Chapter Outlines

Chapter 5      Laboratory Diagnosis of Viral Diseases

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5.1 River's Postulates
  • Modified from Koch's Postulates (proof of bacterial diseases) by T. M. River, 1937.
    • 1. Isolate virus from diseased hosts.
    • 2. Cultivation of virus in host cells.
    • 3. Proof of filterability.
    • 4. Production of a comparable disease when the cultivated virus is used to infect experimental animals.
    • 5. Reisolation of the same virus from the infected experimental animal.
    • 6. Detection of a specific immune response to the virus.
5.2 Cultivation of Viruses in the Laboratory
  • Viruses need a "host" system.
  • Viruses can be grown in:
  • Animals
  • Embryonated eggs
  • Tissue (cell) cultures (preferred method)
  • Virologist's facility
  • Laminar Flow Hoods
    • Contains HEPA filter
  • Inverted light microscopes
    • Observe cytopathic effects (CPEs) of infected cells
  • Rounding/detachment from plastic flask
  • Syncytia/fusion
  • Shrinkage
  • Increased refractility
  • Aggregation
  • Loss of adherence
  • Cell lysis/death
  • Inclusion bodies
  • Hemadsorption test
    • Common Methods Used to Study Viruses in the Research Laboratory
  • Quantitative assays
    • Plaque assays
    • TCID50 assays
  • Tables 5-1 and 5-2
  • Transformation/focus assays
    • Immortalization of cells in culture
    5.3 Viral Diagnostics in the Clinical Laboratory
    • Over 60% of all infectious disease cases seen by a physician are due to viral infections.
    • Quality of patient specimens and their transport to the laboratory is important.
      • Storage and Collection of Biological Specimens for Viral Testing
    • What types of specimens are collected to diagnose?
      • Respiratory tract infections
      • Eye infections
      • Gastrointestinal tract infections
      • Rash
      • CNS (encephalitis and meningitis cases)
      • Genital infections
      • Urinary tract infections
      • Bloodborne infections
    Three General Approaches for Laboratory Diagnosis of Viral Infections
  • Direct detection
  • Virus Isolation
  • Serology
    • Direct Detection
  • Immuno-electron microscopy
  • Immunoflourescence
  • Virus Isolation
  • Cell cultures most sensitive to suspected viruses are inoculated with the clinical specimen
  • Nucleic acid methods
  • PCR (DNA), RT-PCR (RNA)
  • Can be used to detect viruses that are noncultivatable
  • Rapid identification (e.g. RT-PCR- 4 corners outbreak of hantavirus or FRET in the field)
  • Can be used to manage patients (e.g. HIV viral load)
    • Centrifugation Culture (Shell Vial Technique)
  • Used a lot in clinical labs.
    • Viral Serology
  • Enzyme-Linked Immunosorbent Assays (ELISAs)
  • Primary and secondary responses to viral infections
    • IgM (1st exposure)
    • IgG (2nd exposure)
    ELISA Procedures
    • Western Immunoblot
    • Detection of Viral Enzymes
    • Advantages and Disadvantages of Methods
    5.4 Drug Susceptibility Testing
    • Plaque reduction assays
    A New Option in Viral Diagnostics
    • DNA microarrays or DNA chip
      • Can be used to identify novel viruses
      • Detect agents of bioterror
      • Viral pathogenicity genes
      • Patient management
      • Vaccine quality control
    • Procedure is on page 108
    Protein Arrays
    • Spin-off of the DNA chip
    • Chips probed with blood sample from a patient.
    5.5 Laboratory Safety
    • Biosafety Level (BSL) laboratories
      • BSL-1 (minimum containment)
      • BSL-2
      • BSL-3
      • BSL-4 (maximum containment)

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