Inactivation of a norovirus by high-pressure processing

Murine norovirus (strain MNV-1), a propagable norovirus, was evaluated for susceptibility to high-pressure processing. Experiments with virus stocks in Dulbecco's modified Eagle medium demonstrated that at room temperature (20 degrees C) the virus was inactivated over a pressure range of 350 to 450 MPa, with a 5-min, 450-MPa treatment being sufficient to inactivate 6.85 log(10) PFU of MNV-1. The inactivation of MNV-1 was enhanced when pressure was applied at an initial temperature of 5 degrees C; a 5-min pressure treatment of 350 MPa at 30 degrees C inactivated 1.15 log(10) PFU of virus, while the same treatment at 5 degrees C resulted in a reduction of 5.56 log(10) PFU. Evaluation of virus inactivation as a function of treatment times ranging from 0 to 150 s and 0 to 900 s at 5 degrees C and 20 degrees C, respectively, indicated that a decreasing rate of inactivation with time was consistent with Weibull or log-logistic inactivation kinetics. The inactivation of MNV-1 directly within oyster tissues was demonstrated; a 5-min, 400-MPa treatment at 5 degrees C was sufficient to inactivate 4.05 log(10) PFU. This work is the first demonstration that norovirus can be inactivated by high pressure and suggests good prospects for inactivation of nonpropagable human norovirus strains in foods.     

Inactivation of a human norovirus surrogate by high-pressure processing: effectiveness, mechanism, and potential application in the fresh produce industry

Fresh produce is often a high-risk food for norovirus contamination because it can become contaminated at both preharvest and postharvest stages and it undergoes minimal or no processing. Currently, there is no effective method to eliminate the viruses from fresh produce. This study systematically investigated the effectiveness of high-pressure processing (HPP) on inactivating murine norovirus (MNV-1), a surrogate for human norovirus, in aqueous medium and fresh produce. We demonstrated that MNV-1 was effectively inactivated by HPP. More than a 5-log-PFU/g reduction was achieved in all tested fresh produce when it was pressurized at 400 MPa for 2 min at 4°C. We found that pressure, pH, temperature, and food matrix affected the virus survival in foods. MNV-1 was more effectively inactivated at 4°C than at 20°C in both medium and fresh produce. MNV-1 was also more sensitive to HPP at neutral pH than at acidic pH. We further demonstrated that disruption of viral capsid structure, but not degradation of viral genomic RNA, is the primary mechanism of virus inactivation by HPP. However, HPP does not degrade viral capsid protein, and the pressurized capsid protein was still antigenic. Overall, HPP had a variable effect on the sensorial quality of fresh produce, depending on the pressure level and type of product. Taken together, HPP effectively inactivated a human norovirus surrogate in fresh produce with a minimal impact on food quality and thus can provide a novel intervention for processing fruits intended for frozen storage and related products such as purees, sauces, and juices.     

Inactivation of a Norovirus by High-Pressure Processing

Murine norovirus (strain MNV-1), a propagable norovirus, was evaluated for susceptibility to high-pressure processing. Experiments with virus stocks in Dulbecco's modified Eagle medium demonstrated that at room temperature (20°C) the virus was inactivated over a pressure range of 350 to 450 MPa, with a 5-min, 450-MPa treatment being sufficient to inactivate 6.85 log₁₀ PFU of MNV-1. The inactivation of MNV-1 was enhanced when pressure was applied at an initial temperature of 5°C; a 5-min pressure treatment of 350 MPa at 30°C inactivated 1.15 log₁₀ PFU of virus, while the same treatment at 5°C resulted in a reduction of 5.56 log₁₀ PFU. Evaluation of virus inactivation as a function of treatment times ranging from 0 to 150 s and 0 to 900 s at 5°C and 20°C, respectively, indicated that a decreasing rate of inactivation with time was consistent with Weibull or log-logistic inactivation kinetics. The inactivation of MNV-1 directly within oyster tissues was demonstrated; a 5-min, 400-MPa treatment at 5°C was sufficient to inactivate 4.05 log₁₀ PFU. This work is the first demonstration that norovirus can be inactivated by high pressure and suggests good prospects for inactivation of nonpropagable human norovirus strains in foods.     

Inactivation of a human norovirus surrogate, human norovirus virus-like particles, and vesicular stomatitis virus by gamma irradiation

Gamma irradiation is a nonthermal processing technology that has been used for the preservation of a variety of food products. This technology has been shown to effectively inactivate bacterial pathogens. Currently, the FDA has approved doses of up to 4.0 kGy to control food-borne pathogens in fresh iceberg lettuce and spinach. However, whether this dose range effectively inactivates food-borne viruses is less understood. We have performed a systematic study on the inactivation of a human norovirus surrogate (murine norovirus 1 [MNV-1]), human norovirus virus-like particles (VLPs), and vesicular stomatitis virus (VSV) by gamma irradiation. We demonstrated that MNV-1 and human norovirus VLPs were resistant to gamma irradiation. For MNV-1, only a 1.7- to 2.4-log virus reduction in fresh produce at the dose of 5.6 kGy was observed. However, VSV was more susceptible to gamma irradiation, and a 3.3-log virus reduction at a dose of 5.6 kGy in Dulbecco's modified Eagle medium (DMEM) was achieved. We further demonstrated that gamma irradiation disrupted virion structure and degraded viral proteins and genomic RNA, which resulted in virus inactivation. Using human norovirus VLPs as a model, we provide the first evidence that the capsid of human norovirus has stability similar to that of MNV-1 after exposure to gamma irradiation. Overall, our results suggest that viruses are much more resistant to irradiation than bacterial pathogens. Although gamma irradiation used to eliminate the virus contaminants in fresh produce by the FDA-approved irradiation dose limits seems impractical, this technology may be practical to inactivate viruses for other purposes, such as sterilization of medical equipment.     

Enhanced removal of a human norovirus surrogate from fresh vegetables and fruits by a combination of surfactants and sanitizers

Fruits and vegetables are major vehicles for transmission of food-borne enteric viruses since they are easily contaminated at pre- and postharvest stages and they undergo little or no processing. However, commonly used sanitizers are relatively ineffective for removing human norovirus surrogates from fresh produce. In this study, we systematically evaluated the effectiveness of surfactants on removal of a human norovirus surrogate, murine norovirus 1 (MNV-1), from fresh produce. We showed that a panel of surfactants, including sodium dodecyl sulfate (SDS), Nonidet P-40 (NP-40), Triton X-100, and polysorbates, significantly enhanced the removal of viruses from fresh fruits and vegetables. While tap water alone and chlorine solution (200 ppm) gave only <1.2-log reductions in virus titer in all fresh produce, a solution containing 50 ppm of surfactant was able to achieve a 3-log reduction in virus titer in strawberries and an approximately 2-log reduction in virus titer in lettuce, cabbage, and raspberries. Moreover, a reduction of approximately 3 logs was observed in all the tested fresh produce after sanitization with a solution containing a combination of 50 ppm of each surfactant and 200 ppm of chlorine. Taken together, our results demonstrate that the combination of a surfactant with a commonly used sanitizer enhanced the efficiency in removing viruses from fresh produce by approximately 100 times. Since SDS is an FDA-approved food additive and polysorbates are recognized by the FDA as GRAS (generally recognized as safe) products, implementation of this novel sanitization strategy would be a feasible approach for efficient reduction of the virus load in fresh produce.     

Inactivation of Murine Norovirus on a Range of Copper Alloy Surfaces Is Accompanied by Loss of Capsid Integrity

Norovirus is one of the most common causes of acute viral gastroenteritis. The virus is spread via the fecal-oral route, most commonly from infected food and water, but several outbreaks have originated from contamination of surfaces with infectious virus. In this study, a close surrogate of human norovirus causing gastrointestinal disease in mice, murine norovirus type 1 (MNV-1), retained infectivity for more than 2 weeks following contact with a range of surface materials, including Teflon (polytetrafluoroethylene [PTFE]), polyvinyl chloride (PVC), ceramic tiles, glass, silicone rubber, and stainless steel. Persistence was slightly prolonged on ceramic surfaces. A previous study in our laboratory observed that dry copper and copper alloy surfaces rapidly inactivated MNV-1 and destroyed the viral genome. In this new study, we have observed that a relatively small change in the percentage of copper, between 70 and 80% in copper nickels and 60 and 70% in brasses, had a significant influence on the ability of the alloy to inactivate norovirus. Nickel alone did not affect virus, but zinc did have some antiviral effect, which was synergistic with copper and resulted in an increased efficacy of brasses with lower percentages of copper. Electron microscopy of purified MNV-1 that had been exposed to copper and stainless steel surfaces suggested that a massive breakdown of the viral capsid had occurred on copper. In addition, MNV-1 that had been exposed to copper and treated with RNase demonstrated a reduction in viral gene copy number. This suggests that capsid integrity is compromised upon contact with copper, allowing copper ion access to the viral genome.     

Inactivation Kinetics and Mechanism of a Human Norovirus Surrogate on Stainless Steel Coupons via Chlorine Dioxide Gas

Acute gastroenteritis caused by human norovirus is a significant public health issue. Fresh produce and seafood are examples of high-risk foods associated with norovirus outbreaks. Food contact surfaces also have the potential to harbor noroviruses if exposed to fecal contamination, aerosolized vomitus, or infected food handlers. Currently, there is no effective measure to decontaminate norovirus on food contact surfaces. Chlorine dioxide (ClO₂) gas is a strong oxidizer and is used as a decontaminating agent in food processing plants. The objective of this study was to determine the kinetics and mechanism of ClO₂ gas inactivation of a norovirus surrogate, murine norovirus 1 (MNV-1), on stainless steel (SS) coupons. MNV-1 was inoculated on SS coupons at the concentration of 10⁷ PFU/coupon. The samples were treated with ClO₂ gas at 1, 1.5, 2, 2.5, and 4 mg/liter for up to 5 min at 25°C and a relative humidity of 85%, and virus survival was determined by plaque assay. Treatment of the SS coupons with ClO₂ gas at 2 mg/liter for 5 min and 2.5 mg/liter for 2 min resulted in at least a 3-log reduction in MNV-1, while no infectious virus was recovered at a concentration of 4 mg/liter even within 1 min of treatment. Furthermore, it was found that the mechanism of ClO₂ gas inactivation included degradation of viral protein, disruption of viral structure, and degradation of viral genomic RNA. In conclusion, treatment with ClO₂ gas can serve as an effective method to inactivate a human norovirus surrogate on SS contact surfaces.     

Mouse norovirus 1 utilizes the cytoskeleton network to establish localization of the replication complex proximal to the microtubule organizing center

Human noroviruses (family Caliciviridae) are the leading cause of nonbacterial gastroenteritis worldwide. Although Human noroviruses are significant enteric pathogens, there exists no reliable vaccine or therapy to treat infected individuals. To date, attempts to cultivate Human noroviruses within the laboratory have met with little success; however, the related murine norovirus mouse norovirus 1 (MNV-1) has provided an ideal model system to study norovirus replication due to the ease with which the virus is cultivated and the ability to infect a small animal model with this virus. Previously we have identified the association between MNV-1 and components of the host secretory pathway and proposed a role for the viral open reading frame 1 proteins in the replication cycle. Here we describe for the first time a role for cytoskeletal components in early MNV-1 replication events. We show that the MNV-1 utilizes microtubules to position the replication complex adjacent to the microtubule organizing center. Chemical disruption of the microtubule network disperses the sites of MNV-1 replication throughout the cell and impairs production of viral protein and infectious virus. Furthermore, we demonstrate the ability of MNV-1 to redistribute acetylated tubulin to the replication complex and that this association is potentially mediated via the MNV-1 major structural protein, VP1. Transient expression of MNV-1 VP1 exhibited extensive colocalization with both α-tubulin and acetylated tubulin and was observed to alter the distribution of acetylated tubulin in transfected cells. This study highlights the role of the cytoskeleton in early virus replication events and demonstrates the importance of this interaction in establishing the intracellular location of MNV-1 replication complexes.     

Effect of food residues on norovirus survival on stainless steel surfaces

Background

In households and food processing plants, minute food residues left behind from improper cleaning may influence the survivability of human norovirus on surfaces. In this study, the survivability of norovirus on desiccated food residue-attached stainless steel coupons was investigated.

Methodology/Principal Findings

Using murine norovirus-1 (MNV-1) as a surrogate of human norovirus, the survivability of norovirus was investigated on lettuce, cabbage, or ground pork-attached stainless steel coupons. A 6.2 log MPN/ml of MNV-1 infectivity was completely lost at day 30 in residue-free coupons, whereas only a 1.4 log MPN/ml reduction was observed in coupons with residues. Moreover, the disinfective effect of sodium hypochlorite was reduced when residues were present on the coupons.

Conclusions/Significance

This study revealed that the food residues increased the survivability and the resistance to chemicals of norovirus, indicating the need of thorough cleaning in food processing plants and household settings.     

A single-amino-acid substitution in the P2 domain of VP1 of murine norovirus is sufficient for escape from antibody neutralization

Noroviruses cause epidemic outbreaks of acute viral gastroenteritis worldwide, and the number of reported outbreaks is increasing. Human norovirus strains do not grow in cell culture. However, murine norovirus (MNV) replicates in the RAW 264.7 macrophage cell line and thus provides a tractable model to investigate norovirus interactions with host cells. Epitopes recognized by monoclonal antibodies (MAbs) against the human norovirus strains Norwalk virus and Snow Mountain virus (SMV) identified regions in the P domain of major capsid protein VP1 important for interactions with putative cellular receptors. To determine if there was a relationship between domains of MNV VP1 and VP1 of human norovirus strains involved in cell binding, epitope mapping by phage display was performed with an MNV-1-neutralizing MAb, A6.2.1. A consensus peptide, GWWEDHGQL, was derived from 20 third-round phage clones. A synthetic peptide containing this sequence and constrained through a disulfide linkage reacted strongly with the A6.2.1 MAb, whereas the linear sequence did not. Four residues in the A6.2.1-selected peptide, G327, G333, Q334, and L335, aligned with amino acid residues in the P2 domain of MNV-1 VP1. This sequence is immediately adjacent to the epitope recognized by anti-SMV MAb 61.21. Neutralization escape mutants selected with MAb A6.2.1 contained a leucine-to-phenylalanine substitution at position 386 in the P2 domain. The predicted location of these residues on VP1 suggests that the phage peptide and the mutation in the neutralization-resistant viruses may be in close proximity to each other and to residues reported to be important for carbohydrate binding to VP1 of human norovirus strains.     

Efficacies of Sodium Hypochlorite and Quaternary Ammonium Sanitizers for Reduction of Norovirus and Selected Bacteria during Ware-Washing Operations

Cross-contamination of ready-to-eat (RTE) foods with pathogens on contaminated tableware and food preparation utensils is an important factor associated with foodborne illnesses. To prevent this, restaurants and food service establishments are required to achieve a minimum microbial reduction of 5 logs from these surfaces. This study evaluated the sanitization efficacies of ware-washing protocols (manual and mechanical) used in restaurants to clean tableware items. Ceramic plates, drinking glasses and stainless steel forks were used as the food contact surfaces. These were contaminated with cream cheese and reduced-fat milk inoculated with murine norovirus (MNV-1), Escherichia coli K-12 and Listeria innocua. The sanitizing solutions tested were sodium hypochlorite (chlorine), quaternary ammonium (QAC) and tap water (control). During the study, the survivability and response to the experimental conditions of the bacterial species was compared with that of MNV-1. The results showed that current ware-washing protocols used to remove bacteria from tableware items were not sufficient to achieve a 5 log reduction in MNV-1 titer. After washing, a maximum of 3 log reduction in the virus were obtained. It was concluded that MNV-1 appeared to be more resistant to both the washing process and the sanitizers when compared with E. coli K-12 and L. innocua.     

Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages

Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-αβ receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology.     

Replication of Norovirus in Cell Culture Reveals a Tropism for Dendritic Cells and Macrophages

Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-αβ receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology.     

Membrane reactor coupled with electrophoresis for enzymatic production of aspartic acid

Aspartic acid production by aspartase reaction on ammonium fumarate was carried out in a membrane reactor coupled with electrophoresis. A pressurized, stirred vessel attached with an ultrafiltration membrane was used as a membrane reactor. An electric field was applied across the membrane to preferentially remove the product aspartate from the reactor into the permeate stream. The charged molecule, aspartate, is much smaller than the molecular-weight cutoff of the membrane (10(4)) so that the ions would move freely through pores of the membrane. The concentration of aspartate in the permeate stream is determined by the electromigration velocity of the ions and the permeation rate of solvent (water) through the membrane. The permeation rate of solvent could be controlled by the applied pressure, and the migration velocity of the ions could be controlled by the electric field strength applied. The equilibrium conversion of ammonium fumarate to the aspartate was 70%. In the presence of electric field, the aspartase activity was not disturbed. Also, it is shown that the aspartate concentration in the permeate stream was 20% higher than that in the reaction solution with the permeate flow rate of 0.7 mL/min. The steady-state conversion was 60%. Instead of aspartate, aspartic acid can be recovered directly from the permeate stream by controlling the circulation of buffer electrolyte in the anode compartment.     

Murine noroviruses bind glycolipid and glycoprotein attachment receptors in a strain-dependent manner

Human norovirus infections are the most common cause of acute nonbacterial gastroenteritis in humans worldwide, and glycan binding plays an important role in the susceptibility to these infections. However, due to the lack of an efficient cell culture system or small animal model for human noroviruses, little is known about the biological role of glycan binding during infection. Murine noroviruses (MNV) are also enteric viruses that bind to cell surface glycans, but in contrast to their human counterparts, they can be grown in tissue culture and a small animal host. In this study, we determined glycan-binding specificities of the MNV strains MNV-1 and CR3 in vitro, identified molecular determinants of glycan binding, and analyzed infection in vivo. We showed that unlike MNV-1, CR3 binding to murine macrophages was resistant to neuraminidase treatment and glycosphingolipid depletion. Both strains depended on N-linked glycoproteins for binding, while only MNV-1 attachment to macrophages was sensitive to O-linked glycoprotein depletion. In vivo, CR3 showed differences in tissue tropism compared to MNV-1 by replicating in the large intestine. Mapping of a glycan-binding site in the MNV-1 capsid by reverse genetics identified a region topologically similar to the histo-blood group antigen (HBGA)-binding sites of the human norovirus strain VA387. The recombinant virus showed distinct changes in tissue tropism compared to wild-type virus. Taken together, our data demonstrate that MNV strains evolved multiple strategies to bind different glycan receptors on the surface of murine macrophages and that glycan binding contributes to tissue tropism in vivo.     

Murine norovirus 1 infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by STAT1-dependent interferon responses

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

Murine norovirus, a recently discovered and highly prevalent viral agent of mice

Murine norovirus (MNV), a recently discovered viral agent of laboratory mice, is closely related to human norovirus, a contagious pathogen known to cause gastroenteritis. The prototype strain of MNV (MNV-1) was first isolated and characterized in 2003 as a sporadic, lethal pathogen in certain strains of immunocompromised knockout mice. Serological surveillance data from mouse colonies throughout the US and Canada have since shown that MNV is highly prevalent. Because MNV is unique among norovirus strains in its ability to replicate in cell culture, it serves as the most accessible model to elucidate the mechanisms of infection and replication of human norovirus. The author discusses the genetic diversity of MNV, its prevalence, pathology and potential research implications, as well as techniques for detection and eradication of this virus.     

Effects of Abiotic and Biotic Stresses on the Internalization and Dissemination of Human Norovirus Surrogates in Growing Romaine Lettuce

Human norovirus (NoV) is the major causative agent of fresh-produce-related outbreaks of gastroenteritis; however, the ecology and persistence of human NoV in produce systems are poorly understood. In this study, the effects of abiotic and biotic stresses on the internalization and dissemination of two human NoV surrogates (murine norovirus 1 [MNV-1] and Tulane virus [TV]) in romaine lettuce were determined. To induce abiotic stress, romaine lettuce was grown under drought and flood conditions that mimic extreme weather events, followed by inoculation of soil with MNV-1 or TV. Independently, lettuce plants were infected with lettuce mosaic virus (LMV) to induce biotic stress, followed by inoculation with TV. Plants were grown for 14 days, and viral titers in harvested tissues were determined by plaque assays. It was found that drought stress significantly decreased the rates of both MNV-1 and TV internalization and dissemination. In contrast, neither flood stress nor biotic stress significantly impacted viral internalization or dissemination. Additionally, the rates of TV internalization and dissemination in soil-grown lettuce were significantly higher than those for MNV-1. Collectively, these results demonstrated that (i) human NoV surrogates can be internalized via roots and disseminated to shoots and leaves of romaine lettuce grown in soil, (ii) abiotic stress (drought) but not biotic stress (LMV infection) affects the rates of viral internalization and dissemination, and (iii) the type of virus affects the efficiency of internalization and dissemination. This study also highlights the need to develop effective measures to eliminate internalized viruses in fresh produce.     

MDA-5 recognition of a murine norovirus

Noroviruses are important human pathogens responsible for most cases of viral epidemic gastroenteritis worldwide. Murine norovirus-1 (MNV-1) is one of several murine noroviruses isolated from research mouse facilities and has been used as a model of human norovirus infection. MNV-1 infection has been shown to require components of innate and adaptive immunity for clearance; however, the initial host protein that recognizes MNV-1 infection is unknown. Because noroviruses are RNA viruses, we investigated whether MDA5 and TLR3, cellular sensors that recognize dsRNA, are important for the host response to MNV-1. We demonstrate that MDA5-/- dendritic cells(DC) have a defect in cytokine response to MNV-1. In addition, MNV-1 replicates to higher levels in MDA5-/- DCs as well as in MDA5-/- mice in vivo. Interestingly, TLR3-/- DCs do not have a defect in vitro, but TLR3-/- mice have a slight increase in viral titers. This is the first demonstration of an innate immune sensor for norovirus and shows that MDA5 is required for the control of MNV-1 infection. Knowledge of the host response to MNV-1 may provide keys for prevention and treatment of the human disease.