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The zebrafish larvae/embryo model has been shown to support the replication of seven strains (G1.7[P7], GII.2[P16], GII.3[P16], GII.4[P4], GII.4[P16], GII.6[P7], and GII.17[P13]) of human norovirus (HuNoV). However, due to challenges in consistently obtaining HuNoV-positive stool samples from clinical sources, evaluating HuNoV surrogates in this model is highly valuable. This study assesses the potential of zebrafish embryos and larvae as a model for Tulane virus (TuV) replication. Three infection methods were examined: microinjection, immersion, and feeding. Droplet digital PCR was used to quantify viral RNA across all three infection methods. Microinjection of 3 nL of TuV into zebrafish embryos (< 6-h post-fertilization) resulted in significant replication, with viral RNA levels reaching 6.22 logs at 4-day post-infection. In contrast, the immersion method showed no replication after immersing 4-day post-fertilization (dpf) larvae in TuV suspension for 6 h. Similarly, no replication was observed with the feeding method, where Paramecium caudatum loaded with TuV were fed to 4 dpf larvae. The findings indicate that the zebrafish embryo model supports TuV replication through the microinjection method, suggesting that TuV may serve as a useful surrogate for studying HuNoV pathogenesis. Additionally, TuV can be utilized in place of HuNoV in method optimization studies using the zebrafish embryo model, circumventing the limited availability of HuNoV.

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Carpet cleaning guidelines currently do not include the use of an antimicrobial, except after a bodily fluid event. To address this gap, we compared the efficacy of three antimicrobials-two hydrogen peroxide-based (H2O2) products (A and B) and one chlorine-based product (C)-and a steam treatment against two norovirus surrogates, specifically feline calicivirus (FCV) and Tulane virus (TuV). These tests were performed on nylon carpets with either water-permeable or waterproof backing types. The effect of repeated antimicrobial use on carpet properties was also evaluated. For a carpet with water-permeable backing, products A, B, and C achieved a 0.8, 3.1, and 0.9 log10 PFU/coupon reduction of FCV and 0.3, 2.5, and 0.4 log10 TCID50/coupon reduction of TuV, respectively, following a 30 min contact time. For carpet with waterproof backing, only product B achieved a 5.0 log10 PFU/coupon reduction of FCV and >3.0 log10 TCID50/coupon reduction of TuV, whereas products A and C achieved a 2.4 and 1.6 log10 PFU/coupon reduction of FCV and a 1.2 and 1.2 log10 TCID50/coupon reduction of TuV, respectively. Steam treatment achieved a ≥ 5.2 log10 PFU/coupon reduction of FCV and a > 3.2 log10 TCID50/coupon reduction of TuV in 15 seconds on the carpet with both backing types. The repeated use of products A and B decreased the tensile strength of the carpet backing, while use of product B resulted in cracks on carpet fibers. Overall, steam treatment for 15 seconds was efficacious on both carpet types, but only product B achieved efficacy after a 30-minute exposure on the carpet with waterproof backing.IMPORTANCECarpets are common in long-term care facilities, despite its potential as a vehicle for transmission of agents associated with healthcare-associated infections, including human norovirus (NoV). Presently, our understanding of carpet disinfection is limited; hence, there are no commercial antimicrobials against norovirus available for use on carpets. Our findings showed that steam treatment, which minimally affected the properties of carpet fibers and backing, was more efficacious against human norovirus surrogates on carpets compared to the three chemical antimicrobials tested. Additionally, the two surrogates were more sensitive to chemical antimicrobials on the carpet with waterproof backing compared to carpets with water-permeable backing. These findings can inform development of antimicrobials for use on carpets contaminated with human norovirus.

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Human noroviruses are major causes of foodborne outbreaks linked to berries. The overall goal of this study was to investigate the persistence of a human norovirus surrogate, Tulane virus (TV), in berry smoothies and under simulated digestion through the gastrointestinal track. Two types of smoothies were prepared from blueberries and strawberries. Tulane virus was spiked into each smoothie and incubated either at 37 or 4 °C for 2, 60, and 120 min. Furthermore, the virus-spiked smoothies were subjected to sequential oral (2 min), gastric (10 and 60 min), and intestinal (15 and 120 min) digestion according to the standardized INFOGEST model. Quantification of infectious TV was carried out using the TCID50 assay. At 4 °C, in both berry smoothies, TV infectivity did not show significant changes throughout the 120 min period. At 37 °C, TV infectivity showed significant reduction (~0.5 log TCID50/mL) only in blueberry smoothies starting at 60 min. During the oral, gastric, and intestinal digestion phases, the mean log reduction in TV infectivity in blueberry did not exceed ~0.5 log, while infectious TV in strawberry smoothies under all phases was stable. Given the notable stability of infectious viruses in berry smoothies and the gastrointestinal tract, prevention of norovirus contamination of berries is paramount to reduce virus outbreaks linked to berries.

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Human norovirus (HuNoV) is regularly involved in food-borne infections. To detect infectious HuNoV in food, RT-qPCR remains state of the art but also amplifies non-infectious virus. The present study combines pre-treatments, RNase and propidium monoazide, with three molecular analyses, including long-range PCR, to predominantly detect infectious Tulane virus (TuV), a culturable HuNoV surrogate. TuV was exposed to inactivating conditions to assess which molecular method most closely approximates the reduction in infectious virus determined by cell culture (TCID50). After thermal treatments (56 °C/5 min, 70 °C/5 min, 72 °C/20 min), TCID50 reductions of 0.3, 4.4 and 5.9 log10 were observed. UV exposure (40/100/1000 mJ/cm2) resulted in 1.1, 2.5 and 5.9 log10 reductions. Chlorine (45/100 mg/L for 1 h) reduced infectious TuV by 2.0 and 3.0 log10. After thermal inactivation standard RT-qPCR, especially with pre-treatments, showed the smallest deviation from TCID50. On average, RT-qPCR with pre-treatments deviated by 1.1-1.3 log10 from TCID50. For UV light, long-range PCR was closest to TCID50 results. Long-range reductions deviated from TCID50 by ≤0.1 log10 for mild and medium UV-conditions. However, long-range analyses often resulted in qPCR non-detects. At higher UV doses, RT-qPCR with pre-treatments differed by ≤1.0 log10 from TCID50. After chlorination the molecular methods repeatedly deviated from TCID50 by >1.0 log10, Overall, each method needs to be further optimized for the individual types of inactivation treatment.

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Contaminated fomites can lead to hepatitis A virus (HAV) and human norovirus (HuNoV) disease outbreaks. Improved decontamination methods that are user-friendly, cost-effective, and waterless are being researched for sustainability. Traditional ultraviolet light (UV-C) technologies though effective for surface decontamination have drawbacks, using mercury lamps, that pose user-safety risk and environmental hazards. Therefore, UV-C light emitting diode (LED) systems are being designed for delivering required antiviral doses. The objective of this research was to determine the ability of UV-C LED (279 nm) systems to inactivate HuNoV surrogates, feline calicivirus (FCV-F9) and Tulane virus (TV), and HAV on Formica coupons in comparison to UV-C (254 nm) systems. FCV-F9 (∼6 log PFU/mL), TV (∼7 log PFU/mL), or HAV (∼6 log PFU/mL) at 100 µL were surface-spread on sterile Formica coupons (3 × 3 cm2), air-dried, and treated for up to 2.5 min with both systems. Each experiment was replicated thrice. Recovered infectious plaque counts were statistically analyzed using mixed model analysis of variance. FCV-F9, TV, and HAV showed D10 values of 23.37 ± 0.91 mJ/cm2, 16.32 ± 3.6 mJ/cm2, and 12.39 ± 0.70 mJ/cm2 using 279 nm UV-C LED, respectively and D10 values of 9.97 ± 2.44 mJ/cm2, 6.83 ± 1.13 mJ/cm2 and 12.40 ± 1.15 mJ/cm2, respectively with 254 nm UV-C. Higher 279 nm UV-C LED doses were required to cause HuNoV surrogate reduction than 254 nm UV-C, except similar doses with both systems were needed for HAV inactivation on Formica surfaces. It remains critical to measure UV intensity of optical sources and optimize exposure times for desired log reduction on surfaces.

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Human noroviruses (HuNoVs) are a major cause of acute gastroenteritis, contributing significantly to annual foodborne illness cases. However, studying these viruses has been challenging due to limitations in tissue culture techniques for over four decades. Tulane virus (TV) has emerged as a crucial surrogate for HuNoVs due to its close resemblance in amino acid composition and the availability of a robust cell culture system. Initially isolated from rhesus macaques in 2008, TV represents a novel Calicivirus belonging to the Recovirus genus. Its significance lies in sharing the same host cell receptor, histo-blood group antigen (HBGA), as HuNoVs. In this study, we introduce, through cryo-electron microscopy (cryo-EM), the structure of a specific TV variant (the 9-6-17 TV) that has notably lost its ability to bind to its receptor, B-type HBGA-a finding confirmed using an enzyme-linked immunosorbent assay (ELISA). These results offer a profound insight into the genetic modifications occurring in TV that are necessary for adaptation to cell culture environments. This research significantly contributes to advancing our understanding of the genetic changes that are pivotal to successful adaptation, shedding light on fundamental aspects of Calicivirus evolution.

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In the field of chemical engineering and water treatment, the study of viruses, included surrogates, is well documented. Often, surrogates are used to study viruses and their behavior because they can be produced in larger quantities in safer conditions and are easier to handle. In fact, surrogates allow studying microorganisms which are non-infectious to humans but share some properties similar to pathogenic viruses: structure, composition, morphology, and size. Human noroviruses, recognized as the leading cause of epidemics and sporadic cases of gastroenteritis across all age groups, may be mimicked by the Tulane virus. The objectives of this work were to study (i) the ultrafiltration of Tulane virus and norovirus to validate that Tulane virus can be used as a surrogate for norovirus in water treatment process and (ii) the retention of norovirus and the surrogate as a function of water quality to better understand the use of the latter pathogenic viruses. Ultrafiltration tests showed significant logarithmic reduction values (LRV) in viral RNA: around 2.5 for global LRV (i.e., based on the initial and permeate average concentrations) and between 2 and 6 for average LRV (i.e., retention rate considering the increase of viral concentration in the retentate), both for norovirus and the surrogate Tulane virus. Higher reduction rates (from 2 to 6 log genome copies) are obtained for higher initial concentrations (from 101 to 107 genome copies per mL) due to virus aggregation in membrane lumen. Tulane virus appears to be a good surrogate for norovirus retention by membrane processes.

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Ongoing challenges with reproducible human norovirus cultivable assays necessitate the use of surrogates, such as feline calicivirus (FCV-F9) and Tulane virus (TV), during inactivation studies. Chlorine alternates used as control strategies include aqueous and gaseous ozone. This study aimed at determining the inactivation of FCV-F9 and TV by a portable ozone-generating device. FCV-F9 (∼8 log PFU/mL) or TV (∼6 log PFU/mL) in sterile-low-organic matter-containing-water was treated for 0-5 min, or in sterile-water containing newborn calf serum (high-organic matter/protein) for 0-38 min with ∼1 ppm ozone (pH 7-6). Infectivity was determined from triplicate treatments using plaque assays. FCV-F9 titers significantly decreased by 6.07 log PFU/mL after 5 min in ozonated low-organic-matter-containing-water and was non-detectable (≤2 log PFU/mL) after 36 min treatments in high-organic-matter-containing water (p < 0.05). TV titers decreased by 4.18 log PFU/mL after 4 min in ozonated low-organic-matter water (non-detectable after 4.5 min) and were non-detectable after 22.5 min treatments of high-organic-matter-containing water (p < 0.05). Overall, ∼1 ppm aqueous ozone significantly decreased FCV-F9 by >6 log PFU/mL after 5 min, TV to non-detectable levels (≤2 log PFU/mL) after 4.5 min and required longer treatments (>32 and >20 min, respectively) for ≥4 log reduction in high-organic-matter-containing water (p < 0.05). For ozone treatment of both viruses, the linear and Weibull models were similar for low-organic-load water, though the Weibull model was better for the high-organic load water. Prior filtration or organic load removal is recommended before ozonation for increased viral inactivation with decreased treatment-time.

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Contamination of berries and leafy greens with human norovirus (HuNoV) is a major cause of outbreaks of epidemic gastroenteritis worldwide. Using murine norovirus type 1 (MNV-1) and Tulane virus, we studied the possible extension of HuNoV persistence by biofilm-producing epiphytic bacteria on fresh produce. Nine bacterial species frequently found on the surface of berries and leafy greens (Bacillus cereus, Enterobacter cloacae, Escherichia coli, Kocuria kristinae, Lactobacillus plantarum, Pantoea agglomerans, Pseudomonas fluorescens, Raoultella terrigena, and Xanthomonas campestris) were evaluated for the ability to form biofilms in the MBEC Assay Biofilm Inoculator and in 96-well microplates. The biofilm-forming bacteria were further tested for binding MNV-1 and Tulane virus and the ability to protect them against loss of capsid integrity upon exposure to disinfecting pulsed light at a fluence of 11.52 J/cm2. Based on viral reductions, MNV-1 did not benefit from attachment to biofilm whereas Tulane virus was significantly more resistant than the control when attached to biofilms of E. cloacae (P ≤ 0.01), E. coli (P ≤ 0.01), K. kristinae (P ≤ 0.01), P. agglomerans (P ≤ 0.05), or P. fluorescens (P ≤ 0.0001). Enzymatic dispersion of biofilm and microscopic observations suggest that the biofilm matrix composition may contribute to the virus resistance. Our results indicate that direct virus-biofilm interaction protects Tulane virus against disinfecting pulsed light, and that HuNoV on fresh produce therefore might resist such treatment more than suggested by laboratory tests so far. IMPORTANCE Recent studies have shown that bacteria may be involved in the attachment of HuNoV to the surface of fresh produce. Because these foods are difficult to disinfect by conventional methods without compromising product quality, nonthermal nonchemical disinfectants such as pulsed light are being investigated. We seek to understand how HuNoV interacts with epiphytic bacteria, particularly with biofilms formed by bacterial epiphytes, with cells and extracellular polymeric substances, and to determine if it thus escapes inactivation by pulsed light. The results of this study should advance understanding of the effects of epiphytic biofilms on the persistence of HuNoV particle integrity after pulsed light treatment and thus guide the design of novel pathogen control strategies in the food industry.

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Introduction: The impact of water quality on the survival of human norovirus (NoV) was determined in irrigation water field run-off (tail water) and well water from a representative Central Coast vegetable production site in the Salinas Valley, California. Methods: Tail water, well water, and ultrapure water samples were inoculated separately with two surrogate viruses for human NoV-Tulane virus (TV) and murine norovirus (MNV)-to achieve a titer of 1×105 plaque forming units (PFU)/ml. Samples were stored at 11, 19, and 24°C for 28 days. Additionally, inoculated water was applied to soil collected from a vegetable production site in the Salinas Valley or to the surface of growing romaine lettuce leaves, and virus infectivity was evaluated for 28 days in a growth chamber. Results: Virus survival was similar for water stored at 11, 19, and 24°C and there was no difference in infectivity based on water quality. After 28 days, a maximum 1.5 log reduction was observed for both TV and MNV. TV decreased by 1.97-2.26 log and MNV decreased by 1.28- 1.48 logs after 28 days in soil; infectivity was not influenced by water type. Infectious TV and MNV were recovered from lettuce surfaces for up to 7 and 10 days after inoculation, respectively. Across the experiments there was no significant impact of water quality on the stability of the human NoV surrogates. Discussion: Overall, the human NoV surrogates were highly stable in water with a less than 1.5 log reduction over 28 days and no difference observed based on the water quality. In soil, the titer of TV declined by approximately 2 logs over 28 days, while MNV declined by 1 log during the same time interval, suggesting surrogate-specific inactivation dynamics in the soil tested in this study. A 5-log reduction in MNV (day 10 post inoculation) and TV (day 14 post inoculation) was observed on lettuce leaves, and the inactivation kinetics were not significantly impacted by the quality of water used. These results suggest that human NoV would be highly stable in water, and the quality of the water (e.g., nutrient content, salinity, and turbidity) does not significantly impact viral infectivity.

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Human norovirus (HuNoV) has been implicated as the leading cause of foodborne illness worldwide. The ability of HuNoV to persist in water can significantly impact food safety as agriculture and processing water could serve as vehicles of virus transmission. This study focused on the persistence and infectivity of the HuNoV surrogate viruses, murine norovirus (MNV), and Tulane virus (TV), after prolonged storage in diverse environmental water types currently used for agricultural irrigation. In this study, vegetable processing water (VW), brackish tidal surface water (SW), municipal reclaimed water (RW), and pond water (PW) were inoculated with each virus in a 1:10 v/v ratio containing virus at 3.5-4.5 logPFU/mL and stored at 16°C for 100 days. This time and temperature combination was chosen to mimic growing and harvest conditions in the mid-Atlantic area of the United States. Samples were then assayed for the presence of viral RNA using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) approximately weekly throughout the study. Persistence of MNV and TV was not significantly different (p > 0.05) from one another in any water sample (n = 7) or the control (HBSS). However, there was variability observed in viral persistence across water samples with significant differences observed between several water samples. The presence of intact viral capsids enclosing the genomes of MNV and TV were evaluated by an RNase assay coupled with RT-qPCR on specific timepoints and determined to be intact up to and at 100 days after inoculation. TV was also shown to remain infectious in a cell culture assay (TCID50) up to 100 days of incubation. These findings are significant in that the potential for not only detection of enteric viruses can occur long after a contamination event occurs but these viruses may also remain infectious.

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Raw oysters are considered a culinary delicacy but are frequently the culprit in food-borne norovirus (NoV) infections. As commercial depuration procedures are currently unable to efficiently eliminate NoV from oysters, an optimisation of the process should be considered. This study addresses the ability of elevated water temperatures to enhance the elimination of NoV and Tulane virus (TuV) from Pacific oysters (Crassostrea gigas). Both viruses were experimentally bioaccumulated in oysters, which were thereafter depurated at 12 °C and 17 °C for 4 weeks. Infectious TuV and viral RNA were monitored weekly for 28 days by TCID50 and (PMAxx-) RT-qPCR, respectively. TuV RNA was more persistent than NoV and decreased by < 0.5 log10 after 14 days, while NoV reductions were already > 1.0 log10 at this time. For RT-qPCR there was no detectable benefit of elevated water temperatures or PMAxx for either virus (p > 0.05). TuV TCID50 decreased steadily, and reductions were significantly different between the two temperatures (p < 0.001). This was most evident on days 14 and 21 when reductions at 17 °C were 1.3-1.7 log10 higher than at 12 °C. After 3 weeks, reductions > 3.0 log10 were observed at 17 °C, while at 12 °C reductions did not exceed 1.9 log10. The length of depuration also had an influence on virus numbers. TuV reductions increased from < 1.0 log10 after seven days to > 4.0 log10 after 4 weeks. This implies that an extension of the depuration period to more than seven days, possibly in combination with elevated water temperatures, may be beneficial for the inactivation and removal of viral pathogens.

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With the widespread availability of 3D food printing systems for purchase, users can customize their food in new ways. Manufacturer recommendations for cleaning these machines remain untested with regard to the prevention of foodborne pathogen transmission. This study aimed to determine if manufacturer cleaning recommendations for food ink capsules utilized in 3D food printers are adequate to control human norovirus (HuNoV). A HuNoV surrogate, Tulane virus (TuV; ~ 6 log10 PFU/mL), was inoculated onto the interior surface of stainless steel food ink capsules. Capsules were either unsoiled or soiled with one of the following: butter, protein powder solution, powdered sugar solution, or a mixture containing all three food components. The capsules were allowed to dry and then one of three hygienic protocols was applied: manual washing (MW), a dishwasher speed cycle (DSC), or a dishwasher heavy cycle (DHC). The interaction effect between DSC and pure butter was a significant predictor of log reduction (P = 0.0067), with the pure butter and DSC combination achieving an estimated mean log reduction of 4.83 (95% CI 4.13, 5.59). The DSC was the least effective method of cleaning when compared with MW and the DHC. The 3-way interaction effects between wash type, soil, and capsule position were a significant predictor of log reduction (P = 0.00341). Capsules with butter in the DSC achieved an estimated mean log reduction of 2.81 (95% CI 2.80, 2.83) for the front-most position versus 6.35 (95% CI 6.33, 6.37) for the back-most position. Soil matrix, cleaning protocol, and capsule position all significantly impact capsule cleanability and potential food safety risk. The DHC is recommended for all capsules, and the corners should be avoided when placing capsules into the dishwasher. The current study seeks to provide recommendations for users of additive manufacturing and 3D food printing including consumers, restaurants, industry, and regulatory industries.

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Human norovirus (HuNoV) is a pathogenic agent that is frequently associated with foodborne disease outbreaks linked to fresh produce. Within microgreen production systems, understanding of virus transmission routes and persistence is limited. To investigate virus persistence on microgreen leaf surfaces, this study mimicked virus contaminations caused during microgreen handling by farm workers or during overhead irrigation with contaminated water. Specifically, approximately 5 log PFU of Tulane virus (TV)-a HuNoV surrogate-was inoculated on sunflower (SF) and pea shoot (PS) microgreen leaves at 7-day age. The virus reduction on SF was significantly higher than PS (p < 0.05). On day 10, total TV reduction for SF and PS were 3.70 ± 0.10 and 2.52 ± 0.30 log PFU/plant, respectively. Under the environmental scanning electron microscope (ESEM) observation, the leaf surfaces of SF were visually smoother than PS, while their specific effect on virus persistence were not further characterized. Overall, this study revealed that TV persistence on microgreen leaves was plant variety dependent. In addition, this study provided a preliminary estimation on the risk of HuNoV contamination in a microgreen production system which will aim the future development of prevention and control measures.

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The inactivation efficacy by monochloramine for disinfecting gastroenteritis-causing rotaviruses (RV) and Tulane viruses (TV), a surrogate for noroviruses, were evaluated in this study. In addition, the strategies for improving the disinfection efficiency of monochloramine by raising the temperature and sequentially implementing UV irradiation were investigated. The results showed that monochloramine was more effective in the inactivation of TV than RV. Additionally, the inactivation rate constants of RV and TV by monochloramine at 35 °C were improved approximately by 46% and 100%, respectively, compared to those at 25 °C. Moreover, applying UV irradiation before monochloramine enhanced the inactivation efficacy of RV and TV by 63% and 72% compared to monochloramine alone (UV: 6 mJ/cm2, NH2Cl: 60 ppm × min). Furthermore, the synergistic effect was observed during the RV inactivation by the sequential process. Especially, higher than 0.5 log10 reductions of RV VP1 genome contributed to the synergistic effect in sequential treatment, while less than 0.1 log10 reductions of RV VP1 genome were observed during UV alone (13 mJ/cm2) or monochloramine alone (94 ppm × min). The genome damage might be the primary mechanism of generating synergy in sequential treatment for the inactivation of RV. By comparison, no synergistic effect was discovered for the inactivation of TV due to high susceptibility to monochloramine and UV. The findings on the inactivation efficacy and mechanism for improvement will contribute to a wide application of monochloramine for virus inactivation in water treatment and distribution systems.

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Commonly used surface sanitizers often lack activity against human noroviruses (hNoVs). The impact of inactivation versus removal when these products are applied via wiping is poorly characterized. The purpose of this work was to assess the anti-hNoV efficacy of various surface sanitizer chemistries, as applied to a laminate material commonly used for restaurant tabletops, using standard surface assays (ASTM E1053-11) and a newly developed wiping protocol. Four commercially available products with different active ingredient(s) (i.e., ethanol [EtOH], acid + anionic surfactant [AAS], quaternary ammonium compound [QAC], and sodium hypochlorite [NaOCl]) and a water control were evaluated against hNoV GII.4 Sydney, hNoV GI.6, and the cultivable surrogate Tulane virus (TuV). Virus concentration was evaluated using RNase-reverse transcriptase (RT)-quantitative PCR (qPCR) (hNoV) and infectivity assay (TuV). Only the EtOH-based product significantly reduced virus concentration (>3.5 log10 reduction [LR]) by surface assay, with all other products producing ≤0.5 LR. The inclusion of a wiping step enhanced the efficacy of all products, producing complete virus elimination for the EtOH-based product and 1.6 to 3.8 LR for the other chemistries. For hNoVs, no detectable residual virus could be recovered from paper towels used to wipe the EtOH-based product, while high concentrations of virus could be recovered from the used paper towel and the wiped coupon (1.5 to 2.5 log10 lower genome equivalent copies [GEC] compared to control) for the QAC- and AAS-based products and for water. These results illustrate the variability in anti-hNoV activity of representative surface sanitizers and highlights the value of wiping, the efficacy of which appears to be driven by a combination of virus inactivation and removal. IMPORTANCE Human noroviruses (hNoVs) are the leading cause of acute gastroenteritis and food-borne disease worldwide. Noroviruses are difficult to inactivate, being recalcitrant to sanitizers and disinfectants commonly used by the retail food sector. This comparative study demonstrates the variability in anti-hNoV activity of representative surface sanitizers, even those allowed to make label claims based on the cultivable surrogate, feline calicivirus (FCV). It also highlights the importance of wiping in the process of sanitization, which significantly improves product efficacy through the action of physical removal of surface microbes. There is a need for more and better product formulations with demonstrated efficacy against hNoVs, which will likely necessitate the use of alternative cultivable surrogates, such as Tulane virus (TuV). These findings help food safety professionals make informed decisions on sanitizing product selection and application methods in order to reduce the risk of hNoV contamination and transmission in their facilities.

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ABSTRACT: Environmental monitoring (EM) programs are designed to detect the presence of pathogens in food manufacturing environments, with the goal of preventing microbial contamination of food. Nevertheless, limited knowledge exists regarding the influence of environmental conditions on microbial recovery during EM. This study uses a commercially available polyurethane foam EM tool to determine the influence of environmental factors on the recovery of foodborne pathogens. The specific objectives of this study were to determine if environmental conditions and surface composition impact the recovery of sought-after microorganisms found in food processing environments. These data are compared across (i) microorganism type, (ii) surface type, (iii) environmental temperature and relative humidity (RH), and (iv) exposure time. Two bacteria (Listeria monocytogenes and Salmonella Typhimurium) and one human norovirus surrogate (Tulane virus) were inoculated onto three nonporous surfaces (polypropylene, stainless steel, and neoprene). Surfaces were held in an environmental chamber for 24 or 72 h at 30°C with 30% RH, 6°C with 85% RH, and 30°C with 85% RH. Data indicate that microbial recovery from environmental surfaces significantly (P ≤ 0.05) varies by microorganism type, environmental conditions, and exposure time. For instance, all microorganisms were significantly different from each other, with the greatest mean log reduction being Tulane virus and the lesser reduction being L. monocytogenes at 4.94 ± 1.75 log PFU per surface and 2.54 ± 0.91 log CFU per surface, respectively. Overall, these data can be used to improve the effectiveness of EM programs and underscores the need to better comprehend how EM test results are impacted by food manufacturing environmental conditions.

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Proper disinfection of harvested food and water is critical to minimize infectious disease. Grape seed extract (GSE), a commonly used health supplement, is a mixture of plant-derived polyphenols. Polyphenols possess antimicrobial and antifungal properties, but antiviral effects are not well-known. Here we show that GSE outperformed chemical disinfectants (e.g., free chlorine and peracetic acids) in inactivating Tulane virus, a human norovirus surrogate. GSE induced virus aggregation, a process that correlated with a decrease in virus titers. This aggregation and disinfection were not reversible. Molecular docking simulations indicate that polyphenols potentially formed hydrogen bonds and strong hydrophobic interactions with specific residues in viral capsid proteins. Together, these data suggest that polyphenols physically associate with viral capsid proteins to aggregate viruses as a means to inhibit virus entry into the host cell. Plant-based polyphenols like GSE are an attractive alternative to chemical disinfectants to remove infectious viruses from water or food. IMPORTANCE Human noroviruses are major food- and waterborne pathogens, causing approximately 20% of all cases of acute gastroenteritis cases in developing and developed countries. Proper sanitation or disinfection are critical strategies to minimize human norovirus-caused disease until a reliable vaccine is created. Grape seed extract (GSE) is a mixture of plant-derived polyphenols used as a health supplement. Polyphenols are known for antimicrobial, antifungal, and antibiofilm activities, but antiviral effects are not well-known. In studies presented here, plant-derived polyphenols outperformed chemical disinfectants (i.e., free chlorine and peracetic acids) in inactivating Tulane virus, a human norovirus surrogate. Based on data from molecular assays and molecular docking simulations, the current model is that the polyphenols in GSE bind to the Tulane virus capsid, an event that triggers virion aggregation. It is thought that this aggregation prevents Tulane virus from entering host cells.

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The antinoroviral effect of copper ions is well known, yet most of this work has previously been conducted in copper and copper alloy surfaces, not copper ions in solution. In this work, we characterized the effects that Cu ions have on human norovirus capsids' and surrogates' integrity to explain empirical data, indicating virus inactivation by copper alloy surfaces, and as means of developing novel metal ion-based virucides. Comparatively high concentrations of Cu(II) ions (>10 mM) had little effect on the infectivity of human norovirus surrogates, so we used sodium ascorbate as a reducing agent to generate unstable Cu(I) ions from solutions of copper bromide. We found that significantly lower concentrations of monovalent copper ions (∼0.1 mM) compared to divalent copper ions cause capsid protein damage that prevents human norovirus capsids from binding to cell receptors in vitro and induce a greater than 4-log reduction in infectivity of Tulane virus, a human norovirus surrogate. Further, these Cu(I) solutions caused reduction of GII.4 norovirus from stool in suspension, producing about a 2-log reduction of virus as measured by a reverse transcriptase-quantitative polymerase chain reaction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) data indicate substantial major capsid protein cleavage of both GI.7 and GII.4 norovirus capsids, and TEM images show the complete loss of capsid integrity of GI.7 norovirus. GII.4 virus-like particles (VLPs) were less susceptible to inactivation by copper ion treatments than GI.7 VLPs based upon receptor binding and SDS-PAGE analysis of viral capsids. The combined data demonstrate that stabilized Cu(I) ion solutions show promise as highly effective noroviral disinfectants in solution that can potentially be utilized at low concentrations for inactivation of human noroviruses.

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AIMS: To determine the efficacy of a panel of nine EPA-registered disinfectants against two human norovirus (HuNoV) surrogates (feline calicivirus [FCV] and Tulane virus [TuV]) and Clostridioides difficile endospores. METHODS AND RESULTS: Nine EPA-registered products, five of which contained H2 O2 as active ingredient, were tested against infectious FCV, TuV and C. difficile endospores using two ASTM methods, a suspension and carrier test. Efficacy claims against FCV were confirmed for 8 of 9 products. The most efficacious product containing H2 O2 as ingredient achieved a >5.1 log reduction of FCV and >3.1 log reduction of TuV after 5 min, and >6.0 log reduction of C. difficile endospores after 10 min. Of the five products containing H2 O2 , no strong correlation (R2  = 0.25, p = 0.03) was observed between disinfection efficacy and H2 O2 concentration. Addition of 0.025% ferrous sulphate to 1% H2 O2 solution improved efficacy against FCV, TuV and C. difficile. CONCLUSION: Disinfectants containing H2 O2 are the most efficacious disinfection products against FCV, TuV and C. difficile endospores. Product formulation, rather than the concentration of H2 O2 in a product, impacts the efficacy of a disinfection product. SIGNIFICANCE AND IMPACT OF STUDY: H2 O2 -based disinfectants are efficacious against surrogate viruses for HuNoV and C. difficile endospores.

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