Patterns of the within-host evolution of human norovirus in immunocompromised individuals and implications for treatment.

BACKGROUND: Currently, there is no licensed treatment for chronic norovirus infections, but the use of intra-duodenally-delivered immunoglobulins is promising; nevertheless, varying results have limited their wide use. Little is known about the relationship between norovirus genetic diversity and treatment efficacy. METHODS: We analyzed the norovirus within-host diversity and evolution in a cohort of 20 immunocompromised individuals using next-generation sequencing (NGS) and clone-based sequencing of the capsid (VP1) gene. Representative VP1s were expressed and their glycan receptor binding affinity and antigenicity were evaluated. FINDINGS: The P2 domain, within the VP1, accumulated up to 30-fold more non-synonymous mutations than other genomic regions. Intra-host virus populations in these patients tended to evolve into divergent lineages that were often antigenically distinct. Several of these viruses were widely resistant to binding-blocking antibodies in immunoglobulin preparations. Notably, for one patient, a single amino-acid substitution in the P2 domain resulted in an immune-escape phenotype, and it was likely the main contributor to treatment failure. Furthermore, we found evidence for transmission of late-stage viruses between two immunocompromised individuals. INTERPRETATION: The findings demonstrated that within-host noroviruses in chronic infections tend to evolve into antigenically distinct subpopulations. This antigenic evolution was likely caused by the remaining low immunity levels exerted by immunocompromised individuals, possibly undermining antiviral treatment. Our observations provide insights into norovirus (within-host) evolution and treatment. FUNDING: Erasmus MC grant mRACE, the European Union's Horizon 2020 research and innovation program under grant agreement No. 874735 (VEO), and the NWO STEVIN award (Koopmans).

Accessible viral metagenomics for public health and clinical domains with Jovian.

The integration of next-generation sequencing into clinical diagnostics and surveillance initiatives is impeded by the lack of data analysis pipelines that align with privacy legislation and laboratory certification protocols. To address these challenges, we developed Jovian, an open-source, virus-focused, metagenomic analysis workflow for Illumina data. Jovian generates scaffolds enriched with pertinent annotations, including taxonomic classification, combined with metrics needed for quality assessment (coverage depth, average GC content, localization of open reading frames, minority single nucleotide polymorphisms), and incorporates host and disease metadata. Interactive web-based reports with an audit trail are generated. Jovian was employed on four systems, hosted by three institutes, utilizing grid-computers, a high-performance compute singular server, and a Windows10 laptop. All systems yielded identical results with matching MD5sums. Comparison with a commercial online reference tool using viral gastroenteritis samples confirmed the identification of the same pathogens. Jovian provides comparable results to a commercially available online reference tool and generates identical results at different institutes with different IT architectures, proving it is portable and reproducible. Jovian addresses bottlenecks in the deployment of metagenomics within public health and clinical laboratories and has the potential to enhance the breadth of surveillance and testing programs, thereby fostering more effective public health interventions.

Increased circulation of GII.17 noroviruses, six European countries and the United States, 2023 to 2024.

We report an increase in GII.17 norovirus outbreaks and sporadic infections of acute gastroenteritis in Austria, Germany, France, Ireland, the Netherlands, England and the United States during the 2023/24 season. A decrease in GII.4 coincided with GII.17 prevalence increasing to between 17% and 64% of all GII detections. Overall, 84% of the GII.17 strains clustered closely with strains first reported in Romania in 2021 and two new sub-lineages were identified. Norovirus surveillance and molecular characterisation should be prioritised this winter.

Time-series sewage metagenomics distinguishes seasonal, human-derived and environmental microbial communities potentially allowing source-attributed surveillance.

Sewage metagenomics has risen to prominence in urban population surveillance of pathogens and antimicrobial resistance (AMR). Unknown species with similarity to known genomes cause database bias in reference-based metagenomics. To improve surveillance, we seek to recover sewage genomes and develop a quantification and correlation workflow for these genomes and AMR over time. We use longitudinal sewage sampling in seven treatment plants from five major European cities to explore the utility of catch-all sequencing of these population-level samples. Using metagenomic assembly methods, we recover 2332 metagenome-assembled genomes (MAGs) from prokaryotic species, 1334 of which were previously undescribed. These genomes account for ~69% of sequenced DNA and provide insight into sewage microbial dynamics. Rotterdam (Netherlands) and Copenhagen (Denmark) show strong seasonal microbial community shifts, while Bologna, Rome, (Italy) and Budapest (Hungary) have occasional blooms of Pseudomonas-dominated communities, accounting for up to ~95% of sample DNA. Seasonal shifts and blooms present challenges for effective sewage surveillance. We find that bacteria of known shared origin, like human gut microbiota, form communities, suggesting the potential for source-attributing novel species and their ARGs through network community analysis. This could significantly improve AMR tracking in urban environments.

An interlaboratory proficiency test using metagenomic sequencing as a diagnostic tool for the detection of RNA viruses in swine fecal material.

Metagenomic shotgun sequencing (mNGS) can serve as a generic molecular diagnostic tool. An mNGS proficiency test (PT) was performed in six European veterinary and public health laboratories to detect porcine astroviruses in fecal material and the extracted RNA. While different mNGS workflows for the generation of mNGS data were used in the different laboratories, the bioinformatic analysis was standardized using a metagenomic read classifier as well as read mapping to selected astroviral reference genomes to assess the semiquantitative representation of astrovirus species mixtures. All participants successfully identified and classified most of the viral reads to the two dominant species. The normalized read counts obtained by aligning reads to astrovirus reference genomes by Bowtie2 were in line with Kraken read classification counts. Moreover, participants performed well in terms of repeatability when the fecal sample was tested in duplicate. However, the normalized read counts per detected astrovirus species differed substantially between participants, which was related to the different laboratory methods used for data generation. Further modeling of the mNGS data indicated the importance of selecting appropriate reference data for mNGS read classification. As virus- or sample-specific biases may apply, caution is needed when extrapolating this swine feces-based PT for the detection of other RNA viruses or using different sample types. The suitability of experimental design to a given pathogen/sample matrix combination, quality assurance, interpretation, and follow-up investigation remain critical factors for the diagnostic interpretation of mNGS results. IMPORTANCE: Metagenomic shotgun sequencing (mNGS) is a generic molecular diagnostic method, involving laboratory preparation of samples, sequencing, bioinformatic analysis of millions of short sequences, and interpretation of the results. In this paper, we investigated the performance of mNGS on the detection of porcine astroviruses, a model for RNA viruses in a pig fecal material, among six European veterinary and public health laboratories. We showed that different methods for data generation affect mNGS performance among participants and that the selection of reference genomes is crucial for read classification. Follow-up investigation remains a critical factor for the diagnostic interpretation of mNGS results. The paper contributes to potential improvements of mNGS as a diagnostic tool in clinical settings.

Bacteriophage therapy reduces Staphylococcus aureus in a porcine and human ex vivo burn wound infection model.

Burn wounds are a major burden, with high mortality rates due to infections. Staphylococcus aureus is a major causative agent of burn wound infections, which can be difficult to treat because of antibiotic resistance and biofilm formation. An alternative to antibiotics is the use of bacteriophages, viruses that infect and kill bacteria. We investigated the efficacy of bacteriophage therapy for burn wound infections, in both a porcine and a newly developed human ex vivo skin model. In both models, the efficacy of a reference antibiotic treatment (fusidic acid) and bacteriophage treatment was determined for a single treatment, successive treatment, and prophylaxis. Both models showed a reduction in bacterial load after a single bacteriophage treatment. Increasing the frequency of bacteriophage treatments increased bacteriophage efficacy in the human ex vivo skin model, but not in the porcine model. In both models, prophylaxis with bacteriophages increased treatment efficacy. In all cases, bacteriophage treatment outperformed fusidic acid treatment. Both models allowed investigation of bacteriophage-bacteria dynamics in burn wounds. Overall, bacteriophage treatment outperformed antibiotic control underlining the potential of bacteriophage therapy for the treatment of burn wound infections, especially when used prophylactically.

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.