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Title:   Author  item
                                            Kingsley, Daviditem
                                            Meade, Gloriaitem
                                            Richards, Gary
  Submitted to: Applied and Environmental Microbiology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 5/1/2002
                                    Publication  Date: 8/9/2002
                                    Citation: N/AInterpretive Summary: Using molecular biological techniques, hepatitis A virus (HAV) and Norwalk virus were identified in clams imported into the United States from China.  These clams, served at a restaurant in New York State, were associated with an outbreak of Norwalk-like illness with symptoms including vomiting and diarrhea.  The clams were labeled as "cooked",  but appeared raw.  To our knowledge, this is the first report identifying both hepatitis A and Norwalk-like virus in a contaminated food, and this positive test provides a direct link between the implicated clams and viral illness associated with restaurant customers.  Detection of these viruses by a modification of our recently published virus extraction and assay procedure validates this newly developed testing method for shellfish.  Bacterial analysis of clam meats suggests that these shellfish were harvested from waters which were heavily  contaminated with fecal waste.
                                Technical Abstract: Hepatitis A virus (HAV) and Norwalk-like virus (NLV) were detected by reverse transcription-polymerase chain reaction from clams imported into the United States from China. An epidemiological investigation showed that these clams were associated with five cases of Norwalk-like gastroenteritis in New  York State in August, 2000 (FDA Import Alert # 16-50).  They were labeled as "cooked", but appeared raw. Viral RNA extraction was performed using dissected digestive tissues rather than whole shellfish meats, followed by glycine buffer elution, PEG precipitation, Tri-ReagentTM treatment, and purification of poly A RNA using magnetic beads coupled to poly dT oligonucleotides. We identified HAV as genotype I using primer sequences which amplify the VP1/2A junction.  Using RNA-dependant RNA  polymerase-specific primers, we identified the NLV as a genogroup II strain.  Both viruses have high homology to Asian strains.  An analysis of fecal coliforms revealed a most probable number (MPN) of 930,000/100 g of clam meat, which is 400-fold higher than the traditional hygenic standard for shellfish meats.  The evidence indicates that these clams were probably harvested from grossly insanitary waters.


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