Low levels of feces-associated natural virus, simulating virus numbers estimated to exist in moderately polluted shellfish-growing waters, were used to evaluate the effectiveness of depuration as a virus depletion procedure in soft-shell clams. Depuration effectiveness depended upon the numbers of virus bioaccumulated and whether virus was solids associated. Virus uptake was greatest when viruses were solids associated and pollution levels were equivalent or greater than those likely to be found in grossly polluted growing waters. Virtually all bioaccumulated feces-associated natural virus was deposited within either the hepatopancreas or siphon tissues. Viruses usually were eliminated within a 24to 48-h depuration period. Dependence upon depuration of clams to eliminate health hazards of virus etiology involved a risk factor not measureable in the study. The greatest reduction of health risks would come from the routine depuration of clams harvested from growing waters of good sanitary quality.

An improved recovery method and testing strategy were devised for recovery of low numbers of enteric viruses from each of three commercially important shellfish species. Effective recovery of virus depended as much upon details of the test strategy adopted for use of the improved method with each species as on the method itself. The most important test details involved sample composition, pool size, and method of use of cell cultures. Recovery sensitivity measured permitted detection of 25 to 3 plaque-forming units of enteroviruses and 100 to 27 plaque-forming units of reovirus through their recovery in cell culture, with effectivenesses averaging 64 and 46%, respectively. Test samples prepared by the improved recovery method were virtually cytotoxicity free. Optimal recovery of virus on 45-cm2 cell culture monolayers was obtained with 1-ml inocula adsorbed for 2 h. The most effective recovery of virus from shellfish samples was made by a sequential adsorption procedure which allowed equal exposure of an entire sample to each of two or more cell cultures. Removal of nonviral contaminants from test samples by antibiotic treatment was preferable to the use of ether or membrane filtration procedures. Surveillance ofthe sanitary quality of shellfish and shellfish-growing waters has been carried out since 1925 in the United States, when a certification program administered by the U.S. Public Health Service was instituted (1). This monitoring program, which developed into the National Shellfish Sanitation Program (NSSP), was instituted after a typhoid epidemic resulting in 1,500 cases with 150 deaths was attributed to sewage-polluted oysters (13). This epidemic, coming after pmany previously reported outbreaks of enteric disease attributed to shellfish, helped to focus critical attention upon the health hazards associated with the consumption of polluted shellfish. Since then a number of shellfishassociated outbreaks of infectious hepatitis and viral gastroenteritis have been reported. These reports have been reviewed and summarized recently by Gerba and Goyal (5). All of the commercially important shellfish, oysters, hardand soft-shell clams, and mussels, have been incriminated as vehicles for the transmission of enteric virus diseases to humans.

Human viruses present in shellfish harvesting waters are probably in a particulate or feces-associated state, and are of a low order of magnitude. Under simulated conditions, shellfish were exposed to 5- to 10-fold higher virus concentrations than have ever been detected in New Hampshire estuary waters and examined. Usually less than 10 viruses were bioaccumulated by each soft shell clam, and when the shellfish were allowed to depurate in clean water, viruses were eliminated in 48 to 72 h. A small percentage of the shellfish did not depurate completely, a shellfish characteristic consistently found which is probably related to irregular feeding activity. Depuration or relaying of shellfish should reduce microbial contamination, but there is no guarantee that all shellfish will be virus-free.

A method for recovery of small numbers of enteric viruses from oysters and hard and soft shell clams was developed. As few as 3 plaque forming units (PFU) of virus per 100 g of shellfish homogenate could be detected with an overall accuracy of ca. 60 percent in each of the three species tested.