INTRODUCTION TO MEDICAL VIROLOGY

DEFINITION OF A VIRUS

Viruses are organized associations of macromolecules:-
nucleic acid (which carries the blueprint for the replication of progeny virions) contained within a protective shell of protein units .

On its own, a virus may be considered as an inert biochemical complex since it cannot replicate outside of a living cell. Once it has invaded a cell it is able to direct the host cell machinery to synthesize new intact infectious virus particles (virions).
Because viruses are non-motile, they are entirely dependent on external physical factors for chance movement and spread to infect other susceptible cells.

STRUCTURE

Viruses are very small in size (20 - 300 nanometers) and contain either DNA or RNA (not both as in higher forms of life), .

The genome ( DNA or RNA) codes for the few proteins necessary for replication.

• Some proteins are non-structural, eg. nucleic acid polymerases
• and some are structural, ie. they become incorporated and form part of the virion.

Protein building blocks are assembled according to general principles of virus architecture to form a tight "shell" (capsid) inside which the nucleic acid genome lodges for protection. This shell may take the form of a polyhedron (usually icosahedral) or it may be spiral (helical symmetry), or it may be more complex.

Some viruses acquire an outer lipoprotein coat by "budding" through the host cell membranes (nuclear membrane or cytoplasmic membrane) and are thus called enveloped viruses.

All the viral proteins have reactive epitopes which are important for interaction with cellular components during the process of infection and replication. The host's defence mechanisms (cellular and humoral mediated responses) are directed against the viral antigenic epitopes.

View electron micrograph images of various viruses.

Viruses are broadly classified primarily upon the type of genomic nucleic acid, eg. DNA or RNA ,
and then further by the number of strands of nucleic acid (eg. double-stranded DNA, double-stranded RNA or single-stranded RNA, with a positive or negative "sense" of that single strand).
Retroviruses are a special category of RNA viruses that require reverse transcription of their RNA to DNA and then integration of that DNA into the host cell genome before replication can take place. They carry a reverse transcriptase enzyme as part of the virion.

REPLICATION

1. Adsorption

Viruses have reactive sites on their surface which interact with specific receptors on suitable host cells. This is usually a passive reaction (not requiring energy) and the specificity of the reaction between viral protein and host receptor defines and limits the host species as well as the type of cell that is infected (although transfected nucleic acid can by-pass this limitation and extend the host range). Damage to these binding sites (eg. by disinfectants or heat), or blocking by specific antibodies (neutralizing antibodies) can render virions non-infectious.

2. Uptake

After adsorption, the coat of enveloped viruses may fuse with the host cell membrane and release the virus nucleocapsid into the host cytoplasm.
Other viruses may enter the cell by a process of "endocytosis" which involves invagination of the cell membrane to form vesicles in the cell cytoplasm.

3. Uncoating

Refers to the release of the viral genome from its protective capsid to enable the nucleic acid to be transported within the cell and transcribed to form new progeny virions.

4. Genomic activation

Messenger RNA (m-RNA) is transcribed from viral DNA (or formed directly from some RNA viruses) and codes for viral proteins that are translated by the host cell.
" Early" proteins are usually non-structural (eg. DNA or RNA polymerases) and later proteins are structural, eg. capsid proteins, ie. building blocks of the virion.
Nucleic acid replication produces new viral genomes for incorporation into progeny virions.

In general, DNA viruses replicate mainly in the nucleus and RNA viruses mainly in the cytoplasm, but there are exceptions, eg. Pox viruses contain DNA but replicate in the cytoplasm of the host cell.

5. Assembly

Assembly of viral nucleocapsids may take place in the nucleus (eg. herpes virus, adenovirus); in the cytoplasm (eg. polio virus); or at the cell surface, eg. "budding" viruses such as influenza.
Accumulation of virions at sites of assembly may form "inclusions" that are visible in stained cells with the light microscope.

6. Release

Release of new infectious virions is the final stage of replication.
This may occur by by budding from the cell surface, as occurs with many enveloped viruses. In this case capsid proteins and nucleic acid condense directly adjacent to the cell membrane and viral-coded envelope proteins, introduced into the cell membrane, concentrate in the vicinity of capsid aggregates. The membrane surrounding the nucleocapsid then bulges out and becomes "nipped off" to form the new enveloped virion.

Some viruses utilize the cellular secretory pathway to exit the cell. Virus particles enclosed within Golgi-derived vesicles are released to the outside of the cell when the transport vesicle fuses with the cell membrane.

Disintegration or lysis of the infected cell can also result in the release of intact infectious virions.

METHODS OF STUDY OF VIRUSES

Diagnosis of viral infections

Viruses can be studied in a number of direct and indirect ways and all these methods can be applied in a diagnostic situation, ie. is this patient infected with a particular virus? There are two approaches:

1. detection and demonstration of the virus itself; and
2. the study of the host's response to that virus

One of the earliest ways of detecting a virus was by inoculating a susceptible host (laboratory) animal with infectious material derived from a patient or sick animal and then observing that animal for signs of disease. Fertile hens eggs proved useful systems for a number of viruses (especially myxoviruses) and are still used for influenza.
Today, live animals are rarely used as "in vitro" cell cultures have largely replaced them.
(You can see the effects of viruses on cell culture in a separate
illustrated tutorial on CPE)

In recent years "non-cultivable" viruses have been extensively studied by molecular techniques ("genetic engineering").

The structure of different viruses has been elucidated by a range of electron microscopy and x-ray crystallography techniques. Viruses amplified by growth in culture (or in a few special cases, directly from patient specimens without amplification) can be demonstrated by electron microscopy.

Viral antigens can be detected by a wide range of serological techniques utilising polyclonal or monoclonal antibodies. Techniques include precipitation, agglutination, immunofluorescence, ELISA, complement fixation and radio immuno assays. These same techniques, utilising purified viral antigens, can be used to detect specific antibodies to those viruses in the patient's serum. Identification of different classes of antibodies (IgG and IgM) can aid in differentiating between a current infection and immunity.

Some viruses (eg. myxo- and paramyxoviruses, including influenza) have the property of haemagglutination (causing red blood cells to stick together ) which can be used to detect and quantitate the virus (by haemagglutination) or specific antibodies to that virus (haemagglutination inhibition).
Similarly, neutralisation of viral infectivity by antibodies can be used to detect and quantify either virus or specific antibody to that virus.

Modern molecular techniques of both protein chemistry and nucleic acid biochemistry have greatly improved the specificity of virus diagnostic procedures. Methods include:-

• polyacrylamide gel electrophoresis (PAGE) of protein fragments
• western blotting, and identification of specific proteins with labelled probes
• polymerase chain reaction (PCR), to amplify specific segments of viral nucleic acid
• Southern blotting, and DNA hybridisation with labelled probes
• sequencing of portions of the viral genome
• restriction fragment length polymorphisms of viral nucleic acid

Applications

The application of sophisticated molecular technology has enabled the generation of diagnostic assays for viruses that have not yet been visualized or cultured. Hepatitis C virus is the prime example. This RNA virus has never been cultured, but portions of its genome were extracted from blood known to be infectious for hepatitis C. By means of adapted PCR techniques, the nucleotide sequence of the entire viral genome was eventually assembled. Knowing some gene sequences enabled biochemists to synthesise corresponding small portions of proteins (peptides). Some peptides were found to be major antigenic determinants of the virus and these peptides have now been incorporated into commercial ELISA tests designed todetect human antibodies to hepatitis C. The presence of antibodies has been shown to be associated with chronic hepatitis C infection and a high risk of transmitting hepatitis C in blood transfusions. As from 1993, blood transfusion services in South Africa routinely screen all blood donations for hepatitis C antibody.

Molecular biology methods have been used to compare degrees of relatedness of similar organisms and to build phylogenetic trees ("family trees" based on genomic similarities). The ability to detect and sequence portions of a viral genome permits genetic markers of specific sub-strains to be identified. This has led to the new science of genetic epidemiology (ie. disease tracing). For example, health authorities have been able to document the recent spread of the raccoon strain of rabies (as distinct from pre-existing skunk rabies) across the USA . On a global scale, the progression of different genetic strains (genotypes) of polio type 1 (not otherwise distinguishable) can now be followed from one country or continent to another, sometimes replacing pre-existing strains. The strain causing the last recorded polio outbreak in South Africa (Kwazulu, 1988) was previously found in Zimbabwe and was probably imported from there.

STERILISATION AND DISINFECTION

Viruses, especially the enveloped viruses, are generally fairly labile and do not survive too well outside their host cells. However, some (eg. hepatitis B virus) are very resistant to inactivation, and healthcare workers need to take special precautions to avoid transmitting such infections. Means of prevention of the spread of infection, and sterilisation and disinfection of viruses, are very similar to those principles that are applied in bacteriology.

Spread may be by

1) inhalation of aerosolised "droplets";

2) ingestion;

3) direct contact (skin/mucous membrane to skin/mucous membrane), or

4) indirect contact via intermediate "fomites".

Moist heat (autoclaving 120*C x 20 minutes) or dry heat (oven, 180*C for 60 minutes) are effective against all viruses - lesser degrees of heat may inactivate many viruses (eg. simple boiling) but may not reliably inactivate resistant viruses especially if times of exposure are short.
Chemicals: halogens, especially chlorine as hypochlorite are effective against viruses but corrosive on instruments where activated gluteraldehyde ("Cidex") is preferred.
Detergents and lipid solvents inactivate readily the enveloped viruses which need an intact envelope for effective cell adsorption.
Phenolic disinfectants damage proteins and thus inactivate bacteria but do not affect nucleic acids. Phenolics are not recommended for viral disinfection.

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