DETECTION OF HUMAN ENTERIC VIRUSES IN OYSTERS BY IN-VIVO AND IN-VITROAMPLIFICATION OF NUCLEIC-ACIDS

This study describes the detection of enteroviruses and hepatitis A vi rus in 31 naturally contaminated oyster specimens by nucleic acid ampl ification and oligonucleotide probing. Viruses were extracted by adsor ption-elution-precipitation from 50-g oyster samples harvested from an area receiving sewage effluent discharge, Ninety percent of each extr act was inoculated into primate kidney cell cultures for virus isolati on and infectivity assay, Viruses in the remaining 10% of oyster extra ct that was not inoculated into cell cultures were further purified an d concentrated by a procedure involving Freon extraction, polyethylene glycol precipitation, and Pro-Cipitate precipitation, After 3 to 4 we eks of incubation, RNA was extracted from inoculated cultures that wer e negative for cytopathic effects (CPE), These RNA extracts and the RN A from virions purified and concentrated directly from oyster extracts were subjected to reverse transcriptase PCR (RT-PCR) with primer pair s for human enteroviruses and hepatitis A virus, The resulting amplico ns were confirmed by internal oligonucleotide probe hybridization. For the portions of oyster sample extracts inoculated into cell cultures, 12 (39%) were positive for human enteroviruses by CPE and 6 (19%) wer e positive by RT-PCR and oligoprobing of RNA extracts from CPE-negativ e cell cultures: For the remaining sample portions tested by direct RT -PCR and oligoprobing after further concentration, five (about 16%) we re confirmed to be positive for human enteroviruses. Hepatitis A virus was also detected in RNA extracts of two CPE-positive samples by RT-P CR and oligoprobing. Combining the data from all three methods, enteri c viruses were detected in 18 of 31 (58%) samples, Detection by nuclei c acid methods increased the number of positive samples by 50% over de tection by CPE in cell culture, Hence, nucleic acid amplification meth ods increase the detection of noncytopathic human enteric viruses in o ysters.