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Reverse transcription-PCR and hybridization can be used to detect viruses that affect the human gastrointestinal tract in shellfish. Researchers who used these techniques found that mussels were heavily contaminated with astrovirus, rotavirus, enterovirus and Norwalk-like virus. Oysters were less heavily contaminated. Viral contamination was most common in winter.

The primary pathogens related to shellfish-borne gastroenteritis outbreaks are noroviruses. These viruses show persistence in oysters, which suggests an active mechanism of virus concentration. We investigated whether Norwalk virus or viruslike particles bind specifically to oyster tissues after bioaccumulation or addition to tissue sections. Since noroviruses attach to carbohydrates of the histo-blood group family, tests using immunohistochemical analysis were performed to evaluate specific binding of virus or viruslike particles to oyster tissues through these ligands. Viral particles bind specifically to digestive ducts (midgut, main and secondary ducts, and tubules) by carbohydrate structures with a terminal N-acetylgalactosamine residue in an alpha linkage (same binding site used for recognition of human histo-blood group antigens). These data show that the oyster can selectively concentrate a human pathogen and that conventional depuration will not eliminate noroviruses from oyster tissue.

Human astroviruses constitute a significant cause of acute diarrhea in children. Viral transmission occurs via the fecal-oral route, predominantly person-to-person, but the consumption of fecally contaminated water and shellfish has also been implicated. Viral pathogen detection in water, especially, in wastewater, is difficult and PCR is widely used to detect these viruses. Despite the recent development of real-time quantitative PCR, quantification of astroviruses in sewage had not been available up to now. We have developed a method to quantify astroviruses in sewage. For this purpose, we designed a set of primers and a fluorogenic probe located at the 3' -end of the genome of human astroviruses. The amplified region was cloned and the plasmid was transcribed to generate calibration standards for quantification. After validation of the standards, the method was evaluated in artificially contaminated samples. To validate the method on naturally contaminated samples, raw and treated wastewater samples were collected monthly for one year in a sewage treatment plant. Astrovirus genomes were detected in all samples collected at the entrance to the sewage treatment plant, with a mean value of 4.1 x 10(6) astrovirus genomes for 100 ml. Effluents were less strongly contaminated, with a mean value of 1.01 x 10(4) astrovirus genomes. The high prevalence of astroviruses in sewage treatment plant effluents indicates that these plants are not efficiently eliminating the virus. This is a major public health concern and new techniques of depuration are needed. Our method could be effectively used in evaluating new treatment processes to reduce the viral load in the effluent of treatment plants.

Outbreaks of food- and waterborne gastroenteritis are being increasingly reported throughout the world. The analysis of environmental samples by newer diagnostic techniques such as reverse transcription-PCR (RT-PCR) amplification of nucleic acid has begun to identify human enteric viruses (predominantly "Norwalk-like" viruses [NLVs]) as the cause of many of these outbreaks. To streamline NLV detection from environmental samples such as shellfish, we have developed an RT-PCR-oligoprobe amplification and detection method using several new procedures that enable confirmed RT-PCR amplification and product detection in 1 day. The new steps include replacing reverse transcriptase and Taq polymerase with rTth polymerase, a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase, in a single-tube, single-buffer, elevated temperature reaction. An internal standard Norwalk virus (NV) RNA control is added to each RT-PCR to identify sample inhibition, and thermolabile uracil N-glycosylase is incorporated into the reaction to prevent PCR product carryover contamination. Finally, RT-PCR-generated amplicons are detected in microtiter wells using virus-specific biotinylated oligoprobes in an enzyme-linked immunosorbent assay-based format. The DNA enzyme immunoassay is based on the capture of PCR product by biotinylated probes fixed onto individual streptavidin-coated wells. Using this method, low levels of NV were detected in stool and both NLV and hepatitis A virus were detected in bivalve mollusks following bioaccumulation. The method also successfully detected NLV in oysters implicated in an outbreak of NLV gastroenteritis. This method dramatically decreases the time needed for analysis and is amenable to automation.

Developments in the rapid detection of pathogens (PCR and its variations) and molecular typing of strains isolated from the ecosystem illustrate the stimulation of research due to the recent foodborne and waterborne disease outbreaks.