In situ hybridization for quantitative assay of infectious hepatitis A virus.

A method of in situ hybridization using single-stranded RNA probes of opposite polarity for quantitative enumeration of hepatitis A virus (HAV) in infected cells has been developed. Kinetic experiments showed that foci of infected cells appeared as early as day 2 postinfection. The absence of foci in cells examined immediately after virus adsorption indicated that foci detected subsequently were related to viral replication. Foci were detected by hybridization with RNA probes complementary to HAV genomic RNA but not with RNA probes identical to HAV genomic RNA. The number of foci observed was linearly related to the HAV dose inoculated. Focus formation was reduced when a virus inoculum was pretreated with guinea pig anti-HAV hyperimmune serum but not when it was pretreated with preimmune serum. The high resolution of hybridization signals and relative rapidity of the test indicated that this technique will be useful for measuring serum neutralizing antibodies and for quantitative assay of infectious HAV.