Molecular surveillance of enterovirus and norwalk-like virus in oysters relocated to a municipal-sewage-impacted gulf estuary

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 degrees 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.

FIG. 1.

Levels of indicator microorganisms (E. coli and MSC) and the presence or absence of enterovirus and NLV in oyster samples after a 2-week relocation to a Gulf of Mexico estuary impacted by municipal sewage effluents. Sample collections were carried out from November 1999 to December 2001, with no collection between January to July of 2001.

FIG. 2.

Capsid nucleotide sequences of NLV strains of the 95/96-US common subset derived from environmentally polluted oysters. Samples 0300 and 0999 (not listed in Fig. 1 and Table 3) were collected in March of 2000 and September of 1999. Sample 0300 was collected from the same relocation site without information on indicator microorganisms. Sample 0999 was relocated further away from the original site (>30 m) but was contaminated by the same pollution source.