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.

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.

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.

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.

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.

A Targeted Metagenomics Approach to Study the Diversity of Norovirus GII in Shellfish Implicated in Outbreaks.

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.

Metagenomic to evaluate norovirus genomic diversity in oysters: Impact on hexamer selection and targeted capture-based enrichment.

Human virus transmission through food consumption has been identified since many years and the international trade increases the risk of dissemination of viral pathogens. The development of metagenomic approach holds many promises for the surveillance of viruses in food and water. This work aimed to analyze norovirus diversity and to evaluate strain-dependent accumulation patterns in three oyster types by using a metagenomic approach. Different hexamer sets to prime cDNA were evaluated before capture-based approach to enhance virus reads recovery during deep sequencing. The study includes the use of technical replicates of artificially contaminated oysters and the analysis of multiple negatives controls. Results showed a clear impact of the hexamer set used for cDNA synthesis. A set of In-house designed (I-HD) hexamers, selected to lower mollusk amplification, gave promising results in terms of viral reads abundancy. However, the best correlation between CT values, thus concentrations, and number of reads was observed using random hexamers. Random hexamers also provided the highest numbers of reads and allowed the identification of sequence of different human enteric viruses. Regarding human norovirus, different genogroups and genotypes were identified among contigs longer than 500 bp. Two full genomes and six sequences longer than 3600 bases were obtained allowing a precise strain identification. The use of technical triplicates was found valuable to increase the chances to sequence viral strains present at low concentrations. Analyzing viral contamination in shellfish samples is quite challenging, however this work demonstrates that the recovery of full genome or long contigs, allowing clear identification of viral strains is possible.

Contamination of Clams with Human Norovirus and a Novel Hepatitis A Virus in Cameroon.

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.

Complete Genome Sequence of Campylobacter armoricus CA639, Which Carries Two Plasmids, Compiled Using Oxford Nanopore and Illumina Sequencing Technologies.

As determined by a hybrid approach combining Oxford Nanopore MinION and Illumina MiniSeq sequence data, Campylobacter armoricus strain CA639 harbored a circular chromosome of 1,688,169 bp with a G+C content of 28.47% and two plasmids named pCA639-1 and pCA639-2, with lengths of 51,123 and 28,139 bp, and G+C contents of 26.5% and 28.45%, respectively.

Metagenomics-Based Proficiency Test of Smoked Salmon Spiked with a Mock Community.

An inter-laboratory proficiency test was organized to assess the ability of participants to perform shotgun metagenomic sequencing of cold smoked salmon, experimentally spiked with a mock community composed of six bacteria, one parasite, one yeast, one DNA, and two RNA viruses. Each participant applied its in-house wet-lab workflow(s) to obtain the metagenomic dataset(s), which were then collected and analyzed using MG-RAST. A total of 27 datasets were analyzed. Sample pre-processing, DNA extraction protocol, library preparation kit, and sequencing platform, influenced the abundance of specific microorganisms of the mock community. Our results highlight that despite differences in wet-lab protocols, the reads corresponding to the mock community members spiked in the cold smoked salmon, were both detected and quantified in terms of relative abundance, in the metagenomic datasets, proving the suitability of shotgun metagenomic sequencing as a genomic tool to detect microorganisms belonging to different domains in the same food matrix. The implementation of standardized wet-lab protocols would highly facilitate the comparability of shotgun metagenomic sequencing dataset across laboratories and sectors. Moreover, there is a need for clearly defining a sequencing reads threshold, to consider pathogens as detected or undetected in a food sample.

Detection and quantification of noroviruses in shellfish.

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.

Assessment of human enteric viruses in cultured and wild bivalve molluscs.

Standard and real-time reverse transcription-PCR (rRT-PCR) procedures were used to monitor cultured and wild bivalve molluscs from the Ría de Vigo (NW Spain) for the main human enteric RNA viruses, specifically, norovirus (NoV), hepatitis A virus (HAV), astrovirus (AsV), rotavirus (RT), enterovirus (EV), and Aichi virus (AiV). The results showed the presence of at least one enteric virus in 63.4% of the 41 samples analyzed. NoV GII was the most prevalent virus, detected in 53.7% of the samples, while NoV GI, AsV, EV, and RV were found at lower percentages (7.3, 12.2, 12.2, and 4.9%, respectively). In general, samples obtained in the wild were more frequently contaminated than those from cultured (70.6 vs. 58.3%) molluscs and were more readily contaminated with more than one virus. However, NoV GI was detected in similar amounts in cultured and wild samples (6.4 x 10(2) to 3.3 x 10(3) RNA copies per gram of digestive tissue) while the concentrations of NoV GII were higher in cultured (from 5.6 x 10(1) to 1.5 x 10(4) RNA copies per gram of digestive tissue) than in wild (from 1.3 x 10(2) to 3.4 x 10(4) RNA copies per gram of digestive tissue) samples.

Characterization of Norovirus and Other Human Enteric Viruses in Sewage and Stool Samples Through Next-Generation Sequencing.

This study aimed to optimize a method to identify human enteric viruses in sewage and stool samples using random primed next-generation sequencing. We tested three methods, two employed virus enrichment based on the binding properties of the viral capsid using pig-mucin capture or by selecting viral RNA prior to library preparation through a capture using the SureSelect target enrichment. The third method was based on a non-specific biophysical precipitation with polyethylene glycol. Full genomes of a number of common human enteric viruses including norovirus, rotavirus, husavirus, enterovirus and astrovirus were obtained. In stool samples full norovirus genome were detected as well as partial enterovirus genome. A variety of norovirus sequences was detected in sewage samples, with genogroup II being more prevalent. Interestingly, the pig-mucin capture enhanced not only the recovery of norovirus and rotavirus but also recovery of astrovirus, sapovirus and husavirus. Documenting sewage virome using these methods provides information for molecular epidemiology and may be useful in developing strategies to prevent further spread of viruses.

Metavirome Sequencing to Evaluate Norovirus Diversity in Sewage and Related Bioaccumulated Oysters.

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.