Distribution of norwalk virus within shellfish following bioaccumulation and subsequent depuration by detection using RT-PCR

Consumption of raw bivalve mollusks contaminated with pathogens from human feces continues to present a human health risk. The purpose of this study was to monitor the uptake, localization, and removal of Norwalk virus (NV) in shellfish (oyster and clam) tissues by analyzing virus distribution in selected dissected tissues. Live shellfish were allowed to bioaccumulate different input titers of NV for time periods from 4 to 24 h. In some experiments, depuration by shellfish that bioaccumulated NV and Escherichia coli bacteria was allowed to proceed for 24 or 48 hours. Dissected stomach (St), digestive diverticula (DD), adductor muscle (AM), and hemolymph cells (HC) tissues were assayed for NV by the reverse transcription polymerase chain reaction (RT-PCR) method. An internal RNA standard control was added to the RT-PCR to identify the presence of inhibitors to RT-PCR. NV titers in DD tissues before and after depuration were estimated using quantitative RT-PCR end-point dilution. NV was found in the alimentary tract (DD or St) at all concentrations of input virus, but was present more frequently after exposure to higher levels of virus. NV was detected in AM and HC only following exposure to higher levels of virus. In experiments where depuration by oysters was continued for 48 h, depuration of bacteria was efficient (95% reduction of bacteria), but minimal (7%) reduction of NV titers from DD tissues was detected. These findings indicate that NV can localize both within and outside the alimentary tract of shellfish, and NV is poorly depurated using conditions favorable for E. coli depuration.