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.

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.

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.

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.

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.

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.

High pressure processing (HPP) is a powerful non-thermal method for inactivating pathogens. Human norovirus and genetically-related caliciviruses are moderately sensitive to temperatures above 0 °C with >400 MPa (MPa) or higher required to inactivate multiple logs of virus. Sensitivity of murine norovirus (MNV) and Tulane virus (TV) to ice phase transitions was evaluated using ultra low temperature HPP. Identical samples containing MNV or TV were either equilibrated to +1.5 °C (thawed) or -40 °C (frozen) 24 h prior to pressurization. All samples (thawed and frozen) were then placed in a pre-chilled chamber which was then rapidly filled with -40 °C chamber fluid. Samples were immediately pressurized for 5 min at 200, 250 or 300 MPa. Controls were not pressurized. For samples that were thawed and then pressurized in 40 °C chamber fluid, the MNV average log reduction at 200 MPa was 4.4, while >6.1 log reduction (non-detectable) was achieved at 250 and 300 MPa. TV samples averaged 2.3, 5 and 4.3 log reduction at 200, 250, and 300 MPa respectively. For samples that were frozen and then pressurized in 40 °C chamber fluid, the MNV average log reductions were 2.3, 3.2 and 4.2 at 200 MPa, 250 MPa and 300 MPa, respectively, while TV samples averaged 0.81, 2.3 and 1.7 log reductions at 200, 250, and 300 MPa, respectively. Inactivation of TV within oysters at these pressures was also demonstrated. Overall, results indicate that in addition to enhancing inactivation of norovirus surrogates compared to higher temperatures, ultra-cold HPP performed on thawed samples especially enhances inactivation.

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.

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.

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.