Effect of Temperature and Relative Humidity on the Survival of Foodborne Viruses during Food Storage

Millions of people suffer from foodborne diseases throughout the world every year, and the importance of food safety has grown worldwide in recent years. The aim of this study was to investigate the survival of hepatitis A virus (HAV) and viral surrogates of human norovirus (HuNoV) (bacteriophage MS2 and murine norovirus [MNV]) in food over time. HAV, MNV, and MS2 were inoculated onto either the digestive gland of oysters or the surface of fresh peppers, and their survival on these food matrices was measured under various temperature (4°C, 15°C, 25°C, and 40°C) and relative humidity (RH) (50% and 70%) conditions. Inoculated viruses were recovered from food samples and quantified by a plaque assay at predetermined time points over 2 weeks (0, 1, 3, 7, 10, and 14 days). Virus survival was influenced primarily by temperature. On peppers at 40°C and at 50% RH, >4- and 6-log reductions of MNV and HAV, respectively, occurred within 1 day. All three viruses survived better on oysters. In addition, HAV survived better at 70% RH than at 50% RH. The survival data for HAV, MS2, and MNV were fit to three different mathematical models (linear, Weibull, and biphasic models). Among them, the biphasic model was optimum in terms of goodness of fit. The results of this study suggest that major foodborne viruses such as HAV and HuNoV can survive over prolonged periods of time with a limited reduction in numbers. Because a persistence of foodborne virus on contaminated foods was observed, precautionary preventive measures should be performed.     

A Single-Amino-Acid Change in Murine Norovirus NS1/2 Is Sufficient for Colonic Tropism and Persistence

Human norovirus (HuNoV) is the major cause of acute nonbacterial gastroenteritis worldwide but has no clear animal reservoir. HuNoV can persist after the resolution of symptoms, and this persistence may be essential for viral maintenance within the population. Many strains of the related murine norovirus (MNV) also persist, providing a tractable animal model for studying norovirus (NoV) persistence. We have used recombinant cDNA clones of representative persistent (CR6) and nonpersistent (CW3) strains to identify a domain within the nonstructural gene NS1/2 that is necessary and sufficient for persistence. Furthermore, we found that a single change of aspartic acid to glutamic acid in CW3 NS1/2 was sufficient for persistence. This same conservative change also caused increased growth of CW3 in the proximal colon, which we found to be a major tissue reservoir of MNV persistence, suggesting that NS1/2 determines viral tropism that is necessary for persistence. These findings represent the first identified function for NoV NS1/2 during infection and establish a novel model system for the study of enteric viral persistence.     

Survival of Murine Norovirus,Tulane Virus,and Hepatitis A Virus on Alfalfa Seeds and Sprouts during Storage and Germination

Human norovirus (huNoV) and hepatitis A virus (HAV) have been involved in several produce-associated outbreaks and identified as major food-borne viral etiologies. In this study, the survival of huNoV surrogates (murine norovirus [MNV] and Tulane virus [TV]) and HAV was investigated on alfalfa seeds during storage and postgermination. Alfalfa seeds were inoculated with MNV, TV, or HAV with titers of 6.46 ± 0.06 log PFU/g, 3.87 ± 0.38 log PFU/g, or 7.01 ± 0.07 log 50% tissue culture infectious doses (TCID₅₀)/g, respectively. Inoculated seeds were stored for up to 50 days at 22°C and sampled during that storage period on days 0, 2, 5, 10, and 15. Following storage, virus presence was monitored over a 1-week germination period. Viruses remained infectious after 50 days, with titers of 1.61 ± 0.19 log PFU/g, 0.85 ± 0.21 log PFU/g, and 3.43 ± 0.21 log TCID₅₀/g for MNV, TV, and HAV, respectively. HAV demonstrated greater persistence than MNV and TV, without a statistically significant reduction over 20 days (<1 log TCID₅₀/g); however, relatively high levels of genomic copies of all viruses persisted over the testing time period. Low titers of viruses were found on sprouts and were located in all tissues as well as in sprout-spent water sampled on days 1, 3, and 6 following seed planting. Results revealed the persistence of viruses in seeds for a prolonged period of time, and perhaps of greater importance these data suggest the ease of which virus may transfer from seeds to sprouts and spent water during germination. These findings highlight the importance of sanitation and prevention procedures before and during germination.     

Feline Calicivirus,Murine Norovirus,Porcine Sapovirus,and Tulane Virus Survival on Postharvest Lettuce

Human norovirus (HuNoV) is the leading cause of foodborne illnesses, with an increasing number of outbreaks associated with leafy greens. Because HuNoV cannot be routinely cultured, culturable feline calicivirus (FCV), murine norovirus (MNV), porcine sapovirus (SaV), and Tulane virus (TV) have been used as surrogates. These viruses are generated in different cell lines as infected cell lysates, which may differentially affect their stability. Our objective was to uniformly compare the survival of these viruses on postharvest lettuce while evaluating the effects of cell lysates on their survival. Viruses were semipurified from cell lysates by ultrafiltration or ultracentrifugation followed by resuspension in sterile water. Virus survival was examined before and after semipurification: in suspension at room temperature (RT) until day 28 and on lettuce leaves stored at RT for 3 days or at 4°C for 7 and 14 days. In suspension, both methods significantly enhanced the survival of all viruses. On lettuce, the survival of MNV in cell lysates was similar to that in water, under all storage conditions. In contrast, the survival of FCV, SaV, and TV was differentially enhanced, under different storage conditions, by removing cell lysates. Following semipurification, viruses showed similar persistence to each other on lettuce stored under all conditions, with the exception of ultracentrifugation-purified FCV, which showed a higher inactivation rate than MNV at 4°C for 14 days. In conclusion, the presence of cell lysates in viral suspensions underestimated the survivability of these surrogate viruses, while viral semipurification revealed similar survivabilities on postharvest lettuce leaves.     

Protruding domain of capsid protein is necessary and sufficient to determine murine norovirus replication and pathogenesis in vivo

Human noroviruses (HuNoVs) are the major cause of epidemic, nonbacterial gastroenteritis worldwide. Due to the lack of a tractable model system and the inability to grow HuNoVs in cell culture, factors required for the norovirus (NoV) life cycle and pathogenesis in the host remain largely unknown. The discovery of murine norovirus (MNV) and the development of reverse-genetics systems for this virus provide an opportunity to study these aspects of NoV infection in vitro and in vivo. Previous studies identified a single amino acid at residue 296 in the protruding (P) domain of the capsid protein that is responsible for determining the virulence of the MNV clone MNV1.CW1 in 12956/SvEv background STAT1-deficient (STAT1(-/-)) mice. In this report, we identified and characterized another determinant of lethality in the P domain that is necessary and sufficient to determine the replication and pathogenesis of the MNV clones MNV1.CW3 and CR6.STL1 in C57BL/6 background STAT1(-/-) mice. Furthermore, we describe how the role of residue 296 in MNV virulence differs between STAT1(-/-) mouse strains. We also describe potential interactions between subdomains of the P domain, as well as between other virus elements, which facilitate recovery of MNV using a reverse-genetics system.     

Evaluation of Murine Norovirus, Feline Calicivirus, Poliovirus, and MS2 as Surrogates for Human Norovirus in a Model of Viral Persistence in Surface Water and Groundwater

Human noroviruses (NoVs) are a significant cause of nonbacterial gastroenteritis worldwide, with contaminated drinking water a potential transmission route. The absence of a cell culture infectivity model for NoV necessitates the use of molecular methods and/or viral surrogate models amenable to cell culture to predict NoV inactivation. The NoV surrogates murine NoV (MNV), feline calicivirus (FCV), poliovirus (PV), and male-specific coliphage MS2, in conjunction with Norwalk virus (NV), were spiked into surface water samples (n = 9) and groundwater samples (n = 6). Viral persistence was monitored at 25°C and 4°C by periodically analyzing virus infectivity (for all surrogate viruses) and nucleic acid (NA) for all tested viruses. FCV infectivity reduction rates were significantly higher than those of the other surrogate viruses. Infectivity reduction rates were significantly higher than NA reduction rates at 25°C (0.18 and 0.09 log₁₀/day for FCV, 0.13 and 0.10 log₁₀/day for PV, 0.12 and 0.06 log₁₀/day for MS2, and 0.09 and 0.05 log₁₀/day for MNV) but not significant at 4°C. According to a multiple linear regression model, the NV NA reduction rates (0.04 ± 0.01 log₁₀/day) were not significantly different from the NA reduction rates of MS2 (0.05 ± 0.03 log₁₀/day) and MNV (0.04 ± 0.03 log₁₀/day) and were significantly different from those of FCV (0.08 ± 0.03 log₁₀/day) and PV (0.09 ± 0.03 log₁₀/day) at 25°C. In conclusion, MNV shows great promise as a human NoV surrogate due to its genetic similarity and environmental stability. FCV was much less stable and thus questionable as an adequate surrogate for human NoVs in surface water and groundwater.     

Inactivation of murine norovirus, feline calicivirus and echovirus 12 as surrogates for human norovirus (NoV) and coliphage (F+) MS2 by ultraviolet light (254 nm) and the effect of cell association on UV inactivation

Aims: To determine inactivation profiles of three human norovirus (NoV) surrogate viruses and coliphage MS2 by ultraviolet (UV) irradiation and the protective effect of cell association on UV inactivation. Methods and Results: The inactivation rate for cell‐free virus or intracellular echovirus 12 was determined by exposure to 254‐nm UV light at fluence up to 100 mJ cm^?2. The infectivity of murine norovirus (MNV), feline calicivirus (FCV) and echovirus 12 was determined by cell culture infectivity in susceptible host cell lines, and MS2 infectivity was plaque assayed on Escherichia coli host cells. The UV fluencies to achieve 4‐log₁₀ inactivation were 25, 29, 30 and 70 (mJ cm^?2) for cell‐free FCV, MNV, echovirus 12 and MS2, respectively. However, a UV fluence of 85 mJ cm^?2 was needed to inactivate intracellular echovirus 12 by 4 log₁₀. Conclusions: Murine norovirus and echoviruses 12 are more conservative surrogates than FCV to predict the UV inactivation response of human NoV. Intracellular echovirus 12 was 2·8‐fold more resistant to UV irradiation than cell‐free one. Significance and Impact of the Study: Variation in UV susceptibilities among NoV surrogate viruses and a likely protective effect of cell association on virus susceptibility to UV irradiation should be considered for effective control of human NoV in water.     

Year-round prevalence of norovirus in the environment of catering companies without a recently reported outbreak of gastroenteritis

Food handlers play an important role in the transmission of norovirus (NoV) in food-borne outbreaks of gastroenteritis (GE). In a year-round prevalence study, the prevalence of NoV in catering companies without recently reported outbreaks of GE was investigated and compared to the observed prevalence in catering companies with recently reported outbreaks. Swab samples were collected from surfaces in the kitchens and (staff) bathrooms in 832 randomly chosen companies and analyzed for the presence of NoV RNA. In total, 42 (1.7%) out of 2,496 environmental swabs from 35 (4.2%) catering companies tested positive. In contrast, NoV was detected in 147 (39.7%) of the 370 samples for 44 (61.1%) of the 72 establishments associated with outbreaks of gastroenteritis. NoV-positive swabs were more frequently found in winter, in specific types of companies (elderly homes and lunchrooms), and in establishments with separate bathrooms for staff. We found a borderline association with population density but no relation to the number of employees. Sequence analysis showed that environmental strains were interspersed with strains found in outbreaks of illness in humans. Thus, the presence of NoV in catering companies seemed to mirror the presence in the population but was strongly increased when associated with food-borne GE. Swabs may therefore serve as a valuable tool in outbreak investigations for the identification of the causative agent, although results should be interpreted with care, taking into account all other epidemiological data.     

(E)-2-Styrylchromones as potential anti-norovirus agents

Human noroviruses (NoV) are now recognized as the most frequent cause of outbreaks and sporadic cases of acute gastroenteritis. Despite the significant economic impact and considerable morbidity of norovirus disease, no drug or vaccine is currently available to treat or prevent this disease, therefore the discovery of anti-norovirus drugs is urgent.In the present work, a total of 12 structure related chromone and (E)-2-styrylchromones were evaluated for their potential anti-norovirus activity using the murine norovirus (MNV) as a surrogate model for human NoV.From the 12 compounds studied, six (E)-2-styrylchromones were found to have with interesting anti-norovirus activity. The best compounds of the series were (E)-5-hydroxy-2-styrylchromone and (E)-4′-methoxy-2-styrylchromone with an IC₅₀ ≈ 7 μM. A first insight into the mechanism of action of these compounds was possible. An interesting relationship between the anti-norovirus activity and the chemical structure was observed. The present study points out that the (E)-2-styrylchromones skeleton is an important one which deserves to be developed and further explored as new antiviral drugs against NoV.     

Concurrent outbreak of norovirus genotype I and enterotoxigenic Escherichia coli on a U.S. Navy ship following a visit to Lima, Peru

An outbreak of norovirus (NoV) genotype I and Enterotoxigenic Escherichia coli (ETEC) occurred among US Navy Ship personnel following a visit to Lima, Peru, in June 2008. Visiting a specific area in Lima was significantly associated with illness. While ETEC and NoV are commonly recognized as causative agents of outbreaks, co-circulation of both pathogens has been rarely observed in shipboard outbreaks.     

Susceptibility of Murine Norovirus and Hepatitis A Virus to Electron Beam Irradiation in Oysters and Quantifying the Reduction in Potential Infection Risks

Consumption of raw oysters is an exposure route for human norovirus (NoV) and hepatitis A virus (HAV). Therefore, efficient postharvest oyster treatment technology is needed to reduce public health risks. This study evaluated the inactivation of HAV and the NoV research surrogate, murine norovirus-1 (MNV-1), in oysters (Crassostrea virginica) by electron beam (E-beam) irradiation. The reduction of potential infection risks was quantified for E-beam irradiation technology employed on raw oysters at various virus contamination levels. The E-beam dose required to reduce the MNV and HAV titer by 90% (D₁₀ value) in whole oysters was 4.05 (standard deviations [SD], ±0.63) and 4.83 (SD, ±0.08) kGy, respectively. Microbial risk assessment suggests that if a typical serving of 12 raw oysters was contaminated with 10⁵ PFU, a 5-kGy treatment would achieve a 12% reduction (from 4.49 out of 10 persons to 3.95 out of 10 persons) in NoV infection and a 16% reduction (from 9.21 out of 10 persons to 7.76 out of 10 persons) in HAV infections. If the serving size contained only 10² PFU of viruses, a 5-kGy treatment would achieve a 26% reduction (2.74 out of 10 persons to 2.03 out of 10 persons) of NoV and 91% reduction (2.1 out of 10 persons to 1.93 out of 100 persons) of HAV infection risks. This study shows that although E-beam processing cannot completely eliminate the risk of viral illness, infection risks can be reduced.     

VPg-primed RNA synthesis of norovirus RNA-dependent RNA polymerases by using a novel cell-based assay

Molecular studies of human noroviruses (NoV) have been hampered by the lack of a permissive cell culture system. We have developed a sensitive and reliable mammalian cell-based assay for the human NoV GII.4 strain RNA-dependent RNA polymerase (RdRp). The assay is based on the finding that RNAs synthesized by transiently expressed RdRp can stimulate retinoic acid-inducible gene I (RIG-I)-dependent reporter luciferase production via the beta interferon promoter. Comparable activities were observed for the murine norovirus (MNV) RdRp. RdRps with mutations at divalent metal ion binding residues did not activate RIG-I signaling. Furthermore, both NoV and MNV RdRp activities were stimulated by the coexpression of their respective VPg proteins, while mutations in the putative site of nucleotide linkage on VPg abolished most of their stimulatory effects. Sequencing of the RNAs linked to VPg revealed that the cellular trans-Golgi network protein 2 (TGOLN2) mRNA was the template for VPg-primed RNA synthesis. Small interfering RNA knockdown of RNase L abolished the enhancement of signaling that occurred in the presence of VPg. Finally, the coexpression of each of the other NoV proteins revealed that p48 (also known as NS1-2) and VP1 enhanced and that VP2 reduced the RdRp activity. The assay should be useful for the dissection of the requirements for NoV RNA synthesis as well as the identification of inhibitors of the NoV RdRp.     

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.     

Abiotic Stress and Phyllosphere Bacteria Influence the Survival of Human Norovirus and Its Surrogates on Preharvest Leafy Greens

Foodborne outbreaks of human noroviruses (HuNoVs) are frequently associated with leafy greens. Because there is no effective method to eliminate HuNoV from postharvest leafy greens, understanding virus survival under preharvest conditions is crucial. The objective of this study was to evaluate the survival of HuNoV and its surrogate viruses, murine norovirus (MNV), porcine sapovirus (SaV), and Tulane virus (TV), on preharvest lettuce and spinach that were subjected to abiotic stress (physical damage, heat, or flood). We also examined the bacteria culturable from the phyllosphere in response to abiotic stress and in relation to viral persistence. Mature plants were subjected to stressors 2 days prior to inoculation of the viruses on leaves. We quantified the viral RNA, determined the infectivity of the surrogates, and performed bacterial counts on postinoculation days (PIDs) 0, 1, 7, and 14. For both plant types, time exerted significant effects on HuNoV, MNV, SaV, and TV RNA titers, with greater effects being seen for the surrogates. Infectious surrogate viruses were undetectable on PID 14. Only physical damage on PID 14 significantly enhanced HuNoV RNA persistence on lettuce, while the three stressors differentially enhanced the persistence of MNV and TV RNA. Bacterial counts were significantly affected by time and plant type but not by the stressors. However, bacterial counts correlated significantly with HuNoV RNA titers on spinach and with the presence of surrogate viruses on both plant types under various conditions. In conclusion, abiotic stressors and phyllosphere bacterial density may differentially influence the survival of HuNoV and its surrogates on lettuce and spinach, emphasizing the need for the use of preventive measures at the preharvest stage.