Virology And Antivirals

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Virus’s are classified by:

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1. genome content: DNA or RNA
2. morphology: non-enveloped, enveloped; helical, iscosahedral
3. polarity of the genome: (positive or negative stranded)
4. mode of transmission or site of infection: enterovirused, arboviruses)

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Describe viral genomes

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• Viral genomes are DNA OR RNA
• Viral genomes can be linear or circular some RNA viruses have linear, segmented genomes
• All genomes are haploid, except retroviruses (diploid)
• All DNA viruses are double-stranded, except parvoviruses
• All RNA viruses are single-stranded, except reoviruses
• RNA genomes are either (+) stranded or (-) stranded (have polarity; see slides 10, 22)

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Herpes simplex-1 (HSV-1)

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Cold sores, viral encephalitis, opportunistic infections (systemic)

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Herpes simplex-2 (HSV-2)

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Genital ulcers, cold sores, viral meningitis, neonatal infections

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Epstein-Barr virus

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Infectious monculeosis, lymphomas

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cytomegalovirus

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Infectious mononucleosis, neonatal infections, opportunistic infections

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capsids

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Viral nucleic acids are surrounded by protein coats called capsids

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Nucleocapsid

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The genome plus capsid = nucleocapsid

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Capsomeres

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Capsids are built from capsomeres

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Envelopes

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Some viruses have envelopes; this is a lipid bilayer, often supported by a matrix of protein, taken from the host cell

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Virion

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The entire structure of a virus is a virion

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Spikes

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Many viruses have spikes (pentons or peplomers). These are used to bind to cell surface receptors.

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Noneveloped (naked) viruses

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- Enter cell by endocytosis
- stable to temperature, acid, detergents.
- can survive adverse conditions of the gut.
- released from the cell by lysis.
- can spread easily - in dust, droplets.
- antibody response enough for immunity

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Enveloped viruses

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- Some enter cell by endocytosis; have a glycoprotein which mediates fusion of viral and cell membranes
- environmentally labile, sensitive to detergents, drying, acid.
- cannot survive the gastrointestinal tract.
- must stay wet.
- released from the cell by budding.
- spread in large droplets, transfusions, physical contact.
- elicit inflammation due to CTL response.

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Enveloped viruses
Routes by which viruses enter the body

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• Droplet or particle transmission (inhalation)
influenza, common cold, mumps, hemorrhagic fevers
• Oral transmission (contaminated food and drink; saliva)
hepatitis A; viral gastroenteritis; polio
• Direct inoculation (injections; bites; trauma)
viral encephalitis; yellow fever; rabies, hepatitis B & C
• Sexual transmission
herpes simplex, HIV, hepatitis B, papillomaviruses
• Transplacental
Rubella, CMV, herpes simplex, HIV
• Blood-borne
HIV, hepatitis B, C and D, CMV

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Stages of viral replication

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1. attachment to host (absorption)
2. entry into host
3. uncoating
4. genome transport to nucleus
5. Nucleic acid replication (in nucleus for some viruses;
cytoplasm for others) and viral protein synthesis
6. Viral assembly (maturation)
7. Escape from the host cell

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viropexis

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And eveloped virus using endocytosis to enter the cell (ex. Hepatitis C)

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Stages of viral infection

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1. early phase: genome repolication; eclipse period; beginning of latent period; viral uncoating; DNA/RNA replication; protein synthesis
2. late phase: viral assembley; latent phase continues after the eclipse period; can now find intracellular viruses
3. viral release: end of latent period; extracellular viruses

Front 19

Where does Viral DNA/RNA occur?

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Nu cleus, cytosol, both

Front 20

Viral DNA replication strategies

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Parvovirus: ssDNA to dsDNA to either ssDNA virus OR new virus particles
Herpesviruses: dsDNA to mRNA proteins OR new virus particles
Hepadnavirus: (1) RNA to DNA to new virus particles OR (2) mRNA proteins to new virus particles

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Viral RNA replication strategies

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Different mechanisms are used by the different viral strains to make mRNA used to translate viral proteins and/or new virus
1. dsRNA: uses viral RNA polymerase
2. (+)ssRNA: used directly as mRNA (can be thought of as infectious pieces of mRNA)
3. (-)ssRNA: uses viral RNA polymerase
4. (+)retrovirus: uses reverse transcriptase to make dsDNA, which is transcribed by the host to make mRNA

Front 22

How are viruses released from cells?

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The release of viruses is either through cell lysis or budding. Lysis requires the coat to be made and then the cell lyses (by capsid proteins). The coat is made either spontaneously after NA is made or the NA is inserted into a pro-capsid while being synthesized.

Budding can be via plasma membrane or internal membranes. (Herpes uses the inner nuclear membrane) The vessel is then fused with the plasma membrane and released. Envelope glycoproteins are made the same way cell wall glycoproteins are made. Viral proteins can use envelope proteins with a signal sequence incorporated. This signal binds the peptide chain to the rER, enabling it to pass through the membrane. It follows the same path a released protein would (aka via Golgi). The signal can direct which side of the cell the virus is released (orthomyxoviruses bud off apical/outside of epithelial cells, rhabdoviruses bud off inner/basal surface. Retroviruses highjack a regular cellular pathways that release particles.

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How are virus infections diagnosed?

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diagnosis:

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Timeline for viral infections

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Systemic viruses first multiply locally, causing prodromal (initial or local) symptoms (lasting 1-8 days). They then spread, causing fever and major (acute phase) symptoms.
-interferon, local and nonimmune defenses mount on days 1-5
-antibody and cell-mediated immune defenses mount on days 5-8
-inflammatory and immunopathogenesis mounts on day 8, along with symptoms of disease at secondary sites; symptoms of disease at primary site occur on days 1 to 6, after which healing begins

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Prodrome vs acute phase symptoms

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1. measles: Runny nose, cough, watery eyes; primary site is epithelia of oropharynx
Influenza: ‘flu-like’ symptoms; primary site is epithelia of oropharynx
2. measles: rash, high fever, bronchitis; secondary sites include respiratory, lymphatic, brain
Influenza: dry cough, myalgias, prostration; secondary sites include lungs, rately heart and brain

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Means of viral spread to secondary site of infection

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Viremia: by blood
Many others that we don’t have to know

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How do virus’s infect cells in acute infection?

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Most viruses cause acute infections (influenza, polio).
Cytopathic effects-viruses directly kill the cells they infect.
DNA viruses: inhibit cellular DNA synthesis
RNA viruses: shut down RNA and protein synthesis (inhibit transcription)
• inclusion body formation (nuclear and/or cytoplasmic); can have assembled viral particles without DNA b/c they are so fast
• lysis

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How do virus’s infect cells in latent infections?

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Latent (persistent) infections-viruses cause recurrent disease
• no viral replication
• invisible to the immune system
• host infected for life
• cause reactivation infections years after initial infection
Examples are ALL members of the herpes virus family; HIV
Prions [actually not viruses, but historically classified as “slow viruses”] ex. kuru, “mad cow” disease

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How do virus’s infect cells in chronic infections?

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Chronic (“occult”) infections: Virus infection fails to be cleared by the immune system.
Marked by active viral replication with imperfect immune responses.
Pathology occurs months-years after infection, but occurs insidiously (symptoms do not develop until a threshold level of damage occurs).
ex. hepatitis B, hepatitis C, HIV

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Describe some other ways that virus’s cause infection

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Some viral infections are immunosuppressive: Measles, HIV
Some produce proteins to hinder immune responses: all herpesviruses; poxviruses
Some viral infections lead to secondary bacterial infections and sepsis
-Due to epithelial cell damage

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Virus’s that cause cancer

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Some viruses are oncogenic and are strongly associated with human cancers
EPSTEIN-BARR VIRUS: Burkitt’s lymphoma; nasopharyngeal Ca; B cell lymphoma (in AIDS); some Hodgkins’ lymphoma
HEPATITIS C, B, D: primary hepatocellular Ca
HPV (16 & 18): cervical cancer

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Symptoms of viral infections are caused by:

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TNF and interferons cause the flu-like symptoms

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Critical points of viral pathogenesis

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• Proteins for cell surface attachment
• Latency
• Direct cell killing
• Cell transformation (oncogenesis)
• Induction of immunopathogenic responses

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What are the major target mechanisms of antiviral drug therapy

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1. Inhibit viral attachment
2. Inhibit viral-cell fusion
3. Inhibit uncoating
4. Inhibit nucleic acid synthesis
5. Inhibit proteases
6. Inhibit the release of viral particles

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Agents Used to Treat Infections caused by Herpes Simplex Virus (HSV) and Varicella-Zoster Virus (VZV)

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Acyclovir*
Docosanol*
Famciclovir* (converts to peniclovir after oral)
Penciclovir
Trifluridine*
Valacyclovir*

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Acyclovir MOA

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1. An acyclic guanosine derivative
2. Active against HSV-1, HSV-2 and VZV
3. Activation requires a 3-step phosporylation; requires HSV-specific thymidine kinase (initial phosphorlation) and host cell enzymes
4. Acyclovir triphosphate inhibits viral DNA synthesis through 2 mechanisms:
( 1). Competes with deoxyGTP for the viral DNA polymerase
(2.) Chain termination following incorporation into the viral DNA

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Pharmacokinetics of Acyclovir

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• Oral bioavailability is unaffected by food
• IV and topical formulations available
• Diffuse rapidly into most tissues & body fluids
• Cleared mainly via kidney

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Clinical Uses of Acyclovir

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1. ORAL: first episode genital herpes; recurrent genital herpes; genital herpes suppression; varicella; zoster
2. IV: severe HSV infection; herpes encephalitis; neonatal HSV infection
3. TOPICAL: herpes labialis

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Acyclovir MOR and Adverse Rxns

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MOA: alteration of virla thymidine kinase
Adverse Rxns: nausea; diarrhea; headache; ****renal dysfunction or neurologic toxicity (IV); ***adequate hydration should be maintained

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Valacyclovir

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• The L-valyl ester prodrug of acyclovir
• Rapid converted to acyclovir after oral administration
• Clinical Uses (oral)
• Treatment of first or recurrent genital herpes
• More effective in treating VZV than acyclovir***

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Famciclovir

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• Converted to penciclovir after oral administration
• Mechanism of Action: Competitive inhibition of viral DNA polymerase*

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Docosanol (Abreva)

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• A saturated 22-carbon aliphatic alcohol
• Mechanism of Action: Inhibits fusion between plasma membrane and the HSV envelope

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Trifluridine MOA and clinical uses

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MOA: Activation requires phosphorylations by host cell enzymes (but not HSV TK)**; Trifluridine triphosphate competes with TTP for incorporation by viral DNA polymerase
Clinical Uses: Treatment of herpes keratitis and acyclovir-resistant HSV infections
**Not for systemic use (incorporated into both viral and host DNA)

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Agents used to treat CMV (Cytomegalovirus) Infections

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Cidofovir*
Foascarnet*
Ganciclovir*
Valganciclovir

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Ganciclovir MOA and MOR

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 An acyclic guanosine analog
MOA:
• Phosphorylation-dependent drug activation*
• Initial phosphorylation is mediated by the CMV-specific protein kinase phosphotransferase UL97*
• The activated compound competitively inhibit viral DNA polymerase and cause termination of DNA elongation
MOR: mutation in UL97*

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Ganciclovir clinical uses and adverse rxns

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1. Clinical Uses: CMV prophylaxis or CMV retinitis treatment
2. Adverse Rxns:
• Myelosuppression (esp. IV)**
• Nausea, diarrhea, fever, rash, headache, insomnia, peripheral neuropathy, and retinal detachment
• CNS toxicity, hepatotoxicity
• Carcinogenic and embryotoxic at high doses in animals

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Foscarnet MOA, admin, uses, rxns

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1. An inorganic pyrophosphate compound
2. Mechanism of Action: Inhibits viral DNA/RNA polymerase & HIV reverse transcriptase
3. Admin: IV only (poor oral bioavailability); Renal clearance
4. Adverse Rxns: Renal impairment (avoid other nephrotoxic drugs)
• Electrolyte disturbance
• CNS toxicity (seizure)

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Cidofovir MOA, uses, rxns

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1. MOA
• Phosphorylation-dependent activation
• Activated cpd inhibits viral DNA polymerase
2. Clinical Uses:
• Treatment of CMV retinitis
• IV must be administered with probenecid to reduce nephrotoxicity
• Aggressive adjunctive hydration is needed
3. Adverse Rxns: Dose-dependent nephrotoxicity (reduced by saline prehydration)