Virology Lecture 1 POINTLESS Lecture 2 Why study viruses viruses cause disease Viruses are not under control

like bacterial disease are (although antibiotic resistance). Flu such as Avian and swine flu have caused deadly diseases pandemics Frequency of HIV and smoking are most important causes of premature human fatalities SARS also massive There is nto only a cost to life but also an economic cost Viruses can be used to treat disease vaccines Viruses can also be utilized as agents for gene delivery Cancer killing viruses (oncolytic therapy) selectively target cancer cells Immunotherapy generating an immune response against certain antigens such as HIV by presentation of viral vector. phage therapy overcoming antibiotic resistance of bacteria Viruses are unique Viruses are abundant biological forms Least characterised component of ecosphere. e.g. ocean is full of viruses Easier to sequence with the development of shotgun sequencing. Origins of virology may have coevolved throughout our evolution. Those that are particular virolent such as smallpox needed large populations to establish (otherwise lost). Changes in civilisation and society may have cause a rise in viruses (species jump e.g. bird flu) No fossil record of viruses but club foot (polio) and tissue evidence (smallpox) found in mummys Koch and Pasteur proposed Germ theory Koch s postulates - Agent must be regularly associated with disease - Agent must be isolated from diseased individual and grown in culture - disease must be reproduced when pure agent reintroduced - same agent must be re-isolated from experimentally infected host Viruses did not follow koch s postulates because they could not be grown and amplified in culture. Chamberland developed a porcelain filter (0.1uM) capable of separating bacteria from culture medium. Dmitri ivanovski (1892) studied tobacco mosaic virus infected extracts could be passed through filter and caused disease but non viable without host Toxin? diluted and reinfected, no change in effect. Originally viruses thought to be one unique disease but other diseases such as foot and mouth and yellow fever soon found to have same characteristics. Virus definition - small, submicroscopic bags of genes - infectious, obligate - large protein macromolecule encapsulated with a protein cota containing a DNA/RNA genome

- within host cell, the viral genome directs the synthesis - progeny infectious virus particles, virions are formed de novo - the progeny virion assembled during the infectious cycle is the vehicle of transmission Virus look like Sub microscopic (some can be seen however), immunofluorescence or EM can see viruses. viruses are small and simple 30-300nm. Mimivirus has large viral genome 1mB. Lots of viruses have ORF s that are not conserved (60% of ORFs) Viruses are not neccesarily parasites symbiotic relationships exist Grass in yellowstone Together with fungus, can survive extreme temperatures but alone cannot. fungus however must be infected with a certain virus Viral genomes direct the synthesis of new virions T4 lands on surface and inserts itself, pseudorabies enveloped by plasma membrane. Progeny of infectious cycle transmit the genome archael virus ATV undergoes substantial morphogenic changes in extracellular environment at high temperature Mycorviruses infect fungi only infected when fuse with another fungus (no extracellular phase) POlydnavirus integrates into host genome transmitted only vertically (progeny) in host wasp virus pass viral genome but this does not replicate and exists as pro virus virus exists in wasp genome Viruses are non cellular, do not divide, are formed inside a host , do not generate energy, direct their replication from host cell. Lecture 3 Classification of organisms based on initial structural similarities, developmental similarities and finally on genomic sequences. Viruses hard to apply classification on due to variety of structures. Relatedness between close viruses possible, but RNA vs DNA? Possible that viruses have multiple origins Recombination and reassortment in viruses and high rates of mutation. Different selective pressures on different viruses. Inconsistency in naming viruses based on almost anything. (discoverer, replication, shape etc.) Different viruses cause very different symptoms (HSV-1/2) vs VZV

Lwoff, Horne and Tournier (1962) - Nature of the nucleic acid (DNA vs RNA) - symmetry of the protein shell - presence/absence of the lipid membrane - dimensions of the virion and capsid used linnaen system of kingdom phylum order etc.

Baltimore classification Based on - how they replicated - how genes were expressed from viral genome genome type is significant in how genes are expressed. Diversity of viral genomes +- DNA, -DNA, +_RNA, + RNA, - RNA Baltimore identified six classes then seventh class added. arbitrary groups not based on relatedness Classification not adopted by plant or bacterial virologist Type 1: dsDNA HPV and Pox viridae dsDNA have large size variations Can support large viruses because high fidelity, there is proofreading. Type 2: ssDNA parvovirus Type 3: dsDNA rotavirus Type 4: +ssRNA SARS, rubella, yellow fever small viruses. No where near size of DNA viruses Type 5: -ssRNA Rabies, influenza, measles, rabies Type 6: Retro ssRNA HIV Type 7: retro dsDNA hepatitis B ICTV in 2005 500 virologists 3 orders, 73 families, 287 genera, >1950 species Members of species and genera are likely to be related Includes viruses that infect plants, fungi, algae, yeast, bacteria Incorporates Baltimore classification different type of Baltimore classes infect different organisms e.g. fungi vs plants. Baltimore classes are NOT equally represented in fungal, animal, plant and bacterial kingdoms Baltimore classes say NOTHING about virus morphology. Unusual sub-viral infective agents satellite viruses - ssRNA do not encode genes for replication - need coinfection of helper virus to replicate - hepatitis delta virus - eoncodes one protein: for HDV delta antigen - helper virus RNA pol2

Satellite nucleic acids virusoids - requires coinfection; does not encode structural protein tobacco rignspot virus satellite RNA viroids - ssRNA molecules do not encode proteins but do not associate with helper virus, spread mechanically. - replicated by host RNA pol 2 - only described in plants - cause disease by binding to proteins (pkr) Prions - no capsids protein only Prions replicate by the conversion of PrPc in body to PrPsc Lecture 4 Light Microscopy performed live (non invasive), multiple labels (colocalisation), resolution limited to 200nm, no structural information. Deltavision OMX super resolution 3D Sim structured (can see 50nm) Electron microscopy - uses beams of electrons instead of light - scanning and transmission EM (75A), - multiple labels (colocalisation) can use gold particles stuck to antibodies on transmission EM - limited structure resolution Negative staining EM Biological specimens have little contrast use heavy metal (electron dense) stain - distortion of shape due to drying Some structural information

Cryo EM - observation of samples in vitrified water (not dried and distorted) - samples fragile low dose of electron - primary data is low contrast, 3d resolution derives from lots of images. - can be used to map proteins or antigens on surface - cellular receptor for HRV is ICAM - maps density so also reveals internal structures X-ray crystallography sample must be able to form crystals capable of delivering structures in order of 2A - limited to smaller viruses, non enveloped virus - can be mapped onto cryo EM Components of a virion - genome, nucelocapsid, other proteins for replication, tegument, capsid, envelope, surface proteins, polyhedral

Virall capsids must be very stable and provide protection but also disassemble to allow nucleic acid entry. Capsids are composed of multiple subunits (hetero or homo genous)

Coats are often self assembling, minmal free energy Type of capsid symmetry Helical symmetry simplest case : protein subunits attach to nucleic acid genome giving it helical symmetry. - more complex viruses use helical symmetry for the nuclear capsid which is then enwrapped by additional proteins or membranes. -identical environment except for end proteins Example is tobacco mosaic virus. VSV underlying helical symmetry transferred to envelope underlying helical symmetry is not transferred to envelope looks approximately spherical (flu). Icosahedral symmetry - although many viruses appear approximately spherical, they are acutally icosahe dral - very common symmetry found in capsid architecture Icosahedron is 20 equilateral triangles on the sphere with 12 pentagonal vertices 5,3, and 2 fold symmetry. Icosahedral symmetry - 60 asymmetric subunits is the minimum number of units necessary to build a container with icosahedral symmetry - all subunits interact with each other in an identical manner and lie in identical environments - 12 capsomers (pentamers) - minimum fre energy structure Example of T=1 is parvovirus, ssDNA, linear Triangulation number can be 1,3,4,7,9,12 or more. Each face can be subdivided into smaller facets. capsids are predicted to have 60 x T subunits. Pentamers stay the same (12) as T number goes up, but hexamers increase. T=3 Poliovirus Capsids are formed from three protein subunits (60 of each VP1,2,3) genome one +ssRNA T=4 sindbis virus Nucleocapsid formed from single capsid protein T=25 Adenovirus Mixed structure capsids T even phages possess binal symmetry combination of icosahedral and helical symmetry. Complex symmetry Large and complex virions with no recognisible features of symmetry Nucleocapsids are brick shaped and core dumb bell shaped. Naked viruses - protein capsid is exposed to the envrionemnt - usually released from host cell following lysis

- entry recpeotrs part of protein capsid Enveloped viruses - usually released from cell by budding - membrane envelope containing viral and host proteins HIV - protein capsid is enwrapped by the plasma membrane then bud off. Polyhedra cypoviruse and baculoviruses can survive harsh conditions polyhedral are protein crystals that form intracellularly one single protein form a lattice - Very protective but dissolve in alkaline conditions - 2um crystal can contain thousands of infectious particles

Lecture 5 Virus propagation Bacteriophages grown in bacterial cell cultures plant viruses studies primarily by infecting plant hosts, plant cell culture systems not as advanced as animal cell culture systems Initially viruses propagated in animals - Initially serially passaged in children in a ship - variability host immune system is different may get rid of virus - contamination host may have other viral infections - Genetic alterations pressure may cause the virus to change its genes - yellow fever passaging led to development of a safe vaccine Passaging grown adherently in flask Propagation in cell culture - reproducibility (frozen down), fewer variables - can easily manipulate experimentally - can utilize microscopy and recombinant virus technology - HeLa established in 1951, optimisaiton of conditions done in 1960s - not all viruses viable in cell culture (perhaps only certain cells can be infected). Primary cell lines limited life span, fewer effects from passaging, heterogenous e.g. MEFs (mas up a mouse0 time consuming (large mix of different cell types) Particular organ less variation in cells - normal chromosome number and shape, require higher concentrations of growth factors. FACS fluorescence activated cell sorting Continous passaged indefinitely Prkimary cells will reach senescence and die (hayflick limit: 52 divisions) - can be obtained from tumours, viral transformations, oncogenic transformation, mutagenesis, passaging - rapid growth, less dependent on growth factors - homogeneous (clonal)

-susceptible to aneuploidy gain in chromosome number, loss of contact inhibiton (grow multilayer) - lose differentiation characteristics, can be tumorigenic - Mouse cells have longer telomeres and higher telomerase activity HeLA - HPV induced malignant cervical cancer derived - telomerase activity at every cell division Cell growth - FBS (growth factors), carbhoydrates (glucose 4.5g/L), balanced salts (isotonic) - pH indicator (phenolred) - Buffered to 7.2-7.4 with sodium bicarb, HEPES - Amino acids, vitamins (glutamine), Antibiotics, 37c, 5% CO2 DMEM, RPMI Biological safety cabinets class 2. Risk group can be divided into safety levels PC1 low community risk (vaccinia:MVA)- attenuated viruses PC2 moderate risk (Prions, vaccinia, rhinovirus) PC3 High riskto individuals (HIV, yellow fever) PC4 high community risk (ebola) Contaminations - Chemical: metal ions, endotoxins, deposits on glassware - Biological (bacteria, fungal, mycoplasma, viruses,cells) Mycoplasm is parasitic bacteria lacking cell walls detectable only by PCR, changes phenotypes Plaque assay estimating viral titres. Start with monolayer of cells infect cells with serially diluted virus overlay to cells (agarose) blocks virus diffusion, limits infection to neighbours stained with neutral red labels live cells units are expressed as pfu/ml Plaque assay can look at virulence of strain (deletions may form smaller plaques). plaque assay can be used to determine concentration of virus stocks, determine attenuation of virus stock, determine cytopathic effect. Clumping, unviable virus, cell type, attenuation influence pfu and number of virus particles Cytopathic effects - part of the cellular response to fight infection (apoptosis), morphology/phenotype (cell-cell fusion, cell migration) some viruses do not cause cytopathic effects Virus have membrane that can incorporate into plasma membrane two cells can then form syncytia (massive cell to cell fusion).

Lecture 6: Host DNA and RNA polymerases are found in the nucleus, if viruses use these enzymes for genome replication then virus genomes must enter nucleus Some viruses such as pox viruses encode own polymerase. There is temporal control of viral gene expression immediate, early, intermediate, late. Diversity form small genomes Overlapping open reading frames (ORFs) Splice variants, leaky scanning, DsDNA viruses polyomaviridae BK and JC viruses generally asymptomatic, genome is a closed circular dsDNA packed into nucleosomes by cellular histones Gene expression early transcription initiated upon entry of dsDNA genome into the host nucleus, requires host RNA pol II, produces LT antigen At high concentrations Large T antigen inhibits early and activates late gene expression. Host RNApol II generates 5 capped mRNA transcripts all transcripts originate from either side of Ori both starnds used alternative splicing and leaky scanning generates diversity. Differential splicing generates large T and small T antigen (shorter and longer reading frames). Late gene expression capsid proteins VP1, VP2, VP3 Leaky scanning (VP2/3) ribosome recognises AUG and kozak sequence, some sequences with a poor consensus will be skipped and subsequent AUG used for translation initiation. Replication no viral DNA polymerase, genome replication requires LT antigen dsDNA viruses: adenoviridae causes respiratory diseases in humans transmitted predominantly by aerosol. Gene expression transcription initiated upon entry of dsDNA into host nucleus RNA polymerase II does transcription that is subsequently translated. VA-RNA is produced by RNA pol 3 then interacts with cellular proteins Some transcripts are made by RNA polymerase 3 functional mRNA s but not translated. may interact with other RNA s or proteins. inmmediate early transcription of E1A required for early gene expression required for late gene transcription (activated by DNA synthesis and IVA2 and L4). Viral gene expression of Early and Late separated by replication of viral genome. Immediate early gene expression occurs when translation is blocked. E.g. E1A E2A Iva2 L1,L2 replication -->adeno DNA is recognised by pTP (intermediate) viral DNA pol-pTP complex replication. ptP primes synthesis of new strand and remains covalently bound to DNA DNA replicated by strand displacement mechanism. dsDNA viruses: herpesviridae

Family includes genital and oral warts (HSV1/2) genome is linear dsDNA 125-240kb contains between 70-200ORFS. - There are tegument and envelope proteins Gene expression Transcription of intermediate early initiated upon entry entry of dsDNA genome into the host nucleus and requires VP16. VP16 is a tegument protein transcriptiona activator but cant bind DNA must interact with cellular protein Oct-1 Gene expression immediately early (alpha proteins) Early (beta proteins) function in DNA synth Late (y proteins) are structural or function in viral transport Replication - genome encodes complete DNA replication machinery (cellular topoisomerase) circular dna replication via replication fork results in concatemericdna cut up and packaged. Infectious cycle viral dna synthesis separates early from late. dsDNA viruses: poxviridae Family includes variola (small pox) and vaccinia Genome is dsDNA 130-250kb containing 100-200 genes. 12kb ITR (inverted terminal repeat) containing a covalently linked hairpin loop. ORFs are sometimes partially overlapping poxviridae is a very large virus Gene expression transcription occurs outside of nucleus RNA polymerases, capping, polyadenylation must be eoncded in the genome and be present in the virion - transcription will occur in virionpreperations when ATP added - huge genetic cost - early transcription in the core, mRNA extruded out of cores (rna made inside capsids) translation is activated by uncoating

ssDNA viruses parvoviridae ssDNA +DNa or DNA B19 cause of 5th disease childhood rash Adeno-associated associated virus type 2(satellite virus) genomes are linear 4-6kb 2 ORFS ssDNA enters host nucleus converted to dsDNA alternative splicing generates multiple products from 2 ORFS (rep and cap) rep proteins: site specific DNA cleavage and transcription factors transcription dependent on E1A and other proteins transcription by RNA pol II. Replication dependent on host DNA polymerases producing +/-ssDNA packaged into virions genome ends contain DNA hairpins, strand displacement mechanism

dsDNA retrovirus generating DNA from RNA carried out by RTs RTs share structural similarities with DNA/RNA pols. Hepadnaviridae there is a RNA intermediate. hep B 3.2kb, strand is full length partially single stranded (-) multiple overlapping ORFs, every nucleotide encodes for a protein sequence.

Gene expression - protein diversity is generated through multiple transcripts generating different initiation codons (through different mRNA species) - once circular dsDNA genome is repaired in nucleus (partially single stranded) expression of mRNA by host RNApol I Replication variety of mRNA s can be generated doublestranded DNA still exists prgenome RNA is made which is attached to capsid becomes a template for RT to form the dsDNA for CIMV all genomes synthesized by P protein (multifunctional as pol, RT and RNase), reverse transcription produces ssDNA and then dsDNA pregenome degraded. Lecture 7 Normal +ssRNA viruses (not rna retroviruses) can be made into protein by acting as a mRNA -ssRNA cannot be used as a mRNA it s a template for mRNA NA polymerases DNA-dependent DNA pol DNA-dependent, RNA pol RNA-dependent DNA pol RNA-dependent RNA pol

host DNA replication, viral DNA replication generate mRNAs, host and viral viral RT replication of retroviruses viral transcriptases, replicases, replication and gene expression

ssRNA retroviruses: retroviridae incudes HIV Genome is +ssRNA 7-11kb containing 4 main genes (5 gag, pol, pro, env 3 two copies of genome/virion pseudo-diploid only one copy integrated, one copy is sufficient for RT reaction. Gene expression - one example of a +ssRNA genome which does not act as a template + ssRNA genome reverse transcribed in virion following entry resulting in a linear dsDNA - viral genome and integrase protein enter nucleus, site-specific integration into host genome provirus - Expression by host RNA pol II - Genes present in all three reading frames Ribosomal frameshifting 95% lead to gag, 5% lead to gag/pol ribosome jumps between ORFs to a different frame. Gag/pol is then cleaved to generate individual gag, pro, pol proteins Replication - full length genomes made by rnapolII, these are packaged inside progeny virus. ++sRNA viruses: picornaviridae (includes pliovirus, rhinovirus, hep A) - genome is a linear +ssRNA of 7-8kb that contains one single ORF encodes one protein. Gene expression - genome is used as a mRNA translated upon entry of viral genome into host cytoplasm. - polyprotein is then cleaved into multiple products by two viral proteases 2Cpro and 3Cpro

polyprotein viral proteans act in cis (protein cutting itself) and trans (one protein cutting other proteins). picornaviruses don t have 5 cap but are polyadenylated. Have IRES picornavirus infection shuts down translation of capped 5 mRNAs by proteolytically cleaving a ribosome subunit mechanism to shut down host translation. picornavirus genome translated by binding of the ribosome to a region of the 5 UTR called the IRES IRES adopts a secondary structure that imitates tRNA and facilitates binding to ribosome. IRES eIF-4G is cleaved blcking translation of capped 5 mRNA s IRES is unaffected, but cellular translation stopped. IRES can be used experimentally to express multiple proteins from one promoter. doesnto require viral factors Replication THe protein 3D pol is the RNA pol that generates ssRNA from +ssRNA. And then +ssRNA from ssNRA +ssRNA viruses: togaviridae (family includes rubella), genome is 10-13kb +ssRNA and contains 2 ORFS. SUpressionof termination single mRNA prudces multiple proteins with a common N termins but having extended C termini. - normally occurs when normal TRNA s misread stop codon read through sequence surrounding stop codon is important. Expression and replication - +ssRNA acts as a mrna encodes rna pol subgenomicmRNAS made into capsid proteins Sub genomic RNA s are late which are made only after ssRNA is made. Rna virus with a temporally controlled gene expression. -ssRNA viruses (gneome cannot be t ranslated) :orthomyxoviridae viral rna pols packaged inside capsids. -ssRNA viruses: orthomyxoviridae includes influenza A, B and C causative agents of seasonal and pandemic flu. - segmented ssRNA genomes with an overall length of 10-15kb including 6-8 ORFs Expression and replication Virus enters cell segmented genomes released intocytoplasm and transported into nucleus viral RNApol generates mrna from ssRNA using capped 5 ends of host mrna as primers

Viral transcriptase delivered to nucleus (cytoplasm?) made into mrna translated Once translation occurs there is a build up of nucleocapsid protein critical threshold changes viral transcriptase changes makes ssRNA that is not 5 capped or polyadddenylated that is then packaged into virus particles.

dsRNA viruses reoviridae Family includes rotavirus segmented dsRNA genomes coding for 11ORFS over 19-32kb Expression and replication occurs within the cytoplasm mRNAs are identical to +ssRNA genomes mRNA associate with newly formed capsid and complementary ssRNAs are synthesized. genome never leaves the virion shell RNA pol activity cannot be separated from capsid slosh the cells that are infected reoviridae observation under microscope can observe mrna extruding out of capsid

there are 12 pores in capsid which are thought to allow mrna to escape. Satellite viruses: hepatitis D ssRNA satellite viruses coinfection with HBV required. 1.7kb circular genome. Cellular rna pol II will replicate the virus encodes one viruses. Infective genome is described as a viral genome that when transfected into cells lead to production of infectious progeny. those with built in polymerase/replication machinery will not have infective genomes Lecture 8 bacteriophage - MYOviridae Procaryotes differ from eucaryotes They have rigid cell walls with petidoglycans entry by fusion of virus and cell membranes/simple transfer not possible Bacteria lack a nuclear membrane all processes occur in cytoplasm Lack of nuclear membrane means that transcription/translation occur in the same compartment There is a promoter region for binding of rna polymerase sigma factor determines promoter specificity there is then a terminator region Ribosome binds reads start and stop codon Bacteriophages are the viruses of bacteria head and tail structure nto found in other viruses bacteriophage studied in detail before animal/plant viruses easily handled in lab Bacteria grow as single cells easy to follow infection, growth, lysis cycle. Exist as dsDNA (myoviridae), ssDNA (microviridae), dsRNA (cystoviridae) and ssRNA (leviviridae) phage plaques plaques start from a single virion in a lawn of bacteria T even phages capsid head with collar, sheath and tail members. Extended icosahedron structure is nto a regular structure but has an extra 10 faces. Life cycle of T4 Attachment of bacteriophage DNA injection early mRNA made host DNA degraded phage DNA made late RNA made heads and tails made heads filled virions formed hsot cell lysis. Lytic cycle of T even phage Plaque forming units (PFUs) lysing cells causes a massive increase in plaque forming units. there are different ways to represent latent, eclipse, lyse periods. T2,4,6ave different receptors that attach to specific receptors on host. Tail fibre attaches at tips binding is weak but once all six attach strongly bound even if 1-2 detach. browninan movement of head affect angle of tail fibre attachment when base plate attaches tail pins on base plate attach strongly base plate triggers contraction of tail sheath pushes central tube through the cell wall core is pushed through the outer membrane and peptidoglycan but not cytoplasmic membrane DNA goes into periplasm (NOT cytoplasm). There is different specificity of the attachment to receptor on host range Map of T4 genome genes with related functions grouped circular map represents the circular DNA in replicative

form of the chromosome. Baseplate is encoded by lots of genes (30) genes are grouped together in groups for regulatory functions e.g. membrane, tail fiber, tail baseplate etc. T-even phage synthesis of DNA and proteins Host DNA and protein synthesis is stopped T4 DNA is marked by attached glucose molecules. Host DNA degraded but phage DNA escapes degradation because of glucose. Parts of genome are expressed early there are also proteins (sigma factors) that modify host RNA pol THe modified RNA pol now transcribes the late mRNA which encodes structural proteins of the phage head and tail. Enzymes including lysozyme lyse the host cell. THe above changes in expression occur because the promoters differ and during the cycle the RNA polymerase is modified to change promoter specificity. Modification of specific RNA s can be visualized in graph succession waves of polymerase. Expression of T7 genes E. coli cells growing at 30 degrees were infected with T7 proteins were labelled with 35-S methionine before sampling cell lysis sds page with autoradiography. HOST protein synthesis STOPS soon after infection. Linear phage DNA circularises once it enters the cell then replicates as a circle same was as a chromosome Replication fork goes round and round concatemer one strand of genome is nicked at the origin and its 5 end peeled away from the unnicked complement. 1 150 genes, T4 packages DNA from a long double strand generated by rolling circle replication. Headful packaging mechanism head is made first dna is pushed into it Headful is larger than the entire genome packaging starts at one point. THe packaging enzyme complex then packs a headful into another capsid each phage has a genome with a unique starting point but all become the same because of circularization after infection base plate proteins, head porteins, tail fiber proteins packaged separately phage release after 22 minutes the first cells lyse and mature phage are rleased. One phage protein causes damage to host membrane T4 lysozyme gets access to cell wall digests peptidoglycan about 100 phage partiles released. Lecture 9 bacteriophage Alternative cycle Lysogenic cycle Integration into the host genome. Lambda like T4 is a double stranded DNA phage true icosahedral head Lambda has a lytic cycle as well does nto stop host metab Lambda has a linear genome short single strand ends that are complementary but also circularise on infection Lambda has an alternative to the lytic cycle. integrated state is called prophage lysogenic

Rarely is tHeprophage is induced to start functionining and the lytic cycle is followed to lysis. Under some conditions such as UV irradiation most cells of a lysognic strain ar e induced release of pahge The lambda tail has a simple helical symmetry tail with no contractile sheath one short tail fibre for attachment infects E coli and the receptor is the LamB protein. LamB is induced as part of maltose utilisation system Lambda has a linear d/sDNA genome but has 12bp single strand ends that are complementary circularises on infection. Circularisation is spontaneous and the cell ligase joins the ends. site on circle f ormed by joined ends is called the cos site (single strand ends make them cohesive) COMPARE THIS WITH T4 Lambda has a linear D/S DNA chromosome. It is circular within the host and has a circular genetic map. Genome is simpler than T4, has only got 49kb all of transcription is done by three major promoters. Initially the Two PR and PL can code for everything followed later by PR Lmbda phage genome different groups of genes are grouped together, Lysis proteins, head proteins

There are two paths for transcript depending on pathway lytic vs lysogenic pathway In lysogenic pathway head and tail genes are not wanted Homologous recombination integrate circle into large circle of host genome now a prophage not functional and mostly inactive. on infection each infected cell ends up either as a lysognic cell or lytic cell like t4 enter either cycle. Decision is must be made to

Lambda integration and excision After circularization the att site Pp aligns with BB and by recombination is integrated by INT between gal and bio genes. Control region on chromosome two major promoters PL and PR that transcribe in opposite directions these function very early Cro and C1 are very important proteins in deciding lytic vs lysogenic Lytic state is stable until lysis because promoters PL a nd PR are active. Cro is expressed from PR and represses expression fo gene C1. Genes N and Q are expressed give proteins that act as anti terminators allow all genes in major operons to be expressed later PR is activated and head/tail proteins are made. Lysogenic state there is only one phage gene functioning Cl repressor Cl can be expressed from 2 promoters PRE and PRM IN the lysogenic state the Cl gene is expressed from PRM CL repressor blocks activity of PL and PR in the same way lac repssor works There are 2 stable states each determined bye the presence of a specific protein CRo or Cl that blocks the activity of the promoter for the other state.

Cl also represses expression of any infecting lambda phage by blocking the activity of its PL/PR lysogenic bacterium is immune to further infection by lambda.

Lambda has a control region with PL and PR for long leftward and rightward transcripts. After infection PL and PR are recognised by the host RNA poladn transcription starts. After infection PL and PR are recognised by the host RNA polymerase and transcription starts at first only few genes are transcribed CIII, N, cro and CII until N is present CII and CIII activate pRE both C1 and CRO each act to block expression of the other anti termionatorpotein causes transcription to be extended in some cells C1 wins, in other cells CRO wins. REVISE FROM TEXTBOOK Lambda repressor binding operator repressor protein acts as a dimer attached to operator sequences. cro binding functions as a dimer also.
Lambda life cycle - Summary and comparison with T4.
The major difference between lambda and T4 is that T4 manages the various stages of its life cycle by modifying RNA polymerase to turn promotors on and off. The genes involved are in blocks but there are several big operons around the chromosome Lambda has a control region which manages the choice of lytic or lysogenic state by use of repressors and antiterminators. All differences between lytic and lysogenic states are in the activity in the control region. The rest of the genome is expressed from 2 promoters in the control region. The antiterminators N and Q determine the length of the transcript and thus which genes are expressed in the various stages of the lytic cycle. There are no phage encoded sigma factors. The same regulation of early and late mRNA is achieved by different means in Lambda and T4. There are many phages like Lambda and many likd T4 - and also many other variations on the same theme

Lysogenic stage can last indefinitely and many bacteria are lysogens when isolated. THe state can end spontaneously when they enter the lytic cycle not well understood lambda lysogen is irradiated with UV light C1 protein is inactivated repression of PL and PR comes to an end lytic cyel starts Prophage is excised by a reversal of integration INt responsible Bacteriophage play important role in environment documented in oceans large numbers 10^7 per ml on surface. 10 times more numerous than prokaryotic cells. Bacteriophage lysis release nutrients in the cells soluble/particulate Bacteria must increase in growth rates and multiply to keep pace with lysis pahge exert slection pressure Viruses are very important in biogeochemical cycling short circuit flow of carbon and nutrients from phytoplankton and bacteria lysis of cells shunting flux to the pool of dissolved and particulate organic matter -> can be reused by heterotrophic bacteria. Viruses cause lysis of cells converting them to Particular and dissolved organic carbon redues rate at which C sinks from surface layer into deep ocean carbon is retained in surface waters where it is photooxidised faster rate of CO2 build up in the atmosphere than would occur if exported to dep ocean. Lecture 10 immunological and molecular techniques in virology Diagnostic methods detect virus itself (imaging, sereological, activity, nucleic acid) host response to virus (production of antibody) Negative stain EM - virus is collected negatively stained with heavy metal ions (lead, tungsten) sample imaged with TEM Dumbbell shape can rule out certain viruses such as herpes, but not different pox viruses.

Virus can be identified by characteristics such as size, morphology, presence of an enveolope time consuming, low sensitivity, specificity CYtophatic effect viruses induce specific morphological changes : REQUIRES pathlogoist Nuclear shrinking icornaviruses proliferation of nuclear membrane alpha viruses, herpes viruses Vacuoles in the cytoplasm polyomaviruses syncytia paramyxoviruses, coronaviruses cytoplasmic inclusion bodies picornaviruses Expensive lacking specificity, not all viruses can be cultivated Antibodies Can come from patient serum samples, generated in lab May be monoclonal (from mouse) or polyclonal (many species type). DO not discriminate as well due to conservation of homologous proteins

polyclonal antibodies Produced during antigenic stimulation b cells are stimulated (3-5 AA) Stimulated B cells undergo clonal proliferation serum contains a mixture of antibodies Production of polyclonal antisera is cheap, relatively easy, less epitope specific, finite supply. Monoclonal antibodies Single B cell is isolated and immortalised gorwn in tissue culture can often fail extremely useful but expensive, time consuming but limitless supply. Haemagglutination assay detects the presence of virus Many viruses posses the ability bind RBCs only certain viruses can agglutinat e.g. influenza virus because has HA on surface. HA glycoprotein present in lipid envelope of flu virus Can be assayed using HA assay blood cells sediment down into a V if no virus present positive no defined round dot, diffuse cloudy. Haemagglutination inhibition assay Detects the presence of a antibody to a specific virus. Serum containing antibody is added to known quantity of virus and RBC if specific antibody is present HA is blocked. Positive sedimentation into V More specific Complement fixation test Diagnostic test for antibody reactivity that does not rely on ability of virus to induce haemagglutination Complement cascade present in serum presence of antibody, antigen causes membrane lysis. lysis of RBCs can be observed easily add RBC, antibody and free complement lysis IN positive antigen, ab complex reacts and depletes complement. In negative there is no complex, so complement lyses RBC goes pink ELISA quantitiatvely measure antibody reactions use antibody conjugated enzymes

Direct antibody sandwich antibody is adsorbed to well detects antigen antigen then binds

enzyme linked antibody specific for test

Indirect assay antigen is absorbed to well and test antiserum is added. enzyme linked antibody specific for test antibody binds to it colour change. Direct/indirct fluorescence antibody test virus infection can be detected in tissue samples taken from patients Tissue section with antigens direct and indirect fluorescence detected SDS page/ western blot virus are run on SDS page transferred to solid matrix by electrophoresis detection by enzyme linked assay Protein size as determined by WB can be influenced by primary sequence, degradation, cleavage, post translational modification Molecular techniques advancing at a rapid rate high specificity and sensitivity costs are coming down. Can be used to distinguish very closely related viruses can multiplex detect a variety of pathogens simultaneously Sensitivity just a few genomes required for positive signal Upscaled can be upscaled by robotics. PCR specificity achieved by the design of short complementary oligonucleotide primers (15nt+ ) and also taq, dNTP, mg2+ product size vary 100bp 10kb generate a billion molecules from one molecule in a few hours. Denaturing annealing extension Cloning DNA fragments restriction enzymes ligate sequences into plasmids and clone. RT PCR RNa viruses must be converted to dsDNA by reverse transcription prior to PCR. qPCR gives quantitiation of how much is present. sybr green is used to measure amplification of dsDNA binds to dsDNA and undergoes conformational change and exhibits fluorescnence (increases in fluorescence as more ds is bound) very fast, sensitive but expensive. Threshold concentration relationship with log concentration. Lecture 11 plant viruses Plant viruses have different apperances Transmitted by bugs such as aphids, leafhoppers also by pollen, seed and humans Tulip breaking strain changes colour of tulip Plant and animal viruses are not so different However most are +ssRNA Plant virsues express genes for replication, movement, coat protein, transmission, suppression of host defence. THe genomes of plant DNA viruses are circular

THe genomes of plant RNA viruses are linear. RNA doesn t need nuclear transcription Using filtration showed viruses are fundamentally different to bacteria. Wendell Meredith stanely nobel prize crystalized tobacco mosaic virus Conrat found that it was the RNA that caused infection not proteins TMV infects tobacco, tomato and other solanaceae Causes mosaics, mottling and necrosis Transmitteed mechanically infects through wounds Replication - single stranded RNA 3 ORFS (replication, movement and coat) RNA has to survive long enough to be translated 5 cap and 3 poly A tail normally in eukaryotic mrna. Viruses don t have this but TMA Has a 7mG cap that protects 5 end and a TRNA like structure on the 3 end that prevents degradation by exonuclease. Lots of + starnds made from small number of strands movement within the plant is different than animal RNA can move from plasmodesmata connecting individual cells. Core proteins a monomer forms a dimer/trimer and finaly a disk, short helix and a helix. IN movement ORF there is 240nt that forms a stem loop structure that is recognised by helix discs RNA wraps around a groove in the disc. great protection from enzymes Ribosome reads mrna that is still inside coat cotranslational disassembly by ribosome protein coat is pulled apart. Lecture 12 Plant viruses Luteoviridae small genome 6000bp potato leaf virus Important because cause millions of dollars of damage to agriculture. Virus only infects the veins of the plant. vascular tissue sugar moves through the plant via the phloem this I where virus wants to go to this is where aphids feeds through. Aphids then pass the virus through aphids virus enters the body of aphid then enters the system where it enters salivary system when aphid feeds on new plant virus enters the plant. potato leaf is ssRNA with six different genes. Encodes pol gene, coat gene, movement gene and helicase which unwinds dna (replicase formed with pol) there are amber stop codons. There is a subgenomic promoter sequence that makes a sub genomic RNA that encodes of the coat protein. There is a movement protein that is embedded into coat protein leaky scanning allows coat protein to be made Coat protein has poor context ATGa whereas movement protein has good context ATGG

There is amber stop codon after coat protein readhtrough protein (1 in 20) has recpeotrs on the shell readhtrough protein domain directs passage into salivary glands Regulation of polymerase (want large amount of polymerase compared to helicase. When there is a reading of the mrna polymerase slips at the hepta-nucleotide slippery region missing the stop codon reding through the 2nd frame. P0 helps virus protect against being killed off. p0 is in many plant viruses protects against the plant trying to destroy mrna transcript. Double stranded DNA virus enters dicers chop these up into small pieces 21bp and loads one ssRNA into argonaute If there is recognition of ssRNA virus comes in recognition and chops up. Knock out dicers in a plant e.g. DCL2,3,4 look like WT plant infected no more defence against virus very sick

Normaly proteins are ubiqiuitinated and are degraded by proteaseome. P0 recruits UBq pathway but also recruits argonaute argonaute is degraded. Lecture 13 viral diseases in livestock Expanding area of research increasing needs of population for meat. Intensive Farming practices increasing risk Moreopportunites for transfer between hosts (e.g. bird flu) with more animals. Blue tongue disease mygies (flies) transfer disease originally equatorial but global warming move of mygies to asfar north as UK and france. Large economic impact outbreak would cost Australia $6 Cost in loss of export but also in controlling outbreak. One health initiative dedicated imporivng the lives of all species (animals, humans, environments). RInderpest first animal virus eradicated African swine fever virus Highly contagious, fatal, haemmoraghic viral disease of pigs Only member of this family and genus (Genus asfivirus) similar in structure, morphology to iridoviridae similar genomic composition to pox viruses. high conservation of genome between isolates but variation at 5 and 3 ends. Complex, large icosahedral virus 170-190kbp, 150ORFS ASSF capsid structure - DNA containing nucleoid with 30 proteins - matrix shell and ER derived inner membrane, outer evnvelope is acquired d uring virus budding - highly stable survive at least 30 days, and 140 days in some pork products - some farmers feed pigs pork products contamination - inactivated by high termperatures and extreme pH in either direction ASFV distinct serotypes have not been identified because no neutralizing antibodies are produced - 20 different genotypes identified - variation through homologous recombination genetic s hift

Recombination occurs in which 2 strains share genetic information Point mutations, deletions, additions in DNA sequences near the ends of the genome during replication. ASFV strains vary greatly in their virulence high (high mortality), moderate, low ASFV is the Only known DNA arbovirus, arboviruses are transmitted by bugs (ticks). Infection and replication infects domestic and African wild pigs infection via the oral or nsal routes or bites from ticks of ornithodoros genus. - replication primarily in monocytes, macrophages - entry into host cells takes place by receptor mediated endocytosis - replication in cytoplasm where viral factories are formed released by budding ASF-clincal signs fever, ulcers, hamaeorrage Haemmorage is key symptom swelling in spleen and lungs Disease can be peracute dead with no clinical signs acute fatality very high subacute mild and persisting but lower fatality chronic carriers no symptoms, but makes virus African wild pigs are asymptomatic Transmission Direct contac, butes of infected ticks, indirection spread (contaminated clothing, feed) Carrier pigs, including wild pigs are key in maintaining infection in herds and starting fresh outbreaks ASFV are endemic mostly to Africa ASFV diagnostics can use normal tests also haemadsorption test (quite unique to swine fever) red blood cells will aggregate around infected macrophage Differential d iagnosis essential distinguishing between disease that have similar clinical signs e.g. classical swine fever ASFV-control no vaccine available because so complex disease control relies on implementing restricting movement of stock and slaughter. Foot and mouth disease (FMD) highly commuciable viral disease of cattle and cloven hooved ruminants. More than 70 speices Most contagious disease of animals or man Massive economic losses by outbreak Genus aphthovirus, family picornaviridae Small non enveloped icosahedral capsid +ve sense ssRNA 8.4kb 7 different serotypes highly stable in e nvironment up to 3months. Acidic pH, high temperatures, UV can inactivate the virus, desiccation caposid and genome structure - 5 and 3 region, capsid encoding region (3 make up external capsid, 1 lines internal capsid), 8 non structural proteins 3D is viral polymerase.

FMDV has high mutation rate during replication viral RNA pol has high error rate genetic drift (because of mutations) Quasispecies populatin of similar but non identical genomes extensive antigenic variation can generate viruses with varying fitness Infects sheep, cattle and pigs but NOT horses. Infection s via the respiratory tract epithelial cells of oropharynx secondary infection can be in oral cvity and feet Receptor mediated endocytosis using avb6 receptor replication occurs in the cytoplasm lytic cycle Virus can be found in ALL secretions and excreitions from infected animals Transmission direct contact, indirect transmission through fomites, aerosol can be spread over 240km over the sea. Amplifying hosts shed high levels of virus indicator hosts repaid develop clinical signs, often severe maintenance hosts e.g. sheep mild signs delaying diagnosis FMVD clinical signs nearly 100% morbidility rate, fatality is < 2% - vesicles(blisters) by erosison in mouth, feet fever, stringly saliva Animals take 2-3 to recover but can remain carriers reasons for slaughter Carriers live isolates more than 28dpi, non carriers 7-10dpi Viral reservoirs ingenimal centres of lymph nodes could be the cause Can carrier animals infect other animals such as African buffalo not clear. FMD distribution primarily Africa and asia recent outbreak in UK (thought to be from lab leak) FMDV can use a range of molecular techniques but also from saliva (probing) Diagnosis must be differential ( similar to swine vesicular disease, and foot rot) Control spread stop animal and product movement, slaughter infected animals, disinfect contaminated areas, vacciniation.HOWEVER vaccination doesn t protect against different serotypes Hendra virus (HEV) zoonotic virus causing fatal respiratory, neurological disease in horses. Family paramyxoviridae genus henipavirus related to nipah virus spherical virion, -ve sense ssRNA 18.2kb Sensitive to heat and chemical disinfection genome has 6 genes 8ORFS Genome is part of a helicanucleocapsid core containing genome and protein to initiate replication matrix protein layer lipid bilayer envelope (contains 2 glycoproteins. HEV infect and replication cell entry by fusion, replication in cytoplasm -ve strand RNA Is a template for transcription of viral mRNAs release by budding HeV limited to Australia so far Transmission and clinical signs

flying foxes (fruit bats) no clinical sig ns Horses if ingest any contaminated food infected with bat fluids infection Can be asymptomatic during incubation but shedding virus Clinical signs fever, death (70%), loss of balance, respiratory distress Humans direct contact with infected horse respiratory disease encephalitis (death 4 of 7 cases) Transmission must be from bat to horse to human. Diagnosis, using molecular techniques Treatment no drugs or vacciens use intensive care some antivirals are used, but not certain, prognosis not clear Transmission: there are no carriers, si not transmitted by insects or aerosols. Lecture 14 Animal viruses barriers to infection Plasma membrane, cellular compartments, cytoplasm, nucleus Eukaryotic cell binding, lateral movement, signalling plasma membrane is a major barrier to viral entry Once inside cell nuclear membrane is a barrier to entry into the nucleus for replicaton Changing temperature virus will easily bind at 4c and then stay bound even at 37c Initial contact affinity of a virus for cellular target mediated by surface proteins binding e.g. flue virus, bacteriophage binding to cells urfaces Specificyt of surface receptors determines whether virus can bind and infect (e.g. bacteriophage will not infect humans). Change in specificity is amajor determinant of emergence of virus can require a coreceptor for presentation (HIV) Virus receptors will have other functions (otherwise lost in natural selection) Extracellular proteins -> TM proteins, GPI linked, merisylated proteins. There is binding to laminin, collagen etc. Cloning the rhinovirus receptor screening a panel of monoclonal antibodies raised against human antigens, identify antibody able to block rhinovirus infection identified ICAM-1 ICAM-1 functions in cell-cell adhesion and the immune response. binding to this prevented infection. One receptor can be used by multiple viruses to gain access. Poliovirus receptor is PVR glycoprotein of the Ig superfamily cryoEM structure of extracellular domain of PVR bound to capsids PVR binds to cleft in viral capsid Influenza virus receptor HA binds to sialic acid (present ubiquitously) on cell surfaces influenza infection is limited to upper and low respiratory tracts NA cleaves sialic acid. Influenza enters internalized by endosome then uncoated by endosome. HA binds to sialic acid (weak interactions) but large interactsions forms strong interaction sit like this until endosome becomes acidified interaction between HA and sialic to fusion process two plasma membrane fuse

together. segmented genes are then released into cytoplasm. Membrane traffic endocytic pathway endosome is just one pathway. Endocytosis clathrin coated vesciles pinched off clathrin coat is lost drop in pH LCathrin is labelled green (GFP) and v irus labelled red (DID) entry and transport of influenza can be visualized to be recruited into cell.--> then rapidly transported inside cell Paramyxovirus entry at the plasma membrane Two viral proteins are important is entry protein (fusion proteins) are HN and F protein (fusion peptide). HN will land and bind to surface receptor of cell this will cause conformational change in fusion peptide insert into neighbouring membrane plasma membranes fuse ->uncoating and entry of virus particles. Entry of adenovirus Initial binding to fiber receptor subsequent interaction between penton base and integrins triggers endocytosis acidification of endosome triggers uncoating releasing capsid subunits penton base is believed to have lysis activity >ysis activity is restricted only to endosome (otherwise toxic to cell). Entry of poliovirus Enters by endocytosis pH is critical in entry of virus fall in pH generates a conformoational change forms a pore in the endosome into the cytoplasm feeds mrna into cytosol capsids are not disassembled signalling - entry of virus particles often leads to activation of cellular signalling patjhways that have profound implications infection binding to entry receptor can activate clathrin or caveolae transport - growth signalling pathways are activated stimulate cell migration, cell division, regulate gene expression Cytoplasm represents a barrier to diffusion for objects larger than 500kDA cytoplasm is crowded place. Three cytoskeletal networks microtubule, actin networks, intermediate networks Poses major problem for large viruses like vaccinia virus in which transport takes very long Microtubule cytoskeleton play key roles in transport Herpes virus (PRV) tagged with GFP Herpes virus utilize dynein and kinesin to travel much faster throughout cell jumpy transport but overall you do get dominant transport in one direction. Virus are imported into nuclear pore complex by NLS viral proteins use NLS to target their genomes to nucleus some viruses are small enough to enter intact (*parvovirus) capsids are disassembled prior to entry (influenza) wait until nuclear envelope broken down during cell division (retrovirus not HIV) Adeno virus nuclear entry transports to NPC too large to go in binds to NPC adenovirus interacts with histones lead to disassembly of capsids genome transported is released and enters DNA Cell to cell infection cell to cell fusion initial cell viral protein can then infect other cells without extracellular virus (evade antibodies

and immune response). VIrological synapse e.g. HIV dendritic cells go to lymph nodes passed to T cells cytoplasmic bridges pass directly into next cell. Lecture 15 immune response to viral E.g. virus are made Three lines of defense against viruses physical barriers, innate immune response, adaptive immune response Routes of virus entry skin, conjunctiva, tracts Physical barriers skin, tears, low pH PAMPS innate response (immediate 0-4 hours and early induced 4-96 hours). Adapative 96 hours specific immune response designed to eliminate cells Innate receptors no specificity TLR, n formyl receptors, mannose receptors TLRs can recognise things that are only present in microbes or viruses (not present in host) TLR 1,2,6 lipoprotein, lipotechoic acid TLR-4 viral envelope proteins TLR 5 flagellin TLR 3, 7, 8, 9 single stranded RNA TLR activate transcription factors NFkb and IRF-3 gene expression of inflammatory cytokines Prinicple mediators of innate type 1 INF, NK cells and inflammatory such as cytokines. Type 1 IFNs (IFNa and IFnb) inhibit viral infection anti viral state , enhance cytotoxic T cell activity, activate NK cells. anti viral state bind to IFN receptor anti viral state is induced no more viruses produced 2-5 A synthetase and PKR induced activate RNAse and phosphorylate EIF-2 lead to degradation of mrna and no translation IFN induces anti viral state stops cells from becoming infected. IFNs also enhance CTL activity increase expression of MHC class 1 NK cells recognise cells that fail to express MHC class 1 large granular lymphocytes when activated and proliferate in response to IL-12 and IL-15. NK cells and CTL s act differently. NK cells have inhibitory receptor that usually binds to MHC class 1 There is activating recpeotor binding to target ligand on cell when both act/inhibit bound together no death. Cytokines cyotkines are proteins secreted by cells mediators of innate and adaptive immunity. Dendritic cells TNFa stimulates migration of lymph nodes intiation of adaptive immune response

Speicifc adaptive immunity Adaptive immunity is the antigen specific immune response critical host response against virus infection Mediators are antibodies, CTL s, helper T cells.

Entry of microorganism takes type for adaptive immune response to occur immunological memory afterwards Infection occurs at epithelium of tissues resident dendritic cells then move to lymph nodes where they interact with T cells B cells effector functions. Antibodies are proteins which recognise molecular shapes and epitopes Neutralizing antibody bind to virus receptors prevent entry and infection Antibodies (ADCC) binds antigen (viral proteins) on surface of target NK cells recognise antibody and kill cells. MHC class 1 takes virus proteins endogenously presents them on surface where CTL s can act MHC class 2 only on APCs take up exogenous present them to CD4 +ve cells CTL s recognise MHC class 1 cause cell lysis 1) recognise MHC class 1 2) activate CTL 3) CTL granules exocytosed 4) apoptosis of target cell. Immunological synapse CTL and target cell Perforin makes pores in cells -->granzymes then enter then trigger apoptotic pathway CTLs are selective for those cells expressing a SPECIFIC antigen. Helper T cells CD4 T cells recognise antigen peptides presented by MHC class 2 activation of cD4 + killer cells, production of cytokines, help B cells and CTLs timing of the immune response is key Lecture 16 Immunity to viruses Viruses interact with the host to achieve the goal of survival, proliferation, persistence. Viruses have evolved a diverse strategies to evade immune system. infect Immune privileged sites to avoid antigen presentation and immune recognition HSV and VZV infect neurons Establish of a latent dormant infection herpes viruses (limited antigen expression). HSV is an alpha herpesvirus 90% of the human population is HSV1 seropositive HSV1 (oro-facial infections) and HSV2 (genital infections)

HSV pathogenesis sensory endings in skin allows viruses to enter neurons human ganglia contant latent virus. VZV causes chickenpox and shingles Pathogenesis of VZV incubation period primary infection (chicken pox ) latent infection reawaken (shingles). Neurons don t express MHC class 1 Productive infection lytic gene expression, infectious virus released Latent viral genome is present but limited gene expression, lifelong state, no infection

Reactivation virus reawakens from latency Infect and destruction of immune cells e.g. HIV T cells are critical component of adaptive immune CD4 T cells depleted, normal infectous easily handled can be fatal.

HIV kills CD4, induces apoptosis, replicates in CD4 t cells causing lysis during virus exit, triggers CD8 to recognise CD4s and lyse cells (1) evasion of innate immunity Modulating type 1 IFN cytokine receptor mimicry (poxviruses) makes homolog of IFNR that binds the IFN and prevents binding to cell. Intracellular inhibitors of Type 1 IFN signalling (HMCV< adenoviruses, poxviruses) HMCV prevents 2,5adenylatesynthetase from functioning properly. prevents antiviral state Modulating cytokine network Virus can also econde interference with TNF receptor homolog binds TNF and prevents cell surface interaction no proinflammatory response no programmed cell death Evasion of NK cell mediated killing NK cells recognise and lyse cells with MHC class 1 HCMV encodes a MHC class 1 homolog serves as a decoy MHC capatable of presenting antigenic peptides no NK cell activation Mechanism of immune evasion of adaptive response Modulation of antibody response e..g flu and HSV Antigenic variability RNA viruses antigenic drift no longer have capacity to do so Antigenic shift (combination with another flu e.g. swine flu) antibodies no longer work Viral modulation of ADCC HSV infected cells encodes FC receptor caps of viral glycoprotein preventing effector cell from binding Viruses can sabotage MHC class 1 and MHC class 2 pathways Many steps involved including TAP, calnexin, calreticulum, proteasome, CLIP. MHC class 1 all cells express this proteasome and TAP, tapasin important Inhibition of generation of antigenic peptide EBV, HCMV CMVp77 interferes with production of peptides Antigenic peptides transported via the TAP complex HSV, HCMV HSV ICP47 blocks tap mediated transport Inhibiton of MHC class 1 surface expression VZV, HCMV, HIV HIVnef enhances endocytosis of MHC from cell surface Modulation of MHC class 2 Post translation effect inhibition of surface expression of MHC class 2 HIV, HMCV transcriptional effects inhibiton of interferon induced expression of MHC class 2 HCMV, VZV

IFN-y binding jak/stat pathway transcription factor IRF-1 driving gene expression. Modulate of DCs by viruses Mature DCs present antigens need upregulation of MHC class 1,2 CD54, CD40 andCD86, CD80. Delaying T cell recognition by infecting dendritic cell Flow cytometry analysis of immune molecule expression during VZV infection preventing CD80 and 83 from being expressed on surface retention of these molecules in cytoplasm can be observed by fluorescence tagging. CD83 is key in functional mature DC s Virally encoded interleukin 10 IL-10 is an inhibitor of activated macrophages and DCs IL-10 inhibits expression of MHC class 2 and costimulators of DCs some genes such as EBV and HCMV encode a viral homolog Why study virus econded immune evasion May alter vaccine development Characterisation of virally encoded immunomodulators Information about virus host cell interactions Discovery of new immunity associated molecules Lecture 17 hyerpesvirus 100 herpes viruses have been identified Complete genome sequence of 43 determined eight known human HPV Size 120-300nm linear dsDNA up to 250kbp genome is replicated in the nucleus icosahedral capsid symmetry enveloped primary infection inapparent in childhood latent infection followed by remerging infection there are three families (alpha, beta and gamma) Alpha HSV 1 and 2, VZV beta HCMV Gamma EBV, KSHV Ubiquitous infection except HHV-2 and HHV-8 encode enzymes for DNA metabolism, DNA synthesis, protein processing e..g polymerases and kinases Synthesis of DNA and capsid occurs in nucleus Mechanisms of persistence decrease MHC1, inhibiton of apoptosis, immune privileged sites HSV is spread by contact, virus in bodily fluids primary infection is subclinical in most individuals HSV 1 80/90% prevalence causes oral infection HSV 2 10% prevalence genital infections =45% of oral infected individuals and 60% with genital will experience recurrences HSV involved in cold sores and other cutaneous herpes - more serious is meningitis Varicella Zoster virus (chickenpox) Incubation 14-21 days Presents fever and lesions al over the body Complications are rare but occurs more with immunocompromised patients

Complications include v iral pneumonia Recurrence is termed shingles vast majority at over 50 VZV causes postherpetic neuralgia (eeruption of vesicles in dermatome, pain in neurons) HSV-1 pathogenesis primary infection occurs through break n mucus membranes (mouth) Most individuals infected by 1-2 years of age intial infection is usually asymptomatic Virus enters peripheral sensory nerves and migrates along axons life long latent infection with periodic reactivation During latency viral DNA is maintained as an episome (Not integrated) limited expression of specific virus genes Reactivation due to physical or psychological stress virus travels back down sensory nerves Structure of herpesvirus Herpesvirus particles consist of four morpholigcaldifferentialbesturctures Inner core with dsDNA, enclosed by icosahedral capsid shell, surrounded by a layer of 20 different tegument proteins, enclosed in a lipid envelope Herpesvirus capsid determined by cryo EM 2,3,5 fold axes of symmetry, 162 capsomeres (150 hexons and 12 pentons. HSV-1 encodes 90 proteins with different regions 80 proteins which half are essential for growth in cell culture Essential genes encode structural, entry, expression, packaging/replication genes non essential genes fine tuning, overcoming host immune (non essential in cell culture) 13% of all herpesvirus genes appear to have cellular counterparts. Replication cycle of herpesviruses Very similar in neuronal and nonneuronal infection Using EM can visualize various stages of replication cycle. 1. Attachment and entry glycoprotein B and C bind HS chains on cell surface proteoglycans. This facilitates the binding of viral glycoprotein gD to one of its cell surface receptors(HVEM, nectin -1,2) Binding of gD triggers fusion Neuronal cell penetration is pH independent there is also pH dependent endocytosis in non neuronal cells 2.Transport of capsid to nucleus and release of viral DNA After fusion capsid and tegument proteins released into cytosol capsid + subset of getument bind to MT and transported by dynein binding to NLC viral DNA injected 3. Gene expression and DNA replication VP16 tegument protein is involved in immediate early transcription. Entry linear DNA is circularised RNA pol 2 three distinct classes of mRNA s are made a immediate early transcription activator e.g. UL48 b early mRNAs reg proteins, metab and replication proteins y Late mRNAs structural DNA replication is trigged by the interaction of virally-encoded origin binding protein (UL9) Viral helicase protein (ICP8) further unwinds the DNA ATP dependent fashion DNA pol then loads on to the origin to begin new DNA synthesis Genome is nicked and forms a rolling circle template results in concatemers containing many copies of HSV-1 genome

4. Capsid assembly COncatemers is cleaved and packaged contain consensus motifs pac1 and pac2 brought together cleavage site is generated packaged into preformed capsids capsids are all made in nucleus 6 to 7 proteins intiation on portal (being moved) stepwise addition of shell scaffold cleavage. capsidangularisation 5. Primary envelopment/deenvelopment The first budding step occurs at the inner nuclear membrane and involves capsid containing viral genome Results in enveloped primary virions in pernuclear cleft Fusion of primary envelope with outer nuclear membrane deposits deenveloped capsid int cytoplasm o 6. Secondary envelopment at the golgi Addition of tegument to capsids occur in cytoplasm secondary envelopment fowllos with budding into gogli derived vesicles complete enveloped particles are observed within vesicles 7. Release by exocytosis molecular motor kinesin transports to cell surface fusion of the vesicle membrane with the plasma membrane release of mature virions by exocytosis HSV DNA alpha, beta expression DNA replication y gene expression productive infection Latency transcripts are made Function of these transcripts and the mol boil of hsv1 latency remain unclear. Some vaccines and antivrials available e.g. against VZV Vaccines against EBV and HCMV in evelopment Action of acyclovir (Zovirax) nucleoside analogs Competes with dGTP requires VZV/HSV thymidine kinase for phosphorylation monophosphate is then converted to acyclovir triphosphate by kinases ACV-TP incorporated into viral DNA by viral pol incorporated base prevents chain elongation Ganciclovir also used against CMV cidofovir is highly active against CMV. Herpesvirus as vectors fore gene therapy Use defective vectors require helper virus to package and replicate DNA known as amplicons contain minimal HSV sequences Gene replacement vector HSV genomes in which single gene has been replaced by DNA of interest Advantages do not integrate into chromosomes, replicate in high titres, express genes for long periods, can accommodate large DNA inserts Lecture 18 Viral latency Acute infection rapid production of infectious virions rapid resolution and elimination of infection Typical course for viruses such as rhino virus Infections usually brief in immunocompetent hosts Presistent infection unlike acute infection is not cleared effectively by the immune response

There is slow chronic and then there is latent infection Slow crhonic infection production of virus for long time persistent infection (lymphocytic choriomeningitis virus) Another type is measles high production initially body gets rid of most of viruses but not all. if there is weaknening of immune system virus may come back Persistent infection that goes latent After initial acute infection infectious virus is completely cleared by immune response viral genome persists without any dectable infectious virus production highly restricted viral gene expression latent genome is non-infectious periodically virus can re-awaken from latency by a process called reactivation. New infectious virus may or may not cause disease herpesvirus characterised by capacity to establish latent infection. What is viral latency differs from acute infection differs from other types of persistent infection. latent infection is lifelong there is no cure Immune response not capable eliminating the latent pool of infection 3 phases of infection common to all herpes virus Productive lytic gene expressed: infectous virus released Latent viral genome is present but gene expression is limited; lifelong state Reactivation virus reawakens from latency There are 8 herpes virus of the 100 known that have humans as their natural hosts HHV-1 to 8 All establish latent infections alpha- herpes virus Herpes simplex viruses (HSV1 and 2) HSV1 cold sores HSV2 herpes genitalis At some point during acute productive infection virus establishes latent infection in neurons of sensory ganglia travels by retrograde axonal transport to neuronal cell body latent viral genome maintained in an episomal circular configuration. NEURONS are a post mitotic immune privileged cell type. Periodically, virus reactivates latency triggers are stress, UV, menstruation, colds Newly reactivated virus made in neurons can retrace path of primary infection by anterograde axonal transpo rt return to site of primary infection Latency associated transcripts (LATs) the only known viral products LATs do not appear to encode protein LATs accumulate in nuclei of latently infected neurons LATs appear to inhibit apoptosis LATs may inhibit expression of a viral gene (ICP0) which is needed to start replicative cycle promote latency by supressinv HSV genes required for productive infection. VZV latency caused by chickenpox latency in sensory neurons causes shingles Beta- herpesvirus HMCV Ubiquitous about 90% by age 40 Good news mild or asymptomatic in healthy fever; but in mminuosupressed people can be devastating

latent phase plays a critical role in immunosuppressed individuals 1. Productive infection extensive gene expression, novaccine, causes cell death new viral particles CAN be spread to neonates 2. Latent infection viral genome present but highly restricted expression no cure, no antiviral drugs, life long, no replication of new virus can lead to reactivation in transplant Myeloid lineage cells is main site of latent infection monocytes and their progenitors shown to harbour latency when cells differentiate into dendritic cells or macrophages can lead to reactivation CMV latency viral genome exists episomal circular configuration - During natural latent infection 1 in 25k cells are viral DNA positive - only 10 copeis per infected cells - viral gene expression highly restricted, infectious virus cannot be detected Latent infection ultimate immune evasion strategy CMV establishes a life long latent infection in monocytes which will mature ot become APCS CMV able to remain latent in these cells encodes homologues of potent immunosuppressive cytokine human IL-10 viral IL-10 can supress MHC class 2 on latently infected cells suppressing MHC class 2 by latently infected cells renders these cells refractory to recognition by CD4+ Expresses a viral protein during latency which has been shown to suppress (oligoadenylatesynthetaseanti viral pathway). Other viral genes expressed during latency may play important roles not yet defined, only 80/200 are needed for viral replication Gamma herpesvirus EBV pototypic y-herpes virus lymphotropic virus infects primary B lymphocytes transmitted orally by saliva EBV is a tumorigenic hperpesivrus It causes tumour formation during the latent phase of infection of by inducing B cell proliferation. Latent EBV related to various cancers Burkitt slympha common in Africa nasopharyngeal carcinoma common in south china, alaska B cell lymphoma EBV latency Viral genome is maintained in an unintegrated form (episome) EBV has the capcity to replicate every time a B cell divides latent genome maintained in proliferating B cells Ensures the reservoir of latently infected B cells is not diluted by cell division EBV infects fot eh first time target B cell becomes immortalised EBV is found in latent state in 1 to 10^5/6 cells Infection of B cells results in conversion fo about 10% of cells to LCLs

IN these cels virus is present in a latent state no lyticvirus in latent LCLs EBV 6 nuclear proteins, 3 latent membrane proteins expression of small nonpolyadenated RNAs (EBERs) and highly spliced transcripts (BARTS) Different latencies are defined by expression fo different combinations of latency associated viral genes Latency 0,1,2,3 Latency 0 no tumour association Latency 1 (EBNA, EBER) burkitt s lymphoma Latency 2 (LMP, EBNA, EBER, BART) hodgkins lymphoma Latency 3 (EBNA, EBER, BART, LMP) post transplant lymphomas HIV Lentivirus member of retrovirus Infects CD4+ T cells HIV also has a stage that is somewhat like latency HIV latency HAART can suppress HIV replication During HAART resting emmory cd4 cells can support latent infection Latent reservoir is nto cleared by HAART Latency in resting CD4 cells is established early after initial infection of the host very stable (half life 4 years) Persisiting low level replicating HIV

Nave T cells can be infected preintegration latency ; dna is in cytosol which is rapidly degraded clonal proliferation and viral replication CD4 t cells then die IN SOME cases resting memory CD4 T cell and post integration latency CD4 t cell activation Low frequency of latent infection (1 in 10^6 resting memory CD4 cells not recognised by immune system or cleared by antiretrovirals) Lecture 19 retroviruses and HIV Retroviruses enveloped RNA viruses Linear ss+RNA genome 7-10kbp RNA converted to DNA provirus by reverse transcriptase therefore Baltimore class 6 Provirus is integrated into host cell genome once integrated mRNA production is same as for cellular gene 3 Major coding regions - Gag structural proteins - Pol enzymes,RT, intgrease,protease - Env envelope proteins Peyton rous discovered Rous sarcoma virus (RSV) first retrovirus - Temin 1964 DNA synth inhibitor RNA virus doesn t work - Baltimore independently discovered RT in MLV and RSV 1980 HTLV discovered by gallo 1983 HIV isolated discovered in france Retrovirus

- spherical, host derived lipid envelope Simple retrovirus encode only genes needed for replication Compelx encode genes that regulate immune response RNA genome 7-10kb 2 RNA copies per virion (diploid) Provirus is a dsDNA copy of the RNA genome Principal proteins are made as polyproteins then cleaved into functional proteins HIV genome much more complex Multiple splicing and genes Gag gene Matrix gene p17 lines inner surface of virion membrane Capsid gene p24 forms the core of the virus Nucleocapsid p6,7 binds packaging signal, interacts with VPR Pol Gene Can be in same reading frame as gag such as HIV Protease cleaves viral proteins Polymerase and RT converts RNA into DNA provirus Integrase --> facilitates integration Reverse transcriptase 50-100 molecules Composed of 2 polypeptides heterodimer 3 enzymatic activities RT, DNA pol and RNaseH activity digests RNA ina RNA DNA hybrid leaving ssDNA requires a primer like all DNA polymerases done by host derived DNA RT occurs before virus is fully uncoated no proofreading (high mutation) Integration before integration no transcription of mrna 50-100 molecules per virion integrase cleaves hot cell DNA and then ligates into provirus Site of integration is random but some preference for active genes ligation is irreversible. Envelope gene Surface gp120 binds cellular proteins transmembrane gp41- mediates fusion in HIV CD4 is primary receptor, CCR5 is coreceptor HIV accessory genes Vif binds to APOBEC3G allows infection to continue vpr involved in nuclear import nef helps HIV repplication in resting T cells vpu regulates degradation of CD4 Regulatory genes tat HIV promoter rev promotes export of unspliced, single spliced HIV mrnas

LTrs Long terminal repreats at 5 and 3 ends contain unique U and R sequences Unique regions duplicated during RT, function as primer binding site important for integration and regulation of transcription Retroviruses Only diploid viruses only virus whose genome is produced by cellular transcriptional machinery only virus requires specific cellular RNA (tRNA) for replication Only + sense rna virus whose genome does not serve directly as mRNA after infection Oncogenic retroviruses - induce cancer by 3 mechanisms (oncogene capture caries viral oncogene) - insertional mutagenesis (integration of provirus occurs close to proto-oncogene or tumor suppressor gene) - viral gene acts to alter a proto-oncogene/tumor suppressor gene HTLV -1 is known to cause human cancer Asian leucosis virus integrates upstream of cmyc increases synthesis by 100-300x pro-proliferative Human T cell leukaemia virus (HTLV) first human retrovirus discovered, isolate din 1980 4 types HTLV convincingly linked with human disease 6 types of HTLV, A to F, A most prevalent 20 million people infected 90% infected are asymptomatic Transmission occurs via breast feading, fluids, sex Infects T cells thought to spread via cell division not virion production ATL occurs 20-30 years post infection aggressive and shrot survival time Lentiviruses -HIV HIV 1 and 2 and SIV, ther are also no primate BIV, FIV, EIAV Can infect non dividng cells macrophages, lymphocytes Lifelong persistent viremia, high mutation rates 3 stages of infection (acute, latent disease) HIV GRID, AIDS, diagnostic kits in 1980 s Drug s developed in 1987 and dual therapy in 1994/5/6 Origin of HIV likely to be from chimpanzee CIV due to human contact (different species) HIV-1 is far more prevalent than HIV-2 3 groups of HIV-1, 9 subtypes 39 recombinant forms. HIV diversity Replication no proof reading, 1 mutaiton per genome produced each cell produces heaps of virions Creates a swam of quasispecies virus. HIV is transmitted by sex, intravenous, mother to child Diagnosis of HIV ELISA test, then western blot at 22-27 days

Clinical course of infection viral load increases but is brought back down after years will rise as CD4 cells die Initial infection symptoms Mononucleosis like symptoms fever, lethargy, rash, headache 10^7 copies of HIV RNA per ml of plasma In sexual contact CCR5 viruses predominate macrophage cells in mucosa Macrophages are long lived can act as reservoir, Dendritic cells also implicated latent infection Start 3-4 months and last for years- decades very few symptoms Low level viremia CD4 cells decline Viral population heterogeneity increasing Fatigue, susceptible to shingles and fungal infections AIDS Dfined as < 200CD4 t cells Increased quantity of circulating virus lymph node architecture breakdown Viruses using CXCR4 for entry predominate Neurological symptoms may develop Opportunistic infections occur now Antiviral drugs Nucleoside RT inhibitors, non nucleoside RT inhibitors Prootease inhibitors Fusion inhibitors Integrase inhibitors HAART (combination of 4 antivirals) Protease inhibitors + NRTI s Complicated and expensive Reduction in HIV related morbidity and mortality Decreases chance of resistance developing --> but metabolic abnormalities Obstacles to treatment Drug-resistant variatns can be transmitted viral reservoirs drug penetration not always optimal Vaccine design High mutation, recombination, Lecture 20 Influenza Respiratory viruses lots, adenovirus, coronavirus, Flu Lots of different viruses can cause symptoms similar to flu. Influenza is a seasonal sickness strikes in winter sick up to three weeks There are treatments available Influenza orthomyxoviridae

3 genera A,B, C is a ssRNA, 13.6kb and 8 segments Epidemiology Undergoes antigenic drift and shift and is seasonal Transcription/replication in cell nucleus incubation is 1-4 days, large no of virons shed Influenza strains are anmed by Type, Town, isolates, year isolated, major HA/NA (haemagglutinin, neuroamindase) Seasonal in temperatre zones, year round in tropics Pandemic influenza less seasonal in temperature zones 3 main pandemics Spanish, Asian and hongkong Cost of influenza is very high, both medical and economic costs. Influenza rates are skewed in the very young and the very old. Structure Nucleocapsid core SSRNA, 80-120nm Embedded on surface are glycoproteinse, neuraminidase and haemagluttinin (HA1 and HA2) recognise sialic acid receptors in humans. RNP complex made up of polymerase proteins (P1, P2, P3) nucleoprotein and non-structural (NS1 and NS2) matrix proteins Environment and social conditions can produce new viruses JUMP species e.g. bird flu. Increasing populations, travel etc. Antigenic drift Slow gradual change in virus over time no proofreading polymerase in flu viruses belong to the same subtype but show serological cross-reactivity but do not cross-react completely May cause epidemics Antigenic shift major change in circulating virus Either through reassortment (mixing of genes from animal/bird virus with human flu virus) Adaptive mutation changes in genes of an animal influenza virus allowing humans to be infected easily Cause pandemics (Swine flu) H1N1 spanish flu Small change in H1N1 bird flu REassortment allows for genetic variability two species flu merge to form new virus E>g. in pigs have receptors that are susceptible to both human and bird flu. Studying flu grow in vitro in MDCK cells and human kidney cells Invivo grow in eggs Pathogenesis Inhalation of respiratory droplets or contact with mucosal membrane Virus attach to respiratory epithelium receptors via HA inside the nucleus mrna and crna is made viral RNA assembles with nucleoproteins virus release from cell by NA

Matrix protein M1 is used to detect influenza A Clinical features - abrupt onset of fever, sore throat, cough, headache last 7 days on average Complications - pneumonia (viral) - Reye s syndrome in children < 18 - viral encephealitis Influenza vs Cold Chills in flu vs Sore throat/sneezing in cold Flu is abrupt and takes a week to recover Cold is year round Influenza B is milder, less nasal symptoms, mild respiratory infection Avian flu in humans A pandemic can start when three conditions have been met 1) a new influenza virus subtype emerges 2) it infects humans causing serious illness 3) spread easily and sustainable amongst humans H5 chicken incident - 1996 18 people hospitalized in hongknog 6 deaths Culled all poultry no more cases until 2003 (H5N1) bird flu H5N1 binds to alpha 2,3 receptors victims had direct contact with birds entirely avian virus neurological signs, diarrhoea, death. Human-human transmission in h5N1, there seems to be transmission between some humans (close family) but not sustained. Scientists have shown reassortment of H5 and N1 with H3N2 internal proteins cause no transmission demonstrates H and N genes are not the only proteins involved in transmission Swine H1N1 was a mishmash of different genes from a number of viruses IN 2009, influenza cases were substantially higher.due to H1N1 pandemic Labs overloaded when testing viruses. Vacciniation against seasonal Flu A/B fluvax-a purified, inactivated, split virion (split virus) vaccine containing antigens from a number of types predicted to hit that season E.g. in 2011, A California/7/2009 A/perth/16/2009 and B/Brisbane/60/2008 Vaccine is recommended for people at risk Panvax for H1N1 contains only one viral strain. Antiviral treatment Zanamavir and Oseltamavir only effective within 30 horus of symptoms

Lecture 21 Cancer Cell division in multi cellular organism is regulated by signalling pathways Carcinogenesis is the breakdown of the normal mechanisms that control and limit cell proliferation Tumors that reamin isolated and don t spread are benign, spread are malignant (often fatal) Malignant tumours can shed cells that colonise distal parts of organism (metastasis) Control of cell cycle is coordinated by many proteins e.g. cyclins, p53 Cell cycle arrest can be damaged by UV, virus Rous RSV Temin/Rubin 1958 tumour cells release virus virus plays a role in malignant state Transformation of cells in culture Propperties of tnrasformed cells Cells can be propagated indefinitely rapid growth, less dependent on growth factors homogenous easy to grow, susceptible to aneuploidy, sometimes tumorigenic when reintroduced into animals, lose diff characteristics RSV passaged many times isolate tumorigenic virus and non tumorigenic virus RSV temperature sensitive mutant isolated mutation in SRC src was the first oncogene to be discovered v-src and c-src homolog c-src found in chicken acquisition of v-src causes cancer but c-src does not transform why is c-src non transforming src is a cellular tyrosine kinase phosphorylate proteins two tyrosine molecules (one in catalytic domain and C-terminus are key) activity regulated by phosphorylation of these V-src phosphorylation site (iinhibitory site) is lost in v src Many porteins involved in src phosphorylation of hundreds of proteins cause changes in invasion, growth, survival Transducing retrovirus are usually animal in origin replication defective oncogenes when expressed alone may reproduce some/all effects of transformation Oncogenes proto-oncogenes (src, myc, egf, ras) represent cellular counterparts to v-onc that play a role in cancer (activation, overexpression, misexpression) tumorsupresor genes affected (p53) Transducing oncogenic retroviruses: potent efficient in tumour formation Form tumours very quickly Transforming due to capture cellular proto-oncogene Transform cells in culture Transformed cells are not clonal Non-transducing oncogenic retroviruses n-T oncogenic retroviruses caused by insertion of retrovirus altered expression of neighbouring cellular gene associated with haematopoietic cancers in animals Majority of insertions lead to activation of cellular proto-oncogene, dependent on LTR transcription Activity of LTR can determine tumor type A few cases have been characterised to lead to inactivation of tumor suppressor gene deletion of src

non-transducing oncogenic retroviruses Insertion of genome into two exons can cause activation of proto-oncogene Deletion of LTR promoter is porduced Promoter insertion produces a chimera with viral RNA }Enhancer insertion operate over large distance, exerting activation of cellular enhancer = Characteristics latency period of replication Tumours develop more slowly Transforming due to activation/overexpression of neighbouring genes dependent on insertion site Cannot transform cells in culture Transformed cells are clonal all cells in a tumour have the identical insertion site Tumour type can be influenced by tissue specific activity non-transducing, long-latency oncogenic retroviruses E.g. human T cell leukemia causing ATL Classic virus of this category is retrovirus HTLV-1 characterised by long latency period (20-30 years) only 1% of infected individuals develop ATL Insertion events not clonal (no hotspots of insertion) don t capture a cellular oncogene or clonal Not many viral gene expressed in tumors one gneethat is maintained is tax may play a role in transformation. detected by PCR Tax is highly pleiotropic and interacts with many cellular proteins DNA oncogenic viruses Many DNA viruses encode oncogenes not oncogenic in their natural hosts A few DNA viruses are associated with human cancer Can encode oncogenes - those that have clear cellular homologues - those that lack any homology to cellular genes DNA virus oncogenes unlike retrovirus oncogenes often play important roles in viral replication polyomavirus expresses mT binds and activates SRc kinase recruits downstream adaptors Adenovirus expresses two proteins which bind and inactivate various proteins inactivation of tumor suppressor gene Approximately 20% of human cancers have a viral component. Jackalope show rabit horns contained infectious agent able to induce outgrowth of tumours Treeman caused by HPV infections disease outgrowths HPV 80% sexually active women contract HPV skin-skin transmission HPV mostly cleared by immune system can form benign tumours or lead to cervical cancer HPV subtypes predispose to genital warts others to cancer Cervical cancer leading cause of cancer death in the development world PAP smear reduced numbers HPV is detected in 99.7% of all cervical cancers 70% HPV is HPV16 and 18

HPV vaccine L1,2 capsid proteins E1,2 viral replication E4,5 interact with cytokines, growth factors E6,7 oncogenes L1 spontaneously forms virus like particles highly immunogenic protective against HPV infection FDA Unresolved ethical concerns, protection for men, protection following infection?, what about 30% less common strains Oldest animal cell line Canine transmissible veneral tumour spread by sexual activity, sniffing, licking Can be transmitted experimentally only with live cells Tumour cells are clonal ( all cells derived from single cancer that is contagious ) single origin Similar theory postulated by devil factialtumor disease Lecture 22 - prions Prions Infectious proteinacoues particles lack nucleic acids Cause Transmissible Spongiform Encephalopathies (TSEs) TSEs once called slow virus diseases Proteinaceous infectious particle that lacks nucleic acids Prion (proteinaceous-virion) is designated PrPsc Prion protein (prnp) gene found on short arm of chromosome 20 Encodes a protein of 254 amino acids termed PRPc PrPc converted to Prpsc differetnsecondary structure Scrapie - -> TSE of sheep known for 200 years strong genetic component Weight loss, scratching, lack of coordination Kuru progressive neurological disorder found in certain tribes in PNG Predomiantnly affected women and children transmitted by ritual cannibalism Symptoms: unsteadiness, loss of coordination, slurred speech. CJD -rare human disease affected 1 in 1 million individuals - memory loss, dementia, loss of coordination - Iatrogenic transmission recognised in 1970 could be spread by surgical equipment (prion stable) Amyloid fibres and holes in brain Spongiform encephalopathies get their name owing to characteristic holes in thin sections Deposition of amyloid links conventional amyloidosis and TSEs but does not reveal Definitive diagnosis of TSE IHC of post mortem brain tissue, mol gen studies Infection of different parts of the brains causes different diseases Transmission of CJD sporadic CJD somatic mutation or spontaneous conversion of PRPc

Familia CJD - germline mutation Iatrogenic CJD infection from PRPsc contaminated material Variant CJD infection from bovine PRPSc PrPc can be converted by infection to PrPsc, or mutation to PrPm or spontaneous folding to PrPsc PRPc is protease sensitive monomeric PRPsc is protease resistant and forms amyloids Prion hypothesis TSEs are caused by protein only infectious agent autocatalytic process to promote conversion of the endogenous isoform process may involve an unknown protein that possesses a chaperone function PrPsc increased insolubility Nuclear polymerisation model PRPc binds to prpsc filaments becomes converted Template assisted model bind to protein and gets elped Prion hypothesis evidence Robustness, extremely resistant to UV, radiation, heat mouse knockout experiments invitro conversion experiments using recombinant proteins and sequencing size of genomes and dose of radiation that it can survive smaller genome higher dose of radiation tanked Structure of prion protein buried core In vitro conversion of recombinant PRP protease resistant prion protein recovered following 21 cycles of amplification can harvest prpsc and infect animals Prnp mutations associated with inherited prion diseases not found in sporadic CJD - most common is glu200lys Different mutations lead to different phenotype of disease FFI vs CJD In absence of PrPC should render a host resistant to TSes PrPc KO mice display no overt phenotypes develop normally; However there maybe some demyelination abnormalities in peripheral nervous system Prnp knockouts completely resistant Increased expression of prion protein increases speed of onset and severity of disease Variant CJD First repeated in 2 teenagers in UK in 1996 similar to KURU than CJD BSE mad cow due to altered behaviour thought to have emerged due to how meat bone meal waws prepared infectious prion protein survived Ban in bone meal and mechanically recovered meat drastic fall in CJD and bSE (9 year delay). Much earlier onset (28 years) and lasts longer Lecture 23 Controlling viruses Non specific: hygiene and sanitation, quarantine, import controls, notification, decontamination Specific: passive immunisation, vaccination, antiviral compounds Hygeine and sanitation Water purification sewerage treatment oral/faecal viruses

Washing hands after using toilet and before food prep oral/faecal viruses Water used in irrigation. Not coughing/sneezing over people respiratory viruses

Quarantine - separate infected persons or animals with signs of disease Import controls Block animal imports from a country with disease Notification e.g. HIV, FMDV, rabies, Hendra If there is a case must be repoted to government WHO monitor and publicise the global spread of diseases Disinfection Inactivaiton of pathogens on inanimate objects useful in removing both known and unknown pathogens Bleach, acids/alkalines, UV works well across board Animal and vector control - slaughter of infected animals - insecticidal creams, protective clothing, mosquito nets Passive immunisation Injection of Ab as immune serum confers short temr protection prophylactic measure Good screening and fractionation measures essential to ensure no contamination with virus

Vaccination - stimulate a protective immune response, break the cycle of host-host spread - needs to be safe, protective for sustained period, generates neutralising antibody - stable, cheap, easy to administer Types of vaccines - live attenuated Live attenuated strains are devoid of pathogenicity but can still induce a protective response passaging of virus e.g. smallpox (jenner) Passage of virus in an unnatural host cell or host cell polio attenuated in monkey cells in lower temperature Attenuation can be achieved by passaging at lower temperature (greater fitness at low temperature) less fitness when injected. Pros: can replicate in host, antigens intact, inexpensive, activate all phases of immune system, easy admin cons: Reversion, shedding (can be spread), ensuring purity (need to separate from egg), can be problematic for immunocompromised. Killed/inactivated vaccines e.g. polio, flu Pros: Large amounts required boosters adjuvants required epitopes may be changed by denaturing Cons: no chance of reversion, inexpensive, easy to store

Live Recombinant vaccines Gene knockouts deletion of virulence gene Viral vectors cloning viral protein (e.g. coat) into vector transfect cell recombination causes protein to be expressed on vacv surface. Pros: stimulate both cellular and humoral immunity, little chance of reversion, replicate in host cons: adverse reactions to vector virus| Subunit vaccine e.g. HPV L1 (capsomeres grown in yeast) viral antigen presented to the immune system without presence of whole virus single proteins containing epitope of self assembling VLP Pros: no chance of contamination with live virus, useful if viruses are difficult to culture, not GMos Cons: Lack of sufficient immune response, adjuvant may be required, proteins may denature during purificaiton DNA vaccines Dna plasmids encode viral immunogenic proteins Transfected into host cells and viral protein expressed delivery by injection, gene gun pros: induce T/B cell response, non infectious, stable, low cost cons: Antibody responses are generally low, adjuvant required, post translation processing may be required Antiviral compounds Very few anti virals 30 on market mostly for HIV and Herpes Two approaches, rational and combinatorial Adverse effects, lack of information about virus lifecycle Useful agents must inhibit viru specific effects Can only work against active virus not latent virus Many infections are only short duration by symptoms occurring virus is no longer replicating. Producing antiviral compounds Consider bioavailability, pharmockinetics, toxicity must be safe and potent Neuraminidase zanamivir/oseltamavir Neuramindase is released by cleaving sialic acid receptor when exiting By binding neuraminidase virion becomes stuck to cell Tamiflu change to active form when it enters cell : tablet or syrup Common effects: nausea, vomiting, hedache RESISTANCE has developed Relenza inhaled as a powder side effects: bad for respiratory disease people, nausea, headache RESISTANCE appears to be uncommon. Cidofovir monophosphate nucleotide analogue of dCTP IInhibits DNA pol and blocks viral replication Active against CMV< HSV< VZV and hPV can be nephrotoxic Ribavirin Guanosine nucleoside analogue interferes with formation of viral RNA Inhibits inosine monophosphate dehydrogenase stimulates the immune system

Not sure exactly how it works Active against DNA and RNA viruses HSV, Flu, RSV, hep C HIV: nucleoside reverse transcriptase inhibitors Abavacir incorporated into viral DNA : stops function non-nucleoside reverse transcriptase inhibitors neverapine bind non competitively to RT protease inhibitors inhibits HIV proteases saquinavir, ritonavir. Interferons natural antivirals mechanism of the specific immune response Enhance expression of class 1 and class 2 MHC Obstacles in virus control Vaciines that work in lab often fail in field resistant mutants arise Drugs have unexpected side effects Some societies can t afford vaccines, antiviral drugs Lecture 24 emerging viral disease Emerging/reemerginvrial disease causative agent of a new, previously unrecognised infection in a population Agricultural/farming practices influenza Improved sanitation poliovirus Deforestation ebola Blood transfusions HIV Previoius unrecognised polio virus has existed in human population Introduced into a poplation: intraspecies smallpox originated in china approximately 4-5 thousand years ago Substantial selective pressure to develop resistance spread to the america s in 16th century no resistance millions died Rabbits virus was very effective initially in controlling Introduced into a population: interspecies (species jump) influenza A is endemic to wild birds also infect humans New variants arise unpredictability high mortality rate Poliovirus and poliomyelitis Poliovirus is small RNA virus 9kb Most infections cause mild respiratory infections approximately 3% infect cns and cause paralytic poliomyletis clubfoot Polio causes collapse in muscle function need iron lung to assist in breathing first recognised in 19th century outbreaks in US during 20th century REemegence was not due to mutations (retrospective analysis of dna) Improved sanitation led to fewer polio infections people generaly encountered polio virus much later in life Increased transport meant disease could be spread faster

Polio controlled by vaccines now. Seasonal and Pandemic flu Seasonal flu: caused by influenza afflicts 5-20% population/year, 0.1% hyospitalised, 0.01% mortality more infection more immunity. Pandemic flu: caused by newly emerging avian influenza new HA, NA serotypes unprecitable, no immunity, no vaccines e.g. Spanish flu Antigenic shift genomes of different strains mixed Antigenic drift point mutations that alter activity of flu proteins seasonal epidemics E.g. species specificity of sialic acid receptor humans 2,6 and birds 2,3 therefore bird flu is hard to pass onto humans. However pigs have both 2,3 and 2,6 receptors may provide intermediate to adapt H0 cleavage determinant in tropism HA0 must be cleaved by protease to release HA1 and HA2 fusion peptide is primed to mediate fusion Clara is a protease in humans is thought to be limiting infection in lung Birds, highly virulent HA mutatns described H5N1 1 new influenza subtype emerges : shift 2 serious illness in humans: drift 3 easily transmissible between humans (drift) Emergence of flu virus Factors travel, shift, drift Ebola virus Emerginvriuses cause haemorrhagic fevers with high fatality rate Early symptoms depend on host factors incubation 4,10 days development of fever, nausea, headache Symptoms are general misdiagnosed as other deseases such as yellow fever, malaria Symptoms progress internal and external bleeding especially in GIT, liver failure, reduced kidney function, shock, coma, organ failure Death follows 3-21 days following initial symptoms little immune response high varemia Ebola virus -ssRNA 18.9kb 7 proteins, non segmented filamentous structure Marburg virus very similar Marburg virus first identified in germay in workers who had direct contact Outbreaks sporadic and geographicaly confined Ebolavirus outbreak first happened in sudan/zaire 1976 400 case with 88% fatality Diagnosis by PCR, elisa Four subtypes Very high conservation between strains Transmission of Ebola

Direct contact with infected with primates contact with bodily fluids (not easy) NO evidence for aerosol transmission, patients are quarantined and receive therapy Current treatment for EHF no treatment or vaccine Reservoir of Ebola no reliable reservoir local bat species produced mild symptoms characteristics of reservoir Marburg outbreaks associated by their proximaty to bat caves Animal study ELISA and PCR detected positivity in some bats Gorilla and fruit bats compete for resources more frequent outbreaks Factors for emergence deforestation cause proximity of bats and gorillas Climate lead to deforestation and resources bushmeat consumption of meat could lead to infection Poverty not immediately treated/quarantined Dead-end host bird flu zoonotic viral disease that can be transmitted to humans endemic host is an animal humans are DEH because viral disease is nto maintained in human population in Bird flu strain will adapt to new host and establish itself in new reservoir DEH can transmit the viral infection but population cannot act as a reservoir Lecture 25 Overview/Wrapup Origin of viruses no accepted theory most believe viruses have ancient origins (LUCA) early cellular life believed RNA based first viruses were therefore believed to be RNA based Virus first hypothesis viruses evolved independently ( primordial soup) replicative machinery inside soup viruses then moved to parasitic way of life in cellular life Escape hypothesis fragments of RNA ; encoding a coat protein became autonomous can only replicate by entering another cell aided by coat protein (needs LUCA) reduction hypothesis one cell became an endosymbiont and deevolved acquired the ability to transmit between hosts derived from parasitic cells DNA may have been invented by viruses as a mechanism to protect against host defences providing genome stability during transmission