyem (35,241 inhabitants) and Koulamoutou
(16,270 inhabitants), in the north and south, respectively, represent
DOI: http://dx.doi.org/10.3201/eid1904.111925 forested rural regions.

644 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Pandemic Influenza, Gabon

Table. Demographic characteristics of patients and distribution of influenza viruses and other influenza-like illnesses, Gabon, July
2009–June 2011*
Influenza virus types Total no.
Patient and illness data pH1N1 A B pH1N1 + A + B Other† patients
Sex, no. patients
M 33 4 23 60 427 487
F 39 4 28 71 408 479
Median age, y (range) 2 (2 mo–49 y) 45 (9–50 y) 2 (3 mo–41 y) 2 (2 mo–50 y) 1.58 (10 d–82 y) 1.66 (10 d–82 y)
Age group, no. patients
0–23 mo 30 0 20 50 444 494
2–4 y 25 0 17 42 219 261
>4 y 17 5 10 32 147 179
Illness, by year and town
2009
Libreville 3 (33) 6 (67) 0 9 12 21
Franceville 1 (50) 1 (50) 0 2 0 2
Koulamoutou 0 0 0 0 0 0
Oyem 0 1 (100) 0 1 1 2
Total 4 (33) 8 (67) 0 12 13 25
2010
Libreville 16 (70) 0 7 (30) 23 224 247
Franceville 1 (3) 0 34 (97) 35 135 170
Koulamoutou 15 (94) 0 1 (6) 16 42 58
Oyem 11 (85) 0 2 (15) 13 105 118
Total 43 (49) 0 44 (51) 87 506 593
2011
Libreville 11 (92) 0 1 (8) 12 165 177
Franceville 6 (60) 0 4 (40) 10 43 53
Koulamoutou 2 (50) 0 2 (50) 4 32 36
Oyem 6 (100) 0 0 6 76 82
Total 25 (78) 0 7 (22) 32 316 348
Total
Libreville 30 (68) 6 (14) 8 (18) 44 401 445
Franceville 8 (17) 1 (2) 38 (81) 47 178 225
Koulamoutou 17 (85) 0 3 (15) 20 74 94
Oyem 17 (85) 1 (5) 2 (10) 20 182 202
Total 72 (55) 8 (6) 51 (39) 131 835 966
*Values are no. (%) cases except as indicated. pH1N1, pandemic (H1N1) 2009; A, seasonal influenza A; B, seasonal influenza B.
†Influenza-like illnesses other than pH1N1 or seasonal influenza A or B. Age data were missing for 32 patients in this category.

respiratory syncytial virus, human metapneumovirus, and 51 (39%) influenza B (Table; Figure 2). No deaths
parainfluenzavirus (PIV) 1–4, enterovirus, rhinovirus, caused by pH1N1 were reported during the study period.
parechovirus, and human coronavirus (HCoV; strains For the 72 patients infected with pH1N1 virus, median age
OC43, 229E, NL63, and HKU1). Testing protocols are was 2 years (range 2 months–49 years); 76.4% of these
available on request from the authors. Patients who had patients were <4 years of age, and 23.6% were 4–49 years
laboratory-confirmed influenza were contacted several of age. The M:F sex ratio was 0.85.
months after diagnosis to determine outcome. Only 18 patients with pH1N1 harbored another
Nasal swab specimens were collected from 966 respiratory virus; this finding suggests that pH1N1 virus
patients with influenza-like symptoms during July 2009– infection was responsible for the symptoms in all pH1N1
June 2011: 445 from Libreville, 202 from Oyem, 94 virus–infected patients. Among patients with pH1N1,
from Koulamoutou, and 225 from Franceville (Table). we found co-infections with PIV1 (n = 1), PIV3 (n = 2),
Median patient age was 1.66 years (range 10 days–82 PIV4 (n = 1), HCoV 229E (n = 1), HCoV OC43 (n = 1),
years); 81% of these patients were <4 years of age, and respiratory syncytial virus (n = 7), and adenovirus (n = 5).
19% were 4–82 years of age. The M:F sex ratio was 1.02. The first laboratory-confirmed pH1N1 case (case
The number of cases of ILI increased during the 2 rainy 1), in a tourist who resided in the Reunion Islands, was
seasons and decreased during the 2 dry seasons (Figure diagnosed on July 26, 2009 (Figure 2, panel A). On his
2), which is consistent with a study showing an increase trip to Gabon, he had made changeovers in Mauritius and
of the number of influenza cases during the rainy seasons South Africa, 2 countries heavily affected by pH1N1. The
in Senegal (13). patient’s symptoms lasted ≈1 week. The second laboratory-
Among the 966 cases of ILI, 131 (13.6%) were confirmed case (case 2) was detected in Franceville 4
determined to be caused by an influenza virus: 72 (55%) months later, during the short rainy season (Figure 2,
pH1N1, 8 (6%) seasonal influenza A (H1N1 and H3N2), panels A, E). ILI developed in this patient 3 days after his

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 645
DISPATCHES

Figure 2. Clinical and laboratory-confirmed cases of pandemic (H1N1) 2009 (pH1N1), seasonal influenza A (H1N1 and H3N2), seasonal
influenza B, and other influenza-like illnesses (ILI), Gabon, July 2009–June 2011. Bars below chart indicate rainy seasons. *First imported
case; †second imported case; ‡first indigenous cases. A) Gabon; B) Libreville; C) Oyem; D) Koulamoutou; E) Franceville.

arrival in Franceville from France, which was also heavily peak in the Americas and Europe. However, this early
affected by pH1N1 during this time. introduction did not result in continuous virus circulation
The first 2 autochthonous cases were diagnosed on in the rest of the country until the short rainy season in
November 26, 2009 (cases 3), 1 week after the second imported 2010. Only during the 2011 season was there a noteworthy
case of pH1N1, during the short rainy season. Subsequently, increase in case numbers compatible with a pandemic
29 autochthonous cases were detected in Libreville, 17 in wave, suggesting a notable time lag relative to that for
Oyem, 17 in Koulamoutou, and 7 in Franceville (Table). other countries. Our findings indicate that rural tropical
Libreville was the first town with detected pH1N1 cases countries such as Gabon may serve as reservoirs for later
during the rainy season and also had the highest number spread of pH1N1 virus within the country and into other
of pH1N1 cases (Table). The first autochthonous case was countries (14,15).
detected in Oyem in early June 2010; during the 2010 short
rainy season, several pH1N1 cases were detected in Oyem, Acknowledgments
indicating pH1N1 virus dissemination throughout Gabon by We thank Philippe Yaba, André Délicat, and Philipe
that time. A total of 85% of the influenza cases in Oyem were Engandja for technical assistance and the Gabonese Ministry
pH1N1 (Table). A similar pattern of pH1N1 was observed of Health and the health care workers of the medical centers in
in Koulamoutou and Franceville during the short rainy Libreville, Franceville, Koulamoutou, and Oyem for help with
season (Figure 2, panels D, E). The percentage of pH1N1 obtaining samples.
among all influenza cases in Franceville increased from 3%
Centre International de Recherches Médicales de Franceville
in 2010 to 60% in 2011 (Table). We detected no cases of
is supported by the Government of Gabon, Total-Fina-Elf Gabon,
influenza, including pH1N1, in the provinces of Oyem and
and the Ministère des affaires Etrangères et Européennes, France.
Koulamoutou during the first half of 2010.
Seasonal influenza A was diagnosed in Gabon only Ms Lekana-Douki is a PhD student working on infectious
during September–December 2009: 6 cases in Libreville, diseases within the Centre International de Recherches Médicales
1 case in Oyem, and 1 case in Franceville. During the de Franceville Emerging Viral Diseases Unit in Gabon. Her pri-
short rainy season in 2010, the incidence of influenza B mary research focus is the surveillance of influenza-like illness
increased: 8 cases in Libreville, 2 cases in Oyem, 3 cases and the diagnosis of influenza virus in Gabon.
in Koulamoutou, and 38 cases in Franceville (Figure 2;
Table). We detected no cases of co-infection with pH1N1 References
virus and either seasonal influenza A or influenza B viruses.
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DISPATCHES

Response to a World Health Organization guidelines, and vaccines for

dogs (10). The Australian government helped establish a
Rabies Epidemic, direct fluorescent antibody (DFA) test at the Disease Inves-
tigation Center, Denpasar, Bali, and provided supplies for
Bali, Indonesia, emergency dog vaccination. Surveillance was implement-
ed by DFA testing of brain specimens from dogs that died
2008–2011 or were killed after showing signs of rabies and from culled
dogs. This surveillance, although imperfect, proved critical
Anak Agung Gde Putra, Katie Hampson, in tracking rabies spread (Figure).
Janice Girardi, Elly Hiby, Darryn Knobel, In Bali, the first 2 regencies (administrative divisions
I. Wayan Mardiana, Sunny Townsend, below provincial governments) affected by the rabies out-
and Helen Scott-Orr break were Denpasar and Badung. In December 2008, the
Balinese government began culling (using strychnine-laced
Emergency vaccinations and culling failed to contain baits or blow darts) unconfined dogs in areas of Denpasar
an outbreak of rabies in Bali, Indonesia, during 2008–2009. and Badung with confirmed rabies cases and began vac-
Subsequent island-wide mass vaccination (reaching 70%
cinating dogs at fixed posts. The locally manufactured vac-
coverage, >200,000 dogs) led to substantial declines in ra-
bies incidence and spread. However, the incidence of dog
cine required a booster after 3 months. It was estimated
bites remains high, and repeat campaigns are necessary to from a survey in Badung, where the human:dog ratio was
eliminate rabies in Bali. 8.3:1 (5), that 40% of dogs in Badung and Denpasar were
vaccinated during December 2008–March 2009 and that
25% received booster vaccinations by June 2009. Over

R abies was first reported in Indonesia in 1884 and now

occurs in 24 of the country’s 33 provinces (1–3). On
Bali Island, the first cases of rabies in humans and dogs
90% of the dogs in Bali are owned, but most are free-roam-
ing and hard to catch for 1 parenteral vaccination, let alone
booster vaccinations (6,7). Thus, the emergency response
were confirmed in 2008 on Bukit Peninsula (Figure). De- failed to contain the outbreak, and by September 2010, ra-
spite control efforts in 2008–2009, rabies spread across the bies had been confirmed in 221 (30.5%) villages through-
island. In the following 3 years, >130 persons died from out Bali (Table; Figure).
rabies (primarily persons who did not receive postexposure In 2009, the Australian Government donated long-last-
prophylaxis [PEP]) (4), and PEP was given to >130,000 ing vaccines for dogs, but operational funds for administra-
persons with dog bites. This outbreak resulted in consider- tion were unavailable. A local nongovernment organization,
able fear and anxiety and cost >US $17 million. We report the Bali Animal Welfare Association (BAWA), developed
on the outbreak progression and the effect of initial and a technique to improve vaccination coverage by training
subsequently improved control measures. teams to catch dogs with nets. During December 2009–July
2010, 6-person BAWA teams using this technique piloted
The Study door-to-door vaccinations throughout Gianyar Regency,
When the 2008 Bali rabies outbreak began, the island where BAWA is based. The teams vaccinated 48,000 dogs
had no policies for rabies PEP and no dog bite surveil- in Gianyar and 25,000 in nearby Bangli Regency. The
lance, rabies diagnostic facilities, or vaccines for dogs. In World Society for the Protection of Animals donated sup-
response to the outbreak, the Indonesian government pro- plies for this pilot, and BAWA covered operational costs.
vided Bali with postexposure rabies vaccine for humans Surveys of collared (vaccinated) dogs on consecutive days
(Verorab), for intramuscular administration according to after vaccinations indicated 70% coverage in almost all
banjars (subvillages). Beginning in October 2010, BAWA
Author affiliations: Disease Investigation Center, Denpasar, Bali,
teams and Balinese government staff worked together, with
Indonesia (A.A.G. Putra); College of Medical, Veterinary and Life
funding from the World Society for the Protection of Ani-
Sciences, University of Glasgow, Glasgow, United Kingdom (K.
mals, to vaccinate dogs throughout most of Bali, subject to
Hampson, S. Townsend); Bali Animal Welfare Association, Ubud,
the official suspension of culling. By April 2011, a total of
Bali (J. Girardi); World Society for the Protection of Animals, Lon-
249,429 dogs had been vaccinated, with coverage >70%
don, United Kingdom (E. Hiby); Faculty of Veterinary Science, Uni-
in most banjars. During this campaign, dogs in Gianyar
versity of Pretoria, Onderstepoort, South Africa (D. Knobel); Bali
Regency were revaccinated because 18 months had passed
Province Livestock Services, Denpasar, Bali (I.W. Mardiana); and
since the pilot and coverage had declined because of popu-
Faculty of Veterinary Science, University of Sydney, Camden New
lation turnover and movement. A second island-wide cam-
South Wales, Australia (H. Scott-Orr)
paign using these methods was completed in December
DOI: http://dx.doi.org/10.3201/eid1904.120380 2011 by the Balinese government, coordinated by the Food

648 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Response to a Rabies Epidemic, Bali, Indonesia

and Agriculture Organization, and achieved similar cover- Conclusions

age (Table). Rabies was detected in Bali in 2008; it was probably
During 2010, rabies was confirmed in 417 dogs, 2 cats, brought by fishermen from the island of Sulawesi (Indo-
and 3 cows. Of the 417 dogs, 387 (93%) were probably nesia), as happened on the island of Flores (Indonesia)
unvaccinated; 30 had reportedly been vaccinated, but only (3), and subsequently spread throughout the island. Early
9 had a clear vaccination date; 5 were positive for rabies containment attempts by limited fixed-point dog vaccina-
shortly after vaccination and were likely incubating the tion and culling were unsuccessful. This was likely due
disease when vaccinated; and 4 cases were considered vac- to insufficient funding, largely inaccessible free-roaming
cination failures. dog populations with high turnover, limited availability of
When the first island-wide vaccination campaign be- long-lasting dog vaccines (and means to identify vaccinat-
gan in 2010, a total of 140 (19.4%) villages still reported ed dogs), and inconsistent cold chains.
rabies (>1 case in the previous 6 months), and 81 (11.2%) These issues were gradually addressed, and island-
villages that previously reported cases were considered wide vaccinations in 2010 and 2011 approached the
rabies-free (no cases detected for >6 months). In addition, recommended target of 70% coverage (8,9); postvacci-
during this island-wide campaign (October–April 2011), nation surveys of collared dogs enabled better coverage
rabies was detected in 48 previously rabies-free villages. estimates. Considerable coordination was required among
By December 2011, only 30 (4.1%) villages were not con- Bali’s provincial and regency governments, which was fa-
sidered rabies-free (Table). Before island-wide vaccination, cilitated through training and data management systems.
rabies was detected in 10 new villages per month; during Nonetheless, reporting remained challenging due, in part,
the first and second island-wide vaccinations, rabies was to limited infrastructure.
detected in 6.8 and 1.6 villages per month, respectively. Vaccination campaigns reduced rabies incidence and
The monthly number of confirmed cases before mass vac- spread, resulting in decreased attack rates at the regency
cination was also much higher (44.7 cases) than during the level and island-wide. In contrast, culling was ineffective
first (10.8 cases) and second (6.0 cases) mass vaccination in suppressing rabies and can be counterproductive (10).
campaigns, and concomitantly, the island-wide attack rate Although panic led to demand for culling in some loca-
(confirmed rabid dogs per estimated unvaccinated popula- tions, many communities objected because of religious be-
tion) declined from 0.027% to 0.01% (Table). Reported liefs and, especially, when owned (often vaccinated) dogs
dog bites declined slowly, from 6,256 bites per month be- were culled. New puppies were brought to replace culled
fore island-wide vaccination to 4,589 and 4,197 bites per dogs, and some dogs were moved to avoid culls, possibly
month during the first and second vaccination campaigns, resulting in the transportation of infected dogs. Incidence
respectively. However, human deaths from rabies declined declines due to vaccinations reduced the public health threat
from 94 (4.3/month) before island-wide vaccination to 34 and panic that triggered culling; ≈108,000 dogs were culled
(4.8/month) during the first campaign (24/34 persons were before island-wide vaccination, compared with 40,000 dur-
bitten during the prevaccination period) to 9 (1.1/month) ing the 2 vaccination campaigns. However, long-term ac-
deaths during the second campaign. ceptable dog population control is still sought on Bali.

Figure. Timing of confirmed rabies

cases in villages across Bali since the
first case was confirmed on the island
in November 2008. Darker shading
indicates earlier detection according
to the months since the first case was
detected in the index village (marked),
lighter shading indicates later detection,
and white shading indicates no detected
cases by December 2011.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 649
DISPATCHES

Table. Indicators of rabies incidence and spread among the human, dog, and other animal populations before and during mass island-
wide dog vaccination campaigns, Bali, Indonesia, 2008–2011*
During 1st campaign,
Before campaign, Nov Oct 10, 2010– During 2nd campaign,
Indicator 08–Sept 10, 2008 Apr 11, 2011 May 11–Dec 11, 2011
Observation period, mo 22 7 8
Average no. rabid dogs/mo 45 11 6
Average apparent monthly attack rate among dogs, %† 0.03 0.01 0.01
Total no. villages with cases detected among dogs 221 269 282
No. villages with newly detected cases NA 48 13
Rate of spread, no. villages with newly detected dog 10 6.8 1.6
cases/mo
Remaining known villages with cases among dogs, 140 (19.4) 48 (6.6) 30 (4.1)
no. (%)
No. dog bites treated/mo (bites/day) 6,256 (208) 4,589 (153) 4,197 (140)
Human deaths 94 34 9
Estimated no. culled dogs 107,900 40,500 14,000
No. dogs vaccinated (estimated coverage, %) >73,000 (40)‡ 249,429 (>70) 231,155 (>70)
*Rabid dogs correspond to cases confirmed by using the direct fluorescent antibody test. Villages were classified as free from rabies if no cases were
detected for at >6 mo. Coverages were initially estimated from human: dog ratios and subsequently from observations of the proportion of dogs with
collars indicating vaccination. The number of culled dogs was also estimated because some culling was carried out by communities rather than by
government. NA, not applicable.
†Attack rate, confirmed rabid dogs divided by estimated unvaccinated dog population.
‡Data were not available for dogs vaccinated and boosted during the first few months of the outbreak; therefore, only data on vaccinations in Gianyar and
Bangli Regencies are shown.

DFA testing proved an effective surveillance method; Dr Putra is a senior veterinary epidemiologist at the
dog bites were a less sensitive measure. The incidence of Disease Investigation Center, Denpasar, Bali, Indonesia. His
reported bites is higher on Bali than in Indonesian prov- research interests are in the epidemiology and control of zoo-
inces where rabies is endemic; this may reflect heightened notic diseases.
awareness about rabies or be related to the high densities
of humans and dogs. Rabid dogs generally bite without
References
provocation and die <10 days after clinical signs develop
(11); thus, a short observation period (12) may allow more 1. Akoso BT. Rabies in animals in Indonesia. In: Dodet B, Meslin FX,
judicious PEP administration but is often impractical with editors. Rabies control in Asia. Paris: John Libbey Eurotext; 2001.
2. Waltner-Toews D, Maryono A, Akoso BT, Wisynu S, Unruh
unrestrained dogs.
DHA. An epidemic of canine rabies in central Java, Indonesia.
Mass dog vaccinations substantially reduced rabies Prev Vet Med. 1990;8:295–303. http://dx.doi.org/10.1016/0167-
incidence on Bali and must be continued if elimination is 5877(90)90087-X
to be achieved. Further research is needed to assess how 3. Windiyaningsih C, Wilde H, Meslin FX, Suroso T, Widarso HS. The
rabies epidemic on Flores Island, Indonesia (1998–2003). J Med As-
many more campaigns are needed. Improved surveillance
soc Thai. 2004 2004;87:1389–93. PubMed
and control of inter-island dog movement are necessary to 4. Susilawathi NM, Darwinata AE, Dwija IBNP, Budayanti NS, Wi-
prevent further rabies spread within Indonesia. rasandhi GAK, Subrata K, et al. Epidemiological and clinical fea-
tures of human rabies cases in Bali 2008–2010. BMC Infect Dis.
2012;12:81. http://dx.doi.org/10.1186/1471-2334-12-81
Acknowledgments
5. Putra AAG, Gunata IK, Asrama IG. Dog demography in Badung
We are grateful to all agencies and staff involved in the Bali District the Province of Bali and their significance to rabies con-
Rabies Eradication Campaign, including the Central Government trol Buletin Veteriner Balai Besar Veteriner Denpasar. 2011;
of Indonesia; Bali Provincial and Regency Government Livestock XXIII:14–24.
6. Putra AAG, Dartini NL, Faizah, Soegiarto, Scott-Orr H. Surveilans
Services and Public Health Services; the Disease Investigation
seroepidemiologi rabies di Bali. Buletin Veteriner. 2009; XXI
Center, Denpasar; the Australian Government through AusAID 75:52–61.
for donations of dog vaccine and equipment and through the Aus- 7. Putra AAG, Gunata IK, Faizah, Dartini NL, Hartawan DHW, Se-
tralian Centre for International Agricultural Research for tech- tiaji G, et al. Situasi rabies di Bali: enam bulan pasca program
pemberantasan. Buletin Veteriner Balai Besar Veteriner Denpasar.
nical support; the Bali Animal Welfare Association; the World
2009;XXI:13–26.
Society for the Protection of Animals; the World Health Organi- 8. Hampson K, Dushoff J, Cleaveland S, Haydon DT, Kaare M, Packer
zation; and the Food and Agriculture Organization. We also thank C, et al. Transmission dynamics and prospects for the elimination
Prabowo R. Caturroso for support. of canine rabies. PLoS Biol. 2009;7:e53. http://dx.doi.org/10.1371/
journal.pbio.1000053
This work was supported by the Wellcome Trust and the 9. Coleman PG, Dye C. Immunization coverage required to prevent
Medical Research Council, United Kingdom. outbreaks of dog rabies. Vaccine. 1996;14:185–6. http://dx.doi.
org/10.1016/0264-410X(95)00197-9

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 Response to a Rabies Epidemic, Bali, Indonesia

10. World Health Organization. Expert consultation on rabies: first re- Address for correspondence: Anak Agung Gde Putra, Disease Investigation
port. Geneva: the Organization; 2005. Center, Jalan Raya Sesetan 266 Denpasar 80223, Bali, Indonesia; email:
11. Tepsumethanon V, Wilde H, Sitprija V. Ten-day observation of live
dic-denpasar@indo.net.id
rabies suspected dogs. Dev Biol (Basel). 2008;131:543–6.
12. Soenardi. A retrospective epidemiological study of rabies in ani-
The opinions expressed by authors contributing to this
mals and man in central Sumatera, Indonesia. In Proceedings of
journal do not necessarily reflect the opinions of the Centers for
the 4th International Symposium on Veterinary Epidemiology
Disease Control and Prevention or the institutions with which
and Economics, Singapore. Singapore Veterinary Association,
the authors are affiliated.
1986; p 220–3.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 651
DISPATCHES

Genetic and ≈5% of the serosurveyed population in Key West had

evidence of recent DENV infection (4,5). In 2010, addi-
Relatedness of tional dengue cases from Monroe County were reported,
and DENV-1 was isolated from a mosquito pool (6) and a
Dengue Viruses in blood donor from Key West (7); isolates from the mosquito
pool and blood donor appeared to be phylogenetically re-
Key West, Florida, lated (7). This study determined the genetic relatedness of

USA, 2009–2010
the DENV-1 isolates from dengue patients in Key West and
4 other Florida counties during 2009–2010, including the
blood donor and mosquito isolates.
Jorge L. Muñoz-Jordán, Gilberto A. Santiago,
Harold Margolis, and Lillian Stark The Study
During 2009–2010, serum samples from patients with
Sequencing of dengue virus type 1 (DENV-1) strains
suspected dengue were received by the FDOH for dengue
isolated in Key West/Monroe County, Florida, indicate en-
demic transmission for >2 years of a distinct and predomi-
diagnostic testing; the samples came from 16 of Florida’s
nant sublineage of the American–African genotype. DENV-1 67 counties. All samples were tested by using DENV sero-
strains isolated elsewhere in Florida grouped within a sepa- type–specific, real-time RT-PCR (8) and IgM anti-DENV
rate Central American lineage. Findings indicate endemic ELISA (9). Samples with highly positive RT-PCR results
transmission of DENV into the continental United States. were spread onto cultured Ae. albopictus C6/36 cells, and
the presence of virus and genome were confirmed by im-
munofluorescence (10) and RT-PCR, respectively (11). Iso-
D engue is the most common mosquito-borne viral dis-
ease; cases have been reported from ≈100 countries,
and there are indications of increased incidence and sever-
lates were further propagated and viral RNA was extracted
from culture supernatants by using the Universal BioRobot
System (QIAGEN, Valencia, CA, USA). The envelope
ity worldwide (1). The United States has reported year-
glycoprotein (E) gene was amplified (online Technical
round transmission of dengue virus (DENV) in Puerto
Appendix Table 1, wwwnc.cdc.gov/EID/article/19/4/12-
Rico, the US Virgin Islands, and American Samoa and oc-
1295-Techapp1.pdf), and the E gene open-reading frame
casional transmission along the Texas–Mexico border. In
(1,485 bp) was sequenced. All sequences were submitted to
the continental United States, DENV is the most frequent
GenBank; accession numbers are shown in online Techni-
cause of febrile illness among travelers returning from the
cal Appendix Table 2. Multiple sequence alignments were
Caribbean, South America, and Asia (2,3). These frequent
performed by using the MUSCLE module available in
introductions of dengue infections and the increased pres-
MEGA 5 (www.megasoftware.net). Evolutionary history
ence of vectors (i.e., Aedes aegypti and Ae. albopictus
was inferred by using maximum likelihood and phyloge-
mosquitoes) in many US regions may portend the reintro-
netic trees constructed by using neighbor-joining methods.
duction and extended transmission of DENV into the con-
Evolutionary distances were computed, and several E gene
tinental United States.
sequences from GenBank were included in the phylogenet-
In September 2009, the Florida Department of Health
ic tree to support tree topology by genotype (online Techni-
(FDOH) and the Centers for Disease Control and Preven-
cal Appendix Table 2).
tion (San Juan, Puerto Rico) investigated a case of DENV
In 2009, five DENV-1–positive cases were identi-
type 1 (DENV-1) infection in a person (index patient) who,
fied by RT-PCR in Key West. Subsequently, in 2010, the
as confirmed by reverse transcription PCR (RT-PCR), ac-
FDOH tested 195 serum samples by real-time RT-PCR.
quired the virus while traveling to Key West in Monroe
Fifty-six (29%) samples were positive for DENV RNA:
County, Florida, USA. DENV-1 infections were subse-
DENV-1 (37 [66%] samples), DENV-2 (13 [23%] sam-
quently confirmed in 2 Monroe County residents without
ples), DENV-3 (3 [5%] samples), and DENV-4 (3 [5%]
histories of recent travel. In addition, among 13 other cases
samples). Monroe County submitted 73 serum samples,
in the county that were identified by serologic methods, 2
of which 31 (42%) had results positive for recent dengue
were confirmed as DENV-1 infections (4). Thus, a total of
infection: DENV-1 was detected in 22 by RT-PCR, and
5 DENV-1 cases were confirmed in Key West during 2009,
9 had positive IgM anti-DENV ELISA results. No other
DENV serotype was identified in Key West. None of the
Author affiliations: Centers for Disease Control and Prevention, San
DENV-1 patients from Monroe County had a history of
Juan, Puerto Rico (J.L. Muñoz Jordán, G.A. Santiago, H. Margolis);
recent travel to a dengue-endemic region before the on-
and Florida Department of Health, Tampa, Florida, USA (L. Stark)
set of symptoms. Fifteen other Florida counties submitted
DOI: http://dx.doi.org/10.3201/eid1904.121295 serum samples: 13 counties submitted <10 specimens, 1

652 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Dengue Viruses, Key West, Florida

submitted 20–30 specimens, and 1 submitted >60 speci- tested by 1,000 bootstrap replications. This phylogenetic
mens. DENV-1 was found in 15 serum samples from 6 of analysis showed that all the Florida DENV-1 isolates be-
these counties; however, all the patients had a history of long to the American–African genotype (genotype V)
recent international travel. (12,13) together with other viruses isolated throughout the
We sequenced the E gene of 12 DENV-1 strains isolat- Americas (Figure).
ed in Florida during 2009–2010 to determine their genetic Key West DENV-1 viruses grouped among Central
relatedness; of the 12 strains, 8 were from Key West and 1 American viruses, which configure a distinct lineage sepa-
each was from Dade, Broward, Orange, and Pinellas Coun- rate from the Caribbean viruses. This divergence between
ties. In addition, 23 DENV-1 E sequences published in the Central American and Caribbean lineages is well sup-
GenBank, including the 2010 Key West isolates obtained ported by high bootstrap values. Moreover, the Key West
from a blood donor and a mosquito pool (6,7), were used and Monroe County viruses grouped together and indi-
to construct a maximum-likelihood phylogenetic tree. The cated a distinct sublineage supported by a high bootstrap
significance of branch lengths and taxa relationships was value (99%), separating them from viruses isolated in

Figure. Maximum-likelihood phylogenetic tree of dengue virus type 1, including isolates from Key West, Florida, USA, and representative
isolates from 5 genotypes with global geographic distribution. Solid circles, 8 Key West viruses (Monroe County) isolated during 2009–
2010; solid diamonds, isolates from other Florida counties (Dade, Pinellas, Orange, and Broward Counties). Scale bar indicates nucleotide
substitutions per site. Each taxon represents a single virus isolate and is labeled with the geographic origin and collection year. All Florida
viruses were labeled with the county of origin. Boldface taxa labels indicate the Key West lineage and cases not associated with travel.
Isolate KW-Monroe/2009/JQ425068 represents the 2009 Key West outbreak index case virus. Thirty-six envelope glycoprotein gene
sequences obtained from GenBank were included to support tree topology and identify genotypes. All genotypes except the American–
African genotype (V) have been collapsed. Taxa labels and GenBank accession numbers are available in online Technical Appendix Table
2 (wwwnc.cdc.gov/EID/article/19/4/12-1295-Techapp1.pdf).

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 653
DISPATCHES

Dade, Orange, Pinellas, and Broward Counties that were ters for Disease Control and Prevention. His work focuses on im-
more closely related to other Central American viruses proving dengue diagnostics, understanding dengue evolution, and
(Figure 1). One 2009 isolate (IQ425062) from a Key West understanding the host response against dengue infection.
patient is related to this group, suggesting a separate in-
troduction of DENV-1 in Key West. The sequence simi- References
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1. World Health Organization, Special Programme for Re-
<0.9%; however, the evolutionary distance and taxa posi- search and Training in Tropical Diseases. Dengue guidelines
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gin of the Florida DENV-1 strain. Most viruses isolated in 5. Centers for Disease Control and Prevention. Locally acquired den-
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level of genetic similarity among the viruses isolated in Florida, USA, 2010. Emerg Infect Dis. 2011;17:2074–5. http://
Monroe County, their close evolutionary distances, and dx.doi.org/10.3201/eid1711.110419
the lack of recent international travel for the case-patients 7. Añez G, Heisey DA, Espina LM, Stramer SL, Rios M. Phylogenetic
analysis of dengue virus types 1 and 4 circulating in Puerto Rico and
suggest endemic transmission and microevolution of this Key West, Florida, during 2010 epidemics. Am J Trop Med Hyg.
DENV. Conversely, the scattered and separate phyloge- 2012;87:548–53. http://dx.doi.org/10.4269/ajtmh.2012.12-0091
netic positioning of virus strains from patients with travel- 8. Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV.
associated cases from other Florida counties indicates a Rapid detection and typing of dengue viruses from clinical samples
by using reverse transcriptase–polymerase chain reaction. J Clin Mi-
different origin from the majority of Key West isolates. Al- crobiol. 1992;30:545–51.
though the 2009 Broward isolate (JQ425067) is positioned 9. Martin DA, Muth DA, Brown T, Johnson AJ, Karabatsos N, Roehrig
near the Central American lineage, the low bootstrap value JT. Standardization of immunoglobulin M capture enzyme–linked
(53%) does not support lineage ancestry. immunosorbent assays for routine diagnosis of arboviral infections.
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The epidemiologic and phylogenetic evidence sug- 10. Kuno G, Gubler DJ, Velez M, Oliver A. Comparative sensitivity of
gests that the 2010 cases appeared to be a continuation of three mosquito cell lines for isolation of dengue viruses. Bull World
the 2009 outbreak. Unlike cases along the Texas–Mexico Health Organ. 1985;63:279–86.
border (14), all DENV-1 infections in Key West seem to 11. Johnson BW, Russell BJ, Lanciotti RS. Serotype-specific detec-
tion of dengue viruses in a fourplex real-time reverse transcrip-
have been locally acquired. Ae. aegypti mosquitoes collect- tase PCR assay. J Clin Microbiol. 2005;43:4977–83. http://dx.doi.
ed by the FDOH were positive for DENV-1. In addition, org/10.1128/JCM.43.10.4977-4983.2005
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3527(03)59009-1
of DENV (7). Collectively, these findings indicate that en- 13. Holmes EC, Twiddy SS. The origin, emergence and evolutionary
demic DENV-1 was transmitted in Key West over a period genetics of dengue virus. Infect Genet Evol. 2003;3:19–28. http://
of >2 years. dx.doi.org/10.1016/S1567-1348(03)00004-2
14. Ramos MM, Mohammed H, Zielinski-Gutierrez E, Hayden MH,
Lopez JL, Fournier M, et al. Epidemic dengue and dengue hemor-
Acknowledgments rhagic fever at the Texas–Mexico border: results of a household-
We thank Claire Huang for designing the DENV E gene based seroepidemiologic survey, December 2005. Am J Trop Med
amplification primers. We also acknowledge Janae Stovall and Hyg. 2008;78:364–9.
Karen Boroughs for their assistance in E gene sequencing.
Address for correspondence: Jorge L. Muñoz-Jordán, Dengue Branch,
Dr. Muñoz-Jordán is chief of the Molecular Diagnostics Ac- Centers for Disease Control and Prevention, San Juan, Puerto Rico 00969;
tivity, Dengue Branch, Division of Vector-Borne Diseases, Cen- email: ckq2@cdc.gov

654 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Control of (Table 1) in samples from saliva, vesicles, and detached
hooves from pigs with signs typical of FMDV infection
Foot-and-Mouth (i.e., salivation, vesiculation, and ulceration) (7). Samples
from pigs with clinical signs of infection tested positive
Disease during by antibody-detection assay using solid-phase competition
ELISA (PrioCHECK; Prionics, Schlieren, Switzerland) for
2010–2011 the O serotype, excluding liquid-phase blocking ELISA;

Epidemic,
however, antibody tests using nonstructural protein ELI-
SA (VDPro; Jeno Biotech Inc., Chuncheon, South Korea)

South Korea showed negative results (Table 1).

Because many cattle farms were located in the ar-
eas surrounding the pig-farming complex, the virus was
Jong-Hyeon Park, Kwang-Nyeong Lee, detected mainly in cattle during the next 25 days (on-
Young-Joon Ko, Su-Mi Kim, Hyang-Sim Lee, line Technical Appendix Figure 1, wwwnc.cdc.gov/EID/
Yeun-Kyung Shin, Hyun-Joo Sohn, article/19/4/12-1320-Techapp1.pdf). After the first detec-
Jee-Yong Park, Jung-Yong Yeh, Yoon-Hee Lee, tion, the disease spread to 75 cities or counties in 11 prov-
Min-Jeong Kim, Yi-Seok Joo, Hachung Yoon, inces over 144 days, through April 21, 2011; the only prov-
Soon-Seek Yoon, In-Soo Cho, inces not affected were Jeonbuk, Jeonnam, and Jeju (Figure
and Byounghan Kim 1). As soon as an outbreak was reported, animal movement
An outbreak of foot-and-mouth disease caused by sero-
restrictions were imposed, and a 3-km radius protection
type O virus occurred in cattle and pigs in South Korea during zone and 10-km radius surveillance zone were set around
November 2010–April 2011. The highest rates of case and the outbreak area.
virus detection were observed 44 days after the first case FMD spread throughout Gyeongbuk Province until
was detected. Detection rates declined rapidly after culling December 14; at the same time, it spread rapidly to other
and completion of a national vaccination program. regions, including the provinces of Gyeonggi (December
15), Gangwon (December 21), and Incheon (December 23)

F
(Figure 1). For emergency disease control, vaccines were
oot-and-mouth disease (FMD) is a highly conta-
initially administered to cattle in these outbreak areas on
gious disease caused by foot-and-mouth disease vi-
December 25. However, FMD continued to spread into
rus (FMDV; family Picornaviridae, genus Aphthovirus).
additional provinces during January 2011, with outbreaks
FMDV serotypes O, A, and Asia1 are widespread in South-
occurring in Chungnam (January 1), Chungbuk (January
east Asia (1). In South Korea, small-scale outbreaks of
3), Daegu (January 17), and Gyeongnam (January 24). Na-
FMDV infection caused by serotype O occurred in March
tionwide vaccination was implemented on January 13, and
2000, May 2002, and April 2010 (2–5), and an outbreak
the last reported case occurred on April 21 in Youngcheon
caused by serotype A occurred in January 2010 (6). In con-
City, Gyeongbuk Province.
trast, an outbreak during November 2010–April 2011 was
During this outbreak, 153 (73.56%) of 208 farms with
much more widespread (7). We reviewed the progression
suspected cases were confirmed as index points for disease
of this outbreak and methods used to control it, including
transmission into new areas. Of farms with animals show-
culling and vaccination of pigs and cattle.
ing clinical signs, 3,234 (83.98%) of 3,851 had positive
test results for FMDV in animals. Within the affected ar-
The Study
eas, after FMDV infection was confirmed in 1 farm, 295
Clinical signs of FMD in animals appeared on No-
(21.24%) of 1,389 other farms had positive test results. For
vember 23, 2010, in a pig-farming complex in Gyeong-
farms related to the infected farms epidemiologically (e.g.,
buk Province. Reporting to the central government was
by vehicle movement or human contact), 33 (10.68%) of
delayed for ≈1 week because of misdiagnosis caused by
309 had positive test results.
false-negative results from a pen-side antibody kit. FMD-
An FMD vaccine of high potency was imported for
positive test results were confirmed on November 28–29
emergency vaccination; the vaccine used FMDV strain
Author affiliations: Animal, Plant and Fisheries Quarantine and In-
O1 Manisa (8). A postvaccination analysis using serum
spection Agency, Anyang, South Korea (J.-H. Park, K.-N. Lee, Y.-J.
samples collected from vaccinated animals and viruses iso-
Ko, S.-M. Kim, H.-S. Lee, Y.-K. Shin, H.-J. Sohn, J.-Y. Park, J.-Y.
lated in the field showed the vaccine’s high efficacy in the
Yeh, Y.-H. Lee, M.-J. Kim, Y.-S. Joo, H. Yoon, S.-S. Yoon, I.-S. Cho,
field. Cattle in the affected regions were vaccinated first,
B. Kim); and Univeristy of Incheon, Incheon, South Korea (J.-Y. Yeh)
on December 25; later, vaccination was expanded to the
DOI: http://dx.doi.org/10.3201/eid1904.121320 whole cattle population, with vaccination completed by

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 655
DISPATCHES

Table 1. Laboratory diagnosis of FMDV infection in specimens from a pig-farming complex, Gyeongbuk Province, South Korea,
November–December 2010*
Farms in pig Date of sample Antigen detection No. animals No. antibody-positive animals
complex collection Specimen type RT-PCR Antigen ELISA tested SP-O ELISA NSP ELISA
A Nov 28 S, V, H + O serotype 10 2 0
B Nov 28 S, V, H + O serotype 10 2 0
A Nov 29 Serum + ND 90 3 0
B Nov 29 Serum + ND 40 1 0
C Dec 1 Serum – ND 20 0 0
D Dec 1 Serum – ND 20 10 0
E Dec 1 Serum – ND 41 17 7
*FMDV, foot-and-mouth disease virus; RT-PCR, reverse transcription PCR; SP-O, structural protein of FMDV serotype O; NSP, FMDV nonstructural
protein; S, saliva; V, vesicle; H, detached hooves; +, positive; ND, not done; –, negative.

January 31, 2011. Pigs were vaccinated 14 days after the Appendix Figure 2). Some farms that were required to cull
cattle (January 8), and the whole pig population was also livestock because of FMD risk did not undertake the pro-
vaccinated by the end of January. cess in a timely manner, which contributed to a spike in
According to national policy, culling began in Novem- new infections on the 38th–64th days after the outbreak
ber 2010 for all animals on farms with infected animals. began (January 4–31, 2011) (online Technical Appendix
Once vaccination was expanded nationwide in mid-Janu- Figure 3). These new infections, mainly among pigs, oc-
ary 2011, a vaccination-to-live policy was implemented; curred in Chungnam, Chungbuk, Gangwon, Gyeongnam,
that is, vaccinated animals on farms with infected animals and Gyeonggi provinces.
were culled only if the outbreak began within 2 weeks After vaccination and culling were implemented, the
after vaccination but not if the outbreak began >2 weeks number of daily FMD cases decreased gradually. Among
after vaccination. Most culled animals were disposed of cattle, the number of FMD cases began to decrease on
by burial, which was regarded as a suitable method for a the 40th day after the initial outbreak (12 days after the
large-scale outbreak, given its advantage of easy handling first cattle vaccinations). In pigs, the number decreased
within a short time. Approximately 3.48 million animals after the 60th day (18 days after the first pig vaccinations)
(151,425 cattle, 3,318,299 pigs, 8,071 goats, and 2,728 (online Technical Appendix Figure 1). Many animals
deer) were buried at 4,583 burial sites (online Technical also were culled during January 2011 (online Technical

Figure 1. Progress of foot-and-mouth

disease transmission throughout South
Korea during 2010–2011 outbreak.
Circles indicate cases in swine at index
farms; black dots, cases in cattle. A
timeline of case detection is provided
in online Technical Appendix Figure
1 (wwwnc.cdc.gov/EID/article/19/4/1-
1320-Techapp1.pdf). IC, Incheon;
GG, Gyeonggi; GW, Gangwon; CN,
Chungnam; CB, Chungbuk; GB,
Gyeongbuk; GN, Gyeongnam; JN,
Jeonnam; JB, Jeonbuk; DG, Daegu.

656 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Foot-and-Mouth Disease, South Korea

Figure 2. Phylogenetic analysis of

viral protein 1 sequences of serotype
O foot-and-mouth disease viruses
isolated in South Korea (black
dots) and other Asian countries,
2010. The tree was constructed by
using the neighbor-joining method
in MEGA5 (www.megasoftware.
net). Percentages in which the
associated taxa clustered together in
the bootstrap test (1,000 replicates)
are shown next to the branches.
Scale bar indicates nucleotide
substitutions per site. CHA, China;
HKN, Hong Kong; JPN, Japan; RUS,
Russia; SKR or KOR, South Korea;
VN, Vietnam.

Appendix Figure 2), and the number of FMD outbreaks FMDV isolates from Mongolia, Vietnam, and other
decreased to as low as a single index case daily after Janu- countries in Asia largely group into 2 phylogenetic clusters
ary 31, 2011. on the basis of nucleotide similarities (1). To determine the
The outbreak quickly spread nationwide across a large relationship between the South Korea virus strain and those
distance. This rapid spread occurred for several reasons: 1) from other countries in Asia, we analyzed the viral protein 1
the first infection was in a pig-farming complex, and pigs nucleotide sequence of an FMDV virus isolate from the first
excrete the virus in large amounts; 2) detection of the first FMD case, in November 2010. The sequence showed >99%
infection was delayed; 3) FMDV-contaminated feces from identity with the O serotype; this type also matched those
the index pig-farming complex was moved to other prov- found in Gyeonggi Province and another location in Gyeong-
inces to be recycled for use as fuel on November 17, before buk Province during December 2010. However, a group of
the first outbreak; 4) the virus has increased stability dur- FMD viruses identified in South Korea and People’s Repub-
ing the winter months, enabling it to be transmitted more lic of China (group 1) showed 6 amino acid residues of viral
easily; 5) culling of infected animals was not implemented protein 1 different from those of other seasons or countries
quickly enough by affected farms; and 6) the distance be- (Table 2). In addition, among other FMD outbreaks identi-
tween farms in the area was small. fied in neighboring countries, viruses that originated in Chi-
The FMD virus is believed to have entered South Ko- na had the most similar composition in amino acid residues
rea around November 9–16, 2010; the first clinical signs to those from South Korea (Table 2; Figure 2) (1,9).
in pigs appeared on November 23, and serologic investi-
gation found that the time point for FMD infection was Conclusions
November 14. The virus might have been brought into the An outbreak of FMD in South Korea during November
country as a result of a farmer’s trip to Southeast Asia in 2010–April 2011 was caused by serotype O FMDV and
early November. affected ≈3,700 farms; 153 farms were identified as index

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 657
DISPATCHES

Table 2. Comparison of VP1 amino acids of foot-and-mouth disease isolates from South Korea versus viruses originating in other
countries in Southeast Asia, 2010*
Similarities of Alignment of major differences in Genbank
Region and Date of VP1, % VP1 amino acids by position accession
Group and strain Country province collection nt aa 58 139 141 152 157 184 no.
I
O/Andong/SKR/2010 South Andong, Nov 28 Ref Ref S S P Q R T JQ070321
Korea Gyeongbuk
O/PJ/SKR/2010 South Paju, Gyeonggi Dec 15 99.22 100.0 – – – – – – This study
Korea
O/YC/SKR/2010 South Yeoncheon, Dec 15 99.22 100.0 – – – – – – This study
Korea Gyeonggi
O/PC/SKR/2010 South Pyeongchang, Dec 21 99.06 99.53 – – – – – – This study
Korea Gangwon
O/GH/SKR/2010 South Ganghwa, Dec 24 99.22 100.0 – – – – – – This study
Korea Incheon
O/BY/CHA/2010 China Shenzhen, Mar 4 98.75 100.0 – – – – – – JN998085
Guangdong
O/CHA/31/2010 China NA Feb 22 98.90 100.0 – – – – – – JF792356
II
O/DY/CHA/2010 China NA NA 97.65 97.18 – P – – – – HQ652078
O/RUS/Jul 2010 Russia Abagaytuy, Jul 99.06 99.06 – P – – – – JQ070329
Zabajkal’skijkray
III
O/HKN/7/2010 China Hong Kong Feb 22 99.06 99.53 P – – – – – JQ070303
O/HKN/9/2010 China Hong Kong Feb 24 98.9 99.53 P – – – – – JQ070304
IV
O/JPN/MZ1/2010 Japan Miyazaki May 98.90 99.06 – – – P – A AB618503
O/TZ/CHA/2010 China NA NA 98.44 99.06 – – – P – A HQ652081
V
O/VN/YB08/2010 Vietnam Yen Bai Feb 98.28 99.06 P – T – – – HQ26078
O/GZ/CHA/2010 China NA Mar 98.59 98.12 P – T – – – JN998086
O/NC/CHA/2010 China NA NA 97.97 98.59 P – T – – – HQ652080
O/MY/CHA/2010 China NA NA 98.59 99.06 P – T – – – HQ652079
VI
O/KOR/1/2010 South Ganghwa, Apr 8 98.44 99.06 P – – – W – HM143846
Korea Incheon
O/KOR/10/2010 South Cheonyang, Apr 30 98.44 98.59 P – S – W – This study
Korea Chungnam
O/KOR/11/2010 South Cheonyang, May 6 98.44 98.59 P – S – W – This study
Korea Chungnam
*Groups are based on major differences in amino acids. VP1, viral protein 1; nt, nucleotides; aa, amino acids; SKR or KOR, South Korea; ref, referent; –,
no difference; CHA, China; NA, not available; RUS, Russia; HKN, Hong Kong; JPN, Japan; VN, Vietnam.

locations for new outbreaks. A total of 3.48 million suscep- References

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1. Knowles NJ, He J, Shang Y, Wadsworth J, Valdazo-Gonzalez B,
cination program was effective in controlling the outbreak, Onosato H, et al. Southeast Asian foot-and-mouth disease viruses
and FMD incidence declined rapidly after its completion. in Eastern Asia. Emerg Infect Dis. 2012;18:499–501. http://dx.doi.
org/10.3201/eid1803.110908
Acknowledgments 2. Joo YS, An SH, Kim OK, Lubroth J, Sur JH. Foot-and-mouth dis-
We thank the staff of the Disease Control Department at the ease eradication efforts in the Republic of Korea. Can J Vet Res.
2002;66:122–4.
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in collecting samples from animals with suspected FMD cases. primary suspect cases in Korea in 2000. J Vet Med Sci. 2003;65:1–7.
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Plant and Fisheries Quarantine and Inspection Agency’s National et al. Epidemiological characteristics of the 2002 outbreak of
Animal Disease Research Project. foot-and-mouth disease in the Republic of Korea. Transbound
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Quarantine and Inspection Agency on FMD diagnosis and sur-
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veillance, His research interests are development of recombinant disease in 2000 in Korea. Dev Biol (Basel). 2004;119:63–70.
vaccine, antiviral agents, and rapid diagnostic methods for FMD. 6. Park JH, Lee KN, Ko YJ, Kim SM, Lee HS, Park JY, et al. Diag-
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disease A type in the Republic of Korea. Transbound Emerg Dis. and challenge in goats and comparison with sheep. Res Vet Sci.
2012 May 27. Epub ahead of print. 2012;93:1050–9. http://dx.doi.org/10.1016/j.rvsc.2011.10.006
7. Yoon H, Yoon SS, Wee SH, Kim YJ, Kim B. Clinical manifestations 9. Muroga N, Hayama Y, Yamamoto T, Kurogi A, Tsuda T, Tsutsui T.
of foot-and-mouth disease during the 2010/2011 epidemic in the The 2010 foot-and-mouth disease epidemic in Japan. J Vet Med Sci.
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dx.doi.org/10.1111/j.1865-1682.2011.01304.x
8. Madhanmohan M, Nagendrakumar SB, Kumar R, Anilkumar J, Address for correspondence: Jong-Hyeon Park, Animal, Plant and
Manikumar K, Yuvaraj S, et al. Clinical protection, sub-clinical in-
Fisheries Quarantine and Inspection Agency, 175 Anyang-ro, Manangu,
fection and persistence following vaccination with extinction pay-
loads of O(1) Manisa foot-and-mouth disease monovalent vaccine Anyang, Gyeonggido, 430-757, South Korea; email: parkjhvet@korea.kr

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 659
 Photo Quiz
Who Is This Man?

The father of viral oncology, he discovered the first

tumor virus and influenced many later scientists,
including several Nobel laureates.
Is he:
A) Richard Shope C) Simon Flexner
B) Peyton Rous D) Thomas Rivers
E) Ludwik Gross

Decide first. Then turn the page.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 661
PHOTO QUIZ

Francis Peyton Rous

Prasanna Kumar and Frederick A. Murphy

T his is a photograph of Peyton Rous (Francis Peyton

Rous, 1879–1970), who in 1909–11 made 2 seminal
discoveries that are now the foundation blocks of modern
als. It is a spindle-celled sarcoma of a hen, which has thus far
been propagated to the fourth generation….” In his research,
he found that only closely related chickens were susceptible,
virology and oncology. First, he discovered that a malig- but in these chickens, continuous passage of cell-free mate-
nant tumor (a sarcoma in chickens) was transmissible; this rial led to tumors that grew quickly, were more malignant
was the first transmissible solid tumor discovered. Second, than usual, and produced widespread metastases. Rous con-
he found that the tumor-inducing factor could be passed tinued to study the phenomenon he had begun to unravel for
through a Berkefeld ultrafilter known to retain bacteria. In many years, but understanding the mechanistic bases for the
the context of the times, this finding proved that the agent complex natural history of Rous sarcoma virus and related
was a virus, the first of its kind. The virus he discovered viruses had to wait until modern molecular and cellular bio-
is now known as Rous sarcoma virus and has since been logic technologies became available in the 1960s.
studied in many laboratories around the world. In 1934, Rous’ Rockefeller Institute colleague, Rich-
Rous was born in 1879 in Baltimore, Maryland, ard E. Shope, asked him to examine warts on jackrab-
USA, and raised by his widowed mother, who persevered bits that had definitively been shown to be caused by an
in supporting his education. He received bachelor of arts
and doctoral degrees from The Johns Hopkins University
in Baltimore in 1900 and 1905, respectively. After teach-
ing pathology at the University of Michigan, Ann Arbor,
and studying morbid anatomy at Friedrichstadt Municipal
Hospital in Dresden, Germany, in 1909, he took a position
at the Rockefeller Institute for Medical Research in New
York, New York, where he spent the rest of his life. Rock-
efeller was the place in the United States where virology
first emerged as a distinct medical science; from the begin-
ning of his career, Rous was surrounded by great scientists.
Rous’ entry into tumor virology was fortuitous; the
founding director of the Rockefeller Institute, Simon
Flexner, had been interested in oncology but wanted to re-
direct his own work toward polio, which was becoming a
major problem. Rous was hired to continue Flexner’s re-
search into oncology, a subject about which Rous at first
knew nothing.
The beginning of the story behind Rous’ claim to fame
is remarkable: a woman came to the Rockefeller Institute
with a barred Plymouth Rock hen that had a large tumor on
its breast. Rous later wrote, “In this paper is reported the first
avian tumor that has proved transplantable to other individu-
Author affiliations: Mill Creek High School, Hoschton, Georgia, USA
(P. Kumar); and University of Texas Medical Branch, Galveston,
Texas, USA (F.A. Murphy) Figure. Francis Peyton Rous (1879–1970), pictured in 1923, at age
44, in his laboratory at the Rockefeller Institute for Medical Research,
DOI: http://dx.doi.org/10.3201/eid1904.130049 New York, NY, USA. Source: National Library of Medicine.

662 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 PHOTO QUIZ

ultrafilterable virus. This virus was Shope papillomavirus Control and Prevention, Atlanta, Georgia. He is planning on a
(rabbit papillomavirus). When Rous confirmed that the career in medicine.
warts were benign tumors, he was reinvigorated in his in-
Dr Murphy is a professor at the University of Texas Medical
tent to unravel the mysteries of viral oncology. Over the
Branch in Galveston. Formerly, he was chief of the Viral Pathol-
next 30 years, Rous and his colleagues showed that the be-
ogy Branch, then director of the Division of Viral and Rickettsial
nign tumors could progress to malignant carcinomas and
Diseases, and then director of the National Center for Infectious
that chemical carcinogens could interact with the virus—
Diseases at the Centers for Disease Control and Prevention.
further discoveries that formed building blocks for mod-
ern virology. Today, we recognize that ≈20% of human
cancers worldwide have infectious etiologies, for which Suggested Reading
preventive measures such as vaccines have great promise.
1. Andrewes CH. Francis Peyton Rous, 1879–1970. Biogr Mem
Rous’ colleagues, including the scientists René Dubos Fellows R Soc. 1971;17:643–62. http://dx.doi.org/10.1098/
and Charles B. Huggins, lauded Rous’ personal and profes- rsbm.1971.0025
sional qualities, writing that he was gifted with supreme 2. Dubos R. Peyton Rous. J Exp Med. 1979;150:735–7.
intellectual powers, unfailing integrity and honesty, a re- 3. Dulbecco R. Francis Peyton Rous. Biogr Mem Natl Acad Sci.
1976;48:275–306.
markably intuitive sense for the science itself, great perse- 4. Norrby E. Nobel prizes and life sciences. Singapore: World Scien-
verance and work ethic, and an enormous zest for life. One tific Publishing; 2011.
can imagine with wonder Rous in his laboratory and in the 5. Parkin DM. The global health burden of infection-associated can-
legendary lunchroom of the Rockefeller Institute. cers in the year 2002. Int J Cancer. 2006;118:3030–44. http://dx.doi.
org/10.1002/ijc.21731
Rous was duly honored for his masterful work, win- 6. Rous P. A transmissible avian neoplasm (sarcoma of the common
ning the National Medal of Science and membership in the fowl). J Exp Med. 1910;12:696–705. http://dx.doi.org/10.1084/
National Academy of Sciences, the American Philosophi- jem.12.5.696
cal Society, the Royal Society, and other prestigious or- 7. Rous P. A sarcoma of the fowl transmissible by an agent separable
from the tumor cells. J Exp Med. 1911;13:397–411. http://dx.doi.
ganization. In 1966, when he was 87 years old, Rous was org/10.1084/jem.13.4.397
awarded the Nobel Prize in Medicine, an honor he shared 8. Rous P. Nobel lecture: the challenge to man of the neoplastic cell.
with Charles Huggins. After 55 years, the longest “incuba- 1966 [cited 2012 Nov 28]. http://www.nobelprize.org/nobel_prizes/
tion period” in the history of the Nobel Prizes, Rous’ dis- medicine/laureates/1966/rous-lecture.html
9. Rubin H. The early history of tumor virology: Rous, RIF, and
covery had finally been recognized with this honor. In the RAV. Proc Natl Acad Sci U S A. 2011;108:14389–96. http://dx.doi.
end, proof that Rous was just ahead of his time might be org/10.1073/pnas.1108655108
found in the several additional Nobel Prizes awarded since 10. Weiss RA, Vogt PK. 100 years of Rous sarcoma virus. J Exp Med.
1966 to virologists who further unraveled viral oncology. 2011;208:2351–5. http://dx.doi.org/10.1084/jem.20112160

Mr Kumar is a student at Mill Creek High School, Hoschton, Address for correspondence: Prasanna Kumar, Mill Creek High School,
Georgia, USA. He became interested in medical history after 4400 Braselton Hwy, Hoschton, GA 30548, USA; email: pras.sb@
an epidemiology camp experience at the Centers for Disease gmail.com

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 663
ANOTHER DIMENSION

Myth Dispelled
Adam Possner

The flu vaccine cannot

give you the flu, I tell him.
It’s dead virus, there’s
nothing alive about it.
It can’t make you sick.
That’s a myth.
But if we bury it in
the grassy knoll
of your shoulder,
an inch under the stratum
corneum, as sanctioned by
your signature
in a white-coated ceremony
presided over by
my medical assistant
and then mark the grave
with a temporary
non-stick headstone,
the trivalent spirit
of that vaccine
has a 70 to 90 percent
chance of warding off
the Evil One,
and that’s the God’s
honest truth.

Dr Possner is an assistant professor of general internal medi- Address for correspondence: Adam Possner, Medical Faculty
cine at George Washington University in Washington, DC. His Associates, George Washington University, 2150 Pennsylvania
areas of interest include preventive medicine and medical student Ave NW, Suite 5-416 North, Washington, DC 20037, USA; email:
and resident education. apossner@mfa.gwu.edu

Author affiliation: George Washington University, Washington, From JAMA, December 5, 2012. © American Medical Association, 2012.
DC, USA Reprinted with permission.
DOI: http://dx.doi.org/10.3201/eid1904.ET1904

664 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 LETTERS

Novel Serotype of serotypes have spread throughout by using described methods (10) to be
much of continental Europe and parts positive for BTV by serogroup-specific
Bluetongue of the British Isles and Scandinavia, quantitative reverse transcription PCR
Virus, Western precipitating an economically (qRT-PCR) but negative by serotype-
North America devastating epidemic (7). Similarly, specific qRT-PCRs for BTV-10, 11,
ongoing surveillance has identified 13, and 17.
To the Editor: Bluetongue is an novel BTV serotypes in regions to Further analysis using additional
arboviral disease of domestic and wild which it historically has been endemic serotype-specific qRT-PCRs identified
ruminants characterized by vascular (e.g., Australia and the Middle virus in the blood sample as BTV-2.
injury that produces widespread edema East) (2). Climate change may have BTV was isolated in primary bovine
and tissue necrosis (1). Bluetongue contributed to this dramatic recent endothelial cells from blood collected
virus (BTV), the causative agent of expansion in global distribution of from the heifer. Sequence analysis
bluetongue, is the prototype virus BTV, most notably in Europe (8). of the serotype-specific L2 gene of
of the genus Orbivirus in the family Bluetongue was first described the virus isolate confirmed it to be
Reoviridae (2). in the late 19th century among sheep BTV-2 (2), and phylogenetic analyses
BTV occurs throughout brought from Europe to South Africa, showed it to be most closely related to
temperate and tropical areas of the and later in North America in ≈1950 a strain of BTV-2 isolated in Florida
world coincident with the distribution (4). Surveillance in western North in 1999 (Figure). However, sequence
of vector Culicoides spp. midges (3– America since that time has confirmed analysis of the entire genome of the
5). Different midge species transmit that only BTV-10, 11, 13 and 17 virus from California indicated that
different constellations of BTV are present in this region, including it is a reassortant that includes genes
serotypes in distinct global episystems our recent intensive surveillance from BTV-6 and BTV-2. Specifically,
(3,5). For example, C. sonorensis is of sentinel cattle on dairy farms genes encoding the viral protein 1
the principal, if not exclusive, vector throughout California, USA (9,10). polymerase and viral protein 3 major
of BTV serotypes 10, 11, 13, and 17 However, during investigation core protein segregate with those
in much of North America, whereas of an outbreak of acute coronitis and of the US prototype strain of BTV-
C. insignis is the major vector of ulcerative stomatitis among cattle at a 6 (isolated in 2006), but other genes
multiple BTV serotypes (including dairy farm in the northern Sacramento are derived from BTV-2. BTV-2 and
BTV 1–4, 6, 8, 12, 17, 19, 20, and Valley in California in August 2010, a BTV-6 have been isolated only in the
probably others) in the Caribbean blood sample from a heifer was found southeastern United States, which
basin, Central America, and South
America. C. insignis is also found
in the southeastern United States,
and although this species might
have recently expanded its range in
the region, its distribution in North
America remains poorly defined.
Serotypes of BTV other than 10, 11,
13, and 17 are found in areas of the
United States: BTV-2 was first reported
in Florida in 1982. Since 1998, ten
additional serotypes (BTV-1, 3, 5, 6,
9, 12, 14, 19, 22, and 24) have been
identified in the southeastern United
States (6).
Approximately 26 BTV serotypes
have been described and the global
distribution of BTV has recently been
altered (2,4). Coincident with the Figure. Cladogram comparing the L2 genes of different bluetongue virus (BTV) serotypes
invasion of novel BTV serotypes into and global strains of BTV serotype 2 (BTV-2) with that of a virus isolated in northern
California, USA (2-California-2010; GenBank accession nos. JQ822248–JQ822257).
the southeastern United States (6),
Viruses are identified by serotype, country/region of origin, and for isolates of BTV serotype
likely by extension from the adjacent 2, year of identification. Bootstrap percentages are indicated at selected branching points.
Caribbean basin, multiple BTV EHDV1, epizootic hemorrhagic disease of deer virus serotype 1.

665 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
LETTERS

indicates translocation within the Prev Vet Med. 2011;102:107–11http:// In contrast, HEV genotypes 3
United States of reassortant BTV-2. dx.doi.org/10.1016prevetmed.2011.04.005 and 4 are increasingly identified
5. Tabachnick WJ. Culicoides and the
How this virus spread to California global epidemiology of bluetongue virus as causative agents of acute viral
is not known, and its distribution infection. Vet Ital. 2004;40:144–50. hepatitis in industrialized countries
in the United States is uncertain 6. Johnson DJ. Identification of new United (1). Genotypes 1 and 2 are found
because there is no comprehensive States bluetongue types. Proceedings only in humans, whereas genotypes 3
of the United States Animal Health
national BTV surveillance program. Association. 2011;111:209–10. and 4 are associated with food-borne
However, BTV-2 was not detected 7. Saegerman C, Berkvens D, Mellor PS. zoonotic transmission from domestic
previously in California, suggesting Bluetongue epidemiology in the European pigs, wild boar, and deer (1).
that this serotype was recently Union. Emerg Infect Dis. 2008;14:539–44. In addition to these 4 genotypes,
http://dx.doi.org/10.3201/eid1404.071441
introduced into the region or that 8. Purse BV, Brown HE, Harrup L, Mertens HEV-related viruses were detected in
it is uncommon. Identification of PP, Rogers DJ. Invasion of bluetongue and avian, rodent, and bat hosts, which
this novel BTV serotype in western other orbivirus infections into Europe: the formed novel genera within the family
North America emphasizes the role of biological and climatic processes. Hepeviridae (2). In Africa, HEV
Rev Sci Tech. 2008;27:427–42.
need for ongoing entomologic and 9. Osburn BI, McGowan B, Heron B, genotype 1 and 2 strains have been
livestock surveillance, particularly in Loomis E, Bushnell R, Stott JL, et al. identified during HEV epidemics
light of recent changes in the global Epizootiologic study of bluetongue: (3–5). An HEV genotype 3 strain
distribution and nature of BTV virologic and serologic results. Am J Vet was detected in 1 of 40 fecal samples
Res. 1981;42:884–7.
infection (4,6,8). 10. Mayo CE, Barker CM, Mullens BA, from domestic pigs in Kinshasa,
Gerry AC, Mertens PP, Maan S, et al. The Democratic Republic of the Congo,
N. James Maclachlan, combination of abundance and infection and it was suggested that this strain
William C. Wilson, rates of Culicoides sonorensis estimates risk was imported from Belgium to the
of subsequent bluetongue virus infection of
Beate M. Crossley, Democratic Republic of the Congo
sentinel cattle on California dairy farms.
Christie E. Mayo, Vet Parasitol. 2012;187:295–301. http:// by animal trade (6). Therefore, we
Dane C. Jasperson, dx.doi.org/10.1016/j.vetpar.2012.01.004 investigated whether HEV strains of
Richard E. Breitmeyer, and genotype 3 or 4 are circulating among
Annette M. Whiteford Address for correspondence: N. James domestic pigs in Cameroon.
Author affiliations: University of California, Maclachlan, Department of Pathology, During February–March 2012,
Davis, California, USA (N.J. Maclachlan, Microbiology and Immunology, School of a total of 345 liver samples were
B.M. Crossley, C.E. Mayo, R.E. Breitmeyer); Veterinary Medicine, University of California, collected from domestic pigs (age
US Department of Agriculture–Agricultural Davis, CA 95616, USA; email: njmaclachlan@ range 6 months–3 years) in abattoirs
Research Service, Manhattan, Kansas, ucdavis.edu in Douala and Yaoundé, Cameroon,
USA (W.C. Wilson, D.C. Jasperson); and in slaughter slaps (areas) in
and California Department of Food and Bamenda, Cameroon. Pigs were
Agriculture, Sacramento, California, USA mainly of the local breed. In addition,
(A.M. Whiteford) pigs originating from extensive cross-
DOI: http://dx.doi.org/10.3201/eid1904.120347 Hepatitis E Virus breeding (local X landrace and local X
Duroc) were sampled. Liver samples
References
Genotype 3 Strains were collected during post-mortem
in Domestic Pigs, inspection.
1. Maclachlan NJ, Drew CP, Darpel KE,
Worwa G. The pathology and pathogenesis Cameroon Viral RNA was extracted from
of bluetongue. J Comp Pathol.
liver samples by using the RTP DNA/
2009;141:1–16. http://dx.doi.org/10.1016/j. To the Editor: Hepatitis E virus RNA Virus Mini Kit II (STRATEC
jcpa.2009.04.003 (HEV) is a positive-stranded, non- Molecular, Berlin, Germany)
2. Maan S, Maan NS, Nomikou K, Batten C, enveloped RNA virus of the family according to the manufacturer’s
Antony F, Belaganahalli MN, et al. Novel
bluetongue virus serotype from Kuwait.
Hepeviridae that is considered to be instructions. Extracted RNA was
Emerg Infect Dis. 2011;17:886–9. http:// the main causative agent of enterically analyzed for HEV RNA by using 2
dx.doi.org/10.3201/eid1705.101742 transmitted acute hepatitis (1). nested reverse transcription PCRs
3. Gibbs EP, Greiner EC. The epidemiology HEV is classified into 4 genotypes (RT-PCRs) specific for open reading
of bluetongue. Comp Immunol Microbiol
Infect Dis. 1994;17:207–20. http://dx.doi.
(1). HEV genotypes 1 and 2 cause frame 1 (ORF 1) and ORF 2 of
org/10.1016/0147-9571(94)90044-2 large waterborne epidemics of acute HEV (7,8). Nested RT-PCRs and
4. Maclachlan NJ. Bluetongue: History, hepatitis in developing countries, direct sequencing of amplicons were
global epidemiology, and pathogenesis. especially in Africa and Asia (1). performed as described (9). RNA of

666 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 LETTERS

HEV strain Hamburg-HB (GenBank from Japan (JSWINE150-Aom04R; In agreement with distance
accession no. JN986840) was used as GenBank accession no. AB221520) analysis, phylogenetic reconstruction
a positive control for nested RT-PCRs. and Mongolia (swMN06-A1354; using partial nucleotide sequences
HEV RNA was detected in 2 GenBank accession no. AB290105) of ORF 2 (278 nt) showed a close
samples from female pigs in Yaoundé were 90% and 91%, respectively. relationship of strains Yaounde56
(2/139) and 1 sample from a male pig At the amino acid level, the partial and Yaounde94 with HEV genotype
in Bamenda (1/39). All 167 samples RNA-dependent RNA polymerase 3 strains (Figure). However, the HEV
from Douala were negative for HEV sequence (ORF 1) and the partial strains from Cameroon do not cluster
RNA. The sample from Bamenda capsid protein sequence (ORF 2) with the classified HEV genotype 3
showed a positive result for the nested of strain Yaounde56 were identical subtype reference strains (10) in the
RT-PCR specific for HEV ORF 1. to those of HEV genotype 3 strains phylogenetic tree (Figure). These
Genetic distances calculated with HEV/Gt3/HSD40/2009 (GenBank strains cluster within a clade of subtype
partial nucleotide sequences of ORF 1 accession no. AFO71833) from undefined strains and are most closely
(280 nt) and ORF 2 (373 nt) between Germany and swJ12–1 (GenBank related to strain swMN06-A1354 from
strain Yaounde56 and the most closely accession no. BAC66273) from Japan. Mongolia (Figure).
related HEV genotype 3 strains Thus, all mutations were silent. Because the pig production
cycle is shorter than that for cattle,
pig production is a major economic
activity in Cameroon. Most pigs
in Cameroon are local raised, and
extensive cross-breeding is used.
The infection rate of pigs with HEV
genotype 3 strains from Cameroon is
lower than that of pigs from Europe.
Thus, HEV genotype 3 seems to
have an extensive distribution that
includes Africa. Future studies should
investigate how HEV genotype 3
strains contribute to sporadic HEV
cases in Cameroon.
This study was supported by Coorde-
nacão de Aperfeicoamento de Pessoal de
Nível Superior.

Vanessa S. de Paula,
Matthias Wiele,
Afegenwi H. Mbunkah,
Achukwi M. Daniel,
Manchang T. Kingsley,1 and
Jonas Schmidt-Chanasit1
Author affiliations: Fundação Oswaldo
Cruz, Rio de Janeiro, Brazil (V.S. de Paula);
Figure. Phylogenetic analysis of hepatitis E virus (HEV) strains, Cameroon. The Bayesian Bernhard Nocht Institute for Tropical
phylogenetic tree was constructed by using partial nucleotide sequence of open reading
Medicine, Hamburg, Germany (V. Salete
frame 2 (278 nt) of HEV. For each sequence used, the GenBank accession number, strain
designation, source of isolation, country of isolation, and HEV subtype are shown. Multiple de Paula, M. Wiele, J. Schmidt-Chanasit);
nucleotide sequence alignment was analyzed by using the Markov Chain Monte Carlo Centre Pasteur, Yaoundé, Cameroon (A.H.
method implemented in the program MrBayes version 3.0 (http://mrbayes.sourceforge. Mbunkah); and Institute of Agricultural
net/) and applying the general time-reversible substitution model. Posterior probabilities Research for Development, Ngaoundere,
are shown at the nodes of the tree to the right of the slash if >0.5. Bootstrap values
Cameroon (A.M. Daniel, M.T. Kingsley)
calculated from 10,000 replicates are indicated at the nodes of the tree to the left of the
slash. Alignment was analyzed by using the neighbor-joining method and resulted in same DOI: http://dx.doi.org/10.3201/eid1904.121634
tree topology (not shown). Newly described HEV sequences are shown in boldface.
Scale bar indicates evolutionary distance. UK, United Kingdom; USA, United States; DRC,
1
These authors contributed equally to this
Democratic Republic of Congo. article.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 667
LETTERS

References Tropical Medicine, World Health Organization As part of the aforementioned

Collaborating Centre for Arbovirus and molecular epidemiology study
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Fumie Tateno A, Cottontail V, Melim second variable domain of nucleic
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hepatitis E virus-related viruses that form
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2000;7:334–8. http://dx.doi.org/10.1007/
BF02253253
Subtype ON1 dominant circulating RSV genotype,
119 (74%) of 160 RSV isolates were
4. van Cuyck H, Juge F, Roques P. among Children, RSV A. We noted the presence, albeit
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To the Editor: Human respiratory wwwnc.cdc.gov/EID/pdfs/12–1465-
Guthmann JP, Guerin JP, Caron M, et al. syncytial virus (RSV) is a common Techapp.pdf) in specimens collected
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Med Virol. 2005;77:519–21. http://dx.doi.
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6. Kaba M, Colson P, Musongela JP, Tshilolo worldwide (1,2).As part of an RSV health care facilities during February
L, Davoust B. Detection of hepatitis E virus nosocomial transmission study, we 24–April 25, 2012 (Figure and
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was identified during November 2010­– where they received a diagnosis of
dx.doi.org/10.1016/j.meegid.2009.09.011 February 2011 as a novel genotype brochiolitis or bronchiopneumonia
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RG, Reetz J, Schielke A. Detection of a children in a tertiary pediatric hospital With the exception of 1 patient, child
novel hepatitis E-like virus in faeces of
wild rats using a nested broad spectrum
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RT-PCR. J Gen Virol. 2010;91:750–8. 2012. The genotype described in onset of this illness, all ON1 isolates
http://dx.doi.org/10.1099/vir.0.016584-0 Canada was characterized by a 72-nt were community acquired. Seven of
8. Mizuo H, Suzuki K, Takikawa Y, Sugai Y, sequence duplication within the second the 8 ON1 isolates were obtained from
Tokita H, Akahane Y, et al. Polyphyletic
strains of hepatitis E virus are responsible
variable domain of the envelope infants <4 months of age (median 7
for sporadic cases of acute hepatitis in glycoprotein. The 72-nt duplication weeks), who were younger than the
Japan. J Clin Microbiol. 2002;40:3209–18. within the second variable domain 152 children who were not infected
http://dx.doi.org/10.1128/JCM.40.9.3209- in ON1 was the largest sequence with the ONI genotype (median age
3218.2002
9. Pfefferle S, Frickmann H, Gabriel M,
duplication described in this virus (3). 3.5 months). The RSV ON1–infected
Schmitz N, Günther S, Schmidt-Chanasit RSV is divided into 2 genetically children lived within a 2.5-km radius
J. Fatal course of an autochthonous distinct groups, RSV A and B, based of one another (online Technical
hepatitis E virus infection in a patient on the viral envelope glycoprotein Appendix Table). The children who
with leukemia in Germany. Infection.
2012;40:451–4. http://dx.doi.org/10.1007/
nucleotide sequences (4). Sequence were not infected with RSV ON1
s15010-011-0220-7 variability in the C-terminal variable lived in a much wider geographic area;
10. Lu L, Li C, Hagedorn CH. Phylogenetic domain of the glycoprotein gene is >90% lived within an 18-km radius of
analysis of global hepatitis E virus commonly used to determine RSV one another. These spatial associations
sequences: genetic diversity, subtypes and
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phylogeny (3,5). To date, 11 RSV A with disease prevalence suggested that
http://dx.doi.org/10.1002/rmv.482 (ON1, GA1–GA7, SAA1, NA1, and the ON1-infected children represented
NA2) and 17 RSV B (GB1–GB4, a localized cluster of transmission.
Address for correspondence: Jonas Schmidt- SAB1–SAB3, and BA1–BA10) None of the children were infected
Chanasit, Bernhard Nocht Institute for genotypes have been identified (3,6). with HIV, although 3 had antenatal

668 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 LETTERS

Figure. Alignment of deduced amino acid sequences for ON1 isolates from South Africa (patients 1–8, accession nos. JX885730–
JX885737) and Canada (ON67 and ON138) with NA1 isolates (patients 9–12) from South Africa. A) Variable domain sequence copied. B)
Duplicated sequence inserted into variable domain. C) Characteristic amino acid substitutions that distinguish ON1 from NA1. D) Amino
acid substitution (E308K) (position 284 before insertion) that distinguishes between most ON1 isolates from South Africa (patients 2–8)
from those from Canada (ON67 and ON138) ON1.

exposure to HIV. Co-infection with domain, which, similar to ON1, did Study funding was provided by the
adenovirus and rhinovirus was noted not cause serious clinical outcomes University of Cape Town.
in 3 of the patients. Although 3 of (6,8–10). Longitudinal analyses
the patients were hospitalized for during 12 epidemic seasons (1996–
Ziyaad Valley-Omar,
a prolonged period and required 97 through 2007–08) of international
Rudzani Muloiwa,
ventilatory support, the severity and RSV subtype distribution revealed that
Nai-Chung Hu, Brian Eley,
outcome of the RSV ON1 infections since its initial detection in 1999, BA
and Nei-Yuan Hsiao
were similar to RSV infections caused prevalence has gradually increased
Author affiliations: University of Cape Town,
by other genotypes among children of to become the dominant RSV group
Cape Town, South Africa (Z. Valley-Omar,
the same age. B virus genotype in circulation (10).
R. Muloiwa, B. Eley, N.-Y. Hsiao); National
Sequence analyses revealed Because RSV A has traditionally been
Institute for Communicable Diseases, Cape
that ON1 isolates identified in South the dominant RSV type in circulation,
Town (Z. Valley-Omar, N.-C. Hu, N.-Y. Hsiao);
Africa are essentially identical to if the large insertion in ON1 confers
Red Cross War Memorial Children’s Hospital,
those isolated in Canada, possessing similar selection advantage as seen
Cape Town (R. Muloiwa, B. Eley); and National
characteristic amino acid substitutions in BA, the potential dominance of
Health Laboratory, Cape Town (N.-Y. Hsiao)
at positions E232G, T253K, and a single ON1 genotype within this
P314L that distinguish the genotype group might promote bias on RSV DOI: http://dx.doi.org/10.3201/eid1904.121465
from circulating NA1 genotypes (3). type distribution toward RSV A.
However, 7 of 8 ON1 isolates from The novel ON1 genotype was References
South Africa possess a unique E308K first described in Ontario, Canada
(position 284 before insertion) amino (3). Our subsequent findings in South 1. Selwyn BJ. The epidemiology of acute
acid change at the 3′ border of the Africa suggest extensive distribution respiratory tract infection in young
children: comparison of findings from
duplicated gene segment not present of this genotype, which was assumed several developing countries. Coordinated
in the ON1 isolates identified in to have arisen before winter 2010–11 Data Group of BOSTID Researchers. Rev
Canada (Figure). The conservation (3). To understand whether ON1 in Infect Dis. 1990;12(Suppl 8):S870–88.
of the E308K mutation within ≈90% South Africa occurred as a result of http://dx.doi.org/10.1093/clinids/12.
Supplement_S870
of the isolates from South Africa that importation or natural evolution within 2. World Health Organization (WHO).
we studied suggests a possible locally circulating NA1 genotypes, Initiative for Vaccine Research (IVR).
functional role for the positively further research is required. Respiratory syncytial virus and
charged lysine residue. parainfluenza virus. Disease burden.
Geneva: The Organization; 2009.
The capacity of the RSV Acknowledgments
3. Eshaghi A, Duvvuri VR, Lai R, Nadarajah
glycoprotein to accommodate large We thank the staff of the Groote JT, Li A, Patel SN, et al. Genetic variability
insertions and remain functional was Schuur National Health laboratory of human respiratory syncytial virus A
first demonstrated with the RSV B, BA Service, who provided RSV- positive strains circulating in Ontario: a novel
genotype with a 72 nucleotide G gene
genotype (Buenos Aires, Argentina archived cDNA samples. We thank Heidi
duplication. PLoS ONE. 2012;7:e32807.
1999). This genotype contains a 60- Smuts for critical review of the manuscript h t t p : / / d x . d o i . o rg / 1 0 . 1 3 7 1 / j o u r n a l .
nt duplication in the second variable and research guidance. pone.0032807

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 669
LETTERS

4. Johnson PR, Spriggs MK, Olmsted

RA, Collins PL. The G glycoprotein of
Henipaviruses from New Britain and 103 from Lae
were forwarded to the Common-
human respiratory syncytial viruses of and Fruit Bats, wealth Scientific and Industrial Re-
subgroups A and B: extensive sequence
divergence between antigenically Papua New Guinea search Organisation’s Australian Ani-
related proteins. Proc Natl Acad Sci mal Health Laboratory in Geelong,
U S A. 1987;84:5625–9. http://dx.doi. To the Editor: In 2010, detection Australia. Samples from New Britain
org/10.1073/pnas.84.16.5625
5. Cane PA. Molecular epidemiology of of henipavirus (Hendra or Nipah virus) were screened for antibodies against
respiratory syncytial virus. Rev Med and rubulavirus (Tioman or Menangle Hendra virus by virus neutralization
Virol. 2001;11:103–16. http://dx.doi. virus) antibodies in fruit bats in Papua test (VNT) (3). Positive samples were
org/10.1002/rmv.305 New Guinea (PNG) was reported (1). subsequently screened by VNT for an-
6. Dapat IC, Shobugawa Y, Sano Y, Saito R,
Sasaki A, Suzuki Y, et al. New genotypes To explore changes in henipavirus dy- tibodies against Nipah virus. Samples
within respiratory syncytial virus group namics in fruit bats, we compare and from Lae were screened by VNT for
B genotype BA in Niigata, Japan. J Clin contrast this finding with serologic Hendra, Nipah, and Menangle viruses
Microbiol. 2010;48:3423–7. http://dx.doi. findings from 10 years earlier (2; H. (3). A reciprocal VNT titer of >5 was
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7. Peret TC, Hall CB, Schnabel KC, Golub Field et al., unpub. data). considered indicative of antibodies.
JA, Anderson LJ. Circulation patterns of In these earlier studies, blood Of the 20 samples from Madang,
genetically distinct group A and B strains samples were collected from 182 2 (10%) reacted in the Hendra virus
of human respiratory syncytial virus in a wild-caught fruit bats of mixed spe- ELISA. Of the 147 samples from
community. J Gen Virol. 1998;79:2221–9.
8. Trento A, Galiano M, Videla C, Carballal cies, age, and sex from 3 locations in New Britain and Lae that yielded
G, García-Barreno B, Melero JA, et PNG: Madang (1996), New Britain definitive VNT results, 11 (7.5%)
al. Major changes in the G protein (1997), and Lae (1999 ) (2; H. Field yielded neutralizing antibodies to
of human respiratory syncytial virus et al., unpub. data) (Figure). The 20 Hendra virus and 5 (3.4%) to Nipah
isolates introduced by a duplication of 60
nucleotides. J Gen Virol. 2003;84:3115– samples from Madang were collected virus. All samples with antibodies
20. http://dx.doi.org/10.1099/vir.0.19357-0 as blood spots on filter paper and for- against Nipah virus also had anti-
9. Trento A, Viegas M, Galiano M, Videla C, warded to the (then) Department of bodies against Hendra virus; titers
Carballal G, Mistchenko AS, et al. Natural Primary Industries Animal Research against Hendra virus were greater (4
history of human respiratory syncytial
virus inferred from phylogenetic analysis Institute in Brisbane, Australia, where samples) or equivalent (1 sample) to
of the attachment (G) glycoprotein they were eluted and screened by those against Nipah virus. Recipro-
with a 60-nucleotide duplication. J ELISA for antibodies against Hen- cal titers against Hendra virus were
Virol. 2006;80:975–84. http://dx.doi. dra virus (3). Serum from 59 samples 5–160 (median 10) and against Nipah
org/10.1128/JVI.80.2.975-984.2006
10. Trento A, Casas I, Calderón A, Garcia-
Garcia ML, Calvo C, Perez-Breña P, et al.
Ten years of global evolution of the human
respiratory syncytial virus BA genotype
with a 60-nucleotide duplication in the G
protein gene. J Virol. 2010;84:7500–12.
http://dx.doi.org/10.1128/JVI.00345-10

Address for correspondence: Ziyaad Valley-

Omar, National Health Laboratory Service,
NICD Virology, C21 Groote Schuur Hospital,
Main Road Observatory, Cape Town, Western
Cape 7935, South Africa; email: z.valley-
omar@uct.ac.za

Podcasts from

EIDonline Figure. Sampling locations and henipavirus antibody prevalence, Papua New Guinea 1996–
www.cdc.gov/ncidod/EID/podcast/ 1999, among 182 wild-caught fruit bats from Madang (1996), New Britain (1997), and Lae
(1999), Papua New Guinea. HieV, Hendra virus; NiV, Nipah virus; MenV, Menangle virus.

670 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 LETTERS

virus, 5–80 (median 10). None of the the differences is more consistent with Hume Field, Carol E. de Jong,
103 samples from Lae had antibodies a major 10-year change in infection Kim Halpin, and Craig S. Smith
against Menangle virus (Figure). dynamics in these bat populations. Author affiliations: Queensland Centre for
The common and contrasting find- Sendow et al. (4), when reporting Emerging Infectious Diseases, Brisbane,
ings between the study of Breed et al. henipavirus infections in fruit bats in Queensland, Australia (H.E. Field, C.E. de
(1) and the earlier studies are as fol- Indonesia, canvassed the geographic Jong, C.S. Smith); and Life Technologies,
lows. First, the earlier studies identified extent of Hendra virus and Nipah vi- Singapore (K. Halpin)
antibodies against Hendra virus in fruit rus, concluding that a transition prob- DOI: http://dx.doi.org/10.3201/eid1904.111912
bats from multiple locations and spe- ably occurred between Hendra virus in
cies, as did the study by Breed et al. (1). Australia and Nipah virus in Malaysia References
However, in marked contrast, the ear- and beyond. They also concluded that
lier studies found a crude prevalence further research was needed to under- 1. Breed AC, Meng Y, Barr JA, Crameri G,
of antibodies against Hendra virus of stand the nature and stability of the Thalmann CM, Wang LF. Prevalence of
henipavirus and rubulavirus antibodies in
7.8% (13/167) compared with 50% interface between Hendra virus and pteropid bats, Papua New Guinea. Emerg
found by Breed et al. (1). Although this Nipah virus and to investigate the pos- Infect Dis. 2010;16:1997–9. http://dx.doi.
difference could reflect confounding sible presence of unidentified cross- org/10.3201/eid1612.100879
by species, location, or time, when we neutralizing henipaviruses. 2. Mackenzie JS. Emerging viral diseases: an
Australian perspective. Emerg Infect Dis.
controlled for the first 2 by comparing Changed henipavirus dynamics 1999;5:1–8. http://dx.doi.org/10.3201/
only 1 bat species (Pteropus conspicil- in PNG fruit bat populations could eid 0501.990101
latus) from proximate locations (Lae reflect altered population dynamics 3. Daniels P, Ksiazek T, Eaton B. Laboratory
and Madang), the significant differ- (and consequent infection dynamics) diagnosis of Nipah and Hendra virus in-
fections. Microbes Infect. 2001;3:289–95.
ences in antibody prevalence remained: associated with negative ecologic ef- http://dx.doi.org/10.1016/S1286-4579
0 (95% CI 0–23%) in 1999 and 65% fects (e.g., habitat loss, encroachment) (01)01382-X
(95% CI 50%–78%) in 2009. (5). More broadly, such changes might 4. Sendow I, Field HE, Curran J, Darminto,
Second, in the earlier studies, all portend a regional shift in the geo- Morrissy C, Meehan G, et al. Henipavirus
in Pteropus vampyrus bats, Indonesia.
bats (except 1) that had a neutraliz- graphic interface between Hendra and Emerg Infect Dis. 2006;12:711–2. http://
ing antibody titer to Nipah virus had Nipah virus endemicity. dx.doi.org/10.3201/eid1204.051181
a higher neutralizing titer to Hendra More robust interpretation of the 5. Mickleburgh S, Hutson A, Racey P. Old
virus (1 bat had equivalent titers), sug- serologic findings of both studies is World fruit bats: an action plan for their
conservation. Gland (Switzerland): Inter-
gesting that the circulating henipavi- constrained by the lack of henipavirus national Union for Conservation of Na-
rus was more similar to Hendra virus sequence data from PNG and neigh- ture; 1992.
than to Nipah virus. These findings are boring countries. We concur with
supported at a regional level by those Breed et al. (1) that sequencing and Address for correspondence: Hume Field,
reported for nearby Indonesian islands phylogenetic analyses are imperative 39 Kessels Rd, Coopers Plains, Brisbane,
by Sendow et al. (4). However, the if the ecology of henipaviruses in fruit Queensland 4108, Australia; email: hume.
more recent findings of Breed et al. (1) bat populations and the implications field@daff.qld.gov.au
suggest the opposite. Of note, in the for human and livestock health in the
earlier PNG studies, titers against Hen- region are to be fully understood.
dra and Nipah viruses were modest Letters
(median 10) and pose the possibility of Letters commenting on recent articles
Acknowledgments as well as letters reporting cases, out-
cross-neutralization by a related, but We thank Steve Hamilton, Moses breaks, or original research are wel-
undescribed, additional henipavirus. Bockarie, and the Australian Quarantine come. Letters commenting on articles
Third, the earlier studies found no Inspection Services and PNG Veterinary should contain no more than 300
antibodies against Menangle virus in Services for sample collection from New words and 5 references; they are more
the 103 samples; in contrast, Breed et likely to be published if submitted
Britain, Madang, and Lae, respectively; Na- within 4 weeks of the original article’s
al. found 56% (1). Thus, earlier stud- tasha Forrest (then Natasha Smith) and the publication. Letters reporting cases,
ies found no samples with antibodies Australian Animal Health Laboratory for outbreaks, or original research should
against Menangle virus and henipavi- sample testing; and Peter Young for coordi- contain no more than 800 words
rus, in contrast to 36% of samples re- nation of the Hendra virus research project. and 10 references. They may have 1
ported by Breed et al. (1). Figure or Table and should not be di-
vided into sections. All letters should
Although any of these differences Funding was provided by agencies of
contain material not previously pub-
might have multiple interpretations, the Queensland, Australia, and the PNG lished and include a word count.
the collective scope and magnitude of governments.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 671
LETTERS

High Incidence of Biologic Products, Chengdu, China) were tested for antibodies against JE
has been included in the national virus, mumps virus, echoviruses, and
Japanese Expanded Program on Immunization coxsackieviruses (3,4,7). A case of JE
Encephalitis, at no charge. The recommendation for was defined as illness in a person with
Southern China children is vaccination at 8 months IgM against JEV in CSF or serum.
and 2 years of age (5,6). Clinical information was collected by
To the Editor: Japanese To estimate JE incidence in using a standardized chart abstraction
encephalitis virus (JEV) remains a Dehong Prefecture during January form. Linkages to personal identifiers
major source of illness and death in Asia 1–December 31, 2010, we conducted were destroyed.
(1). An estimated 67,900 cases occur an anonymous, unlinked study of A total of 189 eligible patients
each year in Asia; ≈33,900 cases— all cases of encephalitis at the only were enrolled, 150 from Mangshi
half the cases in the world—probably 2 major children’s hospitals in the and 39 from Ruili. Of these, 110
occur in the People’s Republic of region, Dehong Prefecture Hospital (58%) were male and 78 (41%) were
China (2). However, because reporting in Mangshi and Ruili City Hospital in <4 years of age. Enrollment peaked
is incomplete in most countries where Ruili. All eligible patients admitted to during summer (Figure). All patients
JE incidence is high, these estimates these hospitals were included in the were hospitalized within 6 days after
are based on scarce data. In China, a study. Inclusion criteria were age <15 symptom onset. A total of 22 (12%)
study conducted during 2006–2007 in years, residency in Dehong Prefecture, patients were classified as having JE
sentinel hospitals in 1 prefecture each clinical diagnosis of encephalitis, on the basis of IgM, in CSF for 21 and
in Shandong, Hubei, Guangxi, and lumbar puncture performed (routine in serum for 1. Illness onset occurred
Hebei Provinces (all in the eastern half for encephalitis patients at these 2 during May–November (Figure);
of China) found that 9.2% of patients hospitals), and cerebrospinal fluid overall incidence was 10.4 cases per
with acute meningitis and encephalitis (CSF) pleocytosis. After routine 100,000 children <15 years of age.
had JEV; adjusted incidence for each testing was completed, leftover CSF Among these 22 children with JE, 11
prefecture was 0.08–1.58 cases per and serum samples were stored at were male; 20 were from rural areas;
100,000 population (3). Incidence -70°C until further testing, which was 14 were from Mangshi and 8 were
in these 4 prefectures is lower than all conducted at the Chinese Center from Ruili; and 5 were 0–1 years, 6
that among children <14 years of for Disease Control and Prevention in were 2–4 years, and 11 were 5–13 years
age in JE-endemic countries, where Beijing. All CSF specimens were tested of age. JEV vaccination history was
estimated incidence is 5.4 cases per by viral culture in C6/36 and BHK- infrequently recorded in the medical
100,000 population (2). To assess 21 cells (7) and tested for antibodies charts; however, JEV was more likely
the need for strengthening existing against JEV (3,4). Serum samples to be the cause of encephalitis among
JE surveillance and vaccination
programs, we conducted a population-
based study of JE incidence in 1 area
of southern China.
Dehong is a prefecture in
western Yunnan Province, which
borders Myanmar. The JE-susceptible
population of Dehong Prefecture
(residents <15 years of age) is 211,337.
The 2 principal cities of Dehong
Prefecture—Mangshi and Ruili—are
busy commercial centers surrounded
by areas of extensive rice cultivation.
The mosquito vector of JEV, Culex
tritaeniorhynchus, is predominant
during summer (4). During 1988–
2007, JEV vaccination was available
only at certain clinics and only for a
fee; however, since 2008, vaccination
with the live, attenuated SA 14–14–2 Figure. Number of children with encephalitis at 2 hospitals, by etiology and month of symptom
JEV vaccine (Chengdu Institute of onset, Dehong Prefecture, People’s Republic of China, 2010.

672 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 LETTERS

children who received no vaccination Baoqing Dao, Kunhong Li, 7. Xufang Y, Huanyu W, Shihong F, Xiaoyan
(22%, 6/27) than among those with G, Shuye Z, Chunting L, et al. Etiological
Na Li, Zhengliu Yin,
spectrum of clinically diagnosed
unknown vaccination history (10%, Yonghua Liu, Roger Nasci, Japanese encephalitis cases reported in
15/157). Of 5 vaccinated children, 1 Huanyu Wang, Guizhou Province, China, in 2006. J Clin
had JE; however, verification of this and Guodong Liang Microbiol. 2010;48:1343–9. http://dx.doi.
child’s vaccination was not possible. org/10.1128/JCM.01009-09
Author affiliations: Yunnan Institute of
Among 71 children who had no Endemic Disease Control and Prevention,
Address for correspondence: Guodong Liang,
evidence of JE but for whom serum Dali, People’s Republic of China. (Y. Feng,
State Key Laboratory for Infectious Disease
samples were available for testing, 5 H. Zhang, W. Yang, Y. Zhang); National
Prevention and Control, Institute for Viral
had antibodies against mumps virus, Institute for Viral Disease Control and
Disease Control and Prevention, Chinese Center
8 against echoviruses, and 5 against Prevention of the Chinese Center for
for Disease Control and Prevention, Beijing
coxsackieviruses. Viral cultures of CSF Disease Control and Prevention, Beijing,
100052, People’s Republic of China; email:
from all 189 children were negative. China (S. Fu, X. Gao, H. Wang, G. Liang);
gdliang@hotmail.com
Our finding of 10.4 JE cases per Centers for Disease Control and Prevention,
100,000 children <15 years of age in Fort Collins, Colorado, USA (L.R. Petersen,
Dehong Prefecture is higher than the R. Nasci); Dehong Prefecture Center for
estimated incidence of 5.4 cases per Disease Control and Prevention, Mangshi,
100,000 population among children China (B. Zhang, B. Dao, K. Li, N Li);
<14 years of age in JE-endemic and Ruili Center for Disease Control and
countries (2). Nevertheless, the true Prevention, Ruili, China (Z. Yin, Y. Liu) Novel Hantavirus
JE population incidence for Dehong in Field Vole,
Prefecture might be underestimated 1
These authors contributed equally to this
if some children received no medical article.
United Kingdom
care or were admitted to other DOI: http://dx.doi.org/10.3201/eid1904.120137 To the Editor: Hantaviruses
hospitals. Adults were not studied; (family Bunyaviridae) are transmit-
however, ≈90% of JE cases in China ted to humans by inhalation of aero-
References
are reported among children <15 years solized virus in contaminated urine
of age (5,6). Unfortunately, accurate 1. Erlanger TE, Weiss S, Keiser J, Utzinger and feces, mainly from rodents of the
age-adjusted JE vaccination coverage J, Wiedenmayer K. Past, present, and families Cricetidae and Muridae. Al-
data for Dehong Prefecture are not future of Japanese encephalitis. Emerg
Infect Dis. 2009;15:1–7. http://dx.doi.
though infections in rodents are as-
available. Although vaccination org/10.3201/eid1501.080311 ymptomatic, infections in humans can
programs have markedly lowered JE 2. Campbell GL, Hills SL, Fischer M, lead to hemorrhagic fever with renal
incidence in China in recent years Jacobson JA, Hoke CH, Hombach JM, et syndrome and hantavirus cardiopul-
(5,6), the finding of continuing high al. Estimated global incidence of Japanese
encephalitis: a systematic review. Bull
monary syndrome (1).
JE incidence in Dehong Prefecture World Health Organ. 2011;89:766–774. In Europe, 5 rodent-borne hanta-
warrants further attention. http://dx.doi.org/10.2471/BLT.10.085233 viruses have been detected: Dobrava-
3. Yin Z, Wang HY, Yang JY, Luo H, Hadler Belgrade, Saaremaa, Seoul, Puumala,
This work was supported by grants SC, Sandhu HS, et al. Japanese encephalitis
disease burden and clinical features of
and Tula (1,2). The most common
from The Chinese Center for Disease Con-
Japanese encephalitis in four cities in the and widespread hantavirus in Europe
trol and Prevention–US Centers for Dis-
People’s Republic of China. Am J Trop is Puumala virus, which is associated
ease Control and Prevention Cooperative Med Hyg. 2010;83:766–73. http://dx.doi. with the mildest form of hemorrhagic
Agreement (U19-GH000004); the Min- org/10.4269/ajtmh.2010.09-0748
4. Wang J, Zhang H, Sun X, Fu S, Wang H,
fever with renal syndrome (1).
istry of Science and Technology, China
Feng Y, et al. Distribution of mosquitoes In the United Kingdom, only a
(2011CB504702); development grant of the
and mosquito-borne arboviruses in few cases of hantavirus infection in
State Key Laboratory for Infectious Disease Yunnan Province near the China– humans have been reported and con-
Prevention and Control (2008SKLID105); Myanmar–Laos border. Am J Trop Med
Hyg. 2011;84:738–46. http://dx.doi.
firmed serologically, but the caus-
and the National Natural Science Founda-
org/10.4269/ajtmh.2011.10-0294 ative virus species were not identified
tion of China (31070145).
5. Gao X, Nasci R, Liang GD. The neglected (3,4). Subsequent longitudinal stud-
arboviral infections in mainland China. ies reported considerable hantavirus
PLoS Negl Trop Dis. 2010;4:e624. http://
Yun Feng,1 Shihong Fu,1 seropositivity among healthy human
dx.doi.org/10.1371/journal.pntd.0000624
Hailin Zhang, Lyle R. Petersen, 6. Wang H, Li Y, Liang X, Liang G. Japanese cohorts, suggesting past exposure to
Baosen Zhang, Xiaoyan Gao, encephalitis in mainland China. Jpn J hantaviruses or subclinical infection
Weihong Yang, Yuzhen Zhang, Infect Dis. 2009;62:331–6. (3). Serologic surveys of rodents (rats

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 673
LETTERS

and mice) and cats also supported the conditions allowed, blood samples no. JX316009) (online Technical Ap-
presence of a hantavirus indigenous to were collected; otherwise, heart tissue pendix Table). Established reverse
the United Kingdom (3). To determine was collected. Samples, and carcasses transcription PCRs for the medium
whether hantaviruses are circulating that could not be processed within 2 segment were unsuccessful.
in wild rodents in the United King- hours, were stored at −80°C. Comparisons of nucleotide and
dom, we conducted molecular analy- RNA was extracted by using amino acid sequence identities dem-
ses on rodent tissues. TRIzol Reagent (Invitrogen, Life onstrated, as expected, that the Arvico-
From September 2009 through Technologies, Paisley, UK). To detect linae-associated hantaviruses showed
November 2011, a total of 495 wild hantavirus RNA, we used a nested the highest similarity to the UK se-
rodents consisting of 133 brown rats pan-hantavirus reverse transcription quence at the nucleotide (65.7%–
(Rattus norvegicus), 269 wood mice PCR selective for partial polymerase 78.8% for S and 76.6%–77.5% for L)
(Apodemus sylvaticus), 50 house large segment (L) gene sequences (5). and the amino acid (66.4%–86.3% for
mice (Mus musculus), 35 bank voles With the exception of 1 male field S and 80%–88% for L) levels (online
(Myodes glareolus), and 8 field voles vole (B41) collected near Tattenhall, Technical Appendix Table).
(Microtus agrestis) were caught live Cheshire (online Technical Appendix Phylogenetic analyses of partial
across northwestern England (online Figure), all lung samples were nega- L (Figure, panel A) and partial S se-
Technical Appendix Figure, wwwnc. tive for hantavirus RNA. The posi- quences (Figure, panel B) confirm the
cdc.gov/EID/article/19/4/12-1057- tive amplicon was sequenced by us- inclusion of the viral sequence from
Techapp1.pdf). Animals were eutha- ing a BigDye Terminator 3.1v Cycle vole B41 as a distinct member of the
nized in the field by use of isoflurane Sequencing Kit on an ABI3130xl Arvicolinae-associated hantaviruses.
inhalation, according to UK Home Of- genetic analyzer (Applied Biosys- In the partial L tree (Figure, panel
fice Guidelines (http://webarchive.na- tems/Life Technologies, Paisley, UK) A), viral sequence B41 clustered with
tionalarchives.gov.uk/+/http://www. (GenBank accession no. JX316008). Prospect Hill and Tula viruses with
homeoffice.gov.uk/docs/hc193.html). Partial small segment (S) sequences good support, although in the partial
Within 2 hours, kidney, liver, and lung were also recovered from lung RNA S tree (Figure, panel B), B41 seems to
tissues were removed. When field from vole B41 (GenBank accession be more closely related to the Asian

Figure. Bayesian phylogenetic trees constructed by using the models HKY+gamma for partial large segment sequences (n = 19) (A) and
GTR+gamma for partial small segment sequences (n = 39) (B) within BEAST software (6) with Markov chain Monte Carlo chain lengths
of 10 million and strict clock. Optimum substitution models were estimated by using MEGA5 (7). The trees are drawn to scale; branch
lengths are measured in the number of substitutions per site. The numbers at each node are posterior probabilities. All effective sample
size values exceeded 150 for partial L and 1,600 for partial S sequences. The phylogenetic position of virus isolated from field vole
B41 (in boldface) is shown in relation to representative hantaviruses (A) and more closely related Arvicolinae-associated hantaviruses
(B). GenBank accession numbers are shown next to taxonomic names. Scale bars indicate nucleotide substitutions per site. VLAV,
Vladivostok virus; TOPV, Topografov virus; KHAV, Khabarovsk virus; PUUV, Puumala virus; HOKV, Hokkaido virus; MUJV, Muju virus;
PHV, Prospect Hill virus; ISLAV, Isla Vista virus; TULV, Tula virus; LANV, Laguna Negra virus; ANDV, Andes virus; SNV, Sin Nombre virus;
NYV, New York virus.

674 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 LETTERS

Microtus vole–associated hantavirus- in indigenous wildlife in the United DOI: http://dx.doi.org/10.3201/eid1904.121057

es, albeit with low posterior probabil- Kingdom might promote inclusion of
ity values. These differences in tree hantavirus infection in the differential References
topologies probably reflect different diagnosis for patients with acute renal
1. Vaheri A, Henttonen H, Voutilainen L,
compositions of the sequence datasets. failure, undiagnosed febrile illness, Mustonen J, Sironen T, Vapalahti O.
Blood collected from vole B41 and exposure to rodents (4). Hantavirus infections in Europe and their
was positive for hantavirus-specific impact on public health. Rev Med Virol.
2012. 2013;23:35–49.
antibodies (indirect fluorescent anti- Acknowledgments 2. Olsson GE, Leirs H, Henttonen H. Hantavi-
body test that used Puumala antigen) We acknowledge the following col- ruses and their hosts in Europe: reservoirs
(8), suggesting cross-reactivity, as leagues for providing materials and assis- here and there, but not everywhere? Vec-
would be expected for Arvicolinae- tance: Chris Ball, Nicola Williams, Susan tor Borne Zoonotic Dis. 2010;10:549–61.
http://dx.doi.org/10.1089/vbz.2009.0138
associated hantaviruses. Hantavirus Withenshaw, Kikka Kallio, Malcolm Ben- 3. McCaughey C, Hart CA. Hantaviruses. J
RNA was detected in the kidneys but nett, Emma Wise, Denise Marston, Mark Med Microbiol. 2000;49:587–99.
not the liver of vole B41 and not in the Outlaw, Chantal Reusken, and Matt Hartley. 4. Fhogartaigh CN, Newsholme W, Kinirons
lungs, liver, or kidneys of the 7 other M, Tong W. An emerging infectious cause
Financial support was received from of renal impairment in the UK. BMJ Case
field voles. Degenerate cytochrome B
the UK Department for Environment, Food Rep. 2011. pii: bcr0620114326.
gene PCR and sequencing (9) were 5. Klempa B, Fichet-Calvet E, Lecompte
and Rural Affairs project SV3037 and from
used to confirm the morphologic iden- E, Auste B, Aniskin V, Meisel H, et al.
the Research and Policy for Infectious Dis-
tification of the field voles (B41 CytB Hantavirus in African wood mouse, Guin-
ease Dynamics program of the Science and ea. Emerg Infect Dis. 2006;12:838–40.
GenBank accession no. KC222031).
Technology Directorate (US Department of http://dx.doi.org/10.3201/eid1205.051487
The nucleotide and amino acid 6. Drummond AJ, Rambaut A. BEAST:
Homeland Security), the Fogarty Interna-
sequence divergences between B41 Bayesian evolutionary analysis by sam-
tional Center (National Institutes of Health),
and the most related hantaviruses pling trees. BMC Evol Biol. 2007;7:214.
MSD Animal Health, and EU FP7–funded PMID:17996036
correspond to that typically found
Research Infrastructure Grant “European 7. Tamura K, Peterson D, Peterson N, Stech-
between hantavirus species (5). The er G, Nei M, Kumar S. MEGA5: Molecu-
Virus Archive” (no.19 228292). This study
phylogenetic analyses further support lar Evolutionary Genetics Analysis using
was partially funded by EU grant FP7-
B41 as a distinct hantavirus. Thus, maximum-likelihood, evolutionary dis-
261504 EDENext and is catalogued by the tance, and maximum-parsimony methods.
we propose to name this novel virus
EDENext Steering Committee as EDENext Mol Biol Evol. 2011;28:2731–9. http://
Tatenale virus, reflecting the medieval dx.doi.org/10.1093/molbev/msr121
063 (www.edenext.eu).
name of its place of origin. 8. Vaheri A, Vapalahti O, Plyusnin A. How
M. agrestis voles, among the most to diagnose hantavirus infections and de-
Kieran C. Pounder, tect them in rodents and insectivores. Rev
numerous mammals in mainland Brit-
Med Virol. 2008;18:277–88. http://dx.doi.
ain, have not been shown to be primary Michael Begon, Tarja Sironen, org/10.1002/rmv.581
carriers of a specific hantavirus, al- Heikki Henttonen, 9. Schlegel M, Ali HS, Stieger N, Grosch-
though recent studies suggest that they Phillip C. Watts, up MH, Wolf R, Ulrich RG. Molecular
Liina Voutilainen, Olli Vapalahti, identification of small mammal species
might be involved in the maintenance
using novel cytochrome B gene–derived
of Tula virus in Germany (10). Further Boris Klempa, degenerated primers. Biochem Genet.
surveillance is needed to confirm that Anthony R. Fooks, and 2012;50:440–7. http://dx.doi.org/10.1007/
M. agrestis voles are the reservoir hosts Lorraine M. McElhinney s10528-011-9487-8
10. Schmidt-Chanasit J, Essbauer S, Petra-
of Tatenale virus, provide an estimate Author affiliations: University of Liverpool,
ityte R, Yoshimatsu K, Tackmann K,
of virus prevalence, and determine zoo- Liverpool, UK (K.C. Pounder, M. Begon, Conraths FJ, et al. Extensive host shar-
notic risk. Current knowledge of other P.C. Watts); University of Helsinki, Helsinki, ing of central European Tula virus. J
Microtus vole–borne hantaviruses sug- Finland (T. Sironen, L. Voutilainen, O. Va- Virol. 2010;84:459–74. http://dx.doi.
org/10.1128/JVI.01226-09
gests that although they might infect palahti); Finnish Forest Research Institute,
humans, their pathogenic potential is Vantaa, Finland (H. Henttonen, L. Voutilain-
Address for correspondence: Lorraine M.
generally low (1). Future work will in- en); Slovak Academy of Sciences, Bratisla-
McElhinney, Animal Health and Veterinary
volve attempts to isolate Tatenale virus va, Slovakia (B. Klempa); Charité School
Laboratories Agency (Weybridge), Wildlife
and generate its full-genome sequence. of Medicine, Berlin, Germany (B. Klempa);
Zoonoses and Vector-borne Diseases Research
Because hantavirus diseases have Animal Health and Veterinary Laboratories
Group, Woodham Lane, New Haw Addlestone,
such broad clinical features, many Agency, Surrey, UK (A.R. Fooks, L.M. McEl-
Surrey KT15 3NB, UK; email: lorraine.
cases among humans in the United hinney); and University of Liverpool National
mcelhinney@ahvla.gsi.gov.uk
Kingdom might be misdiagnosed. The Consortium for Zoonosis Research, South
confirmation of a novel hantavirus Wirral, UK (A.R. Fooks, L.M. McElhinney)

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 675
LETTERS

Hand, Foot, and

Mouth Disease
Outbreak and
Coxsackievirus A6,
Northern Spain,
2011
To the Editor: Hand, foot, and
mouth disease (HFMD) is an acute,
febrile viral infection characterized
by vesicular exanthema on the
palms of the hands, soles of the
feet, and oral mucosa. The infection
is transmitted through oral and
respiratory secretions, vesicular fluid,
and/or feces of affected persons. The
most common etiologic agents are
coxsackievirus (CV) A16 and human
enterovirus (HEV) 71, but other
HEVs, mainly belonging to species
A, have also been associated with
illness (1). HFMD mainly affects
infants and children <5 years of age.
On May 10, 2011, an outbreak
of HFMD was reported in a daycare
center in the city of Irun in Basque
Country, Spain. Monitoring
subsequently was conducted for
HFMD cases among children in
the health district that contained
the daycare center (a total of 4,540
children <14 years of age). Children
with fever and vesicular rash on
the palms and/or soles and in the
mouth were considered HFMD
patients. Pharyngeal and/or dermal
exudate and/or feces were collected
for virologic confirmation from 37
representative HFMD patients (17
with multiple specimens) selected by
sentinel pediatricians in outpatient
clinics. Viral RNA was extracted
directly from specimens (NucliSENS Figure. Phylogenetic analysis of the partial viral protein 1 gene sequence (positions 2929–
3348, based on strain Shizuoka-18, GenBank accession no. AB678778) of coxsackievirus
Easy-Mag, Bio-Mèrieux, Marcy-
A6 isolated from distinct patients with hand, foot, and mouth disease detected in Irun,
l’Étoile, France) and was used in the Spain, April–September 2011, compared with the Gdula prototype strain and other
amplification methods. Enterovirus representative strains. Black dots indicate the strains in this study (GenBank accession
RNA was detected by an in-house nos. JX845228–JX845243 and KC431245–431253). The tree was constructed by using
real-time PCR that amplified a the neighbor-joining method with 1,000 bootstrap replications and shows bootstrap values
>75%. Genetic distances are based on pairwise analysis by using the Kimura 2-parameter
fragment within the 5′ untranslated
method in MEGA5.1 software (www.megasoftware.net). Bracket indicates strains showing
region by using described primers nucleotide identity >94% and detected in outbreaks during 2008–2011. Scale bar indicates
(2). For genotyping, the viral the number of substitutions per nucleotide position.

676 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 LETTERS

protein 1 gene was amplified by and thus its real incidence cannot be Milagrosa Montes,
using described methods (3), identified. Juncal Artieda, Luis D. Piñeiro,
followed by partial sequencing of Although CVA6 has long been Marina Gastesi,
the obtained amplicons by using the known to cause HFMD (1), it has Inmaculada Diez-Nieves,
3130XL Genetic Analyzer (Applied not usually been considered to play and Gustavo Cilla
Biosystems, Foster City, CA, USA). a major role in this disease. Except Author affiliations: Hospital Universitario
Control measures recommended were in a few countries, CVA6 has been Donostia-Instituto Biodonostia, San
frequent and careful handwashing infrequently detected until recent Sebastián, Spain (M. Montes, L.D. Piñeiro,
with soap and running water by years. However, since 2008, this G. Cilla); Biomedical Research Centre
children and staff and increasing the virus has caused major outbreaks of Network for Respiratory Diseases, San
cleaning of surfaces and objects in HFMD in some countries of eastern Sebastián (M. Montes, G. Cilla); Public
daycare centers and nursery schools. Asia and Europe and, more recently, Health Division of Gipuzkoa, Basque
During April–September 2011, in the United States (4–9); the CVA6 Government, San Sebastián (J. Artieda);
a total of 99 cases of HFMD were strains in this outbreak shared >97% Biomedical Research Centre Network
notified; 53 patients were boys. of nucleotide identities in the viral for Epidemiology and Public Health, San
Twenty-five cases occurred in the protein 1 gene and showed sequence Sebastián (J. Artieda); and Health Centre
daycare center, all before May 13 similarity >94% with the strains of Osakidetza Basque Health Service, Irun,
(attack rate 55.6%), and 74 were that caused these outbreaks. These Spain (M. Gastesi, I. Diez-Nieves)
community acquired, occurring mainly strains segregated in a phylogenetic
DOI: http://dx.doi.org/10.3201/eid1904.121589
after that date. All cases occurred in tree (Figure), supporting the recent
children <4 years of age (median age international spread of emerging
1.8 years; incidence 77 cases/1,000 CVA6 genetic variants (4). In Taiwan References
inhabitants). The highest incidence and Japan, the emergence of these 1. Ang LW, Koh BK, Chan KP, Chua LT,
occurred in children 12–36 months of strains has been associated with a James L, Goh KT. Epidemiology and
age (122.4 cases/1,000 inhabitants). change in the predominant clinical control of hand, foot and mouth disease
In addition to a papulovesicular rash expression of the infections produced in Singapore, 2001–2007. Ann Acad Med
Singapore. 2009;38:106–12.
on the palms, soles, and/or buttocks, by CVA6, from herpangina before 2. Rotbart HA, Sawyer MH, Fast S, Lewinski
89 (90%) HFMD patients showed a 2009 to HFMD in 2010–2011 (7,8). C, Murphy N, Keyser EF, et al. Diagnosis
perioral papulovesicular rash that did The development of a perioral rash of enteroviral meningitis by using PCR
not extend to the rest of the face. None has also been associated to HFMD with a colorimetric microwell detection
assay. J Clin Microbiol. 1994;32:2590–2.
of the children were hospitalized. caused by CVA6 (10). 3. Mirand A, Schuffenecker I, Henquell
Enterovirus was detected in 49 Although the course of HFMD is C, Billaud G, Jugie G, Falcon D, et al.
samples (28 pharyngeal, 2 dermal, usually self-limiting, illness and death Phylogenetic evidence for a recent spread
19 fecal) from 33 HFMD patients. rates vary among outbreaks. Severe of two populations of human enterovirus
71 in European countries. J Gen
For 30 of these patients, the samples illness is more frequent in outbreaks Virol. 2010;91:2263–77. http://dx.doi.
were sufficient for genotyping. CVA6 caused by HEV71 (1); in outbreaks org/10.1099/vir.0.021741-0
was detected in 27 (90%) patients and caused by CVA6 in Taiwan and the 4. Mirand A, Henquell C, Archimbaud
CVA10 in 2 (7%) patients; for 1 patient, United States, the illness affected a C, Ughetto S, Antona D, Bailly JL, et
al. Outbreak of hand, foot and mouth
no genotype was obtained. Seven broader spectrum of skin sites and disease/herpangina associated with
(7%) of the 99 children with HFMD was associated with more severe and coxsackievirus A6 and A10 infections in
were brought for medical assistance extensive rash than was HFMD caused 2010, France: a large citywide, prospective
for onychomadesis during the 9–67 by other coxsackieviruses (7,9). observational study. Clin Microbiol
Infect. 2012;18:E110–8. http://dx.doi.
days after the HFMD episode. In 2 of In conclusion, reports of HFMD org/10.1111/j.1469-0691.2012.03789.x
them, HFMD had been virologically outbreaks associated with CVA6 5. Wu Y, Yeo A, Phoon MC, Tan EL, Poh CL,
confirmed as being caused by CVA6. are increasing. Improved HFMD Quak SH, et al. The largest outbreak of
Our results suggest that CVA6 can surveillance is required, with virus hand; foot and mouth disease in Singapore
in 2008: the role of enterovirus 71 and
cause HFMD outbreaks that develop genotyping as a key element. coxsackievirus A strains. Int J Infect
rapidly and reach a high incidence Dis. 2010;14:e1076–81. http://dx.doi.
in children. Despite the mildness of Acknowledgments org/10.1016/j.ijid.2010.07.006
the disease, the high attack rate in 6. Österback R, Vuorinen T, Linna M, Susi
We thank María M. Yagüe, María
P, Hyypiä T, Waris M. Coxsackievirus A6
the daycare center alarmed families J. Llorens, and Guido Castillo for help and hand, foot, and mouth disease, Finland.
and staff. HFMD is not subject to identifying cases and collecting samples Emerg Infect Dis. 2009;15:1485–8. http://
epidemiologic surveillance in Spain, from this outbreak. dx.doi.org/10.3201/eid1509.090438

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LETTERS

7. Wei SH, Huang YP, Liu MC, Tsou TP, this region, rabies is associated with has a population of >10 million, and
Lin HC, Lin TL, et al. An outbreak poverty and considered a neglected 77% of the population is considered
of coxsackievirus A6 hand, foot,
and mouth disease associated with disease (3). Resolution 19 of the 49th below the national poverty line. In
onychomadesis in Taiwan, 2010. BMC Directing Council of PAHO in 2009 2010, Haiti was devastated by a major
Infect Dis. 2011;11:346. http://dx.doi. regarding neglected diseases and earthquake that affected all sectors,
org/10.1186/1471-2334-11-346 other infections related to poverty set including laboratory diagnosis for
8. Fujimoto T, Iizuka S, Enomoto M,
Abe K, Yamashita K, Hanaoka N, et al. a target for eliminating human rabies rabies (6). After the earthquake,
Hand, foot, and mouth disease caused by transmitted by dogs by 2015. PAHO the country was struck by a cholera
coxsackievirus A6, Japan, 2011. Emerg is currently developing strategies to epidemic. Financial resources have
Infect Dis. 2012;18:337–9. http://dx.doi. assist countries during this period (4). been diverted to control such priorities
org/10.3201/eid1802.111147
9. Centers for Disease Control and Since 2010, a total of 111 human and to provide humanitarian aid. Haiti
Prevention. Notes from the field: severe rabies cases transmitted by bats, and Bolivia heavily depend on technical
hand, foot, and mouth disease associated dogs, and other animal species were cooperation and donations from other
with coxsackievirus A6—Alabama, reported from Latin America and the governments or institutions, and are a
Connecticut, California, and Nevada,
November 2011–February 2012. MMWR Caribbean: 40 transmitted by dogs and high priority for elimination of human
Morb Mortal Wkly Rep. 2012;61:213–4. 63 by bats (Table). Although a major rabies transmitted by dogs (7).
10. Flett K, Youngster I, Huang J, McAdam reduction in human rabies transmitted Another challenge for Latin
A, Sandora TJ, Rennick M, et al. Hand, by dogs was observed in 2010 (only America and the Caribbean is
foot, and mouth disease caused by
coxsackievirus A6. Emerg Infect Dis. 6 cases), the total number of cases development of a common strategy for
2012;18:1702–4. http://dx.doi.org/10. increased to 24 in 2011; most were preventing human rabies transmitted
32 01/eid1810.120813 confirmed by laboratory testing. by bats, especially in remote areas in
The higher risk areas for human the Amazon region (Peru, Ecuador,
Address for correspondence: Gustavo Cilla, rabies transmitted by dogs, for which and Brazil) and Mexico (7), from
Microbiology Service, Hospital Universitario more collaboration and financial which 97% of human rabies cases
Donostia, Paseo Dr Beguiristain s/n, 20014 San support are urgently needed, are were reported during this period.
Sebastián, Spain; email: gustavo.cillaeguiluz@ Haiti, Bolivia, Guatemala, Dominican Since 2000, vampire bats have been
osakidetza.net Republic, and parts of Brazil the leading cause of human rabies
(Maranhão State) and Peru (Puno in Latin America and the Caribbean
Region). Unfavorable conditions in (8). Comparison of data for 2010–
which persons in these areas are living 2012 with data for the previous 3
limit control strategies and maintain years shows a 5.2% increase in bat-
rabies transmission (3). transmitted human rabies, especially
According to the PAHO during 2011, which accounted for
Rabies Update Epidemiologic Surveillance System ≈53% of reports during the past 3
for Latin America for Rabies, during 2010–2012, years (5).
and the Caribbean Bolivia and Haiti had the highest Bats have been identified as
incidence of human rabies transmitted a reservoir for many Lyssavirus
To the Editor: Rabies incidence by dogs in the Western Hemisphere: spp. genotypes, and the geographic
in Latin America and the Caribbean 15% (6/40) and 40% (16/40) of all distribution of variants has been
has decreased and several countries cases, respectively (5). Many factors, associated with climate changes and
(Uruguay, Chile, Costa Rica, Mexico, including national disasters and social, ecologic imbalances. Spread of bats
and Panama) and areas of Peru, Brazil, cultural, and economic factors, have has been facilitated by human-made
and Argentina are free of human rabies interfered with canine rabies control shelters near human dwellings (9).
transmitted by dogs, although there are programs in these countries. Although rabies control in Latin
certain areas to which this disease is still Bolivia has a population of 10 America and the Caribbean has
endemic (1). Coordinated actions for million, and 60.0% of the population is been successful, certain approaches
regional elimination of human rabies considered below the national poverty currently used, such as mass
transmitted by dogs began in 1983 in line. This country has poor suburbs vaccination campaigns for dogs,
Latin America and the Caribbean with on the outskirts of large cities, with postexposure prophylaxis, and
the assistance of the Pan American large populations of unowned dogs epidemiologic surveillance, require
Health Organization (PAHO). This and limited resources to implement improvement in some countries. In
effort has led to an ≈90% reduction dog mass vaccination campaigns and addition, allocation of resources is
of human and canine rabies (2). In animal birth control programs. Haiti needed to enhance national programs

678 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 LETTERS

Table. Cases of human rabies in 10 countries in Latin America and the Caribbean, 2010–2012*
Rabies transmitted by other animals Rabies transmitted by bats Rabies transmitted by dogs
Country 2010 2011 2012† 2010 2011 2012† 2010 2011 2012† Total
Bolivia 0 0 1‡ 0 0 0 0 5 1 7
Brazil 1§ 0 2‡§ 1 0 1 1 2 2 10
Colombia 3¶ 0 0 1 0 0 0 0 0 4
Ecuador 0 0 0 0 12 0 0 0 0 12
Guatemala 0 0 0 0 0 0 0 3 0 3
Haiti 0 0 0 0 0 0 1 13 2 16
Honduras 0 0 0 0 0 0 0 0 1 1
Mexico 0 1# 0 4 2 0 0 0 0 7
Peru 0 0 0 13 19 10 1 1 2 46
Dominican Republic 0 0 0 0 0 0 3 0 2 5
Total 4 1 3 19 33 11 6 24 10 111
*Data were obtained from the Regional Information System of Epidemiologic Surveillance of Rabies in the Americas/Epidemiologic Information System,
Pan American Center for Foot-and-Mouth Disease, Pan American Health Organization–World Health Organization, 2012.
†Data were updated in December 2012.
‡Human rabies transmitted by undetermined animal species (variant hematophagous bat).
§Human rabies transmitted by a marmoset monkey (Callithrix jacchus).
¶Human rabies transmitted by a cat (variant nonhematophagous bat).
#Human rabies transmitted by a skunk.

to eliminate human rabies transmitted information and their commitment to the R9. Elimination of neglected diseases
by dogs. regional human rabies elimination program. and other poverty-related infections.
Washington (DC): The Organization;
PAHO is responsible for 2009 [cited 2013 Jan 21]. http://new.paho.
coordination and technical cooperation Marco A.N. Vigilato, org/hq/index.php?option=com_content&
of the Rabies Elimination Program Ottorino Cosivi, task=view&id=2372&Itemid=1967.
and Operation of the Epidemiologic 5. Pan American Health Organization, Foot-
Terezinha Knöbl, and-Mouth Disease Center, Veterinary
Surveillance System for Rabies. For Alfonso Clavijo, and Public Health Unit. Regional information
the past 60 years, the Pan American Hugo M.T. Silva system for epidemiological surveillance
Center for Foot-and-Mouth Disease/ Author affiliations: Pan American Health
of rabies: SIRVERA/SIEPI; 2012
PAHO has accumulated capabilities [cited 2013 Jan 21]. http://siepi.
Organization, Rio de Janeiro, Brazil (M.A.N. panaftosa.org.br
to develop national programs for Vigilato, O. Cosivi, A. Clavijo, H.M.T. Silva); 6. The World Bank. Working for a world
zoonoses prevention and control, and Universidade de São Paulo, São free of poverty; 2012 [cited 2013 Jan 21].
particularly for rabies elimination in Paulo, Brazil (T. Knöbl)
http://data.worldbank.org
Latin America and the Caribbean. 7. Rupprecht CE, Barrett J, Briggs D, Cliquet
DOI: http://dx.doi.org/10.3201/eid1904.121482 F, Fooks AR, Lumlertdacha B, et al. Can
Strengthening regional, national, rabies be eradicated? Dev Biol (Basel).
and subnational rabies control 2008;131:95–121.
programs must be a priority. The References 8. Schneider MC, Belotto A, Adé MP,
decision in Latin America and the Hendrickx S, Leanes LF, Rodrigues MJ,
1. Organización Panamericana de la Salud et al. Current status of human rabies
Caribbean to eliminate dog-transmitted O. Área de prevención y control de transmitted by dogs in Latin America.
rabies began in 1983 and involved enfermedades. unidad de salud pública Cad Saude Publica. 2007;23:2049–63.
veterinaria. eliminación de la rabia
strong political commitment with http://dx.doi.org/10.1590/S0102-
humana transmitida por perros en América 311X2007000900013
multinational efforts, as well as support Latina: análisis de la situación, año 2004. 9. Carvalho-Costa FA, Tedesqui VL, Jesus
and coordination of other international Washington (DC): La Organization; 2005 Nascimento Monteiro M, Bóia MN.
organizations, nongovernmental [cited 2013 Jan 21]. http://www.paho.org/ Outbreaks of attacks by hematophagous
spanish/ad/dpc/vp/rabia-sit.pdf
organizations, and the private sector. bats in isolated riverine communities in
2. Belotto A, Leanes LF, Schneider MC, the Brazilian Amazon: a challenge to
This interinstitutional collaboration Tamayo H, Correa E. Overview of rabies control. Zoonoses Public Health.
is needed to promote prevention rabies in the Americas. Virus Res. 2012;59:272–7. http://dx.doi.org/10.1111/
and control activities to achieve 2005;111:5–12. http://dx.doi.org/10.10 16/ j.1863-2378.2011.01444.x
j.virusres.2005.03.006
the elimination of human rabies
3. Schneider MC, Aguilera XP, Junior JBS,
transmitted by dogs in the Western Ault SK, Najera P, Martinez J, et al. Address for correspondence: Marco A.N.
Hemisphere by 2015. Elimination of neglected diseases in Latin Vigilato, Veterinary Public Health Unit, Pan
America and the Caribbean: a mapping of American Foot and Mouth Disease Center, Pan
selected diseases. PLoS Negl Trop Dis. American Health Organization, Av. Governador
Acknowledgments 2011;5:e964. http://dx.doi.org/10.1371/
We thank the staff at PAHO and journal.pntd.0000964 Leonel de Moura Brizola, 7778, São Bento,
other collaborating institutions for their 4. Pan American Health Organization, Forty- CEP 25040-004, Duque de Caxias, Rio de
ninth Directing Council. Resolution CD49. Janeiro, Brazil; email: vigilato@paho.org
continuous support in providing accurate

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 679
LETTERS

Serosurvey of certain pathogens and a useful senti- authors in 2010 in QE (not included in
nel for others (3). rabies results), and 11 (all males) had
Dogs for Human, In 2011, serum samples were been castrated by local animal healers
Livestock, and obtained from 116 mixed-breed dogs before serum samples were obtained.
Wildlife Pathogens, during a rabies vaccination cam- The samples were used to test for serop-
paign in and near 3 national parks in revalence rates to rabies virus (RABV),
Uganda southwestern Uganda; the dogs were canine distemper virus (CDV), canine
To the Editor: Domestic dogs >4 months of age and were volun- parvovirus (CPV), Leptospira inter-
live in close association with hu- tarily brought in by their owners (Fig- rogans, Leishmania sp., Toxoplasma
mans and livestock, participating in ure, Appendix, wwwnc.cdc.gov/EID/ gondii, and Neospora caninum (Table).
the transmission of diseases of zoo- article/19/4/12-1143-F1.htm; Table). Seroprevalence rates ranged from 20%
notic, veterinary, and conservation Two of the parks, Bwindi Impenetra- to 100% (Table). CPV seroprevalence
interest (1,2). Most households in ble (BI) and Mgahinga Gorilla (MG), was higher in BI and QE than in MG
Uganda have traditionally kept dogs have some of the most biologically di- (χ2 >12.6, p<0.001); T. gondii serop-
for hunting and for help with herd- verse tropical forests in eastern Africa revalence was higher in BI than in MG
ing, security, and guarding livestock. and are home to mountain gorillas. (Fisher p = 0.002); and RABV serop-
Most dogs receive no prophylactic The third park, Queen Elizabeth (QE), revalence was higher in castrated than
measures (e.g., vaccinations) and is home to populations of protected noncastrated dogs (50% vs. 10%; Fish-
roam freely; this situation exposes carnivores and ungulates. The parks er p = 0.005).
them to pathogens from eating gar- lie within a densely populated rural For humans, the domestic dog is
bage, rodents, and stillborn animals landscape; in some areas, the popula- the main source of exposure to RABV.
and other carcasses and through in- tion is as high as 500 persons/km2. The possibility that the presence of the
halation during scent communica- Of the 116 sampled dogs, 4 had rabies titers in the dog serum samples
tion. Thus, dogs are a reservoir for been vaccinated against rabies by the was due to a previous vaccination can

Table 1. Methodology and seroprevalence for selected pathogens in rural dogs in 3 national parks, Uganda, 2011*
Test, cutoff National park
value, and All 3 parks Queen Elizabeth† Bwindi Impenetrable‡ Mgahinga Gorilla§
(ref) or
commercial Sample Prevalence, Sample Prevalence, Sample Prevalence, Sample Prevalence,
Pathogen kit size % (95% CI) size % (95% CI) size % (95% CI) size % (95% CI)
Rabies FAVN, 0.24 101 19.8 23 21.7 56 19.6 22 16.7
virus¶ IU/mL (4) (12.7–28.6) (9.0–43.3) (11.0–32.0) (5.9–37.2)
CDV c-ELISA, 92 100.0 30 100 39 100 23 100
Ingezim (95.9–100) (88.8–100.0) (91.4–100.0) (85.4–100.0)
Moquillo
IgG#
CPV c-ELISA, 92 65.2 26 80.8 43 76.7 23 26.1
Ingezim (54.9–74.5) (61.7–92.1) (61.7–87.6) (12.0–47.8)
CPV#
Leptospira MAT, 1:200 105 26.7 27 25.9 55 29.1 23 21.7
interrogans** (5) (19.0–36.1) (12.4–46.2) (17.9–42.7) (9.0–43.3)
Leishmania c-ELISA, 92 19.6 26 19.2 43 25.6 23 8.7
sp.†† Ingezim (12.3–29.2) (7.9–38.3) (14.6–40.6) (1.6–27.8)
Leishmania#
Toxoplasma MAT, 1:25 109 90.8 30 90.0 56 98.2 23 73.9
gondii (3) (83.6–95.1) (73.7–97.2) (90.5–99.9) (52.2–88.0)
Neospora c-ELISA, 109 27.5 30 26.7 56 32.1 23 30.4
caninum 30% (3) (19.6–36.6) (13.1–45.0) (21.2–45.5) (14.5–52.2)
*ref, reference; FAVN, fluorescent antibody virus neutralization; CPV, canine parvovirus; c-ELISA, competitive ELISA; CDV, canine distemper virus; MAT,
modified agglutination test.
†0°12 S, 30°0 E (savannah).
‡1°0 S, 29°42 E (tropical forest).
§1°16 S, 29°40 E (tropical forest).
¶Four dogs vaccinated against rabies in Queen Elizabeth are not included in these results.
#Manufactured by Ingenasa, Madrid, Spain.
**Fourteen serovars were investigated. Of the dogs seropositive, 71.5% were seropositive to 1 serovar and 28.5% to 2 serovars. Reacting serovars were
Icterohaemorragiae (42.8% of positive dogs), Canicola (39.2%), Pyrogenes (21.4%), Tarassovi (10.7%), and Gryppothiposa and Australis (7.2% each).
††Antibodies probably correspond to contact with Leishmania donovani.

680 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 LETTERS

be ruled out because the only previ- be the highest reported for dogs world- Javier Millán, Andrea D. Chirife,
ous recent campaign in the area was wide. This protozoon has implications Gladys Kalema-Zikusoka,
conducted by the authors. Antibodies for human and animal health, and dogs, Oscar Cabezón, Jesús Muro,
against RABV in apparently healthy who probably become infected with Ignasi Marco, Florence Cliquet,
dogs have been reported in Africa (6), T. gondii when eating raw meat, are a Luis León-Vizcaíno,
and rabies seems to be not invariably good sentinel for environmental con- Marine Wasniewski,
fatal in dogs. Dogs that have recov- tamination by this parasite. On the other Sonia Almería,
ered from a rabies infection are prone hand, dogs serve as the definitive host and Lawrence Mugisha
to shed RABV in their saliva for long for N. caninum, which is a major cause Author affiliations: Universitat Autònoma
periods (7). Antibodies against RABV of abortions in cattle and causes eco- de Barcelona, Bellaterra, Spain (J. Millán,
were more frequently found in castrat- nomic losses wherever it is enzootic. A.D. Chirife, O. Cabezón, I. Marco, S. Alm-
ed dogs. This finding may be due to an Some of these diseases may also ería); Conservation through Public Health,
increase in virus-related deaths among have implications for the conservation Kampala, Uganda (G. Kalema-Zikusoka);
noncastrated dogs; such dogs tend to be of endangered mountain gorillas. Dis- Ministry of Agriculture, Andorra la Vella,
more aggressive and to roam, so they eases such as leptospirosis, toxoplas- Andorra (J. Muro); Nancy Laboratory for
may come more frequently into contact mosis, and especially, rabies could be Rabies and Wildlife, Malzéville, France
with pathogenic RABV strains. fatal for gorillas, and there are unpub- (F. Cliquet, M. Wasniewski); University of
Results indicate that both CDV lished reports of fights between hunt- Murcia, Murcia, Spain (L. León-Vizcaíno);
and CPV are actively circulating in ing dogs and gorillas. Centre de Recerca en Sanitat Animal, Bel-
the studied dog populations. High Our work should serve as a first laterra, Spain (S. Almería); Makerere Uni-
CDV seroprevalence rates have been step toward the establishment of pre- versity, Kampala (L. Mugisha); and Con-
reported among other rural dog popu- ventive strategies for improvements in servation and Ecosystem Health Alliance,
lations in Africa (8). Sick, debilitated the health of humans and domestic ani- Kampala (L. Mugisha)
pups are at high risk for predation by mals living in rural Uganda and for the DOI: http://dx.doi.org/10.3201/eid1904.121143
wild carnivores, so spillover may take health of the country’s unique wildlife.
place. A dog population exhibiting Tracing the role of dogs in the cycle of References
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MacArthur Foundation and the United tion in orally vaccinated foxes sampled
cases has dramatically increased dur-
States Agency for International Develop- in the fields. Vaccine. 2000;18:3272–9.
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detected during this survey appears to the European Social Fund. antibody by ELISA and RFFIT in

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LETTERS

unvaccinated dogs and in the endan- human growth hormone (hGH) from performed 8 months after illness onset,
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wildlife. Vet Microbiol. 2000;72:217– commercial cadaveric hGH. anoxic insults (Figure). The patient
27. http://dx.doi.org/10.1016/S0378-
1135(99)00207-2 The patient was born at 32 was discharged from a referral hospital
9. Evangelista KV, Coburn J. Leptospira as weeks’ gestation with subsequent with this diagnosis; no postmortem
an emerging pathogen, a review of its bi- developmental delay, agenesis analysis was conducted.
ology, pathogenesis and host immune re- of the corpus callosum, and On the basis of World Health
sponses. Future Microbiol. 2010;5:1413–
25. http://dx.doi.org/10.2217/fmb.10.102 panhypopituitarism. He demonstrated Organization criteria, we conclude
10. Kolaczinski JH, Reithinger R, Worku DT, clinical and laboratory signs of growth that this patient had probable iCJD
Ocheng A, Kasimiro J, Kabatereine N, et hormone deficiency but was denied as a result of hGH treatment (5). The
al. Risk factors of visceral leishmaniasis treatment with hGH through the US patient’s condition was treated with 2
in east Africa, a case-control study in Po-
kot territory of Kenya and Uganda. Int J government–supported National different formulations of commercial
Epidemiol. 2008;37:344–52. http://dx.doi. Hormone and Pituitary Program cadaveric hGH, including one of the
org/10.1093/ije/dym275 (NHPP) because he did not meet the same brands in the same year as that
height requirement. Treatment with of the first reported patient with iCJD
Address for correspondence: Javier Millán, commercial cadaveric hGH began associated with commercial cadaveric
Servei d’Ecopatologia de Fauna Salvatge when he was 5.8 years of age and hGH (3). The patient’s incubation
(SEFaS) (Wildlife Diseases Research Group), continued for 23 months (1983–1985). period (25.5–28 years) is well within
Departament de Medicina i Cirurgia Animals, He received 1.5 units intramuscularly expectations (1).
Universitat Autònoma de Barcelona, 08193 3× per week and was primarily Despite an ongoing active
Bellaterra, Spain; email: syngamustrachea@ treated with Asellacrin (Ares-Serono, surveillance program that identified
hotmail.com Geneva, Switzerland). In early ≈3,500 of ≈4,500 post-1977
1984, for an unspecified duration, he cadaveric hGH recipients in the
received Crescormon (Kabivitrum) US NHPP, all 29 CJD infections in
because of an Asellacrin shortage. NHPP recipients occurred among
Treatment was halted in 1985 because the estimated ≈2,700 pre-1977
Iatrogenic of iCJD concerns and resumed 2 years recipients (1,2). This significant
Creutzfeldt-Jakob later with recombinant hGH. reduction in iCJD was attributed to
Disease from At age 33, 26.5 years (range the 1977 introduction of a highly
25.5–28 years) after the midpoint of selective, column chromatography
Commercial commercial cadaveric hGH treatment, step in the hormone purification
Cadaveric Human dizziness and gait imbalance protocol that can markedly reduce
Growth Hormone developed, causing a fall. The patient’s prion infectivity (1,2). As shown by
mental status also began declining, the many iCJD cases linked to hGH
To the Editor: Iatrogenic and he never returned to his baseline in France, the efficacy of column
Creutzfeldt-Jakob disease (iCJD) is status. Six months after illness chromatography purification steps
an acquired form of prion disease onset, he experienced hallucinations, may vary (1). Commercially derived
that has been declining in incidence weakness of lower extremities, and cadaveric hGH was produced in
since the mid-1990s (1). Worldwide, limb ataxia. Seven months after the different laboratories from those
at least 226 cases of iCJD, including fall, he entered a state of akinetic that produced NHPP-distributed
29 US cases, have been associated mutism; he died 9 months after hGH, and sufficient details regarding
with administration of contaminated symptom onset. A lumbar puncture, sourcing and production methods of

682 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 LETTERS

Figure. Maps showing axial fluid attenuated inversion recovery (FLAIR), diffusion-weighted imaging (DWI), and apparent diffusion
coefficient (ADC) at the level of the basal nuclei (top row) and dorsal frontoparietal cortex (bottom row) of the brain of a 33.8-year-old
man with agenesis of the corpus callosum, schizencephaly, and heterotopia. Note the symmetrical DWI signal hyperintensities in the
striatum and dorsomedial part of the thalami. In addition, DWI signal hyperintensities occurred in the cingulate, precuneus and in the
dysplastic gray matter along the anterior lips of the schizencephalic clefts at the level of the precentral gyri. The signal abnormalities are
associated with decreased diffusivity on ADC maps and are much less prominent on FLAIR images. These findings are highly suggestive
of Creutzfeldt-Jakob disease.

the commercial products are lacking. age at disease onset (33 years) makes Brian S. Appleby, Mei Lu,
Approximately 10,000 persons, this unlikely (6). Alberto Bizzi,
mostly outside the United States, This report suggests that a Michael D. Phillips,
received commercial cadaveric potential risk for iCJD in persons who Sally M. Berri,
hGH produced by Kabivitrum, and received commercial cadaveric hGH Madeleine D. Harbison, and
substantially fewer persons received should be considered. Also, clinicians Lawrence B. Schonberger
product from Ares-Serono (A.F. should not assume that all cadaveric Author affiliations: Cleveland Clinic
Parlow, pers. comm.). Identification hGH administered after 1977 Foundation, Cleveland, Ohio, USA (B.S.
through passive surveillance of 2 carries the same risk for infectivity. Appleby, M. Lu, M.D. Phillips); Instituto Clinico
CJD cases among recipients of such In addition, when CJD is being Humanitas,Milan, Italy (A. Bizzi); Case
hGH further supports a causal, rather considered as a clinical diagnosis, Western Reserve University, Cleveland (S.M.
than chance, association between a history of exposure to cadaveric Berri); Mount Sinai School of Medicine, New
commercial hormone and CJD. It also hGH should always be sought, even York, New York, USA (M.D. Harbison); and
suggests a difference in iCJD risk when patients have normal or tall Centers for Disease Control and Prevention,
between post-1977 NHPP-distributed stature. Finally, we recommend that Atlanta, Georgia, USA (L.B. Schonberger)
hGH and commercial cadaveric hGH. when a clinical diagnosis of CJD is DOI: http://dx.doi.org/10.3201/eid1904.121504
Limitations of this report suspected, but before the patient’s
include the lack of neuropathologic death, the local caregivers, with the References
confirmation and insufficient family, should initiate arrangements
information to strongly implicate a for a postmortem examination 1. Brown P, Brandel J-P, Sato T, Nakamura
single commercial cadaveric hGH to confirm diagnosis (e.g., www. Y, MacKenzie J, Will RG, et al. Iatrogenic
Creutzfeldt-Jakob disease, final assessment.
product as infection source. The report cjdsurveillance.com). Emerg Infect Dis. 2012;18:901–7. http://
of another iCJD case-patient who dx.doi.org/10.3201/eid1806.120116
received Crescormon during the same Acknowledgments 2. Abrams JY, Schonberger LB, Belay ED,
period provides some evidence that We thank the National Prion Disease Maddox RA, Leschek EW, Mills JL, et al.
Lower risk of Creutzfeldt-Jakob disease
the product was the source of prion Pathology Surveillance Center for in pituitary grown hormone recipients
contamination. Although the patient assistance with CSF analyses. initiating treatment after 1977. J Clin
may have had sporadic CJD, his young Endocrinol Metab. 2010; 96:e1966–9.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 683
LETTERS

3. Furtner M, Gelpi E, Kiechl S, Knoflach fever, headache, and myalgia; <1% of 80% neutrophils) and lactate dehy-
M, Zangerl A, Gotwald T, et al. Iatrogenic WNV infections develop into severe drogenase level (522 IU/L), a low C-
Creutzfeldt-Jakob disease 22 years after
human growth hormone therapy: clinical and neuroinvasive disease (1). reactive protein level (0.7 mg/dL), and
radiological features. J Neurol Neurosurg The virus was discovered in 1937 hyponatremia (131 mEq/L). Cerebro-
Psychiatry. 2008; 79:229–31. http://dx.doi. in the West Nile district of Uganda. spinal fluid (CSF) testing showed 90
org/10.1136/jnnp.2007.122267 WNV is endemic to parts of Africa, cells/µL (79% lymphocytes) and glu-
4. Hamlin C, Puoti G, Berri S, Sting E,
Harris C, Cohen M, et al. A comparison Europe, Asia, and the Middle East, cose and protein levels of 72 and 100.9
of tau and 14-3-3 protein in the and since its introduction in New York mg/dL, respectively. Serum obtained
diagnosis of Creutzfeldt-Jakob disease. in 1999, in North America. In Eurasia, on August 15 was sent to the national
Neurology. 2012;79:547–52. http://dx.doi. human WNV infections were first re- reference laboratory at Aristotle Uni-
org/10.1212/WNL.0b013e318263565f
5. World Health Organization. Global ported in Israel and France during the versity (Thessaloniki, Greece), and
surveillance, diagnosis and therapy 1950s–1960s, and the first major out- IgM against WNV was detected by
of human transmissible spongiform break in Romania occurred in 1996 ELISA (WNV IgM Capture DxSelect
encephalopathies: report of a WHO (1). The disease emerged recently and IgG DxSelect; Focus Diagnostics,
consultation. Geneva: The Organization;
1998. in Greece; a large outbreak in 2010 Cypress, CA, USA). IgG was absent.
6. Appleby BS, Appleby KK, Rabins PV. caused neuroinvasive disease in 197 On the second day of hospitalization,
Does the presentation of Creutzfeldt- patients, of whom 33 died (2). Since the patient exhibited seizures (speech
Jakob disease vary by age or presumed 2010, occasional and local epidemics arrest); she was given phenytoin (1/2
etiology? A meta-analysis of the past
10 years. J Neuropsychiatry Clin have been ongoing in Greece, Italy, amp 3×/day intravenously). On Au-
Neurosci. 2007;19:428–35. http://dx.doi. Romania, Hungary, Spain, and the gust 18, the patient was transferred to
org/10.1176/appi.neuropsych.19.4.428 Balkans (3,4). a private hospital. Further treatment
Clinical diagnosis may be diffi- included intravenous fluid, antipyret-
Address for correspondence: Brian S. Appleby, cult because WNV infections resemble ics, antimicrobial drugs, mannitol,
Cleveland Clinic Lou Ruvo Center for Brain other (arbo)viral diseases. Laboratory and oxygen. On August 30, she was
Health, 9500 Euclid Ave/U10, Cleveland, OH diagnosis relies primarily on serologic returned by plane to Belgium.
44195, USA; email: applebb@ccf.org testing. Reverse transcription PCR (RT- CSF obtained 26 days after symp-
PCR) can be used to detect viral RNA tom onset and serum obtained 29 days
during the acute phase of the disease, after symptom onset were sent to the
but its use is hampered by the patient’s Institute of Tropical Medicine (An-
low-level and transient viremia (1). twerp, Belgium) because of its func-
We here describe a confirmed tion as a national reference center for
West Nile Virus case of WNV encephalitis imported Belgium. IgM and IgG against WNV
by a traveler returning from Greece. were detected in both samples by
Infection in Belgian A 73-year-old Belgian woman, who ELISA (Focus Diagnostics) (Table).
Traveler Returning had a medical history of lymphoma, Immunofluorescence assays on serum
from Greece traveled to Kavala city (Macedonia, revealed IgM against WNV only and
Greece). On August 14, 2012, she IgG against West Nile, dengue, yellow
To the Editor: West Nile virus sought treatment at the Kavala Gen- fever, and Japanese encephalitis virus-
(WNV) is an arthropod-borne virus eral Hospital with a 6-day history of es, with the strongest reaction against
that is transmitted to humans by mos- fever, headache, malaise, nausea, con- WNV (Flavivirus Mosaic 1; Euroim-
quitos, primarily of the genus Culex. fusion, decline of consciousness, and mun, Lübeck, Germany). Real-time
Most human infections are asymp- neck stiffness. Results of laboratory RT-PCR (adapted from [5]) on the
tomatic. Clinical symptoms occur in testing on admission demonstrated an serum demonstrated a weak positive
≈20% of case-patients and include increased leukocyte count (9,670/µL; signal. Repeated RNA extraction and

Table. Laboratory results confirming WNV infection of 73-year-old woman, Greece, 2012*†
Sample Date RT-PCR (Ct value) WNV ELISA IgM (ratio) WNV ELISA IgG (ratio) Flavi IFAT IgM Flavi IFAT IgG
Serum Aug 15 Positive (45.47) Positive (25) Negative ND ND
CSF Sep 3 ND Positive (5.16) Positive (2.21) ND ND
Serum Sep 6 Positive (42.87)‡ Positive (4.76) Positive (2.63) WNV positive WNV positive§
*WNV, West Nile virus; RT-PCR, reverse transcription PCR; Ct, cycle threshold; Flavi, flavivirus; IFAT, indirect fluorescent antibody technique; ND, not
done; CSF, cerebrospinal fluid.
†The ELISA is positive if ratio >1.1 for IgM and >1.5 for IgG. The cutoff value for IFAT is 1/10 for both IgG and IgM.
‡Sequencing revealed a 116-bp sequence perfectly matched to the WNV amplicon and is highly suggestive for WNV lineage 2 on the basis of the
presence of 2 specific nucleotides.
§Strongest signal for WNV, weak signal for other flaviviruses (Japanese encephalitis virus, dengue viruses 1–4, yellow fever virus).

684 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 LETTERS

RT-PCR were confirmative (Table). onset in serum, CSF, and urine, re- 5. Linke S, Ellerbrok H, Niedrig M, Nitsche
Sequencing of the RT-PCR product spectively (8–10), and a prolonged A, Pauli G. Detection of West Nile virus
lineages 1 and 2 by real-time PCR. J Virol
confirmed the detection of WNV. Al- period of viremia in immunocompro- Methods. 2007;146:355–8. http://dx.doi.
though the product was short (116 bp), mised patients (9). org/10.1016/j.jviromet.2007.05.021
it was highly suggestive of WNV, lin- 6. Versteirt V, Schaffner F, Garros C,
eage 2. Flemish regional authority in Acknowledgments Dekoninck W, Coosemans M, Van Bortel
W. Introduction and establishment of the
Belgium, national authorities (both in We thank Kathy Demeulemeester, exotic mosquito species Aedes japonicus
Belgium and Greece), and European Elke Gintelenberg, and the laboratory staff japonicus (Diptera: Culicidae) in Bel-
health authorities were notified of the of the serology unit of the Central Labo- gium. J Med Entomol. 2009;46:1464–7.
imported case of WNV encephalitis. ratory for Clinical Biology, Antwerp, for http://dx.doi.org/10.1603/033.046.0632
7. Papa A, Xanthopoulou K, Gewehr S,
According to the case definition of the their excellent technical support. Mourelatos S. Detection of West Nile vi-
European Center for Disease Preven- rus lineage 2 in mosquitoes during a hu-
tion and Control, Stockholm, Sweden, Lieselotte Cnops, Anna Papa, man outbreak in Greece. Clin Microbiol
the patient met the laboratory criteria Infect. 2011;17:1176–80. http://dx.doi.
Frideriki Lagra, org/10.1111/j.1469-0691.2010.03438.x
of having a confirmed case. Philippe Weyers, 8. Briese T, Glass WG, Lipkin WI. Detection
To date, autochthonous WNV Kathleen Meersman, of West Nile virus sequences in cerebro-
infections have not been reported Nicolas Patsouros, and spinal fluid. Lancet. 2000;355:1614–5.
in Belgium, although the presence http://dx.doi.org/10.1016/S0140-
Marjan Van Esbroeck 6736(00)02220-0
of the mosquito vector provides a Author affiliations: National Reference 9. Penn RG, Guarner J, Sejvar JJ, Hartman
potential risk for transmission (6). Center for WNV and Arboviruses–Institute H, McComb RD, Nevins DL, et al. Persis-
This WNV infection was acquired of Tropical Medicine, Antwerp, Belgium (L.
tent neuroinvasive West Nile virus infec-
in Greece (a leading travel destina- tion in an immunocompromised patient.
Cnops, K. Meersman, M. Van Esbroeck); Clin Infect Dis. 2006;42:680–3. http://
tion for tourists from Belgium), spe- National Reference Laboratory for Arbovi- dx.doi.org/10.1086/500216
cifically in the Kavala region, which ruses–Aristotle University Medical School, 10. Murray K, Walker C, Herrington E,
was highly affected by WNV in 2012. Thessaloniki, Greece (A. Papa; Kavala
Lewis JA, McCormick J, Beasley DW,
The lineage responsible for the WNV et al. Persistent infection with West Nile
General Hospital, Kavala, Greece (F. La- virus years after initial infection. J In-
encephalitis was identified as lineage gra); and Saint-Jean Hospital, Brussels, fect Dis. 2010;201:2–4. http://dx.doi.
2, the currently circulating strain in Belgium (P. Weyers, N. Patsouros) org/10.1086/648731
Greece (7). Our report highlights the
DOI: http://doi.dx.org/eid1904.121594
need for physicians and laboratory Address for correspondence: Lieselotte Cnops,
staff to be aware of imported WNV References
Institute of Tropical Medicine, Kronenburgstraat
infections originating from south- 43/3, B-2000 Antwerpen, Belgium: email:
eastern Europe, especially Greece 1. Zeller HG, Schuffenecker I. West Nile vi- lcnops@itg.be
and its neighboring countries, where rus: an overview of its spread in Europe
and the Mediterranean basin in contrast to
recent and recurrent outbreaks have its spread in the Americas. Eur J Clin Mi- The Public Health
occurred (3,4). crobiol Infect Dis. 2004;23:147–56. http:// Image Library (PHIL)
Special attention should be given dx.doi.org/10.1007/s10096-003-1085-1
2. Papa A, Danis K, Baka A, Bakas A, Dou- The Public Health Image
to immunosuppressed and elderly
gas G, Lytras T, et al. Ongoing outbreak Library (PHIL), Centers
patients who are at higher risk of ac- of West Nile virus infections in humans in for Disease Control and
quiring neuroinvasive disease. The Greece, July–August 2010. Euro Surveill. Prevention, contains
73-year-old patient described here was 2010;15:pii19644. thousands of public health-
unconscious when she arrived in Bel- 3. European Center for Disease Control. related images, including high-resolution
Review of the epidemiological situa-
gium. After a short period of relative tion of West Nile virus infection in the
(print quality) photographs, illustrations,
improvement (more reactive and co- European Union, update 19 Septem- and videos.
operative), her condition deteriorated, ber, 2011 [cited 2013 Feb 7]. http:// PHIL collections illustrate current events
and she died on November 23, 2012. www.ecdc.europa.eu/en/publications/ and articles, supply visual content for
Publications/110920_TER_Rapid%20 health promotion brochures, document
The detection of viral RNA 29 days risk%20assessment_WNF.pdf the effects of disease, and enhance
after symptom onset was surprising 4. European Center for Disease Con- instructional media.
but might be explained by the immu- trol. Epidemiological situation of West PHIL Images, accessible to PC and
nocompromised status of the patient. Nile virus infection in the European
Macintosh users, are in the public domain
Union, update, 13 July 2012 [cited 2013
Several studies have reported persis- Feb 7]. http://www.ecdc.europa.eu/en/ and available without charge.
tent WNV RNA for 30 days, 77 days, publications/Publications/1207-TER- Visit PHIL at http://phil.cdc.gov/phil
and even years after the symptom Rapid-risk-assessment-West-Nile-virus.pdf

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 685
LETTERS

Powassan Virus for the other 16 cases. However, many Hepatitis E

case-patients had likely exposure to
Encephalitis, Ixodes scapularis ticks (blacklegged Virus and
Minnesota, USA ticks), the tick species associated with Porcine-derived
To the Editor: Birge and Son-
deer tick virus transmission, and virus- Heparin
es from all POWV-positive tick pools
nesyn report the first death of a Min- were confirmed as deer tick virus by To the Editor: Cases of spo-
nesota resident caused by Powassan sequencing. The distribution of the 2 radic, locally acquired hepatitis E
virus (POWV) (1). However, they lineages in North America is poorly have been increasingly identified in
provide an inaccurate description of understood, and most cases likely go industrialized countries over the last
several critical diagnostic and surveil- undetected without specific POWV few years (1). In this setting, hepatitis
lance issues concerning POWV. surveillance efforts. E is thought to be a zoonotic infec-
The 17 POWV infections detect- tion, with pigs as the primary host.
ed in Minnesota residents from 2008 David F. Neitzel, Ruth Lynfield, Consumption of uncooked or lightly
through 2011 (6 cases were identi- and Kirk Smith cooked pork meat products is thought
fied through 2010, not 8 as reported Author affiliation: Minnesota Department of to be a key route of infection, but oth-
by Birge and Sonnesyn) (Minnesota Health, St. Paul, Minnesota, USA er routes of transmission have been
Department of Health [MDH], unpub. documented (2). For example, there
data) were found through enhanced DOI: http://dx.doi.org/10.3201/eid1904.121651 have been several iatrogenic cases
surveillance. Health alerts to Min- after transfusion of hepatitis E virus
nesota medical providers described References (HEV)–contaminated blood products
POWV as a possible etiologic agent (3) and transplantation of an HEV-
for viral meningitis and encephalitis. 1. Birge J, Sonnesyn S. Powassan virus en- infected donor liver (4). However, in
cephalitis, Minnesota, USA. Emerg In-
Providers consulted with MDH on fect Dis. 2012;18:1669–71. http://dx.doi.
most cases the source and route of in-
suspected cases and submitted serum org/10.3201/eid1810.120621 fection are uncertain.
and cerebrospinal fluid specimens to 2. Hinten SR, Beckett GA, Gensheimer In May 2011, a 42-year-old
MDH. MDH conducted serologic test- KF, Pritchard E, Courtney TM, Sears woman sought care at the Royal
SD, et al. Increased recognition of Pow-
ing for endemic arboviruses (includ- assan encephalitis in the United States,
Cornwall Hospital in Truro, United
ing POWV) and reverse transcription 1999–2005. Vector Borne Zoonotic Dis. Kingdom, for a 1-week history of
PCR (RT-PCR) for flaviviruses and 2008;8:733–40. malaise, diarrhea, nausea, and vom-
POWV. MDH would not have de- 3. Lanciotti RS. Molecular amplification as- iting. Physical examination results
says for the detection of flaviviruses. Adv
tected any POWV infections without Virus Res. 2003;61:67–99. http://dx.doi.
were normal. Her liver function test
enhanced surveillance. Limited field org/10.1016/S0065-3527(03)61002-X results, however, indicated hepatitis:
studies also identified POWV-infected alanine aminotransferase 2,785 IU/L
ticks in 4 Minnesota counties (not 2 as Address for correspondence: David F. Neitzel, (reference range 10–36 IU/L), alka-
reported [1]) (MDH, unpub. data). Minnesota Department of Health, Infectious line phosphatase 319 IU/L (reference
Commercial laboratories do not Disease Epidemiology, 625 Robert St N, PO range 30–130 IU/L), and bilirubin 30
provide testing for POWV, and only Box 64975, St. Paul, MN 55164, USA; email: µmol/L (reference range <21 µmol/L).
a few state health department labo- david.neitzel@state.mn.us HEV IgM and IgG serologic test re-
ratories and the Centers for Disease sults for the patient were positive,
Control and Prevention offer testing. and HEV genotype 3 was identified
Serologic testing (enzyme immuno-
assay with plaque-reduction neutral-
ization testing confirmation) is pre-
Search in her blood by reverse transcription
PCR and sequencing. Other causes
of viral hepatitis and hepatocellular
past issues

EID
ferred (2) because POWV RT-PCRs jaundice, including hepatitis viruses
are not validated, and the short vire- A, B, and C; Epstein-Barr virus; and
mic periods of flaviviruses limit their autoimmune hepatitis, were excluded
usefulness (3). by testing. As with most immunocom-
Few POWV infections are identi- petent persons with HEV, the patient
fied by lineage (prototype vs. deer tick online made an uneventful clinical recovery
virus); Minnesota’s first case in 2008 after 12 weeks, and her liver func-
was identified as a deer tick virus in- www.cdc.gov/eid tion tests returned to normal after
fection, but the lineage was unknown 8 weeks.

686 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 LETTERS

Table. Heparin samples tested for hepatitis E virus, porcine circovirus 2, and porcine parvovirus*
Producer, proprietary name/other Quantity 95% upper
names, batch or lot no. Use Excipient Concentration tested, IU CL, /IU†
Sanofi‡
Clexhane/enoxaparin Injection H2O
ILA01 20 mg/0.2 mL 6,000 0.0006
34751 40 mg/0.4 mL 4,000 0.0009
OLC56 80 mg/0.8 mL 8,000 0.0005
ILA53 60 mg/0.6 mL 6,000 0.0006
OLC07 100 mg/ mL 10,000 0.0004
12255 120 mg/0.8 mL 12,000 0.0003
Pfizer§
Fragmin/dalteparin sodium Injection H2O pH adjusted with
HCl or NaOH
12339A01 5,000 IU/0.2 mL 15,000 0.0002
12338A01 5,000 IU/0.2 mL 15,000 0.0002
12327B01 5,000 IU/0.2 mL 15,000 0.0002
12257A01 5,000 IU/0.2 mL 15,000 0.0002
12444A01 5,000 IU/0.2 mL 10,000 0.0004
12122C01 7,500 IU/0.3 mL 7,500 0.0005
74774D51 10,000 IU/0.4 mL 10,000 0.0004
74871B51 12,500 IU/0.5 mL 25,000 0.0001
74779G51 12,500 IU/0.5 mL 12,500 0.0003
74871B51 12,500 IU/0.5 mL 12,500 0.0003
74743C52 15,000 IU/0.6 mL 30,000 0.0001
74755A51 15,000 IU/0.6 mL 30,000 0.0001
74832A52 15,000 IU/0.6 mL 15,000 0.0002
74832A01 15,000 IU/0.6 mL 15,000 0.0002
X08580 ¶ ¶ 100,000 IU/4 mL 100,000 0.00004
Wockhart#
Monoparin Injection H2O pH adjusted with
HCl or NaOH
PK40319 1,000 IU/mL 20,000 0.0002
3090 1,000 IU/mL 10,000 0.0004
Hepsal Flushing NaCl, H2O, HCl, and
NaOH
5000090 10 IU/mL 120 0.03
91180 50 IU/mL 50 0.07
1069 200 IU/mL 200 0.02
Leo**
Heparin sodium Intravenous Benzyl alcohol, methyl
flushing parahydroxybenzoate,
propyl
parahydroxybenzoate,
sodium citrate, NaCl, and
H2O
DD7314 100 IU/mL 200 0.02
CC4338 100 IU/mL 200 0.02
Celgene††,
Refludan/Lepirudin, 25561611A‡‡ Powder used for Mannitol, NaOH, and H2O 12.5 mg/mL NA NA
solution for
injection/infusion
Total quantity tested NA NA NA 404,270 0.000009
*NA, not applicable.
†The 95% upper confidence limit of the probability of a virus-positive result per IU was calculated on the basis of the quantity tested for each batch. This
was estimated, assuming perfect detection of a Poisson process, by using Fisher exact test. For the pooled result, the upper 95% estimate is 1 per
100,000 IU.
‡Sanofi (Guilford, UK).
§Pfizer (Sandwich, UK).
¶Multidose vials used for injection, Excipients: Benzyl alcohol and H2O.
#Wockhart (Wrexham, UK).
**Leo (Buckinghamshire, UK).
††Celgene (Uxbridge, UK).
‡‡Non-porcine–derived anticoagulant alternative.

The source and route of infection patient. She had not traveled outside fish; and had no workplace, domestic,
in this case was uncertain. A detailed the United Kingdom in the previous 3 or recreational exposure to pigs or
in-person assessment of potential months. She rarely ate pork products their effluent. However, 4 weeks be-
risk factors was undertaken with the (well cooked bacon only); ate no shell- fore symptom onset, the patient had

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 687
Page 1 of 1
LETTERS

acute appendicitis for which she un- Health Organization HEV RNA DOI: http://dx.doi.org/10.3201/eid1904.121792
derwent an uneventful laparoscopic standard (http://whqlibdoc.who.int/
appendectomy and was hospitalized hq/2011/WHO_BS_2011.2175_eng. References
for 2 days. During hospitalization pdf). In addition, we tested the hepa-
1. Kamar N, Bendall R, Abravanel F, Xia
she received no blood products, but, rin samples for porcine circovirus 2 N, Ijaz S, Izopet J, et al. Hepatitis E.
as prophylaxis for thromboembolic (PCV2), an identified adventitious Lancet. 2012;379:2477–88. http://dx.doi.
disease, she received 2 doses (5,000 agent of several rotavirus vaccines (8) org/10.1016/S0140-6736(11)61849-7
IU each) of low–molecular weight and porcine parvovirus (PPV) (9), a 2. Scobie L, Dalton HR. Hepatitis E:
source and route of infection, clinical
heparin (Fragmin [dalteparin sodium]; known contaminate of porcine clot- manifestations and new developments. J
Pfizer, Sandwich, UK) by subcutane- ting factor hyate:C (10). Although Viral Hepat. 2013;20:1–11. http://dx.doi.
ous injection. All heparins used in Eu- samples were tested in parallel with org/10.1111/jvh.12024
rope and North America are isolated PCV2- and PPV- positive spiked con- 3. Colson P, Coze C, Gallian P, Henry M, De
Micco P, Tamalet C. Transfusion-associat-
from porcine intestinal mucosa (5). trols, we were unable to calculate the ed hepatitis E, France. Emerg Infect Dis.
The exact purification methods used LOD for these assays because interna- 2007;13:648–9. http://dx.doi.org/10.3201/
by heparin manufacturers are deemed tional standards are not available for eid1304.061387
commercially sensitive and not in the these viruses. 4. Schlosser B, Stein A, Neuhaus R, Paul
S, Ramez B, Kruger DH, et al. Liver
public domain, so it is impossible to All samples tested negative for transplant from a donor with occult
evaluate whether the isolation process HEV, PCV2, and PPV (Table), which HEV infection induced chronic hepa-
would be sufficient to remove or in- would indicate the patient’s source titis and cirrhosis in the recipient. J
activate any contaminating HEV. The of HEV infection is unlikely to have Hepatol. 2012;56:500–2. http://dx.doi.
org/10.1016/j.jhep.2011.06.021
virus is known to be acid and alkaline been the heparin. However, we cannot 5. Alban S. The ‘precautionary principle’
stable; heat sensitivity varies, depend- rule out low-level viral contamination as a guide for future drug development.
ing on strain and heating conditions, below the sensitivity of the assay. We Eur J Clin Invest. 2005;35(Suppl 1):33–
although heating at 60°C for 1 hour also cannot exclude that the negative 44. http://dx.doi.org/10.1111/j.0960-13
5X.2005.01455.x
is generally sufficient to achieve 96% test results were related to the Pois- 6. Emerson SU, Arankalle VA, Purcell RH.
inactivation (6). To our knowledge, no son effect. Given that all samples ana- Thermal stability of hepatitis E virus. J
investigation has determined whether lyzed were negative for all 3 viruses Infect Dis. 2005;192:930–3. http://dx.doi.
clinical-grade heparin could contain tested, it seems likely that the heparin org/10.1086/432488
7. Jothikumar N, Cromeans TL, Robertson
viral contaminants. Thus, we hypoth- manufacturing process is sufficient to BH, Meng XJ, Hill VR. A broadly reac-
esized that the heparin the patient re- remove viral contaminants. However, tive one-step real-time RT-PCR assay
ceived might have been the source of this may not necessarily be the case for the rapid and sensitive detection of
her HEV infection. for other porcine-derived products, hepatitis E virus. J Virol Methods. 2006;
131:65–71. http://dx.doi.org/10.1016/j.j
To examine this possibility, we such as porcine insulin, factor VIII C, viromet.2005.07.004
screened multiple batches of hospital pancreatin, and poractant alfa. Further 8. Gilliland SM, Forrest L, Carre H, Jenkins
pharmacy–grade heparin for the pres- investigation is warranted to exclude A, Berry N, Martin J, et al. Investigation
ence of HEV, including batches of these products as potential sources of of porcine circovirus in human vaccines.
Biologicals. 2012;40:270–7. http://dx.doi.
dalteparin sodium that were in use at HEV infection. org/10.1016/j.biologicals.2012.02.002
the hospital when the patient received 9. Lau SKP, Woo PCY, Tse H, Fu CTY, Au
treatment for appendicitis. Before Acknowledgment Wk, Chen XC, et al. Identification of
testing, the samples were ultracentri- novel porcine and bovine parvoviruses
We thank the Chief Scientist Office closely related to human parvovirus 4. J
fuged to concentrate any contaminat- of Scotland who funded this work under Gen Virol. 2008;89:1840–8. http://dx.doi.
ing virus and enable the removal of project ETM/32. org/10.1099/vir.0.2008/000380-0
excipients, which could inhibit the as- 10. Soucie JM, Erdman DD, Evatt BL,
say. We tested samples by quantitative Anderson LJ, Torok TJ, El-Jamil M,
reverse transcription PCR (7) in paral- C. Crossan, L. Scobie, et al. Investigation or porcine parvo-
virus among persons with hemophilia
lel with positive World Health Orga- J. Godwin, J.G. Hunter,
receiving Hyate:C porcine factor VIII
nization HEV RNA standard spiked T. Hawkes, and H.R. Dalton concentrate. Transfusion. 2000;40:
controls, which showed the limit of Author affiliations: Glasgow Caledonian 708–11. http://dx.doi.org/10.1046/j.1537-
University, Glasgow, Scotland, UK (C. 2995.2000.40060708.x
detection (LOD) to be 500 IU/mL, re-
gardless of the heparin’s excipient or Crossan, L. Scobie, J. Godwin); Royal
concentration. This LOD is within the Cornwall Hospital, Truro, UK (J.G. Hunter, Address for correspondence: H. Dalton, Royal
range used by collaborating laborato- T. Hawkes, H.R. Dalton); and University of Cornwall Hospital, Truro, UK; email: harry.
ries in the establishment of the World Exeter Medical School, Truro (H.R. Dalton) dalton@rcht.cornwall.nhs.uk

688 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 LETTERS

Human Enterovirus Because we could not amplify of age (BCH2859A, BCH2892A,

EV-C104 by using primers specific BCH2894A, and BCH3034A) and a
Genotype C104, for the viral protein (VP) 1 region (9), 30-year-old man (PUMCH12286).
China we used a reverse transcription PCR BLAST analysis (www.ncbi.nlm.nih.
to amplify the 5′ -untranslated region/ gov) of sequences of these amplicons
To the Editor: Human VP4/VP2 gene (10). Amplicons of showed that the 590-nt sequences had
enteroviruses (EVs) are small, ≈600 bp were obtained for samples 94.0% identity with that of the EV-
nonenveloped RNA viruses belonging from 5 patients: 4 boys 2–11 months C104 prototype strain CL-12310945
to the family Picornaviridae.
Approximately 100 EV genotypes
have been identified. Recently,
EV68 epidemics in respiratory tract
infections (RTIs) have been reported
worldwide (1,2). Moreover, rarely
detected EVs (e.g., EV-C104 and
EV-C109) have been increasingly
identified in patients with RTIs (3–7),
indicating a possible association of
EVs with respiratory syndromes.
Little is known about the role of
EV-C104 in RTIs. EV-C104 has been
reported in 3 countries: Switzerland
(7 children with pneumonia or otitis
media) (3), Italy (3 adults and 4
children with RTIs) (4,7), and Japan (1
adult with an upper RTI [URTI]) (5).
We report additional EV-C104 strains
in 4 children with lower RTIs and in 1
adult with a URTI in China.
To identify EV infections, we
collected nasopharyngeal aspirates
from 3,108 children (1,963 boys) <14
years of age (median age 12 months;
age range 0.3–168 months) who had
lower RTIs at admission to Beijing
Children’s Hospital during March
2007–February 2012. Throat and nasal
swab specimens were also collected
from 9,232 adults (4,140 men) >15
years of age (median age 35.3 years;
age range 15–97 years) with acute
RTIs who received treatment at Peking
Union Medical College Hospital
during August 2006–February 2012.
All samples were screened for Figure. Phylogenetic tree of human enteroviruses (EVs) for nucleotide sequences of the
viral protein (VP) 4/VP2 gene region (435 nt, corresponding to nt positions 654–1,088 of EV-
influenza virus, parainfluenza virus C104 prototype strain CL-12310945 [EU840733]), People’s Republic of China, March 2007–
type 1–4, respiratory syncytial virus February 2012. The tree was generated with 1,000 bootstrap replicates. Neighbor-joining
(RSV), coronaviruses (229E, NL63, analysis of targeted nucleotide sequence was performed by using the Kimura 2-parameter
HKU1, and OC43), metapneumovirus, model with the Molecular Evolutionary Genetics Analysis software version 4.0 (www.
adenovirus, rhinovirus, bocavirus, and megasoftware.net/). Each strain detected in this study is indicated by a black circle and
a specific identification code (BCH/PUMCH), followed by the patient number. Enterovirus
EVs (8). Overall, 37 (1.2%) children 68, cocksackievirus (CV) A2, and echovirus (E) 3 (GenBank accession nos. AY426531,
and 158 (1.7%) adults were positive AY421760, and AY302553) were used as outgroups. PV, poliovirus. EV-C denotes the EV
for EV. species to which EV-C104 belongs. Scale bar indicates evolutionary distance.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 689
LETTERS

(EU840733). The amplicon contained expectoration for 1 day before a URTI Mérieux, Lyon, France (G. Vernet, G.
155 nt in the 5′-untranslated region, was diagnosed. EV-C104 was the only Paranhos-Baccalà)
207 nt in VP4, and 228 nt in VP2. virus detected in this patient. DOI: http://dx.doi.org/10.3201/eid1904.121435
Phylogenetic analysis of VP4/VP2 We compared relative viral loads
sequences showed that the 5 sequences for all viruses in the 5 patients and References
obtained in this study (GenBank quantified viral load of EV-C104 and
accession nos. JX560522–JX560526) other viruses by using real-time PCR 1. Centers for Disease Control and
belonged to genotype EV-C104 within (methods available upon request). Prevention. Clusters of acute respiratory
illness associated with human enterovirus
the EV-C species (Figure). Median viral load in the 5 patients 68—Asia, Europe, and United States,
Virus isolation for EV-C104 was 2.4 ×106 RNA copies/mL (range 2008–2010. MMWR Morb Mortal Wkly
with Vero and H1-HeLa cells was 5.6 × 104–7.0 × 106 copies RNA/mL Rep. 2011;60:1301–4.
unsuccessful. Although we screened (Table, Appendix, wwwnc.cdc.gov/ 2. Xiang Z, Gonzalez R, Wang Z, Ren L,
Xiao Y, Li J, et al. Coxsackievirus A21,
591, 797, 459, 664 and 597 samples EID/article/19/4/12-1435-T1.htm). enterovirus 68, and acute respiratory
from children for 5 consecutive years Overall, we found few (5/12,340) tract infection, China. Emerg Infect Dis.
and 1,765, 1,978, 1,350, 1,562, 1,573, EV-C104–positive specimens. All 2012;18:821–4. http://dx.doi.org/10.3201/
and 1,004 samples from adults for 6 EV-C104–positive children were co- eid1805.111376
3. Tapparel C, Junier T, Gerlach D, Van-Belle
consecutive years, we did not detect infected with RSV or adenoviruses S, Turin L, Cordey S, et al. New respiratory
EV-C104 strains until November (high viral loads) in our study. The enterovirus and recombinant rhinoviruses
2011–February 2012. role of EV-C104 in RTIs needs to among circulating picornaviruses. Emerg
Nucleotide identity of the EV- be further studied. Nevertheless, the Infect Dis. 2009;15:719–26. http://dx.doi.
org/10.3201/eid1505.081286
C104 sequences from this study was finding of EV-C104–positive adults 4. Piralla A, Rovida F, Baldanti F, Gerna
99.5%–100% among BCH strains and with high viral loads in China (3.9 G. Enterovirus genotype EV-104 in
97.7%–98.0% between the PUMCH ×106 RNA copies/mL) and Italy (2.0 humans, Italy, 2008–2009. Emerg Infect
strain and the BCH strains. Deduced × 106 RNA copies/mL) (7) indicates Dis. 2010;16:1018–21. http://dx.doi.
org/10.3201/eid1606.091533
amino acid sequences in VP4/ a possible association between 5. Kaida A, Kubo H, Sekiguchi J, Hase A,
VP2 among the BCH strains were EV-C104 with RTIs. Our data Iritani N. Enterovirus 104 infection in
identical, albeit for 1 aa difference also confirm a wide distribution of adult, Japan, 2011. Emerg Infect Dis.
for the PUMCH strain (BCH strains EV-C104. 2012;18:882–3. http://dx.doi.org/10.3201/
eid1805.111890
had Pro110, but the PUMCH strain This study was supported by the 6. Yozwiak NL, Skewes-Cox P, Gordon
had Leu110, which was consistent National Major Science and Technology A, Saborio S, Kuan G, Balmaseda A,
with strains detected in children and Project for the Control and Prevention
et al. Human enterovirus 109: a novel
adults in Italy). Deduced amino acid interspecies recombinant enterovirus
of Major Infectious Diseases in China isolated from a case of acute pediatric
sequences for all 5 strains isolated (2012ZX10004-206), the National respiratory illness in Nicaragua. J
in this study had 97.9%–100.0% Science Foundation for Outstanding Virol. 2010;84:9047–58. http://dx.doi.
identity with those from Switzerland, Young Scientists (81225014), and the
org/10.1128/JVI.00698-10
Italy, and Japan. BCH strains were 7. Piralla A, Lilleri D, Sarasini A, Marchi
Fondation Mérieux. A, Zecca M, Stronati M, et al. Human
community acquired because these 4 rhinovirus and human respiratory
patients came from different cities and enterovirus (EV68 and EV104)
were admitted to different wards on Zichun Xiang, Zhengde Xie, infections in hospitalized patients in
different dates. Zhong Wang, Lili Ren, Yan Xiao, Italy, 2008–2009. Diagn Microbiol
Linlin Li, Guy Vernet, Infect Dis. 2012;73:162–7. http://dx.doi.
The 4 EV-C104–positive boys org/10.1016/j.diagmicrobio.2012.02.019
all had fever and cough for >10 days Gláucia Paranhos-Baccalà,
8. Ren L, Gonzalez R, Wang Z, Xiang
before their hospitalization. Chest Kunling Shen, and Z, Wang Y, Zhou H, et al. Prevalence
radiographs showed increased lung Jianwei Wang of human respiratory viruses in
Author affiliations: Ministry of Health adults with acute respiratory tract
markings or patchy shadows diagnosed infections in Beijing, 2005–2007. Clin
Key Laboratory of Systems Biology of
as pneumonia or bronchopneumonia. Microbiol Infect. 2009;15:1146–53.
Pathogens, Beijing, People’s Republic of
RSV or adenovirus was also detected h t t p : / / d x . d o i . o r g / 1 0 . 1111 / j . 1 4 6 9 -
China (Z. Xiang, L. Ren, L. Li, J. Wang); 0691.2009.02746.x
in 3 of the boys. The fourth boy was
Institute of Pathogen Biology, Beijing (Z. 9. Nix WA, Oberste MS, Pallansch MA.
positive for parainfluenza virus type Sensitive, seminested PCR amplification
Xiang, L. Ren, Y. Xiao, L. Li, J. Wang);
1, adenovirus, and bocavirus. Clinical of VP1 sequences for direct identification
Beijing Children’s Hospital, Beijing (Z. Xie,
outcomes for all 4 children were of all enterovirus serotypes from original
K. Shen); Peking Union Medical College clinical specimens. J Clin Microbiol.
favorable. The EV-C104–positive
Hospital, Beijing (Z. Wang); and Fondation 2006;44:2698–704. http://dx.doi.org/10.
man had fever, chills, pantalgia, and 1128/JCM.00542-06

690 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 LETTERS

10. Savolainen C, Blomqvist S, Mulders although antiviral prophylaxis is rou- 4. Fuentes A, Gamerl S. Disproportion-
MN, Hovi T. Genetic clustering of all tinely prescribed to persons bitten by ate participation by age/sex classes in
102 human rhinovirus prototype strains: aggressive interactions between long-
serotype 87 is close to human enterovirus rhesus monkeys, there is not a single tailed macaques (Macaca fascicularis)
70. J Gen Virol. 2002;83:333–40. report of herpes B virus infection in a and human tourists at Padangtegal Mon-
human outside the laboratory/zoo con- key Forest, Bali, Indonesia. Am J Pri-
Address for correspondence: Jianwei Wang, text, although thousands of persons matol. 2005;66:197–204. http://dx.doi.
org/10.1002/ajp.20138
9 Dong Dan San Tiao, Dongcheng District, are likely bitten by macaques in Asia 5. Jones-Engel L, Engel GA, Schillaci
Beijing 100730, People’s Republic of China; every year (3,4). MA, Aida Rompis A, Putra A, Suary-
email: wangjw28@163.com In contrast, zoonotic transmission ana KG, et al. Primate-to-human retro-
of simian foamy virus, a retrovirus viral transmission in Asia. Emerg Infect
Dis. 2005;11:1028–35. http://dx.doi.
ubiquitous in nonhuman primates, has org/10.3201/eid1107.040957
been shown to occur from macaques 6. Monkeys with herpes B virus culled at a
to humans, probably through monkey safari park. Commun Dis Rep CDR Wkly.
bites, although this virus has not been 2000;10:99,102.

Monkey Bites shown to cause disease in humans (5).

Address for correspondence: Lisa Jones-
Although it is advisable to avoid con-
among US Military tact with monkeys, risk for disease
Engel, University of Washington–National

Members, transmission should be placed in proper

Primate Research Center, 1705 Pacific St NE,
HSB I-039, Seattle, WA 98195, USA; email:
Afghanistan, 2011 perspective. Exaggerating risks of bites
jonesengel@wanprc.org
has, in the past, led to irrational culling
To the Editor: We take serious of entire populations of macaques (6).
issue with the dispatch by Mease and
Baker on monkey bites among US mil- Gregory A. Engel,
itary members in Afghanistan during Agustin Fuentes,
2011 (1). In particular, we are troubled Benjamin P.Y.-H. Lee,
by the first paragraph. The dispatch Michael A. Schillaci, and In Response: In response to the
opens by listing bites from rhesus ma- Lisa Jones-Engel letter by Engel et al. (1), we concur
caques (Macaca mulatta) as one of Author affiliations: Swedish Family Medi- that combat-related deaths and illness
the many risks faced by military per- cine, Seattle, Washington, USA (G.A. are a greater risk than monkey bites for
sonnel deployed to Afghanistan. Al- Engel); University of Notre Dame, South deployed military personnel. Further-
though technically a true statement, it Bend, Indiana, USA (A. Fuentes); National more, we agree that risk for monkey
is misleading in its perspective. Since Parks Board, Singapore (B.P.Y.-H. Lee); bites should be considered in perspec-
2001, ≈2,000 US soldiers have died University of Toronto Scarborough, Toronto, tive with other risks faced by deployed
in Afghanistan and another ≈18,000 Ontario, Canada (M.A. Schillaci); Univer- personnel. We also believe that action
have been wounded in action (2). The sity of Washington, Seattle (G.A. Engel, L. taken to decrease macaque popula-
authors juxtapose this toll with minor Jones-Engel) tions in response to risks mentioned
injuries incurred by 10 soldiers who DOI: http://dx.doi.org/10.3201/eid1904.121505 would be irrational and inappropriate;
flouted explicit rules prohibiting con- in a country affected by war, wildlife
tact with pet monkeys. References conservation efforts are needed. We
None of the bitten soldiers were did not intend to imply that the rabies-
reported to have sequelae. Further- 1. Mease LE, Baker KA. Monkey bites associated death mentioned in our ar-
among US military members, Afghanistan,
more, the first paragraph leaves the 2011. Emerg Infect Dis. 2012;18:1647–9.
ticle was caused by contact with a ma-
impression that a US Army soldier http://dx.doi.org/10.3201/eid1810.120419 caque (2). As reported elsewhere, the
who died of rabies while serving in 2. US Department of Defense. Casualty patient likely contracted rabies from a
eastern Afghanistan may have con- status: Operation Iraqi Freedom; Op- dog bite (3).
eration New Dawn; Operation Enduring
tracted the disease from a macaque. Freedom. November 6, 2012 [cited 2012
Nonetheless, we believe that risk
This finding would be an extremely Nov 7]. http://www.defense.gov/news/ for monkey bites deserves the atten-
unlikely occurrence. casualty.pdf tion of deployed medical providers.
We have yet to see a single credi- 3. Engel GA, Jones-Engel L, Schillaci MA, Risks for bacterial infection and major
Suaryana KG, Putra A, Fuentes A. Hu-
ble report of macaque-to-human trans- man exposure to herpesvirus B–seroposi-
local trauma are critical for any ma-
mission of rabies. In fact, we have yet tive macaques, Bali, Indonesia. Emerg caque bite. We acknowledge that risk
to see a report of naturally acquired ra- Infect Dis. 2002;8:789–95. http://dx.doi. for contracting viral disease (rabies or
bies infection in a macaque. Similarly, org/10.3201/eid0808.010467 B virus infection) from macaques in

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 691
LETTERS

the wild is probably low, but we be- exacerbating underreporting and dis- DOI: http://dx.doi.org/10.3201/eid1904.121876
lieve that it merits consideration. couraging deployed personnel from
Social conditions in Afghanistan seeking needed care. We believe that References
have prevented any substantial rabies the role of command support and re-
1. Engel GA, Fuentes A, Lee BPY-H, Schil-
prevention program. Consequently, sponsibility cannot be overempha- laci MA, Jones-Engel L. Monkey bites
prevalence of rabies among wild ani- sized in preventing deployed person- among US military members, Afghani-
mals and pets is unknown and could nel from interacting with local animals stan, 2011. Emerg Infect Dis. 2013;19:69/.
2. Mease LE, Baker KA. Monkey bites
be higher than in other countries. (5). We thank Engel et al. for provid-
among US military members, Afghanistan,
Absence of any documented human ing additional perspective on the risk 2011. Emerg Infect Dis. 2012;18:1647–9.
deaths from B virus in a country with a for monkey bites to personnel de- http://dx.doi.org/10.3201/eid1810.120419
developing medical system and a high ployed in Afghanistan. 3. Centers for Disease Control and Preven-
tion. Imported human rabies in a U.S.
mortality rate does not confirm ab-
Army soldierNew York, 2011. MMWR
sence of risk. B virus has been shown Acknowledgment Morb Mortal Wkly Rep. 2012;61:302–5.
to be fatal in other areas, particularly This study was conducted exclusive- 4. Huff JL, Barry PA. B-virus (Cercopith-
in countries with greater medical diag- ly as part of our service as active duty US ecine herpesvirus 1) infection in humans
and macaques: potential for zoonotic dis-
nostic capacity (4). Army officers. ease. Emerg Infect Dis. 2003;9:246–50.
Engel et al. suggest that recorded http://dx.doi.org/10.3201/eid0902.020272
monkey bites occurred because af- 5. Chretien JP. Protecting service mem-
Luke E. Mease
fected persons flouted rules prohibit- bers in war–non-battle morbidity and
and Katheryn A. Baker command responsibility. N Engl J Med.
ing contact with local animals. Given
Author affiliations: Army Health Clinic, 2012;366:677–9. http://dx.doi.org/10.1056/
the unpredictable nature of operations NEJMp1112981
Dugway Proving Ground, Utah, USA (L.E.
in Afghanistan, it is impossible to
Mease); and General Leonard Wood Army
determine fault for the animal bites Address for correspondence: Luke E. Mease,
Community Hospital, Fort Leonard Wood,
detailed. Furthermore, blaming bite Army Health Clinic, 5116 Kister Ave, Dugway
Missouri, USA (K.A. Baker)
victims may be counterproductive, UT 84022, USA: email: luke.mease@us.army.mil

etymologia
Syncytium [sin-sish´e-əm]

F rom the Greek syn (together) and kytos (receptacle,

vessel), a multinucleate mass of protoplasm pro-
duced by the merging of cells. Respiratory syncytial virus
Morris originally called the new agent “chimpanzee
coryza agent,” although when Chanock et al. confirmed
that the agent caused respiratory illness in humans, it
was discovered in 1956 by Morris et al., who isolated it was renamed because “the striking characteristic of these
from a group of chimpanzees with respiratory symptoms. viruses is the production of syncytial areas in tissue culture.”

Sources
1. Chanock R, Finberg L. Recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (CCA). II. Epidemio-
logic aspects of infection in infants and young children. Am J Hyg. 1957;66:291–300.
2. Dorland’s Illustrated Medical Dictionary. 32nd ed. Philadelphia: Elsevier Saunders; 2012.
3. Hall CB. Respiratory syncytial virus. In: Mandell GL, Bennett JE, Dolin R, editors. Principles and practices of infectious diseases. 7th ed.
Philadelphia: Churchill Livingstone; 2010. p. 2207–21.

Address for correspondence: Ronnie Henry, Centers for Disease Control and Prevention, 1600 Clifton Rd NE, Mailstop E03, Atlanta, GA
30333, USA; email: boq3@cdc.gov

DOI: http://dx.doi.org/10.3201/eid1904.ET1904

692 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 BOOK REVIEW

The Foundations are clear advantages to having the Hippocrates’ observations in 400 bce
hard copy in hand, in a place where it to the 2010 declaration of the global
of Virology: can be picked up and absorbed a little eradication of rinderpest. Particularly
Discoverers and at a time. given the origin of new, emerging, and
Discoveries, The full title of the book reflects reemerging viral infections today, it is
the content; it is a chronology of im- valuable to see pictures and stories to
Inventors and ages of discoverers, developers, and remind us of the exceptional crossover
Inventions, inventors, alongside their discoveries, between human and veterinary virolo-
Developers and developments, and inventions. The gy. Because of the author’s longstand-
book is physically laid out in a land- ing involvement in virology since the
Technologies scape format, usually with multiple 1960s, his explanations for discover-
Frederick A. Murphy photographs on each page, featuring ies of the past 5 decades provide a
scientists, their institutions, spot maps unique sense of context.
Infinity Publishing, West from major epidemics, electron mi- This visual account of history
Conshohocken, Pennsylvania,
crographs of then-emerging viruses, makes obvious the sparse involve-
USA, 2012
and graphics from landmark publica- ment of women in virology until the
ISBN-10: 0741473658 tions. Along the bottom of each page late 1900s. However, Murphy made
ISBN-13: 978-0741473653 is printed in large font the year, names a good effort to acknowledge women
of contributors, and their historic con- when possible. He also did a good job
Pages: 536; Price US $119.95 tributions. This bottom line layout acknowledging seminal forces world-
History of science is not a priority serves as an effective design enabling wide that shaped the development of
of most virologists. However, if given a quick orientation to time, with the op- virology: influential books, journals,
visually engaging coffee-table book of tion for most entries of reading a more societies, conferences, databases,
the persons and discoveries that shaped in-depth explanation. It also makes it even the Google search engine.
the field of virology since Hippocrates, easy to skip to different points in time, Murphy’s website (www.utmb.
curious scientists will inevitably start an efficiency advantage over Mur- edu/virusimages) is surely one of the
paging through history. For this reason, phy’s online resources. Other books most comprehensive and publically
every chairperson of virology depart- on the history of virology are a dense accessible virology and history of vi-
ments worldwide should leave a copy read (and lack all the fun pictures). rology resources available. But having
of Fred Murphy’s The Foundations of Murphy’s selection of images finger-tip access to this scientist’s cof-
Virology in the break room. highlights his conviction that science fee-table book is a worthwhile invest-
Professor Murphy has a long and of viral diseases predates the concept ment for any infectious disease spe-
distinguished career in emerging vi- of the specificity of disease causation cialist interested in medical history.
ruses from the perspective of public and depends on initial discoveries
about bacteria and bacterial diseases. Sharon Bloom
health and academic bench science.
Readers can thus expect to see images Author affiliation: Centers for Disease Con-
Murphy has made his 536-page book
of Pasteur, Koch, and many others trol and Prevention, Atlanta, Georgia, USA
available as a downloadable, lower-
resolution, eBook on his website, who laid the groundwork for infec- DOI: http://dx.doi.org/10.3201/eid1904.130054
which for years has also been an enor- tious disease sciences. Although in
mous open-access library resource for his Foreword, Murphy disclaims that Address for correspondence: Sharon Bloom,
virologists. Because of numerous re- his selection of names and discoveries Division of Global HIV/AIDS, Center for
quests to have hard copies printed for was “completely arbitrary,” his book Global Health, Centers for Disease Control and
fingertip access, Murphy has made this leaves out few major discoveries in Prevention, 1600 Clifton Rd NE, Mailstop E41,
book available in paperback. There human and veterinary virology, from Atlanta, GA 30333, USA; email: sab0@cdc.gov

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 693
ABOUT THE COVER

Egon Schiele (1890–1918) Self-Portrait with Physalis (1912) (detail) Oil and opaque color on wood (32.2 cm × 39.8 cm) Leopold
Museum, Vienna, Austria, www.leopoldmuseum.org

Pale horse, pale rider done taken my lover away1

Polyxeni Potter

“I t simply divided my life, cut across it like that. So

that everything before that was just getting ready, and
after that I was in some strange way altered, really,” said
with orange flecks in them, and his hair the color of a hay-
stack when you turn the weathered top back to the clear
straw beneath,” Adam, who had “never had a pain in his
Katherine Anne Porter about her nearly fatal encounter life that he [could] remember,” had died.
with the Spanish flu. “It took me a long time to go out and Porter, the consummate storyteller, used words to ex-
live in the world again.” Years later, in a thinly disguised press the devastating effects of illness on her life. Across
autobiographical novel, she laid out not just her own trau- the world, in Austria, Egon Schiele, whose self-portrait
matic run-in with death, the pale rider, but also a rare liter- graces this month’s cover, in similar circumstances, found
ary account of the 1918 flu pandemic in the United States no comfort in words, despite his own poetic nature. When
and the unprecedented human loss. his wife was dying of the flu, he was unable to articulate
For her recollection of the pandemic, Porter had to his feelings. In a letter to his mother, he coolly speculated
rely on fragments of memory before her illness and after that Edith would probably not survive. But he used art to
her recovery. These fragments involved the landlady; her express his devastation. He made several sketches of his
beloved fiancé Adam; and fatal flu in Denver, Colorado, wife during the last 2 days of her life. In these sketches,
during World War I, which killed many young men needed the lines were fluid and sensitive, the colors subdued, the
for battle. “I tell you, they must come for her now or I’ll put format understated. All these features, a departure from his
her on the sidewalk.” “They can’t get an ambulance, and usual provocative style, reflected the emotional stability
there aren’t any beds. And we can’t find a doctor or nurse. found in his life with Edith. His last work was a portrait
They’re all busy.” “It’s as bad as anything can be … all the of his wife, who died the following day. She was 6 months
theaters and nearly all the shops and restaurants are closed, pregnant. He died 3 days later.
and the streets have been full of funerals all day and ambu- Even before his fatal encounter with the flu, Schiele
lances all night.” “Two cherry flavored pills,” “orange juice was acquainted with adversity. His early years were marred
and ice cream,” “coffee in a thermos bottle.” “The men are by a troubled relationship with his mother, poor academic
dying like flies out there …. This funny new disease. Sim- performance, and the loss of his father, a provincial railroad
ply knocks you into a cocked hat.” stationmaster, to tertiary syphilis, when Egon was 14 years
Porter wakes up from her illness to find that Adam, old. “I don’t know whether there is anyone else at all who
“tall and heavily muscled in the shoulders, narrow in the remembers my noble father with such sadness.” The elder
waist and flanks,” handsome Adam with “his eyes pale tan
1
Negro spiritual, in Katherine Anne Porter’s "Pale Horse, Pale
Author affiliation: Centers for Disease Control and Prevention,
Rider". See also “Go Down Death: A Funeral Sermon” in God’s
Atlanta, Georgia, USA
Trombones: Seven Negro Sermons in Verse at http://docsouth.unc.
DOI: http://dx.doi.org/10.3201/eid1904.AC1904 edu/southlit/johnson/johnson.html

694 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 ABOUT THE COVER

Schiele had kept his condition from his 17-year-old bride, lives in a short time than any other disease in history, yet
Egon’s mother. Her first three babies were stillborn. The because it was intertwined with the “Great War,” the horror
fourth child died at age 10 of meningitis, a complication of it in human terms may not have been adequately chron-
of late-onset congenital syphilis. Egon was the first boy icled. Some have called it the “forgotten pandemic”—but
to survive. “I shall be the fruit which after its decay will not those who work in public health.
leave behind eternal life; therefore how great must be your Many strides have been made in flu prevention and
joy―to have borne me?” Egon wrote to his mother. His control since 1918: better understanding of the virus, its
penchant for grandiosity and certain physical features in his distribution in nature, its presence in animals and birds,
early self-portraits led some to wonder whether he might some of its virulence factors, how it mutates, how it is dis-
have also been infected. tributed in tissues, how it is transmitted. We now have pre-
Schiele lived his life at an accelerated pace. He started vention programs, vaccines, and antiviral drugs. But many
to draw as a child and was enrolled in the Vienna Acad- still die, and the threat of another pandemic lurks.
emy of Fine Arts at age 16. He became a protégé of Gustav Dramatic tension captured in literature and art prevents
Klimt, a strong early influence. Once when asked if young us from forgetting the dead and the grave pandemics of his-
Schiele’s drawings showed talent, Klimt responded, “Much tory. Schiele did his part by immortalizing the faces of his
too much.” The gifted but troublesome student would soon beloved persons—very much like Porter, who in her ver-
go off on his own and form the New Artists group. Later, sion of the spiritual “Pale Horse, Pale Rider,” contends that
when Klimt was struck down by the flu, Schiele made sev- death takes away the singer’s lover, mother, siblings, and
eral portraits of him on his deathbed. eventually over the course of several verses the entire fam-
Schiele went on to serve in the military; to have high- ily, “But not the singer, not yet,” “Death always leaves one
profile love affairs; get arrested and be thrown in jail; and singer to mourn.” That’s to ensure remembrance, which ap-
before his own untimely death, make a proper and by all ac- plies as well in public health, where to prevent the next
counts promising marriage. All along, he grew as an artist pandemic, it pays to remember and study the past ones.
and achieved an expressionist style focused on feelings and
their interpretation. He was drawn to the unconventional Bibliography
and controversial, and his hundreds of self-portraits were
penetrating and disquieting. His exaggerated lines, unreal- 1. Comini A. Egon Schiele: portraits. Berkeley (CA): University of
California Press; 1974.
istic shapes, and intense colors invoked human situations 2. Fuller TL, Gilbert M, Martin V, Cappelle J, Hosseini P, Njabo KY, et
with a candor that many found disturbing. During his brief al. Predicting hotspots for influenza reassortment. Emerg Infect Dis.
career he created more than 3,000 works on paper, some 2013;19:581–8.
300 paintings. Despite the edginess, his artistic reputation 3. Kallir J. Egon Schiele: life and work. New York: Harry N Abrams,
Inc.; 2003.
grew, he was offered commissions, and his work sold well. 4. Knafo D. Egon Schiele: a self in creation: a psychoanalytic study of
In interpreting any self-portrait, it is tempting to draw the artist’s self-portraits. Madison (NJ): Fairleigh Dickinson Univer-
clues from the artist’s life. During his short career, Schiele sity Press; 1993.
was alienated and vulnerable and in the midst of World 5. Kuster SP, Coleman BL, Raboud J, McNeill S, De Serres G, Gubbay
J, et al. Risk factors for influenza infection among health care work-
War I and pandemic flu. Self-Portrait with Physalis, prob- ers during 2009 pandemic, Toronto, Ontario, Canada. Emerg Infect
ably his best-known self-portrait, shows him at the peak Dis. 2013;19:606–15.
of his creativity in 1912, his most productive year, when 6. Lekana-Douki SE, Mouinga-Ondémé A, Nkoghe D, Drosten C,
his expressionist style had matured. Clues for interpreting Drexler JF, Kazanji M, et al. Early introduction and delayed dis-
semination of pandemic influenza, Gabon. Emerg Infect Dis.
a self-portrait can also be drawn from facial expressions, 2013;19:644–47. http://dx.doi.org/10.3201/eid1904.111925
gestures, and props within the painting—what the artist al- 7. Lucie-Smith E. Lives of the great 20th-century artists. London:
lows us to see. In this self-portrait, the arms and body are Thames and Hudson; 1999.
severely cropped, only the cocked head and shoulders are 8. Porter KA. Pale horse, pale rider: three short novels. New York: Har-
court, Brace and Co.; 1939.
shown. The one-eyed stare challenges the viewer. So do the 9. Chandra S. Deaths associated with influenza pandemic of 1918–19,
pursed lips. This is a laconic composition, though a the- Japan. Emerg Infect Dis. 2013;19:616–22. http://dx.doi.org/10.3201/
atrical one, what with its intensely colored lampion fruit eid1904.120103
and dreamy sentimentality. Despite the scattered character 10. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning
TG. Characterization of the 1918 influenza virus polymerase genes.
clues, there is one thing we will never know from Schiele’s Nature. 2005;437:889–93. PubMed http://dx.doi.org/10.1038/na-
Self-Portrait with Physalis, and that is what would have ture04230
happened if he had not died at age 28 of pandemic flu.
When the pandemic ended, which took away even her Address for correspondence: Polyxeni Potter, EID Journal, Centers for
“decrepit hound and silver kitten,” there was, Porter wrote, Disease Control and Prevention, 1600 Clifton Rd NE, Mailstop D61,
a “dazed silence.” The “Great Pandemic” claimed more Atlanta, GA 30333, USA; email: pmp1@cdc.gov

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 695
 Earning CME Credit
To obtain credit, you should first read the journal article. After reading the article, you should be able to answer the following,
related, multiple-choice questions. To complete the questions (with a minimum 70% passing score) and earn continuing
medical education (CME) credit, please go to www.medscape.org/journal/eid. Credit cannot be obtained for tests completed
on paper, although you may use the worksheet below to keep a record of your answers. You must be a registered user on
Medscape.org. If you are not registered on Medscape.org, please click on the New Users: Free Registration link on the left
hand side of the website to register. Only one answer is correct for each question. Once you successfully answer all post-
test questions you will be able to view and/or print your certificate. For questions regarding the content of this activity, con-
tact the accredited provider, CME@medscape.net. For technical assistance, contact CME@webmd.net. American Medical
Association’s Physician’s Recognition Award (AMA PRA) credits are accepted in the US as evidence of participation in CME
activities. For further information on this award, please refer to http://www.ama-assn.org/ama/pub/category/2922.html. The
AMA has determined that physicians not licensed in the US who participate in this CME activity are eligible for AMA PRA
Category 1 Credits™. Through agreements that the AMA has made with agencies in some countries, AMA PRA credit may
be acceptable as evidence of participation in CME activities. If you are not licensed in the US, please complete the ques-
tions online, print the certificate and present it to your national medical association for review.

Article Title
Serotype IV and Invasive Group B Streptococcus Disease
in Neonates, Minnesota, USA, 2000–2010
CME Questions
1.You are seeing a 2-day-old male infant with fever 3. What were the most common serotypes of GBS
to 38.3°C and some irritability with poor feeding. among infected children in the current study?
You suspect that this child might have group B
Streptococcus (GBS) infection. Which of the following A. Ib, II
statements regarding GBS infection among infants is B. III, Ia
most accurate? C. IV, Ib
D. IV, V
A. E
 arly-onset (EO) GBS infection is defined by
infection between birth and day 6 of life 4. What should you consider regarding infection with
B. L
 ate-onset (LO) GBS infection is defined by serotype IV GBS among children in the current study?
infection between 1 to 3 months of life
 creening for GBS in the third trimester has had
C. S A. Type IV was associated with higher rates of LO vs EO
no effect on the prevalence of EO disease among disease
infants B. The mortality rate associated with type IV infection
D. S
 creening for GBS in the third trimester has was 50%
primarily reduced the prevalence of LO disease C. Type IV disease produced higher degrees of fever
among infants compared with other serotypes
D. Type IV disease was associated with higher rates of
2. What should you consider regarding the antimicrobial resistance in 2010 compared with other
epidemiology of GBS disease in the current study? serotypes

A. There were more LO vs EO cases of GBS disease

B. Early-onset GBS was almost always associated with
cerebrospinal fluid infection
C. Most infants with EO disease were full-term
D. There were no cases of GBS disease after infants
were at least 3 months old
Activity Evaluation
1. The activity supported the learning objectives.
Strongly Disagree Strongly Agree
1 2 3 4 5
2. The material was organized clearly for learning to occur.
Strongly Disagree Strongly Agree
1 2 3 4 5
3. The content learned from this activity will impact my practice.
Strongly Disagree Strongly Agree
1 2 3 4 5
4. The activity was presented objectively and free of commercial bias.
Strongly Disagree Strongly Agree
1 2 3 4 5

696 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013
 Earning CME Credit
To obtain credit, you should first read the journal article. After reading the article, you should be able to answer the following,
related, multiple-choice questions. To complete the questions (with a minimum 70% passing score) and earn continuing
medical education (CME) credit, please go to www.medscape.org/journal/eid. Credit cannot be obtained for tests completed
on paper, although you may use the worksheet below to keep a record of your answers. You must be a registered user on
Medscape.org. If you are not registered on Medscape.org, please click on the New Users: Free Registration link on the left
hand side of the website to register. Only one answer is correct for each question. Once you successfully answer all post-
test questions you will be able to view and/or print your certificate. For questions regarding the content of this activity, con-
tact the accredited provider, CME@medscape.net. For technical assistance, contact CME@webmd.net. American Medical
Association’s Physician’s Recognition Award (AMA PRA) credits are accepted in the US as evidence of participation in CME
activities. For further information on this award, please refer to http://www.ama-assn.org/ama/pub/category/2922.html. The
AMA has determined that physicians not licensed in the US who participate in this CME activity are eligible for AMA PRA
Category 1 Credits™. Through agreements that the AMA has made with agencies in some countries, AMA PRA credit may
be acceptable as evidence of participation in CME activities. If you are not licensed in the US, please complete the ques-
tions online, print the certificate and present it to your national medical association for review.

Article Title
Risk Factors for Influenza among Health Care Workers
during 2009 Pandemic, Toronto, Ontario, Canada
CME Questions
1. A 40-year-old nurse at your local hospital presents 3. The patient in question 1 asks you if her risk of
to your office for a physical examination. She reports influenza is higher due to her occupation. You inform
a one-day history of fever. That morning, you received her that:
a notice that we are currently in the first wave of a
pH1N1 pandemic. This is defined as periods for which A. Her risk is higher compared to non-healthcare workers
the weekly proportion of respiratory specimens which (HCWs)
yielded pH1N1 was greater than: B. Her risk is lower compared to non-HCWs
C. There is no difference in risk compared to non-HCWs
A. 1% D. None of the above is correct
B. 5%
C. 10% 4. What other risk factors should you inquire about the
D. 25% patient in question 1 that would increase her risk of
influenza?
2. You suspect the patient in question 1 may have
pH1N1 when she reported the following symptoms: A. Performing aerosol-generating medical procedure
B. Recent pH1N1 vaccination
A. Current urinary tract infection symptoms C. High adherence to hand hygiene recommendations
B. Sore throat and cough D. Working in the pediatric ward
C. Runny nose and headache
D. Answers B and C

Activity Evaluation
1. The activity supported the learning objectives.
Strongly Disagree Strongly Agree
1 2 3 4 5
2. The material was organized clearly for learning to occur.
Strongly Disagree Strongly Agree
1 2 3 4 5
3. The content learned from this activity will impact my practice.
Strongly Disagree Strongly Agree
1 2 3 4 5
4. The activity was presented objectively and free of commercial bias.
Strongly Disagree Strongly Agree
1 2 3 4 5

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 697
 NEWS & NOTES

Upcoming
Infectious
Disease
Upcoming Issue
Activities
Transmission of Mycobacterium tuberculosis Beijing Strains,
Alberta, Canada, 1991–2007
2013
Foodborne Transmission of Bovine Spongiform Encephalopathy to
May 1–4, 2013
Nonhuman Primates The Society for Healthcare
Epidemiology of America (SHEA)
Populations at Risk for Alveolar Echinococcosis, France Spring 2013 Conference
Atlanta, GA, USA
WHO International Standard to Harmonize Assays for Detection of http://shea2013.org
Hepatitis E Virus RNA

Full-Genome Deep Sequencing and Phylogenetic Analysis of September 5–10, 2013

Novel Human Betacoronavirus
Options for the Control
Targeted Surveillance for Zoonotic Virus Discovery of Influenza VIII
Cape Town, South Africa
http://www.isirv.org
Changing Severity of Influenza A(H1N1)pdm09 Infection in
Hospitalized Patients in First Postpandemic Season, Germany
September 10–13, 2013
Severe Fever with Thrombocytopenia Syndrome Virus Infection
among Domesticated Animals, China ICAAC 2013
(Interscience Conference
Campylobacter coli Outbreak among Men Who Have Sex with on Antimicrobial Agents and
Men, Quebec, Canada, 2010–2011 Chemotherapy)\
Denver, Colorado, USA
Delayed Diagnosis of Chronic Q Fever and Cardiac Valve Surgery http://www.icaac.org

Treatment of Tularemia in Patient with Chronic Graft-versus-Host Announcements

Disease To submit an announcement, send an
email message to EIDEditor (eideditor@
cdc.gov). Include the date of the event, the
Scrub Typhus Outbreak, Northern Thailand. 2006–2007 location, the sponsoring organization(s),
and a website that readers may visit or a
Rickettsia parkeri Infection Detected from Eschar Swab Specimens telephone number or email address that
readers may contact for more information.
Contaminated Ventilator Air Flow Sensor and Bacillus cereus Announcements may be posted on the
Colonization of Newborns journal Web page only, depending on the
event date.
Mapping Environmental Suitability for Malaria Transmission,
Greece
The opinions expressed by authors
contributing to this journal do not
Complete list of articles in the May issue at necessarily reflect the opinions of
http://www.cdc.gov/eid/upcoming.htm the Centers for Disease Control and
Prevention or the institutions with which
the authors are affiliated.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 699