Viruses and what they do - An overview Wednesday, August 25, 2010

Viruses (Encyclopedia Britannica) ..infectious agents of small size and simple composition that can multiply only in living cells of animals, plants and bacteria. Viruses are obligate parasites that are metabolically inert when they are outside their hosts. They all rely, to varying extents, on the metabolic processes of their hosts to reproduce themselves. The viral diseases we see are due to the effects of this interaction between the virus and its host cell (and/or the host’s response to this interaction). obligate parasites - since viruses rely to varying extents on the host cell’s metabolic processes it has been difficult to develop anti-viral agents that are specifically targeted to viral mechanisms. The development of antiviral agents has therefore lagged behind the development of anti-bacterial agents (antibiotics). However, in the last few decades much progress has been made and we now have reliable anti-virals against many human pathogens such as HIV, herpesviruses and influenza viruses. None have been approved for veterinary use although some antivirals designed for people can be used (off label) quite effectively to control infections in animals (eg. acylovir or Valacyclovir to control equine herpesvirus type 1). The viral diseases we see are due to the effects of this interaction between the virus and the host-cell (and/or the host’s response to this interaction) - cell destruction and/or loss of function. Cell destruction -> inflammation -> cytokines -> clinical signs

Viral Genomes Nucleic Acid Single Stranded DNA Double Stranded Positive “simple composition” - viruses, in their extracellular state are made up of the genome (nucleic acid, either DNA or RNA), protein and, sometimes, the main structure is surrounded by a lipo-protein envelope. The genome can be either single or double stranded. In the case of RNA it can have either a positive (can function as mRNA) or a negative (must be converted to a positive strand mRNA before translation). In some cases the genome is RNA but must be converted to a DNA copy in the infected cell before virus replication can begin. Cells are less able to “proof-read” RNA than DNA. RNA viruses are therefore generally more changeable. This leads to phenomenon such as antigenic drift, change in virulence and tissue specificity. Single Stranded RNA Negative RNA DNA

SS RNA genomes +ve (sense) and -ve (anti-sense) RNA genomes Positive (sense) Negative (anti-sense) AUG GCA CGA met ala arg UAC CGU GCU

Virion Capsid capsomeres adeno herpes capsid “naked” virus particle or Virion envelope capsomeres enveloped Virus or Virion Capsid virus made up of protein coat called capsid + genome. capsid - made up of capsomeres Capsid. Capsids, when they contain a genome are sometimes called nucleocapsids virion - infectious unit some viruses are enveloped - in such cases virion is made up of nucleocapsid + envelope since nucleocapsid, by itself would not be infectious other viruses are naked enveloped viruses are generally more sensitive to inactivation -> spread by close contact. Naked viruses are more difficult to inactivate. Implications for spread and disinfectants

Proteins produced by viruses Structural proteins Non-structural proteins Structural proteins are proteins that make up the virion For all viruses these proteins mediate the initial stages of infection - getting the virus into its host cell - attachment to the cell as well as penetration of the cell membrane. For other viruses the virion acts as a “tool kit” and has proteins for specialized functions such as enzymes needed to begin the program for infection, replicating the genome, modifying the host cell so that it is dedicated to replicating the virus. Non-structural proteins - proteins produced in virus-infected cells which are not a part of the infectious virion. Enzymes needed to make precursors of DNA or RNA, DNA or RNA polymerases, proteins that reprogram the host cell and proteins that either suppress the host’s defenses or protect infected cells from the immune system. Non-structural proteins often determine virulence and pathology. For instance, Obenauer and others recently discovered (Science, Jan 26, 2006) that avian influenza viruses (such as H5N1), which often cause severe disease in people, have a non-structural protein that is different from that of flu viruses that have adapted to humans. This difference may allow the avian viruses to activate signaling pathways in human cells leading to “cytokine storms” which are thought to be responsible for the severe, often fatal disease caused by these viruses. Immune response against non-structural proteins can be used to distinguish between an animal that has been vaccinated with a vaccine containing inactivated virus and one that has been exposed to a disease causing virus (important for diseases such as Foot and Mouth where exposed animals must be culled).

Some viral shapes papillomavirus adenovirus 100 nm parvovirus Viruses are too small to be seen by light microscopy. To visualize viruses the sample is stained with metal containing dyes. The dye enters the crevasses of the structures and is excluded from the tightly packed molecules. An electron beam is then shown on the sample. Electron dense areas (impregnated with dyes) show up dark. Areas the electrons can penetrate show up as white. The electron microscope is often used as the first step to identifying an unknown virus in a sample. It allows one to identify which family a virus in the sample belongs. adenovirus “naked” viruses 100 nm parvovirus 1 nm = 1 millionth of a mm 100 nm = 1 ten thousandth of a mm

Some viral shapes influenzavirus parainfluenza virus herpesvirus 1 nm = 1 millionth of a mm 100 nm = 1 ten thousandth of a mm 100 nm influenzavirus parainfluenza virus Enveloped viruses herpesvirus poxvirus

Taxonomy What is it? On what is it based? Is it important? Do I need to remember all the details? Taxonomy - from tassein (Greek for to classify) or taxis (Gr for order, arrangement) and nomos (law). The science of classifying living things. Viral taxonomy is an attempt to bring order to the large number of viruses discovered in the last eighty years. Based on appearance of virus in EM, structure, type of nucleic acid and genetic homology. It has more practical purposes as well. All members of a group often have similar biological properties. Knowing something about the biological properties of a group sometimes tells you something about how a newly discovered member might behave. For instance, all herpesviruses have the same structure and look the same in electron micrographs. They also all become latent in their hosts and are reactivated periodically. A new member (the Brazilian Mugwump virus) would therefore also be expected to cause recurrent reactivations. Most members of the genus Flavivirus (West Nile Virus) are spread by insects. If a new virus, that has physical and chemical properties of flaviviruses, is discovered the chances are that it is spread by insects as well. Based on - viral genome, morphology, biological properties, serological relationships International Committee on Taxonomy of Viruses

Viruses with ss DNA genomes porcine circovirus Circoviridae canine parvovirus-2 Parvoviridae feline panleukopenia virus porcine parvovirus (SMEDI)

Viruses with ds DNA genomes papillomaviruses Papovaviridae adenoviruses Adenoviridae equine herpesviruses -1,4 bovine herpesvirus-1,2 Herpesviridae porcine cytomegalovirus malignant catarrhal fever virus Poxviridae poxviruses African swine fever virus african swine fever virus

Viruses with ds RNA genomes rotaviruses Reoviridae bluetongue virus african horse sickness Birnaviridae infectious bursal disease (chickens) infectious pancreatic necrosis (salmonid fish)

Viruses with +ve RNA genomes foot and mouth disease virus Picornaviridae porcine enteroviruses Caliciviridae feline calicivirus Coronaviridae coronaviruses Arteriviridae equine arterivirus, PRRS Flaviviridae flaviviruses (WNV) pestiviruses (BVD) Togaviridae equine encephalitis viruses

Viruses with -ve RNA genomes influenzaviruses Orthomyxoviridae parainfluenza virus canine distemper virus Paramyxoviridae Hendra, Nipah viruses respiratory syncytial virus Rhabdoviridae rabies virus vesicular stomatitis virus Filoviridae Ebola virus Bunyaviridae Haantan virus

Viruses with reverse transcriptase feline leukemia virus Retroviridae feline, bovine immunodeficiency viruses bovine, avian leukosis viruses caprine arthritis-encephalitis virus Hepadnaviridae

Antigenic classification: Serotypes and Groups How new serotypes arise – gradual changes in external proteins due to pressure by neutralizing antibodies

no selective pressure on internal proteins external viral proteins antibodies to all viral proteins antibodies to external proteins neutralize virus

selective pressure on external viral proteins antibodies to all viral proteins antibodies to external proteins neutralize virus selective pressure forces selection of virions with slightly different external proteins

virus, including changed virus passed on to new host selective pressure on external viral proteins antibodies to all viral proteins antibodies to external proteins neutralize virus selective pressure forces slight change in external proteins

process repeated, over time….. neutralizes neutralizes neutralizes neutralizes does not neutralize serum from original cat NOTE: Only external proteins change. Internal proteins do not change

process repeated, over time same serotype new serotype neutralizes neutralizes neutralizes neutralizes does not neutralize serum from original cat NOTE: Only external proteins change. Internal proteins do not change

Serotype - all isolates of a virus that can be neutralized by a common antiserum are said to belong to the same serotype. …..because of changes in external protein (internal proteins do not change) external proteins are called TYPE SPECIFIC antigens internal proteins are called GROUP SPECIFIC antigens

process repeated, over time same serotype new serotype different serotypes same group

Groups, types (sero-types), isolates and ‘strains’ Type -A specific antigen Type -A Type - B Group specific antigen isolate Type - C isolate - every time a laboratory isolates and grows a virus and does some basic characterization (identification) that virus is referred to as the isolate. If an isolate is characterized further (serological and biological properties) and is used in experiments it is sometimes referred to as a “strain”. The term “serological property” refers to the antigenic characterization of a virus. When a virus infects an animal the animal eventually produces antibodies against all the viral proteins - proteins that lie on the outside of the virion and proteins that are inside the nucleocapsid. Proteins on the outside of the virion allow it to attach and enter host cells and antibody directed against these proteins can neutralize the infectivity of the virion by blocking this process. Serotype - all isolates of a virus that can be neutralized by a common antiserum are said to belong to the same serotype. Since “neutralization” involves the external proteins of a virion the serotype is dictated by these proteins. For instance, feline leukemia viruses (FeLV) come in one of three serotypes - A, B and C. All FeLv isolates that can be neutralized by Anti-A serum are said to belong to serotype A. Isolates that are not neutralized by Anti-A serum belong to either serotype B or C, and so on. While the external proteins of viruses of different serotypes may be antigenically different, the proteins on the inside may be antigenically similar. For instance the inner proteins of FeLV of all three serotypes are similar and react in immunological tests to antibodies against the inner protein. All viruses that react to antibodies directed against the inner protein are said to belong to the same group. The inner protein is called the group specific antigen while the external proteins are called the type-specific antigens. Group Type - C specific antigen

Group and type specific antigens “naked” virus (eg FMDV) enveloped virus (eg influenza, FeLV) group specific antigen

Serotypes and neutralizing antibody (eg. FMDV) C O SAT1 SAT2 SAT3 Asia serotypes of FMD virus receptor implications - vaccination with one serotype or prior infection with one serotype does not protect against infection by another serotype. receptor binding protein on viral surface antibodies against receptor binding protein of serotype A will neutralize viruses of serotype A but not of serotype C

example - influenza type specific antigen serotype H1 serotype H5 group specific antigen test based on group specific antigen will detect all three vaccination against one serotype will not protect against others

Infection of a cell Stage Biological implications Host defenses Drug intervention Steps in virus infection 1) attachment - a specific interaction between a protein present on the surface of the virion (capsid, or for enveloped viruses - in the envelope) and a molecule on the surface of the host cell. Eg - membrane associated lactase on intestinal epithelial cells are receptors for rotaviruses,CD4 and coreceptor CCR5 are receptors for HIV, sialic acid for influenza viruses. Because the interaction between the virus and the host cell is specific some viruses can only infect particular species or organs (host or tissue specificity). Viruses must change viral protein to alter host-range. Conversely, genetic mutations in host protein can lead to resistance. Eg CCR5D32 mutation in northern European populations and resistance to HIV (see notes for slide - Genetic Resistance). Incidentally the CCD5D32 mutation may not be without cost - a report by Glass et al. in the Jan 23, 2006 issue of the Jn of Exp Med (203:35) report that the mutation is over represented in patients with severe or fatal West Nile Virus disease (1% in normal population vs 4to 8%). The deletion may also make individual more susceptible to severe disease by related viruses (for recent review see Klein, J Inf Dis. 2008, 197:183). Attachment is one step which is prevented in the immune animal. Antibodies directed against viral proteins that mediate attachment neutralize viral infectivity. 2) entry - The act of virus binding to its receptor may trigger the cell to take up the virus. Eg. The binding of Coxsackie B virus to decay accelerating factor (DAF) on epithelial surfaces causes actin rearrangement and stimulates the transfer of the virus into the cell in caveolin vesicles. 3) Uncoating - The viral genome is released from the nucleocapsid. For some viruses this process can be interrupted (influenza and amantadine). 4) gene expression - The viral genome is expressed to make non-structural proteins and structural proteins. By exploiting differences between cellular and viral enzymes drugs can be designed to inhibit viruses that make their own enzymes eg. acyclovir (or valacyclovir) is converted to the prodrug - acyclovir-monophosphate by herpesvirus thymidine kinase but the cellular enzyme does not recognize ACV as a substrate. Viral proteins are expressed on the surface of infected cells - as 5) replication of the viral genome. This step can also be interrupted by anti-viral drugs eg phosphonoformate - herpesviruses. 6)packaging of viral genomes in nucleocapsids. 7) release from the cell. This can be by budding from the cell surface for enveloped viruses or by death of the cell for un-enveloped viruses. Neuraminidase inhibitors such as Oseltamivir (Tamiflu) and Zanamivir (Relenza) inhibit the release of influenza A and B viruses from the cell.

Distribution of the CCR532 mutation in human populations from PLoS Biology, Nov 2005

Errors in replication lead to “quasispecies” persistent infection Due to the error-prone nature of viral enzymes that replicate the genomes of RNA viruses, a population of such viruses is not represented by a single genotype but is a collection of related sequences and can be called a quasi-species. The variant viruses collectively contribute to the characteristics of the population. Although variations or mutations in individual genomic RNA molecules may sometimes be detrimental to virus multiplication, when cells are simultaneously infected with several different mutants they complement each other. Vignuzzi and others recently (Nature Jan 2006) tested the benefits for the virus of this by replacing the RNA polymerase of poliovirus, which normally makes many mistakes, with an enzyme that replicates RNA with high fidelity. They found that while the virus with the high fidelity polymerase replicated in cultured cells as well as the wild-type virus, in animals it rapidly lost its neurovirulence. The neurovirulence could be restored if the mutant was allowed to replicate in the presence of chemical mutagens, which compensated for the fidelity of the mutant polymerase. mixture of variant viruses (quasispecies)

inclusion bodies Inclusion bodies are abnormal structures within cells that are the result of virus infection. For many families of viruses the staining characteristics and appearance of inclusion bodies are characteristic and can be used as a way of identifying the virus. In general DNA viruses lead to nuclear inclusion bodies while RNA viruses cause cytoplasmic bodies. There are exceptions to this - paramyxoviruses (canine distemper) produce both nuclear and cytoplasmic inclusion bodies while cytomegalovirus, a member of the herpesvirus group and a DNA virus can produce cytoplasmic bodies. Some inclusion bodies are acidophilic or eosinophilic (stain pink with eosin), others are basophilic (stain blue with hematoxylin). Some, such as those of rabies virus represent aggregates of replicating virus. Others represent abnormal cellular processes brought about as a result of virus infection. The intranuclear bodies in herpesvirus infected cells are characteristic. They look like a dark mass in the cell nucleus surrounded by a clear zone. They are a fixation artifact caused by retraction of chromatin in the nucleus which leaves a clear zone between the shrunk chromatin and the nuclear membrane. Is this cell infected with an RNA or a DNA virus?

Release of virus or by budding (without Release by lysis of cell death of cell, non-cytopathic) Release by lysis of cell (cytopathic)