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Noroviruses are a major agent of acute gastroenteritis in humans, but host cell requirements for efficient replication in vitro have not been established. We engineered a human intestinal cell line (designated mCD300lf-hCaco2) expressing the murine norovirus (MNV) receptor, mouse CD300lf to become fully permissive for MNV replication. To explore the replicative machinery and host response of these cells, we performed a single-cell RNA sequencing (scRNA-seq) transcriptomics analysis of an MNV infection over time. Marked similarities were observed between certain global features of MNV infection in human cells compared to those previously reported in mouse cells by whole population transcriptomics such as downregulation of ribosome biogenesis, mitochondrial dysfunction, and cell cycle preference for G1. Our scRNA-seq analysis allowed further resolution of an infected cell population into distinct clusters with varying levels of viral RNA and interferon-stimulated gene ISG15 transcripts. Cells with high viral replication displayed downregulated ribosomal protein small (RPS) and large (RPL) genes and mitochondrial complexes I, III, IV, and V genes during exponential viral propagation. Ferritin subunit genes FTL and FTH1 were also downregulated during active MNV replication, suggesting that inhibition of iron metabolism may increase replication efficiency. Consistent with this, transcriptional activation of these genes with ferric ammonium citrate and overexpression of FTL lowered virus yields. Comparative studies of cells that support varying levels of norovirus replication efficiency, as determined by scRNA-seq may lead to improved human cell-based culture systems and effective viral interventions.IMPORTANCEHuman noroviruses cause acute gastroenteritis in all age groups. Vaccines and antiviral drugs are not yet available, in part, because it is difficult to propagate the viruses causing human disease in standard laboratory cell culture systems. In contrast, a norovirus found in mice [murine norovirus (MNV)] replicates efficiently in murine-based cell culture and has served as a model system. In this study, we established a new human intestinal cell line that was genetically modified to express the murine norovirus receptor so that the human cells became permissive to murine norovirus infection. We then defined the host response to MNV infection in the engineered human cell line at a single-cell resolution and identified cellular genes associated with the highest levels of MNV replication. This study may lead to the improvement of the current human norovirus cell culture systems and help to identify norovirus-host interactions that could be targeted for antiviral drugs., Competing Interests: The authors declare no conflict of interest.

Norovirus infection can cause gastrointestinal disease in humans. Development of therapies and vaccines against norovirus have been limited by the lack of a suitable and reliable animal model. Here we established rhesus macaques as an animal model for human norovirus infection. We show that rhesus macaques are susceptible to oral infection with human noroviruses from two different genogroups. Variation in duration of virus shedding (days to weeks) between animals, evolution of the virus over the time of infection, induction of virus-specific adaptive immune responses, susceptibility to reinfection and preferential replication of norovirus in the jejunum of rhesus macaques was similar to infection reported in humans. We found minor pathological signs and changes in epithelial cell surface glycosylation patterns in the small intestine during infection. Detection of viral protein and RNA in intestinal biopsies confirmed the presence of the virus in chromogranin A-expressing epithelial cells, as it does in humans. Thus, rhesus macaques are a promising non-human primate model to evaluate vaccines and therapeutics against norovirus disease., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Importance: Noroviruses are an important cause of chronic diarrhea in patients with compromised immune systems. Presently, there are no effective therapies to clear the virus, which can persist for years in the intestinal tract. The goal of our study was to develop a better understanding of the norovirus strains that are associated with these long-term infections. With the remarkable diversity of norovirus strains detected in the immunocompromised patient cohort we studied, it appears that most, if not all, noroviruses circulating in nature may have the capacity to establish a chronic infection when a person is unable to mount an effective immune response. Our work is the most comprehensive genetic data set generated to date in which near full-length genomes from noroviruses associated with chronic infection were analyzed by high-resolution next-generation sequencing. Analysis of this data set led to our discovery that certain patients in our cohort were shedding noroviruses that could be subdivided into distinct haplotypes or populations of viruses that were co-evolving independently. The ability to track haplotypes of noroviruses during chronic infection will allow us to fine-tune our understanding of how the virus adapts and maintains itself in the human host, and how selective pressures such as antiviral drugs can affect these distinct populations., Competing Interests: The authors declare no conflict of interest.

Acute gastroenteritis caused by human noroviruses (HuNoVs) is a significant global health and economic burden and is without licensed vaccines or antiviral drugs. The GII.4 HuNoV causes most epidemics worldwide. This virus undergoes epochal evolution with periodic emergence of variants with new antigenic profiles and altered specificity for histo-blood group antigens (HBGA), the determinants of cell attachment and susceptibility, hampering the development of immunotherapeutics. Here, we show that a llama-derived nanobody M4 neutralizes multiple GII.4 variants with high potency in human intestinal enteroids. The crystal structure of M4 complexed with the protruding domain of the GII.4 capsid protein VP1 revealed a conserved epitope, away from the HBGA binding site, fully accessible only when VP1 transitions to a "raised" conformation in the capsid. Together with dynamic light scattering and electron microscopy of the GII.4 VLPs, our studies suggest a mechanism in which M4 accesses the epitope by altering the conformational dynamics of the capsid and triggering its disassembly to neutralize GII.4 infection., (© 2023. Springer Nature Limited.)

Rotaviruses (RVs) preferentially replicate in the small intestine and frequently cause severe diarrheal disease, and the following enteric infection generally induces variable levels of protective systemic and mucosal immune responses in humans and other animals. Rhesus rotavirus (RRV) is a simian RV that was previously used as a human RV vaccine and has been extensively studied in mice. Although RRV replicates poorly in the suckling mouse intestine, infection induces a robust and protective antibody response. The recent availability of plasmid only-based RV reverse genetics systems has enabled the generation of recombinant RVs expressing foreign proteins. However, recombinant RVs have not yet been experimentally tested as potential vaccine vectors to immunize against other gastrointestinal pathogens in vivo . This is a newly available opportunity because several live-attenuated RV vaccines are already widely administered to infants and young children worldwide. To explore the feasibility of using RV as a dual vaccine vector, we rescued replication-competent recombinant RRVs harboring bicistronic gene segment 7 that encodes the native RV nonstructural protein 3 (NSP3) protein and a human norovirus (HuNoV) VP1 protein or P domain from the predominant genotype GII.4. The rescued viruses expressed HuNoV VP1 or P protein in infected cells in vitro and elicited systemic and local antibody responses to HuNoV and RRV following oral infection of suckling mice. Serum IgG and fecal IgA from infected suckling mice bound to and neutralized both RRV and HuNoV. These findings have encouraging practical implications for the design of RV-based next-generation multivalent enteric vaccines to target HuNoV and other human enteric pathogens.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Norovirus is a major cause of acute gastroenteritis worldwide. Over 30 different genotypes, mostly from genogroup I (GI) and II (GII), have been shown to infect humans. Despite three decades of genome sequencing, our understanding of the role of genomic diversification across continents and time is incomplete. To close the spatiotemporal gap of genomic information of human noroviruses, we conducted a large-scale genome-wide analyses that included the nearly full-length sequencing of 281 archival viruses circulating since the 1970s in over 10 countries from four continents, with a major emphasis on norovirus genotypes that are currently underrepresented in public genome databases. We provided new genome information for 24 distinct genotypes, including the oldest genome information from 12 norovirus genotypes. Analyses of this new genomic information, together with those publicly available, showed that (i) noroviruses evolve at similar rates across genomic regions and genotypes; (ii) emerging viruses evolved from transiently-circulating intermediate viruses; (iii) diversifying selection on the VP1 protein was recorded in genotypes with multiple variants; (iv) non-structural proteins showed a similar branching on their phylogenetic trees; and (v) contrary to the current understanding, there are restrictions on the ability to recombine different genomic regions, which results in co-circulating populations of viruses evolving independently in human communities. This study provides a comprehensive genetic analysis of diverse norovirus genotypes and the role of non-structural proteins on viral diversification, shedding new light on the mechanisms of norovirus evolution and transmission., Competing Interests: The authors have declared that no competing interests exist.

Human noroviruses are a major cause of diarrheal illness, but pathogenesis is poorly understood. Here, we investigate the cellular tropism of norovirus in specimens from four immunocompromised patients. Abundant norovirus antigen and RNA are detected throughout the small intestinal tract in jejunal and ileal tissue from one pediatric intestinal transplant recipient with severe gastroenteritis. Negative-sense viral RNA, a marker of active viral replication, is found predominantly in intestinal epithelial cells, with chromogranin A-positive enteroendocrine cells (EECs) identified as a permissive cell type in this patient. These findings are consistent with the detection of norovirus-positive EECs in the other three immunocompromised patients. Investigation of the signaling pathways induced in EECs that mediate communication between the gut and brain may clarify mechanisms of pathogenesis and lead to the development of in vitro model systems in which to evaluate norovirus vaccines and treatment.

Background: Chronic norovirus infection in immunocompromised patients can be severe, and presently there is no effective treatment. Adoptive transfer of virus-specific T cells has proven to be safe and effective for the treatment of many viral infections, and this could represent a novel treatment approach for chronic norovirus infection. Hence, we sought to generate human norovirus-specific T cells (NSTs) that can recognize different viral sequences., Methods: Norovirus-specific T cells were generated from peripheral blood of healthy donors by stimulation with overlapping peptide libraries spanning the entire coding sequence of the norovirus genome., Results: We successfully generated T cells targeting multiple norovirus antigens with a mean 4.2 ± 0.5-fold expansion after 10 days. Norovirus-specific T cells comprised both CD4+ and CD8+ T cells that expressed markers for central memory and effector memory phenotype with minimal expression of coinhibitory molecules, and they were polyfunctional based on cytokine production. We identified novel CD4- and CD8-restricted immunodominant epitopes within NS6 and VP1 antigens. Furthermore, NSTs showed a high degree of cross-reactivity to multiple variant epitopes from clinical isolates., Conclusions: Our findings identify immunodominant human norovirus T-cell epitopes and demonstrate that it is feasible to generate potent NSTs from third-party donors for use in antiviral immunotherapy., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

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.

Human norovirus (HuNoV) is the leading cause of acute nonbacterial gastroenteritis. Vaccine design has been confounded by the antigenic diversity of these viruses and a limited understanding of protective immunity. We reviewed 77 articles published since 1988 describing the isolation, function, and mapping of 307 unique monoclonal antibodies directed against B cell epitopes of human and murine noroviruses representing diverse Genogroups (G). Of these antibodies, 91, 153, 21, and 42 were reported as GI-specific, GII-specific, MNV GV-specific, and G cross-reactive, respectively. Our goal was to reconstruct the antigenic topology of noroviruses in relationship to mapped epitopes with potential for therapeutic use or inclusion in universal vaccines. Furthermore, we reviewed seven published studies of norovirus T cell epitopes that identified 18 unique peptide sequences with CD4- or CD8-stimulating activity. Both the protruding (P) and shell (S) domains of the major capsid protein VP1 contained B and T cell epitopes, with the majority of neutralizing and HBGA-blocking B cell epitopes mapping in or proximal to the surface-exposed P2 region of the P domain. The majority of broadly reactive B and T cell epitopes mapped to the S and P1 arm of the P domain. Taken together, this atlas of mapped B and T cell epitopes offers insight into the promises and challenges of designing universal vaccines and immunotherapy for the noroviruses.

We estimated the prevalence of astrovirus, sapovirus, and norovirus among patients enrolled in research protocols and receiving medical care at the Clinical Center of the National Institutes of Health, Bethesda, MD, a clinical research hospital with a large immunocompromised patient population. We identified patients whose fecal specimens were submitted to the Clinical Center for testing on the Biofire FilmArray Gastrointestinal Panel from September 15, 2015 through November 30, 2016. Among 442 patients with fecal specimens submitted for multiplex testing, 11% had norovirus identified, 2% had astrovirus, and 2% had sapovirus. Like norovirus, astrovirus was detected in multiple sequential samples from a single patient, consistent with chronic infection or the occurrence of multiple reinfections. Coinfection with non-viral gastrointestinal pathogens was detected in 31% of patients with positive results for norovirus, astrovirus, or sapovirus. Norovirus remains common in this immunocompromised patient population, and both sapovirus and astrovirus are present., (Copyright © 2018. Published by Elsevier Inc.)

A luciferase immunoprecipitation systems (LIPS) assay was developed to define the antigenic specificity and titer of antibodies directed against human norovirus (HuNoV). Recombinant proteins, expressed by plasmid constructs encoding Renilla luciferase (Ruc) fused to the full-length HuNoV major capsid protein (VP1) (Ruc-antigen), were generated for ten HuNoV strains. In addition, subdomain constructs Ruc-Shell (S) and Ruc-Protruding (P) were engineered for a representative GII.4 norovirus (strain GII.4/2006b). The LIPS assay measured antibody levels in a well-defined panel of HuNoV-specific sera, and the results were compared to an ELISA standard. In hyperimmune sera, the LIPS produced titers similar to or higher than those measured by the ELISA of HuNoV-specific antibodies. The specificity of antibodies in various sera was profiled by LIPS with a panel of diverse Ruc-antigens containing full-length HuNoV VP1 proteins or VP1 subdomains, and the assay detected both specific and cross-reactive antibodies. Competition assays, in which antibodies were pre-incubated with one or more intact VLPs representing different genotypes, proved useful in further assessment of the antibody specificity detected by LIPS in complex polyclonal sera. The profiling of HuNoV-specific antibodies in the high-throughput LIPS format may prove useful in defining the strength or specificity of the adaptive immune response following natural infection or vaccination., (Published by Elsevier B.V.)

The emergence of pandemic GII.4 norovirus (NoV) strains has been proposed to occur due to changes in receptor usage and thereby to lead to immune evasion. To address this hypothesis, we measured the ability of human sera collected between 1979 and 2010 to block glycan binding of four pandemic GII.4 noroviruses isolated in the last 4 decades. In total, 268 sera were investigated for 50% blocking titer (BT 50 ) values of virus-like particles (VLPs) against pig gastric mucin (PGM) using 4 VLPs that represent different GII.4 norovirus variants identified between 1987 and 2012. Pre- and postpandemic sera (sera collected before and after isolation of the reference NoV strain) efficiently prevented binding of VLP strains MD145 (1987), Grimsby (1995), and Houston (2002), but not the Sydney (2012) strain, to PGM. No statistically significant difference in virus-blocking titers was observed between pre- and postpandemic sera. Moreover, paired sera showed that blocking titers of ≥160 were maintained over a 6-year period against MD145, Grimsby, and Houston VLPs. Significantly higher serum blocking titers (geometric mean titer [GMT], 1,704) were found among IgA-deficient individuals than among healthy blood donors (GMT, 90.9) ( P < 0.0001). The observation that prepandemic sera possess robust blocking capacity for viruses identified decades later suggests a common attachment factor, at least until 2002. Our results indicate that serum IgG possesses antibody-blocking capacity and that blocking titers can be maintained for at least 6 years against 3 decades of pandemic GII.4 NoV. IMPORTANCE Human noroviruses (NoVs) are the major cause of acute gastroenteritis worldwide. Histo-blood group antigens (HBGAs) in saliva and gut recognize NoV and are the proposed ligands that facilitate infection. Polymorphisms in HBGA genes, and in particular a nonsense mutation in FUT2 (G428A), result in resistance to global dominating GII.4 NoV. The emergence of new pandemic GII.4 strains occurs at intervals of several years and is proposed to be attributable to epochal evolution, including amino acid changes and immune evasion. However, it remains unclear whether exposure to a previous pandemic strain stimulates immunity to a pandemic strain identified decades later. We found that prepandemic sera possess robust virus-blocking capacity against viruses identified several decades later. We also show that serum lacking IgA antibodies is sufficient to block NoV VLP binding to HBGAs. This is essential, considering that 1 in every 600 Caucasian children is IgA deficient., (Copyright © 2017 American Society for Microbiology.)

Molecular and serological data suggest that noroviruses (NoVs) might be transmitted between humans and domestic carnivores. In this study we screened an age-stratified collection of canine sera (n=516) by using an ELISA assay based on virus-like particles (VLPs) of human NoVs GII.4 and GIV.1 and carnivore NoVs GIV.2 and GVI.2. Antibodies against GII.4 and GIV.1 human NoVs and GIV.2 and GVI.2 NoVs from carnivores were identified in dog sera (13.0%, 67/516) suggesting their exposure to homologous and heterologous NoVs. Analysis of the trends of age-class prevalence showed a gradual increase in the positive rate from 9.0% and 7.0%, in young dogs <1year of age to 15.0% in dogs older than 12 years, for GII.4 and GVI.2 NoVs, respectively. A significant difference in the IgG distribution by age classes was observed for GIV.1 NoVs, with the highest rate of antibodies (7.0%) in the age group <1year and the lowest (1.0%) in the age-classes 7-9 (P=0.049). High correlation between the reactivity to GII.4 and GVI.2 NoVs was observed, likely due to conserved epitopes in the capsid structure., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Noroviruses are major pathogens associated with acute gastroenteritis worldwide. Their RNA genomes are diverse, with two major genogroups (GI and GII) comprised of at least 28 genotypes associated with human disease. To elucidate mechanisms underlying norovirus diversity and evolution, we used a large-scale genomics approach to analyze human norovirus sequences. Comparison of over 2000 nearly full-length ORF2 sequences representing most of the known GI and GII genotypes infecting humans showed a limited number (≤5) of distinct intra-genotypic variants within each genotype, with the exception of GII.4. The non-GII.4 genotypes were comprised of one or more intra-genotypic variants, with each variant containing strains that differed by only a few residues over several decades (remaining "static") and that have co-circulated with no clear epidemiologic pattern. In contrast, the GII.4 genotype presented the largest number of variants (>10) that have evolved over time with a clear pattern of periodic variant replacement. To expand our understanding of these two patterns of diversification ("static" versus "evolving"), we analyzed using NGS the nearly full-length norovirus genome in healthy individuals infected with GII.4, GII.6 or GII.17 viruses in different outbreak settings. The GII.4 viruses accumulated mutations rapidly within and between hosts, while the GII.6 and GII.17 viruses remained relatively stable, consistent with their diversification patterns. Further analysis of genetic relationships and natural history patterns identified groupings of certain genotypes into larger related clusters designated here as "immunotypes". We propose that "immunotypes" and their evolutionary patterns influence the prevalence of a particular norovirus genotype in the human population., Competing Interests: The authors have declared that no competing interests exist.

Norovirus (NoV) infections are a significant health burden to society, yet the lack of reliable tissue culture systems has hampered the development of appropriate antiviral therapies. Here we show that the NoV NS3 protein, derived from murine NoV (MNV), is intimately associated with the MNV replication complex and the viral replication intermediate double-stranded RNA (dsRNA). We observed that when expressed individually, MNV NS3 and NS3 encoded by human Norwalk virus (NV) induced the formation of distinct vesicle-like structures that did not colocalize with any particular protein markers to cellular organelles but localized to cellular membranes, in particular those with a high cholesterol content. Both proteins also showed some degree of colocalization with the cytoskeleton marker β-tubulin. Although the distribution of MNV and NV NS3s were similar, NV NS3 displayed a higher level of colocalization with the Golgi apparatus and the endoplasmic reticulum (ER). However, we observed that although both proteins colocalized in membranes counterstained with filipin, an indicator of cholesterol content, MNV NS3 displayed a greater association with flotillin and stomatin, proteins known to associate with sphingolipid- and cholesterol-rich microdomains. Utilizing time-lapse epifluorescence microscopy, we observed that the membrane-derived vesicular structures induced by MNV NS3 were highly motile and dynamic in nature, and their movement was dependent on intact microtubules. These results begin to interrogate the functions of NoV proteins during virus replication and highlight the conserved properties of the NoV NS3 proteins among the seven Norovirus genogroups., Importance: Many mechanisms involved in the replication of norovirus still remain unclear, including the role for the NS3 protein, one of seven nonstructural viral proteins, which remains to be elucidated. This study reveals that murine norovirus (MNV) NS3 is intimately associated with the viral replication complex and dsRNA. We observed that the NS3 proteins of both MNV and Norwalk virus (NV) induce prominent vesicular structures and that this formation is dependent on microtubules and cellular cholesterol. Thus, this study contributes to our understanding of protein function within different Norovirus genogroups and expands a growing knowledge base on the interaction between positive-strand RNA [(+)RNA] viruses and cellular membranes that contribute to the biogenesis of virus-induced membrane organelles. This study contributes to our understanding of viral protein function and the ability of a viral protein to recruit specific cellular organelles and lipids that enable replication., (Copyright © 2017 American Society for Microbiology.)

To determine whether the norovirus strain GII.17 recently detected in Maryland, USA, (Hu/GII.17/Gaithersburg/2014/US) is spreading globally, we characterized the genome. High similarity with the norovirus GII.17 that caused recent outbreaks in Asia indicates that the same strain was present in the United States during the 2014-15 norovirus season (winter).

Reverse genetics systems constitute one of the most important and powerful tools to study the molecular biology of viruses. We developed a new strategy for the recovery of murine norovirus from a single plasmid in which a bacteriophage T7 RNA polymerase (T7pol) promoter for transcription and an EMCV IRES for efficient translation were engineered immediately upstream of the viral genome. Infectious noroviruses were recovered following transfection of the newly designed plasmid into nonpermissive BHK-21 and HEK293T cell lines that were engineered to express T7pol constitutively. Recovery of the virus did not require the presence of a ribozyme at the 3'-end of the virus genome. The strategy worked also for the efficient recovery of feline calicivirus in these normally nonpermissive cell types. This simplified reverse genetics approach may be broadly applicable to other caliciviruses., (Published by Elsevier B.V.)

Noroviruses (NoVs) of genogroup IV (GIV) (Alphatron-like) cause infections in humans and in carnivorous animals such as dogs and cats. We screened an age-stratified collection of serum samples from 535 humans in Italy, using virus-like particles of genotypes GIV.1, circulating in humans, and GIV.2, identified in animals, in ELISA, in order to investigate the prevalence of GIV NoV-specific IgG antibodies. Antibodies specific for both genotypes were detected, ranging from a prevalence of 6.6% to 44.8% for GIV.1 and from 6.8% to 15.1% for GIV.2 among different age groups. These data are consistent with a higher prevalence of GIV.1 strains in the human population. Analysis of antibodies against GIV.2 suggests zoonotic transmission of animal NoVs, likely attributable to interaction between humans and domestic pets. This finding, and recent documentation of human transmission of NoVs to dogs, indicate the possibility of an evolutionary relationship between human and animal NoVs.

Unlabelled: Human norovirus infection is the most common cause of viral gastroenteritis worldwide. Development of an effective vaccine is required for reducing norovirus outbreaks. The inability to grow human norovirus in cell culture has hindered the development of live-attenuated vaccines. To overcome this obstacle, we generated a recombinant Newcastle disease virus (rNDV)-vectored experimental norovirus vaccine by expressing the capsid protein (VP1) of norovirus strain VA387. We compared two different NDV vectors, a conventional rNDV vector and a modified rNDV vector, for their efficiencies in expressing VP1 protein. Our results showed that the modified vector replicated to higher titers and expressed higher levels of VP1 protein in DF1 cells and in allantoic fluid of embryonated chicken eggs than did the conventional vector. We further demonstrated that the VP1 protein produced by rNDVs was able to self-assemble into virus-like particles (VLPs) that are morphologically similar to baculovirus-expressed VLPs. Evaluation of their immunogenicity in mice showed that the modified rNDV vector induced a higher level of IgG response than those induced by the conventional vector and by the baculovirus-expressed VLPs. The rNDV vectors predominantly induced IgG2a subclass antibody for the Th1 response, and specifically, high levels of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-2 (IL-2) were detected in splenocytes. In addition, the modified rNDV vector induced a higher level of fecal IgA response in mice than did baculovirus-expressed VLPs. Our findings suggest that the rNDV vector is an efficient system to produce cost-effective VLPs in embryonated chicken eggs and has the potential to be used as a live-attenuated vaccine in humans., Importance: Noroviruses are the major cause of viral gastroenteritis worldwide. Currently, effective vaccines against norovirus infection are not available. In this study, we have evaluated Newcastle disease virus (NDV) as a vaccine vector for norovirus. Our results suggest that NDV can be used not only as a cost-effective method for large-scale production of norovirus-like particle vaccines but also as a live-attenuated vectored vaccine., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Human noroviruses are a major cause of epidemic and sporadic gastroenteritis worldwide and can chronically infect immunocompromised patients. Efforts to develop effective vaccines and antivirals have been hindered by the uncultivable nature and extreme genetic diversity of human noroviruses. Although they remain a particularly challenging pathogen to study, recent advances in norovirus animal models and in vitro cultivation systems have led to an increased understanding of norovirus molecular biology and replication, pathogenesis, cell tropism, and innate and adaptive immunity. Furthermore, clinical trials of vaccines consisting of nonreplicating virus-like particles have shown promise. In this review, we summarize these recent advances and discuss controversies in the field, which is rapidly progressing toward generation of antiviral agents and increasingly effective vaccines., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We investigated sequential episodes of acute norovirus gastroenteritis in a young child within an 11-month period. The infections were caused by 2 distinct genotypes (GII.4 and GII.6). Failure to achieve cross-protective immunity was linked to absence of an enduring and cross-reactive mucosal immune response, a critical consideration for vaccine design.

Noroviruses (NV) are the most common cause of acute gastrointestinal illness in the United States and worldwide. The development of specific antiviral countermeasures has lagged behind that of other viral pathogens, primarily because norovirus disease has been perceived as brief and self-limiting and robust assays suitable for drug discovery have been lacking. The increasing recognition that NV illness can be life-threatening, especially in immunocompromised patients who often require prolonged hospitalization and intensive supportive care, has stimulated new research to develop an effective antiviral therapy. Here, we propose a path forward for evaluating drug therapy in norovirus-infected immunocompromised individuals, a population at high risk for serious and prolonged illness. The clinical and laboratory features of norovirus illness in immunocompromised patients are reviewed, and potential markers of drug efficacy are defined. We discuss the potential design of clinical trials in these patients and how an antiviral therapy that proves effective in immunocompromised patients might also be used in the setting of acute outbreaks, especially in confined settings such as nursing homes, to block the spread of infection and reduce the severity of illness. We conclude by reviewing the current status of approved and experimental compounds that might be evaluated in a hospital setting., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Genotype II.3 (GII.3) noroviruses are a major cause of sporadic gastroenteritis, particularly in children. The greater incidence of GII.3 noroviruses in the pediatric population compared to the adult demographic suggests development of herd immunity to this genotype, possibly as a consequence of limited evolution of immune epitopes. This study aimed to identify and characterize immune epitopes on the GII.3 capsid protein and to determine the level of immune cross-reactivity within the genotype. A panel of seven GII.3 virus-like particles (VLPs), representing norovirus strains isolated during 1975 to 2008, was tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with human sera and a rabbit anti-GII.3 strain-specific polyclonal serum generated against the 2008 GII.3 VLP. Immunoprecipitation of protease-digested GII.3 VLPs and sequencing of bound peptides via mass spectrometry were used to locate epitopes on the capsid. Two epitopes were investigated further using Mimotopes technology. Serum binding studies demonstrated complete intragenotype GII.3 cross-reactivity using both human and rabbit serum. Six immunoreactive regions containing epitopes were located on the GII.3 capsid protein, two within each capsid domain. Epitopes in the S and P1 domains were highly conserved within GII.3 noroviruses. P2 domain epitopes were variable and contained evolutionarily important residues and histo-blood group antigen (HBGA) binding residues. In conclusion, anti-GII.3 antibody-binding epitopes are highly cross-reactive and mostly conserved within GII.3 strains. This may account for the limited GII.3 prevalence in adults and suggests that a GII.3 strain may be a valuable inclusion in a multivalent pediatric targeted VLP vaccine. Exploration of norovirus immune epitopes is vital for effective vaccine design. IMPORTANCE This study represents an important contribution to the understanding of norovirus immunology in a pediatric genotype. The high cross-reactivity and conservation of GII.3 epitopes suggest development of herd immunity against GII.3 and indicate that a GII.3 strain would be a valuable inclusion in a pediatric targeted multivalent vaccine. Immunological understanding of pediatric norovirus strains is important since norovirus vaccines will likely target high-risk groups such as the pediatric population.

Noroviruses belong to a genus of genetically diverse viruses within the family Caliciviridae and cause acute gastroenteritis in humans and animals. They are subdivided into genogroups, each of which further segregates into genotypes. Until recently, a new genotype was based on a defined pairwise distance cutoff of complete VP1 sequences, but with the increasing number of available norovirus sequences, this cutoff is no longer accurate, and sequences in the public database have been misclassified. In this paper, we demonstrate that the pairwise distance cutoff method can no longer be used and outline a phylogenetic approach to classify noroviruses. Furthermore, we propose a dual nomenclature using both ORF1 and VP1 sequences, as recombination is common and recognizing recombinant viruses may be relevant. With the continuing emergence of new norovirus lineages, we propose to coordinate nomenclature of new norovirus genotypes through an international norovirus working group.

Noroviruses are major pathogens associated with acute gastroenteritis. They are diverse viruses, with at least six genogroups (GI-GVI) and multiple genotypes defined by differences in the major capsid protein, VP1. This diversity has challenged the development of broadly cross-reactive vaccines as well as efficient detection methods. Here, we report the characterization of a broadly cross-reactive monoclonal antibody (MAb) raised against the capsid protein of a GII.3 norovirus strain. The MAb reacted with VLPs and denatured VP1 protein from GI, GII, GIV and GV noroviruses, and mapped to a linear epitope located in the inner shell domain. An alignment of all available VP1 sequences showed that the putative epitope (residues 52-56) is highly conserved across the genus Norovirus. This broadly cross-reactive MAb thus constitutes a valuable reagent for the diagnosis and study of these diverse viruses.

We report the solution structures of the VPg proteins from feline calicivirus (FCV) and murine norovirus (MNV), which have been determined by nuclear magnetic resonance spectroscopy. In both cases, the core of the protein adopts a compact helical structure flanked by flexible N and C termini. Remarkably, while the core of FCV VPg contains a well-defined three-helix bundle, the MNV VPg core has just the first two of these secondary structure elements. In both cases, the VPg cores are stabilized by networks of hydrophobic and salt bridge interactions. The Tyr residue in VPg that is nucleotidylated by the viral NS7 polymerase (Y24 in FCV, Y26 in MNV) occurs in a conserved position within the first helix of the core. Intriguingly, given its structure, VPg would appear to be unable to bind to the viral polymerase so as to place this Tyr in the active site without a major conformation change to VPg or the polymerase. However, mutations that destabilized the VPg core either had no effect on or reduced both the ability of the protein to be nucleotidylated and virus infectivity and did not reveal a clear structure-activity relationship. The precise role of the calicivirus VPg core in virus replication remains to be determined, but knowledge of its structure will facilitate future investigations.

Norovirus genotype II.3 (GII.3) strains are a major cause of sporadic gastroenteritis. Intergenic recombination between the capsid and RNA-dependent RNA polymerase (RdRp) genes is common and results in the acquisition of an alternative RdRp genotype. This study aimed to explore the evolution of the GII.3 capsid gene, focusing on the influence of intergenic recombination. The capsid genes from six GII.3 norovirus strains, collected from Australian children between 2001 and 2010, were sequenced and aligned with 66 GII.3 capsid sequences from GenBank, spanning 1975 to 2010. The GII.3 capsid gene evolved at a rate of 4.16 × 10(-3) to 6.97 × 10(-3) nucleotide substitutions/site/year from 1975 to 2010 and clustered into five temporally sequential lineages. Clustering of the GII.3 capsid gene sequences was associated with intergenic recombination and switches between RdRp genotypes GII.3, GII.a, GII.b, GII.12, and an undefined ancestral RdRp. Comparison of the substitution rate of the GII.3 and GII.b RdRps suggested that RdRp switching allows a higher evolutionary rate, leading to increased genetic diversity and adaptability. Alignment of GII.3 capsid sequences revealed 36 lineage-specific conserved amino acid substitutions, four of which were under positive selection. Many conserved substitutions were within predicted antibody binding regions and close to host attachment factor binding sites. In conclusion, evolution of GII.3 noroviruses was primarily driven by intergenic recombination. The acquisition of new RdRps may lead to a faster mutation rate and increased genetic diversity, improving overall GII.3 fitness.

Prompted by the discovery of new gastrointestinal viruses, the NIH, NIAID and WHO investigated the etiology of acute diarrhea that occurred from 1976-1979 in a global cohort of infants and young children. Rotaviruses were found to be major pathogens worldwide, whereas the Norwalk virus could not be detected using a radioimmunoassay. The aim of this study is to re-evaluate the role and diversity of rotaviruses and noroviruses in the original cohort using more sensitive current technologies. Stools collected from Asia, Africa, and South America (n = 485) were evaluated for viral genotypes by RT-PCR and sequencing. Rotaviruses were detected in 28.9% and noroviruses in 9.7% of the specimens, with G1 rotaviruses and GII noroviruses accounting for the majority of each respective virus. Various strains in this study predated the currently assigned dates of discovery for their particular genotype, and in addition, two noroviruses (KL45 and T091) could not be assigned to current genotypes. Phylogenetic analyses demonstrated a relative constancy in circulating rotavirus genotypes over time, with several genotypes from this study becoming established in the current repertoire of viral species. Similarly, GII noroviruses have maintained dominance, with GII.4 noroviruses continuing as a predominant genotype over time. Taken together, the complex molecular epidemiology of rotaviruses and noroviruses circulating in the 1970's is consistent with current patterns, an important consideration in the design of multivalent vaccines to control these viruses.

Noroviruses are major etiological agents of acute viral gastroenteritis. In 2002, a GII.4 variant (Farmington Hills cluster) spread so rapidly in the human population that it predominated worldwide and displaced previous GII.4 strains. We developed and characterized a panel of six monoclonal antibodies (MAbs) directed against the capsid protein of a Farmington Hills-like GII.4 norovirus strain that was associated with a large hospital outbreak in Maryland in 2004. The six MAbs reacted with high titers against homologous virus-like particles (VLPs) by enzyme-linked immunoassay but did not react with denatured capsid protein in immunoblots. The expression and self-assembly of newly developed genogroup I/II chimeric VLPs showed that five MAbs bound to the GII.4 protruding (P) domain of the capsid protein, while one recognized the GII.4 shell (S) domain. Cross-competition assays and mutational analyses showed evidence for at least three distinct antigenic sites in the P domain and one in the S domain. MAbs that mapped to the P domain but not the S domain were able to block the interaction of VLPs with ABH histo-blood group antigens (HBGA), suggesting that multiple antigenic sites of the P domain are involved in HBGA blocking. Further analysis showed that two MAbs mapped to regions of the capsid that had been associated with the emergence of new GII.4 variants. Taken together, our data map antibody and HBGA carbohydrate binding to proximal regions of the norovirus capsid, showing that evolutionary pressures on the norovirus capsid protein may affect both antigenic and carbohydrate recognition phenotypes.

Noroviruses, a major cause of acute gastroenteritis worldwide, present antigenic diversity that must be considered for the development of an effective vaccine. In this study, we explored approaches to increase the broad reactivity of virus-like particle (VLP) norovirus vaccine candidates. The immunogenicity of a GII.4 "Consensus" VLP that was engineered from sequences of three genetically distinct naturally occurring GII.4 strains was examined for its ability to induce cross-reactive immune responses against different clusters of GII.4 noroviruses. Rabbits immunized with GII.4 Consensus VLPs developed high serum antibody titers against VLPs derived from a number of distinct wild-type GII.4 viruses, including some that had been circulating over 30 years ago. Because the sera exhibited low cross-reactivity with antigenically distinct GI norovirus strains, we investigated the serum antibody response to a bivalent vaccine formulation containing GI.1 (Norwalk virus) and GII.4 Consensus VLPs that was administered to animals under varying conditions. In these studies, the highest homologous and heterologous antibody titers to the bivalent vaccine were elicited following immunization of animals by the intramuscular route using Alhydrogel (Al(OH)(3)) as adjuvant. Our data indicate that the use of both genetically engineered norovirus VLPs that incorporate relevant epitopes from multiple strains and multivalent vaccine formulations increase the breadth of the immune response to diverse variants within a genotype and, thus, prove helpful in the rational design of VLP-based vaccines against human noroviruses., (Published by Elsevier Ltd.)

Host resistance to viral infection requires type I (α/β) and II (γ) interferon (IFN) production. Another important defense mechanism is the degradative activity of macroautophagy (herein autophagy), mediated by the coordinated action of evolutionarily conserved autophagy proteins (Atg). We show that the Atg5-Atg12/Atg16L1 protein complex, whose prior known function is in autophagosome formation, is required for IFNγ-mediated host defense against murine norovirus (MNV) infection. Importantly, the direct antiviral activity of IFNγ against MNV in macrophages required Atg5-Atg12, Atg7, and Atg16L1, but not induction of autophagy, the degradative activity of lysosomal proteases, fusion of autophagosomes and lysosomes, or the Atg8-processing protein Atg4B. IFNγ, via Atg5-Atg12/Atg16L1, inhibited formation of the membranous cytoplasmic MNV replication complex, where Atg16L1 localized. Thus, the Atg5-Atg12/Atg16L1 complex performs a pivotal, nondegradative role in IFNγ-mediated antiviral defense, establishing that multicellular organisms have evolved to use portions of the autophagy pathway machinery in a cassette-like fashion for host defense., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Noroviruses are the most common cause of epidemic gastroenteritis. Genotype II.3 is one of the most frequently detected noroviruses associated with sporadic infections. We studied the evolution of the major capsid gene from seven archival GII.3 noroviruses collected during a cross-sectional study at the Children's Hospital in Washington, DC, from 1975 through 1991, together with capsid sequence from 56 strains available in GenBank. Evolutionary analysis concluded that GII.3 viruses evolved at a rate of 4.16 × 10(-3) nucleotide substitutions/site/year (strict clock), which is similar to that described for the more prevalent GII.4 noroviruses. The analysis of the amino acid changes over the 31-year period found that GII.3 viruses evolve at a relatively steady state, maintaining 4% distance, and have a tendency to revert back to previously used residues while preserving the same carbohydrate binding profile. In contrast, GII.4 viruses demonstrate increasing rates of distance over time because of the continued integration of new amino acids and changing HBGA binding patterns. In GII.3 strains, seven sites acting under positive selection were predicted to be surface-exposed residues in the P2 domain, in contrast to GII.4 positively selected sites located primarily in the shell domain. Our study suggests that GII.3 noroviruses caused disease as early as 1975 and that they evolve via a specific pattern, responding to selective pressures induced by the host rather than presenting a nucleotide evolution rate lower than that of GII.4 noroviruses, as previously proposed. Understanding the evolutionary dynamics of prevalent noroviruses is relevant to the development of effective prevention and control strategies.

Antibody prevalence studies in laboratory mice indicate that murine norovirus (MNV) infections are common, but the natural history of these viruses has not been fully established. This study examined the extent of genetic diversity of murine noroviruses isolated from healthy laboratory mice housed in multiple animal facilities within a single, large research institute- the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIAID-NIH) in Bethesda, Maryland, U.S. Ten distinct murine norovirus strains were isolated from various tissues and feces of asymptomatic wild type sentinel mice as well as asymptomatic immunodeficient (RAG 2(-/-)) mice. The NIH MNV isolates showed little cytopathic effect in permissive RAW264.7 cells in early passages, but all isolates examined could be adapted to efficient growth in cell culture by serial passage. The viruses, although closely related in genome sequence, were distinguishable from each other according to facility location, likely due to the introduction of new viruses into each facility from separate sources or vendors at different times. Our study indicates that the murine noroviruses are widespread in these animal facilities, despite rigorous guidelines for animal care and maintenance.

Noroviruses are a major cause of epidemic gastroenteritis in children and adults, and GII.4 has been the predominant genotype since its first documented occurrence in 1987. This study examined the evolutionary dynamics of GII.4 noroviruses over more than three decades to investigate possible mechanisms by which these viruses have emerged to become predominant. Stool samples (n = 5,424) from children hospitalized at the Children's Hospital in Washington, DC, between 1974 and 1991 were screened for the presence of noroviruses by a custom multiplex real-time reverse transcription-PCR. The complete genome sequences of five GII.4 noroviruses (three of which predate 1987 by more than a decade) in this archival collection were determined and compared to the sequences of contemporary strains. Evolutionary analysis determined that the GII.4 VP1 capsid gene evolved at a rate of 4.3 x 10(-3) nucleotide substitutions/site/year. Only six sites in the VP1 capsid protein were found to evolve under positive selection, most of them located in the shell domain. No unique mutations were observed in or around the two histoblood group antigen (HBGA) binding sites in the P region, indicating that this site has been conserved since the 1970s. The VP1 proteins from the 1974 to 1977 noroviruses contained a unique sequence of four consecutive amino acids in the P2 region, which formed an exposed protrusion on the modeled capsid structure. This protrusion and other observed sequence variations did not affect the HBGA binding profiles of recombinant virus-like particles derived from representative 1974 and 1977 noroviruses compared with more recent noroviruses. Our analysis of archival GII.4 norovirus strains suggests that this genotype has been circulating for more than three decades and provides new ancestral strain sequences for the analysis of GII.4 evolution.

Human noroviruses (family Caliciviridae) are the leading cause of nonbacterial gastroenteritis worldwide. Despite the prevalence of these viruses within the community, the study of human norovirus has largely been hindered due to the inability to cultivate the viruses ex vivo and the lack of a small-animal model. In 2003, the discovery of a novel murine norovirus (MNV-1) and the identification of the tropism of MNV-1 for cells of a mononuclear origin led to the establishment of the first norovirus tissue culture system. Like other positive-sense RNA viruses, MNV-1 replication is associated with host membranes, which undergo significant rearrangement during infection. We characterize here the subcellular localization of the MNV-1 open reading frame 1 proteins and viral double-stranded RNA (dsRNA). Over the course of infection, dsRNA and the MNV-1 RNA-dependent RNA polymerase (NS7) were observed to proliferate from punctate foci located in the perinuclear region. All of the MNV-1 open reading frame 1 proteins were observed to colocalize with dsRNA during the course of infection. The MNV-1 replication complex was immunolocalized to virus-induced vesicle clusters formed in the cytoplasm of infected cells. Both dsRNA and MNV-1 NS7 were observed to localize to the limiting membrane of the individual clusters by cryo-immunoelectron microscopy. We show that the MNV-1 replication complex initially associates with membranes derived from the endoplasmic reticulum, trans-Golgi apparatus, and endosomes. In addition, we show that MNV-1 replication is insensitive to the fungal metabolite brefeldin A and consistently does not appear to recruit coatomer protein complex I (COPI) or COPII component proteins during replication. These data provide preliminary insights into key aspects of replication of MNV-1, which will potentially further our understanding of the pathogenesis of noroviruses and aid in the identification of potential targets for drug development.

Noroviruses (NVs) are recognized as a major cause of nonbacterial gastroenteritis in humans. Studies of the human NVs continue to be hampered by the inability to propagate them in any cell culture system. Until recently, most data concerning NV replication were derived from studies of feline calicivirus and rabbit hemorrhagic disease virus, which are cultivable members of the family Caliciviridae. From such studies, it was proposed that caliciviruses induce apoptosis to facilitate the dissemination of viral progeny in the host. The discovery that MNV type 1 (MNV-1) grows in RAW264.7 cells provided the first cell culture system for use in studying the role of apoptosis in NV infection. We first showed that MNV-1 replication triggered apoptosis in infected RAW264.7 cells and then demonstrated that cell death was associated with activation of caspase-9 and caspase-3 through the mitochondrial pathway. This process was dependent on virus replication, since inactivated virus failed to induce signs of apoptosis. In order to better understand the apoptotic process induced by MNV-1 infection of RAW264.7 cells, we investigated the expression profiles of MNV-1-infected versus mock-infected cells. Survivin, a member of the inhibitor of apoptosis protein family, was found to be significantly downregulated in an inverse relationship with the virus genome replication. This study showed that, unlike other viruses that upregulate survivin, MNV-1 is the first virus found to downregulate the levels of survivin. We observed that MNV-1 replication in RAW264.7 cells activated caspases, resulting in apoptosis through the mitochondrial pathway, possibly as a result of downregulation of survivin.

Noroviruses are an important cause of non-bacterial epidemic gastroenteritis, but no specific antiviral therapies are available. We investigated the inhibitory effect of phosphorodiamidiate morpholino oligomers (PMOs) targeted against norovirus sequences. A panel of peptide-conjugated PMOs (PPMOs) specific for the murine norovirus (MNV) genome was developed, and two PPMO compounds directed against the first AUG of the ORF1 coding sequence near the 5'-end of the genome proved effective in inhibiting MNV replication in cells. A consensus PPMO (designated Noro 1.1), designed to target the corresponding region of several diverse human norovirus genotypes, decreased the efficiency of protein translation in a cell-free luciferase reporter assay and inhibited Norwalk virus protein expression in replicon-bearing cells. Our data suggest that PPMOs directed against the relatively conserved 5'-end of the norovirus genome may show broad antiviral activity against this genetically diverse group of viruses.

Caliciviruses have a positive strand RNA genome covalently-linked at the 5'-end to a small protein, VPg. This study examined the biochemical modification of VPg by the ProPol form of the polymerase of human norovirus strain MD145 (GII.4). Recombinant norovirus VPg was shown to be nucleotidylylated in the presence of Mn2+ by MD145 ProPol. Phosphodiesterase I treatment of the nucleotidylylated VPg released the incorporated UMP, which was consistent with linkage of RNA to VPg via a phosphodiester bond. Mutagenesis analysis of VPg identified Tyrosine 27 as the target amino acid for this linkage, and suggested that VPg conformation was important for the reaction. Nucleotidylylation was inefficient in the presence of Mg2+; however the addition of full- and subgenomic-length MD145 RNA transcripts led to a marked enhancement of the nucleotidylylation efficiency in the presence of this divalent cation. Furthermore, evidence was found for the presence of an RNA element near the 3'-end of the polyadenylated genome that enhanced the efficiency of nucleotidylylation in the presence of Mg2+.

Background: Noroviruses are enterically transmitted and are a frequent cause of gastroenteritis, affecting 23 million people annually in the United States. We describe a norovirus outbreak and its control in a tertiary care hospital during February-May 2004., Methods: Patients and health care workers met the case definition if they had new onset of vomiting and/or diarrhea during the outbreak period. Selected stool samples were tested for norovirus RNA. We also determined outbreak costs, including the estimated lost revenue associated with unit closures, sick leave, and cleaning expenses., Results: We identified 355 cases that affected 90 patients and 265 health care workers and that were clustered in the coronary care unit and psychiatry units. Attack rates were 5.3% (7 of 133) for patients and 29.9% (29 of 97) for health care workers in the coronary care unit and 16.7% (39 of 233) for patients and 38.0% (76 of 200) for health care workers in the psychiatry units. Thirteen affected health care workers (4.9%) required emergency department visits or hospitalization. Detected noroviruses had 98%-99% sequence identity with representatives of a new genogroup II.4 variant that emerged during 2002-2004 in the United States (e.g., Farmington Hills and other strains) and Europe. Aggressive infection-control measures, including closure of units and thorough disinfection using sodium hypochlorite, were required to terminate the outbreak. Costs associated with this outbreak were estimated to be $657,644., Conclusions: The significant disruption of patient care and cost of this single nosocomial outbreak support aggressive efforts to prevent transmission of noroviruses in health care settings.

Murine norovirus (MNV) is a recently discovered infectious agent in mice and may be the most common naturally occurring infection of laboratory mice in North America. In 2005, we surveyed the Swiss Webster female sentinel mice in our institute's research facilities. Of the 4 facilities surveyed, 3 had sentinel mice that were positive for MNV antibodies, whereas our largest facility (which only receives mice directly from select vendors or by embryo rederivation directly into the facility) was apparently MNV-free. However, testing of sentinel mice in this large facility 1 y later found that 2% of the animals had developed MNV-specific antibodies. In a recently opened fifth facility, a serologic survey in 2006 identified MNV-antibody-positive Tac:SW sentinel mice that had received bedding from experimental mice on the same rack quadrant. Reverse transcription- polymerase chain reaction analysis of feces from the cages of these mice showed evidence for shedding of MNV. These sentinel mice were used to study the fecal excretion, antibody development, gross lesions upon necropsy, histopathology, and immunohistochemistry of the viral infection. None of the MNV-antibody-positive sentinel mice exhibited clinical signs or gross lesions, but these mice excreted virus in feces and developed antibodies to MNV. Histopathologic lesions consisted only of a few hepatic inflammatory foci in each liver section, some of which were immunoreactive with antibodies to MNV. MNV viral antigens also were present in the mesenteric lymph nodes.

Murine norovirus (MNV) is presently the only member of the genus Norovirus in the Caliciviridae that can be propagated in cell culture. The goal of this study was to elucidate the proteolytic processing strategy of MNV during an authentic replication cycle in cells. A proteolytic cleavage map of the ORF1 polyprotein was generated, and the virus-encoded 3C-like (3CL) proteinase (Pro) mediated cleavage at five dipeptide cleavage sites, 341E/G342, Q705/N706, 870E/G871, 994E/A995, and 1177Q/G1178, that defined the borders of six proteins with the gene order p38.3 (Nterm)-p39.6 (NTPase)-p18.6-p14.3 (VPg)-p19.2 (Pro)-p57.5 (Pol). Bacterially expressed MNV 3CL Pro was sufficient to mediate trans cleavage of the ORF1 polyprotein containing the mutagenized Pro sequence into products identical to those observed during cotranslational processing of the authentic ORF1 polyprotein in vitro and to those observed in MNV-infected cells. Immunoprecipitation and Western blot analysis of proteins produced in virus-infected cells demonstrated efficient cleavage of the proteinase-polymerase precursor. Evidence for additional processing of the Nterm protein in MNV-infected cells by caspase 3 was obtained, and Nterm sequences 118DRPD121 and 128DAMD131 were mapped as caspase 3 cleavage sites by site-directed mutagenesis. The availability of the MNV nonstructural polyprotein cleavage map in concert with a permissive cell culture system should facilitate studies of norovirus replication.

Murine norovirus (MNV) was recently discovered in Rag2-/-/Stat1-/- mice in a U.S. medical research facility. Presently, little is known concerning the epidemiology and natural history of this virus. We studied the pathology of naturally occurring MNV infection in 28 immunodeficient mice of several different genotypes (Rag1-/-/IFNgamma R-/-, OT1 Rag1-/-/IFNgamma R-/-, OT2 Rag1-/-/IFNgamma R-/-, Rag1-/-/Stat1-/-, and Rag2-/-) that were maintained in two U.S. research facilities. The mice were selected for study because sentinel mice housed in their holding rooms had been identified as positive for MNV-specific antibodies during routine screening for infectious agents. Our data indicate that in certain lines of immunodeficient mice, MNV can establish a disseminated infection that is characteristically associated with inflammation in multiple tissues, including liver (hepatitis), lung (focal interstitial pneumonia) and the peritoneal and pleural cavities. In addition, MNV can establish an asymptomatic infection in the mesenteric lymph nodes of Rag2-/- mice. Further studies are needed to determine whether MNV presents a confounding variable in immunological, toxicological and pathological studies in mice naturally infected with MNV.

Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-alphabeta receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology., Competing Interests: The authors have declared that no conflicts of interest exist.

Stool specimens from 156 Maryland nursing home residents, who became ill during 20 outbreaks of gastroenteritis from November 1987 through February 1988, were analyzed. All tested negative for astroviruses, enteroviruses, Group A rotaviruses, Sapporo-like caliciviruses, and enteric bacteria (i.e., Salmonella, Clostridium, and Shigella species). Eighty-two (52%) were positive for Norwalk-like viruses (NLVs), members of the family Caliciviridae. Six distinct genetic clusters within genogroups I and II of the NLVs were detected; a genogroup II (GII) virus closely related to the Camberwell virus in the NLV GII/4 genetic cluster was the predominant strain. Serologic evidence of infection with > or = 1 NLV was detected in 61 (56%) of 109 patients tested against 3 NLV antigens (i.e., Norwalk, Hawaii, and Toronto viruses). Sixteen (80%) outbreaks met the definition for an NLV outbreak. Taken together with a retrospective analysis of bacterial gastroenteritis in this same setting, these data support a major role for NLVs as etiologic agents of gastroenteritis in elderly persons.