Viruses 2


Viruses 2

Viruses 2

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Diagnosis Specimen for Viral Diagnosis

  • Direct and indirect ways
    • Symptoms
    • Virus isolation (direct) - immunofluorescence
    • Viral antigens/virus - haemagglutination inhibition test, neutralisation, complement fixation test
    • Antivirus antibodies - immunofluorescence, ELISA
    • Molecular biology - PCR
  • Biological specimen for viral diagnosis
    • Nasopharynx swab, nasopharynx aspirate, throat swab, lesion swab, eye swab, rectal swab, blood, saliva, tissue biopsy, cerbral spinal fluid (CSF), urine, stool
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Virus Cultivation and Multiplication

  • Viruses have to be cultured in the presence of the host cell
  • This often makes research/diagnosis difficult
  • Plaque assay
    • Grow a lawn of host cells on dish
    • Add virus
    • Virus replicate and lyse host cells
    • Viral plaques: cytopathic effect
    • Count the number of plaques
    • Results of assay expressed as plaque forming units (pfu)
    • Count of infectivity not number of viruses
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  • Detection of host antibodies against virus
  • Viral haemagglutination
  • Some viruses (influenze, mumps and measles) have the property of haemagglutination (causing red blood cells to stick together)
  • Presence of anti-virus antibodies in patient serum can inhibit reaction
  • Haemagglutination Inhibition Assay (HI test) - Influenza
    • Measure how well antibodies bind to influenza viruses
    • Red blood cells (turkey, guinea pigs) in a solution will sink to the bottom of the assay well and form a red dot at the bottom
    • When an influenza virus is added to the red blood cell solution, the virus' haemagglutinin surface proteins bind to multiple red blood cells and keep red blood cells suspended
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  • Antibodies can be used to inhibit, detect and quantify virus
  • Detection of an antiviral antibody using Enzyme-Linked Immunosorbent Assay (ELISA) - uses antibodies and colour change to identify a substance 
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Molecular Biology and Diagnostic Virology

  • Modern molecular techniques of both protein chemistry and DNA/RNA biochemistry
  • Improve the specificity of virus diagnostic procedures
  • Methods include:
    • Viral DNA hybridisation with labelled probes
    • Sequencing of portions of the viral genome
    • Polymerase chain reaction (PCR)
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Antiviral Chemotherapy

  • Targets
    • Virus specific enzymes
    • Life cycle process
  • Nucleoside Reverse Transcriptase Inhibitors (NRTIs)
    • Reverse transcriptase is the number one target for drug therapy
    • Most highly active antiretroviral therapy (HAART) therapies contain a reverse transcriptase inhibitor component
    • Well-known NRTIs for HIV treatment include zidovudine (AZT) and tenofovir but also abacavir, didanosine (DDL), emtricitabine (FTC), lamivudine (3TC), stavudine (d4T)
    • AZT: Zidovudine (Azidothymidine): by mouth 500-600mg daily divided in 2-3 doses
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  • DNA is synthesised from four deoxynucleotide triphosphates
  • AZT is a nucleoside analogue - it resembles the nucleotides that are the building blocks of DNA
  • When AZT gets used to build DNA, it prevents any more from being made - it blocks further extension of the growing DNA chain
  • When reverse transcriptase is creating new DNA it 'accidentally' inserts a molecule of zidovudine (or tenfovir) rather than a nucleoside
  • This terminates the DNA chain and no more can be appended to it. Other nucleoside analogues which inhibit reverse transcriptase are ddI (Didanosine) and d4T (Stavudine)
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Non-Nucleoside Reverse Transcriptase Inhibitors (N

  • NNRTIs also targets the HIV reverse transcriptase
  • NNRTIs attach to the reverse transcriptase and affect the activity of the enzyme by restricting its mobility and making it unable to function
  • Antivirals include efavirenz, etravirine, neviparine, rilpivirine
  • Efavirenz (Sustiva): 600mg once daily
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Protease Inhibitors

  • Viral proteinsase (protease): an enzyme that cleaves peptide/polypeptide/protein chains
  • Protease cleaves the polypeptide into functional proteins - mature virus
  • HIV protease inhibitors binds to enzyme active site
  • HIV virus unable to process proteins
  • Atazanavir (Reyataz): 300mg daily
  • Darunavir, fosamprenavir, indinavir, Lopinavir, ritonavir, saquinavir, tipranavir
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Integrase Inhibitor

  • Viral integrase: an enzyme that allows the transfer of HIV cDNA to cellular DNA
  • Dolutegravir (Trivicay): 50mg once daily
  • Raltegravir (Isentress): 400mg twice daily
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Entry Inhibitor

  • Fusion inhibitor: an enzyme that prevent HIV envelope fusion
  • Enfuvirtide (Fuzeon): 90mg twice daily (subcutaneous injection)
  • Receptor antagonist: entry inhibitor; a negative allosteric modulator of the CCR5 receptor
  • C-C chemokine receptor type 5, also known as CCR5 or CD195, is a protein on the surface of white blood cells that is involved in the immune system as it acts as a receptor for chemokines (chemotactic cytokines)
  • Maraviroc (Celsentri): 300mg twice daily
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Inhibitor of Viral DNA Polymerase

  • Viral DNA polymerase: enzyme that creates DNA molecules by assembling nucleotides - essential for viral DNA synthesis
  • Acilovir is a guanosine analogue
  • Aciclovir is converted by viral thymidine kinase to aciclovir monophosphate which is then converted by host cell kinases to aciclovir triphosphate (ACV-TP)
  • ACV-TP competitively inhibits and inactivates HSV-specific DNA polymerases
  • Aciclovir (Zovirax): 200mg 5 times daily for 5 days
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Neuraminidase Inhibitors

  • Neuraminidase inhibitor: prevent the release of the virions (slow progression of infection)
  • Oseltamivir is a competitive inhibitor of the activity of the viral neuraminidase
  • It is a prodrug as it needs to be hydrolysed in the liver to be active (oseltaminivir carboxylate)
  • Oseltamivir (Tamiflu): 75mg once daily for 10 days
    • must be taken within 48 hours that symptoms arise
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Types of Vaccine

  • Inactivated vaccines
    • Poliovirus, influenze, hepatitis A virus, rabies
  • Attenuated vaccines
    • yellow fever, measles, rubella, mumps (MMR)
    • more durable immunological responses and preferred type for healthy adults
    • may not be safe for use in immunocompromised individual
  • Protein subunit vaccines
    • Hepatitis B virus; papillomavirus, infleunza (haemagglutinin, neuraminidase)
    • a viral fragment can elicit an immune response
    • protein subunit are usually produced in a bacteria/yeast (genetic engineering)
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  • Polio (poliomyelitis) mainly affects children under 5 years of age
  • One in 200 infections leads to irreversible paralysis. Among those paralysed, 5-10% die when their breathing muscles become immobilised
  • Polio cases have decreased by >99% since 1988, from an estimated 350,000 cases then to 416 reported cases in 2013
  • In 2014, only 3 countries (Afghanistan, Nigeria and Pakistan) remain polio-endemic, down from more than 125 in 1988
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