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Noroviruses are the leading cause of severe childhood diarrhea and foodborne disease worldwide. While they are a major cause of disease in all age groups, infections in the very young can be quite severe, with annual estimates of 50,000–200,000 fatalities in children under 5 years old. In spite of the remarkable disease burden associated with norovirus infections, very little is known about the pathogenic mechanisms underlying norovirus diarrhea, principally because of the lack of tractable small animal models. The development of the murine norovirus (MNV) model nearly two decades ago has facilitated progress in understanding host–norovirus interactions and norovirus strain variability. However, MNV strains tested thus far either do not cause intestinal disease or were isolated from extraintestinal tissue, raising concerns about translatability of research findings to human norovirus disease. Consequently, the field lacks a strong model of norovirus gastroenteritis. Here we provide a comprehensive characterization of a new small animal model system for the norovirus field that overcomes prior weaknesses. Specifically, we demonstrate that the WU23 MNV strain isolated from a mouse naturally presenting with diarrhea causes a transient reduction in weight gain and acute self-resolving diarrhea in neonatal mice of several inbred mouse lines. Moreover, our findings reveal that norovirus-induced diarrhea is associated with infection of subepithelial cells in the small intestine and systemic spread. Finally, type I interferons (IFNs) are critical to protect hosts from norovirus-induced intestinal disease whereas type III IFNs exacerbate diarrhea. This latter finding is consistent with other emerging data implicating type III IFNs in the exacerbation of some viral diseases. This new model system should enable a detailed investigation of norovirus disease mechanisms.

Intestinal microbiota have profound effects on viral infections locally and systemically. While they can directly influence enteric virus infections, there is also an increasing appreciation for the role of microbiota-derived metabolites in regulating virus infections. Because metabolites diffuse across the intestinal epithelium and enter circulation, they can influence host response to pathogens at extraintestinal sites. In this review, we summarize the effects of three types of microbiota-derived metabolites on virus infections. While short-chain fatty acids serve to regulate the extent of inflammation associated with viral infections, the flavonoid desaminotyrosine and bile acids generally regulate interferon responses. A common theme that emerges is that microbiota-derived metabolites can have proviral and antiviral effects depending on the virus in question. Understanding the molecular mechanisms by which microbiota-derived metabolites impact viral infections and the highly conditional nature of these responses should pave the way to developing novel rational antivirals.

Human norovirus (HNoV) is the most prevalent causative agent of gastroenteritis worldwide. Infection results in a self-limiting disease that can become chronic and severe in the immunocompromised, the elderly, and infants.

The COVID-19 pandemic has resulted in 153 million infections and 3.2 million deaths as of May 2021. While effective vaccines are being administered globally, there is still a great need for antiviral therapies as potentially antigenically distinct SARS-CoV-2 variants continue to emerge across the globe. Viruses require host factors at every step in their life cycle, representing a rich pool of candidate targets for antiviral drug design. To identify host factors that promote SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human coronavirus OC43. While we identified multiple genes and functional pathways that have been previously reported to promote human coronavirus replication, we also identified a substantial number of novel genes and pathways. Of note, host factors involved in cell cycle regulation were enriched in our screens as were several key components of the programmed mRNA decay pathway. Finally, we identified novel candidate antiviral compounds targeting a number of factors revealed by our screens. Overall, our studies substantiate and expand the growing body of literature focused on understanding key human coronavirus-host cell interactions and exploit that knowledge for rational antiviral drug development.One Sentence SummaryGenome-wide CRISPR screens identified host factors that promote human coronavirus infection, revealing novel antiviral drug targets.

Evidence has accumulated to demonstrate that the intestinal microbiota enhances mammalian enteric virus infections1. For example, we and others previously reported that commensal bacteria stimulate acute and persistent murine norovirus infections2-4. However, in apparent contradiction of these results, the virulence of murine norovirus infection was unaffected by antibiotic treatment. This prompted us to perform a detailed investigation of murine norovirus infection in microbially deplete mice, revealing a more complex picture in which commensal bacteria inhibit viral infection of the proximal small intestine while simultaneously stimulating the infection of distal regions of the gut. Thus, commensal bacteria can regulate viral regionalization along the intestinal tract. We further show that the mechanism underlying bacteria-dependent inhibition of norovirus infection in the proximal gut involves bile acid priming of type III interferon. Finally, the regional effects of the microbiota on norovirus infection may result from distinct regional expression profiles of key bile acid receptors that regulate the type III interferon response. Overall, these findings reveal that the biotransformation of host metabolites by the intestinal microbiota directly and regionally impacts infection by a pathogenic enteric virus.

Interferons (IFNs) are key controllers of viral replication, with intact IFN responses suppressing virus growth and spread. Using the murine norovirus (MNoV) system, we show that IFNs exert selective pressure to limit the pathogenic evolutionary potential of this enteric virus. In animals lacking type I IFN signaling, the nonlethal MNoV strain CR6 rapidly acquired enhanced virulence via conversion of a single nucleotide. This nucleotide change resulted in amino acid substitution F514I in the viral capsid, which led to >10,000-fold higher replication in systemic organs including the brain. Pathogenicity was mediated by enhanced recruitment and infection of intestinal myeloid cells and increased extraintestinal dissemination of virus. Interestingly, the trade-off for this mutation was reduced fitness in an IFN-competent host, in which CR6 bearing F514I exhibited decreased intestinal replication and shedding. In an immunodeficient context, a spontaneous amino acid change can thus convert a relatively avirulent viral strain into a lethal pathogen., Author summary The evolution of viruses both within a single host and during transmission between hosts often leads to novel characteristics as mutations develop, with increased lethality or transmissibility being particularly concerning potential outcomes. Here, using a novel in vivo experimental evolution strategy with intestinal pathogen murine norovirus (MNoV), we identified a single nucleotide mutation that consistently and exclusively arose when mice lacking interferon signaling, a key aspect of the early innate immune response, were infected intracranially with a nonlethal strain of MNoV. This mutation was both sufficient and necessary for viral virulence, conferring virulence by enhanced infection of immune cells to facilitate systemic spread, representing a shift in the cell types infected by the virus. Conversely, this mutation was also associated with more limited infection in the intestine, suggesting a fitness cost. Our work identifies key immunological constraints on the evolution of virulence and provides specific mechanistic insights into viral pathogenesis.

Human noroviruses are the leading cause of severe childhood diarrhea worldwide, yet we know little about their pathogenic mechanisms. Murine noroviruses cause diarrhea in interferon-deficient adult mice but these hosts also develop systemic pathology and lethality, reducing confidence in the translatability of findings to human norovirus disease. Herein we report that a murine norovirus causes self-resolving diarrhea in the absence of systemic disease in wild-type neonatal mice, thus mirroring the key features of human norovirus disease and representing a norovirus small animal disease model in wild-type mice. Intriguingly, lymphocytes are critical for controlling acute norovirus replication while simultaneously contributing to disease severity, likely reflecting their dual role as targets of viral infection and key components of the host response., Here the authors show that a murine norovirus causes self-resolving diarrhea in the absence of systemic disease in wild-type neonatal mice and show that lymphocytes affect disease in a dual manner. The data suggests that neonatal mice are a promising model for further studies.

Human noroviruses are the leading cause of foodborne gastroenteritis worldwide and disease outbreaks have been linked to contaminated surface waters as well as to produce consumption. Noroviruses are extremely stable in water and their presence is being detected with increasing frequency, yet there are no viable methods for reducing norovirus contamination in environmental water. Despite this, there is little knowledge regarding the physical and chemical factors that influence the environmental persistence of this pathogen. This study evaluated the impact of common chemical and physical properties of surface water on the stability of murine norovirus and examined the effect of food-safe chitosan microparticles on infectivity of two human norovirus surrogates. While chemical additives had a minor impact on virus survival, chitosan microparticles significantly reduced infectious titers of both murine norovirus and MS2 bacteriophage.

Human noroviruses are the leading cause of severe childhood diarrhea worldwide yet we know very little about their pathogenic mechanisms. Murine noroviruses cause diarrhea in interferon-deficient adult mice but these hosts also develop systemic pathology and lethality, reducing confidence in the translatability of findings to human norovirus disease. Herein we report that a murine norovirus causes self-resolving diarrhea in the absence of systemic disease in wild-type neonatal mice, thus mirroring the key features of human norovirus disease and representing a robust norovirus small animal disease model. Intriguingly, lymphocytes are critical for controlling acute norovirus replication while simultaneously contributing to disease severity, likely reflecting their dual role as targets of viral infection and key components of the host response.

Noroviruses are the leading cause of food-borne gastroenteritis outbreaks and childhood diarrhoea globally, estimated to be responsible for 200,000 deaths in children each year 1-4 . Thus, reducing norovirus-associated disease is a critical priority. Development of vaccines and therapeutics has been hindered by the limited understanding of basic norovirus pathogenesis and cell tropism. While macrophages, dendritic cells, B cells and stem-cell-derived enteroids can all support infection of certain noroviruses in vitro 5-7 , efforts to define in vivo norovirus cell tropism have generated conflicting results. Some studies detected infected intestinal immune cells 8-12 , other studies detected epithelial cells 13 , and still others detected immune and epithelial cells 14-16 . Major limitations of these studies are that they were performed on tissue sections from immunocompromised or germ-free hosts, chronically infected hosts where the timing of infection was unknown, or following non-biologically relevant inoculation routes. Here, we report that the dominant cellular targets of a murine norovirus inoculated orally into immunocompetent mice are macrophages, dendritic cells, B cells and T cells in the gut-associated lymphoid tissue. Importantly, we also demonstrate that a norovirus can infect T cells, a previously unrecognized target, in vitro. These findings represent the most extensive analyses to date of in vivo norovirus cell tropism in orally inoculated, immunocompetent hosts at the peak of acute infection and thus they significantly advance our basic understanding of norovirus pathogenesis.

Human noroviruses are a leading cause of gastroenteritis, and so, vaccine development is desperately needed. Elucidating viral mechanisms of immune antagonism can provide key insight into designing effective immunization platforms. We recently revealed that B cells are targets of norovirus infection. Because noroviruses can regulate antigen presentation by infected macrophages and B cells can function as antigen-presenting cells, we tested whether noroviruses regulate B-cell-mediated antigen presentation and the biological consequence of such regulation. Indeed, murine noroviruses could prevent B-cell expression of antigen presentation molecules and this directly correlated with impaired control of acute infection. In addition to B cells, acute control required MHC class I molecules, CD8+ T cells, and granzymes, supporting a model whereby B cells act as antigen presenting cells to activate cytotoxic CD8+ T cells. This immune pathway was active prior to the induction of antiviral antibody responses. As in macrophages, the minor structural protein VP2 regulated B-cell antigen presentation in a virus-specific manner. Commensal bacteria were not required for the activation of this pathway and ultimately only B cells were required for the clearance of viral infection. These findings provide new insight into the role of B cells in stimulating antiviral CD8+ T-cell responses.

Noroviruses constitute a family of ubiquitous and highly efficient human pathogens. In spite of decades of dedicated research, human noroviruses remain a major cause of gastroenteritis and severe diarrheal disease around the world. Recent findings have begun to unravel the complex mechanisms that regulate norovirus pathogenesis and persistent infection, including the important interplay between the virus, the host immune system, and commensal bacteria. Herein, we will summarize recent research developments regarding norovirus cell tropism, the use of M cells, and commensal bacteria to facilitate norovirus infection, and virus, host, and bacterial determinants of persistent norovirus infections. J. Med. Virol. 88:1837-1843, 2016. © 2016 Wiley Periodicals, Inc.

In this issue of Immunity, Kweon and colleagues (2016) report a novel antiviral cocktail approach to specifically reduce the virome component of the intestinal microbiota. Using this strategy, they generate compelling evidence that the virome provides protection from gut inflammatory conditions.

Human noroviruses are a leading cause of gastroenteritis across the globe, but the pathogenic mechanisms responsible for disease are not well established. The availability of a murine norovirus model system provides the opportunity to elucidate viral and host determinants of virulence in a natural host. For example, previous studies have revealed that the protruding domain of the murine norovirus capsid protein VP1, specifically residue 296 of VP1, regulates virulent infection. We identified a panel of nonsynonymous mutations in the open reading frame 2 (ORF2) gene encoding VP1 that arose in persistently infected mice and tested whether these mutations conferred phenotypic changes to viral replication and virulence. Consistent with previous studies, we demonstrate that a glutamic acid at position 296 results in attenuation. For the first time, we also demonstrate that a lysine at this position is sufficient to confer virulence on an otherwise attenuated murine norovirus strain. Moreover, our studies reveal a direct correlation between the efficiency of viral replication in B cells and virulence. These data are especially striking because mutations causing reduced B cell replication and attenuation had minimal effects on the ability of the virus to replicate in macrophages. Thus, norovirus infection of B cells may directly contribute to disease outcome. IMPORTANCE Human noroviruses are a major global cause of disease, yet we know very little about their pathogenic mechanisms. The availability of a murine norovirus model system facilitates investigation of noroviruses in a natural host organism and the identification of viral and host determinants of pathogenesis. We have identified a panel of mutations arising in the viral capsid protein VP1 during persistent infection of mice. Our data reveal that the protruding domain of VP1 regulates the ability of the virus to replicate in B cells, and this directly correlates with virulence. Importantly, mutations impairing B cell infection had minimal effects on macrophage infection, revealing a potentially critical role for B cell infection in norovirus pathogenesis.

Noroviruses are a leading cause of gastroenteritis outbreaks globally. Several lines of evidence indicate that noroviruses can antagonize or evade host immune responses, including the absence of long-lasting immunity elicited during a primary norovirus exposure and the ability of noroviruses to establish prolonged infections that are associated with protracted viral shedding. Specific norovirus proteins possessing immune antagonist activity have been described in recent years although mechanistic insight in most cases is limited. In this review, we discuss these emerging strategies used by noroviruses to subvert the immune response, including the actions of two nonstructural proteins (p48 and p22) to impair cellular protein trafficking and secretory pathways; the ability of the VF1 protein to inhibit cytokine induction; and the ability of the minor structural protein VP2 to regulate antigen presentation. We also discuss the current state of the understanding of host and viral factors regulating the establishment of persistent norovirus infections along the gastrointestinal tract. A more detailed understanding of immune antagonism by pathogenic viruses will inform prevention and treatment of disease.

Noroviruses are a leading cause of gastroenteritis worldwide. Although infections in healthy individuals are self-resolving, immunocompromised individuals are at risk for chronic disease and severe complications. Chronic norovirus infections in immunocompromised hosts are often characterized by long-term virus shedding, but it is unclear whether this shed virus remains infectious. We investigated the prevalence, genetic heterogeneity, and temporal aspects of norovirus infections in 1140 patients treated during a 6-year period at a pediatric research hospital. Additionally, we identified 20 patients with chronic infections lasting 37 to &gt, 418 days. Using a new human norovirus in vitro assay, we confirmed the continuous shedding of infectious virus for the first time. Shedding lasted longer in male patients and those with diarrheal symptoms. Prolonged shedding of infectious norovirus in immunocompromised hosts can potentially increase the likelihood of transmission, highlighting the importance of isolation precautions to prevent nosocomial infections.

Noroviruses (NoVs) are highly prevalent, positive-sense RNA viruses that infect a range of mammals, including humans and mice. Murine noroviruses (MuNoVs) are the most prevalent pathogens in biomedical research colonies, and they have been used extensively as a model system for human noroviruses (HuNoVs). Despite recent successes in culturing HuNoVs in the laboratory and a small animal host, studies of human viruses have inherent limitations. Thus, owing to its versatility, the MuNoV system—with its native host, reverse genetics, and cell culture systems—will continue to provide important insights into NoV and enteric virus biology. In the current review, we summarize recent findings from MuNoVs that increase our understanding of enteric virus pathogenesis and highlight similarities between human and murine NoVs that underscore the value of MuNoVs to inform studies of HuNoV biology. We also discuss the potential of endemic MuNoV infections to impact other disease models.

Since 1996, there have been at least six human norovirus pandemics. All of the pandemic strains are genetically related, segregating in the genogroup II, genotype 4 (GII.4) cluster within the Norovirus genus. Evidence indicates that these strains are closely related but antigenically distinct, supporting immune-driven viral evolution. Thus, norovirus vaccines will likely require periodic reformulation to protect from newly emergent strains. A major obstacle is that the reservoir of emergent strains is unknown. Noroviruses display tight species specificity and there is no evidence supporting zoonotic transmission, so an animal reservoir is considered unlikely. Moreover, available data indicate minimal viral diversity in most natural human infections. In this Gem, we discuss the widely speculated idea that chronically infected immunocompromised individuals are norovirus reservoirs and provide a rationale for the theory that elderly and malnourished hosts may also represent norovirus reservoirs.

Human noroviruses are a leading cause of gastroenteritis worldwide but research on these important enteric pathogens has long been restricted by their uncultivability. Extensive efforts to infect intestinal epithelial cells with murine and human noroviruses in vitro have been thus far unsuccessful while murine noroviruses efficiently and lytically infect innate immune cells including macrophages and dendritic cells. We have recently discovered that murine and human noroviruses infect B cells in vitro. The nature of B cell infection was distinct from innate immune cell infection in that mature B cells were infected noncytopathically in contrast to the lytic infection of macrophages and dendritic cells. Human norovirus infection of B cells was facilitated by commensal bacteria expressing an appropriate histo-blood group antigen. Importantly, we used the mouse model of norovirus infection to confirm that Peyer's patch B cells are infected, and that commensal bacteria stimulate infection, in vivo.

Currently, there are only limited data available on rates of major diagnostic categories of illnesses among Haitian children. We have established a cohort of 1,245 students attending schools run by the Christianville Foundation in the Gressier/Leogane region of Haiti, for whom our group provides primary medical care. Among 1,357 clinic visits during the 2012-2013 academic year, the main disease categories (with rates per 1,000 child years of observation) included acute respiratory infection (ARI) (385.6 cases/1,000 child years of observation), gastrointestinal complaints (277.8 cases/1,000 child years), febrile illness (235.0 cases/1,000 child years), and skin infections (151.7 cases/1,000 child years). The most common diarrheal pathogen was enteroaggregative Escherichia coli (present in 17% of children with diarrhea); Vibrio cholerae O1 and norovirus were the next most common. Our data highlight the importance of better defining etiologies for ARI and febrile illnesses and continuing problems of diarrheal illness in this region, including mild cases of cholera, which would not have been diagnosed without laboratory screening.

Human noroviruses (HuNoVs) are a leading cause of foodborne disease and severe childhood diarrhea, and they cause a majority of the gastroenteritis outbreaks worldwide. However, the development of effective and long-lasting HuNoV vaccines and therapeutics has been greatly hindered by their uncultivability. We recently demonstrated that a HuNoV replicates in human B cells, and that commensal bacteria serve as a cofactor for this infection. In this protocol, we provide detailed methods for culturing the GII.4-Sydney HuNoV strain directly in human B cells, and in a coculture system in which the virus must cross a confluent epithelial barrier to access underlying B cells. We also describe methods for bacterial stimulation of HuNoV B cell infection and for measuring viral attachment to the surface of B cells. Finally, we highlight variables that contribute to the efficiency of viral replication in this system. Infection assays require 3 d and attachment assays require 3 h. Analysis of infection or attachment samples, including RNA extraction and RTRT-qPCR, requires similar to 6 h.

Norovirus infection in humans typically results in acute gastroenteritis but may also occur in many animal species. Noroviruses are recognized as one of the most common causes of acute gastroenteritis in the world, being responsible for almost 20% of all cases. Despite their prevalence and impact, our knowledge of the norovirus life cycle and the pathological processes associated with norovirus-induced disease is limited. Whilst infection of the intestine is the norm, extraintestinal spread and associated pathologies have also been described. In addition, long-term chronic infections are now recognized as a significant cause of morbidity and mortality in the immunocompromised. This review aims to summarize the current state of knowledge with respect to norovirus pathology and the underlying mechanisms that have been characterized to date. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

RNA viruses can recombine their genetic material during co-infection. However, the in vivo frequency of co-infections is unclear. In this issue of Cell Host & Microbe, Erickson et al. (2018) demonstrate that an enteric RNA virus concentrates itself through multi-virion binding to bacteria, thus increasing genetic recombination and virus adaptability.

The intestinal microbiota exerts a marked influence in the mammalian host, both during homeostasis and disease. However, until very recently, there has been relatively little focus on the potential effect of commensal microorganisms on viral infection of the intestinal tract. In this Progress article, I review the recent advances that elucidate the mechanisms by which enteric viruses use commensal bacteria to enhance viral infectivity. These mechanisms segregate into two general categories: the direct facilitation of viral infection, including bacterial stabilization of viral particles and the facilitation of viral attachment to host target cells; and the indirect skewing of the antiviral immune response in a manner that promotes viral infection. Finally, I discuss the implications of these interactions for the development of vaccines and novel therapeutic approaches.

Human noroviruses are significant emerging pathogens, causing the majority of non-bacterial gastroenteritis outbreaks worldwide. The recent discovery of 30 murine norovirus strains is beginning to facilitate a detailed investigation of norovirus pathogenesis. Here, we have performed an in vivo comparative analysis of two murine norovirus strains, MNV-1 and MNV-3. In immunocompetent mice, MNV-1 caused modest intestinal pathology whereas MNV-3 was attenuated compared to MNV-1. Surprisingly though, MNV-3 reached higher titers in intestinal tissue than MNV-1. MNV-3 also displayed attenuation in mice deficient in the critical interferon signaling molecule STAT-1, demonstrating that MNV-3 attenuation is not a result of increased interferon sensitivity. Importantly, MNV-3-infected mice lost weight and developed gastric bloating and diarrhea in STAT1(-/-) mice, from which all animals recovered. This disease profile recapitulates several key features of acute gastroenteritis experienced by people infected with a human norovirus.

Human noroviruses are responsible for more than 95% of nonbacterial epidemic gastroenteritis worldwide. Both onset and resolution of disease symptoms are rapid, suggesting that components of the innate immune response are critical in norovirus control. While the study of the human noroviruses has been hampered by the lack of small animal and tissue culture systems, our recent discovery of a murine norovirus (MNV) and its in vitro propagation have allowed us to begin addressing norovirus replication strategies and immune responses to norovirus infection. We have previously demonstrated that interferon responses are critical to control MNV-1 infection in vivo and to directly inhibit viral replication in vitro. We now extend these studies to define the molecular basis for interferon-mediated inhibition. Viral replication intermediates were not detected in permissive cells pretreated with type I interferon after either infection or transfection of virion-associated RNA, demonstrating a very early block to virion production that is after virus entry and uncoating. A similar absence of viral replication intermediates was observed in infected primary macrophages and dendritic cells pretreated with type I IFN. This was not due to degradation of incoming genomes in interferon-pretreated cells since similar levels of genomes were present in untreated and pretreated cells through 6 h of infection, and these genomes retained their integrity. Surprisingly, this block to the translation of viral proteins was not dependent on the well-characterized interferon-induced antiviral molecule PKR. Similar results were observed in cells pretreated with type II interferon, except that the inhibition of viral translation was dependent on PKR. Thus, both type I and type II interferon signaling inhibit norovirus translation in permissive myeloid cells, but they display distinct dependence on PKR for this inhibition.

Human noroviruses are the major cause of nonbacterial epidemic gastroenteritis worldwide. However, little is known regarding their pathogenesis or the immune responses that control them because until recently there has been no small animal model or cell culture system of norovirus infection. We recently reported the discovery of the first murine norovirus, murine norovirus 1 (MNV-1), and its cultivation in macrophages and dendritic cells in vitro. We further defined interferon receptors and the STAT-1 molecule as critical in both resistance to MNV-1-induced disease in vivo and control of virus growth in vitro. To date, neither histopathological changes upon infection nor viral replication in wild-type mice has been shown. Here we extend our studies to demonstrate that MNV-1 replicates and rapidly disseminates to various tissues in immunocompetent mice and that infection is restricted by STAT1-dependent interferon responses at the levels of viral replication and virus dissemination. Infection of wild-type mice is associated with histopathological alterations in the intestine (mild inflammation) and the spleen (red pulp hypertrophy and white pulp activation); viral dissemination to the spleen, liver, lung, and lymph nodes; and low-level persistent infection in the spleen. STAT-1 inhibits viral replication in the intestine, prevents virus-induced apoptosis of intestinal cells and splenocytes, and limits viral dissemination to peripheral tissues. These findings demonstrate that murine norovirus infection of wild-type mice is associated with initial enteric seeding and subsequent extraintestinal spread, and they provide mechanistic evidence of the role of STAT-1 in controlling clinical norovirus-induced disease.

Human noroviruses (HuNoVs) cause a majority of gastroenteritis outbreaks across the globe and are the leading cause of severe childhood diarrhea and foodborne disease outbreaks in the United States [1,2]. In impoverished countries, they are estimated to cause over one million clinic visits and 200,000 deaths in young children annually [3]. However, the mechanisms used by noroviruses (NoVs) to infect the intestinal tract and cause disease are not well understood, primarily due to the paucity of cell culture and animal model systems. Recent major advances in developing such models now leave the field poised to tackle these critical questions. The goal of this opinion article is to propose a working model of early steps involved in intestinal infection by NoVs. In this model, NoVs bind carbohydrates on the surface of specific members of the intestinal microbiota and/or enterocytes and are then transcytosed across the intestinal epithelial barrier to gain access to their target immune cells. Evidence supporting each step of this model will be discussed. We also include a brief discussion of how NoVs cause disease as it relates to our model. NoVs Are Transcytosed Across Enterocytes in the Absence of Viral Replication HuNoV and murine NoVs (MuNoV) are transcytosed across intestinal epithelial cells in vitro [4,5], although they have not been shown to productively infect these cells in immunocompetent hosts (reviewed in [6]). Transcytosis of MuNoV across polarized murine intestinal epithelial cell monolayers does not disrupt tight junctions, is enhanced by B cell coculture, and is mediated by cells with characteristics of microfold (M) cells [4], a specialized cell type within the intestine responsible for sampling particulate antigen [7]. In a similar system, HuNoV virus-like particles were visualized on the basolateral side of cell nuclei from polarized Caco-2 cells [5], suggesting transport of particles through epithelial cells. However, whether particles were released from cells, whether particle transport modulated tight junction integrity, or whether a specialized cell type such as M cells mediated this process was not investigated. The importance of M cells for the efficient initiation of MuNoV infection in vivo was subsequently demonstrated by infecting mice depleted of M cells and observing reductions in viral titers in the intestine [8]. Furthermore, this partial, in contrast to complete, reduction of MuNoV infection in M cell-depleted mice suggests the presence of additional viral uptake routes across the intestinal barrier. Reovirus, a double-stranded RNA virus that infects enterocytes, similarly requires M cells for efficient infection [8], and other enteric pathogens also exploit M cells to infect the host [9]. Hence, we speculate that similar mechanisms are used by HuNoV to cross the intestinal epithelial barrier (Fig 1). Open in a separate window Fig 1 A working model for NoV intestinal infection. Multiple studies demonstrate that NoVs bind carbohydrates. These carbohydrates are expressed on enterocytes and secreted into the gut lumen. Furthermore, enteric bacteria can express similar carbohydrates. NoVs may bind to such carbohydrates in any of these contexts (1). NoVs are then transcytosed across the intestinal epithelium via M cells (2) and additional as-yet-to-be-identified pathways. Following transcytosis, NoVs infect dendritic cells, macrophages, and B cells (3). Depending on the species, infection can occur in the presence or absence of carbohydrates. Free carbohydrates or bacterially expressed carbohydrates may be cotranscytosed with the virus. Immune cell infection and putative concomitant viral-bacterial antigen presentation during NoV infections could have significant consequences on the nature and magnitude of antiviral immune responses.

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, 341 E/G 342 , Q 705 /N 706 , 870 E/G 871 , 994 E/A 995 , and 1177 Q/G 1178 , 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 118 DRPD 121 and 128 DAMD 131 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.

Bacteria help norovius infect B cells Stomach ache, nausea, diarrhea—many people know the sort of gastrointestinal havoc norovirus can wreak. Despite this, norovirus biology remains unclear, because human norovirus cannot be grown in culture. Jones et al. now report that with the help of bacteria, human norovirus can infect cultured B cells (see the Perspective by Robinson and Pfeiffer). To infect B cells, human norovirus required the presence of gut bacteria that expressed proteins involved in determining blood type. Mouse norovirus also infected B cells, and the treatment of mice with antibiotics protected them from norovirus infection. Science , this issue p. 755 ; see also p. 700

Human noroviruses are the primary cause of severe childhood diarrhea in the United States, and they are of particular clinical importance in pediatric populations in the developing world. A major contributing factor to the general increased severity of infectious diseases in these regions is malnutrition—nutritional status shapes host immune responses and the composition of the host intestinal microbiota, both of which can influence the outcome of pathogenic infections. In terms of enteric norovirus infections, mucosal immunity and intestinal microbes are likely to contribute to the infection outcome in substantial ways. We probed these interactions using a murine model of malnutrition and murine norovirus infection. Our results reveal that malnutrition is associated with more severe norovirus infections as defined by weight loss, impaired control of norovirus infections, reduced antiviral antibody responses, loss of protective immunity, and enhanced viral evolution. Moreover, the microbiota is dramatically altered by malnutrition. Interestingly, murine norovirus infection also causes changes in the host microbial composition within the intestine but only in healthy mice. In fact, the infection-associated microbiota resembles the malnutrition-associated microbiota. Collectively, these findings represent an extensive characterization of a new malnutrition model of norovirus infection that will ultimately facilitate elucidation of the nutritionally regulated host parameters that predispose to more severe infections and impaired memory immune responses. In a broad sense, this model may provide insight into the reduced efficacy of oral vaccines in malnourished hosts and the potential for malnourished individuals to act as reservoirs of emergent virus strains., IMPORTANCE Malnourished children in developing countries are susceptible to more severe infections than their healthy counterparts, in particular enteric infections that cause diarrhea. In order to probe the effects of malnutrition on an enteric infection in a well-controlled system devoid of other environmental and genetic variability, we studied norovirus infection in a mouse model. We have revealed that malnourished mice develop more severe norovirus infections and they fail to mount effective memory immunity to a secondary challenge. This is of particular importance because malnourished children generally mount less effective immune responses to oral vaccines, and we can now use our new model system to probe the immunological basis of this impairment. We have also determined that noroviruses evolve more readily in the face of malnutrition. Finally, both norovirus infection and malnutrition independently alter the composition of the intestinal microbiota in substantial and overlapping ways.

Whether or not primary norovirus infections induce protective immunity has become a controversial issue, potentially confounded by the comparison of data from genetically distinct norovirus strains. Early human volunteer studies performed with a norovirus-positive inoculum initially led to the conclusion that primary infection does not generate long-term, protective immunity. More recently though, the epidemiological pattern of norovirus pandemics has led to the extrapolation that primary norovirus infection induces herd immunity. While these are seemingly discordant observations, they may in fact reflect virus strain-, cluster-, or genogroup-specific differences in protective immunity induction. Here, we report that highly genetically related intra-cluster murine norovirus strains differ dramatically in their ability to induce a protective immune response: Primary MNV-3 infection induced robust and cross-reactive protection, whereas primary MNV-1 infection induced modest homotypic and no heterotypic protection. In addition to this fundamental observation that intra-cluster norovirus strains display remarkable differences in protective immunity induction, we report three additional important observations relevant to norovirus:host interactions. First, antibody and CD4+ T cells are essential to controlling secondary norovirus infections. Second, the viral minor structural protein VP2 regulates the maturation of antigen presenting cells and protective immunity induction in a virus strain-specific manner, pointing to a mechanism by which MNV-1 may prevent the stimulation of memory immune responses. Third, VF1-mediated regulation of cytokine induction also correlates with protective immunity induction. Thus, two highly genetically-related norovirus strains displayed striking differences in induction of protective immune responses, strongly suggesting that the interpretation of norovirus immunity and vaccine studies must consider potential virus strain-specific effects. Moreover, we have identified immune (antibody and CD4+ T cells) and viral (VP2 and possibly VF1) correlates of norovirus protective immunity. These findings have significant implications for our understanding of norovirus immunity during primary infections as well as the development of new norovirus vaccines., Author Summary Human noroviruses are a significant cause of gastroenteritis outbreaks worldwide and likely the leading cause of severe childhood diarrhea. An efficacious norovirus vaccine would have a major impact on human health but will undoubtedly be confounded by several roadblocks. First, the norovirus genus is highly genetically, and potentially antigenically, diverse. Second, it is currently unclear whether human noroviruses elicit lasting protective immunity upon natural infection. Here, we test the hypothesis that noroviruses display virus strain-specific differences in their stimulation of protective immunity. Indeed, our results reveal that two highly genetically related murine norovirus strains differ dramatically in their stimulation of protective immune responses. Moreover, we demonstrate that antibody and CD4+ T cells are absolutely essential to protecting from a secondary norovirus infection. Finally, we have revealed two viral correlates of protective immunity induction – VF1-mediated cytokine antagonism and VP2-dependent inhibition of antigen presenting cell maturation. Collectively, this information not only offers a potential explanation for the seemingly discordant results regarding human norovirus protective immunity but it also brings to light a previously unrecognized complexity in developing an efficacious human norovirus vaccine – individual virus strains may differ significantly in their interactions with the host immune system and thus in their immunogenicity.

Noroviruses are responsible for a large majority of non-bacterial gastroenteritis outbreaks worldwide. They are considered to be the leading cause of foodborne disease outbreaks and are classified as category B biodefense agents. Noroviruses pose huge medical and economic burdens to the global community: While they display a rapid course of infection in healthy adults, several risk groups including children, the elderly, and immunocompromised individuals are susceptible to more severe and prolonged infections. Moreover, noroviruses can affect any semiclosed community including nursing homes, schools, disaster relief situations, vacation-based and military settings, and they are a major cause of hospital closures for the purpose of outbreak control. Elucidating the host immune responses contributing to the control of norovirus infections is essential to advance our understanding of norovirus pathogenesis and eventually to the design of effective anti-norovirus therapeutic strategies. Recent work on both human and murine noroviruses has unequivocally demonstrated that type I interferon responses, and in particular STAT1-mediated signaling events, are essential for norovirus control. This regulation is mediated at least in part by direct inhibition of norovirus replication in infected cells. The pattern recognition receptor MDA5 contributes to norovirus recognition, although other as-yet-unidentified host receptors must also be involved. Host genes induced upon type I interferon signaling target norovirus translation independent of the well-characterized antiviral molecule PKR. There is suggestive indirect evidence that noroviruses can delay the induction of type I interferon, but may not encode mechanisms to antagonize interferon-mediated signaling. Future mechanistic studies aimed at elucidating the precise interferon-dependent mechanisms by which host cells target norovirus replication may contribute to the design of effective anti-norovirus therapeutic strategies modeled on this activity. Furthermore, there are ongoing efforts to determine whether exogenous interferon can be used therapeutically in the context of norovirus infections.

Human noroviruses in the family Caliciviridae are a major cause of epidemic gastroenteritis. They are responsible for at least 95% of viral outbreaks and over 50% of all outbreaks worldwide. Transmission of these highly infectious plus-stranded RNA viruses occurs primarily through contaminated food or water, but also through person-to-person contact and exposure to fomites. Norovirus infections are typically acute and self-limited. However, disease can be much more severe and prolonged in infants, elderly, and immunocompromised individuals. Norovirus outbreaks frequently occur in semi-closed communities such as nursing homes, military settings, schools, hospitals, cruise ships, and disaster relief situations. Noroviruses are classified as Category B biodefense agents because they are highly contagious, extremely stable in the environment, resistant to common disinfectants, and associated with debilitating illness. The number of reported norovirus outbreaks has risen sharply since 2002 suggesting the emergence of more infectious strains. There has also been increased recognition that noroviruses are important causes of childhood hospitalization. Moreover, noroviruses have recently been associated with multiple clinical outcomes other than gastroenteritis. It is unclear whether these new observations are due to improved norovirus diagnostics or to the emergence of more virulent norovirus strains. Regardless, it is clear that human noroviruses cause considerable morbidity worldwide, have significant economic impact, and are clinically important emerging pathogens. Despite the impact of human norovirus-induced disease and the potential for emergence of highly virulent strains, the pathogenic features of infection are not well understood due to the lack of a cell culture system and previous lack of animal models. This review summarizes the current understanding of norovirus pathogenesis from the histological to the molecular level, including contributions from new model systems.

Human noroviruses in theCaliciviridaefamily are the major cause of nonbacterial epidemic gastroenteritis worldwide. Primary human norovirus infection does not elicit lasting protective immunity, a fact that could greatly affect the efficacy of vaccination strategies. Little is known regarding the pathogenesis of human noroviruses or the immune responses that control them because there has previously been no small-animal model or cell culture system of infection. Using the only available small-animal model of norovirus infection, we found that primary high-dose murine norovirus 1 (MNV-1) infection fails to afford protection against a rechallenge with a homologous virus. Thus, MNV-1 represents a valuable model with which to dissect the pathophysiological basis for the lack of lasting protection against human norovirus infection. Interestingly, the magnitude of protection afforded by a primary MNV-1 infection inversely correlates with the inoculum dose. Future studies will elucidate the mechanisms by which noroviruses avoid the induction of protective immunity and the role played by the inoculum dose in this process, ultimately translating this knowledge into successful vaccination approaches.

Norwalk-like caliciviruses (Noroviruses) cause over 90% of nonbacterial epidemic gastroenteritis worldwide, but the pathogenesis of norovirus infection is poorly understood because these viruses do not grow in cultured cells and there is no small animal model. Here, we report a previously unknown murine norovirus. Analysis of Murine Norovirus 1 infection revealed that signal transducer and activator of transcription 1–dependent innate immunity, but not T and B cell–dependent adaptive immunity, is essential for norovirus resistance. The identification of host molecules essential for murine norovirus resistance may provide targets for prevention or control of an important human disease.