Noroviruses (NoV), which are members of the family Caliciviridae, are the most important cause of outbreaks of acute gastroenteritis worldwide and are commonly found in shellfish grown in polluted waters. In the present study, we developed broadly reactive one-step TaqMan reverse transcription (RT)-PCR assays for the detection of genogroup I (GI) and GII NoV in fecal samples, as well as shellfish samples. The specificity and sensitivity of all steps of the assays were systematically evaluated, and in the final format, the monoplex assays were validated by using RNA extracted from a panel of 84 stool specimens, which included NoV strains representing 19 different genotypes (7 GI, 11 GII, and 1 GIV strains). The assays were further validated with 38 shellfish cDNA extracts previously tested by nested PCR. Comparison with a recently described real-time assay showed that our assay had significantly higher sensitivity and was at least as sensitive as the nested PCR. For stool specimens, a one-step duplex TaqMan RT-PCR assay performed as well as individual genogroup-specific monoplex assays. All other enteric viruses examined were negative, and no cross-reaction between genogroups was observed. These TaqMan RT-PCR assays provide rapid (less than 90 min), sensitive, and reliable detection of NoV and should prove to be useful for routine monitoring of both clinical and shellfish samples.

The contamination of bivalve shellfish with norovirus from human fecal sources is recognized as an important human health risk. Standardized quantitative methods for the detection of norovirus in molluscan shellfish are now available, and viral standards are being considered in the European Union and internationally. This 2-year systematic study aimed to investigate the impact of the application of these methods to the monitoring of norovirus contamination in oyster production areas in the United Kingdom. Twenty-four monthly samples of oysters from 39 United Kingdom production areas, chosen to represent a range of potential contamination risk, were tested for norovirus genogroups I and II by using a quantitative real-time reverse transcription (RT)-PCR method. Norovirus was detected in 76.2% (643/844) of samples, with all sites returning at least one positive result. Both prevalences (presence or absence) and norovirus levels varied markedly between sites. However, overall, a marked winter seasonality of contamination by both prevalence and quantity was observed. Correlations were found between norovirus contamination and potential risk indicators, including harvesting area classifications, Escherichia coli scores, and environmental temperatures. A predictive risk score for norovirus contamination was developed by using a combination of these factors. In summary, this study, the largest of its type undertaken to date, provides a systematic analysis of norovirus contamination in commercial oyster production areas in the United Kingdom. The data should assist risk managers to develop control strategies to reduce the risk of human illness resulting from norovirus contamination of bivalve molluscs.T he contamination of bivalve shellfish with norovirus from human fecal sources is recognized as a major human health risk (21). Risk assessment and management currently rely on the use of Escherichia coli as an indicator of fecal (sewage) contamination in shellfish (3). However, this approach has been repeatedly demonstrated to inadequately indicate the risk from human enteric viruses (2,8,16). Over the last decade, considerable progress has been made toward the development of sensitive quantitative detection methods for norovirus in molluscan shellfish, and there are numerous reports describing various approaches to virus testing (19,24,25). All available methods are based on the detection of virus genomes by using molecular techniques (PCR), as the successful cultivation of norovirus by conventional virological techniques has been reported only rarely (33, 34) and is not currently feasible for application to foodstuffs. At present, no internationally accepted reference method exists; however, the European Committee on Normalization (CEN) has an active working group addressing the development of an ISO standard method for the detection of norovirus and hepatitis A virus (HAV) in foodstuffs, including bivalve shellfish (22). The method is scheduled for publication in 2012, and publication will be followed by an internatio...

The human health risk associated with the consumption of molluscan shellfish grown in sewage-contaminated waters is well established. Noroviruses, which cause gastroenteritis, are the principal agents of shellfish-related illness. Fecal-indicator quality standards based on Escherichia coli are well established in Europe and elsewhere. However, norovirus outbreaks after consumption of shellfish meeting these standards still occur, and the need to improve consumer health protection is well recognized. Alternative approaches proposed include direct monitoring of viral pathogens and the use of alternative indicator organisms capable of providing a better indication of virus risk. This study applies a recently developed TaqMan PCR assay to assess norovirus contamination in shellfish. Comparison was made with E. coli as the existing sanitary standard and a male-specific RNA bacteriophage as a possible alternative. Two commercial pacific oyster (Crassostrea gigas) harvesting areas were monitored over a 31-month period. The results show peaks of norovirus contamination in both areas during winter months, with average levels approximately 17 times higher in oysters sampled October to March than during the remainder of the year, consistent with epidemiological data for the United Kingdom showing oyster-associated illness is confined to winter months. While there was no apparent association with E. coli, an association between levels of norovirus contamination and the male-specific RNA bacteriophage was noted, with average norovirus levels over 40 times higher in samples with male-specific RNA bacteriophage counts of >1,000 PFU/100 g than in samples with <100 PFU/100 g. Overall, these results suggest that norovirus monitoring in shellfish production areas could be an effective strategy for reduction of virus risk.

Hepatitis A virus (HAV) and norovirus are important agents of food-borne human viral illness, with common vehicles including bivalve molluscan shellfish, soft fruit and various vegetables. Outbreaks of viral illness due to contamination of the surfaces of foods, or food preparation surfaces by for example infected food handlers are also common. Virus analysis of food matrices can contribute towards risk management for these hazards and a two-part technical specification for determination of Hepatitis A virus and norovirus in food matrices (ISO/TS 15216:2013) was published jointly by the European Committee for Standardisation and the International Organization for Standardization in 2013. As part of the European Mandate No. M381 to validate 15 standards in the field of food microbiology, an international validation study involving 18 laboratories from 11 countries in Europe was conducted between 2012 and 2014. This study aimed to generate method characteristics including limit of detection, limit of quantification, repeatability and reproducibility for ISO 15216 - Part 1, the method for quantification, in seven food matrices. The organization and results of this study, including observations that led to improvements in the standard method are presented here. After its conclusion, the method characteristics generated were added to the revised international standard, ISO 15216-1:2017, published in March 2017.

Norovirus is the principal agent of bivalve shellfish–associated gastroenteric illness worldwide. Numerous studies using PCR have demonstrated norovirus contamination in a significant proportion of both oyster and other bivalve shellfish production areas and ready-to-eat products. By comparison, the number of epidemiologically confirmed shellfish-associated outbreaks is relatively low. This suggests that factors other than the simple presence or absence of virus RNA are important contributors to the amount of illness reported. This study compares norovirus RNA levels in oyster samples strongly linked to norovirus or norovirus-type illness with the levels typically found in commercial production areas (non–outbreak-related samples). A statistically significant difference between norovirus levels in the two sets of samples was observed. The geometric mean of the levels in outbreak samples (1,048 copies per g) was almost one order of magnitude higher than for positive non–outbreak-related samples (121 copies per g). Further, while none of the outbreak-related samples contained fewer than 152 copies per g, the majority of positive results for non–outbreak-related samples was below this level. These observations support the concept of a dose-response for norovirus RNA levels in shellfish and could help inform the establishment of threshold criteria for risk management.

Globally, Hepatitis E virus (HEV) causes over 20 million cases worldwide. HEV is an emerging and endemic pathogen within economically developed countries, chiefly resulting from infections with genotype 3 (G3) HEV. G3 HEV is known to be a zoonotic pathogen, with a broad host range. The primary source of HEV within more economically developed countries is considered to be pigs, and consumption of pork products is a significant risk factor and known transmission route for the virus to humans. However, other foods have also been implicated in the transmission of HEV to humans. This review consolidates the information available regarding transmission of HEV and looks to identify gaps where further research is required to better understand how HEV is transmitted to humans through food.

Wastewater-based epidemiology has become an important tool for the surveillance of SARS-CoV-2 outbreaks. However, the detection of viruses in sewage is challenging and to date there is no standard method available which has been validated for the sensitive detection of SARS-CoV-2. In this paper, we describe a simple concentration method based on polyethylene glycol (PEG) precipitation, followed by RNA extraction and a one-step quantitative reverse transcription PCR (qRT-PCR) for viral detection in wastewater. PEG-based concentration of viruses is a simple procedure which is not limited by the availability of expensive equipment and has reduced risk of disruption to consumable supply chains. The concentration and RNA extraction steps enable 900–1500× concentration of wastewater samples and sufficiently eliminates the majority of organic matter, which could inhibit the subsequent qRT-PCR assay. Due to the high variation in the physico-chemical properties of wastewater samples, we recommend the use of process control viruses to determine the efficiency of each step. This procedure enables the concentration and the extraction the DNA/RNA of different viruses and hence can be used for the surveillance of different viral targets for the comprehensive assessment of viral diseases in a community.

Bivalve shellfish have the capacity to accumulate norovirus (NoV) from waters contaminated with human sewage. Consequently, shellfish represent a major vector for NoV entry into the human food chain, leading to gastrointestinal illness. Identification of areas suitable for the safe cultivation of shellfish requires an understanding of NoV behaviour upon discharge of municipal-derived sewage into coastal waters. This study exploited the potential of edible mussels (Mytilus edulis) to accumulate NoV and employed the ISO method for quantification of NoV within mussel digestive tissues. To evaluate the spatial spread of NoV from an offshore sewage discharge pipe, mesh cages of mussels were suspended from moorings deployed in a 9 km grid array around the outfall. Caged mussels were retrieved after 30 days and NoV (GI and GII), total coliforms and E. coli enumerated. The experimentally-derived levels of NoV GI and GII in mussels were similar with total NoV levels ranging from 7 × 10 to 1.6 × 10 genome copies g shellfish digestive gland (ΣGI + GII). NoV spread from the outfall showed a distinct plume which matched very closely to predictions from the tidally-driven effluent dispersal model MIKE21. A contrasting spatial pattern was observed for coliforms (range 1.7 × 10 to 2.1 × 10 CFU 100 g shellfish tissue) and E. coli (range 0-1.2 × 10 CFU 100 g shellfish tissue). These data demonstrate that hydrodynamic models may help inform effective exclusion zones for bivalve harvesting, whilst coliform/E. coli concentrations do not accurately reflect viral dispersal in marine waters and contamination of shellfish by sewage-derived viral pathogens.