Persistence of foodborne viruses on various frozen berries initially frozen at different temperatures and stored for up to two years.

Inactivation of hepatitis E virus and murine norovirus during smoking treatments alone and an industrial warm smoking with additional heat treatment of pork sausages

Protocol for virome characterization in low-volume respiratory samples from broiler chickens.

Human norovirus is the leading cause of gastroenteritis worldwide. Norovirus exhibits remarkable genetic diversity. Understanding the impact of genetic diversity on infection and immunity has been challenging due to the difficulties in in vitro cultivation and the current lack of a small animal model. Murine norovirus (MNV) has emerged as a premier model system to investigate norovirus biology. Here, we identify TRIM47 as a host restriction factor that potently inhibits MNV infection in a strain-dependent manner. We determine that TRIM47 expression inhibits an early stage of the viral life cycle of the MNV strain CR6 (MNVCR6), while the replication of the closely related strain CW3 (MNVCW3) is not restricted by TRIM47. MNVCW3 causes an acute infection that spreads beyond intestinal tissues and is lethal to immunodeficient mice. In contrast, MNVCR6 fails to spread to systemic tissues but establishes a persistent infection by infecting intestinal tuft cells. Using a forward genetic screen, we determine that genetic variation within the nonstructural protein 1 (NS1) accounts for this differential sensitivity of MNV strains to TRIM47. While most TRIM-containing proteins promote the ubiquitination and degradation of their targets, TRIM47 does neither. Instead, TRIM47 promotes the deubiquitination of the NS1/2 precursor protein. Our data provide new insight into a potential antiviral gene and mechanistic insight into norovirus evolution that may impact viral tropism.IMPORTANCEViruses exist as genetically heterogeneous populations. Understanding the contribution of viral genetic variation to infection outcomes is critical in predicting emerging viruses and their variants. Noroviruses are genetically diverse, but human norovirus has been technically challenging to study. In this study, we use the model system murine norovirus to identify a viral strain-specific restriction mechanism where a host gene can specifically restrict one strain of the virus but has no impact on a closely related strain. Dissecting the mechanism of this specificity provides insight into viral diversity and possible host restriction pathways.

Abstract Description Human noroviruses (Caliciviridae family) are the leading cause of gastroenteritis worldwide. Murine norovirus (MNV) has been used as a model system to study human noroviruses due to its robust replication in vitro and in a mouse model. Calicivirus replication involves translation of a single non-structural polyprotein complex from its ssRNA genome, which is then cleaved into individual non-structural proteins (NS1-7) by its protease NS6. Previously, we found that a mutation (MNV NS6F182C) which decreased the efficiency of NS6-NS7 cleavage was able to overcome restriction imposed by Trim7 but resulted in attenuation of the virus. We used Trim7 as a tool to uncover suppressor mutations in this attenuated MNV system (MNV NS6F182C) via viral passaging. We identified NS4V11I as the only mutation that arose in this screen. Norovirus NS4 is a part of the viral replication complex and forms double membrane vesicles in host cells. Previous reports have shown that overexpression of NS4 activates the cGAS-Sting pathway by promoting the release of mitochondrial DNA. While NS4 is considered to have immunomodulatory functions, little is known of the function of NS4 during norovirus infection. Therefore, we leverage viral genetics tools to study differences in replication kinetics of WT MNV and MNV NS4V11I and an unbiased proteomics screen to determine protein interactors of NS4 that will provide critical insight into new mechanisms of viral antagonists to the immune system. Topic Categories Viral Immunology (VIR)

The spillover of highly pathogenic avian influenza (HPAI) to dairy cattle and its presence at high titers in raw milk has created a concern that consumption of dairy products made from unpasteurized milk could result in human exposure. We examined whether H1N1, as a surrogate for HPAI viruses, and murine norovirus (MNV), as a surrogate for unenveloped enteric viruses, can survive the cheese-making process by making cream cheese-style and feta-style cheeses from artificially inoculated raw milk. We used the modified ISO 15216 method for viral extraction, which has a limit of detection of 3.0-3.7 log for infectious H1N1 in soft cheeses. Infectious H1N1 virus was detected in the curd from both types of cheeses and in the whey from cream cheese but not in the whey from feta cheese. Infectious MNV particles were detected in the curd and in the whey for both types of cheeses. To study the effect of salt, pH, aging, and microbiome of various unpasteurized milk cheeses on viral survival, we artificially inoculated different commercial unpasteurized milk cheeses with 7 log PFU of H1N1 and 6 log PFU of MNV and examined viral survival over an 8 week period at 4°C. We observed that H1N1 survived for 8 weeks, with an average of 2.6, 2.75, and 4.0 log reductions on cheddar, washed rind firm cheese, and semisoft brie-like cheese, respectively (P < 0.001). The level of infectious H1N1 fell below the limit of detection in cream cheese after 2 weeks of inoculation. It is noteworthy that the sensory qualities of soft and semisoft cheeses deteriorated in 2 weeks, but the sensory qualities of firm cheeses did not change drastically, demonstrating that, as expected, they endure the aging process better than the soft and semisoft cheeses. Furthermore, the limit of detection for soft cheeses is approximately 1 log lower compared to firm cheeses, and the extraction method has a lower efficiency for soft cheeses compared to firm cheeses. MNV survived in firm cheeses for 8 weeks with only 1 log reduction on cheddar cheese and 2 log reductions on the washed rind firm cheese (P < 0.05). The data obtained in this study could help with the risk assessment of dairy products made from unpasteurized milk.IMPORTANCEThe rapid spread of H5N1 viruses among dairy cattle in the United States and the viral shedding at high titers raised concern regarding the safety of dairy products, specifically cheeses made with unpasteurized milk. In this study, we demonstrated that murine norovirus and influenza virus can survive the cheese-making and aging process. Therefore, it is recommended that milk contaminated with viruses be heat-treated to ensure safety.

ABSTRACTThis study evaluated the effectiveness of washing potatoes inoculated with Escherichia coli, Listeria monocytogenes, and murine norovirus (MNV) through agitation with peroxyacetic acid (PAA) and chlorine dioxide. Potential cross‐contamination during simultaneous washing of inoculated and uninoculated potatoes and the effect of water reuse were also assessed. E. coli showed a reduction of 1.86–2.51 log colony‐forming units (CFU)/potato when treated with 40 ppm PAA and 1.98–2.97 log CFU/potato when treated with 80 ppm chlorine dioxide (ClO2), both for 1–5 min. L. monocytogenes showed a reduction of 2.59 log CFU/potato when treated with 120 ppm PAA for 1 min and 2.55 log CFU/potato when treated with 80 ppm ClO2 for 5 min. MNV showed a reduction of 2.28 log CFU/potato when treated with ≥40 ppm PAA for 5 min; however, no significant reduction was noted on treatment with ClO2. During tap water washing, cross‐contamination with E. coli and L. monocytogenes occurred at 3.01 and 3.22 log CFU/potato, respectively; however, MNV was not detected because of its higher LOD. PAA completely inhibited E. coli transfer (below the LOD), whereas ClO2 allowed limited transfer (0.83–1.30 log CFU/potato). However, L. monocytogenes transfer persisted in both treatments (2.74–3.46 log CFU/potato), showing only partial mitigation compared to tap water washing. Reuse of wash water significantly increased cross‐contamination with E. coli and L. monocytogenes (p < 0.05); however, MNV transfer was not observed under any condition. For eradicating other types of microorganisms, the concentration must be determined according to the characteristics of each microorganism.Practical ApplicationsThis study revealed that washing potatoes with disinfectants like PAA or ClO2 can reduce harmful bacteria and viruses. PAA was effective even at concentrations below the regulatory limits. Hence, it may help prevent cross‐contamination during bulk washing processes in food production facilities and kitchen environments.

Inhibitory mechanism of cinnamon, clove, and thyme essential oils against murine norovirus and their anti-noroviral effect in suspension and on food-contact surfaces.

Introduction: Reference tests are crucial for ensuring the comparability and quality assurance of test results in the field of chemical disinfectant testing. For testing virucidal activity, EN 14476:2019 specifies formaldehyde as reference substance. However, due to toxicological and technical concerns, formaldehyde needs to be replaced with new and more suitable reference substances.In order to find the replacement, in 2023-24 an international ring trial was conducted by the European Standardization Committee TC 216 WG 1 (chemical disinfectants and antiseptics, working group human medicine) with 17 participating laboratories. The goal was to evaluate the suitability of these substances for optimization of existing standards.Materials and methods: The study assessed the stability of the test viruses used in the disinfection test – specifically, Modified vaccinia virus Ankara, Adenovirus type 5, Murine Norovirus S99, Minute Virus of Mice, Poliovirus type 1 strain LSc-2ab and Bovine Enterovirus type 1 – to two newly selected reference substances: glutaraldehyde and peracetic acid. The study tested reproducibility and repeatability based on a predefined test protocol. Results: The results of the two tested reference substances – glutaraldehyde and peracetic acid – provide new reference substance ranges for relevant test viruses. The findings are incorporated into the revision of the European standards, ensuring the quality assurance of efficacy testing in virucidal testing.

Noroviruses are the leading cause of gastroenteritis outbreaks in humans worldwide. Their unique properties ensure stability over extended periods under adverse conditions, which enhances their risk as food and water contaminants. In recent years, intensive research has focused on the natural antimicrobial potential of plant metabolites as disinfectants against environmental pathogens. The oregano essential oil (OEO) has gained attention due to its valuable properties, including antimicrobial, antioxidant, antiviral, and antifungal activities. However, the susceptibility of OEO to degradation and oxidation under environmental or storage conditions, coupled with its low water solubility, has limited its practical applications. Nanoencapsulation has emerged as a promising strategy to overcome these limitations by prolonging shelf life, improving stability, enabling controlled release, and expanding its potential uses. In this study, we evaluated the virucidal potential of chitosan-based polymeric nanoparticles incorporating Origanum vulgare essential oil against murine norovirus 1 (MNV-1) for food and environmental applications. To assess the virucidal effect of the OEO nanoparticles, the reduction in viral infectivity was determined by comparing the TCID50/mL values of untreated viral suspensions with those treated with the tested compounds at varying concentrations. The results demonstrated effective viral inactivation at all tested concentrations, with the undiluted formulation (40mg/mL incorporated OEO) achieving the highest inactivation rate (99.72%). The blank formulation showed no significant virucidal activity, while the pure OEO exhibited cytotoxicity at most tested concentrations. These findings support the development of a biotechnological disinfectant with potential applications in both environmental and controlled conditions.

Use of soiled bedding sentinels (SBS) for rodent colony health monitoring is limited by inconsistent pathogen detection, reliance on live animals, high costs, and labor intensity. Sentinel-free soiled bedding (SFSB) offers a viable alternative for all rodent housing systems, overcoming limitations by using PCR testing of matrices exposed to soiled bedding. As an alternative, a matrix may be exposed to all cages via direct colony dredging (DCD). This study compared pathogen detection and costs between SFSB, DCD, and SBS for mice housed in individually-ventilated cage rack system cages. For each study rack, SFSB was performed with one matrix shaken in composite soiled bedding, while DCD was performed with a second matrix exposed to all soiled cages on the rack using a dredging method. We hypothesized that the SFSB and DCD matrices would detect Rodentibacter heylii, Rodentibacter pneumotropicus, Helicobacter typhlonius, Helicobacter mastomyrinus, Helicobacter hepaticus, Helicobacter bilis, Helicobacter rodentium, Helicobacter ganmani, and murine norovirus (MNV) with equal or superior efficacy to SBS, at a comparable or reduced program cost. All SBS failed to detect R. heylii, R. pneumotropicus, H. typhlonius, H. mastomyrinus, H. hepaticus, H. bilis, H. rodentium, and H. ganmani when tested by fecal PCR, and 25% failed to detect MNV when tested via serology. In contrast, SFSB and DCD matrices detected MNV, R. heylii, R. pneumotropicus, H. typhlonius, H. mastomyrinus, H. hepaticus, H. bilis, H. rodentium, and H. ganmani even with low pathogen prevalence, although neither method achieved 100% detection. DCD had negative ergonomic, workflow, and labor challenges compared with SFSB. Overall, SFSB and DCD had reduced costs and superior pathogen detection compared with SBS, while SFSB provided the most efficient and user-friendly approach for health monitoring by this institution.

Beyond cold chain: Exploring bacterial-viral interactions and disinfection strategies for norovirus persistence on organic lettuce under temperature fluctuations.

Health monitoring of rodent colonies has traditionally used live animal (LA) sampling by means such as the use of soiled bedding sentinels (SBS), with the associated expenditure of labor, supplies, and animals. In the spirit of the 3Rs, sentinel-free (SF) approaches are becoming more common. PCR testing of environmental samples is replacing traditional SBS-based testing for routine health monitoring of rodent colonies. Passive sampling of in-cage media exposed to pooled, soiled bedding is effective for detecting some common rodent pathogens. We hypothesized that PCR testing of commercially available media exposed to soiled bedding would be as effective as sampling SBS, or SBS combined with samples from colony animals, for detecting several enzootic organisms of mice (Mus musculus) within our facility. Media were placed in IVC cages and exposed to pooled dirty bedding from all cages on a rack side at biweekly cage changes during a 3-mo period. PCR results of the SF soiled bedding-exposed media were compared with results from feces, pelt, and oral swabs from SBS with and without SBS combined with 10 randomly sampled colony animals from the same rack side over the same period. Detection rates were similar for murine norovirus and Staphylococcus xylosus using SF testing compared with SBS with and without direct colony samples. Five organisms, Proteus mirabilis, Rodentibacter heylii, Staphylococcus aureus, Klebsiella oxytoca, and Klebsiella pneumoniae, were detected by SF testing, but not by LA samples. Demodex musculi, Entamoeba, Proteus mirabilis, Helicobacter spp., and Rodentibacter heylii were detected at significantly higher rates by SF testing compared with SBS with and without colony animal samples. SF testing detected organisms of zoonotic concern (S. aureus, K. pneumoniae) that were undetected by LA testing. SF testing detected organisms at similar rates during 2 consecutive quarters. We conclude that PCR testing of media exposed to pooled soiled bedding effectively detects these common enzootic organisms.

Viruses can rapidly adapt and evolve to new, unfavorable environments due to their decreased replication fidelity, large reproductive index, and short life cycle. Often these adaptations that enable increased fitness in a new, specialized environment comes with a trade-off of decreased fitness in a standard, general environment. Understanding the tradeoffs of generalist and specialist viruses has provided important insight into vaccine development, mechanism of action of antivirals, and function of viral proteins. Here, we sought to identify how a specialist murine norovirus (MNV) could be converted to a generalist without a simple reversion of a genetic mutation. Previously, we found that a mutation in MNV (NS6F182C) overcame restriction by host protein Trim7 but decreased the efficiency of viral polyprotein NS6–7 cleavage and resulted in attenuation of this specialist virus. Here, we find that a single valine-to-isoleucine mutation in MNV non-structural protein NS4 (NS4V11I) is sufficient to rescue the attenuated replication of specialist NS6F182C over multiple cycles of replication. However, NS4V11I did not affect the defective polyprotein cleavage but instead the NS4V11I mutation facilitates faster viral spread in vitro independent of interferon signaling. The emergence of this mutation in NS4V11I suggests an unappreciated connection between NS4 and NS6 during norovirus replication and provides a system to define the unknown role of norovirus NS4 during infection.

Norovirus–bacterial interactions influence viral replication and immune responses, yet the molecular details that mediate binding of these viruses to commensal bacteria are unknown. Studies with other enteric viruses have revealed that LPS and other lipid/carbohydrate structures facilitate virus–bacterial interactions, and it has also been shown that human noroviruses (HuNoVs) can interact with histo-blood group antigen (HBGA)-like compounds on the surface of bacterial cells. Based on these findings, this study hypothesized that carbohydrate-based compounds were the ligands that facilitated binding of both human and murine noroviruses (MNV) to bacteria. Using glycan microarrays, competitive inhibition assays, and a panel of bacterial mutants, the project assessed the influence of specific glycans on viral attachment to bacteria. Protein-based interactions were also examined. The results supported previous work which demonstrated that HuNoVs strongly bind HBGA-like glycans, while MNV displayed distinct binding to other glycans including aminoglycosides and fucosylated structures. Ultimately, this work demonstrates that HuNoVs have more limited binding requirements for bacterial attachment compared to MNV, and the MNV binding to bacteria may involve both specific structures as well as electrostatic interactions. Given the importance of commensal bacteria during viral infection, defining the molecular mechanisms that mediate virus–bacteria interactions is critical for understanding infection dynamics and may be useful in the development of disease therapeutics and novel technologies for viral detection from food and environmental sources.

Fermented foods have been shown to exert positive effects on gut health and immune function. However, the potential of fermented foods to enhance the bioavailability of bioactive compounds and support the growth of the beneficial microbial community’s key factors in antiviral immunity remains less explored. In this review, we show that probiotic-fermented food improves the bioactive compound contents and is increasingly studied by basic and clinical researchers. Bioactive compounds, including phenolic, alkaloids, terpenoids, flavonoids, stilbenes, coumarins, tannins, anthocyanidins, flavones, isoflavonoids, and polyphenols, are increased in the probiotic fermentation conditions. Additionally, beneficial bacteria such as Lactobacilli, Bifidobacteria, Pediococcus, and Weissella are also restored in the fermented foods. These bioactive compounds, combined with a functional microbiota, play a role in preventing viral infections by targeting influenza, noroviruses (NoVs), Murine norovirus-1 (MNV-1), and COVID-19, while also stimulating the immune function of the host. It was suggested that clinical and pre-clinical investigations are required to explore the dose-response and duration efficacy of probiotic fermented foods against viral infections.

Immunohistochemical analysis of the distribution and quantification of internalized Escherichia coli, Listeria monocytogenes, and murine norovirus in intact and damaged potatoes.

Evaluation of nucleic acid extraction methods for recovery of Cyclospora cayetanensis, Salmonella enterica, and murine norovirus from water and sludge.

Viruses encounter a range of selective pressures, but inefficiencies during replication can be masked. To uncover factors that limit viral replication, we used forward genetics to enrich for a murine norovirus (MNV) mutant with faster replication. We sequentially harvested the earliest progeny in cultured cells and identified a single amino acid change in the viral NS3 protein, K40R, that was sufficient to enhance replication speed. We found that the NS3-K40R virus induced earlier cell death and viral egress compared with wild-type virus. Mechanistically, NS3-K40R protein disrupted membranes more efficiently than wild-type NS3 protein, potentially contributing to increased mitochondrial dysfunction and cell death. Immunodeficient mice infected with NS3-K40R virus had increased titers, suggesting that increasing egress did not reduce fitness in vivo. Overall, by using a forward genetic approach, we identified a previously unknown inefficiency in norovirus egress and provide new insights into selective pressures that influence viral replication and evolution.

Standard food detection methods do not distinguish between infectious and non-infectious human norovirus leading to uncertainty in the management of a norovirus positive food sample. These methods also require expensive RT-qPCR-based equipment and reagents. In contrast, CRISPR-based, compared to RT-qPCR-based, detection methods are generally less expensive and yield similar sensitivity and specificity. Our goal was to detect norovirus with an intact capsid, a proxy for infectivity, through a CRISPR-Cas13a-based detection method together with an RNase-capsid integrity assay. We termed this assay: Foodborne RNA-virus Enzymatic Sensing for High-throughput on fresh produce (CRISPR FRESH) reflecting its potential to detect infectious or potentially infectious virus particles. Our CRISPR FRESH method detected murine norovirus (MNV-1), with an intact capsid, at a limit of detection of 2.59 log10 gc/25g (5 gc/rx). This method did not cross-react with other targets (synthetic DNA targets for hepatitis A virus; human norovirus GI, GII; rotavirus). Compared with RT-qPCR, CRISPR FRESH showed an increased sensitivity when detecting low copy numbers of RNase-pre-treated MNV-1 in lettuce and blueberries samples. Viral detection with the RT-qPCR assay is quantifiable while the CRISPR assay is present/absent. This report describes a CRISPR-based detection of potentially infectious viruses in food samples.