Human Sapovirus Replication in Human Intestinal Enteroids.

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 log10-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.

Species-Specific N-Glycomes and Methylation Patterns of Oysters Crassostrea gigas and Ostrea edulis and Their Possible Consequences for the Norovirus-HBGA Interaction.

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.

Use of Human Intestinal Enteroids to Evaluate Persistence of Infectious Human Norovirus in Seawater.

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.

Depuration and Relaying: A Review on Potential Removal of Norovirus from Oysters.

Pollution of coastal waters can result in contamination of bivalve shellfish with human enteric viruses, including norovirus (NoV), and oysters are commonly implicated in outbreaks. Depuration is a postharvest treatment involving placement of shellfish in tanks of clean seawater to reduce contaminant levels; this review focuses on the efficacy of depuration in reducing NoV in oysters. There have been many NoV outbreaks from depurated oysters containing around 103 genome copies/g oyster tissue, far exceeding the median infectious dose (ID50). Half of the published NoV reduction experiments showed no decrease in NoV during depuration, and in the remaining studies it took between 9 and 45.5 d for a 1-log reduction-significantly longer than commercial depuration time frames. Surrogate viruses are more rapidly depurated than NoV; the mean number of days to reduce NoV by 1 log is 19, and 7.5 d for surrogates. Thus, surrogates do not appear to be suitable for assessing virological safety of depurated oysters; data on reduction of NoV infectivity during depuration would assist evaluations on surrogate viruses and the impact of methods used. The longer persistence of NoV highlights its special relationship with oysters, which involves the binding of NoV to histo-blood group-like ligands in various tissues. Given the persistence of NoV and on-going outbreaks, depuration as currently performed appears ineffective in guaranteeing virologically safe oysters. Conversely, relaying oysters for 4 wk is more successful, with low NoV concentrations and no illnesses associated with products. The ineffectiveness of depuration emphasizes the need for coastal water quality to be improved to ensure oysters are safe to eat.

High Proportion of Asymptomatic Infections in an Outbreak of Hepatitis E Associated With a Spit-Roasted Piglet, France, 2013.

BACKGROUND: On 11 December 2013, 3 clustered cases of hepatitis E were reported on a French coastal island. Individuals had taken part in a wedding meal that included a spit-roasted piglet. The piglet had been stuffed with a raw stuffing partly made from the liver. Investigations were carried out to identify the vehicle of contamination and evaluate the dispersion of the hepatitis E virus (HEV) in the environment. METHODS: A questionnaire was administered to 98 wedding participants who were asked to give a blood sample. Cases were identified by reverse transcription-polymerase chain reaction and serological tests. A retrospective cohort study was conducted among 38 blood-sampled participants after the exclusion of 14 participants with evidence of past HEV infection. Relative risks (RR) and 95% confidence intervals were calculated based on food consumed at the wedding meal using univariate and multivariable Poisson regressions. Phylogenetic analyses were performed to compare the clinical HEV strains. Strains were detected in the liquid manure sampled at the farm where the piglet was born and in the untreated island wastewater. RESULTS: Seventeen cases were identified, 70.6% were asymptomatic. Acute HEV infection was independently associated with piglet stuffing consumption (RR = 1.69 [1.04-2.73], P = .03). Of clinical strains from the index cases, veterinary and environmental HEV strains were identical. CONCLUSIONS: Our investigation attributed this large HEV outbreak to the consumption of an undercooked pig liver-based stuffing. After infection, the cases became a temporary reservoir for HEV, which was detected in the island's untreated wastewater.

Digital PCR for Quantifying Norovirus in Oysters Implicated in Outbreaks, France.

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.

Tulane Virus as a Potential Surrogate To Mimic Norovirus Behavior in Oysters.

Oyster 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.

Bioaccumulation efficiency, tissue distribution, and environmental occurrence of hepatitis E virus in bivalve shellfish from France.

Hepatitis 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.

Environmental detection of genogroup I, II, and IV noroviruses by using a generic real-time reverse transcription-PCR assay.

Norovirus is the most common agent implicated in food-borne outbreaks and is frequently detected in environmental samples. These viruses are highly diverse, and three genogroups (genogroup I [GI], GII, and GIV) infect humans. Being noncultivable viruses, real-time reverse transcription-PCR (RT-PCR) is the only sensitive method available for their detection in food or environmental samples. Selection of consensus sequences for the design of sensitive assays has been challenging due to sequence diversity and has led to the development of specific real-time RT-PCR assays for each genogroup. Thus, sample screening can require several replicates for amplification of each genogroup (without considering positive and negative controls or standard curves). This study reports the development of a generic assay that detects all three human norovirus genogroups on a qualitative basis using a one-step real-time RT-PCR assay. The generic assay achieved good specificity and sensitivity for all three genogroups, detected separately or in combination. At variance with multiplex assays, the choice of the same fluorescent dye for all three probes specific to each genogroup allows the levels of fluorescence to be added and may increase assay sensitivity when multiple strains from different genogroups are present. When it was applied to sewage sample extracts, this generic assay successfully detected norovirus in all samples found to be positive by the genogroup-specific RT-PCRs. The generic assay also identified all norovirus-positive samples among 157 archived nucleic acid shellfish extracts, including samples contaminated by all three genogroups.

Oysters and norovirus: something special?

Shellfish can be a vector for human pathogens. Despite regulation based on enteric bacteria, shellfish are still implicated in viral outbreaks. Oysters are the most common shellfish associated with outbreaks, and noroviruses, which cause acute gastroenteritis, are the most frequently identified pathogen in these outbreaks. Analysis of shellfish-related outbreak data worldwide shows an unexpected high proportion of genogroup I strains. Recent studies performed in vitro, in vivo and in the environment indicate that oysters are not just a passive filter, but can selectively accumulate norovirus strains based on virus carbohydrate ligands shared with humans. These observations may help explain the GI/GII bias observed in shellfish-related outbreaks compared to other outbreaks.

Looking into sewage: how far can metagenomics help to detect human enteric viruses?

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.

Porcine Epidemic Diarrhea Virus, Surrogate for Coronavirus Decay Measurement in French Coastal Waters and Contribution to Coronavirus Risk Evaluation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in infected patients mainly displays pulmonary and oronasal tropism; however, the presence of the virus has also been demonstrated in the stools of patients and consequently in wastewater treatment plant effluents, raising the question of the potential risk of environmental contamination (such as seawater contamination) through inadequately treated wastewater spillover into surface or coastal waters even if the environmental detection of viral RNA alone does not substantiate risk of infection. Therefore, here, we decided to experimentally evaluate the persistence of the porcine epidemic diarrhea virus (PEDv), considered as a coronavirus representative model, in the coastal environment of France. Coastal seawater was collected, sterile-filtered, and inoculated with PEDv before incubation for 0 to 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 persistence of coronaviruses in coastal environments and contributes to risk evaluation, not only for SARS-CoV-2 persistence, but also for other coronaviruses, specifically enteric coronaviruses from livestock. IMPORTANCE 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. Our findings are of interest to researchers and authorities seeking to monitor 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.

Impact of xynthia tempest on viral contamination of shellfish.

Viral contamination in oyster and mussel samples was evaluated after a massive storm with hurricane wind named "Xynthia tempest" destroyed a number of sewage treatment plants in an area harboring many shellfish farms. Although up to 90% of samples were found to be contaminated 2 days after the disaster, detected viral concentrations were low. A 1-month follow-up showed a rapid decrease in the number of positive samples, even for norovirus.

Development of passive samplers for the detection of SARS-CoV-2 in sewage and seawater: Application for the monitoring of sewage.

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 membranes 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 optimize 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 correlated 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.

SARS-CoV-2 Whole-Genome Sequencing Using Oxford Nanopore Technology for Variant Monitoring in Wastewaters.

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.

Calicivirus removal in a membrane bioreactor wastewater treatment plant.

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.

Strain-dependent norovirus bioaccumulation in oysters.

Noroviruses (NoVs) are the main agents of gastroenteritis in humans and the primary pathogens of shellfish-related 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.

[Norovirus and oysters: from the land to the sea!] / Norovirus et huîtres : de la terre à la mer !

Human fecal wastes contain a large variety of viruses that can enter the environment through discharge of waste materials from infected individuals. Despite this high diversity introduced into the environment by human fecal pollution, noroviruses have been recognized as the primary cause of disease in association with consumption of contaminated shellfish. To explain bivalve mollusk contamination, several factors including human epidemiology, virus persistence through sewage treatment plant and shellfish uptake may be suggested. Considering different outbreaks described in the literature, the most common route for transmission is accidental contamination after heavy rainfall, when extra loads cause an overflow and release of untreated sewage into the aquatic environment. Outbreak analysis also demonstrates the impact on shellfish consumption of some viral strain transmission and thus their impact on molecular epidemiology, especially for norovirus. To limit shellfish contamination and thus to protect the consumer, the most desirable and effective option is to reduce the viral input.

Optimisation of a PMAxx™-RT-qPCR Assay and the Preceding Extraction Method to Selectively Detect Infectious Murine Norovirus Particles in Mussels.

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 log10 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 and Animal RNA Virus Diversity Detected by Metagenomics in Cameroonian Clams.

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.