VIRUS REPLICATION E-mail Web site PETER H. RUSSELL, BVSc, PhD, FRCPath, MRCVS Department of Pathology and Infectious Diseases, The Royal Veterinary College, Royal College Street, London NW1 OTU. E-mail Web site This lecture is the part of THE VIROLOGY COURSE, 2000 (The aim of this foundation course is to link the structure, diagnosis and control of the different families of veterinary viruses in an up-to-date and well-paced manner). COURSE OBJECTIVES - hyperlinks to course.htm BOOK LIST - hyperlink to book.htm USEFUL WEBSITES- hyperlink to websites.htm INDEX OF ABBREVIATIONS - hyperlink to index.htm

Objectives Students should be able to: describe in outline how to cultivate viruses list the stages of virus replication in a host cell with reference to how these differ between RNA and DNA viruses. explain the effects that viruses have on cells.

Viruses grow and kill cells, this results in cell death in vitro and necrotic lesions in-vivo

HOSTS FOR VIRUS CULTIVATION 1) EXPERIMENTAL ANIMALS, 4 uses (a) Diagnosis: only now used if cell cultures do not work e.g. African Horse Sickness virus in suckling mice. (b) Pathogenesis studies: to prove that a particular virus causes a specific disease, Koch’s postulates, and to establish which tissues are richest in virus for diagnosis. (c) Vaccine development: to prove efficacy. Laboratory animals may be used if a lab. animal model is available and appropriate, before trials in the target species. (d) Serology: horses, rabbits & goats used to provide virus-specific antibodies. Mice for monoclonal antibodies.

2) FERTILE HEN’S EGGS FERTILE HEN’S EGGS, used at 10 days, half way through embyonation. Some mammalian viruses, e.g. influenza viruses, as well as chickem viruses grow in them. Viruses from the mucosa grow inside the allantoic cavity eg influenza. Viruses from the skin grow on its surface eg pox.

3) CELL CULTURES CELL CULTURES, widely used since the advent of antibiotics and sterile air cabinets. Not all viruses grow in all cell lines although viruses can often grow in cells from several different species.

Production of cell cultures: The tissue, eg. kidney, testis, thyroid, is made into a single cell suspension by limited digestion with a proteolytic enzyme such as trypsin. The single cells are grown in glass or plastic bottles in buffered medium consisting of physiologic salts, an energy source (eg glucose), amino acids, antibiotics and usually animal serum. The cells adhere to glass & plastic surfaces, divide and form a confluent monolayers. After several days these cells must be split into 4-12 flasks by disrupting the monolayer back into single cells using trypsin with EDTA. This is called "passaging the cells". Cultures after the first passage from a tissue are called primary cell cultures, at the second pass, secondary cell cultures.

Continuous cell lines. Most cell cultures die after about 10 passages, some change into continuous cell lines. These are used whenever possible in virology but they tend to be the dedifferentated fast-growing cells with either epitheloid or fibroblastic conformation. Certain fussy viruses need primary cells eg african swine fever in porcine macrphages.

VIRUS ISOLATION Bacteria-free samples, eg a nasal swab or diseased spleen, combined with antibiotics, are inoculated into animals, eggs or cells, which are known to support the suspected virus. These examined for cell death or lesions in comparison to uninoculated controls and positive controls several days later. Second and third serial passages are often required to increase the level of virus eg to recover a low level of virus from an equine influenza nasal swab in eggs.

GROWTH: One virus enters a susceptible cell and produces 100-3000 progeny overnight Replication in vitro results in a cytopathic effect which spreads until all the cells in the vessel are dead Replication in-vivo results in necrotic lesions whose location depend on virus tropism. Lesions are then limited by nonspecifc immunity eg interferon

The stages in virus infection are : 1) Attachment 2) Penetration and uncoating 3) Formation of viral messenger RNA 4) Formation of new genomes 5) Formation of new protein 6) Assembly. 7) Release 8) Latency. 1) Attachment: the virus meets and then binds to a cell surface receptor, this explains why viruses often only infect certain species or tissues eg liver compared to neurones 2) Penetration and uncoating: the virus is enveloped by an endosome and is degraded and its nucleic acid escapes into the cytoplasm, no infectious virus remains 3) Formation of messenger RNA: Viruses have their own polymerase proteins which force the cell to make a huge amount of viral mRNA and also nucleic acid. This may involve steps which the host cell cannot do e.g. RNA to mRNA or RNA to DNA. DNA viruses make mRNA in the cell nucleus. RNA virus make mRNA in the cytoplasm. Some RNA viruses have -RNA genomes. Retroviridae are the exception. They transcribe double-stranded DNA which is then ligated by a viral integrase into the host cell chromosome as a chain of viral genes

The stages in virus infection are : 1) Attachment 2) Penetration and uncoating 3) Formation of viral messenger RNA 4) Formation of new genomes 5) Formation of new protein 6) Assembly. 7) Release 8) Latency. 4) Formation of new genomes which may be aided by early proteins which are either viral polymerases, see above, or promoting cell division to provide new cells for the madly dividing viruses. 5) Formation of new protein is always on the host cell ribosomes. Early proteins are viral enzymes e.g. polymerases (see above) or viral growth factors which stimulate cell division to provide new host cells for virus. Late proteins are the structural proteins e.g. capsids and spikes 6) Assembly. Nucleocapsid of DNA viruses are assembled the nucleus. Nucleocapsid of RNA viruses are assembled the nucleus. The notable exception is the DNA poxviruses which assemble the cytoplasm. Nucelocapsid 'factories' can be seen as inclusion bodies by light microscopy. Glycoprotein spikes insert into the cell-surface plasma membrane. 7) Release. Release of many particles at once when the cell dies and then bursts. Or each enveloped virus particle gradually buds from the cell surface. 8) Latency - certain viruses, the herpes and retro's, form ± DNA during replication and this can remain latent in the nucleus for years but then become reactivated to make new particles during immunosuppression. The notable exception are the DNA poxviruses which do all their replicate in the cytoplasm

THE EFFECTS THAT VIRUSES HAVE ON CELLS In diagnosis a cytopathic effect (cpe) indicates the presence of a virus but virus-specific detection e.g. by immunofluorescence or neutralisation is necessary to prove which virus is present Certain viruses have no cytopathic effect on cells eg the leukaemia viruses and are detected by electron microscopy (very laborious) or immunostaining.

Cytopathic effects: cell lysis syncytia transformation. Cell lysis. Most viruses take over the cell's replicative machinery. The cell dies after 1-2 days when it contains masses of virus particles and is unable to repair itself. The masses of virus particles appear as inclusion bodies by staining. A common problem in diagnosis is that the tissue homogenate is toxic . If the toxicity is of viral origin the medium above the dying cells can usually be diluted at least one millionfold and then kill new cell cultures

Syncytia. Viruses with a second envelope spike called fusion (F) glycoprotein as well as an attachment spike, e.g. canine distemper virus, make the plasma membranes of adjacent cells fuse to each other and to the lipid bilayer of the virus. These cells can eventually become multinucleate syncytia with about 30 nucleii before the replicating virus kills them. Fusion is also a means of virus spreading between cells. Antibody to fusion protein determines the success of vaccination

Transformation The cells stop being flat but round up and start dividing uncontrollably to become piles of round cells eg after infection by feline sarcoma virus. Similar cells can also be cultured from some virus-induced tumours e.g. Feline leukaemia virus, Mareks disease of chickens. The virus does not kill the cells. 3 different mechanisms are involved in viral oncogenesis: i)DNA viruses produce early proteins and gene promoters which bind to DNA and stimulate uncontolled cell division without virus particle production. eg bovine papilloma virus and Marek’s disease. ii)Leukaemia viruses turn on host cellular proto-oncogenes which are adjacent to where their proviral DNA is integrated (termed insertional mutagenesis). This is a rare event and may never happen in the lifetime of the animal. (iii)The sarcoma viruses have an oncogene in their own genome. They cause transformation in-vitro and rapid fibrosarcomas in-vivo and are very rare.

VIRUS INFECTIVITY Infectivity is relevant to diagnosis to find out which tissues contain most virus and therefore are best for sampling. 100 infectious units of virus are often used to tests for neutralising antibody in a serum sample. Method, TCID50 and pfu The virus suspension is diluted out in ten-fold dilutions steps (log10) because a suspension may contain 108 infectious virions per 0.1ml. Replicates of each dilution are inoculated into cells in microwells or hens eggs. The infectivity is the dilution at which 50% of the cultures have become infected within a week. This is called the tissue culture infectious dose 50 (TCID50 per 0.1ml.) similar to the LD50 in pharmacology. In a plaque assay virus growth is confined to the area of infection by a semi-solid overlay eg agar in the medium. If there were 50 plaques at a dilution of 1:10,000 using 0.1ml of inoculum the number of plaque forming units, (pfu) is 5 x 105 plaques per 0.1ml of inoculum and the virus could be diluted 1/5000 for a virus neutralisation test..

PRESERVATION OF VIRUS INFECTIVITY Viruses usually remain infectious for several weeks at 4degC providing they are not contaminated with bacteria or fungi. To to retain stocks they are frozen at -70degC or freeze-dried. The failure to maintain the cold chain is a common reason for vaccine breakdowns in the tropics. The enveloped viruses with helical nucleocapsids are the most labile, the poxviruses and small icosahedral viruses are best able to survive in dust or fomites (greek for inanimate wooden objects)

Summary Viruses grow in susceptible hosts eg cells, eggs or animals Viruses utilise the host cells and its enzymes to make several hundred particles overnight. Certain viruses have their own enzymes because they undergo steps which the host cell does not, eg RNA to RNA All viruses must attach to host cell receptors. This often determines their host range. After entry viruses uncoat to release their nucleic acid and are not infectious at this stage. The internal virus proteins are assembled in discrete areas which appear as inclusion bodies. These are usually in the nucleus for DNA viruses and in the cytoplasm for RNA viruses. Viruses leave the cell when it dies although some enveloped viruses bud-off living cells Certain viruses become latent and integrated in-vivo and reactivate later or cause tumours. Virus replication results in cell death with pyknosis or rounding, syncytium formation then a slower death, no effect, or transformation Virus infectivity can be measured by TCID50 or pfu Samples must be kept cold and sterile before assay