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Le virus de l’hépatite A et les norovirus humains sont les principaux agents viraux causant des maladies d’origine alimentaire mondialement. Contrairement aux bactéries, une contamination des aliments par ces virus n’est pas facilement détectable et ceux-ci y sont présents en très faible quantité. Malheureusement, une faible concentration de ces virus sur les aliments suffit à provoquer une infection. Afin d’effectuer une détection sensible et éviter des éclosions, il est primordial d’effectuer une concentration de ces virus. L’objectif de ce projet était donc de développer deux méthodes de concentration, l’une possédant une affinité spécifique basée sur les anticorps spécifiques et l’autre possédant une affinité à large spectre utilisant l’apolipoprotéine H. La méthode basée sur les anticorps a été élaborer à partir d’autres méthodes similaires présenté dans la littérature. La méthode basée sur l’apolipoprotéine H, pour sa part, est méthode élaborer et optimiser durant ce projet de recherche. La caractérisation de l’affinité de l’apolipoprotéine H et son utilisation dans une méthode de concentration virale étant très peu détaillé par la communauté scientifique. Étant donné que les résultats obtenus pour la méthode de concentration basée sur les anticorps spécifiques étaient insatisfaisants. En effet, la sensibilité de la méthode ainsi que ça répétabilité était faible. Les efforts ont été dirigés vers la méthode de concentration basée sur l’apolipoprotéine H qui présentait des résultats prometteurs. À ce jour, aucune étude n’a démontré une affinité entre le virus de l’hépatite A et l’apolipoprotéine H et l’applicabilité de cette méthode à concentrer ces virus dans les aliments. Dans ce projet de recherche, la méthode basée sur l’apolipoprotéine H a été testée sur des fraises et des framboises fraîches et congelées contaminés expérimentalement par le virus de l’hépatite A et le norovirus humain GII.4 et comparée à la méthode référence ISO 15216 : 1. / Hepatitis A virus and human noroviruses are the major viral agents causing foodborne illness worldwide. Unlike bacteria, food contamination by these viruses is not easily detectable and they are present in very small quantities. Unfortunately, a low concentration of these viruses on food is enough to cause infection. In order to carry out a sensitive detection and avoid outbreaks, it is essential to carry out a concentration of these viruses. The objective of this project was therefore to develop two concentration methods, one with specific affinity based on specific antibodies and the other with broad-spectrum affinity using apolipoprotein H.The antibody-based method has been developed from other similar methods presented in the literature. The method based on apolipoprotein H, for that part, is a method developed and optimized during this research project. The characterization of the affinity of apolipoproteinH and its use in a method of viral concentration being very little detailed by the scientific community. Since the results obtained for the concentration method based on specific antibodies were unsatisfactory. Indeed, the sensitivity of the method as well as its repeatability was low. Efforts were directed towards the concentration method based on apolipoprotein H which showed promising results. To date, no study has demonstrated an affinity between the hepatitis A virus and apolipoprotein H and the applicability of this method to concentrate these viruses in food. In this research project, the method based on apolipoprotein H wastested on fresh and frozen strawberries and raspberries experimentally contaminated with the hepatitis A virus and the human norovirus GII.4 and compared with the ISO reference method. 15216: 1.

Human rotavirus (HRV) and human norovirus (HuNoV) are the most common causative agents of acute gastroenteritis- (AGE) related morbidity and mortality around the world. Gnotobiotic (Gn) pigs are the ideal large-animal model that allows for accurate, and precise, preclinical evaluation of vaccine efficacy. Similarities in gastrointestinal anatomy, physiology, and immune system allows for direct translation of results from Gn pigs to humans. Commercially available HRV vaccines perform significantly poorer in low- and middle- income countries as compared with developed countries. Non-replicating rotavirus vaccines (NRRVs) have been proposed as a viable solution to the problems facing currently available live-, attenuated oral vaccines and evaluation of a NRRV was the first research project in this dissertation. Three doses of a novel parenterally administered nanoparticle-based RV vaccine, P24-VP8*, adjuvanted with Al(OH)3 adjuvant, was able to prime VP8*-specific mucosal and systemic T cell responses (IFN-γ producing CD4+ and CD8+ T cells), and to induce strong systemic B cell responses (IgA, IgG and serum neutralizing antibodies). A significant reduction in the mean diarrhea duration, fecal virus shedding titers, and significantly lower fecal cumulative consistency scores was observed among vaccinated pigs demonstrating the efficacy of the vaccine against RV infection and diarrhea. Next, we determined the median infectious dose (ID50) and median diarrhea dose (DD50) of the GII.4/2003 Cin-1 variant of HuNoV in Gn pigs to better standardize the pig model for HuNoV vaccine evaluation. Gn pigs were inoculated with 7 different doses of Cin-1 at 33-34 days of age. Pigs were monitored daily from post-inoculation day (PID) 1 to 7, for fecal virus shedding and fecal consistency to evaluate the virus infectiousness and associated diarrhea. The Log10 ID50 and DD50 were determined based on various mathematical models to be between 3.11 to 3.76, and 3.37 to 4.87 RNA copies, respectively. The Beta-Poisson was identified to be the best-fitting statistical model for estimating both the ID50 and DD50 of Cin-1. Determining the ID50 of the challenge virus strain is crucial for identifying the true infectiousness of HuNoVs and for accurate evaluation of protective efficacies in pre-clinical studies of therapeutics, vaccines and other prophylactics using this reliable animal model. The lack of an easily reproducible cell culture model for HuNoV has significantly delayed the development of effective vaccines. There is still no HuNoV vaccine available. Currently, the vaccine development efforts are mostly based on genetically engineered virus-like particles (VLPs) comprised of the major HuNoV capsid protein VP1. We tested the immunogenicity of a novel tetravalent VLP vaccine containing 4 major HuNoV genotypes (GI.1, GII.3, GII.4 and GII.17) using Gn pigs and evaluated its protective efficacy when challenged with GII.4 Cin-1 HuNoV. Three doses of the VLP vaccine with Al(OH)3 adjuvant administered to Gn pigs intramuscularly (IM), induced high levels of VLP-specific serum IgA and IgG antibody and hemagglutination inhibition antibody responses in the vaccinated pigs. VLP-specific IFN-γ producing CD4+ and CD8+ T cells were also elevated among vaccinated pigs at post-challenge day (PCD) 7 in the spleen and blood, but not in the ileum. However, the vaccinated pigs were not protected from infection and diarrhea when challenged with any one of the three different doses (2 x 105, 8 x 104, and 2 x 104 genome RNA copies) of Cin-1 HuNoV. These results indicated that the IM tetravalent VLP vaccine was highly immunogenic, but the presence of high levels of immune effectors induced by the vaccine were not sufficient for protecting the Gn pigs from Cin-1 challenge. Amino acid (aa) sequence analysis showed that the GII.4 Sydney 2012 strain which was included in the VLP vaccine, had 23 aa substitutions in the major receptor binding domain (P2) compared to the Cin-1, a GII.4 Farmington Hills 2002 strain. Our findings, for the first time, provided in vivo experimental evidence for the total lack of cross-genogroup, cross-genotype and cross-variant protection among HuNoV. This finding has importance implications for HuNoV vaccine development. HuNoV vaccines have to include multiple variants and have to be routinely updated in order to ensure sustained protection among the population. Together these three studies in this dissertation demonstrate the versatility of Gn pigs as a reliable large animal model for studying the pathogenesis and immunity of enteric viruses and the evaluation of immunogenicity and protective efficacy of novel enteric viral vaccines. / Doctor of Philosophy / People of all age groups are susceptible to acute gastroenteritis (AGE), a condition characterized by sudden onset of diarrhea, nausea and abdominal cramps. The two most important viral pathogens responsible for causing AGE are rotavirus (RV) and norovirus (NoV). Gnotobiotic (Gn) pigs have been valuable in helping us understand the mechanism of infection, pathogenesis, immunity and have played a key role in the expediting development of novel vaccines and therapeutics against both of these viruses. Live oral RV vaccines are available but they are not very effective in low income countries where the vaccines are needed the most. Next generation parenteral vaccines are proposed to improve the RV vaccine efficacy. Our first study showed that a nanoparticle-based intramuscular (IM) RV vaccine effectively reduced the duration and severity of human RV infection and diarrhea in Gn pigs. Secondly, we examined in detail the infectivity of HuNoV and identified accurately using different mathematical models on how much virus would be required to infect and cause diarrhea in naïve Gn pigs. This knowledge would greatly help in the accurate assessment of the efficacy of NoV vaccines. Third, we evaluated the immunogenicity and protective efficacy of a tetravalent IM NoV vaccine in Gn pigs. Although the vaccine was highly immunogenic, it did not confer any protection against infection and diarrhea upon challenge with the NoV at different doses. NoVs are so diverse that one year we might be infected with one strain and a few years later, we might be infected again with another strain, even though they belong to the same genotype, and experience the same symptoms. This is because, changes brought about due to mutation in the virus capsid protein allow the viruses to hide from neutralizing antibodies induced by previous infection or vaccination as we have revealed in this study. NoV diversity and lack of cross protection need to be taken into consideration during vaccine development. This thesis shows how Gn pigs can be used to study these components in order to further maximize our ability to understand and combat enteric viral diseases.

Norovirus and rotavirus are the most common viral causes of acute gastroenteritis among all age groups and in children under 5 years of age, respectively. Understanding the pathogenesis of the virus and correlates of protective immunity is fundamental to developing effective prevention and treatment strategies. Gnotobiotic (Gn) pigs are an attractive animal model for studying enteric viruses due to their similarities to humans, particularly in regards to the immune system and gastrointestinal anatomy and physiology. Here, to establish a reliable Gn pig model of human norovirus (HuNoV) infection and disease, we determined the median infectious dose (ID50) of a GII.4 2006b variant in pigs. We also evaluated the effects of age and administration of the cholesterol-lowering drug simvastatin on susceptibility to NoV infection. In neonatal pigs (4-5 days of age, the ID50 was determined to be 2.74 x 103 viral RNA copies. The ID50 was increased in 33-34 day old pigs (6.43 x 104), but decreased to <2.74 x 103 following simvastatin treatment in the same age group. Overall, the development of diarrhea, fecal virus shedding and small intestinal cytopathological changes confirmed the usefulness of the Gn pig as an appropriate animal model for studying HuNoVs. We also utilized the well-established Gn pig model of human rotavirus (HRV) infection and disease to evaluate adjunctive treatment options for HRV-induced diarrhea. We demonstrated that the anti-secretory drug racecadotril was capable of diminishing clinical signs of HRV infection and shortening duration of illness. Reduced dehydration in the racecadotril-treated pigs was evident by the significant gain in body weight compared to controls during the course of the study. We also determined that a high dose of the probiotic Lactobacillus acidophilus NCFM (LA) was able to reduce RV diarrhea severity and duration compared to a low dose. The difference in therapeutic potential was attributed to divergent effects in innate immunity pre- and post-challenge. High dose of LA (HiLA) induced an anti-inflammatory dendritic cell (DC) profile, characterized primarily by upregulation of TLR2 expression and production of cytokine IL-10. Conversely, low dose of LA (LoLA) upregulated TLR3 and TLR9 and increased secretion of cytokine IL-6. Additionally, HiLA induced both IFN-alpha and TNF-alpha responses in DCs, but LoLA was only able to increase the frequency of TNF-alpha-producing DCs. These results provide further support of Gn pigs as a highly applicable animal model for studying pathogenesis, innate immunity and therapeutics of human enteric viruses. / Ph. D.

Human noroviruses (HuNoVs) are the leading cause of viral epidemic acute gastroenteritis and responsible for the deaths of over 200,000 children each year worldwide. HuNoV research has been hampered by the long absence of a readily reproducible cell culture system and a suitable small animal model, while gnotobiotic (Gn) pigs have been a unique animal model for understanding HuNoV pathogenesis and immunity, as well as evaluating vaccine and therapeutics. Recent reports of HuNoVs infection and replication in B cells supplemented with commensal bacteria Enterobacter cloacae and in Blab/c mice deficient in RAG/IL2RG have gained extensive attention, and my studies utilized the well-established Gn pig model to investigate the effects of these two interventions on HuNoV infection. Surprisingly, the colonization of E. cloacae inhibited HuNoV infectivity in Gn pigs, evidenced by the significantly reduced HuNoV shedding in feces and HuNoV titers in intestinal tissues and blood compared to control pigs. Moreover, HuNoV infection of enterocytes but not B cells was observed with or without E. cloacae colonization, indicating B cells were not a target cell type for HuNoV in Gn pigs. On the other hand, using RAG2/IL2RG deficient pigs generated by CRISPR/Cas9 system, with confirmed severe combined immunodeficiency, I evaluated the effects of host immune responses on HuNoV infection. Compared to wild-type Gn pigs, longer HuNoV shedding was observed in RAG2/IL2RG deficient pigs (16 versus 27 days), and higher HuNoV titers were detected in intestinal tissues and contents and in blood, indicating increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs. In addition, I evaluated dietary interventions including probiotics and rice bran using Gn pig model of HuNoV infection and diarrhea. While the colonization of probiotic bacteria Lactobacillus rhamnosus GG (LGG) and Escherichia coli Nissle 1917 (EcN) in Gn pigs completely inhibited HuNoV fecal shedding, the two cocktail regimens, in which rice bran feeding started either 7 days prior to or 1 day after viral inoculation in the LGG+EcN colonized Gn pigs, exhibited dramatic anti-HuNoV effects, including reduced incidence and shorter duration of diarrhea, as well as shorter duration of virus fecal shedding. The anti-HuNoV effects of the cocktail regimens were associated with the enhanced IFN-𝛾⁺ T cell responses, increased production of intestinal IgA and IgG, and longer villus length. Taken together, my dissertation work improves our understanding of HuNoV infection and immunity, and further supports for Gn pigs as a valuable model for future studies of human enteric virus infection, host immunity, and interventions. / Ph. D. / Human noroviruses (HuNoVs) are the leading cause of viral epidemic acute gastroenteritis. Using the gnotobiotic pig model of HuNoV infection and diarrhea, we found that (1) the colonization of a commensal bacterium E. cloacae inhibited HuNoV infectivity, and B cells were not a target cell type for HuNoV in gnotobiotic pigs. (2) Increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs, which had severe combined immunodeficiency. (3) The dietary supplementation of rice bran and colonization of two probiotic bacteria significantly reduced HuNoV infectivity and diarrhea, and the beneficial effects were associated with enhanced intestinal immunity and health. Taken together, the dissertation work improves our understanding of HuNoV infection and immunity, and further supports for gnotobiotic pigs as a valuable model for future studies of human enteric virus infection, host immunity, and interventions.

Le virus de l'hépatite A et les norovirus humains sont les principaux agents viraux causant des maladies d'origine alimentaire mondialement. Contrairement aux bactéries, une contamination des aliments par ces virus n'est pas facilement détectable et ceux-ci y sont présents en très faible quantité. Malheureusement, une faible concentration de ces virus sur les aliments suffit à provoquer une infection. Afin d'effectuer une détection sensible et éviter des éclosions, il est primordial d'effectuer une concentration de ces virus. L'objectif de ce projet était donc de développer deux méthodes de concentration, l'une possédant une affinité spécifique basée sur les anticorps spécifiques et l'autre possédant une affinité à large spectre utilisant l'apolipoprotéine H. La méthode basée sur les anticorps a été élaborer à partir d'autres méthodes similaires présenté dans la littérature. La méthode basée sur l'apolipoprotéine H, pour sa part, est méthode élaborer et optimiser durant ce projet de recherche. La caractérisation de l'affinité de l'apolipoprotéine H et son utilisation dans une méthode de concentration virale étant très peu détaillé par la communauté scientifique. Étant donné que les résultats obtenus pour la méthode de concentration basée sur les anticorps spécifiques étaient insatisfaisants. En effet, la sensibilité de la méthode ainsi que ça répétabilité était faible. Les efforts ont été dirigés vers la méthode de concentration basée sur l'apolipoprotéine H qui présentait des résultats prometteurs. À ce jour, aucune étude n'a démontré une affinité entre le virus de l'hépatite A et l'apolipoprotéine H et l'applicabilité de cette méthode à concentrer ces virus dans les aliments. Dans ce projet de recherche, la méthode basée sur l'apolipoprotéine H a été testée sur des fraises et des framboises fraîches et congelées contaminés expérimentalement par le virus de l'hépatite A et le norovirus humain GII.4 et comparée à la méthode référence ISO 15216 : 1. / Hepatitis A virus and human noroviruses are the major viral agents causing foodborne illness worldwide. Unlike bacteria, food contamination by these viruses is not easily detectable and they are present in very small quantities. Unfortunately, a low concentration of these viruses on food is enough to cause infection. In order to carry out a sensitive detection and avoid outbreaks, it is essential to carry out a concentration of these viruses. The objective of this project was therefore to develop two concentration methods, one with specific affinity based on specific antibodies and the other with broad-spectrum affinity using apolipoprotein H. The antibody-based method has been developed from other similar methods presented in the literature. The method based on apolipoprotein H, for that part, is a method developed and optimized during this research project. The characterization of the affinity of apolipoprotein H and its use in a method of viral concentration being very little detailed by the scientific community. Since the results obtained for the concentration method based on specific antibodies were unsatisfactory. Indeed, the sensitivity of the method as well as its repeatability was low. Efforts were directed towards the concentration method based on apolipoprotein H which showed promising results. To date, no study has demonstrated an affinity between the hepatitis A virus and apolipoprotein H and the applicability of this method to concentrate these viruses in food. In this research project, the method based on apolipoprotein H was tested on fresh and frozen strawberries and raspberries experimentally contaminated with the hepatitis A virus and the human norovirus GII.4 and compared with the ISO reference method. 15216: 1.

Thèse ou mémoire avec insertion d’articles. / La consommation d'aliments contaminés par le norovirus humain, tels que les baies et les légumes feuillus, est une cause majeure de gastro-entérite épidémique dans le monde. Par conséquent, les stratégies de contrôle visant à limiter l'adhésion du norovirus à la surface des produits végétaux ont fait l'objet de plusieurs études. Pendant la dernière décennie, les interactions entre les virus entériques et les bactéries commensales de l'intestin des mammifères ont été étudiées. Cependant, peu de travaux ont été réalisés sur les communautés bactériennes associées à la surface des baies et des légumes feuillus et leur rôle dans la transmission du norovirus humain. D'ailleurs, certaines bactéries de la flore commensale des végétaux sont connues pour leur capacité à former des biofilms dans lesquels des substances analogues aux récepteurs des norovirus sont sécrétées. La présence de biofilms bactériens pourrait ainsi contribuer à l'adhésion du norovirus à la surface des végétaux et favoriser sa persistance. Puisque les produits végétaux sont délicats et susceptibles d'être endommagés, les traitements conventionnels ne sont pas toujours appropriés pour inactiver les virus sans compromettre la qualité des aliments. Dans ce mémoire, neuf bactéries associées à la surface des baies et des légumes feuillus sous forme de biofilms ont été étudiées selon leur effet protecteur sur la persistance de deux virus modèles du norovirus humain, le norovirus murin (MNV-1) et le virus Tulane, après un traitement d'inactivation par la lumière pulsée à une fluence de 11,52 J/cm². Aucune différence significative n'a été détectée dans les réductions du titre viral de MNV-1 en présence de biofilms par rapport au MNV-1 seul après le traitement par la lumière pulsée. Le virus Tulane a montré une résistance plus élevée au traitement par la lumière pulsée lorsqu'il était en présence des biofilms formés par Enterobacter cloacae, Escherichia coli, Kocuria kristinae, Pantoae agglomerans et Pseudomonas fluorescens. / Consumption of contaminated food with human norovirus, such as fresh berries and leafy vegetables, is a major cause of epidemic gastroenteritis worldwide. Consequently, several studies have focused on strategies to prevent norovirus attachment to fresh produce. Over the last decade, interactions between enteric viruses and commensal bacteria of the mammalian gut have been investigated, but limited work has been done on the bacterial communities associated with the surface of fresh produce and their role in norovirus transmission. Some bacterial species can form a matrix of extracellular polymeric substances, called biofilm, in which molecules like norovirus receptors can be secreted. Thus, bacterial biofilms associated with the surface of berries and leafy vegetables could interplay with norovirus through direct interactions and modulate its attachment and persistence. Since fresh produce are susceptible to damage, conventional treatments are not always appropriate to inactivate viruses without compromising food quality. Therefore, there is an urgent need to understand how norovirus interacts with epiphytic bacteria, and more specifically with bacterial biofilms. Nine bacterial species were studied in terms of their protective effect on the persistence and infectivity of two norovirus surrogates, murine norovirus 1 (MNV-1) and Tulane virus, when subjected to a pulsed light inactivation treatment at a fluence of 11.52 J/cm². No significant difference was detected in reductions of MNV-1 when attached to bacterial biofilms compared with the control whereas Tulane virus showed a significantly higher resistance to pulsed light treatment when attached to biofilms formed by Enterobacter cloacae, Escherichia coli, Kocuria kristinae, Pantoae agglomerans, and Pseudomonas fluorescens.