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Human noroviruses are a major cause for gastroenteritis outbreaks. Filter-feeding bivalve molluscs, which accumulate noroviruses in their digestive tissues, are a typical vector for human infection. RT-qPCR, the established method for human norovirus detection in food, does not allow discrimination between infectious and non-infectious viruses and can overestimate potentially infectious viral loads. To develop a more accurate method of infectious norovirus load estimation, we combined intercalating agent propidium monoazide (PMAxx™)-pre-treatment with RT-qPCR assay using in vitro-cultivable murine norovirus. Three primer sets targeting different genome regions and diverse amplicon sizes were used to compare one-step amplification of a short genome fragment to three two-step long-range RT-qPCRs (7 kbp, 3.6 kbp and 2.3 kbp amplicons). Following initial assays performed on untreated infectious, heat-, or ultraviolet-inactivated murine noroviruses in PBS suspension, PMAxx™ RT-qPCRs were implemented to detect murine noroviruses subsequent to their extraction from mussel digestive tissues; virus extraction via anionic polymer-coated magnetic beads was compared with the proteinase K-dependent ISO norm. The long-range RT-qPCR process detecting fragments of more than 2.3 kbp allowed accurate estimation of the infectivity of UV-damaged murine noroviruses. While proteinase K extraction limited later estimation of PMAxx™ pre-treatment effects and was found to be unsuited to the assay, magnetic bead-captured murine noroviruses retained their infectivity. Genome copies of heat-inactivated murine noroviruses differed by 2.3 log 10 between RT-qPCR and PMAxx™-RT-qPCR analysis in bivalve molluscs, the PMAxx™ pre-treatment allowing a closer approximation of infectious titres. The combination of bead-based virus extraction and PMAxx™ RT-qPCR thus provides a more accurate model for the estimation of noroviral bivalve mollusc contamination than the conjunction of proteinase K extraction and RT-qPCR and has the potential (once validated utilising infectious human norovirus) to provide an added measure of security to food safety authorities in the hazard assessment of potential bivalve mollusc contamination.

Human noroviruses are recognized as the leading worldwide cause of sporadic and epidemic viral gastroenteritis, causing morbidity and mortality in impoverished developing countries and engendering enormous economic losses in developed countries. Transmitted faecal-orally, either via person-to-person contact, or by consumption of contaminated foods or water, norovirus outbreaks are often reported in institutional settings or in the context of communal dining. Bivalve molluscs, which accumulate noroviruses via filter feeding and are often eaten raw or insufficiently cooked, are a common food vehicle implicated in gastroenteritis outbreaks. The involvement of bivalve molluscs in norovirus outbreaks and epidemiology over the past two decades are reviewed. The authors describe how their physiology of filter feeding can render them concentrated vehicles of norovirus contamination in polluted environments and how high viral loads persist in molluscs even after application of depuration practices and typical food preparation steps. The global prevalence of noroviruses in bivalve molluscs as detected by different monitoring efforts is determined and the various methods currently utilized for norovirus extraction and detection from bivalve matrices described. An overview of gastroenteritis outbreaks affirmatively associated with norovirus-contaminated bivalve molluscs as reported in the past 18 years is also provided. Strategies for risk reduction in shellfish contamination and subsequent human infection are discussed., (© 2019 Blackwell Verlag GmbH.)

Bivalve shellfish may accumulate contaminants of public health concern including pathogenic bacteria and viruses. Microbiological monitoring of production areas is based on faecal coliforms in water in the USA and Escherichia coli in bivalve molluscs in the European Union. E. coli is known to reflect contamination with Salmonella enterica but not necessarily with other human pathogens such as enteric viruses. A structured field study was undertaken at three locations in order to investigate the geographical and temporal variability of E. coli and norovirus (NoV). Total norovirus concentration differed significantly by both sampling site and sampling date. A significant correlation was found between total NoV concentration and E. coli concentration by sample, but not with E. coli in seawater. The results have implications for the establishment of sampling plans for NoV in harvesting areas and potentially also for the approach taken to classification based on faecal indicator bacteria., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

European Legislation has fixed microbiological, chemical and biotoxicological limits for shellfish but no limits for viruses. In the present study we report the results of an investigation on Salmonella spp., Escherichia coli, Hepatitis A virus (HAV) and Norovirus (NoV) contamination in 59 bivalve shellfish collected during the years 2011-2012 in Southern Italy. All the samples of Mytilus galloprovincialis and of Solen marginatus were negative for HAV whereas 6.8% of them were positive for Norovirus GI (NoVGI) and 11.9% positive for Norovirus GII (NoVGII). Samples were also negative for Salmonella spp., while 16 of them (27%) were positive for E. coli. No correlation was found between E. coli and NoV contamination in bivalve molluscs. Moreover, the Competent Authorities are advised to take into serious consideration additional measures for the legislation in force in order to guarantee the consumer's health.

While exports of oysters from Japan are increasing in recent years, it is necessary to establish a pre-export testing implementation system in Japan using ISO 15216 method, which is used internationally as a testing method for norovirus in oyster. However, NucliSENS miniMAG (bio-Merieux), an RNA extraction device widely used in Europe, has already been discontinued and is not available in Japan. In this study, we verified the substitutability of Mealstrom 8 (TANBead) as an alternative suggested in a previous report, using pacific oyster (Crassostrea gigas), which is commonly consumed in Japan. Moreover, we verified the substitutability of the three reagent kits available in Japan for RNA UltraSense one-step quantitative RT-PCR system (Thermo Fisher Scientific), which is the reagent recommended by the o伍 cial protocol of ISO 15216 method. As a result, viral RNA was extracted more efficiently by Maelstrom 8 than by the NucliSENS miniMAG. And of the three reagent kits, CEERAMTOOLS noroGI-GII@ceeramTools (bioMerieux) was shown to have the same performance of gene detection as UltraSense. These results are expected to contribute to establishment of the pre-export testing system for oysters using ISO 15216 method in Japan. [ABSTRACT FROM AUTHOR]

Norovirus (NoV) and Hepatitis A virus (HAV) are the most important viral hazards associated with human illness following consumption of contaminated bivalve molluscs. The effectiveness of the current EU criteria for heat processing of bivalve molluscs (i.e. raising the temperature of the internal mollusc flesh to at least 90°C for a minimum of 90 seconds) was evaluated using predictive microbiology. A HAV thermal inactivation model was developed based on literature data in mollusc matrices during isothermal heat treatment. Application of the developed model demonstrated that the 90°C-90 s requirement may lead to significantly different virus inactivation depending on the commercial process design. This shows the need for the establishment of a Performance Criterion for bivalve molluscs heat processing which will assure a common specified level of consumer protection. A risk-based approach is described that allows for an effective processing design providing a more transparent and objective relation between the thermal processing targets and public health. Model simulations demonstrate that the F-value is a more appropriate Process Criterion than a single time-temperature combination since it enables the food business operators to design a process that is compliant with the safety requirements while at the same time achieving a desired product quality. [ABSTRACT FROM AUTHOR]

European Legislation has fixed microbiological, chemical and biotoxicological limits for shellfish but no limits for viruses. In the present study we report the results of an investigation on Salmonella spp., Escherichia coli, Hepatitis A virus (HAV) and Norovirus (NoV) contamination in 59 bivalve shellfish collected during the years 2011-2012 in Southern Italy. All the samples of Mytilus galloprovincialis and of Solen marginatus were negative for HAV whereas 6.8% of them were positive for Norovirus GI (NoVGI) and 11.9% positive for Norovirus GII (NoVGII). Samples were also negative for Salmonella spp., while 16 of them (27%) were positive for E. coli. No correlation was found between E. coli and NoV contamination in bivalve molluscs. Moreover, the Competent Authorities are advised to take into serious consideration additional measures for the legislation in force in order to guarantee the consumer's health.

The European Union Rapid Alert System for Food and Feed (EU RASFF) database is an invaluable instrument for analyzing notifications involving norovirus in food. The aim of this work was to carry out a thorough research of the alert and border rejection notifications submitted in the RASFF database from its onset until 31 August 2017. Some conclusions of interest were: (i) Denmark, France, Italy, the Netherlands and Norway have contributed the majority of alert notifications as notifying countries, (ii) France and Serbia have been cited more often in alert notifications as countries of origin, (iii) Italy and Spain have submitted the majority of border rejection notifications, (iv) Third Countries implicated more frequently in border rejection notifications for norovirus in bivalve molluscs were Vietnam and Tunisia, whereas in fruits and vegetables were China and Serbia, (v) “risk dispersion” from norovirus-contaminated food was narrow since, in just over half of all alert notifications and all of the border rejection notifications, only up to three countries were involved, and (vi) both raw (oysters and berries) and cooked (mussels) food products can present a health risk to consumers. The information retrieved from the RASFF database on norovirus-contaminated food could prove helpful in the planning of future norovirus risk analysis endeavors.

Different sources were consulted to obtain information on the occurrence of viruses in bivalve molluscs on the European market. Twenty-six peer-reviewed articles were identified reporting on the molecular detection of viral RNA in 4,260 samples in total. The data obtained will be presented geographically on virus types detected, the origin and treatment of the shellfish, and the detection technique applied. The data demonstrate that viral RNA can be detected in shellfish from polluted areas, in depurated shellfish as well as those for human consumption. The European Rapid Alert System for Food and Feed (RASFF) database was consulted as another source. Twenty-eight notifications were identified on the presence of hepatitis A virus or norovirus in shellfish on the European market. The most recent report of the European laboratory network was referred to, to gain insight into the laboratory capability at present for the analyses of shellfish on the presence of viruses. Approximately 67% of 27 participating laboratories obtained intended results for all samples, consisting of lenticules loaded with 103 copies norovirus (genogroup I (GGI) and/or genogroup II (GGII)) and/or 1 × 105–8 × 104 copies of hepatitis A virus. From 1993, there has been a continuous development of molecular detection techniques and tools have been described to ensure quality assurance. End product testing will, however, not be achievable. As depuration has been shown not to be effective for the complete elimination of viruses, shellfish should not be in contact with faecal contaminated water in order to minimise the risk of shellfish-transmittable viral diseases. [ABSTRACT FROM AUTHOR]