During the halcyon days of the Rockefeller Foundation's Virus Research Program through the 1950s and 1960s hundreds of previously unknown arboviruses were isolated from arthropods, wild birds and mammals, domesticated animals, and humans by intracerebral inoculation of infant mice. The suckling mouse is a particularly sensitive host, still used by some laboratories for diagnostic purposes today, but is progressively being replaced by cultured cells. Vero or BHK-21 cells are the most commonly used vertebrate cell lines, but invertebrate cell lines such as C6/36 from the mosquito Acdes albojnctus tend to be more sensitive. Cytopathic effects do not regularly occur. The isolate is identified either by immunofluorescence (using the fixed monolayer), or hemagglutination inhibition, complement fixation, or EIA (on the culture supernatant), to place the virus in the correct serogroup, followed by neutralization using appropriate monoclonal antibodies to characterize the species. Apart lrom brain or other tissue sampled at autopsy, the only appropriate specimen for isolation of virus is blood taken while the patient is still viremic. Since the probability of isolating virus drops markedly after the first couple of days of illness, serology should also be used to avoid missing the diagnosis.
During an epidemic, an IgM capture EIA is routinely employed to detect antiviral IgM in a single specimen of serum; such antibodies are almost always detectable during or before the second week of the illness. However, because of serological cross-reactions between alphaviruses it is important to confirm the diagnosis by demonstrating a rise in neutralizing antibody in paired sera.
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