Variant profiling after whole genome sequencing. Omicron sub-lineage BA.2 was predominant (>83%). BA.4 and BA.5 were emergent variants. Rare mutations of SARS-CoV-2 detected. The same representativeness of both sampling methods.

Investigating potential zoonotic viruses in animal reservoirs is crucial to anticipate viral emergence. Seals can represent large populations of coastal mammals with unknown consequences on the microbiological quality of their surrounding environment. To assess this, we conducted a metaviromics analysis of feces collected from two species of seals in the North-Western Atlantic (Saint-Pierre et Miquelon archipelago). We focused on the Caliciviridae family, which regroups several genera with viruses infecting humans and other mammals, including marine mammals, but none identified in seals (Phocidae). Among the assembled sequences identified as Caliciviridae, there were four known genera (norovirus, sapovirus, vesivirus, and salovirus) and unknown, distantly related viruses. Complete or nearly-complete genomes could be assembled for each genus. Norovirus and sapovirus sequences from seals were diverse and likely represent several new genogroups or genotypes. Seal vesivirus formed a monophyletic group, representing a potential new species related to the canine vesivirus. Salovirus, which are fish viruses, were likely diet-derived, like the distant sequences which exhibited the hallmarks of caliciviruses and were more closely related to fish and reptile viruses. In conclusion, seals are a reservoir for a large diversity of Caliciviridae, some related to norovirus or sapovirus genotypes known to infect humans, and their impact on the quality of coastal water or shellfish should be further assessed. This study expands the knowledge on Caliciviridae genetic diversity and circulation in marine mammals.

Human noroviruses are a major cause of foodborne outbreaks worldwide. Filter-feeding shellfish, such as oysters, can bioaccumulate these viruses in their digestive tissue when grown in sewage-impacted coastal areas and are often implicated in norovirus foodborne outbreaks. Despite the high sensitivity of current molecular assays, these methods for norovirus detection in shellfish fail to distinguish between infectious and non-infectious particles. Assessing norovirus infectivity in shellfish remains a challenge due to the lack of suitable isolation methods that maintain capsid integrity. In this study, a protocol for isolating infectious norovirus from oyster tissues, based on chloroform-butanol elution and polyethylene glycol concentration (CB-PEG), was optimized for the recovery of human norovirus GI and GII. While CB-PEG method recovered various norovirus GI and GII genotypes, it was less efficient at the genomic level than a protocol based on proteinase K elution (adapted from ISO 15216) and showed genotype-dependent viral recovery rates. By optimizing the flocculation step, we improved the method’s compatibility with human intestinal enteroid (HIE) cultures. Using this approach, we successfully quantified infectious norovirus GII.3 titers recovered from artificially-contaminated live oysters. Interestingly, infectious virus was better isolated following a freezing step of the digestive tissues, with titers ranging from 13 to 40 TCID50/mL for positive samples. In conclusion, this study established an optimized methodological approach for the relative quantification of infectious norovirus GII.3 in shellfish, paving the way for future research on viral persistence and inactivation strategies in this foodstuff.

Human sewage is the main source of contamination of environmental waters with human enteric viruses, that can contaminate food such as shellfish. Metagenomic represents a new way of analyzing viral diversity through an a priori massive parallel sequencing approach. However, the precise identification of enteric viruses in sewage or shellfish matrices, is still challenging due to the low viral load, large diversity of viral genera and the large amounts of matrix masking viral sequences. This work compared three commercial kits using capture-based enrichment during the library preparation, for the diversity of detected enteric viruses and for the identification of viral strains in sewage and shellfish samples, focusing on four families impacting human health. Triplicate libraries were prepared for each sample and each kit. All three kits allowed the characterization of a variety of viral genera. In sewage samples, a large number of long contigs was obtained allowing a precise identification of more than 35 strains. In shellfish samples, long contigs were rarer but allowed the identification of one human astrovirus and one norovirus strains. Of the tested kits, one displayed lower variation between replicates, allowed to sequence a higher diversity of viruses from the four families of interest and yielded a higher number of nearly-whole genomes.

Bivalve molluscan shellfish have been consumed for centuries. Being filter feeders, they may bioaccumulate some microorganisms present in coastal water, either naturally or through the discharge of human or animal sewage. Despite regulations set up to avoid microbiological contamination in shellfish, human outbreaks still occur. After providing an overview showing their implication in disease, this review aims to highlight the diversity of the bacteria or enteric viruses detected in shellfish species, including emerging pathogens. After a critical discussion of the available methods and their limitations, we address the interest of technological developments using genomics to anticipate the emergence of pathogens. In the coming years, further research needs to be performed and methods need to be developed in order to design the future of surveillance and to help risk assessment studies, with the ultimate objective of protecting consumers and enhancing the microbial safety of bivalve molluscan shellfish as a healthy food.

SARS-CoV-2 in infected patient mainly display pulmonary and oronasal tropism however, the presence of the virus has also been demonstrated in stools of patients and consequently in wastewater treatment plant effluents, questioning the potential risk of environmental contamination (such as seawater contamination) through inadequately treated wastewater spill-over into surface or coastal waters. The environmental detection of RNA alone does not substantiate risk of infection, and evidence of an effective transmission is not clear where empirical observations are lacking.Therefore, here, we decided to experimentally evaluate the persistence and infectious capacity of the Porcine epidemic diarrhea virus (PEDv), considered as a coronavirus representative model and SARS-CoV-2 surrogate, in the coastal environment of France. Coastal seawater was collected, sterile-filtered, and inoculated with PEDv before incubation for 0–4 weeks at four temperatures representative of those measured along the French coasts throughout the year (4, 8, 15, and 24°C). The decay rate of PEDv was determined using mathematical modeling and was used to determine the half-life of the virus along the French coast in accordance with temperatures from 2000 to 2021.We experimentally observed an inverse correlation between seawater temperature and the persistence of infectious viruses in seawater and confirm that the risk of transmission of infectious viruses from contaminated stool in wastewater to seawater during recreational practices is very limited. The present work represents a good model to assess the risk of transmission of not only SARS-CoV-2 but may also be used to model the risk of other coronaviruses, specifically enteric coronaviruses.Importance This present work is a follow up addressing the question of the persistence of coronavirus in marine environment owing to the fact that SARS-CoV-2 is regularly detected in wastewater treating plan and the coastal environment is particularly at risk since it is subjected to increasing anthropogenic pressure and is the final receiver of surface waters and treated or sometimes insufficiently depurated waste waters. Our findings are of interest to researchers and authorities seeking to monitor SARS-CoV-2 and also enteric coronaviruses in the environment, either in tourist areas or in regions of the world, where centralized systems for wastewater treatment are not implemented, and more broadly, to the scientific community involved in “One Health” approaches.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThis work is supported by the Agence Nationale de la Recherche and the Fondation de France (ANR RA-Covid wave 5, 00109676), the Region Pays de la Loire (order 2020-12887), by an internal funding from Ifremer General Direction (SARS-CoV-2 action plan) and the European project VEO (H2020, SC1-2019-874735).### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesI confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Human sapoviruses (HuSaVs), like human noroviruses (HuNoV), belong to the Caliciviridae family and cause acute gastroenteritis in humans. Since their discovery in 1976, numerous attempts to grow HuSaVs in vitro were unsuccessful until 2020, when these viruses were reported to replicate in a duodenal cancer cell-derived line. Physiological cellular models allowing viral replication are essential to investigate HuSaV biology and replication mechanisms such as genetic susceptibility, restriction factors, and immune responses to infection. In this study, we demonstrate replication of two HuSaV strains in human intestinal enteroids (HIEs) known to support the replication of HuNoV and other human enteric viruses. HuSaVs replicated in differentiated HIEs originating from jejunum, duodenum and ileum, but not from the colon, and bile acids were required. Between 2h and 3 to 6 days postinfection, viral RNA levels increased up from 0.5 to 1.8 log(10)-fold. Importantly, HuSaVs were able to replicate in HIEs independent of their secretor status and histo-blood group antigen expression. The HIE model supports HuSaV replication and allows a better understanding of host-pathogen mechanisms such as cellular tropism and mechanisms of viral replication. IMPORTANCE Human sapoviruses (HuSaVs) are a frequent but overlooked cause of acute gastroenteritis, especially in children. Little is known about this pathogen, whose successful in vitro cultivation was reported only recently, in a cancer cell-derived line. Here, we assessed the replication of HuSaV in human intestinal enteroids (HIEs), which are nontransformed cultures originally derived from human intestinal stem cells that can be grown in vitro and are known to allow the replication of other enteric viruses. Successful infection of HIEs with two strains belonging to different genotypes of the virus allowed discovery that the tropism of these HuSaVs is restricted to the small intestine, does not occur in the colon, and replication requires bile acid but is independent of the expression of histo-blood group antigens. Thus, HIEs represent a physiologically relevant model to further investigate HuSaV biology and a suitable platform for the future development of vaccines and antivirals.

The impact of human sewage on environmental and food contamination constitutes an important safety issue. Indeed, human sewage reflects the microbiome of the local population, and a variety of human viruses can be detected in wastewater samples. Being able to describe the diversity of viruses present in sewage will provide information on the health of the surrounding population health and will help to prevent further transmission. Metagenomic developments, allowing the description of all the different genomes present in a sample, are very promising tools for virome analysis. However, looking for human enteric viruses with short RNA genomes which are present at low concentrations is challenging. In this study we demonstrate the benefits of performing technical replicates to improve viral identification by increasing contig length, and the set-up of quality criteria to increase confidence in results. Our approach was able to effectively identify some virus sequences and successfully describe the viral diversity. The method yielded full genomes either for norovirus, enterovirus and rotavirus, even if, for these segmented genomes, combining genes remain a difficult issue. Developing reliable viromic methods is important as wastewater sample analysis provides an important tool to prevent further virus transmission by raising alerts in case of viral outbreaks or emergence.

The present work addresses the question of the persistence of coronavirus in marine environments because SARS-CoV-2 is regularly detected in wastewater treatment plants, and the coastal environment, subjected to increasing anthropogenic pressure and the final receiver of surface waters and sometimes insufficiently depurated wastewater, is particularly at risk. The problem also arises in the possibility of soil contamination by CoV from animals, especially livestock, during manure application, where, by soil impregnation and runoff, these viruses can end up in seawater.

Noroviruses, the major cause of acute viral gastroenteritis, are known to bind to histo-blood group antigens (HBGAs), including ABH groups and Lewis-type epitopes, which decorate the surface of erythrocytes and epithelial cells of their host tissues. The biosynthesis of these antigens is controlled by several glycosyltransferases, the distribution and expression of which varies between tissues and individuals. The use of HBGAs as ligands by viruses is not limited to humans, as many animal species, including oysters, which synthesize similar glycan epitopes that act as a gateway for viruses, become vectors for viral infection in humans. Here, we show that different oyster species synthesize a wide range of N-glycans that share histo-blood A-antigens but differ in the expression of other terminal antigens and in their modification by O-methyl groups. In particular, we show that the N-glycans isolated from Crassostrea gigas and Ostrea edulis exhibit exquisite methylation patterns in their terminal N-acetylgalactosamine and fucose residues in terms of position and number, adding another layer of complexity to the post-translational glycosylation modifications of glycoproteins. Furthermore, modeling of the interactions between norovirus capsid proteins and carbohydrate ligands strongly suggests that methylation has the potential to fine-tune the recognition events of oysters by virus particles.

Since the beginning of the Coronavirus Disease-19 (COVID-19) pandemic, multiple Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) mutations have been reported and led to the emergence of variants of concern (VOC) with increased transmissibility, virulence or immune escape. In parallel, the observation of viral fecal shedding led to the quantification of SARS-CoV-2 genomes in wastewater, providing information about the dynamics of SARS-CoV-2 infections within a population including symptomatic and asymptomatic individuals. Here, we aimed to adapt a sequencing technique initially designed for clinical samples to apply it to the challenging and mixed wastewater matrix, and hence identify the circulation of VOC at the community level. Composite raw sewage sampled over 24 h in two wastewater-treatment plants (WWTPs) from a city in western France were collected weekly and SARS-CoV-2 quantified by RT-PCR. Samples collected between October 2020 and May 2021 were submitted to whole-genome sequencing (WGS) using the primers and protocol published by the ARTIC Network and a MinION Mk1C sequencer (Oxford Nanopore Technologies, Oxford, United Kingdom). The protocol was adapted to allow near-full genome coverage from sewage samples, starting from ∼5% to reach ∼90% at depth 30. This enabled us to detect multiple single-nucleotide variant (SNV) and assess the circulation of the SARS-CoV-2 VOC Alpha, Beta, Gamma, and Delta. Retrospective analysis of sewage samples shed light on the emergence of the Alpha VOC with detection of first co-occurring signature mutations in mid-November 2020 to reach predominance of this variant in early February 2021. In parallel, a mutation-specific qRT-PCR assay confirmed the spread of the Alpha VOC but detected it later than WGS. Altogether, these data show that SARS-CoV-2 sequencing in sewage can be used for early detection of an emerging VOC in a population and confirm its ability to track shifts in variant predominance.

Recent studies have shown that passive sampling is a promising tool for SARS-CoV-2 detection for wastewater-based epidemiology (WBE) application. We have previously developed passive sampling of viruses using polymer mem-branes in seawater. Even though SARS-CoV-2 was not detected yet in seawater, passive sampling could be optimized for future application in coastal areas close to wastewater treatment plant (WWTP). The aim of this study was to opti-mize passive sampling of SARS-CoV-2 in sewage and seawater by selecting a suitable membrane, to determine whether the quantities of virus increase over time, and then to determine if passive sampling and traditional sampling are cor-related when conducted in a wastewater treatment plant. Nylon and Zetapor allowed the detection of heat inactivated SARS-CoV-2 and of the Porcine Epidemic Diarrhea Virus (PEDV), a coronavirus surrogate, in wastewater and seawater spiked with these 2 viruses, showing an increase in detection between 4 h and 24 h of immersion and significantly higher recoveries of both viruses with nylon in seawater (15%) compared to wastewater (4%). On wastewater samples, both membranes detected the virus, the recovery rate was of about 3% for freshly collected samples, and no significant difference was found between SARS-CoV-2 genome concentration on Zetapor and that in water. In sewage spiked seawater, similar concentrations of genome were found on both membranes, with a mean recovery rate of 16% and 11% respectively for nylon and Zetapor. A 3-weeks monitoring with passive sampler allowed the detection of viruses in the influent of a WWTP with a frequency of 100% and 76% for SARS-CoV-2 and norovirus GII respectively. Passive and traditional sampling gave the same evolution of the SARS-CoV-2 concentration over time. All these results confirmed the interest of passive sampling for virus detection and its potential application for monitoring in the wastewater system for targeted public health actions.

Little data on the persistence of human norovirus infectivity are available to predict its transmissibility. Using human intestinal enteroids, we demonstrate that 2 human norovirus strains can remain infectious for several weeks in seawater. Such experiments can improve understanding of factors associated with norovirus survival in coastal waters and shellfish.

EFSA Supporting PublicationsVolume 19, Issue 6 7348E External scientific reportOpen Access Application of Next Generation Sequencing on Norovirus-contaminated oyster samples Joanna Ollivier, Corresponding Author Joanna Ollivier biohaw@efsa.europa.eu Ifremer, Microbiology laboratory, Nantes, FranceCorrespondence:biohaw@efsa.europa.euSearch for more papers by this authorJames Lowther, James Lowther Centre for Environment, Fisheries and Aquaculture Science, Weymouth, UKSearch for more papers by this authorMarion Desdouits, Marion Desdouits Ifremer, Microbiology laboratory, Nantes, FranceSearch for more papers by this authorJulien Schaeffer, Julien Schaeffer Ifremer, Microbiology laboratory, Nantes, FranceSearch for more papers by this authorCandice Wacrenier, Candice Wacrenier Ifremer, Microbiology laboratory, Nantes, FranceSearch for more papers by this authorBas B. Oude Munnink, Bas B. Oude Munnink Erasmus University Medical Center, Rotterdam, the NetherlandsSearch for more papers by this authorAlban Besnard, Alban Besnard Ifremer, Microbiology laboratory, Nantes, FranceSearch for more papers by this authorFrederico Mota Batista, Frederico Mota Batista Centre for Environment, Fisheries and Aquaculture Science, Weymouth, UKSearch for more papers by this authorTina Stapleton, Tina Stapleton Centre for Environment, Fisheries and Aquaculture Science, Weymouth, UKSearch for more papers by this authorAnna Charlotte Schultz, Anna Charlotte Schultz National Food Institute, Technical University of Denmark, Lyngby, Denmark Anna Charlotte Schultz left the DTU and the consortium in February 2020.Search for more papers by this authorFrank Aarestrup, Frank Aarestrup National Food Institute, Technical University of Denmark, Lyngby, DenmarkSearch for more papers by this authorMarion Koopmans, Marion Koopmans Erasmus University Medical Center, Rotterdam, the NetherlandsSearch for more papers by this authorMiranda de Graaf, Miranda de Graaf Erasmus University Medical Center, Rotterdam, the NetherlandsSearch for more papers by this authorSoizick Le Guyader, Soizick Le Guyader Ifremer, Microbiology laboratory, Nantes, FranceSearch for more papers by this author Joanna Ollivier, Corresponding Author Joanna Ollivier biohaw@efsa.europa.eu Ifremer, Microbiology laboratory, Nantes, FranceCorrespondence:biohaw@efsa.europa.euSearch for more papers by this authorJames Lowther, James Lowther Centre for Environment, Fisheries and Aquaculture Science, Weymouth, UKSearch for more papers by this authorMarion Desdouits, Marion Desdouits Ifremer, Microbiology laboratory, Nantes, FranceSearch for more papers by this authorJulien Schaeffer, Julien Schaeffer Ifremer, Microbiology laboratory, Nantes, FranceSearch for more papers by this authorCandice Wacrenier, Candice Wacrenier Ifremer, Microbiology laboratory, Nantes, FranceSearch for more papers by this authorBas B. Oude Munnink, Bas B. Oude Munnink Erasmus University Medical Center, Rotterdam, the NetherlandsSearch for more papers by this authorAlban Besnard, Alban Besnard Ifremer, Microbiology laboratory, Nantes, FranceSearch for more papers by this authorFrederico Mota Batista, Frederico Mota Batista Centre for Environment, Fisheries and Aquaculture Science, Weymouth, UKSearch for more papers by this authorTina Stapleton, Tina Stapleton Centre for Environment, Fisheries and Aquaculture Science, Weymouth, UKSearch for more papers by this authorAnna Charlotte Schultz, Anna Charlotte Schultz National Food Institute, Technical University of Denmark, Lyngby, Denmark Anna Charlotte Schultz left the DTU and the consortium in February 2020.Search for more papers by this authorFrank Aarestrup, Frank Aarestrup National Food Institute, Technical University of Denmark, Lyngby, DenmarkSearch for more papers by this authorMarion Koopmans, Marion Koopmans Erasmus University Medical Center, Rotterdam, the NetherlandsSearch for more papers by this authorMiranda de Graaf, Miranda de Graaf Erasmus University Medical Center, Rotterdam, the NetherlandsSearch for more papers by this authorSoizick Le Guyader, Soizick Le Guyader Ifremer, Microbiology laboratory, Nantes, FranceSearch for more papers by this author First published: 15 June 2022 https://doi.org/10.2903/sp.efsa.2022.EN-7348 Question number:: EFSA-Q-2019-00153 Disclaimer:: The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender procedure. The present document is published complying with the transparency principle to which the Authority is subject. It may not be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Bivalve molluscan shellfish (BMS) contamination with gastroenteritis viruses such as norovirus (NoV) is recognized as a significant public health risk worldwide. These foodborne epidemics are characterized by very low viral concentrations in the implicated foods, and by diverse viruses or multiple NoV strains originating from human sewage, resulting in different strains (co-)infecting the consumers. Next-generation sequencing (NGS) offers promising means to describe the diversity of strains present in BMS or to retrace transmission chains in outbreak settings, but their sensitivity and reproducibility remained to be assessed for this application. In this work, we evaluated the ability of three promising NGS methods to sequence diverse NoV in environmental or food BMS and human stool samples. Using laboratory-prepared samples of known NoV composition, we evaluated the sensitivity, reproducibility, repeatability and selectivity of metabarcoding, capture-based metagenomics and long amplicon sequencing, considering representative NoV strains from genogroup I and II, and the impact of the BMS matrix. The metabarcoding, with separate amplification of polymerase and capsid gene segments followed by Illumina sequencing, was the most sensitive method. It was applied to a selection of 212 BMS samples collected through the European Commission's NoV baseline survey (BLS), demonstrating a high diversity of NoV sequences found in the BMS which reflect the diversity of NoV strains circulating in the European human population. Besides, a capture-based metagenomics with enrichment of vertebrate viruses was applied on 20 of these BLS samples as well as 20 BMS linked to outbreaks and 10 related human stool samples. In BMS, it yielded NoV sequences compatible with the genomes identified in stool samples, but they were too short to allow definitive confirmation of the infection source. The present report describes NGS methods, including the bioinformatic pipelines, applicable to molecular epidemiology of NoV in BMS, their current limitations and expected outcomes. Supporting Information Filename Description efs37348e-sup-0001-Annex_A.xlsxExcel 2007 spreadsheet , 255.9 KB efs37348e-sup-0002-Annex_B.xlsxExcel 2007 spreadsheet , 4.6 MB efs37348e-sup-0003-Annex_C.xlsxExcel 2007 spreadsheet , 79.3 KB efs37348e-sup-0004-Annex_D.pdfPDF document, 116 KB Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Volume19, Issue6June 20227348E ReferencesRelatedInformation

The emergence and worldwide spread of SARS-CoV-2 raises new concerns and challenges regarding possible environmental contamination by this virus through spillover of human sewage, where it has been detected. The coastal environment, under increasing anthropogenic pressure, is subjected to contamination by a large number of human viruses from sewage, most of them being non-enveloped viruses like norovirus. When reaching coastal waters, they can be bio-accumulated by filter-feeding shellfish species such as oysters. Methods to detect this viral contamination were set up for the detection of non-enveloped enteric viruses, and may need optimization to accommodate enveloped viruses like coronaviruses (CoV). Here, we aimed at assessing methods for the detection of CoV, including SARS-CoV-2, in the coastal environment and testing the possibility that SARS-CoV-2 can contaminate oysters, to monitor the contamination of French shores by SARS-CoV-2 using both seawater and shellfish. Using the porcine epidemic diarrhea virus (PEDV), a CoV, as surrogate for SARS-CoV-2, and Tulane virus, as surrogate for non-enveloped viruses such as norovirus, we assessed and selected methods to detect CoV in seawater and shellfish. Seawater-based methods showed variable and low yields for PEDV. In shellfish, the current norm for norovirus detection was applicable to CoV detection. Both PEDV and heat-inactivated SARS-CoV-2 could contaminate oysters in laboratory settings, with a lower efficiency than a calicivirus used as control. Finally, we applied our methods to seawater and shellfish samples collected from April to August 2020 in France, where we could detect the presence of human norovirus, a marker of human fecal contamination, but not SARS-CoV-2. Together, our results validate methods for the detection of CoV in the coastal environment, including the use of shellfish as sentinels of the microbial quality of their environment, and suggest that SARS-CoV-2 did not contaminate the French shores during the summer season.

Many recent pandemics have been recognized as zoonotic viral diseases. While their origins remain frequently unknown, environmental contamination may play an important role in emergence. Thus, being able to describe the viral diversity in environmental samples contributes to understand the key issues in zoonotic transmission. This work describes the use of a metagenomic approach to assess the diversity of eukaryotic RNA viruses in river clams and identify sequences from human or potentially zoonotic viruses. Clam samples collected over 2years were first screened for the presence of norovirus to verify human contamination. Selected samples were analyzed using metagenomics, including a capture of sequences from viral families infecting vertebrates (VirCapSeq-VERT) before Illumina NovaSeq sequencing. The bioinformatics analysis included pooling of data from triplicates, quality filtering, elimination of bacterial and host sequences, and a deduplication step before de novo assembly. After taxonomic assignment, the viral fraction represented 0.8–15% of reads with most sequences (68–87%) remaining un-assigned. Yet, several mammalian RNA viruses were identified. Contigs identified as belonging to the Astroviridae were the most abundant, with some nearly complete genomes of bastrovirus identified. Picobirnaviridae sequences were related to strains infecting bats, and few others to strains infecting humans or other hosts. Hepeviridae sequences were mostly related to strains detected in sponge samples but also strains from swine samples. For Caliciviridae and Picornaviridae , most of identified sequences were related to strains infecting bats, with few sequences close to human norovirus, picornavirus, and genogroup V hepatitis A virus. Despite a need to improve the sensitivity of our method, this study describes a large diversity of RNA virus sequences from clam samples. To describe all viral contaminants in this type of food, and being able to identify the host infected by viral sequences detected, may help to understand some zoonotic transmission events and alert health authorities of possible emergence.

Shellfish constitute an important protein source but may be contaminated by viruses from various origins. A study performed on clams collected in Cameroon showed a high prevalence of norovirus and hepatitis A virus. After sequencing, the hepatitis A virus showed similarities with the genotype V simian strains.

Human noroviruses (NoV) cause epidemics of acute gastroenteritis (AGE) worldwide and can be transmitted through consumption of contaminated foods. Fresh products such as shellfish can be contaminated by human sewage during production, which results in the presence of multiple virus strains, at very low concentrations. Here, we tested a targeted metagenomics approach by deep-sequencing PCR amplicons of the capsid (VP1) and polymerase (RdRp) viral genes, on a set of artificial samples and on shellfish samples associated to AGE outbreaks, to evaluate its advantages and limitations in the identification of strains from the NoV genogroup (G) II. Using artificial samples, the method allowed the sequencing of most strains, but not all, and displayed variability between replicates especially with lower viral concentrations. Using shellfish samples, targeted metagenomics was compared to Sanger-sequencing of cloned amplicons and was able to identify a higher diversity of NoV GII and GIV strains. It allowed phylogenetic analyses of VP1 sequences and the identification, in most samples, of GII.17[P17] strains, also identified in related clinical samples. Despite several limitations, combining RdRp- and VP1-targeted metagenomics is a sensitive approach allowing the study NoV diversity in low-contaminated foods and the identification of NoV strains implicated in outbreaks.

Foodborne viral infections rank among the top 5 causes of disease, with noroviruses and hepatitis A causing the greatest burden globally. Contamination of foods by infected food handlers or through environmental pollution are the main sources of foodborne illness, with a lesser role for consumption of products from infected animals. Viral partial genomic sequencing has been used for more than two decades to track foodborne outbreaks and whole genome or metagenomics next-generation-sequencing (NGS) are new additions to the toolbox of food microbiology laboratories. We discuss developments in the field of targeted and metagenomic NGS, with an emphasis on application in food virology, the challenges and possible solutions towards future routine application.