Development of a broad-spectrum therapeutic Fc-nanobody for human noroviruses.

Human norovirus was discovered more than five decades ago and is a widespread cause of outbreaks of acute gastroenteritis. There are no approved vaccines or antivirals currently available. However, norovirus inhibitors, including capsid-specific monoclonal antibodies (Mabs) and nanobodies, have recently shown promising results. Several Mabs and nanobodies were found to inhibit norovirus replication using a human intestinal enteroid (HIE) culture system and/or could block norovirus attachment to histo-blood group antigen (HBGA) co-factors. In our pursuit to develop a single broad-spectrum norovirus therapeutic, we continued our analysis and development of a cross-reactive and HBGA interfering nanobody (NB26). To improve NB26 binding capacity and therapeutic potential, we conjugated NB26 onto a human IgG Fc domain (Fc-NB26). We confirmed that Fc-NB26 cross-reacts with genetically diverse GII genotype capsid protruding (P) domains (GII.8, GII.14, GII.17, GII.24, GII.26, and GII.NA1) using a direct enzyme-linked immunosorbent assay. Furthermore, X-ray crystallography structures of these P domains and structures of other GII genotypes reveal that the NB26 binding site is largely conserved, validating its broad reactivity. We showed that Fc-NB26 has ~100-fold higher affinity toward the norovirus P domain compared to native NB26. We also found that both NB26 and Fc-NB26 neutralize human norovirus replication in the HIE culture system. Furthermore, the mode of inhibition confirmed that like NB26, Fc-NB26 caused norovirus particle disassembly and aggregation. Overall, these new findings demonstrate that structural modifications to nanobodies can improve their therapeutic potential.IMPORTANCEDeveloping vaccines and antivirals against norovirus remains a challenge, mainly due to the constant genetic and antigenic evolution. Moreover, re-infection with genetically related and/or antigenic variants is not uncommon. We further developed our leading norovirus nanobody (NB26) that indirectly interfered with norovirus binding to HBGAs, by converting NB26 into a dimeric Fc-linked Nanobody (Fc-NB26). We found that Fc-NB26 had improved binding affinity and neutralization capacity compared with native NB26. Using X-ray crystallography, we showed this nanobody engaged highly conserved capsid residues among genetically diverse noroviruses. Development of such broadly reactive potent therapeutic nanobodies delivered as a slow-releasing prophylactic could be of exceptional value for norovirus outbreaks, especially for the prevention or treatment of severe acute gastroenteritis in high-risk groups such as the young, elderly, and immunocompromised.

The attachment factors and attachment receptors of human noroviruses.

Human noroviruses (HuNoVs) are the leading etiological agent causing the worldwide outbreaks of acute epidemic non-bacterial gastroenteritis. Histo-blood group antigens (HBGAs) are commonly acknowledged as cellular receptors or co-receptors for HuNoVs. However, certain genotypes of HuNoVs cannot bind with any HBGAs, suggesting potential additional co-factors and attachment receptors have not been identified yet. In addition, food items, such as oysters and lettuce, play an important role in the transmission of HuNoVs. In the past decade, a couple of attachment factors other than HBGAs have been identified and analyzed from foods and microbiomes. Attachment factors exhibit potential as inhibitors of viral binding to receptors on host cells. Therefore, it is imperative to further characterize the attachment factors for HuNoVs present in foods to effectively control the spread of HuNoVs within the food chain. This review summarizes the potential attachment factors/receptors of HuNoVs in humans, foods, and microbiome.

Genetic Diversity and Phylogenetic Relationship of Human Norovirus Sequences Derived from Municipalities within the Sverdlovsk Region of Russia.

Human noroviruses (HuNoVs) are highly contagious pathogens responsible of norovirus-associated acute gastroenteritis (AGE). GII.4 is the prevailing HuNoV genotype worldwide. Currently there are no studies on the molecular monitoring and phylogenetic analysis of HuNoVs in the territory of the Sverdlovsk region; therefore, it is not possible to objectively assess their genetic diversity. The aim of the study is to carry out genotyping and phylogenetic analysis of HuNoVs in the Sverdlovsk region from 2022 to 2023. Fecal samples (n = 510) were collected from children suffering from HuNoV-AGE in municipalities of the Sverdlovsk region and the capsid genotype was determined by amplifying the ORF1/ORF2 junction. Of the 196 HuNoVs typed, which represent 38% of the studied samples, the largest share of HuNoV genotypes belong to the GII genogroup-86%, followed by the GI genogroup-14%. Noroviruses GII.4 and GII.17 were the co-dominant capsid genotypes (33.2% each). Phylogenetic analysis demonstrates that the identified sequences on the territory of the Sverdlovsk region have the smallest genetic distance, which gives grounds for their unification into a common cluster. Routine monitoring and phylogenetic analysis of circulating norovirus pathogens spectrum will enable timely tracking of HuNoVs genetic diversity and evolutionary events. This will lead to the development of more effective anti-epidemic measures, ultimately reducing the burden of infectious diseases.

The reversible activation of norovirus by metal ions.

Murine norovirus (MNV) undergoes extremely large conformational changes in response to the environment. The T = 3 icosahedral capsid is composed of 180 copies of ~58-kDa VP1 comprised of N-terminus (N), shell (S), and C-terminal protruding (P) domains. At neutral pH, the P domains are loosely tethered to the shell and float ~15 Å above the surface. At low pH or in the presence of bile salts, the P domain drops onto the shell and this movement is accompanied by conformational changes within the P domain that enhance receptor interactions while blocking antibody binding. While previous crystallographic studies identified metal binding sites in the isolated P domain, the ~2.7-Å cryo-electron microscopy structures of MNV in the presence of Mg2+ or Ca2+ presented here show that metal ions can recapitulate the contraction observed at low pH or in the presence of bile. Further, we show that these conformational changes are reversed by dialysis against EDTA. As observed in the P domain crystal structures, metal ions bind to and contract the G'H' loop. This movement is correlated with the lifting of the C'D' loop and rotation of the P domain dimers about each other, exposing the bile salt binding pocket. Isothermal titration calorimetry experiments presented here demonstrate that the activation signals (bile salts, low pH, and metal ions) act in a synergistic manner that, individually, all result in the same activated structure. We present a model whereby these reversible conformational changes represent a uniquely dynamic and tissue-specific structural adaptation to the in vivo environment.IMPORTANCEThe highly mobile protruding domains on the calicivirus capsids are recognized by cell receptor(s) and antibodies. At neutral pH, they float ~15 Å above the shell but at low pH or in the presence of bile salts, they contract onto the surface. Concomitantly, changes within the P domain block antibody binding while enhancing receptor binding. While we previously demonstrated that metals also block antibody binding, it was unknown whether they might also cause similar conformational changes in the virion. Here, we present the near atomic cryo-electron microscopy structures of infectious murine norovirus (MNV) in the presence of calcium or magnesium ions. The metal ions reversibly induce the same P domain contraction as low pH and bile salts and act in a synergistic manner with the other stimuli. We propose that, unlike most other viruses, MNV facilely changes conformations as a unique means to escape immune surveillance as it moves through various tissues.

A study on the occurrence of human enteric viruses in salad vegetables and seafood and associated health risks for consumers in Mauritius.

Norovirus (NOV) and hepatitis A virus (HAV) are human enteric viruses of major concern worldwide. Salad vegetables and molluscan shellfish are highly susceptible to contamination by NOV and HAV and can pose a health threat when consumed raw. The objective of this study was to determine the occurrence of NOV and HAV in lettuce, watercress, tomatoes, and oysters using the enzyme-linked immunosorbent assay and assess the health risks associated with the consumption of these commodities by semiquantitative risk assessment. The occurrence of NOV in vegetables ranked in the following decreasing order: lettuce (36%) > watercress (16%) > tomatoes (4%). However, HAV was more frequently detected in watercress (56%), compared to lettuce or tomatoes (12%). Additionally, NOV was detected in oysters (60%). The risk assessment exercise pointed to a medium-risk score of contracting a foodborne illness of viral origin for consumers eating fresh watercress or oysters. Future research will ascertain the presence of these enteric viruses in a broader range of food commodities.

Blockade Antibody Responses in Human Subjects Challenged with a New Snow Mountain Virus Inoculum.

Background: Noroviruses (NoVs) are a leading cause of non-bacterial gastroenteritis in young children and adults worldwide. Snow Mountain Virus (SMV) is the prototype of NoV GII genotype 2 (GII.2) that has been developed as a viral model for human challenge models, an important tool for studying pathogenesis and immune response of NoV infections and for evaluating NoV vaccine candidates. Previous studies have identified blockade antibodies that block the binding of NoV virus-like particles (VLPs) to histo-blood group antigens (HBGAs) as a surrogate for neutralization in human Norwalk virus and GII.4 infections but little is known about SMV blockade antibodies. Methods: In this secondary data analysis study, blockade antibodies were characterized in pre-challenge and post-challenge serum samples from human subjects challenged with a new SMV inoculum. The correlation between blockade antibody geometric mean antibody titers (GMTs) and SMV-specific serum IgG/IgA GMTs were examined after stratifying the subjects by infection status. A linear mixed model was applied to test the association between HBGA blockade antibody concentrations and post-challenge days accounting for covariates and random effects. Results: Laboratory results from 33 SMV inoculated individuals were analyzed and 75.7% (25/33) participants became infected. Serum SMV-specific blockade antibodies, IgA, and IgG were all significantly different between infected and uninfected individuals beginning day 15 post-challenge. Within infected individuals, a significant correlation was observed between both IgG and IgA and blockade antibody concentration as early as day 6 post-challenge. Analysis of blockade antibody using the linear mixed model showed that infected individuals, when compared to uninfected individuals, had a statistically significant increase in blockade antibody concentrations across the post-challenge days. Among the post-challenge days, blockade antibody concentrations on days 15, 30, and 45 were significantly higher than those observed pre-challenge. The intraclass correlation coefficient (ICC) analysis indicated that the variability of blockade antibody titers is more observed between individuals rather than observations within subjects. Conclusions: These results indicate that HBGA-blockade antibody GMTs are generated after SMV challenge and the blockade antibodies were still detectable at day 45 post-challenge. These data indicate that the second generation of SMV inoculum is highly effective.

Rapid and sensitive on-site detection of SARS-CoV-2 RNA from environmental surfaces using portable laboratory devices.

IMPORTANCE: This study presents the development of a highly sensitive on-site method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA on various surfaces, including doorknobs and tables. Identifying SARS-CoV-2 RNA on these surfaces can be crucial in guiding decision-making for implementing non-pharmaceutical interventions, such as zoning strategies, improving ventilation, maintaining physical distancing, and promoting increased hand hygiene practices. Moreover, the on-site detection system can facilitate the swift initiation of mitigation responses in non-laboratory settings, including long-term care facilities and schools. The protocols established in this study offer a comprehensive approach for achieving both sensitivity and rapidity in on-site SARS-CoV-2 RNA detection. Furthermore, since the RT-qPCR assay serves as the gold standard for detecting viral RNAs, the developed protocol holds potential for application to other viruses, including enteroviruses and noroviruses.

Benchmarking State-of-the-Art Approaches for Norovirus Genome Assembly in Metagenome Sample.

A recently published article in BMCGenomics by Fuentes-Trillo et al. contains a comparison of assembly approaches of several noroviral samples via different tools and preprocessing strategies. It turned out that the study used outdated versions of tools as well as tools that were not designed for the viral assembly task. In order to improve the suboptimal assemblies, authors suggested different sophisticated preprocessing strategies that seem to make only minor contributions to the results. We have reproduced the analysis using state-of-the-art tools designed for viral assembly, and we demonstrate that tools from the SPAdes toolkit (rnaviralSPAdes and coronaSPAdes) allow one to assemble the samples from the original study into a single contig without any additional preprocessing.

Molecular and Genetics-Based Systems for Tracing the Evolution and Exploring the Mechanisms of Human Norovirus Infections.

Human noroviruses (HuNoV) are major causes of acute gastroenteritis around the world. The high mutation rate and recombination potential of noroviruses are significant challenges in studying the genetic diversity and evolution pattern of novel strains. In this review, we describe recent advances in the development of technologies for not only the detection but also the analysis of complete genome sequences of noroviruses and the future prospects of detection methods for tracing the evolution and genetic diversity of human noroviruses. The mechanisms of HuNoV infection and the development of antiviral drugs have been hampered by failure to develop the infectious virus in a cell model. However, recent studies have demonstrated the potential of reverse genetics for the recovery and generation of infectious viral particles, suggesting the utility of this genetics-based system as an alternative for studying the mechanisms of viral infection, such as cell entry and replication.

Epidemiological investigation of norovirus infections in Punjab, Pakistan, through the One Health approach.

Introduction: Norovirus, mainly associated with acute gastroenteritis, is very contagious and can affect a vast range of species ranging from cattle, pigs, dogs, mice, cats, sheep, and lions to humans. It is a foodborne pathogen that mainly transmits through the fecal-oral route. Methods: This is the first-ever study conducted in Lahore and Sheikhupura districts of Punjab, Pakistan, to investigate noroviruses through the One Health approach. From January 2020 to September 2021, 200 fecal samples were collected from clinical cases of hospitalized patients and 200 fecal samples from sick animals at veterinary hospitals and local farms. In addition, 500 food and beverage samples were collected from street vendors and retail stores. A predesigned questionnaire was used to assess the risk factors and clinical characteristics of sick people and animals. Results and discussion: Overall, 14% of the human clinical samples were positive by RT-PCR for genogroup GII. All bovine samples were negative. Food and beverage samples were tested in pools, resulting in sugarcane juice samples positive for genogroup GII. Previous contact with acute gastroenteritis patients, sex, and presence of vomiting were found to be significant risk factors (p ≤ 0.05). The substantial number of diarrhea cases associated with noroviruses calls for additional studies to investigate the epidemiology and transmission and to improve surveillance.

Viral Gastroenteritis: Sickness Symptoms and Behavioral Responses.

Viral infections have a major impact on physiology and behavior. The clinical symptoms of human rotavirus and norovirus infection are primarily diarrhea, fever, and vomiting, but several other sickness symptoms, such as nausea, loss of appetite, and stress response are never or rarely discussed. These physiological and behavioral changes can be considered as having evolved to reduce the spread of the pathogen and increase the chances of survival of the individual as well as the collective. The mechanisms underlying several sickness symptoms have been shown to be orchestrated by the brain, specifically, the hypothalamus. In this perspective, we have described how the central nervous system contributes to the mechanisms underlying the sickness symptoms and behaviors of these infections. Based on published findings, we propose a mechanistic model depicting the role of the brain in fever, nausea, vomiting, cortisol-induced stress, and loss of appetite.

An oral NoV-rAd5 vaccine with built-in dsRNA adjuvant elicits systemic immune responses in mice.

Norovirus (NoV) is an enteric pathogen notorious for causing epidemics of acute gastroenteritis. An effective vaccine against NoV is therefore urgently needed. A short double-stranded RNA (dsRNA) has been described that acts as a retinoic-acid-inducible gene-I agonist to induce the production of type I interferon; it also exhibits adjuvant activity. Using built-in dsRNA of different lengths (DS1 and DS2), we developed a recombinant adenovirus 5 (rAd5) expressing NoV VP1, and evaluated its immunogenicity following oral administration in a mouse model. An in vitro study demonstrated that the dsRNA adjuvants significantly enhanced VP1 protein expression in infected cells. The oral administration of both rAd5-VP1-DS vaccines elicited high serum levels of VP1-specific IgG and blocking antibodies, as well as strong and long-lasting mucosal immunity. There was no apparent difference in immunostimulatory effects in immunised mice between the two dsRNA adjuvants. This study indicates that an oral NoV-rAd5 vaccine with a built-in dsRNA adjuvant may be developed to prevent NoV infection in humans.

Development and Characterization of Synthetic Norovirus RNA for Use in Molecular Detection Methods.

Background: Reference materials are essential for the quality assurance of molecular detection methods. We developed and characterized synthetic norovirus GI and GII RNA reference materials. Methods: Norovirus GI and GII RNA sequences including the ORF1-ORF2 junction region were designed based on 1,495 reported norovirus sequences and synthesized via plasmid preparation and in vitro transcription. The synthetic norovirus GI and GII RNAs were evaluated using six commercial norovirus detection kits used in Korea and subjected to homogeneity and stability analyses. A multicenter study involving five laboratories and using four commercial real-time PCR norovirus detection assays was conducted for synthetic norovirus RNA characterization and uncertainty measurements. Results: The synthetic norovirus GI and GII RNAs were positively detected using the six commercial norovirus detection kits and were homogeneous and stable for one year when stored at -20°C or -70°C. All data from the five laboratories were within a range of 1.0 log copies/µL difference for each RNA, and the overall mean concentrations for norovirus GI and GII RNAs were 7.90 log copies/µL and 6.96 log copies/µL, respectively. Conclusions: The synthetic norovirus GI and GII RNAs are adequate for quality control based on commercial molecular detection reagents for noroviruses with high sequence variability. The synthetic RNAs can be used as reference materials in norovirus molecular detection methods.

Functional and structural characterization of Norovirus GII.6 in recognizing histo-blood group antigens.

Noroviruses (NoVs) are the primary cause of acute gastroenteritis worldwide. Histo-blood group antigens (HBGAs) are receptors or attachment factors that affect the prevalence and host susceptibility of NoVs. GII.6 NoV is one of the predominant genotypes in humans, which recognizes the type ABO secretor of HBGAs. However, the structural basis of GII.6 NoV's interaction with HBGAs receptors remains elusive. In this study, we investigated the binding features of the GII.6 strain to HBGAs using saliva- and glycan-ELISA assays and characterized the molecular basis of the GII.6 virus that recognizes H disaccharide. We showed that the GII.6 â€‹P domain recognized some A and O secretor's saliva samples, most B secretor's saliva samples, and H disaccharide antigen, but did not bind non-secretors' saliva. Further, we determined the crystal structures of GII.6 and its complex with H disaccharides at 1.7 â€‹Å, revealing that the P domain of GII.6 shares the conventional binding interface and mode of GII HBGAs. Single residue mutations at the GII.6-H binding sites could inhibit the binding of GII.6 to HBGAs, demonstrating that the interaction residues were crucial in maintaining NoV-glycan integrity. Finally, structural and sequence analyses showed that the major residues of the GII.6-H interaction were conserved among NoVs in the GII genogroup. Taken together, our study characterized the functional and structural features of GII.6 that allow it to interact with HBGAs, and shed light on NoV evolution, epidemiology, and anti-viral drug development.

A systematic review and meta-analysis indicates a substantial burden of human noroviruses in shellfish worldwide, with GII.4 and GII.2 being the predominant genotypes.

Human noroviruses (HuNoVs) have been found as the leading cause of acute gastroenteritis outbreaks in all age groups and are significantly correlated with the consumption of shellfish. In this study, the contamination of HuNoVs in shellfish was estimated through a systematic review and meta-analysis. Studies on the contamination of HuNoVs in shellfish were searched from PubMed, Web of Science, Embase, and Cochrane Library from January 2000 to August 2021. A total of 75 studies were included, and the pooled HuNoVs prevalence in shellfish was 29% (95% CI: 23-35) worldwide. As revealed by the results of the subgroup meta-analysis, the prevalence of dominant genogroup was variable, and 4% (95% CI: 3-6), 13% (95% CI: 10-17), with 7% (95% CI: 4-11) of the samples, respectively, contaminated by GI alone, GII alone, and GI&GII. The HuNoVs prevalence of shellfish in Europe, America, and Asia was 33% (95% CI: 24-43), 24% (95% CI: 7-47), and 27% (95% CI: 18-35), respectively, while only 10% (95% CI: 5-17) in Africa. Furthermore, the prevalence of HuNoVs in shellfish was the highest in spring (35%, 95% CI: 23-49) and winter (35%, 95% CI: 22-50), and the lowest in summer (11%, 95% CI: 5-18). Oysters, clams, and mussels had comparable HuNoVs prevalence of 28% (95% CI: 20-37), 27% (95% CI: 16-39) and 24% (95% CI: 17-32), respectively. The prevalence of HuNoVs in shellfish from harvest areas and markets was 30% (95% CI: 23-38) and 30% (95% CI: 19-41), respectively. The results of this study suggest a substantial burden of HuNoVs in shellfish worldwide, with GII.4 (92.86%) and GII.2 (46.43%) as the predominant genotypes. This study provides information regarding the contamination of HuNoVs in shellfish worldwide, which will contribute to the development of appropriate control measures to prevent shellfish-related HuNoVs gastroenteritis.

Blockade Antibody Responses in Human Subjects Challenged with a New Snow Mountain Virus Inoculum.

Background: Noroviruses (NoVs) are a leading cause of non-bacterial gastroenteritis in young children and adults worldwide. Snow Mountain Virus (SMV) is the prototype of NoV GII genotype 2 (GII.2) that has been developed as a viral model for human challenge studies, an important tool for studying pathogenesis and immune response of NoV infections and for evaluating NoV vaccine candidates. Previous studies have identified blockade antibodies that block the binding of NoV virus-like particles (VLPs) to histo-blood group antigens (HBGAs) as a surrogate for neutralization in human Norwalk virus and GII.4 infections but little is known about SMV blockade antibodies. Methods: In this secondary data analysis study, blockade antibodies were characterized in pre-challenge and post-challenge serum samples from human subjects challenged with a new SMV inoculum. The correlation between blockade antibody geometric mean antibody titers (GMTs) and SMV-specific serum IgG/IgA GMTs were examined after stratifying the subjects by infection status. A linear mixed model was applied to test the association between HBGA blockade antibody concentrations and post-challenge days accounting for covariates and random effects. Results: Laboratory results from 33 SMV inoculated individuals were analyzed and 75.7% (25/33) participants became infected. Serum SMV-specific blockade antibodies, IgA, and IgG were all significantly different between infected and uninfected individuals beginning day 15 post-challenge. Within infected individuals, a significant correlation was observed between both IgG and IgA and blockade antibody concentration as early as day 6 post-challenge. Analysis of blockade antibody using the linear mixed model showed that infected individuals, when compared to uninfected individuals, had a statistically significant increase in blockade antibody concentrations across the post-challenge days. Among the post-challenge days, blockade antibody concentrations on days 15, 30, and 45 were significantly higher than those observed pre-challenge. The intraclass correlation coefficient (ICC) analysis indicated that the variability of blockade antibody titers is more observed between individuals rather than within subjects. Conclusions: These results indicate that HBGA-blockade antibody GMTs are generated after SMV challenge and the blockade antibodies were still detectable at day 45 post-challenge. These data indicate that the second-generation of SMV inoculum is highly effective.

Identification of compelling inhibitors of human norovirus 3CL protease to combat gastroenteritis: A structure-based virtual screening and molecular dynamics study.

Human noroviruses (NV) are the most prevalent cause of sporadic and pandemic acute gastroenteritis. NV infections cause substantial morbidity and death globally, especially amongst the aged, immunocompromised individuals, and children. There are presently no authorized NV vaccines, small-molecule therapies, or prophylactics for humans. NV 3 C L protease (3CLP) has been identified as a promising therapeutic target for anti-NV drug development. Herein, we employed a structure-based virtual screening method to screen a library of 700 antiviral compounds against the active site residues of 3CLP. We report three compounds, Sorafenib, YM201636, and LDC4297, that were revealed to have a higher binding energy (BE) value with 3CLP than the control (Dipeptidyl inhibitor 7) following a sequential screening, in-depth molecular docking and visualization, physicochemical and pharmacological property analysis, and molecular dynamics (MD) study. Sorafenib, YM201636, and LDC4297 had BEs of -11.67, -10.34, and -9.78 kcal/mol with 3CLP, respectively, while control had a BE of -6.38 kcal/mol. Furthermore, MD simulations of the two best compounds and control were used to further optimize the interactions, and a 100 ns MD simulation revealed that they form stable complexes with 3CLP. The estimated physicochemical, drug-like, and ADMET properties of these hits suggest that they might be employed as 3CLP inhibitors in the management of gastroenteritis. However, wet lab tests are a prerequisite to optimize them as NV 3CLP inhibitors.

Norovirus GII.3[P12] Outbreak Associated with the Drinking Water Supply in a Rural Area in Galicia, Spain, 2021.

On 30 September 2021, the city council of Muxia, Spain (population of 4,564 inhabitants), reported an unusual increase of patients with acute gastroenteritis (AGE). Because geographically widespread villages belonging to the same water supply were affected, a waterborne outbreak was suspected. Overall, 115 probable cases were ascertained during epidemiological investigations carried out by the local health authority (attack rate, 5.7%); the age range was 0 to 92 years, and 54% were female. The main symptoms were vomiting (78.1%) and diarrhea (67.5%). Primary cases peaked on 29 September and subsided on 1 October, compatible with a point-source outbreak followed by possible secondary cases until 7 October. We conducted an unmatched case-control study using phone surveys. The case-control study included 62 cases and 46 controls. Univariate analysis showed that cases had a higher exposure to tap water through direct consumption (odds ratio [OR] = 86; 95% confidence interval [CI], 18 to 409) or vegetable washing (OR = 27; 95% CI, 7 to 98). Norovirus GII was detected in two terminal points of the water supply system, and 14 cases were laboratory confirmed after detection of GII in stool samples. A unique genotype (GII.3[P12]) was identified in stool samples. On 1 October, a tap water ban was put in place and the water was purged and chlorinated. The rapid increase in the number of cases and its decline after implementing control measures suggested a waterborne point-source outbreak among the residents of Muxia sharing the same water distribution system. IMPORTANCE Noroviruses are likely to be underrecognized in most suspected waterborne outbreaks. Therefore, effective norovirus detection and the early recognition of water as a possible source of infection are important to reduce morbidity as appropriate steps are taken to control the source. In our study, we combined epidemiological, environmental, and microbiological investigations to demonstrate that it was a waterborne outbreak caused by norovirus. Metagenomic sequencing in one norovirus-positive stool sample confirmed norovirus etiology and the absence of other potential pathogens. Detection of fecal indicator bacteria and the fact that the drinking water was not chlorinated suggest a breakdown in chlorination as the cause of the outbreak. This outbreak investigation also demonstrated the importance of timely communication to the public about the risk linked to tap water consumption.