Molecular Surveillance of Enterovirus andNorwalk-Like Virus in Oysters Relocated to a Municipal-Sewage-ImpactedGulfEstuary

Abstract

An 18-month survey was conducted to examine the prevalence of enteric viruses and their relationship to indicators in environmentally polluted shellfish. Groups of oysters, one group per 4 weeks, were relocated to a coastal water area in the Gulf of Mexico that is impacted by municipal sewage and were analyzed for enteroviruses, Norwalk-like viruses (NLV), and indicator microorganisms (fecal coliform, Escherichia coli, and male-specific coliphages). The levels of indicator microorganisms were consistent with the expected continuous pollution of the area. Fourteen of the 18 oyster samples were found by reverse transcription (RT)-PCR to harbor NLV and/or enterovirus sequences. Of the four virus-negative oysters, three had exposure to water temperatures of >29°C. Concomitant with these findings, two of these four oysters also accumulated the lowest levels of coliphages. PCR primers targeting pan-enteroviruses and the NLV 95/96-US common subset were utilized; NLV sequences were detected more frequently than those of enteroviruses. Within the 12-month sampling period, NLV and enterovirus sequences were detected in 58 and 42%, respectively, of the oysters (67% of the oysters tested were positive for at least one virus) from a prohibited shellfish-growing area approximately 30 m away from a sewage discharge site. Eight (4.6%) of the 175 NLV capsid nucleotide sequences were heterogeneous among the clones derived from naturally polluted oysters. Overall, enteric viral sequences were found in the contaminated oysters throughout all seasons except hot summer, with a higher prevalence of NLV than enterovirus. Although a high percentage of the oysters harbored enteric viruses, the virus levels were usually less than or equal to 2 logs of RT-PCR-detectable units per gram of oyster meat.Virus infection accounts for two-thirds of the 13.8 million food-borne illnesses each year in the United States for which the pathogen is known (25). Major food vehicles associated with viral gastrointestinal diseases are shellfish (12,21,30,31), fresh produce and produce products (17,28,32), and readyto-eat deli food (1). Shellfish and fresh produce have been fecally contaminated in the field before or during harvest. Illegal overboard sewage discharges into shellfish harvesting waters was the most probable cause for recent major U.S. outbreaks (4, 37). Norwalk-like virus (NLV), recently renamed norovirus, was largely responsible for U.S. shellfish-borne viral gastroenteritis in the 1990s (8,19,22,35) and has been classified into different genogroups (16,39). Subgrouping of NLV strains has been carried out based upon (i) amino acid sequences of the open-reading frame 2 capsid region (2), resulting in 15 genetic clusters for genogroups I and II, (ii) the NLV capsid N/S domain (18), etc. Importantly, NLV genogroup II (among four genogroups) was identified predominantly in the 1990s outbreaks (10,11,13,20,24,41), and the 95/96-US subset of genotype II (cluster 4) was responsible for many outbreaks in the U.S. and elsewhere (15,29,40,41).Successfu...

Rapid and Sensitive Detection of Noroviruses by Using TaqMan-Based One-Step Reverse Transcription-PCR Assays and Application to Naturally Contaminated Shellfish Samples

“…Hence, introduction of routine norovirus monitoring of shellfish would be highly advisable, but most reported conventional RT-PCR assays are unable to detect all human noroviruses (36). Although nested PCR assays have been successfully employed for amplification of low levels of virus found in naturally contaminated shellfish (6,21,22,25,26,31), there is currently no international approved standardized method, and to our knowledge, no laboratory offers an accredited service for detection of NoV in shellfish.…”

Human and Animal Enteric Caliciviruses in Oysters from Different Coastal Regions of the United States

“…In France HuNoVs were detected in 23% of samples collected from authorized harvesting areas (European Community category B) during a 3-year study, and in England NoVs were detected in 27% of samples collected from a highly polluted area (34,36). In a study performed in the United States in 2003, 52% of samples that were previously depurated and relocated to a prohibited shellfish-growing area were positive for HuNoVs (52). Our results showed a higher percentage of positive samples than the percentages detected in other studies, although the samples came from approved shellfishgrowing areas based on the criteria used for bacterial contamination (fecal coliform level).…”

Meta-Analysis of the Reduction of Norovirus and Male-Specific Coliphage Concentrations in Wastewater Treatment Plants

“…During this feeding process, each bivalve mollusc may filter 20 to 90 liters of water per day and bioaccumulate a variety of microorganisms, including viruses and bacteria that are associated with pollution sources (8,(10)(11)(12). Significantly, molluscan shellfish have been found to retain viruses to a greater extent and for much longer periods than they do bacteria (8,13,14). Bivalve molluscs, therefore, may become contaminated with NoV when they are grown in harvesting areas contaminated with human wastes.…”

“…More than half of the norovirus outbreaks attributed to the consumption of bivalve molluscs in the United States during the years from 2001 to 2008 are believed to have originated from contamination during production or processing (7). Bivalve molluscan shellfish typically grow in estuaries, which may contain NoV-contaminated human fecal material from municipal wastewater outfalls, combined sewer overflow, or nonpoint sources of pollution, including human waste discharged from marine vessels (8,9). Bivalve molluscan shellfish feed on algae from the surrounding water.…”