Virology

Introduction
• Viruses contribute significantly to the global burden of infectious
disease.
• We experience countless infections throughout their lives, with
particularly high frequency in early childhood. While most of these
are mild, viruses may cause severe disease in susceptible individuals,
such as the mal-nourished, immune-compromised, the very old and
the very young. Recent years have also seen the emergence of new
viral diseases such as HIV, SARS and "swine flu" (H1N1 pandemic
influenza A).
• Viruses are uniquely different from the many uni-cellular micro-
organisms you have studied so far. Protozoa, yeasts, bacteria,
mycoplasmas, rikettsiae and chlamydiae are all living organisms with the
following features in common:
• They are all cells
• They store their genetic information as DNA
• Within their cell, they contain all the organelles necessary for producing
energy and synthesizing proteins, carbohydrates, cell wall structures etc.
• Replicate by means of binary fission
Introduction Continued - VIRUSES
• Viruses do not share these properties. They are not cells. They are very simple structures
consisting essentially of a nucleic acid genome, protected by a shell of protein. They are
metabolically inert and can only replicate once they are inside a host cell.

• The genome consists of only one type of nucleic acid: either RNA or DNA. Most DNA viruses are
double stranded and most RNA viruses have a single stranded (ss) genome. A ssRNA genome
may be either positive sense (this means that it can be used as mRNA to make proteins) or
negative sense.
• Negative sense RNA is complimentary to mRNA, in other words, it has to be copied into mRNA.
The viral genome codes only for the few proteins necessary for replication: some proteins are
non-structural e.g. polymerase and some are structural, i.e. they form part of the virion
structure.

• They have no organelles.

• They are very small, sizes range from 20 to 200 nm. This is beyond the resolving power of the
light microscope.
Terminology
• Virion = virus particle

• Capsid = protein shell which surrounds and protects the genome. It is built up of multiple
(identical) protein sub-units called capsomers. Capsids are either icosahedral or tubular in
shape

• Nucleocapsid = genome plus capsid

• Envelope = lipid membrane which surrounds some viruses. It is derived from the plasma
membrane of the host cell.

• Peplomers = proteins found in the envelope of the virion. They are usually glycosylated and are
thus more commonly known as glycoproteins.
Viral replication
Viral replication
• Viruses are the ultimate parasite. They are totally dependent on a
host cell to replicate (make more copies of itself). While the sequence
of events varies somewhat from virus to virus, the general strategy of
replication is similar:
Viral replication
• Adsorption: The surface of the virion contains structures that interact with
molecules (receptors) on the surface of the host cell. This is usually a passive
reaction (not requiring energy), but highly specific. It is the specificity of the
reaction between viral protein and host receptor that defines and limits the
host species and type of cell that can be infected by a particular virus.
Damage to the binding sites on the virion or blocking by specific antibodies
(neutralization) can render virions non-infectious.

• Uptake: The process whereby the virion enters the cell. It occurs either as a
result of fusion of the viral envelope with the plasma membrane of the cell
or else by means of endocytosis.

• Uncoating: Once inside the cell, the protein coat of the virion dissociates
and the viral genome is released into the cytoplasm.
• Early phase
• Once the genome is exposed, transcription of viral mRNA and
translation of a number of non-structural ("early") proteins takes
place. The function of these is to replicate the viral genome.

• Genome replication
• Multiple copies of the viral genome are synthesized by a viral
polymerase (one of the "early" proteins).
Late phase

• Transcription and translation of viral mRNA and synthesis of the

structural ("late") proteins which are needed to make new virions.

• Assembly of new virions

• Assembly of new viral capsids takes place either in the nucleus (e.g.
herpesviruses) or in the cytoplasm (e.g. poliovirus) of the cell, or
sometimes, just beneath the cell surface (e.g. budding viruses such as
influenza). The proteins self assemble and a genome enters each new
capsid.
Release of progeny virions

• Release of new infectious virions is the final stage of replication. This

may occur either by budding from plasma membrane or else by
disintegration (lysis) of the infected cell. Some viruses use the
secretory pathway to exit the cell: virus particles enclosed in golgi-
derived vesicles are released to the outside of the cell when a
transport vesicle fuses with the cell membrane.
Disease
Disease
• Viruses are capable of infecting all types of living organism from
bacteria to humans, (including plants and insects!). A major factor
that controls which cell type a virus can infect (cell tropism) is the
presence (on the cell surface) of the appropriate receptor, to which
the virus must attach in order to gain entry into the cell.
 Disease
• Viruses enter the body by
• inhalation,
• ingestion,
• sexual intercourse
• or inoculation through the skin or mucous membranes.
• Infection may also sometimes be passed from a mother to her foetus transplacentally
(vertical transmission).
• Once a virus has gained entry into the body, infection may either remain localised to the
site of entry (an example of this is influenza where the virus remains confined to the
respiratory tract),
• or it may cause a disseminated infection. Here, the virus replicates initially at the site of
entry, but then enters the blood (viraemia) or lymphatics and spreads throughout the body
(e.g. Measles).
• Other viruses such as Rabies and Herpes Simplex may replicate locally initially, then enter
nerve endings and travel up the axon to infect the central nervous system.
Disease
• The term incubation period defines the time from exposure to an
organism to the onset of clinical disease. In general, viruses that
cause localized infections have short incubation periods (<7 days),
while in disseminated infections, the incubation period tends to be
longer.
Disease
• Both viral and host factors contribute to clinical disease during the course of a viral
infection.
• Host immune cells release interferons and other cytokines which induce the symptoms
of fever and malaise.
• Tissue specific damage may be due to virus-induced lysis of infected cells or due to
inflammation and destruction of infected cells by the host's immune response.
• Because viruses replicate intracellularly, recovery from a viral infection requires the
action of specific cytotoxic T lymphocytes which recognize and eliminate virus-infected
cells.
• Virus-specific antibody levels rise during the course of the infection, but antibody plays
only a limited role in recovery from an established infection for most viruses.
Nonetheless specific antibody plays a very important role in preventing re-infection of
the host with the same virus.
IMMUNOLOGY
• An effective immune response can eliminate most viruses from the
body and thus most viral infections are short lived.
• However, there are certain viruses that are able to evade the immune
response and establish persistent infections in their host.
• The most famous example of such a virus is HIV, but there are many
others.
• Viruses use a variety of strategies to evade the immune system. On
the whole, these persistent infections are asymptomatic and only
manifest clinically if the patient becomes immuno-compromised.
Viruses and cancer:

• About 15% of human cancers are caused by viruses. Certain persistent

viruses survive in the host by transforming the cells they infect (inducing
infected cells to proliferate). However, the virus infection is only the first
step in the pathway to malignancy and only a small percentage of
infected people actually get cancer.

• Common virus-induced cancers include: carcinoma of the cervix (Human

papillomavirus), liver cancer (hepatitis B and C), Kaposi sarcoma (human
herpesvirus 8) and Burkitts lymphoma (Epstein Bar virus).
Disinfection and inactivation
• Heat - Most are inactivated at 56 °C for 30 minutes or at 100 °C for a few
seconds
• Drying - Variable; enveloped viruses are rapidly inactivated.
• Ultra-violet - irradiation Inactivates viruses
• Organic solvents - (Chloroform, Ether, Alcohol) Enveloped viruses are
inactivated; those without are resistant.
• Oxidizing and reducing agents - Viruses are inactivated by formaldehyde,
chlorine, iodine and hydrogen peroxide
• Phenols - Most viruses are resistant
Laboratory diagnosis
Four approaches to confirming a
viral infection
Namely:
• serology: demonstrating an antibody response in a patient's serum
• direct detection of viral antigens in a clinical sample
• virus culture
• viral nucleic acid detection
• Serologic vs virologic testing
Electron Microscopy

• Viruses are very small and cannot be visualized by light microscopy.

Historically the electron microscope was very useful in defining the
morphology of many human viruses.
• However, it is not a tool that is routinely used to identify viruses in a
diagnostic setting. This is because viruses are usually present in very
small numbers in clinical specimens and other contaminating material
tends to obscure their presence.
 Demonstration of virus-infected cells in clinical samples by labelled antibodies.

• This technique is commonly used to identify the causative agent in a patient with a
respiratory infection, caused by viruses such as RSV, Influenza or Adenovirus.

• Infected cells synthesize and express viral proteins (antigens). The presence of these can be
detected using specific mono-clonal or poly-clonal antibodies labelled with fluorescence (a
green dye). The antibody binds to the cells if they express the corresponding antigen. The
cells can then be visualized by examination under a fluorescent microscope. Positive cells
fluoresce a bright green color.

• The limitation of the test is that you have to know what virus you are looking for.
• The advantage is that one can get a very rapid answer as to which virus is causing the
problem.
Culture

• Viruses can only replicate in living cells. Therefore to culture them in vitro one must provide
them with living cells. In the past it was common to use laboratory animals, or chick embryos
to grow viruses, but these have largely been replaced by the use of cell monolayers.

• The clinical sample is inoculated into a test tube containing a glass cover slip on which a cell
monolayer is growing. Replicating viruses change the appearance of the cells to induce a
cytopathic effect. Different viruses cause different types of cytopathic effects. Only some
medically important viruses can be cultured.

• Immunofluorescence: Another way to identify a virus growing in a cell culture is to add

fluorosceine labelled monoclonal antibodies to likely viruses to the cell sheet and examine
under a fluorescent microscope.
Cell Culture
• Primary Cell Line
• Semi-continuous
• Continuous
Molecular techniques

• Nucleic acid amplification techniques such as polymerase chain

reaction (PCR) can be used to detect viral genomes in clinical material.
The same technique can be used to detect any DNA sequence (viral,
bacterial or other). To detect RNA, an initial reverse transcription step
is performed (converts RNA into cDNA). After this, PCR can be
performed. Molecular assays are very sensitive (able to detect only a
few viruses in a clinical sample.) They can also be used to measure the
amount of virus (viral load) in a patient's sample.
 Antibody assays:

• An acute or recent infection may be confirmed by demonstrating the

presence of specific IgM in a single serum sample, or showing a sero-
conversion or rise in titre of specific IgG in paired sera.
• In general, the presence of IgG and the absence of IgM, is indicative
of past infection or immunity.
• These days, antibody assays are usually tested by means of the
enzyme-linked immuno-assay (ELISA) technique.
OLDER Techniques
• CFT
• HI

• Determine total antibody

• Acute vs convalescent
• Titer cut off
– RNA Viruses

e.g., poliovirus, flaviviruses (including hepatitis C

virus)
(+) Strand RNA Viruses
Single Strand RNA Viruses with Two
Identical Strands
Single Strand RNA Viruses with Two
Identical Strands
(-) Strand Non segmented RNA
Viruses

paramyxoviruses,
rhabdoviruses, filoviruses)
(-) Strand Segmented RNA Viruses
Double Stranded RNA Viruses
DNA Viruses
Single-Strand DNA Viruses
Partially Double Stranded DNA
Viruses

hepadnaviruses
References
• http://www.columbia.edu/itc/hs/medical/pathophys/id/2004/lecture
/notes/viral_rep_Hammer.pdf
• http://www.virology.uct.ac.za/teachindex.html