Molecular characterisation of norovirus contamination in wastewater and oysters

Abstract

Bivalve molluscan shellfish (BMS), such as oysters, when grown in waters impacted by human wastewater may bioaccumulate human enteric viruses such as Norovirus (NoV). NoV is the leading agent of foodborne viral gastroenteritis worldwide. A wide variety of different genotypes of NoV are believed to circulate in the community. This research project involved the quantification and identification of NoV genotypes in wastewater, oysters and in NoV outbreak related faecal samples. The overall aim of the project was to establish a link between NoV genotypes circulating in the community (through analysis of municipal wastewater) and their uptake in oysters. This link was further related to the risk to consumers by investigating NoV concentrations and genotypes in oysters and faecal samples related to two outbreaks of gastroenteritis. During the initial part of this study, NoV concentrations and genotypes were determined in oyster samples collected monthly over a 2-year period from a commercial harvest area. Despite ongoing E. coli analysis indicating a category B classification under EU regulations, the area was closed for harvesting because of previous implication in a number of NoV related outbreaks. Total NoV (GI and GII) concentrations in oysters ranged from <LOQ to 20,080 genome copies g(-1) of digestive tissue (DT) and displayed a strong seasonal trend, greater concentrations occurring from October to March and correlated with the known increased seasonal prevalence of NoV illness in the population. Multiple NoV genotypes were identified in oysters from the harvest area; NoV GI.4, GI.3, GI.2, GII.4, GII.b, GII.2, GII.12, and GII.e. This study demonstrated that E. coli monitoring alone is not sufficient to assess the NoV contamination of shellfish harvesting area. Moreover, NoV genotypes changed over the study period, and unexpectedly high concentrations of NoV GI were present. The application of the nested RT-PCR targeting the part of the RNA-depended RNA polymerase (RdRp) was suitable for genotyping, but its usefulness was insuffiecint to distinguish between all GII.4 variants. To determine the pathway of NoV strains present in the community and those subsequently accumulated in oysters, weekly samples of influent, secondary treated effluent, and oysters adjacent to a wastewater treatment (WWT) outfall were collected during the peak period of NoV community infections during 2010. A nested RT-PCR assay for NoV was used for phylogenetic analysis of NoV strains based on the N/S capsid domain of the genome. Over a 13 week study period, a total of 931 laboratory-confirmed cases of NoV GII infection were recorded in comparison to 16 cases of NoV GI. Despite the few cases of NoV GI reported, concentrations of NoV GI were similar to NoV GII detected in influent and effluent wastewater, and subsequently in oysters. The dominant strain implicated in NoV outbreaks in Ireland at this time was NoV GII.4 variant 2010. However, in addition to detecting NoV GII.4 variant 2010, multiple genotypes of NoV GI (GI.1, GI.4, GI.5, GI.6, and GI.7), NoV GII (GII.3, GII.4, GII.6, GII.7, GII.12, GII.13, and GII.17), and four putative NoV recombinant strains were identified in the wastewater and oyster samples. Different NoV genotype profiles were observed in influent, effluent and oysters possibly indicating differing survival characteristics or selective bioaccumulation by oysters of different NoV strains. This study demonstrated that a wide variety of NoV GI and GII strains is present in effluent wastewaters and may be potentially accumulated by oysters, highlighting that the role of oysters as a vector of multiple NoV GI and GII strains, including recombinants. In addition, it was demonstrated that the nested RT-PCR targeting the N/S domain of NoV genome allowed for better discrimination between strains, including GII.4 variants, and the recombinant identification than the nested RT-PCR targeting the part of RdRp gene. Finally, the RT-qPCR and genotyping procedures previously implemented into the laboratory were used to investigate two separate oyster-related NoV outbreaks that occurred in Ireland in 2010 and 2012. In both outbreaks, NoV concentration in oysters exceeded 1000 genome copies g-1 DT. In addition, highly similar or identical NoV sequences were detected in the faeces of individuals with gastroenteritis and in oysters that were served in restaurants and directly associated with illness. In faecal samples, GII.13 was the only genotype implicated in outbreak 1, detected using direct sequencing, whereas multiple genotypes were detected in outbreak 2, following the application of cloning procedures. This study demonstrated that various genotypes are present in oyster samples containing high NoV concentrations, and therefore, cloning of faecal samples is vital to fully characterise the causative NoV genotype in outbreak investigations. Overall the findings from this thesis demonstrate that NoV genotypes detected in oysters can change over time, and in general are reflective of strains circulating in community. NoV genotype profiles detected in the influent, effluent and oyster samples varied indicating differing survival characteristics through the treatment process or preferential accumulation of some genotypes in oysters. Phylogenetic analysis of NoV sequences can be used as a supportive tool in tracking the origin of NoV oyster-related outbreaks. However, cloning of nested PCR products is recommended prior to sequencing of the NoV-positive faecal samples since multiple NoV GI and GII genotypes can be identified in the faeces of individuals. Oysters containing NoV concentrations in excess of 1000 genome copies g-1 DT were responsible for both gastroenteritis outbreaks investigated in this study. Concentrations of NoV in the closed harvest area frequently exceeded these values and would probably have made people ill if consumed. Therefore, NoV monitoring using RT-qPCR can be used to monitor NoV contamination within shellfish harvesting areas at risk of human wastewater pollution and be used for risk management purposes.