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.     

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.     

Lack of correlation between virus barosensitivity and the presence of a viral envelope during inactivation of human rotavirus, vesicular stomatitis virus, and avian metapneumovirus by high-pressure processing

High-pressure processing (HPP) is a nonthermal technology that has been shown to effectively inactivate a wide range of microorganisms. However, the effectiveness of HPP on inactivation of viruses is relatively less well understood. We systematically investigated the effects of intrinsic (pH) and processing (pressure, time, and temperature) parameters on the pressure inactivation of a nonenveloped virus (human rotavirus [HRV]) and two enveloped viruses (vesicular stomatitis virus [VSV] and avian metapneumovirus [aMPV]). We demonstrated that HPP can efficiently inactivate all tested viruses under optimal conditions, although the pressure susceptibilities and the roles of temperature and pH substantially varied among these viruses regardless of the presence of a viral envelope. We found that VSV was much more stable than most food-borne viruses, whereas aMPV was highly susceptible to HPP. When viruses were held for 2 min under 350 MPa at 4°C, 1.1-log, 3.9-log, and 5.0-log virus reductions were achieved for VSV, HRV, and aMPV, respectively. Both VSV and aMPV were more susceptible to HPP at higher temperature and lower pH. In contrast, HRV was more easily inactivated at higher pH, although temperature did not have a significant impact on inactivation. Furthermore, we demonstrated that the damage of virion structure by disruption of the viral envelope and/or capsid is the primary mechanism underlying HPP-induced viral inactivation. In addition, VSV glycoprotein remained antigenic although VSV was completely inactivated. Taken together, our findings suggest that HPP is a promising technology to eliminate viral contaminants in high-risk foods, water, and other fomites.     

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.     

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 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.     

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.     

Grape seed extract for control of human enteric viruses

Grape seed extract (GSE) is reported to have many pharmacological benefits, including antioxidant, anti-inflammatory, anticarcinogenic, and antimicrobial properties. However, the effect of this inexpensive rich source of natural phenolic compounds on human enteric viruses has not been well documented. In the present study, the effect of commercial GSE, Gravinol-S, on the infectivity of human enteric virus surrogates (feline calicivirus, FCV-F9; murine norovirus, MNV-1; and bacteriophage MS2) and hepatitis A virus (HAV; strain HM175) was evaluated. GSE at concentrations of 0.5, 1, and 2 mg/ml was individually mixed with equal volumes of each virus at titers of ～7 log(10) PFU/ml or ～5 log(10) PFU/ml and incubated for 2 h at room temperature or 37°C. The infectivity of the recovered viruses after triplicate treatments was evaluated by standardized plaque assays. At high titers (～7 log(10) PFU/ml), FCV-F9 was significantly reduced by 3.64, 4.10, and 4.61 log(10) PFU/ml; MNV-1 by 0.82, 1.35, and 1.73 log(10) PFU/ml; MS2 by 1.13, 1.43, and 1.60 log(10) PFU/ml; and HAV by 1.81, 2.66, and 3.20 log(10) PFU/ml after treatment at 37°C with 0.25, 0.50, and 1 mg/ml GSE, respectively (P < 0.05) in a dose-dependent manner. GSE treatment of low titers (～5 log(10) PFU/ml) at 37°C also showed viral reductions. Room-temperature treatments with GSE caused significant reduction of the four viruses, with higher reduction for low-titer FCV-F9, MNV-1, and HAV compared to high titers. Our results indicate that GSE shows promise for application in the food industry as an inexpensive novel natural alternative to reduce viral contamination and enhance food safety.     

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.     

Efficacy and Mechanisms of Murine Norovirus Inhibition by Pulsed-Light Technology

Pulsed light is a nonthermal processing technology recognized by the FDA for killing microorganisms on food surfaces, with cumulative fluences up to 12 J cm⁻². In this study, we investigated its efficacy for inactivating murine norovirus 1 (MNV-1) as a human norovirus surrogate in phosphate-buffered saline, hard water, mineral water, turbid water, and sewage treatment effluent and on food contact surfaces, including high-density polyethylene, polyvinyl chloride, and stainless steel, free or in an alginate matrix. The pulsed-light device emitted a broadband spectrum (200 to 1,000 nm) at a fluence of 0.67 J cm⁻² per pulse, with 2% UV at 8 cm beneath the lamp. Reductions in viral infectivity exceeded 3 log₁₀ in less than 3 s (5 pulses; 3.45 J cm⁻²) in clear suspensions and on clean surfaces, even in the presence of alginate, and in 6 s (11 pulses; 7.60 J cm⁻²) on fouled surfaces except for stainless steel (2.6 log₁₀). The presence of protein or bentonite interfered with viral inactivation. Analysis of the morphology, the viral proteins, and the RNA integrity of treated MNV-1 allowed us to elucidate the mechanisms involved in the antiviral activity of pulsed light. Pulsed light appeared to disrupt MNV-1 structure and degrade viral protein and RNA. The results suggest that pulsed-light technology could provide an effective alternative means of inactivating noroviruses in wastewaters, in clear beverages, in drinking water, or on food-handling surfaces in the presence or absence of biofilms.     

High-pressure inactivation of human norovirus virus-like particles provides evidence that the capsid of human norovirus is highly pressure resistant

Human norovirus (NoV) is the leading cause of nonbacterial acute gastroenteritis epidemics worldwide. High-pressure processing (HPP) has been considered a promising nonthermal processing technology to inactivate food- and waterborne viral pathogens. Due to the lack of an effective cell culture method for human NoV, the effectiveness of HPP in inactivating human NoV remains poorly understood. In this study, we evaluated the effectiveness of HPP in disrupting the capsid of human NoV based on the structural and functional integrity of virus-like particles (VLPs) and histo-blood group antigen (HBGA) receptor binding assays. We found that pressurization at 500 to 600 MPa for 2 min, a pressure level that completely inactivates murine norovirus and feline calicivirus, was not sufficient to disrupt the structure and function of human NoV VLPs, even with a holding time of 60 min. Degradation of VLPs increased commensurate with increasing pressure levels more than increasing time. The times required for complete disruption of human NoV VLPs at 700, 800, and 900 MPa were 45, 15, and 2 min, respectively. Human NoV VLPs were more resistant to HPP in their ability to bind type A than type B and O HBGAs. Additionally, the 23-nm VLPs appeared to be much more stable than the 38-nm VLPs. Taken together, our results demonstrated that the human NoV capsid is highly resistant to HPP. While human NoV VLPs may not be fully representative of viable human NoV, destruction of the VLP capsid is highly suggestive of a typical response for viable human NoV.     

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.     

Inactivation of murine norovirus 1, coliphage phiX174, and Bacteroides [corrected] fragilis phage B40-8 on surfaces and fresh-cut iceberg lettuce by hydrogen peroxide and UV light

In this study, the inactivating properties of liquid hydrogen peroxide (L-H(2)O(2)), vaporized hydrogen peroxide (V-H(2)O(2)), UV light, and a combination of V-H(2)O(2) and UV light were tested on murine norovirus 1 (MNV-1) and bacteriophages (φX174 and B40-8) as models for human noroviruses. Disinfection of surfaces was examined on stainless steel discs based on European Standard EN 13697 (2001). For fresh-produce decontamination, a mixture of the viruses was inoculated onto shredded iceberg lettuce and treated after overnight incubation at 2°C. According to our results, L-H(2)O(2) (2.1%) was able to inactivate MNV-1 and φX174 on stainless steel discs by approximately 4 log(10) units within 10 min of exposure, whereas for B40-8, 15% of L-H(2)O(2) was needed to obtain a similar reduction in 10 min. Only a marginal reduction (≤1 log(10) unit after 5 min of exposure) by V-H(2)O(2) (2.52%) was achieved for the tested model viruses, although in combination with UV light, a 4-log(10)-unit decrease within 5 min of treatment was observed on stainless steel discs. Similar trends were observed for the decontamination of shredded iceberg lettuce, but the viral decline was reduced. These results demonstrated that both L-H(2)O(2) and a combination of V-H(2)O(2) and UV light can be used for norovirus inactivation on surfaces; V-H(2)O(2) (2.52%) in combination with UV light is promising for decontamination of fresh produce with much less consumption of water and disinfectant.     

Thermal Inactivation of Enteric Viruses and Bioaccumulation of Enteric Foodborne Viruses in Live Oysters (Crassostrea virginica)

Human enteric viruses are among the main causative agents of shellfish-associated outbreaks. In this study, the kinetics of viral bioaccumulation in live oysters and the heat stabilities of the predominant enteric viruses were determined both in tissue culture and in oyster tissues. A human norovirus (HuNoV) GII.4 strain, HuNoV surrogates (murine norovirus [MNV-1], Tulane virus [TV]), hepatitis A virus (HAV), and human rotavirus (RV) bioaccumulated to high titers within oyster tissues, with different patterns of bioaccumulation for the different viruses. We tested the thermal stability of each virus at 62, 72, and 80°C in culture medium. The viruses can be ranked from the most heat resistant to the least stable as follows: HAV, RV, TV, MNV-1. In addition, we found that oyster tissues provided protection to the viruses during heat treatment. To decipher the mechanism underlying viral inactivation by heat, purified TV was treated at 80°C for increasing time intervals. It was found that the integrity of the viral capsid was disrupted, whereas viral genomic RNA remained intact. Interestingly, heat treatment leading to complete loss of TV infectivity was not sufficient to completely disrupt the receptor binding activity of TV, as determined by the porcine gastric mucin–magnetic bead binding assay. Similarly, HuNoV virus-like particles (VLPs) and a HuNoV GII.4 strain retained some receptor binding ability following heat treatment. Although foodborne viruses have variable heat stability, 80°C for >6 min was sufficient to completely inactivate enteric viruses in oysters, with the exception of HAV.     

Survival of viruses on fresh produce, using MS2 as a surrogate for norovirus

AIMS: To study the survival and removal of viruses from fresh fruit and vegetables using the bacteriophage MS2 as a potential surrogate for noroviruses. METHOD AND RESULTS: Survival of MS2 in buffer and on fresh produce was studied at 4, 8 and 22 degrees C. At 4 and 8 degrees C a reduction of <1 log10 was observed after 50 days in buffer; however a reduction in excess of 1 log10 occurred within 9 days at 22 degrees C. Similar results were obtained with fresh produce with virus survival times exceeding the shelf life of the produce. In washing experiments, using a chlorine wash (100 ppm), in all but one case <1.5 log10 MS2 bacteriophage was removed from fruit and vegetables. The mean across all produce types was 0.89 log10. With potable water, reduction was lower (0.3 log mean across all produce types). CONCLUSIONS: MS2 survived for prolonged periods, both in buffer and on fresh produce, at temperatures relevant to chilled foods. It was not removed effectively by chlorine washing. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophage MS2 has been evaluated as a potential surrogate for noroviruses on fresh produce. Experimental results together with current knowledge of norovirus resistance and survival indicate that MS2 could be used as an effective surrogate in future evaluations.