Theodore G. Metcalf

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.

Uptake and Depletion of Particulate-Associated Polioviruses by the Soft Shell Clam

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.

Removal of indigenous rotaviruses during primary settling and activated-sludge treatment of raw sewage

Abstract An eight month study of indigenous rotavirus removal during primary settling and activated sludge treatment of raw sewage was made in a plant in Houston, Texas treating 1.5 million gal day −1 . An average reduction of 44–55% was obtained by primary settling and a 93–99% reduction was achieved in final chlorinated effluents. Composite sampling at 1 h intervals over a 24 h period indicated average removals of 85% compared to a misleading 6% indicated by one set of grab samples of raw sewage and effluent collected simultaneously. Quantification of rotaviruses was made by immunofluorescent foci counts 24 h after addition of sample concentrates to coverslip cultures of fetal rhesus kidney cells. Rotaviruses varied from 40–5101 −1 of raw sewage and from 0 to 25 in the final chlorinated effluent.

Cooperative Study of Methods for the Recovery of Enteric Viruses from Shellfish

Ten investigators actively involved in shellfish virology agreed to participate in a methods study. Each participant was given the freedom of choice as to method and cell cultures used. Considerable variation was noted in the number of viruses recovered from six 100-g representative samples. All the methods appeared to detect polioviruses but difficulty was encountered in detecting low levels of coxsackie-viruses and echoviruses. The glasswool filtration-hydroextraction method and modifications of the Sobsey method were the most effective techniques for detecting and quantitating virus in this study.

A Method for Recovery of Viruses from Oysters and Hard and Soft Shell Clams

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.