Interferons and tuft cell numbers are bottlenecks for persistent murine norovirus infection.

Noroviruses (NoVs) are a leading cause of viral gastroenteritis. Despite global clinical relevance, our understanding of how host factors, such as antiviral cytokines interferons (IFNs), modulate NoV population dynamics is limited. Murine NoV (MNoV) is a tractable in vivo model for the study of host regulation of NoV. A persistent strain of MNoV, CR6, establishes a reservoir in intestinal tuft cells for chronic viral shedding in stool. However, the influence of host innate immunity and permissive cell numbers on viral population dynamics is an open question. We generated a pool of 20 different barcoded viruses (CR6BC) by inserting 6-nucleotide barcodes at the 3' position of the NS4 gene and used this pool as our viral inoculum for in vivo infections of different mouse lines. We found that over the course of persistent CR6 infection, shed virus was predominantly colon-derived, and viral barcode richness decreased over time irrespective of host immune status, suggesting that persistent infection involves a series of reinfection events. In mice lacking the IFN-λ receptor, intestinal barcode richness was enhanced, correlating with increased viral intestinal replication. IL-4 treatment, which increases tuft cell numbers, also increased barcode richness, indicating the abundance of permissive tuft cells to be a bottleneck during CR6 infection. In mice lacking type I IFN signaling (Ifnar1-/-) or all IFN signaling (Stat1-/-), barcode diversity at extraintestinal sites was dramatically increased, implicating different IFNs as critical bottlenecks at specific tissue sites. Of interest, extraintestinal barcodes were overlapping but distinct from intestinal barcodes, indicating that disseminated virus represents a distinct viral population than that replicating in the intestine. Barcoded viruses are a valuable tool to explore the influence of host factors on viral diversity in the context of establishment and maintenance of infection as well as dissemination and have provided important insights into how NoV infection proceeds in immunocompetent and immunocompromised hosts.

Murine norovirus mutants adapted to replicate in human cells reveal a post-entry restriction.

RNA viruses lack proofreading in their RNA polymerases and therefore exist as genetically diverse populations. By exposing these diverse viral populations to selective pressures, viruses with mutations that confer fitness advantages can be enriched. To examine factors important for viral tropism and host restriction, we passaged murine norovirus (MNV) in a human cell line, HeLa cells, to select mutant viruses with increased fitness in non-murine cells. A major determinant of host range is expression of the MNV receptor CD300lf on mouse cells, but additional host factors may limit MNV replication in human cells. We found that viruses passaged six times in HeLa cells had enhanced replication compared with the parental virus. The passaged viruses had several mutations throughout the viral genome, which were primarily located in the viral non-structural coding regions. Although viral attachment was not altered for the passaged viruses, their replication was higher than the parental virus when the entry was bypassed, suggesting that the mutant viruses overcame a post-entry block in human cells. Three mutations in the viral NS1 protein were sufficient for enhanced post-entry replication in human cells. We found that the human cell-adapted MNV variants had reduced fitness in murine BV2 cells and infected mice, with reduced viral titers. These results suggest a fitness tradeoff, where increased fitness in a non-native host cell reduces fitness in a natural host environment. Overall, this work suggests that MNV tropism is determined by the presence of not only the viral receptor but also post-entry factors. IMPORTANCE: Viruses infect specific species and cell types, which is dictated by the expression of host factors required for viral entry as well as downstream replication steps. Murine norovirus (MNV) infects mouse cells, but not human cells. However, human cells expressing the murine CD300lf receptor support MNV replication, suggesting that receptor expression is a major determinant of MNV tropism. To determine whether other factors influence MNV tropism, we selected for variants with enhanced replication in human cells. We identified mutations that enhance MNV replication in human cells and demonstrated that these mutations enhance infection at a post-entry replication step. Therefore, MNV infection of human cells is restricted at both entry and post-entry stages. These results shed new light on factors that influence viral tropism and host range.

Human norovirus cultivation systems and their use in antiviral research.

Human norovirus (HuNoV) is a major cause of acute gastroenteritis and foodborne diseases, affecting all age groups. Despite its clinical needs, no approved antiviral therapies are available. Since the discovery of HuNoV in 1972, studies on anti-norovirals, mechanism of HuNoV infection, viral inactivation, etc., have been hampered by the lack of a robust laboratory-based cultivation system for HuNoV. A recent breakthrough in the development of HuNoV cultivation systems has opened opportunities for researchers to investigate HuNoV biology in the context of de novo HuNoV infections. A tissue stem cell-derived human intestinal organoid/enteroid (HIO) culture system is one of those that supports HuNoV replication reproducibly and, to our knowledge, is most widely distributed to laboratories worldwide to study HuNoV and develop therapeutic strategies. This review summarizes recently developed HuNoV cultivation systems, including HIO, and their use in antiviral studies.

Intestinal tuft cell immune privilege enables norovirus persistence.

The persistent murine norovirus strain MNVCR6 is a model for human norovirus and enteric viral persistence. MNVCR6 causes chronic infection by directly infecting intestinal tuft cells, rare chemosensory epithelial cells. Although MNVCR6 induces functional MNV-specific CD8+ T cells, these lymphocytes fail to clear infection. To examine how tuft cells promote immune escape, we interrogated tuft cell interactions with CD8+ T cells by adoptively transferring JEDI (just EGFP death inducing) CD8+ T cells into Gfi1b-GFP tuft cell reporter mice. Unexpectedly, some intestinal tuft cells partially resisted JEDI CD8+ T cell-mediated killing-unlike Lgr5+ intestinal stem cells and extraintestinal tuft cells-despite seemingly normal antigen presentation. When targeting intestinal tuft cells, JEDI CD8+ T cells predominantly adopted a T resident memory phenotype with decreased effector and cytotoxic capacity, enabling tuft cell survival. JEDI CD8+ T cells neither cleared nor prevented MNVCR6 infection in the colon, the site of viral persistence, despite targeting a virus-independent antigen. Ultimately, we show that intestinal tuft cells are relatively resistant to CD8+ T cells independent of norovirus infection, representing an immune-privileged niche that can be leveraged by enteric microbes.

Strain-specific requirements of susceptibility to rhesus enteric calicivirus infection.

Recently, we identified the coxsackie and adenovirus receptor (CAR) as the entry receptor for rhesus enteric calicivirus (ReCV) isolate FT285 and demonstrated that co-expression of the CAR and the type B histo-blood group antigen (HBGA) is required to convert the resistant CHO cell line susceptible to infection. To address whether the CAR is also the functional entry receptor for other ReCV isolates and the requirement for specific HBGAs or other glycans, here we used a panel of recombinant CHO cell lines expressing the CAR and the type A, B, or H HBGAs alone or in combination. Infection studies with three diverse ReCV strains, the prototype GI.1 Tulane virus (TV), GI.2 ReCV-FT285, and GI.3 ReCV-FT7, identified that cell surface expression of the CAR is an absolute requirement for all three strains to promote susceptibility to infection, while the requirement for HBGAs varies among the strains. In addition to the CAR, ReCV-FT285 and TV require type A or B HBGAs for infection. In the absence of HBGAs, TV, but not Re-CV FT285, can also utilize sialic acids, while ReCV-FT7 infection is HBGA-independent and relies on CAR and sialic acid expression. In summary, we demonstrated strain-specific diversity of susceptibility requirements for ReCV infections and that CAR, type A and B HBGA, and sialic acid expression control susceptibility to infection with the three ReCV isolates studied. Our study also indicates that the correlation between in vitro HBGA binding and HBGAs required for infection is relatively high, but not absolute. This has direct implications for human noroviruses.IMPORTANCEHuman noroviruses (HuNoVs) are important enteric pathogens. The lack of a robust HuNoV cell culture system is a bottleneck for HuNoV cell culture-based studies. Often, cell culture-adapted caliciviruses that rapidly replicate in conventional cell lines and recapitulate biological features of HuNoVs are utilized as surrogates. Particularly, rhesus enteric caliciviruses (ReCVs) display remarkable similarities, including the primate host, clinical manifestation of gastroenteritis, genetic/antigenic diversity, and reliance on histo-blood group antigens (HBGAs) for attachment. While the HuNoV entry receptor(s) is unknown, the coxsackie and adenovirus receptor (CAR) has recently been identified as the ReCV entry receptor. Here, we identified the CAR, the type A and B HBGAs, and sialic acids as critical cell surface molecules controlling susceptibility to ReCV infections. The CAR is required for all ReCV isolates studied. However, the requirement for the different carbohydrate molecules varies among different ReCV strains. Our findings have direct implications for HuNoVs.

Human intestinal enteroids and predictive models validate the operational limits of sanitizers used for viral disinfection of vegetable process wash water.

Vegetables are globally associated with a considerable number of foodborne outbreaks caused by viral infections, specifically human norovirus. In fresh produce industry, washing represents a critical step for food safety as process wash water (PWW) needs to be maintained at appropriate microbial quality to prevent water-mediated cross-contamination. This study aimed to explore the disinfection efficacy of chlorine (free chlorine, FC), chlorine dioxide (ClO2) and peracetic acid (PAA) in PWW against infectious human norovirus and Tulane virus (TV). First, we tested the extent of TV inactivation in baby leaf, bell pepper, and vegetables mix PWW and monitored the viral decay by cell culture. Then, inactivation kinetics were defined for infectious human norovirus exposed to FC, ClO2 and PAA in baby leaves PWW using the human intestinal enteroids (HIE) system. Finally, kinetic inactivation models were fitted to TV reduction and decay of sanitizers to aid the implementation of disinfection strategies. Results showed that >8 log10 human norovirus and 3.9 log10 TV were inactivated by 20 ppm FC within 1 min; and by 3 ppm ClO2 in 1 min (TV) or 5 min (norovirus). PAA treatment at 80 ppm reduced ca. 2 log10 TV but not completely inactivated the virus even after 20 min exposure, while 5 min treatment prevented norovirus replication in HIE. TV inactivation in PWWs was described using an exponential decay model. Taking these data together, we demonstrated the value of applying the HIE model to validate current operational limits for the most commonly used sanitizers. The inactivation kinetics for human norovirus and TV, along with the predictive model described in this study expand the current knowledge to implement post-harvest produce safety procedures in industry settings.

Heat stability of foodborne viruses - Findings, methodological challenges and current developments.

Heat treatment of food represents an important measure to prevent pathogen transmission. Thus far, evaluation of heat treatment processes is mainly based on data from bacteria. However, foodborne viruses have gained increasing attention during the last decades. Here, the published literature on heat stability and inactivation of human norovirus (NoV), hepatitis A virus (HAV) and hepatitis E virus (HEV) was reviewed. Data for surrogate viruses were not included. As stability assessment for foodborne viruses is often hampered by missing infectivity assays, an overview of applied methods is also presented. For NoV, molecular capsid integrity assays were mainly applied, but data from initial studies utilizing novel intestinal enteroid or zebrafish larvae assays are available now. However, these methods are still limited in applicability and sensitivity. For HAV, sufficient cell culture-based inactivation data are available, but almost exclusively for one single strain, thus limiting interpretation of the data for the wide range of field strains. For HEV, data are now available from studies using pig inoculation or cell culture. The results of the reviewed studies generally indicate that NoV, HAV and HEV possess a high heat stability. Heating at 70-72 °C for 2 min significantly reduces infectious titers, but often does not result in a >4 log10 decrease. However, heat stability greatly varied dependent on virus strain, matrix and heating regime. In addition, the applied method largely influenced the result, e.g. capsid integrity assays tend to result in higher measured stabilities than cell culture approaches. It can be concluded that the investigated foodborne viruses show a high heat stability, but can be inactivated by application of appropriate heating protocols. For HAV, suggestions for safe time/temperature combinations for specific foods can be derived from the published studies, with the limitation that they are mostly based on one strain only. Although significant improvement of infectivity assays for NoV and HEV have been made during the last years, further method development regarding sensitivity, robustness and broader applicability is important to generate more reliable heat inactivation data for these foodborne viruses in future.

Murine norovirus infection of macrophages induces intrinsic apoptosis as the major form of programmed cell death.

Human norovirus is the leading cause of acute gastroenteritis worldwide, however despite the significance of this pathogen, we have a limited understanding of how noroviruses cause disease, and modulate the innate immune response. Programmed cell death (PCD) is an important part of the innate response to invading pathogens, but little is known about how specific PCD pathways contribute to norovirus replication. Here, we reveal that murine norovirus (MNV) virus-induced PCD in macrophages correlates with the release of infectious virus. We subsequently show, genetically and chemically, that MNV-induced cell death and viral replication occurs independent of the activity of inflammatory mediators. Further analysis revealed that MNV infection promotes the cleavage of apoptotic caspase-3 and PARP. Correspondingly, pan-caspase inhibition, or BAX and BAK deficiency, perturbed viral replication rates and delayed virus release and cell death. These results provide new insights into how MNV harnesses cell death to increase viral burden.

In vitro virucidal activity of nebulized citrate-complexed silver nanoparticles against equine herpesvirus-1 and murine norovirus.

Viruses can be involved in respiratory disorders in horses, with limited therapeutic options. Citrate-complexed silver nanoparticles (C-AgNP) have shown bactericidal properties after in vitro nebulization. The aim of the present study was to assess the virucidal activity of C-AgNP after in vitro instillation or nebulization on equine herpesvirus-1 (EHV-1) and murine norovirus (MNV), the latter used as surrogate for small non-enveloped viruses. Both viruses were instilled or nebulized with C-AgNP of increasing concentrations, and titres were determined via TCID50 method. We demonstrated efficient inactivation of enveloped EHV-1 following instillation and nebulization of C-AgNP (infectivity losses of ≥ three orders of magnitude). While tenacious MNV was inactivated via 2000 ppm C-AgNP instillation, nebulized C-AgNP did not lead to reduction in MNV titres. Nebulization of C-AgNP may represent a novel virucidal therapeutic approach in horses. Further investigations are needed to assess its safety and effective concentrations for in vivo use.

Norovirus MLKL-like protein initiates cell death to induce viral egress.

Non-enveloped viruses require cell lysis to release new virions from infected cells, suggesting that these viruses require mechanisms to induce cell death. Noroviruses are one such group of viruses, but there is no known mechanism that causes norovirus infection-triggered cell death and lysis1-3. Here we identify a molecular mechanism of norovirus-induced cell death. We found that the norovirus-encoded NTPase NS3 contains an N-terminal four-helix bundle domain homologous to the membrane-disruption domain of the pseudokinase mixed lineage kinase domain-like (MLKL). NS3 has a mitochondrial localization signal and thus induces cell death by targeting mitochondria. Full-length NS3 and an N-terminal fragment of the protein bound the mitochondrial membrane lipid cardiolipin, permeabilized the mitochondrial membrane and induced mitochondrial dysfunction. Both the N-terminal region and the mitochondrial localization motif of NS3 were essential for cell death, viral egress from cells and viral replication in mice. These findings suggest that noroviruses have acquired a host MLKL-like pore-forming domain to facilitate viral egress by inducing mitochondrial dysfunction.

Antiviral Effects of Quillaja saponaria Extracts Against Human Noroviral Surrogates.

Aqueous extracts of Quillaja saponaria Molina are US FDA approved as food additives in beverages with known antiviral activity. Due to lack of commercially available vaccines against human noroviruses (HNoVs), alternate methods to prevent their spread and the subsequent emergence of variant strains are being researched. Furthermore, HNoVs are not yet culturable at high enough titers to determine inactivation, therefore surrogates continue to be used. This research analyzed the effect of aqueous Quillaja saponaria extracts (QE) against HNoV surrogates, Tulane virus (TV), murine norovirus (MNV-1), and feline calicivirus (FCV-F9) at room temperature (RT) and 37 °C. Viruses (~ 5 log PFU/mL) were individually treated with 1:1 or 1:5 (v/v) diluted QE (pH ~ 3.75), malic acid control (pH 3.0) or phosphate-buffered saline (pH 7.2, as control) at 37 °C or RT for up to 6 h. Individual treatments were replicated three times using duplicate plaque assays for each treatment. FCV-F9 at ~ 5 log PFU/mL was not detectable after 15 min by 1:1 QE at 37 °C and RT. At RT, 1:5 QE lowered FCV-F9 titers by 2.05, 2.14 and 2.74 log PFU/mL after 0.5 h, 1 h and 2 h, respectively. MNV-1 showed marginal reduction of < 1 log PFU/mL after 15 min with 1:1 or 1:5 QE at 37 °C without any significant reduction at RT, while TV titers decreased by 2.2 log PFU/mL after 30 min and were undetectable after 3 h at 37 °C. Longer incubation with higher QE concentrations may be required for improved antiviral activity against MNV-1 and TV.

CLIC and membrane wound repair pathways enable pandemic norovirus entry and infection.

Globally, most cases of gastroenteritis are caused by pandemic GII.4 human norovirus (HuNoV) strains with no approved therapies or vaccines available. The cellular pathways that these strains exploit for cell entry and internalization are unknown. Here, using nontransformed human jejunal enteroids (HIEs) that recapitulate the physiology of the gastrointestinal tract, we show that infectious GII.4 virions and virus-like particles are endocytosed using a unique combination of endosomal acidification-dependent clathrin-independent carriers (CLIC), acid sphingomyelinase (ASM)-mediated lysosomal exocytosis, and membrane wound repair pathways. We found that besides the known interaction of the viral capsid Protruding (P) domain with host glycans, the Shell (S) domain interacts with both galectin-3 (gal-3) and apoptosis-linked gene 2-interacting protein X (ALIX), to orchestrate GII.4 cell entry. Recognition of the viral and cellular determinants regulating HuNoV entry provides insight into the infection process of a non-enveloped virus highlighting unique pathways and targets for developing effective therapeutics.

Current decontamination challenges and potentially complementary solutions to safeguard the vulnerable seafood industry from recalcitrant human norovirus in live shellfish: Quo Vadis?

Safeguarding the seafood industry is important given its contribution to supporting our growing global population. However, shellfish are filter feeders that bioaccumulate microbial contaminants in their tissue from wastewater discharged into the same coastal growing environments leading to significant human disease outbreaks unless appropriately mitigated. Removal or inactivation of enteric viruses is very challenging particularly as human norovirus (hNoV) binds to specific histo-blood ligands in live oyster tissue that are consumed raw or lightly cooked. The regulatory framework that sets out use of clean seawater and UV disinfection is appropriate for bacterial decontamination at the post-harvest land-based depuration (cleaning) stage. However, additional non-thermal technologies are required to eliminate hNoV in live shellfish (particularly oysters) where published genomic studies report that low-pressure UV has limited effectiveness in inactivating hNoV. The use of the standard genomic detection method (ISO 15, 216-1:2017) is not appropriate for assessing the loss of infectious hNoV in treated live shellfish. The use of surrogate viral infectivity methods appear to offer some insight into the loss of hNoV infectiousness in live shellfish during decontamination. This paper reviews the use of existing and potentially other combinational treatment approaches to enhance the removal or inactivation of enteric viruses in live shellfish. The use of alternative and complementary novel diagnostic approaches to discern viable hNoV are discussed. The effectiveness and virological safety of new affordable hNoV intervention(s) require testing and validating at commercial shellfish production in conjunction with laboratory-based research. Appropriate risk management planning should encompass key stakeholders including local government and the wastewater industry. Gaining a mechanistic understanding of the relationship between hNoV response at molecular and structural levels in individually treated oysters as a unit will inform predictive modeling and appropriate treatment technologies. Global warming of coastal growing environments may introduce additional contaminant challenges (such as invasive species); thus, underscoring need to develop real-time ecosystem monitoring of growing environments to alert shellfish producers to appropriately mitigate these threats.

Interferon-Driven Immune Dysregulation in Common Variable Immunodeficiency-Associated Villous Atrophy and Norovirus Infection.

PURPOSE: About 15% of patients with common variable immunodeficiency (CVID) develop a small intestinal enteropathy, which resembles celiac disease with regard to histopathology but evolves from a distinct, poorly defined pathogenesis that has been linked in some cases to chronic norovirus (NV) infection. Interferon-driven inflammation is a prominent feature of CVID enteropathy, but it remains unknown how NV infection may contribute. METHODS: Duodenal biopsies of CVID patients, stratified according to the presence of villous atrophy (VA), IgA plasma cells (PCs), and chronic NV infection, were investigated by flow cytometry, multi-epitope-ligand cartography, bulk RNA-sequencing, and RT-qPCR of genes of interest. RESULTS: VA development was connected to the lack of intestinal (IgA+) PC, a T helper 1/T helper 17 cell imbalance, and increased recruitment of granzyme+CD8+ T cells and pro-inflammatory macrophages to the affected site. A mixed interferon type I/III and II signature occurred already in the absence of histopathological changes and increased with the severity of the disease and in the absence of (IgA+) PCs. Chronic NV infection exacerbated this signature when compared to stage-matched NV-negative samples. CONCLUSIONS: Our study suggests that increased IFN signaling and T-cell cytotoxicity are present already in mild and are aggravated in severe stages (VA) of CVID enteropathy. NV infection preempts local high IFN-driven inflammation, usually only seen in VA, at milder disease stages. Thus, revealing the impact of different drivers of the pathological mixed IFN type I/III and II signature may allow for more targeted treatment strategies in CVID enteropathy and supports the goal of viral elimination.

Bioaccumulation Dynamic by Crassostrea gigas Oysters of Viruses That Are Proposed as Surrogates for Enteric Virus Contamination in Environmental Samples.

Oysters are filter-feeders and retain sewage-derived pathogens in their organs or tissues. Since most enteric viruses involved in outbreaks cannot grow in cell culture, studies using viral surrogate models are essential. Some species are proposed as surrogates for enteric viruses in environmental samples, including in bivalve mollusk samples, such as murine norovirus type 1 (MNV-1) and somatic (as φX) or F-specific coliphages (as MS2) bacteriophages. This study evaluated the tissue distribution of viral surrogates for enteric virus contamination after their bioaccumulation by Crassostrea gigas. Oyster tissues were analyzed for the distribution of viral surrogates (MNV-1, φX-174, and MS2) in digestive tissue (DT), gills (GL), and mantle (MT) after 4, 6, and 24 h of experimental bioaccumulation. MNV-1 had higher counts at 6 h in DT (1.2 × 103 PFU/g), followed by GL and MT (9.5 × 102 and 3.8 × 102 PFU/g, respectively). The bacteriophage φX-174 had a higher concentration in the MT at 4 and 6 h (3.0 × 102 PFU/g, in both) and MS2 in the GL after 24 h (2.2 × 102 PFU/g). The bioaccumulation pattern of MNV-1 by oysters was similar to the other enteric viruses (more in DT), while that of phages followed distinct patterns from these. Since the MNV-1 is bioaccumulated by C. gigas and is adapted to grow in cell culture, it is an important tool for bioaccumulation and viral inactivation tests in oysters. Although bacteriophage bioaccumulation was not similar to enteric viruses, they can be indicated for viral bioaccumulation analysis, analyzing MT and GL, since they do not bioaccumulate in DT.

Tuft-cell-intrinsic and -extrinsic mediators of norovirus tropism regulate viral immunity.

Murine norovirus (MNoV) is a model for human norovirus and for interrogating mechanisms of viral tropism and persistence. We previously demonstrated that the persistent strain MNoVCR6 infects tuft cells, which are dispensable for the non-persistent strain MNoVCW3. We now show that diverse MNoV strains require tuft cells for chronic enteric infection. We also demonstrate that interferon-λ (IFN-λ) acts directly on tuft cells to cure chronic MNoVCR6 infection and that type I and III IFNs signal together via STAT1 in tuft cells to restrict MNoVCW3 tropism. We then develop an enteroid model and find that MNoVCR6 and MNoVCW3 similarly infect tuft cells with equal IFN susceptibility, suggesting that IFN derived from non-epithelial cells signals on tuft cells in trans to restrict MNoVCW3 tropism. Thus, tuft cell tropism enables MNoV persistence and is determined by tuft cell-intrinsic factors (viral receptor expression) and -extrinsic factors (immunomodulatory signaling by non-epithelial cells).

Human Norovirus Efficiently Replicates in Differentiated 3D-Human Intestinal Enteroids.

Human norovirus (HNoV) accounts for one-fifth of all acute viral gastroenteritis worldwide and an economic burden of ~$60 billion globally. The lack of treatment options against HNoV is in part due to the lack of cultivation systems. Recently, a model of infection in biopsy-derived human intestinal enteroids (HIE) has been described: 3D-HIE are first dispersed in 2D-monolayers and differentiated prior to infection, resulting in a labor-intensive, time-consuming procedure. Here, we present an alternative protocol for HNoV infection of 3D-HIE. We found that 3D-HIE differentiated as efficiently as 2D-monolayers. In addition, immunofluorescence-based quantification of UEA-1, a lectin that stains the villus brush border, revealed that ~80% of differentiated 3D-HIE spontaneously undergo polarity inversion, allowing for viral infection without the need for microinjection. Infection with HNoV GII.4-positive stool samples attained a fold-increase over inoculum of ~2 Log10 at 2 days postinfection or up to 3.5 Log10 when ruxolitinib, a JAK1/2-inhibitor, was added. Treatment of GII.4-infected 3D-HIE with the polymerase inhibitor 2'-C-Methylcytidine (2CMC) and other antivirals showed a reduction in viral infection, suggesting that 3D-HIE are an excellent platform to test anti-infectives. The transcriptional host response to HNoV was then investigated by RNA sequencing in infected versus uninfected 3D-HIE in the presence of ruxolitinib to focus on virus-associated signatures while limiting interferon-stimulated gene signatures. The analysis revealed upregulated hormone and neurotransmitter signal transduction pathways and downregulated glycolysis and hypoxia-response pathways upon HNoV infection. Overall, 3D-HIE have proven to be a highly robust model to study HNoV infection, screen antivirals, and to investigate the host response to HNoV infection. IMPORTANCE The human norovirus (HNoV) clinical and socio-economic impact calls for immediate action in the development of anti-infectives. Physiologically relevant in vitro models are hence needed to study HNoV biology, tropism, and mechanisms of viral-associated disease, and also as a platform to identify antiviral agents. Biopsy-derived human intestinal enteroids are a biomimetic of the intestinal epithelium and were recently described as a model that supports HNoV infection. However, the established protocol is time-consuming and labor-intensive. Therefore, we sought to develop a simplified and robust alternative model of infection in 3D enteroids that undergoes differentiation and spontaneous polarity inversion. Advantages of this model are the shorter experimental time, better infection yield, and spatial integrity of the intestinal epithelium. This model is potentially suitable for the study of other pathogens that infect intestinal cells from the apical surface but also for unraveling the interactions between intestinal epithelium and indigenous bacteria of the human microbiome.

Intestinal Norovirus Binding Patterns in Nonsecretor Individuals.

Human norovirus (HuNoV) infection is associated with an active FUT2 gene, which characterizes the secretor phenotype. However, nonsecretor individuals are also affected by HuNoV infection although in a lesser proportion. Here, we studied GII.3, GII.4, and GII.17 HuNoV interactions in nonsecretor individuals using virus-like particles (VLPs). Only GII.4 HuNoV specifically interacted with nonsecretor saliva. Competition experiments using histo-blood group antigen (HBGA)-specific monoclonal antibodies (MAbs) demonstrate that GII.4 VLPs recognized the Lewis a (Lea) antigen. We also analyzed HuNoV VLP interactions on duodenum tissue blocks from healthy nonsecretor individuals. VLP binding was observed for the three HuNoV genotypes in 10 of the 13 individuals, and competition experiments demonstrated that VLP recognition was driven by an interaction with the Lea antigen. In 3 individuals, binding was restricted to either GII.4 alone or GII.3 and GII.17. Finally, we performed a VLP binding assay on proximal and distal colon tissue blocks from a nonsecretor patient with Crohn's disease. VLP binding to inflammatory tissues was genotype specific since GII.4 and GII.17 VLPs were able to interact with regenerative mucosa, whereas GII.3 VLP was not. The binding of GII.4 and GII.17 HuNoV VLPs was linked to Lea in regenerative mucosae from the proximal and distal colon. Overall, our data clearly showed that Lea has a pivotal role in the recognition of HuNoV in nonsecretors. We also showed that Lea is expressed in inflammatory/regenerative tissues and interacts with HuNoV in a nonsecretor individual. The physiological and immunological consequences of such interactions in nonsecretors have yet to be elucidated. IMPORTANCE Human norovirus (HuNoV) is the main etiological agent of viral gastroenteritis in all age classes. HuNoV infection affects mainly secretor individuals where ABO(H) and Lewis histo-blood group antigens (HBGAs) are present in the small intestine. Nonsecretor individuals, who only express Lewis (Le) antigens, are less susceptible to HuNoV infection. Here, we studied the interaction of common HuNoV genotypes (GII.3, GII.4, and GII.17) in nonsecretor individuals using synthetic viral particles. Saliva binding assays showed that only GII.4 interacted with nonsecretor saliva via the Lewis a (Lea) antigen Surprisingly, the three genotypes interacted with nonsecretor enterocytes via the Lea antigen on duodenal tissue blocks, which were more relevant for HuNoV/HBGA studies. The Lea antigen also played a pivotal role in the recognition of GII.4 and GII.17 particles by inflammatory colon tissue from a nonsecretor Crohn's disease patient. The implications of HuNoV binding in nonsecretors remain to be elucidated in physiological and pathological conditions encountered in other intestinal diseases.

Selective Polyprotein Processing Determines Norovirus Sensitivity to Trim7.

Noroviruses are a leading cause of gastroenteritis worldwide, yet the molecular mechanisms of how host antiviral factors restrict norovirus infection are poorly understood. Here, we present a CRISPR activation screen that identifies mouse genes which inhibit murine norovirus (MNV) replication. Detailed analysis of the major hit Trim7 demonstrates a potent inhibition of the early stages of MNV replication. Leveraging in vitro evolution, we identified MNV mutants that escape Trim7 restriction by altering the cleavage of the viral NS6-7 polyprotein precursor. NS6, but not the NS6-7 precursor, directly binds the substrate-binding domain of Trim7. Surprisingly, the selective polyprotein processing that enables Trim7 evasion inflicts a significant evolutionary burden, as viruses with decreased NS6-7 cleavage are strongly attenuated in viral replication and pathogenesis. Our data provide an unappreciated mechanism of viral evasion of cellular antiviral factors through selective polyprotein processing and highlight the evolutionary tradeoffs in acquiring resistance to host restriction factors. IMPORTANCE To maximize a limited genetic capacity, viruses encode polyproteins that can be subsequently separated into individual components by viral proteases. While classically viewed as a means of economy, recent findings have indicated that polyprotein processing can spatially and temporally coordinate the distinct phases of the viral life cycle. Here, we present a function for alternative polyprotein processing centered on immune defense. We discovered that selective polyprotein processing of the murine norovirus polyprotein shields MNV from restriction by the host antiviral protein Trim7. Trim7 can bind the viral protein NS6 but not the viral precursor protein NS6-7. Our findings provide insight into the evolutionary pressures that define patterns of viral polyprotein processing and uncover a trade-off between viral replication and immune evasion.

Bovine natural antibody IgM inhibits the binding of human norovirus protruding domain to its HBGA receptors.

Human norovirus (HuNoV) is the primary viral pathogen that causes acute gastroenteritis (AGE) in humans. The protruding (P) domain of HuNoV interacts with cell surface histo-blood group antigens (HBGAs) to initiate infection. Owing to the lack of an effective in vitro culture method and a robust animal model, our understanding of HuNoVs is limited, and as a result, there are no commercial vaccines or antivirals available at present against the virus. In an attempt to develop a preventative measure, we previously identified that bovine colostrum (bCM) contains functional factors that inhibit the binding of HuNoV P domain to its HBGA receptors. In this study, a candidate functional factor in bCM was identified as immunoglobulin M (IgM) using mass spectrometry, followed by database comparison. The natural antibody IgM was further verified to be a functional protein that inhibited HuNoV P protein binding to HBGA receptors through receptor-binding inhibition experiments using bCM, commercial IgM, and fetal bovine serum. Our findings provide a foundation for future development of natural IgM into an antiviral drug, which may help to prevent and/or treat HuNoV infection.