Virology 9: Active Viral Infection

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13 Terms

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Fluorescent antibody test

  1. Detects Ag

  2. Apply a fluorescently labeled, virus-specific Ab

  3. The Ab binds the matching Ag in cells or tissue

  4. A fluorescence microscope reveals the staining pattern


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Lateral flow immunoassay

  1. Sample carries Ag and labeled Ab along the strip

  2. A capture Ab traps the Ag complex at the test line

  3. The control line checks that the strip has run


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Enzyme-linked immunosorbent assay (ELISA)

  1. Anti-viral antibodies (capture and secondary): usually mAbs against different epitopes of the viral protein being captured

  2. Secondary antibody labeled with enzyme that will react with the substrate to produce a chromogenic reaction

  3. Does not require infectious virus


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PCR

Amplifies a defined DNA target

  1. Primers define the sequence to amplify

  2. DNA polymerase extends primers using nucleotides (dNTPs)

  3. For RNA, make complementary DNA (cDNA) by reverse transcription


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Conventional PCR

End-point test

  1. Amplification: exponential growth, then reduced efficiency and a plateau

  2. Band intensity does not reliably measure starting viral load


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qPCR

  1. Monitors amplification during cycling

  2. Fluorescence is recorded during PCR cycles

  3. Detection uses a probe or a DNA-binding dye

  4. For RNA targets, use RT-qPCR


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Ct

When fluorescence crosses the threshold in qPCR

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20, interchangeable, infectious virus

  1. Ct 20 vs. Ct 32

    1. With the same assay and comparable specimens, Ct [20/32] usually means more starting target

    2. Ct values from different assays or specimen types are not directly ___

    3. Ct does not by itself prove ___ or explain disease severity


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Recoverable infectious virus

What does a positive result support for virus isolation?

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Viral antigen detected

What does a positive result support for FA, IFA, and antigen ELISA?

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Target DNA/RNA detected

What does a positive result support for PCR / RT-qPCR?

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Host antibody detected

What does a positive result support for serology?

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Virus isolation

What is the only test in this lecture that requires an infectious virus?