A Comprehensive Review for the Surveillance of Human Pathogenic Microorganisms in Shellfish.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Can shellfish be used to monitor SARS-CoV-2 in the coastal environment?

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.