Evaluation of murine norovirus persistence in environments relevant to food production and processing.

Attachment of noroviruses to stainless steel and their inactivation, using household disinfectants.

The metabolic pathways of central carbon metabolism, glycolysis and oxidative phosphorylation (OXPHOS), are important host factors that determine the outcome of viral infections and can be manipulated by some viruses to favor infection. However, mechanisms of metabolic modulation and their effects on viral replication vary widely. Herein, we present the first metabolomics and energetic profiling of norovirus-infected cells, which revealed increases in glycolysis, OXPHOS, and the pentose phosphate pathway (PPP) during murine norovirus (MNV) infection. Inhibiting glycolysis with 2-deoxyglucose (2DG) in macrophages revealed that glycolysis is an important factor for optimal MNV infection, while inhibiting the PPP and OXPHOS showed a relatively minor impact of these pathways on MNV infection. 2DG affected an early stage in the viral life cycle after viral uptake and capsid uncoating, leading to decreased viral protein production and viral RNA. The requirement of glycolysis was specific for MNV (but not astrovirus) infection, independent of the type I interferon antiviral response, and unlikely to be due to a lack of host cell nucleotide synthesis. MNV infection increased activation of the protein kinase Akt, but not AMP-activated protein kinase (AMPK), two master regulators of cellular metabolism, implicating Akt signaling in upregulating host metabolism during norovirus infection. In conclusion, our findings suggest that the metabolic state of target cells is an intrinsic host factor that determines the extent of norovirus replication and implicates glycolysis as a virulence determinant. They further point to cellular metabolism as a novel therapeutic target for norovirus infections and improvements in current human norovirus culture systems.IMPORTANCE Viruses depend on the host cells they infect to provide the machinery and substrates for replication. Host cells are highly dynamic systems that can alter their intracellular environment and metabolic behavior, which may be helpful or inhibitory for an infecting virus. In this study, we show that macrophages, a target cell of murine norovirus (MNV), increase glycolysis upon viral infection, which is important for early steps in MNV infection. Human noroviruses (hNoV) are a major cause of gastroenteritis globally, causing enormous morbidity and economic burden. Currently, no effective antivirals or vaccines exist for hNoV, mainly due to the lack of high-efficiency in vitro culture models for their study. Thus, insights gained from the MNV model may reveal aspects of host cell metabolism that can be targeted for improving hNoV cell culture systems and for developing effective antiviral therapies.

Free chlorine as hypochlorite is recommended to decontaminate fecally contaminated surfaces to control human norovirus (NoV). We evaluated the efficacy of sodium hypochlorite to decontaminate GII.4 NoV and three surrogates of human NoVs, feline calicivirus (FCV), murine norovirus (MNV), and coliphage MS2, on a fecally soiled stainless steel surface. Reduction of infectivity of FCV, MNV, and MS2 was measured by plaque assay and the decline of genomic copy numbers of GII.4 NoV by reverse transcriptase-polymerase chain reaction. Sodium hypochlorite solution at 5000 ppm could inactivate FCV by 3 log(10) plaque forming units after approximately 1.9 minutes of contact time, but required longer exposure times of 3.2 and 4.5 minutes to reduce MNV and MS2 by 3 log(10), respectively. However, detection of viral RNA by reverse transcriptase-polymerase chain reaction assay may not be reliable to estimate the effectiveness of sodium hypochlorite against human NoV. Of three NoV surrogates, FCV is not the most resistant of the virus tested for inactivation by hypochlorite and thus is not the worst-case model for estimating NoV inactivation. Although the use of 5000 ppm of hypochlorite for fecally soiled surfaces is effective, it may require longer exposure times of ≥3 minutes to control NoVs. Surface precleaning before hypochlorite disinfection is recommended to initially reduce the fecal organic load for better virus inactivation and should be a part of the environmental hygiene response measures during an NoV outbreak or where NoV fecal contamination of environmental surfaces is likely or suspected to be present.

Background: Human Norovirus (HuNoV) is the leading cause of infectious gastroenteritis and food-borne outbreaks, responsible for over 220K deaths annually. Given difficulties studying HuNoV, Murine Norovirus (MNV) has been used as a model for norovirus (NoV) pathophysiology. A major barrier to understand how NoV causes diarrhea is the absence of diarrhea in adult wild-type (WT) mice, which have asymptomatic MNV infections. While several KO mouse strains (e.g., STAT1) have been employed to recapitulate disease, these mice often have exaggerated, systemic, and lethal disease courses that imperfectly match human disease. Very recently, a new model of MNV that appears to recapitulate symptomatic HuNoV infection has been established. This model involves the oral gavage of three- or four-day-old (d.o.) WT BALB/c mice with high-concentrations of MNV resulting in a diarrheal phenotype. Current studies were undertaken to understand the ion transport basis of MNV diarrhea using this novel model. Methods: Three to four d.o. BALB/c pups were gavaged with either 1x108 plaque-forming units (PFU) of MNV-1 (CW3) or PBS alone followed by sacrifice at 48 hours-post-gavage (hpg). Small and large intestines were harvested for RNA and immunofluorescence (IF) analysis of multiple ion transporters that have been linked to diarrhea in other disease models. Results: MNV-gavaged pups had loose, orange stools, consistent with a diarrheal phenotype observed previously with this model. MNV RNA was detectable in small and large intestinal tissue for CW3-gavaged vs mock-gavaged pups (p-values <0.05) demonstrating successful infection with MNV. MNV infection had no significant effect on transcript levels of various electrolyte and nutrient transporters including DRA, NHE3, PAT-1, Na-K-ATPase, or SGLT-1. However, we found significant increases in CFTR and Vasoactive intestinal peptide receptor 1 (VPAC1) transcripts (p <0.05 and 0.01, respectively) and a significant increase in CFTR IF staining in frozen sections of the SI over mock (p<0.01). We did not find a significant increase in VIP transcript, VPAC1 protein expression or immunofluorescent staining, nor nuclear localization of pCREB in SI frozen sections. Conclusion: Using this novel neonatal WT BALB/c MNV mouse model to evaluate the effect of infection on gene transcription, and protein expression and localization of ion transporters commonly implicated in diarrhea, we observed significant increases in CFTR and VPAC1 SI gene expression in CW3-gavaged pups with negligible changes in transcription of all other transporters examined in this study. In addition, apical and total CFTR staining increased significantly without changes in VPAC1 or pCREB nuclear localization in frozen SI sections of CW3-gavaged pups compared to mock, suggesting that MNV causes a CFTR-mediated secretory diarrhea in the absence of changes in VPAC1 protein expression, localization, or activation. These studies were supported by the Department of Veterans Affairs, Veterans Heath Administration, Office of Research and Development, Biomedical Laboratory Research and Development: Merit Review Award: BX002011 (PKD), and VA Senior Research Career Scientist Award:1IK6BX005242 (PKD). The studies were also supported by NIH/NIDDK grants, R01 DK54016 and DK92441 (PKD). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The effect of high hydrostatic pressure (HPP) was evaluated for inactivation of murine norovirus (MNV), a propagable norovirus (NoV), and human NoV genogroup II.4. Inactivation of MNV was assessed by viral culturing (50% tissue culture infectious dose [TCID(50)]) and real-time reverse-transcription-polymerase chain reaction (RT-qPCR), whereas NoV survival was determined only by RT-qPCR. A treatment of 450 MPa for 15 min at 45°C was sufficient to inactivate 6.5 log(10) of infectious MNV in culture medium as determined by TCID(50). Further, the inactivation of MNV was enhanced when pressure was applied at an initial temperature of 25°C. On the other hand, a baroprotective effect was observed when MNV suspensions were supplemented with 10 mM of CaCl(2). A 400 MPa treatment at 45°C inactivated >5 log(10) of infectious MNV, whereas the addition of CaCl(2) increased the pressure resistance of MNV, with <0.5 log(10) reduction observed. MNV decay as determined by TCID(50) was generally greater than that determined by RT-qPCR; for instance, MNV genomes were detected even after 15 min treatment at 450 MPa, with <0.5 log(10) reduction. Experiments with NoV suspensions showed that all tested HPP treatments reduced the numbers of NoV by <0.5 log(10) units as determined by RT-qPCR. Additionally, RNA of human NoV was more resistant to certain HPP treatments than the RNA of MNV.

Two classes of viruses, namely members of the Potyviridae and Caliciviridae, use a novel mechanism for the initiation of protein synthesis that involves the interaction of translation initiation factors with a viral protein covalently linked to the viral RNA, known as VPg. The calicivirus VPg proteins can interact directly with the initiation factors eIF4E and eIF3. Translation initiation on feline calicivirus (FCV) RNA requires eIF4E because it is inhibited by recombinant 4E-BP1. However, to date, there have been no functional studies carried out with respect to norovirus translation initiation, because of a lack of a suitable source of VPg-linked viral RNA. We have now used the recently identified murine norovirus (MNV) as a model system for norovirus translation and have extended our previous studies with FCV RNA to examine the role of the other eIF4F components in translation initiation. We now demonstrate that, as with FCV, MNV VPg interacts directly with eIF4E, although, unlike FCV RNA, translation of MNV RNA is not sensitive to 4E-BP1, eIF4E depletion, or foot-and-mouth disease virus Lb protease-mediated cleavage of eIF4G. We also demonstrate that both FCV and MNV RNA translation require the RNA helicase component of the eIF4F complex, namely eIF4A, because translation was sensitive (albeit to different degrees) to a dominant negative form and to a small molecule inhibitor of eIF4A (hippuristanol). These results suggest that calicivirus RNAs differ with respect to their requirements for the components of the eIF4F translation initiation complex.

Ethanol is a popular agent for preventing cross-contamination with human norovirus (NoV) on stainless steel (STS) surfaces that are used for food preparation and manufacture. A polynomial equation was used to evaluate the anti-viral efficacy of ethanol by feline calicivirus (FCV) and murine norovirus (MNV) as NoV surrogates. The level of FCV VR-782 and MNV on stainless steel surfaces were measured at room temperature over 24 h posttreatment with various concentrations (0–70%) of ethanol for different treatment times (0–10 mins). The amount of FCV and MNV that survived on STS after 24 h were 1.60±0.01 and 1.23±0.04 log TCID50/coupon, respectively. FCV and MNV had a higher resistance to STS surfaces than Escherichia coli, which was used as a representative comparative pathogenic bacterium. The polynomial equations predicting the inactivation of FCV and MNV were as follows: FCV (log TCID50/coupon)= +0.19379+0.067282x1+0.058945x2−8.57143E-004x1x2−1.44483E-003x1 2−3.51935E-004x2 2 (x1: time and x2: concentration); and MNV (log TCID50/coupon)=+1.08790+0.65635x1+0.077860x2−1.47143E-003x1x2−0.024552x12−6.56158E-004 x2 2 (x1: time and x2: concentration). Therefore, these polynomial equation models for reduction of FCV and MNV could be used to predict the minimum concentration of ethanol and exposure time required to control human NoV on food contact surfaces.

BackgroundHuman norovirus (NoV) causes more than 80% of nonbacterial gastroenteritis in Europe and the United States. NoV transmission via contaminated surfaces may be significant for the spread of viruses. Therefore, measures for prevention and control, such as surface disinfection, are necessary to interrupt the dissemination of human NoV. Murine norovirus (MNV) as a surrogate for human NoV was used to study the efficacy of active ingredients of chemical disinfectants for virus inactivation on inanimate surfaces.MethodsThe inactivating properties of different chemical biocides were tested in a quantitative carrier test with stainless steel discs without mechanical action. Vacuum-dried MNV was exposed to different concentrations of alcohols, peracetic acid (PAA) or glutaraldehyde (GDA) for 5 minutes exposure time. Detection of residual virus was determined by endpoint-titration on RAW 264.7 cells.ResultsPAA [1000 ppm], GDA [2500 ppm], ethanol [50% (v/v)] and 1-propanol [30% (v/v)] were able to inactivate MNV under clean conditions (0.03% BSA) on the carriers by ≥ 4 log10 within 5 minutes exposure time, whereas 2-propanol showed a reduced effectiveness even at 60% (v/v). Furthermore, there were no significant differences in virus reduction whatever interfering substances were used. When testing with ethanol, 1- and 2-propanol, results under clean conditions were nearly the same as in the presence of dirty conditions (0.3% BSA plus 0.3% erythrocytes).ConclusionProducts based upon PAA, GDA, ethanol and 1-propanol should be used for NoV inactivation on inanimate surfaces. Our data provide valuable information for the development of strategies to control NoV transmission via surfaces.

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.

Norovirus (NoV) is the most common viral cause of acute gastroenteritis worldwide. Vitamin A has demonstrated the potential to protect against gastrointestinal infections. However, the effects of vitamin A on human norovirus (HuNoV) infections remain poorly understood. This study aimed to investigate how vitamin A administration affects NoV replication. We demonstrated that treatment with retinol or retinoic acid (RA) inhibited NoV replication in vitro based on their effects on HuNoV replicon-bearing cells and murine norovirus-1 (MNV-1) replication in murine cells. MNV replication in vitro showed significant transcriptomic changes, which were partially reversed by retinol treatment. RNAi knockdown of CCL6, a chemokine gene that was downregulated by MNV infection but upregulated by retinol administration, resulted in increased MNV replication in vitro. This suggested a role of CCL6 in the host response to MNV infections. Similar gene expression patterns were observed in the murine intestine after oral administration of RA and/or MNV-1.CW1. CCL6 directly decreased HuNoV replication in HG23 cells, and might indirectly regulate the immune response against NoV infection. Finally, relative replication levels of MNV-1.CW1 and MNV-1.CR6 were significantly increased in CCL6 knockout RAW 264.7 cells. This study is the first to comprehensively profile transcriptomes in response to NoV infection and vitamin A treatment in vitro, and thus may provide new insights into dietary prophylaxis and NoV infections.

The effects of heat (72°C) and UV-light (254 nm) on the infectivity of murine norovirus-1 (MNV-1) and human norovirus (NoV) are reported. The infectivity of MNV-1 was measured using standard cell-culture plaque assays and was compared with a specific duplex RT-qPCR for MNV-1, which targets two fragments of the MNV genome, one 142 bp (short-range, sRT-qPCR) and a second 4.6 kb (long-range, loRT-qPCR) upstream from the reverse transcription (RT) priming site (poly-A tail). After the heat treatment of MNV neither the sRT- nor loRT-qPCR correlated with decreased infectivity as assessed by the plaque assay. However, after UV-exposure, the lo-qPCR showed decreased qPCR amplification, which became more pronounced with increased UV-exposure time, and reflected decreased infectivity as shown in the plaque assay. The sRT-qPCR amplification was not affected by UV-exposure. Similar results were obtained when challenging non-culturable human GI and GII NoV with UV-light. Using previously published RT-qPCR assays for GI and GII NoV, no decrease in RT-qPCR amplification was seen when using the respective reverse primers of the RT-qPCR assay in the RT reaction, whereas a significant decrease in qPCR amplification occurred when the RT reaction was primed at the poly-A tail about 2.3 kb from the qPCR amplification site. The results indicate that, in contrast to RT-qPCR with a long-range RT, RT-qPCR with a short-range RT does not reflect genomic integrity and therefore viral infectivity. Furthermore, human NoV appears to be highly sensitive to UV-irradiation which could lead to more efficient decontamination procedures.

The integrated stress response (ISR) is a cellular response system activated upon different types of stresses, including viral infection, to restore cellular homeostasis. However, many viruses manipulate this response for their own advantage. In this study, we investigated the association between murine norovirus (MNV) infection and the ISR and demonstrate that MNV regulates the ISR by activating and recruiting key ISR host factors. We observed that during MNV infection, there is a progressive increase in phosphorylated eukaryotic initiation factor 2α (p-eIF2α), resulting in the suppression of host translation, and yet MNV translation still progresses under these conditions. Interestingly, the shutoff of host translation also impacts the translation of key signaling cytokines such as beta interferon, interleukin-6, and tumor necrosis factor alpha. Our subsequent analyses revealed that the phosphorylation of eIF2α was mediated via protein kinase R (PKR), but further investigation revealed that PKR activation, phosphorylation of eIF2α, and translational arrest were uncoupled during infection. We further observed that stress granules (SGs) are not induced during MNV infection and that MNV can restrict SG nucleation and formation. We observed that MNV recruited the key SG nucleating protein G3BP1 to its replication sites and intriguingly the silencing of G3BP1 negatively impacts MNV replication. Thus, it appears that MNV utilizes G3BP1 to enhance replication but equally to prevent SG formation, suggesting an anti-MNV property of SGs. Overall, this study highlights MNV manipulation of SGs, PKR, and translational control to regulate cytokine translation and to promote viral replication.IMPORTANCE Viruses hijack host machinery and regulate cellular homeostasis to actively replicate their genome, propagate, and cause disease. In retaliation, cells possess various defense mechanisms to detect, destroy, and clear infecting viruses, as well as signal to neighboring cells to inform them of the imminent threat. In this study, we demonstrate that the murine norovirus (MNV) infection stalls host protein translation and the production of antiviral and proinflammatory cytokines. However, virus replication and protein translation still ensue. We show that MNV further prevents the formation of cytoplasmic RNA granules, called stress granules (SGs), by recruiting the key host protein G3BP1 to the MNV replication complex, a recruitment that is crucial to establishing and maintaining virus replication. Thus, MNV promotes immune evasion of the virus by altering protein translation. Together, this evasion strategy delays innate immune responses to MNV infection and accelerates disease onset.

Detection of viable murine norovirus using the plaque assay and propidium-monoazide-combined real-time reverse transcription-polymerase chain reaction

Human norovirus (NoV) is a member of the calicivirus family, can cause nausea, vomiting, abdominal pain and diarrhea as the main clinical symptoms of acute gastroenteritis. Human norovirus infection can be popular in the world, all ages, causing serious burden of disease. Due to the lack of suitable small animal models, there is still a lack of understanding of human immune to norovirus infection and pathogenesis. Murine norovirus (MNV) was originally isolated in immune deficient mice, and causes infection and epidemic in mice. MNV provides an alternative model to study human norovirus infection and the host intestinal immune mechanism. This article will elaborate on two aspects of innate immunity and adaptive immunity. Key words: Murine norovirus; Infection; Immune response

Dendritic cells (DCs) are permissive to murine norovirus (MNV) infection in vitro and in vivo. However, their roles during infection in vivo are not well defined. To determine the role of DCs during infection, conventional DCs were depleted from CD11c-DTR mice and infected with a persistent MNV strain. Viral titres in the intestine and secondary lymphoid organs were determined at early time points during infection, and anti-MNV antibody responses were analysed later during infection. Depletion of conventional DCs resulted in increased viral loads in intestinal tissues, impaired generation of antibody responses, and a failure of MNV to efficiently infect lymphoid tissues. These data suggest that DCs play multiple roles in MNV pathogenesis, in both innate immunity and the efficient generation of adaptive immune responses against MNV, as well as by promoting the dissemination of MNV to secondary lymphoid tissues. This is the first study to probe the roles of DCs in controlling and/or facilitating a norovirus infection in vivo and provides the basis for further studies aimed at defining mechanisms by which DCs control MNV replication and promote viral dissemination.