Virology Midterm Ii

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Poxviridae

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Large dsDNA virus, enveloped. Cytoplasmic replication. Contains small pox virus (variola virus). Infects invertebrates and vertebrates. Brick shaped. Virion contains active enzymes needed to start DNA replication and early viral gene expression:Incl.enzymes for nt and mRNA synthesis

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Orthopoxviruses (Poxviridae)

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Large linear dsDNA concatenated genomes (180+ kbp -- up to 200 genes). Encodes RNA pol, has 5' capping enzyme, and has 3' poly A polymerase. Have telomere-like tandem repeats to allow replication of linear DNA. Replicates in cytoplasm.

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Primase

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Makes the RNA primer

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Adenoviruses

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Linear dsDNA genome; 20-36 kbp Icosahedral virions, spiked, non enveloped, 70-90 nm + 9-77 nm spikes i.e., medium size genome and virions. Can only replicate in a dividing cell.

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Adenovirus claims to fame

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(1) discovery of mRNA splicing (Nobel prize);employs alternate splicing to yield many partially overlapping RNAs (2) 5´-terminal genome-linked protein (protein-primed DNA synthesis)

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E3

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In Adenoviruses. Causes immune disruption. If in non-susceptible host, often E3 loses it's function due to lack of selection.

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VA RNAs

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dsRNA decoys that interfere with the interferon and RNAi responses (found in Adenoviruses).

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Adenovirus DNA replication

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Not circularized, so it needs to find a place to start. Pre-terminal protein binds to DNA ends; a Ser residue forms a phosphodiester to CTP, which presents a 3´-OH for priming. Adenovirus DNA Polymerase used for synthesis (as well as its own SSBs).

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Parvoviridae

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Smallest DNA virus ~ 5.5 kb ssDNA, linear2-3 genes, but differential splicing allow up to ~10proteins to be made. Small icosahedral virions, ~20 nm,non-enveloped. Cannot stimulate cell division. Restricted to dividing cells - dependent upon mitotically active cells. Often in immunesystem cells. Often infected Human B19 virus has initial replication in upperrespiratory tract; spreads to erythroid cells

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Densovirinae

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Virusesof insects

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Parvovirus

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Hosts aredogs, other mammals and birds (Parvovirinae)

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Dependovirus

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Requireshelper virus (adeno, herpes or papilloma) e.g., Adeno-associated viruses (AAV); 90% of adults have Abs. (Parvovirinae).

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Erythrovirus

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B19- human host: ‘fifth disease’ rash. Mild, common, ~50% of adults are seropositive. Occasional seriousdisease. Causes non-specific flu-likesymptoms. Can cause hydrops (fetal edema). Infection of adults can result in chronicjoint inflammation ( Parvovirinae).

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Gag

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(Group specific antigen): nucleocapsid,martix, capsidtranslated fromgenome-length mRNA, followed by proteolytic processing by viral protease

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Pol

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reverse transcriptase, RNAse H, and integrase (retroviruses)

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Pro

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(protease), can be initially fused as polyprotein with gag or pol, or separate; aspartate protease (2 aspartates in active site); acts as dimer - requires high concentration of proteins (e.g. within virion) for dimer partners to bepresent•This prevents thefragmentation of proteins before assembly

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RTase

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RNA-dependent (or DNA dependent) DNA Polymerase

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RNase H

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RNase H degrades RNA in a DNA:RNAhybrid

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Env

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(envelope), a precursor protein that iscleaved by host furin protease in the ER to yield the envelope SU (surface) andTM (transmembrane) proteins

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Read Through (RT)

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Read through; stop codon occasionally suppressed and can result in a long fusion protein (inefficient, only a few percent).

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Frame Shift (FS)

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Frame shift; ribosome slips one nucleotide back. Allows continued translation.

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Picornaviridae

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-7.5kb

-Non-env, spherical 30 nm

-Poliovirus, human rhinovirus, hepatitis A, foot and mouth disease

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Smallpox as a biowarfare agent

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-Easy togrow
-Does notrequire refrigeration
-Respiratoryspread10-14 dayincubation period-Psychologicaleffects
-Environmentalstability
-Otherpoxviruses with similar pathology…..

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Why a bioterrorist might consider releasing smallpox in theUS?

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-Susceptiblepopulace – no longer vaccinated-Vaccinedepleted - vaccine no longer stock piled -Vaccineside-effects – therefore people are reluctant to be vaccinated

-Noantiviral drug

-Potentialglobal catastrophe

However, how coulda bioterrorist guarantee that the infection would not spread to alliedcountries or people?

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Myxomatosis Virus (MYXV, Poxviridae)

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Afterdetermining that MYXV would not adversely affect domestic stock or native animals, MYXV introduced toAustralia in 1950 killed 95% of feral rabbits in 10 years This ledto strong selection for lower virulence in virus, facilitating transmission,and genetic resistance in rabbits. Overall mortalityrate was greatly reduced by within a few years after introduction.

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Pseudotyping

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Process of producingviruses or viral vectors in combination with foreign viral envelope proteins

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Controlling Rabies

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Vaccinate wildanimals – protect humans and domestic animals. Contaminatebait with a recombinant vaccinia virus expressing a rabies virus glycoprotein. Vaccinia infects but does not cause disease in a wide variety of animals. Because the vaccinia virus expresses the rabies proteins, the animals becomeimmune to rabies. 12 million doses of RABORALV-RG® vaccine are used each year.

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Terminal inverted repeats

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Present in Parvoviridae. Allow ends to snap back and those creates a hydroxy group for the polymerase to bind to and replicate the DNA.

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Gene therapy

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Adenovirus - can package up to 5 kb of DNA, can integrate Problems: people may be immune because ofprevious exposure

Herpesviruses - large genome, esp. CMV, with capacity for geneinserts

Baculovirus - insect virus,large DNA genome, does not infect vertebrates,but can deliver DNA into cells

If vector inserted near oncogene, can causeelevated expression. This caused the immume cells toproliferate -> Leukemia

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Hepadnaviridae

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-DNA -> RNA -> DNA (pararetrovirus)

-Tinygenome (3.2 kb)

-Envelopedvirions; multiple serotypes

-Productionof large amount of particles lacking DNA

-Tropismfor liver, lymphocytes,pancreas

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HBV (Hepatitis B Virus) Genes and Gene Products

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Genomeencodes four overlapping genes:

-S(envelope or surface antigen) (3 forms/lengths: L, M, S)

-C(core protein) makesicosahedral capsid

-P (DNApolymerase that also has reverse transcriptase activity)

-X (trans-activating protein) X enhances the replication of HBV

-ORF overlap greatly, allowing for smaller genome size

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Replication of HBV

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1.Partially DS DNA genome is transported to nucleus & converted to covalently closed circular DNA

2. Transcribed by cellular RNApolymerase + virus factor

3.mRNAs and pre-genomic (pg)RNA

4.pgRNA is packaged into nucleocapsid, reverse transcribed (-) strand andpartial plus strand are made (RT and RNAse H activity)

5. Bud out through ER and obtain envelope

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Protein P (Hepadnaviruses)

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Piscomprised of terminal protein[tp] (with tyroosine -OH), reversetranscriptase [rt], and RNAse H domain.

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Togaviridae and Flaviviridae

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(+)single-stranded RNA genomes

-10-12kbsingleRNA genome

-components

-Cytoplasmic replication (capping enzyme)

-spherical,enveloped

-mostlyarboviruses with wide host range

-Viralfamilies with many similar properties but not closely related genetically

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Togaviridae diseases

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Alphavirus-> Chikungunya

Rubivirus -> Rubella -> German measles

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Alphavirus genome (Togaviridae)

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-Poly proteins (nsP = non structural protein), e = envelope, c = capsid.

-CAP and poly-A tail.

- + strand synthesis, can start at 3' end or internally (to make subgenomic RNA).

-Important proteins: RNA-dependent RNA polymerase, helicase, capping enzyme (methyl transferase).

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Flaviviridae genera

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Flavivirus, Hepacivirus, Pestivirus

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Flaviviridae diseases

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Flavivirus -> Zika, Dengue 1-4, Yellow Fever

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Flavivirus genome

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-Poly protein, 5' CAP, NO POLY-A tail, similar arrangement to picornaviruses (structural proteins first, then non-structural proteins).

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IRES

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internal ribosomal entry site; nucleotide sequence that allows for translation initiation in the middle of a messenger RNA (mRNA) sequence as part of the greater process of protein synthesis. Present in hepacivirus and picornaviruses

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Virokines

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(Poxviridae) mimiccytokines – competitive inhibitors

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E1A andE1B

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(Adenoviruses) Oncogenes in animal hosts; Forcecell into cell cycle S phase by binding Rband p53 tumor suppressors. E1Aalso inhibits transcription of interferon-stimulated genes E1Bblocks apoptosis.

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The gag/pro/pol

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Made from a single mRNA, by translationinvolving frameshifting and/or stop codon suppression (read-through), followedby proteolytic maturation

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Slippery sequence

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7nt sequences of reiterated nucleotides upstream of ahairpin (leads to frame shift in retroviruses)

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HIVPathogenesis

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1. Entry atsites of M cells or trauma (STDs) M (microfold) cells are in Peyer’s patches in gutlining and specialized for antigen uptake (lymphoid cells) 2. Transit to lymph nodes by binding to C-type lectins on dendritic cells 3. 3. Peak CD4+ Tcell infection days 4-74. Viremiapeaks day 145. All lymphoidtissues infected by day 23

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HIV Detection

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Generally do a serological test(antigen/antibody combination immunoassay), then differentiation assay andfinally RTPCR (if necessary).

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How is HIV so effective at escaping immune control?

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1.Reverse transcriptase is highly errorprone - no proof reading; high mutation rate leads to antigenic drift of gp120,can occur during a multi-year infection2.Heavy glycosylation ofgp120 evades antibody detection .i.e. decorated with common sugars (notunique antigens)3.Loss of CD4+ helper cells that arerequired for CD8 killer cell and antibody responses4.Direct infection and destruction of thecells of the immune system5. Proviruses arepresent (latency-reversing drugs are being developed to cause HIV geneexpression from provirus and detection of infected by the immune system)

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HAART

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Doubleortriple therapy(highlyactive antiretroviral therapy)

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HIV-1

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closelyrelated to SIV present in chimpanzees

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HIV-2

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closelyrelated to SIV present in sooty mangabeys

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protein P

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(Piscomprised of terminal protein[tp], Reversetranscriptase [rt] and RNAse H domain; in hepadnaviruses

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Why wasit difficult to develop tissue culture infections for HBV?

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Onlyin hepatocytes, difficult toculture because differentiated state wasn’t stable.

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What arethe serious clinical outcomes of HBV infection?

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Carcinoma

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What isnotable about the BHV vaccine?

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SubunitVaccine

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Whatviral particles are produced by infection?

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Emptycapsids.

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What is apoint of similarity between HBV and adenovirus?

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Proteincovalently linked to genome and used as a primer

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CD4 CELLS

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HIV replicates in them

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Picornavirus genome replication

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-Requires viralpolymerase (RdRp)

-Error rates ~ 1 in 10^4-10^5

-Eukaryotic (+) strandvirus replication is associated with host membranes, which form “spherule”compartments about the size of a virion.

-Replication proceeds by transcribing full-length (-) RNA from the (+) genome, followed byfull-length (+) from the (-) intermediate.

-usual ratio of (+) to(-) strands in an infection is 20:1 to 50:1.

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Enterovirus (poliovirus)

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-Enter via aerosol or ingestion

-Virus replicates in oropharynx

-Virus replicates in Meyer's patches.

-Causes viremia (either Polio, Echo and/or Cox)

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Polio Attenuated vaccinestrain (Sabin)

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- Confers strong systemic immunity

- Inexpensive, oral delivery

- Herd immunity (vaccine excreted)

- Potentialto revert to virulent strain

- Requirescold chain in storage/delivery

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Polio Killed virus vaccine(Salk)

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-Safe (no reversion)

- Can be used in immuno-compromisedpatients

- Systemic immunity

- Intramuscularinjection with repeat doses

- More expensive

- No mucosal immunity in gut: can’t eradicate wild polio

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Footand Mouth Disease Virus

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-Picornaviridae

• Extremely contagiousinfection of cloven-hoofed animals, incl. deer, antelopes, camels, pigs; can be inapparent in sheep: large wild reservoir

-High fever, blistersin mouth and hooves

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polyprotein

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(Picornaviridae) translationmakes a large precursor polyprotein thatis cleaved to yield multipleproducts by a viral protease

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Genome segmentation

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: division of genomeinto >1 RNA

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Subgenomic mRNA

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alongside genomereplication, partial genome transcription producesshorter 3´-coterminal mRNAs