Diagnostic Virology

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Viruses grown in lab in . . .

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cell cultures

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best time to collect viral samples

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early in acute phase when it is still being shed

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enveloped viruses are (stable/unstable) outside host

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unstable

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Virus transport medium (VTM) components (3)

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protein (usually serum)
pH buffer
Antimicrobials

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Storage relating to temperature

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store on ice but do NOT freeze

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cytology

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microscopic observation of tissue for cytopathologic effect (CPE)

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syncytia

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multinucleated giant cells formed by cell fusion

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inclusion bodies

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histologic changes caused by viruses

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Owl's-eye inclusion bodies (and prominent example)

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basophilic nuclear IB
(CMV)

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Cowdry Type I IB (and example)

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eosinophilic nuclear (herpes simplex, varicella)

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Negri bodies

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cytoplasmic, round/oval (rabies)

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primary cell culture

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use trypsin or collagenase to separate cells, grow as monolayers, limited number of times culture can be passed

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diploid cell lines

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single cell type that can be passed many but finite amount of times

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tumor cell lines/immortalized cell lines

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single cell type, passed infinitely

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Plaque

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visible colony of lysed cells in a CPE

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heterologous interference

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one virus prevents replication with another

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hemadsorption

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RBCs bind to surface of infected cells in culture

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hemagglutination

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viral particles released agglutinate RBCs, forming clumps

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hemagglutination inhibition (HI)

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blocking agglutination with specific antibodies against a virus

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Tissue culture dose (TCD-50)

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dilution of virus causing CPE in 50% of cells

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Lethal dose (LD50)

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dilution of virus that kills 50% of a set of test animals

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Infectious Dose (ID50)

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dilution of a virus that initiates detectable symptoms, antibody, or other response in 50% of test animals

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Plaque forming units (PFU)

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ONE virion/cell, only neighboring cells infected (forming colony), CPE visible, 10-fold dilution series

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Advantage/disadvantage of PFU

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Advantage: quantitative
Disadvantage: must grow in culture, too slow, and need lots of virus

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antibody used in serologic testing

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IgM

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Seroconversion postive reaction

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must see 4 fold increase or greater in titer in the convalescent serum compared to the acute serum (p. 32)

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Direct immunofluorescence

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primary Abs linked to fluorescent molecule bind to viral Ag

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Indirect immunofluorescence

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primary Abs are bound to cells w/ viral Ags, fluorescent 2ndary Abs added

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Enzyme Immunoassay (EIA)

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enzyme-conjugated Ab used w/ a substrate that is converted to a color when Ab is bound

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ELISA - Ab detection

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viral Ag attached to surface and then add patients Abs, then use enzyme-linked 2ndary Abs

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ELISA - Ag detection

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Abs for specific virus attached to surface, then add patients serum, then add enzyme-linked 2ndary Abs against predicted Ag

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Radioimmunoassay (RIA)

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like ELISA but with radiolabeled Ab

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Latex agglutination (LA)

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beads coated with Ag or viral Ab used to bind patients serum; if they clump, get a positive reaction

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shell vial (glass tube)

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enhances detection of viable virus particle

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Western blot

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Agar, filter paper, probe with Abs, add enzyme-linked Abs, positive rxn = color bands

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Dot-blot for western blot

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instead of using electrophoresis, just place proteins on defined spots

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advantages of using genomic detection of viruses (6)

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1) if virus does NOT cause CPE
2) low levels of virus
3) Abs/Ags cross-react w/ other viruses
4) done rapidly
5) sensitive (goes with 2)
6) specific (goes with 3)

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in situ Hybridization

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fixed biopsy specimens are probed directly with DNA probes. Shows cellular localization. Good for slowly replicating viruses.

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Southern blot

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DNA w/ electrophoresis

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Northern blot

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RNA w/ electrophoresis

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reverse transcriptase PCR (RT-PCR)

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amplify RNA

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real-time PCR

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quantitative analysis of RNA/DNA; compare level of fluorescence over time

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Branched-chain DNA assay

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Use PCR to detect small amounts of DNA/RNA sequences --> use it for HIV