Metagenomic Surveillance of Viral Gastroenteritis in a Public Health Setting.

Norovirus is the primary cause of viral gastroenteritis (GE). To investigate norovirus epidemiology, there is a need for whole-genome sequencing and reference sets consisting of complete genomes. To investigate the potential of shotgun metagenomic sequencing on the Illumina platform for whole-genome sequencing, 71 reverse transcriptase quantitative PCR (RT-qPCR) norovirus positive-feces (threshold cycle [CT], <30) samples from norovirus surveillance within The Netherlands were subjected to metagenomic sequencing. Data were analyzed through an in-house next-generation sequencing (NGS) analysis workflow. Additionally, we assessed the potential of metagenomic sequencing for the surveillance of off-target viruses that are of importance for public health, e.g., sapovirus, rotavirus A, enterovirus, parechovirus, aichivirus, adenovirus, and bocaparvovirus. A total of 60 complete and 10 partial norovirus genomes were generated, representing 7 genogroup I capsid genotypes and 12 genogroup II capsid genotypes. In addition to the norovirus genomes, the metagenomic approach yielded partial or complete genomes of other viruses for 39% of samples from children and 6.7% of samples from adults, including adenovirus 41 (N = 1); aichivirus 1 (N = 1); coxsackievirus A2 (N = 2), A4 (N = 2), A5 (N = 1), and A16 (N = 1); bocaparvovirus 1 (N = 1) and 3 (N = 1); human parechovirus 1 (N = 2) and 3 (N = 1); Rotavirus A (N = 1); and a sapovirus GI.7 (N = 1). The sapovirus GI.7 was initially not detected through RT-qPCR and warranted an update of the primer and probe set. Metagenomic sequencing on the Illumina platform robustly determines complete norovirus genomes and may be used to broaden gastroenteritis surveillance by capturing off-target enteric viruses. IMPORTANCE Viral gastroenteritis results in significant morbidity and mortality in vulnerable individuals and is primarily caused by norovirus. To investigate norovirus epidemiology, there is a need for whole-genome sequencing and reference sets consisting of full genomes. Using surveillance samples sent to the Dutch National Institute for Public Health and the Environment (RIVM), we compared metagenomics against conventional techniques, such as RT-qPCR and Sanger-sequencing, with norovirus as the target pathogen. We determined that metagenomics is a robust method to generate complete norovirus genomes, in parallel to many off-target pathogenic enteric virus genomes, thereby broadening our surveillance efforts. Moreover, we detected a sapovirus that was not detected by our validated gastroenteritis RT-qPCR panel, which exemplifies the strength of metagenomics. Our study shows that metagenomics can be used for public health gastroenteritis surveillance, the generation of reference-sets for molecular epidemiology, and how it compares to current surveillance strategies.

Capturing the SARS-CoV-2 infection pyramid within the municipality of Rotterdam using longitudinal sewage surveillance.

Despite high vaccination rates in the Netherlands, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to circulate. Longitudinal sewage surveillance was implemented along with the notification of cases as two parts of the surveillance pyramid to validate the use of sewage for surveillance, as an early warning tool, and to measure the effect of interventions. Sewage samples were collected from nine neighborhoods between September 2020 and November 2021. Comparative analysis and modeling were performed to understand the correlation between wastewater and case trends. Using high resolution sampling, normalization of wastewater SARS-CoV-2 concentrations, and 'normalization' of reported positive tests for testing delay and intensity, the incidence of reported positive tests could be modeled based on sewage data, and trends in both surveillance systems coincided. The high collinearity implied that high levels of viral shedding around the onset of disease largely determined SARS-CoV-2 levels in wastewater, and that the observed relationship was independent of variants of concern and vaccination levels. Sewage surveillance alongside a large-scale testing effort where 58 % of a municipality was tested, indicated a five-fold difference in the number of SARS-CoV-2-positive individuals and reported cases through standard testing. Where trends in reported positive cases were biased due to testing delay and testing behavior, wastewater surveillance can objectively display SARS-CoV-2 dynamics for both small and large locations and is sensitive enough to measure small variations in the number of infected individuals within or between neighborhoods. With the transition to a post-acute phase of the pandemic, sewage surveillance can help to keep track of re-emergence, but continued validation studies are needed to assess the predictive value of sewage surveillance with new variants. Our findings and model aid in interpreting SARS-CoV-2 surveillance data for public health decision-making and show its potential as one of the pillars of future surveillance of (re)emerging viruses.

Rise and fall of SARS-CoV-2 variants in Rotterdam: Comparison of wastewater and clinical surveillance.

Monitoring of SARS-CoV-2 in wastewater (WW) is a promising tool for epidemiological surveillance, correlating not only viral RNA levels with the infection dynamics within the population, but also to viral diversity. However, the complex mixture of viral lineages in WW samples makes tracking of specific variants or lineages circulating in the population a challenging task. We sequenced sewage samples of 9 WW-catchment areas within the city of Rotterdam, used specific signature mutations from individual SARS-CoV-2 lineages to estimate their relative abundances in WW and compared them against those observed in clinical genomic surveillance of infected individuals between September 2020 and December 2021. We showed that especially for dominant lineages, the median of the frequencies of signature mutations coincides with the occurrence of those lineages in Rotterdam's clinical genomic surveillance. This, along with digital droplet RT-PCR targeting signature mutations of specific variants of concern (VOCs), showed that several VOCs emerged, became dominant and were replaced by the next VOC in Rotterdam at different time points during the study. In addition, single nucleotide variant (SNV) analysis provided evidence that spatio-temporal clusters can also be discerned from WW samples. We were able to detect specific SNVs in sewage, including one resulting in the Q183H amino acid change in the Spike gene, that was not captured by clinical genomic surveillance. Our results highlight the potential use of WW samples for genomic surveillance, increasing the set of epidemiological tools to monitor SARS-CoV-2 diversity.

Normalisation of SARS-CoV-2 concentrations in wastewater: The use of flow, electrical conductivity and crAssphage.

Over the course of the Corona Virus Disease-19 (COVID-19) pandemic in 2020-2022, monitoring of the severe acute respiratory syndrome coronavirus 2 ribonucleic acid (SARS-CoV-2 RNA) in wastewater has rapidly evolved into a supplementary surveillance instrument for public health. Short term trends (2 weeks) are used as a basis for policy and decision making on measures for dealing with the pandemic. Normalisation is required to account for the dilution rate of the domestic wastewater that can strongly vary due to time- and location-dependent sewer inflow of runoff, industrial discharges and extraneous waters. The standard approach in sewage surveillance is normalisation using flow measurements, although flow based normalisation is not effective in case the wastewater volume sampled does not match the wastewater volume produced. In this paper, two alternative normalisation methods, using electrical conductivity and crAssphage have been studied and compared with the standard approach using flow measurements. For this, a total of 1116 24-h flow-proportional samples have been collected between September 2020 and August 2021 at nine monitoring locations. In addition, 221 stool samples have been analysed to determine the daily crAssphage load per person. Results show that, although crAssphage shedding rates per person vary greatly, on a population-level crAssphage loads per person per day were constant over time and similar for all catchments. Consequently, crAssphage can be used as a quantitative biomarker for populations above 5595 persons. Electrical conductivity is particularly suitable to determine dilution rates relative to dry weather flow concentrations. The overall conclusion is that flow normalisation is necessary to reliably determine short-term trends in virus circulation, and can be enhanced using crAssphage and/or electrical conductivity measurement as a quality check.

Population-based screening in a municipality after a primary school outbreak of the SARS-CoV-2 Alpha variant, the Netherlands, December 2020-February 2021.

An outbreak of SARS-CoV-2 Alpha variant (Pango lineage B.1.1.7) was detected at a primary school (School X) in Lansingerland, the Netherlands, in December 2020. The outbreak was studied retrospectively, and population-based screening was used to assess the extent of virus circulation and decelerate transmission. Cases were SARS-CoV-2 laboratory confirmed and were residents of Lansingerland (November 16th 2020 until February 22th 2021), or had an epidemiological link with School X or neighbouring schools. The SARS-CoV-2 variant was determined using variant PCR or whole genome sequencing. A questionnaire primarily assessed clinical symptoms. A total of 77 Alpha variant cases were found with an epidemiological link to School X, 16 Alpha variant cases linked to the neighbouring schools, and 146 Alpha variant cases among residents of Lansingerland without a link to the schools. The mean number of self-reported symptoms was not significantly different among Alpha variant infected individuals compared to non-Alpha infected individuals. The secondary attack rate (SAR) among Alpha variant exposed individuals in households was 52% higher compared to non-Alpha variant exposed individuals (p = 0.010), with the mean household age, and mean number of children and adults per household as confounders. Sequence analysis of 60 Alpha variant sequences obtained from cases confirmed virus transmission between School X and neighbouring schools, and showed that multiple introductions of the Alpha variant had already taken place in Lansingerland at the time of the study. The alpha variant caused a large outbreak at both locations of School X, and subsequently spread to neighbouring schools, and households. Population-based screening (together with other public health measures) nearly stopped transmission of the outbreak strain, but did not prevent variant replacement in the Lansingerland municipality.

Community-based SARS-CoV-2 testing in low-income neighbourhoods in Rotterdam: Results from a pilot study.

Background: High incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and low testing uptake were reported in low-income neighbourhoods in Rotterdam. We aimed to improve willingness and access to testing by introducing community-based test facilities, and to evaluate the effectiveness of a rapid antigen detection test (RDT). Methods: Two to eleven test facilities operated consecutively in three low-income neighbourhoods in Rotterdam, offering the options of walk-in or appointments. Background characteristics were collected at intake and one nasopharyngeal swab was taken and processed using both RDT and reverse transcription polymerase chain reaction (RT-PCR). Visitors were asked to join a survey for evaluation purposes. Results: In total, 19 773 visitors were tested - 9662 (48.9%) without an appointment. Walk-in visitors were older, lived more often in the proximity of the test facilities, and reported coronavirus disease (COVID-19)-related symptoms less often than by-appointment visitors. For 67.7% of the visitors, this was the first time they got tested. A total of 1211 (6.1%) tested SARS-CoV-2-positive with RT-PCR, of whom 309 (25.5%) were asymptomatic. Test uptake increased among residents of the pilot neighbourhoods, especially in the older age groups, compared to people living in comparable neighbourhoods without community-based testing facilities. RDT detected asymptomatic individuals with 71.8% sensitivity, which was acceptable in this high prevalence setting. Visitors reported positive attitudes towards the test facilities and welcomed the easy access. Conclusions: Offering community-based SARS-CoV-2 testing seems a promising approach for increasing testing uptake among specific populations in low-income neighbourhoods.

Profiling of humoral immune responses to norovirus in children across Europe.

Norovirus is a leading cause of epidemic acute gastroenteritis. More than 30 genotypes circulate in humans, some are common, and others are only sporadically detected. Here, we investigated whether serology can be used to determine which genotypes infect children. We established a multiplex protein microarray with structural and non-structural norovirus antigens that allowed simultaneous antibody testing against 30 human GI and GII genotypes. Antibody responses of sera obtained from 287 children aged < 1 month to 5.5 years were profiled. Most specific IgG and IgA responses were directed against the GII.2, GII.3, GII.4, and GII.6 capsid genotypes. While we detected antibody responses against rare genotypes, we found no evidence for wide circulation. We also detected genotype-specific antibodies against the non-structural proteins p48 and p22 in sera of older children. In this study, we show the age-dependent antibody responses to a broad range of norovirus capsid and polymerase genotypes, which will aid in the development of vaccines.

Clinical and In Vitro Evidence Favoring Immunoglobulin Treatment of a Chronic Norovirus Infection in a Patient With Common Variable Immunodeficiency.

BACKGROUND: Immunocompromised individuals can become chronically infected with norovirus, but effective antiviral therapies are not yet available. METHODS: Treatments with nitazoxanide, ribavirin, interferon alpha-2a, and nasoduodenally administered immunoglobulins were evaluated sequentially in an immunocompromised patient chronically infected with norovirus. In support, these components were also applied to measure norovirus inhibition in intestinal enteroid cultures in vitro. Viral RNA levels were determined in fecal and plasma samples during each treatment and viral genomes were sequenced. RESULTS: None of the antivirals resulted in a reduction of viral RNA levels in feces or plasma. However, during ribavirin treatment, there was an increased accumulation of virus genome mutations. In vitro, an effect of interferon alpha-2a on virus replication was observed and a genetically related strain was neutralized effectively in vitro using immunoglobulins and post-norovirus-infection antiserum. In agreement, after administration of immunoglobulins, the patient cleared the infection. CONCLUSIONS: Intestinal enteroid cultures provide a relevant system to evaluate antivirals and the neutralizing potential of immunoglobulins. We successfully treated a chronically infected patient with immunoglobulins, despite varying results reported by others. This case study provides in-depth, multifaceted exploration of norovirus treatment that can be used as a guidance for further research towards norovirus treatments.

Prevalence and circulation patterns of SARS-CoV-2 variants in European sewage mirror clinical data of 54 European cities.

For community-level monitoring, the European Commission under the EU Sewage Sentinel System recommends wastewater-based SARS-CoV-2 surveillance. Tracking SARS-CoV-2 variants in a community is pivotal for appropriate public health response. Genome sequencing of SARS-CoV-2 in wastewater samples for tracking variants is challenging, often resulting in low coverage genome sequences, thereby impeding the detection of the SARS-CoV-2 mutations. Therefore, we aimed at high-coverage SARS-CoV-2 genome sequences from sewage samples which we successfully accomplished. This first pan-European surveillance compared the mutation profiles associated with the variants of concerns: B.1.1.7, P.1, B.1.351 and B.1.617.2 across 20 European countries, including 54 municipalities. The results highlight that SARS-CoV-2 variants detected in the wastewater samples mirror the variants profiles reported in clinical data. This study demonstrated that >98% coverage of SARS-CoV-2 genomic sequences is possible and can be used to track SARS-CoV-2 mutations in wastewater to support identifying variants circulating in a city at the community level.

Droplet digital RT-PCR to detect SARS-CoV-2 signature mutations of variants of concern in wastewater.

Wastewater surveillance has shown to be a valuable and efficient tool to obtain information about the trends of COVID-19 in the community. Since the recent emergence of new variants, associated with increased transmissibility and/or antibody escape (variants of concern), there is an urgent need for methods that enable specific and timely detection and quantification of the occurrence of these variants in the community. In this study, we demonstrate the use of RT-ddPCR on wastewater samples for specific detection of mutation N501Y. This assay enabled simultaneous enumeration of lineage B.1.351 (containing the 501Y mutation) and Wild Type (WT, containing 501N) SARS-CoV-2 RNA. Detection of N501Y was possible in samples with mixtures of WT with low proportions of B.1.351 (0.5%) and could accurately determine the proportion of N501Y and WT in mixtures of SARS-CoV-2 RNA. The application to raw sewage samples from the cities of Amsterdam and Utrecht demonstrated that this method can be applied to wastewater samples. The emergence of N501Y in Amsterdam and Utrecht wastewater aligned with the emergence of B.1.1.7 as causative agent of COVID-19 in the Netherlands, indicating that RT-ddPCR of wastewater samples can be used to monitor the emergence of the N501Y mutation in the community. It also indicates that RT-ddPCR could be used for sensitive and accurate monitoring of current (like K417N, K417T, E484K, L452R) or future mutations present in SARS-CoV-2 variants of concern. Monitoring these mutations can be used to obtain insight in the introduction and spread of VOC and support public health decision-making regarding measures to limit viral spread or allocation of testing or vaccination.

A luciferase-based approach for measuring HBGA blockade antibody titers against human norovirus.

BACKGROUND: Noroviruses are the most common cause of viral gastroenteritis worldwide, yet there is a deficit in the understanding of protective immunity. Surrogate neutralization assays have been widely used that measure the ability of antibodies to block virus-like particle (VLP) binding to histo-blood group antigens (HBGAs). However, screening large sample sets against multiple antigens using the traditional HBGA blocking assay requires significant investment in terms of time, equipment, and technical expertise, largely associated with the generation of purified VLPs. METHODS: To address these issues, a luciferase immunoprecipitation system (LIPS) assay was modified to measure the norovirus-specific HBGA blockade activity of antibodies. The assay (designated LIPS-Blockade) was validated using a panel of well-characterized homotypic and heterotypic hyperimmune sera as well as strain-specific HBGA blocking monoclonal antibodies. RESULTS: The LIPS-Blockade assay was comparable in specificity to a standard HBGA blocking protocol performed with VLPs. Using time-ordered patient sera, the luciferase-based approach was also able to detect changes in HBGA blocking titers following viral challenge and natural infection with norovirus. CONCLUSION: In this study we developed a rapid, robust, and scalable surrogate neutralization assay for noroviruses that circumvented the need for purified VLPs. This LIPS-Blockade assay should streamline the process of large-scale immunological studies, ultimately aiding in the characterization of protective immunity to human noroviruses.

Monitoring SARS-CoV-2 Circulation and Diversity through Community Wastewater Sequencing, the Netherlands and Belgium.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become a major global health problem, and public health surveillance is crucial to monitor and prevent virus spread. Wastewater-based epidemiology has been proposed as an addition to disease-based surveillance because virus is shed in the feces of ≈40% of infected persons. We used next-generation sequencing of sewage samples to evaluate the diversity of SARS-CoV-2 at the community level in the Netherlands and Belgium. Phylogenetic analysis revealed the presence of the most prevalent clades (19A, 20A, and 20B) and clustering of sewage samples with clinical samples from the same region. We distinguished multiple clades within a single sewage sample by using low-frequency variant analysis. In addition, several novel mutations in the SARS-CoV-2 genome were detected. Our results illustrate how wastewater can be used to investigate the diversity of SARS-CoV-2 viruses circulating in a community and identify new outbreaks.

Human Noroviruses Attach to Intestinal Tissues of a Broad Range of Animal Species.

Human noroviruses are the most common nonbacterial cause of gastroenteritis outbreaks, with new variants and genotypes frequently emerging. The origin of these new viruses is unknown; however, animals have been proposed as a potential source, as human noroviruses have been detected in animal species. Here, we investigated the potential of animals to serve as a reservoir of human noroviruses by testing norovirus attachment to formalin-fixed intestinal tissues of a range of potential reservoir animals. We set up a novel method to study norovirus binding using fluorescein isothiocyanate (FITC)-labeled virus-like particles (VLPs). In humans, noroviruses interact with histo-blood group antigens (HBGAs), carbohydrates that are expressed, among others, on the epithelial lining of the gastrointestinal tract. In animals, this interaction is not well understood. To test if virus binding depends on HBGAs, we characterized the HBGA phenotype in animal tissues by immunohistochemistry. With the exception of the black-headed gull and the straw-colored fruitbat, we observed the attachment of several human norovirus genotypes to the intestinal epithelium of all tested animal species. However, we did not find an association between the expression of a specific HBGA phenotype and virus-like particle (VLP) attachment. We show that selected human noroviruses can attach to small-intestinal tissues across species, supporting the hypothesis that human noroviruses can reside in an animal reservoir. However, whether this attachment can subsequently lead to infection needs to be further assessed.IMPORTANCE Noroviruses are a major cause of acute gastroenteritis in humans. New norovirus variants and recombinants (re)emerge regularly in the human population. From animal experiments and surveillance studies, it has become clear that at least seven animal models are susceptible to infection with human strains and that domesticated and wild animals shed human noroviruses in their feces. As virus attachment is an important first step for infection, we used a novel method utilizing FITC-labeled VLPs to test for norovirus attachment to intestinal tissues of potential animal hosts. We further characterized these tissues with regard to their HBGA expression, a well-studied norovirus susceptibility factor in humans. We found attachment of several human strains to a variety of animal species independent of their HBGA phenotype. This supports the hypothesis that human strains could reside in an animal reservoir.

Detection of Norovirus Variant GII.4 Hong Kong in Asia and Europe, 2017-2019.

We report a new norovirus GII.4 variant, GII.4 Hong Kong, with low-level circulation in 4 Eurasia countries since mid-2017. Amino acid substitutions in key residues on the virus capsid associated with the emergence of pandemic noroviruses suggest that GII.4 Hong Kong has the potential to become the next pandemic variant.

Improving Hand Hygiene Compliance in Nursing Homes: Protocol for a Cluster Randomized Controlled Trial (HANDSOME Study).

BACKGROUND: Hand hygiene compliance is considered the most (cost-)effective measure for preventing health care-associated infections. While hand hygiene interventions have frequently been implemented and assessed in hospitals, there is limited knowledge about hand hygiene compliance in other health care settings and which interventions and implementation methods are effective. OBJECTIVE: This study aims to evaluate the effect of a multimodal intervention to increase hand hygiene compliance of nurses in nursing homes through a cluster randomized controlled trial (HANDSOME study). METHODS: Nursing homes were randomly allocated to 1 of 3 trial arms: receiving the intervention at a predetermined date, receiving the identical intervention after an infectious disease outbreak, or serving as a control arm. Hand hygiene was evaluated in nursing homes by direct observation at 4 timepoints. We documented compliance with the World Health Organization's 5 moments of hand hygiene, specifically before touching a patient, before a clean/aseptic procedure, after body fluid exposure risk, after touching a patient, and after touching patient surroundings. The primary outcome is hand hygiene compliance of the nurses to the standards of the World Health Organization. The secondary outcome is infectious disease incidence among residents. Infectious disease incidence was documented by a staff member at each nursing home unit. Outcomes will be compared with the presence of norovirus, rhinovirus, and Escherichia coli on surfaces in the nursing homes, as measured using quantitative polymerase chain reaction. RESULTS: The study was funded in September 2015. Data collection started in October 2016 and was completed in October 2017. Data analysis will be completed in 2020. CONCLUSIONS: HANDSOME studies the effectiveness of a hand hygiene intervention specifically for the nursing home environment. Nurses were taught the World Health Organization's 5 moments of hand hygiene guidelines using the slogan "Room In, Room Out, Before Clean, After Dirty," which was developed for nursing staff to better understand and remember the hygiene guidelines. HANDSOME should contribute to improved hand hygiene practice and a reduction in infectious disease rates and related mortality. TRIAL REGISTRATION: Netherlands Trial Register (NTR6188) NL6049; https://www.trialregister.nl/trial/6049. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/17419.

SARS-CoV-2 productively infects human gut enterocytes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause coronavirus disease 2019 (COVID-19), an influenza-like disease that is primarily thought to infect the lungs with transmission through the respiratory route. However, clinical evidence suggests that the intestine may present another viral target organ. Indeed, the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is highly expressed on differentiated enterocytes. In human small intestinal organoids (hSIOs), enterocytes were readily infected by SARS-CoV and SARS-CoV-2, as demonstrated by confocal and electron microscopy. Enterocytes produced infectious viral particles, whereas messenger RNA expression analysis of hSIOs revealed induction of a generic viral response program. Therefore, the intestinal epithelium supports SARS-CoV-2 replication, and hSIOs serve as an experimental model for coronavirus infection and biology.

Norovirus outbreak in a natural playground: A One Health approach.

Norovirus constitutes the most frequently identified infectious cause of disease outbreaks associated with untreated recreational water. When investigating outbreaks related to surface water, a One Health approach is insightful. Historically, there has been a focus on potential contamination of recreational water by bird droppings and a recent publication demonstrating human noroviruses in bird faeces suggested this should be investigated in future water-related norovirus outbreaks. Here, we describe a One Health approach investigating a norovirus outbreak in a natural playground. On social media, a large amount of waterfowl were reported to defecate near these playground premises leading to speculations about their potential involvement. Surface water, as well as human and bird faecal specimens, was tested for human noroviruses. Norovirus was found to be the most likely cause of the outbreak but there was no evidence for transmission via waterfowl. Cases had become known on social media prior to notification to the public health service underscoring the potential of online media as an early warning system. In view of known risk factors, advice was given for future outbreak investigations and natural playground design.

Novel opportunities for NGS-based one health surveillance of foodborne viruses.

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.

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.

Updated classification of norovirus genogroups and genotypes.

Noroviruses are genetically diverse RNA viruses associated with acute gastroenteritis in mammalian hosts. Phylogenetically, they can be segregated into different genogroups as well as P (polymerase)-groups and further into genotypes and P-types based on amino acid diversity of the complete VP1 gene and nucleotide diversity of the RNA-dependent RNA polymerase (RdRp) region of ORF1, respectively. In recent years, several new noroviruses have been reported that warrant an update of the existing classification scheme. Using previously described 2× standard deviation (sd) criteria to group sequences into separate clusters, we expanded the number of genogroups to 10 (GI-GX) and the number of genotypes to 48 (9 GI, 27 GII, 3 GIII, 2 GIV, 2 GV, 2 GVI and 1 genotype each for GVII, GVIII, GIX [formerly GII.15] and GX). Viruses for which currently only one sequence is available in public databases were classified into tentative new genogroups (GNA1 and GNA2) and genotypes (GII.NA1, GII.NA2 and GIV.NA1) with their definitive assignment awaiting additional related sequences. Based on nucleotide diversity in the RdRp region, noroviruses can be divided into 60 P-types (14 GI, 37 GII, 2 GIII, 1 GIV, 2 GV, 2 GVI, 1 GVII and 1 GX), 2 tentative P-groups and 14 tentative P-types. Future classification and nomenclature updates will be based on complete genome sequences and will be coordinated and disseminated by the international norovirus classification-working group.