Detection of Hepatitis A Virus and Other Enteroviruses in Environmental Samples Using Gene Probe Methods

Abstract

Sensitive and specific methods are needed to detect hepatitis A virus (HAV) and other human enteroviruses in environmental samples such as drinking water and foods. Clones of cDNA representing the 5′ noncoding regions in the HAV and coxsackievirus B3 (CB3) genomes were ligated into T7/SP6 RNA transcription vectors. 32P-labeled T7 transcripts (ssRNA) of HAV and CB3 cDNA detected genomic (viral) RNA. The HAV probe showed no homology with intracellular RNAs from 6 primate cell lines or the 13 enteroviruses tested but hybridized with all 7 strains of HAV tested at levels as low as 500–1000 PFU. The CB3 probe did not react with intracellular RNAs from 6 primate cell lines or HAV RNA but reacted with all 13 enteroviruses tested. The probes were used to detect HAV and other enteroviruses in water samples after virus amplification in cell culture. HAV was detected using the ssRNA probe in samples that were negative for HAV by direct RIA and were not positive by immunoassays until several additional weeks of cell culture propagation. The CB3 ssRNA probe detected enteroviruses in a number of samples that were negative for cytopathic effects in inoculated cell cultures. Preliminary studies have shown increased detection sensitivity for enteroviruses by polymerase chain reaction amplification of target viral RNA prior to probing.