Murine Norovirus Research Articles

TRIMming viral infection: identification and characterization of the novel antiviral TRIM7

Abstract Noroviruses (NoVs) are the leading cause of acute viral gastroenteritis worldwide, causing up to 700 million infections and 200,000 deaths annually, and treatment is limited to supportive care as there are currently no targeted antivirals nor vaccines against NoVs. Therefore, it is critical to discover novel antivirals and develop targeted interventions in order to improve outcomes. Here we have used a novel genome-wide CRISPR activation screening approach to identify host genes that can inhibit murine norovirus (MNoV) infection or replication. A substantial number of high scoring gene candidates acted in interferon and immune regulation signaling networks, but several were not previously associated with antiviral activity or immunity. TRIM7, the strongest screen hit, was a little studied E3 ubiquitin ligase associated with metabolism and some cancers, yet its expression served as a powerful antiviral in an MNoV-infected cell. Since our initial screen, TRIM7 has begun to emerge as a strategic player in both positive and negative regulation of the innate immune response during infection. We have since characterized the antiviral mechanism of action of TRIM7 against MNoV in vitro, and have found TRIM7 to be acting early in the viral life cycle to limit viral protein production. Co-immunoprecipitation and in vitro viral evolution experiments have identified MNoV NS6 (viral protease) and NS7 (RNA-dependent RNA polymerase) as important targets of the antiviral activity of TRIM7, and also point to key residues of interest associated with MNoV resistance to TRIM7. Finally, we have investigated the relevance of TRIM7 during in vivo infection using knock-in mouse lines to determine tissue specificity and potency of the antiviral effect.

A Systematic Review on the Efficacy of Vaporized Hydrogen Peroxide as a Non-Contact Decontamination System for Pathogens Associated with the Dental Environment.

Aerosol generation and a wide range of pathogens originating from the oral cavity of the patient contaminate various surfaces of the dental clinic. The aim was to determine the efficacy of vaporized hydrogen peroxide fogging on pathogens related to the dental environment and its possible application in dentistry. PICOS statement (Population, Intervention, Comparison/Control, Outcome and Study design statement) was used in the review. Six electronic databases were searched for articles published from 2010 to 2020. Articles written in English reporting vaporized hydrogen peroxide on pathogens deemed to be relevant to the dental environment were assessed. The quality of the studies was assessed using the risk-of-bias assessment tool designed for the investigation of vaporized hydrogen peroxide application in dentistry. A total of 17 studies were included in the qualitative synthesis. The most commonly reported single bacterial pathogen was Methicillin-resistant Staphylococcus aureus in five studies, and the viruses Feline calicivirus, Human norovirus, and Murine norovirus were featured in three studies. The results of the studies reporting the log kill were sufficient for all authors to conclude that vaporized hydrogen peroxide generation was effective for the assessed pathogens. The studies that assessed aerosolized hydrogen peroxide found a greater log kill with the use of vaporized hydrogen peroxide generators. The overarching conclusion was that hydrogen peroxide delivered as vaporized hydrogen peroxide was an effective method to achieve large levels of log kill on the assessed pathogens. The hydrogen peroxide vapor generators can play a role in dental bio-decontamination. The parameters must be standardized and the efficacy assessed to perform bio-decontamination for the whole clinic. For vaporized hydrogen peroxide generators to be included in the dental bio-decontamination regimen, certain criteria should be met. These include the standardization and efficacy assessment of the vaporized hydrogen peroxide generators in dental clinics.

Virucidal Efficacy of Household Dishwashers

Not only since SARS-CoV-2, have transmission routes of viruses been of interest. Noroviruses e.g., can be transmitted via smear infection, are relatively stable in the environment and very resistant to chemical disinfection. Some studies determined the virucidal efficacy of laundering processes, but few studies focused on the virucidal efficacy of dishwashing processes. Here, especially consumer related conditions are of interest. Households for example are a hotspot of norovirus infection and thus a sufficient reduction of these and other viruses from dishes must be insured to avoid an infection via this route. The likelihood of such an event should not be underestimated, since it was shown that the washing machine can be a reservoir for the transmission of extended spectrum beta-lactamase producing bacteria in newborns. Although viruses do not replicate in these devices a transmission via contaminated cutlery e.g., cannot be excluded. Using a consumer related approach to determine the virucidal efficacy of dishwashers, we found a combination of a bleach containing dishwasher detergent, a cleaning temperature of 45 °C for 45 min and a rinsing temperature of 50 °C, to be sufficient to reduces viral titer of bovine corona virus, murine norovirus and modified vaccinia virus by 4.8, 4.2 and 3.8 logarithmic stages respectively.

Reduction of Norovirus Surrogates Alone and in Association with Bacteria on Leaf Lettuce and Tomatoes During Application of Aqueous Ozone.

Retail foodservice establishments (FSE) frequently utilize washes with sanitizing agents during fresh produce preparation. This study evaluated the efficacy of ozonated water on the inactivation of viruses, bacteria, and viruses in association with bacteria on produce surfaces. Boston bibb lettuce (BB) and cherry tomatoes were spot inoculated with viruses (murine norovirus (MNV) and MS2 bacteriophage), bacteria (Enterobacter cloacae and Bacillus cereus), or MNV associated with E. cloacae or B. cereus. Following inoculation, produce was held at 4°C for 90min (virus, virus + bacteria) or 24h (virus, bacteria) prior to treatment. A batch wash ozone sanitation system (BWOSS) was prepared with ice (3-5°C) and 0.5ppm initial ozone concentration or no ozone. Produce samples were treated for 40min with an ozonated water (0.86-0.99ppm) or water-only wash with samples taken every 10min. Samples were processed for microbial recovery, and plaque forming units (PFU) and colony forming units remaining on the produce were determined. Although microbial reductions of 99 to 99.99% were achieved during ozone treatments, few statistically significant differences (P > 0.1) were detected when comparing the ozonated water to water-only wash. Notably, a significant difference (P = 0.009) in log reduction of MNV + bacteria and MNV alone on BB was observed after 40min ozonated water wash. Specifically, MNV with B. cereus achieved a 1-log greater reduction (2.60 log PFU/ml) compared to MNV alone (1.63 log PFU/ml). Overall, washing produce in ozonated water did not significantly increase microbial inactivation compared to water alone under the conditions presented here. Variables impacting ozone wash effectiveness should be considered when implementing produce wash sanitation systems within FSE.

Emerging Pathogenic Unit of Vesicle-Cloaked Murine Norovirus Clusters is Resistant to Environmental Stresses and UV254 Disinfection

An individual virion was long believed to act as an independent infectious unit in virology, until the recent discovery of vesicle-cloaked virus clusters which has greatly challenged this central paradigm. Vesicle-cloaked virus clusters (also known as viral vesicles) are phospholipid-bilayer encapsulated fluid sacs that contain multiple virions or multiple copies of viral genomes. Norovirus is a global leading causative agent of gastroenteritis, and the reported prevalence of vesicle-cloaked norovirus clusters in stool has raised concerns whether the current disinfection, sanitation, and hygiene practices can effectively control environmental pollution by these pathogenic units. In this study, we have demonstrated that vesicle-cloaked murine norovirus (MNV-1) clusters were highly persistent under temperature variation (i.e., freeze-thaw) and they were partially resistant to detergent decomposition. MNV-1 vesicles were 1.89-3.17-fold more infectious in vitro than their free virus counterparts. Most importantly, MNV-1 vesicles were up to 2.16-times more resistant to UV254 disinfection than free MNV-1 at a low viral load in vitro. Interestingly, with the increase of the viral load, free MNV-1 and MNV-1 vesicles showed equivalent resistance to UV254 disinfection. We show that the increased multiplicity of infection provided by vesicles is in part responsible for these attributes. Our study, for the first time, sheds light on the environmental behavior of vesicle-cloaked virus clusters as unique emerging pathogenic units. Our study highlights the need to revisit current paradigms of disinfection, sanitation, and hygiene practices for protecting public health.

Effects of Calcination Temperature on the Phase Composition, Photocatalytic Degradation, and Virucidal Activities of TiO2 Nanoparticles.

The application of TiO2 nanoparticles in the photocatalytic treatment of chemically or biologically contaminated water is an attractive, albeit unoptimized, method for environmental remediation. Here, TiO2 nanoparticles with mixed brookite/rutile phases were synthesized and calcined at 300–1100 °C to investigate trends in photocatalytic performance. The crystallinity, crystallite size, and particle size of the calcined materials increased with calcination temperature, while the specific surface area declined significantly. The TiO2 phase composition varied: at 300 °C, mixed brookite/rutile phases were observed, whereas a brookite-to-anatase phase transformation occurred above 500 °C, reaching complete conversion at 700 °C. Above 700 °C, the anatase-to-rutile phase transformation began, with pure rutile attained at 1100 °C. The optical band gaps of the calcined TiO2 nanoparticles decreased with rising calcination temperature. The mixed anatase/rutile phase TiO2 nanoparticles calcined at 700 °C performed best in the photocatalytic degradation of methylene blue owing to the synergistic effect of the crystallinity and phase structure. The photocatalytic virus inactivation test demonstrated excellent performance against the MS2 bacteriophage, murine norovirus, and influenza virus. Therefore, the mixed anatase/rutile phase TiO2 nanoparticles calcined at 700 °C may be considered as potential candidates for environmental applications, such as water purification and virus inactivation.

Norovirus evolution in immunodeficient mice reveals potentiated pathogenicity via a single nucleotide change in the viral capsid.

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.

Comparison of antiviral effect of oxidizing disinfectants against murine norovirus-1 and hepatitis A virus on fresh root vegetables

This study compared the effectiveness between sodium hypochlorite (NaOCl) and its alternatives—chlorine dioxide (ClO2) and peroxyacetic acid (PAA)—against murine norovirus-1 (MNV−1) and hepatitis A virus (HAV) contamination on carrot and lotus root. ClO2 treatment at 123–300 and 170–300 ppm for 3 min reduced MNV-1 by 1–2.51 log PFU/mL (carrot) and 1–1.83 log unit (lotus root), respectively. ClO2 treatment at > 270 ppm also achieved >1 log HAV reduction for both samples, and PAA treatment at 180–500 and 280–500 ppm for 3 min decreased MNV-1 and HAV by 1–3 and 1–2 log units, respectively. NaOCl showed the lowest antiviral effect among the three disinfectants. Enteric viruses were more difficult to inactivate on lotus root than carrot because of the starch as organic matter. No significant differences (P > 0.05) in the physicochemical qualities (surface color, hardness, pH) of carrot and lotus root were observed after treatment, even at the maximum concentration applied. Such findings suggest the use of 270 ppm ClO2 or 280 ppm PAA in the fresh root vegetable industry to control norovirus and HAV without changing food quality.

NMR Experiments Shed New Light on Glycan Recognition by Human and Murine Norovirus Capsid Proteins

Glycan–protein interactions are highly specific yet transient, rendering glycans ideal recognition signals in a variety of biological processes. In human norovirus (HuNoV) infection, histo-blood group antigens (HBGAs) play an essential but poorly understood role. For murine norovirus infection (MNV), sialylated glycolipids or glycoproteins appear to be important. It has also been suggested that HuNoV capsid proteins bind to sialylated ganglioside head groups. Here, we study the binding of HBGAs and sialoglycans to HuNoV and MNV capsid proteins using NMR experiments. Surprisingly, the experiments show that none of the norovirus P-domains bind to sialoglycans. Notably, MNV P-domains do not bind to any of the glycans studied, and MNV-1 infection of cells deficient in surface sialoglycans shows no significant difference compared to cells expressing respective glycans. These findings redefine glycan recognition by noroviruses, challenging present models of infection.

Effectiveness of water and sanitizer washing solutions for removing enteric viruses from blueberries

The presence of enteric viruses on fresh berries poses a health risk to consumers. After berries are picked, a washing step can reduce microbial counts on fruit surfaces to increase their safety. This study compared the effectiveness of tap water, commercial solutions of fruit and vegetable sanitizer composed of peroxyacetic acid and hydrogen peroxide (PAA) and neutral electrolyzed oxidizing water (NEW) for washing fresh blueberries that had been inoculated with either murine norovirus (MNV-1), bacteriophage MS2, bovine rotavirus (boRV) or hepatitis A virus (HAV). Initially, inactivation levels were measured after blueberries were washed for 1, 3, 5, 10 and 20 min. Subsequently, the effect of combining up to three sequential 1-min washes was evaluated. Washes with water lasting up to 20 min reduced MNV-1, MS2 and HAV populations by 1–2 log10 (PFU) and boRV by less than 4 log10. Increasing washing time did not have a significant effect, except where NEW washes were increased by up to 3 min for MS2 and by 10 min for boRV, with viral titers decreasing by > 4 log10 and >5 log10, respectively. Viral counts were further reduced through additional washes as follows: reduction of MS2 with water, of MNV-1 with NEW and HAV with PAA. PAA achieved significant and rapid inactivation of boRV (>6 log10 PFU after 1 min), MNV-1 (>4 log10 after 10 min) and MS2 (>3 log10 after 3 min). HAV was moderately affected (<3 log10 after 20 min or 3 log10 after 3 washes). NEW was effective at inactivating MNV-1 (>4 log10 after 1 min), MS2 (>4 log10 after 3 min) and HAV (>4 log10 after 1 min). For boRV, washing time needed to be increased to 10 min or three sequential washes were needed to reach >5 log10 reduction. These results highlight the efficiency of PAA at reducing infectivity of MNV-1, MS2 bacteriophage and boRV, and NEW to inactivate the four viruses.

Effect of Plant-Derived Proteases on Infectivity of Tulane Virus, Murine Norovirus, and Hepatitis A Virus

Plant-derived proteases, bromelain, papain, and ficin, are broad-acting enzymes with generally recognized as safe status for foods and have current application in several food industries. These proteases have also been reported to have antimicrobial properties. This study investigated the efficacy of commercially prepared bromelain, papain, and ficin, individually and combined (2,500 ppm of crude extract), for inactivation of hepatitis A virus (HAV) and human norovirus surrogates, Tulane virus (TV), and murine norovirus (MNV). Various treatment temperatures (45, 50, or 55°C), times (10 or 60 min), and pH values (5.5 or 7.0) in the presence of cysteine (2 mM) were evaluated. Inactivation was assessed by infectivity in plaque assay for TV and MNV and by median tissue culture infective dose for HAV. No reduction in infectious TV or HAV was attributed to the plant-derived proteases at any of the conditions tested. Infectious MNV was reduced by 1 to 3 log PFU/mL; the most effective treatment was bromelain at pH 7 and 50°C for 10 min. A time course study with MNV in bromelain at 50°C indicated that a 2-log PFU/mL reduction could be achieved within 6 min, but extended treatment of 15 min was still insufficient to eliminate infectious MNV. The lack of or limited efficacy of bromelain, papain, and ficin on HAV, TV, and MNV, even at elevated temperatures and exposure times, suggests the plant-derived proteases are not commercially applicable for inactivation of virus on commodities or materials that could not also withstand mild heat treatment. The variable susceptibilities observed between TV and MNV illustrate limitations in utilization of surrogates for predicting pathogen behavior for a structure-specific treatment.

Electron Beam Susceptibility of Enteric Viruses and Surrogate Organisms on Fruit, Seed and Spice Matrices

The objective of this study was to use high-energy electron beam (HEEB) treatments to find surrogate microorganisms for enteric viruses and to use the selected surrogates as proof of concept to investigate low-energy electron beam (LEEB) treatments for enteric virus inactivation at industrial scale on frozen blueberries. Six food matrices inoculated with HAV (hepatitis A virus), MNV S99 (murine norovirus), bacteriophages MS2 and Qβ, and Geobacillus stearothermophilus spores were treated with HEEB at 10 MeV using 4, 8 and 16 kGy doses. G. stearothermophilus spores showed the highest inactivation on all matrices except on raisins, with a dose-dependent effect. HAV reached the maximum measurable log10 reduction (> 3.2 log10) when treated at 16 kGy on raisins. MNV showed the highest resistance of all tested microorganisms, independent of the dose, except on frozen blueberries. On frozen blueberries, freeze-dried raspberries, sesame seeds and black peppercorns, HAV showed a mean inactivation level in between those of MS2 and G. stearothermophilus. Based on this, we selected both surrogate organisms as first approximation to estimate HAV inactivation on frozen blueberries during LEEB treatment at 250 keV using 16 kGy. Reductions of 3.1 and 1.3 log10 were measured for G. stearothermophilus spores and MS2, respectively, suggesting that a minimum reduction of 1.4 log10 can be expected for HAV under the same conditions.

AN AUTOPHAGY PROTEIN ATG16L1 PREVENTS NECROPTOSIS BY REGULATING INTERFERON SIGNALING IN THE INTESTINAL EPITHELIUM

ATG16L1, an essential component of autophagy, is one of the susceptibility genes of Crohn’s disease. We previously showed mice with decreased expression of Atg16L1 and Crohn’s disease patients homozygous for ATG16L1T300A risk alleles develop Paneth cell abnormalities in the intestinal epithelium. Moreover, the abnormalities were dependent on murine norovirus (MNV) infection in mice. Most recently, we reported mice with deletion of ATG16L1 in the intestinal epithelial cells (Atg16L1ΔIEC) are more susceptible to disease following intestinal injury than wild type mice when they are infected with MNV. Also, we revealed intestinal organoids derived from Atg16L1ΔIEC mouse are susceptible to TNFa-induced necroptosis. However, how ATG16L1 protects IECs against TNFa, and whether the susceptibility is preserved in human IECs harboring ATG16L1T300A risk alleles remains unclear.

Impact of chlorine dioxide and electron-beam irradiation for the reduction of murine norovirus in low-salted “jogaejeotgal”, a traditional Korean salted and fermented clam

Clam jeotgal, called “jogaejeotgal,” is a Korean fermented seafood product with, generally, a high amount of added salt to inhibit the growth of pathogenic microorganisms. This study aimed to evaluate the efficacy of chlorine dioxide (ClO2) and sodium hypochlorite (NaOCl) against murine norovirus 1 (MNV-1), a surrogate for human norovirus, in salt-fermented clam, jogaejeotgal. The sequential effect of ClO2 and electron-beam (e-beam) irradiation on the inactivation of MNV-1 was also investigated. Treatments of up to 300 ppm ClO2 and 1000 ppm NaOCl were used to determine the disinfectant concentrations at which more than 1 log (90%) MNV-1 inactivation occurred. The sequential treatment of ClO2 (50–300 ppm) and e-beam (1–5.5 kGy) was performed after storage at 4 °C for 7 days. There was a 1.9-log reduction of the virus in seasoned clams irradiated at 5.5 kGy after ClO2 treatment at 300 ppm. No significant change (p > 0.05) in physicochemical quality was observed after the combined treatment, suggesting the potential for the use of a combined treatment using ClO2 (300 ppm) and e-beam (5.5 kGy) in the jeotgal manufacturing industry for the reduction of norovirus.

Survival and Inactivation by Advanced Oxidative Process of Foodborne Viruses in Model Low-Moisture Foods

Enteric viruses, such as human norovirus (NoV) and hepatitis A virus (HAV), are the major causes of foodborne illnesses worldwide. These viruses have low infectious dose, and may remain infectious for weeks in the environment and food. Limited information is available regarding viral survival and transmission in low-moisture foods (LMF). LMFs are generally considered as ready-to-eat products, which undergo no or minimal pathogen reduction steps. However, numerous foodborne viral outbreaks associated with LMFs have been reported in recent years. The objective of this study was to examine the survival of foodborne viruses in LMFs during 4-week storage at ambient temperature and to evaluate the efficacy of advanced oxidative process (AOP) treatment in the inactivation of these viruses. For this purpose, select LMFs such as pistachios, chocolate, and cereal were inoculated with HAV and the norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV), then viral survival on these food matrices was measured over a four-week incubation at ambient temperature, by both plaque assay and droplet-digital RT-PCR (ddRT-PCR) using the modified ISO-15216 method as well as the magnetic bead assay for viral recovery. We observed an approximately 0.5 log reduction in viral genome copies, and 1 log reduction in viral infectivity for all three tested viruses following storage of select inoculated LMFs for 4 weeks. Therefore, the present study shows that the examined foodborne viruses can persist for a long time in LMFs. Next, we examined the inactivation efficacy of AOP treatment, which combines UV-C, ozone, and hydrogen peroxide vapor, and observed that while approximately 100% (4 log) inactivation can be achieved for FCV, and MNV in chocolate, the inactivation efficiency diminishes to approximately 90% (1 log) in pistachios and 70% (< 1 log) in cereal. AOP treatment could therefore be a good candidate for risk reduction of foodborne viruses from certain LMFs depending on the food matrix and surface of treatment.

Antibody Titers and Seroconversion Kinetics of Outbred Swiss and Heterozygous Nude Soiled-bedding Sentinels for Murine Norovirus and Mouse Hepatitis Virus.

Sentinel animals remain a common means of evaluating rodent health in research colonies. An evaluation of our sentinel program revealed that using Crl:CD1(ICR)-Elite (CD1-E) mice was expensive, occasionally disrupted by limited supply, and minimally responsive to the 3Rs. This evaluation prompted us to explore the use of CRL:NU-Foxn1nu/+ (Het-nude) mice as soiled-bedding sentinel (SBS) animals. Het-nude mice are a byproduct of breeding outbred athymic nude mice and are reared in isolators, with similar health status as CD1-E. Het-nude mice have a thymus, but may have smaller thymic size and fewer bone marrow stem cells than do wildtype controls, suggesting that Het-nude mice might not be immunologically normal. This study compared the antibody titer and seroconversion kinetics of Het-nude and CD1-E SBS to murine norovirus (MNV) and mouse hepatitis virus (MHV). Het-nude and CD1-E female SBS (n = 22 mice of each stock) were housed continuously on soiled bedding collected from MNV-positive or MNV- and MHV-positive colonies at cage changes. Blood was collected for serology at 3, 9 and 12 to 19 wk after the start of soiled bedding exposure. Antibody titers to MNV or MHV did not differ significantly between Het-nude and CD1-E mice. A significant relationship was found between weeks of exposure and titer levels with an increase in titer over the testing period. This study supports the possible use of Het-nude mice as SBS, given that their antibody responses to MNV and MHV are equivalent to those of CD1-E mice.

Antioxidant and anti-norovirus properties of aqueous acetic acid macromolecular extracts of edible brown macroalgae

Previous studies reported the activity of fucoidans and polyphenols (phlorotannins) extracted from brown algae against enveloped viruses. The aim of this study was to investigate the effects of brown macroalgae on the non-enveloped norovirus. Aqueous acetic acid macromolecular (>3 kDa) extracts (AAEs) were obtained from ten samples of eight brown macroalgal species. Saccharides and sulphate composition, weight-averaged molecular weight, and total phenolic content (TPC) of the AAEs were measured to estimate their fucoidan and polyphenol content. Murine RAW264.7 macrophage cells and the human norovirus surrogate murine norovirus-1 (MNV-1) were used for the anti-norovirus assay. One hour of direct exposure to TPC-rich AAEs from Sargassum horneri (Sh), Eisenia bicyclis (Eb), Cladosiphon okamuranus (Co), and Undaria pinnatifida inactivated MNV-1. In contrast, pre-treatment of RAW264.7 cells for 20 h with AAEs from another U. pinnatifida sample, Analipus japonicus, and Co protected RAW264.7 cells against MNV-1 infection. The AAEs from Co contained high TPC and sulphated fucans (fucoidans) might be the most promising anti-norovirus agents as they directly inactivated MNV-1 and protected RAW264.7 cells from it. The AAEs of phlorotannin-rich Sh and Eb are also potentially effective at direct inactivating MNV-1.

AN AUTOPHAGY PROTEIN ATG16L1 PREVENTS NECROPTOSIS BY REGULATING INTERFERON SIGNALING IN THE INTESTINAL EPITHELIUM

Abstract Introduction ATG16L1, an essential component of autophagy, is one of the susceptibility genes of Crohn’s disease. We previously showed mice with decreased expression of Atg16L1 and Crohn’s disease patients homozygous for ATG16L1T300A risk alleles develop Paneth cell abnormalities in the intestinal epithelium. Moreover, the abnormalities were dependent on murine norovirus (MNV) infection in mice. Most recently, we reported mice with deletion of ATG16L1 in the intestinal epithelial cells (Atg16L1ΔIEC) are more susceptible to disease following intestinal injury than wild type mice when they are infected with MNV. Also, we revealed intestinal organoids derived from Atg16L1ΔIEC mouse are susceptible to TNFa-induced necroptosis. However, how ATG16L1 protects IECs against TNFa, and whether the susceptibility is preserved in human IECs harboring ATG16L1T300A risk alleles remains unclear. Aims In this study, we first examined the mechanism by which ATG16L1-deficiency renders IECs susceptible to necroptosis. Next, we investigated whether a specific ISG might be involved in the increased susceptibility. Finally, we tested whether human intestinal organoids harboring ATG16L1T300A risk alleles are susceptible to TNFa-induced necroptosis. Methods &amp; Results We performed RNA-Seq analysis using small intestinal organoids derived from Atg16L1ΔIEC and control mouse, and found 49 genes were upregulated in naïve Atg16L1ΔIEC organoids (Figure 1). Interestingly, most of them were the genes known as interferon-stimulated genes (ISGs) representing a type I IFN (IFN-I) signature. Importantly, we found Atg16L1ΔIEC organoids pretreated with JAK-STAT inhibitor Ruxolitinib displayed better viability and decreased level of necroptosis-related proteins when they were stimulated with TNFa. Moreover, we found protein kinase R (PKR) was increased in naïve Atg16L1ΔIEC organoids, and inhibition of PKR significantly protected Atg16L1ΔIEC organoids from TNFa. Finally, we generated human intestinal organoids using biopsy specimens mostly collected from Crohn’s disease patients. We compared the viability between individuals with 2 copies vs 0 or 1 copy of ATG16L1T300A risk allele, and found organoids derived from ATG16L1T300A homozygous individuals exhibited heightened susceptibility to TNFa (Figure 2 A and B). Moreover, we tested the effect of Ruxolitinib as well as multiple necroptosis inhibitors on human organoids, and found all the inhibitors successfully protected ATG16L1T300A homozygous organoids from TNFa-induced cell death. Conclusion Our data indicate ATG16L1 prevents necroptosis by regulating interferon signaling in both mouse and human IECs, and suggest inhibition of JAK-STAT as well as necroptosis pathway could be promising therapeutic options for Crohn’s disease patients with ATG16L1T300A risk alleles.

CD300lf Conditional Knockout Mouse Reveals Strain-Specific Cellular Tropism of Murine Norovirus.

Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout (CD300lfF/F ) mouse to elucidate the cell tropism of persistent and nonpersistent strains of murine norovirus. Using this mouse model, we demonstrated that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoVCR6) in vivo In contrast, the nonpersistent MNoV strain CW3 (MNoVCW3) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (LysM Cre+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (CD19 Cre), neutrophils (Mrp8 Cre), and dendritic cells (CD11c Cre) did not affect MNoVCW3 viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoVCW3 to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoVCR6; that myelomonocytic cells are a major, but not exclusive, target cell of MNoVCW3; and that STAT1 signaling restricts the cellular tropism of MNoVCW3 This study provides the first genetic system for studying the cell type-specific role of CD300lf in norovirus pathogenesis.IMPORTANCE Human noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use murine norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a Cd300lf conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoVCR6 requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for nonpersistent MNoVCW3 infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoVCW3 infection. Mortality associated with the MNoVCW3 strain in Stat1-/- mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism in vivo.

Detection and whole genome sequencing of murine norovirus in animal facility in Italy

Viruses belonging to the genus Norovirus (NoV) of the family Caliciviridae are the major cause of acute viral gastroenteritis worldwide. NoVs are classified into 10 genogroups (GI-GX), and those belonging to the genogroup GV are able to infect several species of rodents. To evaluate the circulation of MNV among mice housed in an Italian facility, sampling was performed over two separate periods, in 2011, and 3 years later in 2014. During the two samplings, 75 fecal samples were collected from healthy mice housed in the animal facility and subjected to RT-PCR for viral detection. After the analysis, 41/75 animals (54.6%) resulted positive for the presence of MNV in feces. Nucleotide sequencing revealed the presence of two MNV variants co-circulating in both 2011 and 2014. One MNV strain was isolated on RAW264.7 cell line, and subjected to full genome sequencing. Our study showed that the murine noroviruses are widespread in the investigated animal facility, despite guidelines for animal care and maintenance. Full genome sequence analysis of the MNV strain described in this study showed a correlation with other strains circulating in Europe. Understanding the molecular epidemiology of this virus should give insight into its natural history and evolution in mice.