Noroviruses (NoVs) are a cause of nonbacterial acute gastroenteritis affecting all ages. NoV infections result in over 200,000 pediatric deaths in developing countries annually. Vaccine development has been hindered by the lack of cell culture systems and small animal models; thus, vaccine development has relied upon recombinant VP1 capsid proteins, such as virus-like particles (VLPs) and P particles. P particles are a novel vaccine candidate derived from expression of the VP1 protruding (P) domain, while VLPs require expression of the full-length VP1. My studies utilize a gnotobiotic (Gn) pig model of human NoV infection and diarrhea to evaluate the protective efficacy and T cell responses induced by P particles and to compare them with prior NoV infection (NoVPO) and VLPs. Gn pigs received 100 µg of P particles (LoPP) or VLPs, 250 µg P particles (HiPP), or adjuvants only intranasally at post-inoculation day (PID) 0, 10, and 21. Monophosphoryl lipid A and chitosan were used as mucosal adjuvants. At PID 28, a subset of pigs were orally challenged with 10 median infectious doses (ID50) NoV. NoVPO, LoPP, HiPP, and VLPs provided partial protection from diarrhea (83%, 47%, 60%, and 60% protection rates, respectively). Only NoVPO and HiPP provided protection from shedding (49% and 60% protection rates, respectively) and also reduced the number of CD25- regulatory T cells (Tregs) in duodenum following challenge. NoV primary infection induced an overall pro-Treg and low, transient Th1 response. LoPP induced stronger overall T cell responses compared to VLPs, including activated CD4+ T cells and duodenal CD8+IFN-γ+ T cells, suggesting that P particles are more immunogenic than VLPs. I also evaluated the effects of simvastatin, a cholesterol-reducing drug that increases NoV infectivity, on P particle vaccine efficacy. Simvastatin abolished P particle-induced protection and significantly increased diarrhea severity. Simvastatin reduced total numbers of duodenal mononuclear cells, IFN-γ+ T cells pre-challenge, and Tregs post-challenge, indicating that simvastatin impairs development of immune system and immune responses. Findings from these studies elucidate potential mechanisms behind P particle-induced immunity and reveal the negative effects of simvastatin on NoV-induced protective immunity. The knowledge will facilitate the development of effective NoV vaccines. / Ph. D.

Les vecteurs permettant la transmission des infections nosocomiales ne sont pas toujours bien identifiés. Les risques représentés par les virus aéroportés dans les milieux de soins doivent être étudié davantage. Ce mémoire présente des méthodologies pour détecter la présence de virus aéroportés et évaluer leur résistance dans cet état. Le virus influenza, le norovirus et le virus respiratoire syncytial y sont à l’étude. Deux techniques d’échantillonnage furent utilisées soit un échantillonnage à sec (NIOSH 251) et un échantillonneur liquide (Coriolis µ®). Les ADNc viraux furent détectés par qPCR. La résistance des norovirus à l’aérosolisation a été démontrée à l’aide d’un virus-modèle, le norovirus murin. La première détection des norovirus dans l’air de milieux de soins y est décrite. La présence du virus influenza dans l’air a aussi été démontrée. Le virus respiratoire syncytial n’a pu être mis en évidence dans l’air. Les virus pathogènes peuvent être aéroportés et représenter un risque infectieux. / The route of transmission of healthcare associated infections is not always well defined. The airborne dissemination of influenza virus, norovirus and respiratory syncytial virus has to be assessed. This thesis presents methodologies to detect airborne viruses and some innovative way to assess their resistance through the airborne route. Two sampling techniques were used, more precisely a dry sampling using a NIOSH 251 impactor and a liquid sampling using a Corriolis µ®. Viral cDNA was detected by real-time PCR. To assess the resistance of norovirus through the air route we used a cultivable experimental model; the murine norovirus. This thesis presents the first detection of airborne norovirus in a healthcare setting. Influenza virus was detected in the air of an emergency department and in the room of influenza positive patient. Respiratory syncytial virus could not be detected in an airborne state. Pathogenic virus can be disseminated through the airborne route and could represent an infectious risk.

Thèse ou mémoire avec insertion d'articles. / Titre de l'écran-titre (visionné le 23 octobre 2023) / Le norovirus humain (HuNoV) et le virus de l'hépatite A (VHA) ont été détectés plusieurs fois dans les petits fruits, tandis que le HuNoV, le VHA et le virus de l'hépatite E (VHE) ont été associés à des épisodes de contamination des huîtres et des moules dans les dernières années. Ces virus sont principalement transmis par la voie féco-orale, mais le VHE peut également être transmis par voie zoonotique. Le HuNoV et le VHA peuvent contaminer les aliments via l'eau, notamment lors de l'irrigation avec de l'eau contaminée ou en cas de déversement dans les zones de production. De plus, le VHE peut être transmis aux aliments par les fèces d'animaux contaminés. Cependant, les connaissances sur la contamination des aliments cultivés au Québec par ces virus sont limitées. Le but de ce projet était d'évaluer la contamination des canneberges par le HuNoV et le VHA ainsi que celle des huîtres par le HuNoV, le VHA et le VHE, entre deux zones de production. Pour ce faire, 234 échantillons de canneberges ont été récoltés chez 44 producteurs québécois et 260 huîtres ont été prélevées près de la côte, et 260 huîtres au large de la côte des Îles-de-la-Madeleine, chaque échantillon étant représenté par 10 huîtres. L'ARN viral a été détecté et quantifié par détection moléculaire, en respectant la méthodologie de la méthode ISO 15216-1 : 2017. Parmi les 234 échantillons de canneberges testés, seulement trois étaient positifs au HuNoV GI (1,28 %). Aucun des 52 échantillons d'huîtres n'a été considéré comme positif pour le HuNoV, le VHA ou le VHE. Ces résultats suggèrent que le risque de contamination par des canneberges et des huîtres cultivées au Québec est faible et que la contamination des huîtres près de la côte n'est pas significativement différente de celle des huîtres au large de la côte. / Human norovirus (HuNoV), hepatitis A virus (HAV) and hepatitis E virus (HEV) have been the cause of many global epidemics, such as those caused by HuNoV and HAV in berries or even HuNoV, HAV and HEV in shellfish in recent years. These viruses are primarily transmitted through the fecal-oral route, although HEV can also be transmitted through zoonotic routes. HuNoV and HAV can contaminate food through water, especially during irrigation with contaminated water when spilled in production areas. In addition, HEV can be transmitted to food through contaminated water but also when in contact with contaminated animal feces. However, limited information is available on the contamination of food grown in Quebec by these foodborne viruses. The objective of this project was to evaluate the levels of HuNoV and HAV contamination in cranberries, as well as levels of HuNoV, HAV and HEV contamination in oysters, between two production areas. For this purpose, 234 cranberry samples were collected from 44 Quebec producers. As for molluscs, 260 oysters were obtained near the coast and 260 oysters were collected offshore of Magdalen Islands, each sample consisting of 10 oysters. The presence of viral RNA was detected and quantified using molecular detection methods in accordance with ISO 15216-1:2017. Among the 234 cranberry samples tested, only three were positive for HuNoV GI (1.28%). None of the 52 oyster samples were found to be positive for HuNoV, HAV or HEV. These findings suggest that the risk of contamination of cranberries and oysters cultivated in Quebec is low, and that there is no significant difference in contamination between oysters near the coast and those offshore in this project.

La majorité des gastroentérites non-bactériennes sont causées par les norovirus, qui se transmettent principalement par les surfaces, les aliments et l’eau. La plupart des désinfectants domestiques sont inefficaces contre ces virus à l’exception de l’acide hypochloreux qui, cependant, perd une partie de son activité et génère des sous-produits toxiques en présence de matières organiques. Des méthodes de désinfection alternatives, efficaces et plus sécuritaires sont aujourd’hui nécessaires pour lutter contre la propagation des norovirus. Ce projet de recherche a porté sur deux approches prometteuses, les acides peroxycarboxyliques et les lumières pulsées. Dans un premier temps, quatre acides peroxycarboxyliques ont été évalués en fonction de leur concentration et du temps de contact. Les acides monoperoxycarboxyliques, à savoir les acides peracétique et perpropionique, ont été les plus efficaces en réduisant la charge virale d’environ 4 log10 après un traitement de 50 mg L-1 pendant 5 minutes. Ces molécules ont conservé cette activité contre des norovirus déposés sur de l’acier inoxydable et du PVC, propres ou sales, ainsi qu’immobilisés dans un biofilm artificiel. Dans le cas de l’acide peracétique, les expérimentations ont démontré que cette inactivation résultait principalement de dommages causés sur l’ARN viral, probablement par l’intermédiaire de radicaux libres. À forte concentration, les protéines de la capside étaient aussi altérées. Dans un deuxième temps, les norovirus ont été traités par une lumière pulsée (200 à 1000 nm ; 0,69 J cm-2 par impulsion). Trois impulsions (1,6 seconde) ont diminué la charge virale d’environ 4 log10 dans tous les milieux testés (tampon salin, eaux dures, minérale et usées) à l’exception des eaux turbides. A la turbidité maximum (1000 NTU), la réduction était de 2.4 log10. Cette technologie est aussi efficace pour la désinfection de surfaces propres, même en présence d’un biofilm artificiel (4 log10 après 7 impulsions), mais est affectée par la présence de matières protéiques. Nous avons démontré que les lumières pulsées inactivaient les norovirus en induisant des dommages à la fois sur l’ARN viral et l’intégrité des particules. En conclusion, ces résultats attestent l’efficacité de ces techniques et contribuent à une meilleure compréhension de leur principe d'action. / The majority of non-bacterial gastroenteritis is caused by norovirus, which is transmitted primarily through surfaces, food and water. Most household disinfectants are ineffective against these viruses with the exception of the bleach which, however, loses partially its activity and generates toxic residues in the presence of organic matter. Alternative, effective and safer methods of disinfection are necessary to hamper the spread of norovirus. This research project focused on two promising approaches, namely peroxycarboxylic acids and pulsed light. On the one hand, four peroxycarboxylic acids were evaluated based on their concentration and contact time. Monoperoxycarboxylic acids, namely peracetic and perpropionic acids, were the most effective by reducing the viral load of about 4 log10 after a treatment with 50 mg L-1 for 5 minutes. These molecules maintained their activity against noroviruses attached on stainless steel and PVC, clean or dirty, and entrapped in an artificial biofilm. In the case of peracetic acid, we showed that this inactivation was mainly due to damages on the RNA, probably through free radicals. At high concentrations, the capsid was also altered. On the other hand, noroviruses were treated with pulsed light (200-1000 nm; 0.69 J cm 2 per pulse). Three pulses (1.6 seconds) decreased viral load of approximately 4 log10 in all media tested (buffered saline, hard water, mineral water and sewage treatment effluent) with the exception of turbid water. At the maximum turbidity (1000 NTU), the reduction was 2.4 log10. This technology is also effective to disinfect clean surfaces even in the presence of an artificial biofilm (4 log10 after 7 pulses), but is affected by the presence of proteinaceous material. We demonstrated that the pulsed light inactivated norovirus by inducing damage to both the RNA and the integrity of the particles. In conclusion, these results demonstrate the effectiveness of these approaches and contribute to a better understanding of their mechanism of action.

Même si les traitements thermiques ou la désinfection par les oxydants ont démontré leur efficacité virucide, les mécanismes liés à la perte du caractère infectieux ne sont pas connus. Ceci pose un réel problème d’interprétation de la présence de génome viral en matière de risque infectieux dans les aliments. Ce travail de thèse a pour objectif d’étudier l’évolution des propriétés de surface (charge et hydrophobie) de virus modèles, bactériophage MS2 et norovirus murin, au cours de l’inactivation par la chaleur, l’hypochlorite de sodium et l’ozone. Pour nos deux virus, nous démontrons l’existence d’une température critique au-delà de laquelle la particule virale se déstructure en libérant son génome. Un simple traitement à la RNase permettrait alors de ne détecter que des virus infectieux par biologie moléculaire. Le traitement thermique implique aussi une augmentation de l’hydrophobie soulignant des modifications conformationnelles de la capside. L’hypochlorite de sodium ne modifie que peu les propriétés de surface mais des phénomènes d’oxydation ont lieu au niveau de la capside puisque la charge du bactériophage MS2 est légèrement modifiée. Ces modifications diminuent la résistance thermique du virus. Nous démontrons un effet synergique de l’hypochlorite de sodium et la chaleur sur le bactériophage MS2 (inactivation, RNase et hydrophobie). Quant à l’ozone gazeux, nous soulignons son intérêt pour le traitement virucide des aliments fragiles. Ainsi, ce travail précise les mécanismes d’inactivation des virus et ouvre de nouvelles perspectives tant pour discriminer les virus infectieux et non-infectieux que pour proposer l’exploration de nouveaux traitements technologiques / Although heat treatments or disinfections by oxidants have proven their virucidal efficiencies, mechanisms related to the loss of infectivity are not known. This statement could lead to a misinterpretation of the presence of viral genome on infection risk for humans in food matrices. This thesis aimed to study the evolution of surface properties (charge and hydrophobicity) for model viruses, bacteriophage MS2 and murine norovirus, during the heat, sodium hypochlorite and ozone inactivations. For both viruses, the existence of a critical temperature beyond which the viral particle was disrupted and released its genome was demonstrated. Simple treatment with RNase would then only detect infectious virus by molecular biology. The heat treatment also involved a transient increase in the hydrophobicity which highlighted conformational changes of the viral capsid. Sodium hypochlorite slightly modified the surface properties but oxidation phenomena occurred onto capsid since the bacteriophage MS2 charge has changed a little. These changes decreased the thermal resistance of the virus. Synergistic effects of both sodium hypochlorite and heat were observed on the inactivation of MS2 phages, the sensitivity of their genome to RNases and the increase in hydrophobicity of remaining infectious particles. Regarding gaseous ozone, we underlined its interest in the case of virucidal treatment of fragile food matrices. Therefore, this work specified the virus inactivation mechanisms and opened up new perspectives to discriminate infectious from non-infectious viruses but also to propose the exploration of new technological processes

Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. Despite the significant disease and economic burden, currently there are no licensed vaccines or antivirals. The understanding of norovirus biology has been hampered by the inability to cultivate HuNoV in cell culture. To establish a tissue culture system, infectious HuNoVs were purified from clinical stool samples. HuNoV replication was tested in different cell types. The B-cell and intestinal organoids culture systems were validated. In addition, using organoids culture a DNA-based reverse genetic system was shown to recover infectious HuNoV. Due to the challenges associated with cultivating HuNoV, murine norovirus (MNV) was used as a surrogate system to understand the role of eIF4E phosphorylation in norovirus pathogenesis, and VP1-RdRp interaction in regulating viral genome replication. MNV infection results in the phosphorylation of the translation initiation factor eIF4E, re-programming host-cell translation during infection. Inhibiting eIF4E phosphorylation reduces MNV replication in cell culture suggesting a role in viral replication. A mouse model with eIF4E S209A, a phosphor-ablative mutation, was established to understand the role of eIF4E phosphorylation in MNV pathogenesis. In vitro and in vivo characterisations demonstrated that eIF4E phosphorylation may have multiple roles in norovirus-host interactions, but overall has little impact on MNV pathogenesis. The shell domain (SD) of norovirus major capsid protein VP1 interacts with viral RNA-dependent RNA polymerase (RdRp) in a genogroup-specific manner to enhance de novo initiation of RdRp, and to promote negative-strand RNA synthesis. To understand how VP1 regulates norovirus genome replication, chimeric MNVs with genogroup-specific residues mutagenised were characterised in vitro and in vivo. A single amino acid mutation was shown to destabilise viral capsid. SDs with reduced VP1-RdRp interaction showed less capacity to stimulate RdRp, resulting in delayed virus replication. In vivo, the replication of an MNV-3 with homologous mutations was abolished, highlighting the crucial role of this interaction.

Les rotavirus et les norovirus sont les principaux agents étiologiques des gastro-entérites en Tunisie. Pendant l’hiver 2011-2012, nous avons collecté les selles et les salives de 114 enfants âgés de moins de 6 ans, souffrant de gastro-entérites et admis à l’Hôpital Fattouma Bourguiba de Monastir. L’analyse des salives a montré que la cohorte se répartissait entre 79% de sécréteur et 21% de non-sécréteur (absence d’antigène dans les salives). Parmi les sécréteurs, les individus du groupe O étaient les plus représentés (42%) suivis des groupes A (30%), B (21%) et AB (7%) alors que 96% des patients étaient positifs pour l’antigène Lewis. Pour 98 patients, l’analyse génétique du sang a montré que le gène FUT2 se répartissait entre 77.6% de sécréteur (Se+/Se+ et Se+/se-) et 22.4% de non-sécréteurs (se-/se-, N=22).L’analyse des fèces a montré que les rotavirus et les norovirus étaient responsables respectivement de 22% et 31% des cas, les infections mixtes représentant 6% des cas. Parmi les norovirus, le génotype GII.3 était prédominant (69% de tous les NoV) tandis que le génotype G9P[8] était le plus fréquemment détecté de tous les rotavirus (37,5%). Les rotavirus ont été détectés chez les individus sécréteurs (N=28) mais aussi chez 4 patients non-sécréteurs (3 souches G9P[8] et une souche G3P[8]). Nous n’avons pas observé de distribution particulière des rotavirus en fonction des antigènes A, B et H parmi les enfants sécréteurs. En revanche, nous avons constaté que l'infection à rotavirus ne s’était produite que chez les individus positifs pour l’antigène Lewis (P=0.017). La présence de génotype P[8] chez des non-sécréteurs est inédite, elle a été confirmée par le séquençage du segment correspondant à VP8* de ces rotavirus.La majorité des infections à norovirus a été détectée chez les patients sécréteurs et cela sans distribution particulière en fonction des antigènes A, B, H et Lewis. Cinq GII.3, un GII.1 et un norovirus de génotype GII.7 ont été détectés chez les non-sécréteurs, Lewis-positifs. La production de particules de synthèse (VLP) de norovirus GII.3 en baculovirus à partir des selles d’un des patients non-sécréteurs nous a permis de tester les échantillons salivaires de toute la cohorte. L’absence d’attachement de ces VLP sur les salives des non-sécréteurs montre que la présence ou l’absence des antigènes de groupe ne reflète pas nécessairement le pouvoir infectieux des norovirus chez les jeunes enfants. Les résultats obtenus sur les rotavirus et les norovirus suggèrent qu’ils existent des voies alternatives aux antigènes de groupe sanguin pour l’attachement des rotavirus et des norovirus dans l’intestin. / Rotavirus and norovirus are the main aetiological agents of gastroenteritis in Tunisia. Stool specimens and saliva were collected from children younger than 6 years of age, admitted to the Fattouma Bourguiba Hospital (Monastir, Tunisia) for gastroenteritis during the winter 2011-12. Saliva analysis showed that 79% and 21% patients had secretor and non-secretor phenotypes, respectively. Group O blood type was predominant (42%) followed by groups A (30%), B (21%) and AB (7%), whilst 96% of the patients were positive for Lewis antigen. For 98 patients, blood samples were available and were used for FUT2 genotyping. 77.6% of the cohort were secretor (Se+/Se+ and Se+/se-) and 22.4% were non-secretor (se-/se-).Rotavirus and norovirus were found alone in 22% and 31% of the stool specimens, respectively. Mixed rotavirus-norovirus infections accounted for 6% of the cases. GII.3 noroviruses were predominant among the noroviruses whilst the G9P[8] genotype was predominant for the rotaviruses.Rotaviruses were detected in secretor (N=28) as well as in non-secretor individuals (three G9P[8] strains and one G3P[8]). No significant association was found between ABO antigens or the secretor status and RV infection. Inversely, we observed that RV infection always occurred in Lewis-positive patients (P=0.017). The presence of the P[8] genotype was confirmed by sequencing part of the VP8* coding region.There was no significant association between norovirus infection and ABO antigens and the FUT2 genotype. Five GII.3, one GII.1 and one GII.7 noroviruses were found in Lewis-positive non-secretor patients. Virus-like particles from a GII.3 norovirus infecting a non-secretor patient from the cohort were expressed in baculovirus and used for binding assay with the 114 saliva samples of the study group. VLP binding with non-secretor saliva was negative and suggested that saliva binding assay might not reflect norovirus infectivity. Overall, our data suggested that rotavirus and norovirus infection might involve non-HBGA binding pathways as well as the canonical HBGA ligands.

Enteric caliciviruses were detected in humans and animals incoming into food chain such as porcine and bovine species. Caliciviruses have a single stranded, positive, polyadenylated RNA genome. They share properties of high environmental stability, high excretion load and high genetic variation linked to point mutations and recombination. These properties have allowed the formulation of hypothesis about zoonotic transmission or animal reservoir for human strains. The aim of the thesis, composed of four studies, was to investigate the zoonotic risk associated to animal enteric caliciviruses. In a first study, circulation of both sapoviruses and noroviruses was evidenced by molecular detection in Belgian pig farms. They were detected in both asymptomatic animals or in piglets showing clinical signs of enteritis. In a second study, bovine noroviruses were molecularly detected in Belgian cattle. Strains phylogenetically related to those of the genotype 2 were predominant. In the same study, seroprevalence against bovine norovirus infection in cattle was investigated by indirect ELISA. Antigens included in the ELISA were virus-like particules obtained in the baculovirus system by expressing the capsid protein of a strain isolated by the laboratory during a previous study. Apparent seroprevalence was high (93.2%), confirming previous results about apparent molecular prevalence in diarrheic calves (7.5%). In a third study about molecular detection of bovine noroviruses, diagnostic strategy was revised in order to improve the detection of genotype 1 strains and to deal with opportunity of recombination events. Bovine norovirus recombinant strains and also, surprisingly, some sequences genetically related to bovine kobuviruses were detected. In a fourth study, attachment factors and internalization pathways for genotype 2 bovine noroviruses were studied with an original quantitative method based on flow cytometry analysis. Along with a galactosyl residue that seems to be essential, a sialic acid residue was also showed to be implied in the binding of genotype 2 bovine noroviruses or in a posterior step. Internalisation pathways related to lipid rafts and to macropinocytosis were found. Together the results have contributed to the analysis of the zoonotic risk associated to enteric animal caliciviruses in the Belgian epidemiological situation. According to these results, the zoonotic risk seems to be low as no sequences genetically related to the human ones were detected. However, some results suggest to maintain a certain degree of vigilance. Indeed, molecular detection showed the co circulation of both bovine and porcine noroviruses in Belgian farms, implicating that hazard exposition exists and could be high in the Belgian epidemiological situation. Morevover, circulation of recombinant strains in the overall population of the bovine norovirus strains implies that this phenomenon was included in the risk assessment. Infection pressures and high prevalences for human and animal strains in a closed epidemiological context as the Belgian one could increase the risk of interspecific recombination. Finally, the sialic acid residue, possibly involved in the binding of genotype 2 bovine noroviruses, and a poorly specific internalisation pathway as macropinocytosis could also favor interspecies barrier crossing. In the current knowledge, zoonotic risk associated to animal enteric caliciviruses can be communicated as low but a degree of vigilance has to be retained, associated for example to observation of genetic evolution in the populations of human and animal strains.

Human norovirus (NoV) causes gastroenteritis worldwide and has been associated with a number of produce related outbreaks. The design of effective inactivation and prevention procedures requires an understanding of virus survival in environments applicable to the production and processing of fresh produce. To evaluate the extent of NoV risk from farm to fork, the survival of murine norovirus (MNV), a surrogate for human NoV, was studied on stainless steel disks, soil and in bottled water for 42 days and on lettuce for 15 days in the laboratory. Stability experiments were then conducted on farm during one lettuce planting/harvest cycle, for 4 weeks. MNV stability was tested at room temperature in the laboratory or under ambient conditions on the farm. A one log reduction in virus titre was achieved after 30 days in water, 4 days on lettuce, 15 days on stainless steel disks, 12 days on loamy and sandy soil. For farm testing, infectious virus was recovered from both soil and lettuce on the day of inoculation. Although infectious virus was not recovered at later time points, the viral genomes were detected for up to four weeks. The observed long-term persistence of NoV, under both laboratory and field conditions, provides valuable information for developing risk assessments and control procedures to limit the possibility for NoV transmission in the food supply.

Human norovirus (NoV) causes gastroenteritis worldwide and has been associated with a number of produce related outbreaks. The design of effective inactivation and prevention procedures requires an understanding of virus survival in environments applicable to the production and processing of fresh produce. To evaluate the extent of NoV risk from farm to fork, the survival of murine norovirus (MNV), a surrogate for human NoV, was studied on stainless steel disks, soil and in bottled water for 42 days and on lettuce for 15 days in the laboratory. Stability experiments were then conducted on farm during one lettuce planting/harvest cycle, for 4 weeks. MNV stability was tested at room temperature in the laboratory or under ambient conditions on the farm. A one log reduction in virus titre was achieved after 30 days in water, 4 days on lettuce, 15 days on stainless steel disks, 12 days on loamy and sandy soil. For farm testing, infectious virus was recovered from both soil and lettuce on the day of inoculation. Although infectious virus was not recovered at later time points, the viral genomes were detected for up to four weeks. The observed long-term persistence of NoV, under both laboratory and field conditions, provides valuable information for developing risk assessments and control procedures to limit the possibility for NoV transmission in the food supply.