An Introduction To Virology

Importance Viruses are important to vets as they are a cause of disease in domestic, farmed and wild animals. Disease in these animals can cause concern in terms of welfare, conservation and economic loss. Further importance is given by both the possibility and reality of zoonotic diseases as many emerging diseases (both human and animal) initiate from animal disease. As viruses can mutate extremely quickly there is a huge threat to humans from zoonotic diseases emerging which we do not fully understand how to treat and so viral patterns and processes are closely studied to provide research data. The molecular biology of viruses is important to vets as understanding the structure, replication and genetics can greatly assist in the design and interpretation of diagnostic tests, and increase our knowledge of the underlying science of therapies and prophylactic regimes. Another important aspect is being able to educate clients with regards to viral outbreaks, for instance different virions can survive for different lengths of time outside a host cell, this is important in relation to disinfectants and the restocking time of a building. research scientists are starting to be able to manipulate viruses for our own purposes, hijacking their natural processes to be of benefit rather than harm (gene therapy).

Key Concepts The structural and nonstructural features of viruses define the cells and hosts they can infect and influence the diagnosis and control of viral disease. Viruses rely on hosts for their replication- this is what causes the symptoms of the disease. Viruses are a quasispecies, they are clouds of closely related genetic material with a common consensus sequence, however they are primed for change. Viruses are dynamic, new diseases are always emerging and so it is important to be vigilant. Vaccines must be constantly reviewed to confirm that they still control the new strains of a virus.

Intended learning objectives Describe the components of a virion, their role in the life cycle and their interaction with the host. Provide an overview of virus replication and be able to outline replication of a specific virus Describe mechanisms by which viral variants arise and selection pressures which favour the emergence of new virus strains

What are Viruses Viruses are sub microbial particles made up of nucleic acid contained within a protein shell. They can infect bacteria and plants, and animals. They are significantly different to bacteria in a number of fundamental ways: they cannot be treated with antibiotics (antibiotics administered for FHV but only to counter the secondary bacterial infection), they cannot replicate outside of a host cell and they are a factor of 10 -1000 smaller. A recent development in the study of viruses has discovered Pandora Viruses. These are huge viruses which contain upward of 2000 genes (2500kbps) and infect amoebas. They also have the ability to influence the reproduction of other viruses within the same host.

Virion structure The innermost layer of a virion is the genetic material which can be made of either RNA (single or double stranded) or DNA. Nucleic acids can either be positive sense or negative sense or mixed. Positive sense means it is the same orientation as mRNA. The genetic material in some virions is segmented while in others it is a continuous unbroken piece. The viruses which affect mammals and birds typically have between 2 and 300 kbps however larger genomes are currently being discovered. The genes code for virus proteins, control protein production and contains the elements necessary for the replication and genome packaging. The genetic material is encased in the protein shell known as the capsid (made of structural proteins). Together the genome and capsid are known as the nucleocapsid. The nucleocapsid facilitates entry into the host cells and delivery of the nucleic acid to the necessary location. All virus strains have in common the symmetry of their nucleocapsids. One of 2 symmetrical patterns is always shows: icosahedral or helical. An icosahedral symmetrical nucleocapsid has 20 faces, each a perfect equilateral triangle. As such is displays 2,3 and 5 fold rotational symmetry. This structure encloses the maximum volume for the minimum surface area. Helical nucleocapsids are coiled like a phone cord. They are typically seen in simple viruses with small genomes to provide protection without the need to encode for multiple capsid proteins. All animal viruses with a helical symmetry are enveloped. symmetry in the capsid exists to allow the virus genome to remain extremely small, while being economic with the amount of work it must do to protect itself. A third optional layer called the envelope is sometimes present, this is made of a lipid bilayer acquired from (different places within) the host cell along with envelope glycoproteins which act as anti receptors. The type of glycoprotein present affects the type of cell that the virion can enter as it interacts with cell receptors and so affects the symptoms exhibited. the type of cells a virus can enter is known as its tropism. Glycoproteins also act as targets for the immune system as antibodies bind to them, some viruses however have the ability to trick the immune system into leaving it alone. The diagnosis of viral disease relies heavily on these glycoproteins. The antigenic nature of viruses is made up on internal and external components. this corresponds to the humoral and cell mediated immune responses the body can exhibit.

Stability of viruses Non-enveloped viruses tend to be more hardy than their enveloped counterparts, for example foot and mouth is much hardier than influenza. Different viruses have dramatically different abilities to survive outside and inside of a host. They are all sensitive to pH, desiccation, lipid solvents, and detergents, however different viruses can withstand different ranges and lengths of exposure.

Virus classification Virus taxonomy is determined by the international committee on the taxonomy of viruses. Historically they have been categorized by their chemical characteristics, genome type, replication strategy, diseases, vectors, geographical distribution and their host species, however now their nucleotide sequence is used to determine their nearest relatives. The main class is the family (name ends with viridae), followed by subfamily (ending in virinae), followed by genus (virus) and finally strain (virus). For example herpesviridae alphaherpesvirinae varicelovirus equid herpesvirus 1

Outcomes of viral infection When a virion interacts with a host cell there can be one of three outcomes. The first possibility is a productive interaction, where the host cells are permissive. this results in replication of the virion and its progeny being released (the cell may or may not be killed as a result). The other extreme is an abortive interaction, where the cells are nonpermissive. This would result in no further virion particles being released. The middle ground and last option is that there may be a restrictive or latent outcome, the viral genome is partially expressed leading to intermittent production of the virus. This can result in diseases lying dormant for several years before causing clinical signs at times of immunosuppression or stress. Latent infections are associated with causing cancers and are extremely hard to diagnose and treat. To continue their genetic line a successfully a virion must infect a permissive host cell. the tropism of the virus determines what kind of symptoms are exhibited, and can be known as its pathogenesis.

Attachment The virion attaches to the host cell via a lock and key mechanism. The glycoprotein anti receptors bind to complementary receptors on the cell surface membrane. after the initial attachment there is then a stabilisation process which locks the virion into place.

Entry/penetration the virion can enter the host cell by 3 methods. the first is receptor mediated endocytosis as happens with the influenza virus. The second is membrane fusion, the envelope of the virion fuses with the phospholipid bilayer leaving the nucleocapsid to travel inside the cell. The final method is translocation. A vesicle is formed around the virion by the phospholipid bilayer, transporting it deeper inside the cell.

Uncoating Once inside the host cell the virus must shed its capsid to expose its genetic material. In some instances this occurs in the cytoplasm and in others the nucleus, depending on the site of replication. Some viruses need only partial uncoating before they can be transcribed.

Genome replication The production of viral mRNAs and genomic copies requires polymerases. Some viruses use the polymerases already present in the host cell's nucleus, these are DNA dependant DNA polymerases and DNA dependant RNA polymerases. A virus can only make use of these if it's genome is DNA based and the host cell is currently undergoing replication (this is the only time theses enzymes are present in large enough numbers). There are many advantages to this method, the virus's genome does not need to contain the genes to produce polymerases in huge quantities and there is no associated energy cost or the virus. Some viruses however have an RNA genome, these are replicated in the cytoplasm as they must first produce their own RNA dependant polymerases such as reverse transcri ptase for retroviruses. Virus coded polymerases are much less accurate as they lack the ability to properly proof read the polymer they are producing. this results in a larger number of mistakes than in host cell polymerization and so more mutation. Most viruses which replicate in the nucleus use host polymerases as they are DNA based while most RNA based viruses replicate in the cytoplasm and produce their own polymerases (exceptions are pox and flu).

Assembly Small simple viruses without envelopes assemble themselves by an automated process triggered by high concentrations of the components associating with the produced nucleic acid. larger more complex viruses however require scaffolding proteins to obtain their shape, then the viral proteins can attach themselves.

Exit and maturation There are 3 methods by which the newly produced virions can leave the host cell. Firstly they can simply accumulate in huge numbers and literally burst the cell, killing it. Secondly they can bud off of the cell gathering an envelope and glycoproteins as they go or they can leave by exocytosis in a vesicle created by the host cell.

Effects of viruses on cells There are several mechanisms by which the virus can damage the host cell. While producing viral nucleic acid, the host is not producing its own DNA, not transcribing it and not producing any proteins for itself. Upon leaving the host cell the virus can damage cell membranes and induce apoptosis, killing the cell. The worst affect the virus can have is altering the gene expression and cellular signalling of the host cell inducing cytopathic changes and creating the possibility of the cell becoming cancerous.

Routes of infection A virus must gain entry to a host before it can hope to replicate. They can gain entry through any barrier with the environment including the respiratory tract, ingestion, skin trauma, the urogenital tract and the conjunctiva. viruses have specific hosts they are best at infecting and specific tissues within that host which are more susceptible. this gives rise to the viral tropism. The infection caused can be either local or systemic depending on whether or not the virions are contained within a tissue or spread into the blood/lymph/neuronal system.

Host defences The host defends itself by a number of physical and chemical barriers. The biggest physical barrier is the skin, but also included are the mucus, hair and stomach acid. Most mature animals have an accumulated resistance to most common viruses within their environment through exposure to them. Some of this immunity is passed on via colostrum to young and some immunity is innate (genetic). the extent to which the host is immune coupled with the virus characteristics (virulence/load) determine the outcome of the infection.

Effects on host The main effect on the host is caused through cell/tissue destruction which can be direct or indirect. direct cell damage is caused by cells bursting or initiating apoptosis, while indirect damage can be caused by secondary infections for example from bacteria A secondary effect upon the host can be from virus induced immunopathology where an immune complex formation results in permanent damage to the host. Viral oncogenesis is another possibility, the virus changes the host DNA causing the expression of cancerous genes for example bovine papillomavirus. In short any viral infection has the potential to lead to secondary infection by immune suppressing the host leading to it being easier for other microbes to take hold. The most dangerous type of infected animals is one with an inapparent infection as the effects are undetectable, but the virus can still be spread to other animals. A subclinical animal with still be effected by becoming less producing, being less fertile and less productive but there will be no real way to ascertain the cause. the infection seen in a herd is often only the tip of the iceberg.

Spread of viruses The tropism of a virus determines the source of spread to other animals, a respiratory virus is more likely to be spread by coughing or sneezing than by fecal contact.

Detection of viruses Viruses can be detected by a number of methods, the virus particles themselves may be detected or it may be the antigens, presence of nucleic acid or antibodies which are raised which are detected.

Treatment Treatment of viruses is difficult , you can treat the symptoms with husbandry (fluids, comfort, nutrition) or by antibiotics to treat any secondary bacterial infections. Another line of attack is to inhibit virus replication and its effects. This can be done by using antiviral drugs which can be general or virus specific (tamiflu), immune system modulators, post exposure vaccines (rabies) or passive immunisation(immunoglobulin).

Prevention To prevent viral outbreaks you should vaccinate the target and/or reservoir species if possible. Otherwise you are left with only husbandry methods such as isolation, eradication of reservoirs, legislation and disease surveillance.

Genetic change During replication in viruses genetic changes occur. There is a balance between stability (maintenance of the virus genotype from generation to generation) and variation (emergence of new viral strains with new biological properties as a consequence of mutation and selection). Genetic change can occur in 2 ways, spontaneous mutation and genetic exchange between viruses (and rarely between virus and host cell).

Mutation and selection Mutation is a change in the nucleotide sequence of a genome caused by an error while copying the parental template. The error could be a point mutation in either DNA or RNA, the chances of a mutation happening in DNA are much lower (10-10 to 10-11) while the chances of a mutation while copying RNA are more like 10-3 to 10-4. This happens because for RNA viruses, the host cell's own polymerases cannot be used to replicate the genome and so new ones must be made, these have less ability for proof reading and so more mistakes slip through. Point mutations such as these result in very small changes (insertions/deletions/rearrangements affecting protein coding or regulatory regions) to the genome (the majority of which are actually lethal), larger changes occur as an artefact of genetic exchange between 2 viruses, or a virus and a cell (rare), this happens though recombination/reassortment. Successful point mutations result in a cloud of quasispecies forming, all of which share a consensus sequence. If you took the genome of the original infecting virus, then compared it to the new mutated viruses emerging from the host, the average of each base would still match the original sequence, but subtle differences overall would be present. The Mechanism for gene transfer requires 2 viruses of different genotypes to infect the same host cell, the 2 genotypes being reproduced can become confused, leading to a small number of recombinant new genotypes. These new genotypes emerge from the host with all the other viruses of both genomes which entered, they are significantly in the minority. One of the methods by which this occurs is recombination, this just means a swapping over of homologous sequences, the second is reassortment, where whole segments of the genome are swapped (can only occur in segmented DNA such as influenza). There are 4 possible outcomes of any kind of mutation. They can be lethal, silent, provide a persistent disadvantage to survival or a persistent advantage. Where a significant advantage is created, the mutated strains become established as selection pressure will favour it. Changing environmental pressures effect which strains become established, these can include the immunity of the target population, antigenic variation, a new host, drug use and altered population dynamics.

Influenza A Influenza A is part of the orthomyxovirus family. It is an enveloped protein with glycoproteins of the types H (hemagglutinin) and N (neuraminidase). The genome is divided into 8 linear segments, H and N are coded for by different segments. Hemagglutinin is the major antigen for neutralising antibodies and binding to host receptors while neuraminidase is responsible for the release of progeny viruses from the cell surface. Changes to H and N can occur by antigenic drift and shift. Antigenic drift is a spontaneous mutation in the surface antigens in response to a particular selection pressure, it is the reason behind the 'flu vaccine needing to be updated every year to protect against new strains of seasonal flu. Antigenic shift it the exchange of genome segments between 2 influenza viruses and has the potential to produce a major antigenic change. Antigenic shift is responsible for new strains of pandemic flu rather than seasonal flu, for instance it could be responsible for an avian flu being able to infect humans. Man is "naive" of H% antigens and so has no resistance to them, this means that any 'flu virus with H5 can be a potential pandemic. Pigs can become infected with Swine, Avian and Human flu and so act as a mixing vessel for new strains. Avian 'flu has a 60% mortality rate in humans, however it does not spread well between humans. Asia is especially vulnerable to animal transmitted 'flu as they have lots of small holdings and live animal markets.