Virus Misc.txt

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emm64
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143010
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Virus Misc.txt
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2012-03-21 21:51:40
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Virus Micro
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Virus Micro
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  1. Viral diagnosis
    • infectivity
    • visualization
    • detection of components
    • evaluation of immune response (serology)
  2. Cytopathic Effect (CPE)
    • - virus-induced morphological changes of cells
    • - such as cell rounding, shrinkage, syncytia…
    • - visible by light microscopy
    • Characteristic of each virus but not all cause CPE
    • Most viruses take 5-10 days to show CPE
  3. Plaques
    • Cytopathic effect (CPE)
    • - focal areas of cell destruction
    • Each plaque originates from one infectious virus
    • Can be used to quantitate infectious virus particles
  4. Syncytia
    • multinuclear cell, fusion of multiple cells
    • Caused by the fusion protein of paramyxoviruses
    • - measles virus
  5. Hemadsoption
    • - Red blood cells adhere to the infected cells
    • - viral protein, hemagglutinin, bind red blood cells
    • - influenza, mumps, parainfluenza
    • Cells infected by influenza, mumps, parainfluenza express viral proteins (hemagglutinin) on the surface and stick to red blood cells

  6. Infectivity
    • (need live virus in the specimen):
    • Gold standard for virus detection
    • Highly sensitive
    • Problems
    • - Need for technical expertise
    • - Generally long incubation period (5-10 days)
    • - Some viruses cannot grow in cell culture
    • - Some viruses do not produce CPE
    • - the identify of the virus still requires additional tests
    • - Not for point-of-care testing
  7. Visualization
    • Each virus has its own unique shape
    • Limitation
    • Poor sensitivity ( require >10^6 viral particles/ml)
    • - viral gastroenteritis (at the peak of diarrhea, >10^10 in feces)
    • Require skilled staff
    • Costly and complicated instrumentation
    • Not a routine diagnosis method, but a great research tool and a useful approach to identify unknown viruses
  8. Electron microscopy
    • SARS – crown shape, a novel coronavirus
  9. Detection of viral components
    • Viral proteins
    • - capsid
    • - glycoprotein
    • - nucleoprotein
    • Nucleic acids
    • - RNA/ DNA
    • Require antibodies
    • - Immunoassay, ELISA (enzyme-linked immunosorbent assay)
    • - immunofluorescence, western blot
    • Protein activity
    • - enzymatic function
    • - hemagglutination (binding to red blood cells)
  10. Immunoassay of viral proteins
    • require two antibodies: detection and capture
  11. ELISA
  12. Rapid detection
    • allows for early administration of anti-viral treatment
    • can be performed at point-of-care settings but not hign sensitivity

  13. Western blot
    • Using antibodies against viral protein for detection
    • HIV-infected cells
    • anti-HIV capsid Ab
  14. Immunofluorescence assay
    • Using antibodies against viral protein for detection
    • Influenza A infected epithelial cells
    • anti –influenza A Ab (fluorscent green)
  15. Detecting activities of viral proteins
    • (less common)
    • Enzymatic activity
    • - Reverse transcriptase (Retrovirus) – copy RNA into DNA
    • - Neuraminidase (Influenza) – cleaves sialic acid
    • Hemagglutination
    • – hemagglutinin, binds to red blood cells
    • - Influenza, Mumps, Parainfluenza
  16. HA test
    • Titer=32 HA units
    • Hemagglutination assay. Seven different samples of influenza virus, numbered 1 through 7 at the left, were serially diluted as indicated at the top, mixed with chicken red blood cells (RBC), and incubated on ice for 1 to 2 hours. Wells in the bottom row contain no virus. Agglutinated RBCs coat wells evenly, in contrast to nonagglutinated cells, which form a distinct button at the bottom of the well. The HA titer, shown at the right, is the last dilution that shows complete hemagglutination activity
  17. Detection – viral nucleic acids
    • Using labeled DNA (RNA) sequence to detect viral genomes
    • Classical (no amplification)
    • - In situ hybridization HPV
    • Amplification by polymerase chain reaction (PCR)
    • - primary tool
    • - great sensitivity
    • - problems: false positive, contamination
  18. Virus-specific antibody
    • Part of body’s protective immune response
    • indicates a past or recent infection or
    • chronic infection (retrospective)
  19. Using viral proteins for detection of antibody
    • ELISA
    • IFA (Immunofluorescence assay)
    • Western blot
    • Neutralization
    • - some antibodies can inhibit viral infection
    • Hemagglutination inhibition assay (IFA)
    • - some antibodies target hemagglutinin and can block its activity
  20. Indirect ELISA
  21. Neutralization
    • (virus-specific antibody blocks the virus to infect cells, thus to inhibit CPE)
  22. Assays for infection
    • During disease stage (acute phase)
    • choosing anti-viral therapy and deciding prevention
    • - PCR of viral nucleic acids
    • - ELISA of viral proteins
    • During chronic infection or after resolution
    • blood screening and epidemiology studies
    • - serological studies of virus-specific antibodies
  23. Neutralizing antibodies
    • - natural host responses to virus infection
    • - bind to viral envelope proteins and blocks the binding to cells
    • - pooled human immuneglobulin
    • - e.g. Hepatitis A virus, Hepatitis B virus, Rabies
  24. Uncoating targeting drugs
    • Pleconaril – picornaviruses (Polio, common cold viruses) blocking release of viral genomes
    • Amantadine – influenza A virus
    • Rimantadine blocking virus release from vesicles into cells
  25. Nucleic Acid Synth targeting
    • Most anti-viral drugs target to this step.
    • Viruses encode their own enzymes for this step.
    • - DNA polymerase, DNA  DNA
    • - Reverse transcriptase, RNA  DNA
    • - RNA polymerase, RNA  RNA
    • Most drugs of this type are nucleoside analogues.
    • DNA Polymerase->Herpesviruses (HSV, VZV, CMV)
    • Reverse Transcriptase->HIV Hep B
    • RNA Polyerase->RSV Hep C
  26. Protein synthesis targeting
    • Interferons (α and )
    • Body’s natural responses to infection
    • Actions
    • – Degradation of viral mRNA
    • - Inhibition of protein synthesis
    • - Enhance host immune response
    • Used to treat chronic HBV and HCV infection
  27. Assembly and release targets
    • protease inhibitor
    • - HIV, assembly
    • Neuraminidase inhibitor
    • - Influenza (H1N1), release
    • - Tamiflu

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