Noroviruses (NoVs) are the most common viral agents of acute gastroenteritis in humans, and high concentrations of NoVs are discharged into the environment. As these viruses are very resistant to inactivation, the sanitary consequences are contamination of food, including molluscan shellfish. There are four major problems with NoV detection in shellfish samples: low levels of virus contamination, the difficulty of efficient virus extraction, the presence of interfering substances that inhibit molecular detection, and NoV genetic variability. The aims of this study were to adapt a kit for use with a method previously shown to be efficient for detection of NoV in shellfish and to use a one step real-time reverse transcription-PCR method with addition of an external viral control. Comparisons of the two methods using bioaccumulated oysters showed that the methods reproducibly detected similar levels of virus in oyster samples. Validation studies using naturally contaminated samples also showed that there was a good correlation between the results of the two methods, and the variability was more attributable to the level of sample contamination. Magnetic silica very efficiently eliminated inhibitors, and use of extraction and amplification controls increased quality assurance. These controls increased the confidence in estimates of NoV concentrations in shellfish samples and strongly supported the conclusion that the results of the method described here reflected the levels of virus contamination in oysters. This approach is important for food safety and is under evaluationfor European regulation.

Noroviruses (NoVs) are the main agents of gastroenteritis in humans and the primary pathogens of shellfishrelated outbreaks. Some NoV strains bind to shellfish tissues by using carbohydrate structures similar to their human ligands, leading to the hypothesis that such ligands may influence bioaccumulation. This study compares the bioaccumulation efficiencies and tissue distributions in oysters (Crassostrea gigas) of three strains from the two principal human norovirus genogroups. Clear differences between strains were observed. The GI.1 strain was the most efficiently concentrated strain. Bioaccumulation specifically occurred in digestive tissues in a dose-dependent manner, and its efficiency paralleled ligand expression, which was highest during the cold months. In comparison, the GII.4 strain was very poorly bioaccumulated and was recovered in almost all tissues without seasonal influence. The GII.3 strain presented an intermediate behavior, without seasonal effect and with less bioaccumulation efficiency than that of the GI.1 strain during the cold months. In addition, the GII.3 strain was transiently concentrated in gills and mantle before being almost specifically accumulated in digestive tissues. Carbohydrate ligand specificities of the strains at least partly explain the strain-dependent bioaccumulation characteristics. In particular, binding to the digestive-tube-specific ligand should contribute to bioaccumulation, whereas we hypothesize that binding to the sialic acid-containing ligand present in all tissues would contribute to retain virus particles in the gills or mantle and lead to rapid destruction.

To evaluate membrane bioreactor wastewater treatment virus removal, a study was conducted in southwest France. Samples collected from plant influent, an aeration basin, membrane effluent, solid sludge, and effluent biweekly from October 2009 to June 2010 were analyzed for calicivirus (norovirus and sapovirus) by real-time reverse transcription-PCR (RT-PCR) using extraction controls to perform quantification. Adenovirus and Escherichia coli also were analyzed to compare removal efficiencies. In the influent, sapovirus was always present, while the norovirus concentration varied temporally, with the highest concentration being detected from February to May. All three human norovirus genogroups (GI, GII, and GIV) were detected in effluent, but GIV was never detected in effluent; GI and GII were detected in 50% of the samples but at low concentrations. In the effluent, sapovirus was identified only once. An adenovirus titer showing temporal variation in influent samples was identified only twice in effluent. E. coli was always below the limit of detection in the effluent. Overall, the removal of calicivirus varied from 3.3 to greater than 6.8 log units, with no difference between the two main genogroups. Our results also demonstrated that the viruses are blocked by the membrane in the treatment plant and are removed from the plant as solid sludge.

Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, evolution, and transmission of individual antibiotic resistance genes (ARGs) is essential to develop sustainable strategies combatting this threat. Here, we use metagenomic sequencing to analyse ARGs in 757 sewage samples from 243 cities in 101 countries, collected from 2016 to 2019. We find regional patterns in resistomes, and these differ between subsets corresponding to drug classes and are partly driven by taxonomic variation. The genetic environments of 49 common ARGs are highly diverse, with most common ARGs carried by multiple distinct genomic contexts globally and sometimes on plasmids. Analysis of flanking sequence revealed ARG-specific patterns of dispersal limitation and global transmission. Our data furthermore suggest certain geographies are more prone to transmission events and should receive additional attention.

cOyster contamination by noroviruses is an important health and economic problem. The present study aimed to compare the behaviors of Norwalk virus (the prototype genogroup I norovirus) and two culturable viruses: Tulane virus and mengovirus. After bioaccumulation, tissue distributions were quite similar for Norwalk virus and Tulane virus, with the majority of viral particles detected in digestive tissues, while mengovirus was detected in large amounts in the gills and mantle as well as in digestive tissues. The levels of persistence of all three viruses over 8 days were comparable, but clear differences were observed over longer periods, with Norwalk and Tulane viruses displaying rather similar half-lives, unlike mengovirus, which was cleared more rapidly. These results indicate that Tulane virus may be a good surrogate for studying norovirus behavior in oysters, and they confirm the prolonged persistence of Norwalk virus in oyster tissues. Shellfish are filter feeders that can accumulate different types of pathogens from human fecal pollution and were identified as vectors for human enteric pathogen transmission more than a century ago. We have known for almost 40 years that bacteria and viruses show differences in terms of concentration and accumulation in and depuration from contaminated shellfish (1). Nowadays the problem of viral contamination has become dominant, and over the last 10 years about 40% of RASFF (Rapid Alert System for Food and Feed) notifications are related to the detection of norovirus (NoV) in oysters (2). Improvements in detection methods, increased epidemiological surveillance, and efforts by authorities to improve the quality of products put on the market have contributed to better recognition of viral contamination. These improvements have assisted in identifying that increases in human populations in coastal areas, as well as climate change, inducing heavy rainfall and associated sewage overflows, constitute risk factors for shellfish contamination (3, 4).Among human enteric viruses, NoVs are recognized as the leading cause of epidemics and sporadic cases of gastroenteritis in all age groups of humans (5, 6). NoVs of human origin are excreted in large quantities by ill people, but they may also be present in asymptomatic, healthy individuals (7). As a consequence, they are discharged in large numbers into sewage, and due to their resistance to inactivation, they are frequently detected in wastewater treatment plant effluent and in surface waters (8-10). Sewage treatment which incorporates new technologies, such as membrane filtration, contributes to decreasing the numbers of microorganisms discharged into the coastal environment (11, 12), but this does not prevent accidental contamination. Depuration of shellfish, which was developed to eliminate bacteria, does not efficiently eliminate viruses that persist for several weeks or months in bivalve tissues (13,14). As a consequence, in most cases of contamination, the only risk management option to prevent consumer infections is the clo...

dHepatitis E virus (HEV), an enteric pathogen of both humans and animals, is excreted by infected individuals and is therefore present in wastewaters and coastal waters. As bivalve molluscan shellfish are known to concentrate viral particles during the process of filter feeding, they may accumulate this virus. The bioaccumulation efficiencies of oysters (Crassostrea gigas), flat oysters (Ostrea edulis), mussels (Mytilus edulis), and clams (Ruditapes philippinarum) were compared at different time points during the year. Tissue distribution analysis showed that most of the viruses were concentrated in the digestive tissues of the four species. Mussels and clams were found to be more sensitive to sporadic contamination events, as demonstrated by rapid bioaccumulation in less than 1 h compared to species of oysters. For oysters, concentrations increased during the 24-h bioaccumulation period. Additionally, to evaluate environmental occurrence of HEV in shellfish, an environmental investigation was undertaken at sites potentially impacted by pigs, wild boars, and human waste. Of the 286 samples collected, none were contaminated with hepatitis E virus, despite evidence that this virus is circulating in some French areas. It is possible that the number of hepatitis E viral particles discharged into the environment is too low to detect or that the virus may have a very short period of persistence in pig manure and human waste.

Metagenomic sequencing is a promising method to determine the virus diversity in environmental samples such as sewage or shellfish. However, to identify the short RNA genomes of human enteric viruses among the large diversity of nucleic acids present in such complex matrices, method optimization is still needed. This work presents methodological developments focused on norovirus, a small ssRNA non-enveloped virus known as the major cause of human gastroenteritis worldwide and frequently present in human excreta and sewage. Different elution protocols were applied and Illumina MiSeq technology were used to study norovirus diversity. A double approach, agnostic deep sequencing and a capture-based approach (VirCapSeq-VERT) was used to identify norovirus in environmental samples. Family-specific viral contigs were classified and sorted by SLIM and final norovirus contigs were genotyped using the online Norovirus genotyping tool v2.0. From sewage samples, 14 norovirus genogroup I sequences were identified of which six were complete genomes. For norovirus genogroup II, nine sequences were identified and three of them comprised more than half of the genome. In oyster samples bioaccumulated with these sewage samples, only the use of an enrichment step during library preparation allowed successful identification of nine different sequences of norovirus genogroup I and four for genogroup II (>500 bp). This study demonstrates the importance of method development to increase virus recovery, and the interest of a capture-based approach to be able to identify viruses present at low concentrations.

Using samples from oysters clearly implicated in human disease, we quantified norovirus levels by using digital PCR. Concentrations varied from 43 to 1,170 RNA copies/oyster. The analysis of frozen samples from the production area showed the presence of norovirus 2 weeks before consumption.