DETECTION OF HEPATITIS-A VIRUS IN MERCENARIA-MERCENARIA BY COUPLED REVERSE TRANSCRIPTION AND POLYMERASE CHAIN-REACTION

Hepatitis A virus (HAV) is a major cause of infectious hepatitis in hu mans. In this respect, bivalve mollusks pose a major health concern be cause they are filter feeders and can concentrate the virus up to 900- fold from contaminated water. Detection of HAV has been hampered becau se wild-type HAV grows poorly if at all in cell culture. Here we descr ibe a technique for the detection of HAV in shellfish based on reverse transcription coupled with the polymerase chain reaction. RNA is isol ated from hard-shell clam tissue and reverse transcribed with avian my eloblastosis virus reverse transcriptase. A portion of the cDNA pool i s then amplified with primers specific for HAV. In experiments with an in vitro-synthesized HAV transcript, we were able to detect HAV seque nce in the presence of a 200-million-fold excess of shellfish RNA. Whe n intact virus was added to shellfish tissue before the isolation of R NA, the method was capable of detecting 10 viral RNA molecules in a re action mixture.