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.

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.

Les norovirus sont maintenant reconnus mondialement comme étant la cause majeure de maladies d'origine alimentaire. Deux causes majeures expliqueraient la recrudescence des cas d'empoisonnements alimentaires d'origine virale: leur très grande stabilité dans l'environnement facilitant ainsi leur transfert et leur résistance à la majorité des approches de lutte contre les microorganismes pathogènes tels que la désinfection chimique. L'objectif général de ce projet était d'étudier et de caractériser le phénomène d'attachement des norovirus humain et murin aux surfaces inertes en fonction des différents paramètres physico-chimiques (pH et humidité relative) et d'évaluer l'impact de ce phénomène sur la sensibilité des norovirus aux désinfectants chimiques domestiques. Les résultats obtenus ont démontré un meilleur attachement à pH acide ou neutre peu importe l'humidité relative. On a d'ailleurs observé un attachement moindre lorsque le norovirus murin était sous des conditions de pH 9 et de faible humidité relative comparativement au norovirus humain où aucune différence n'a été observée selon les différentes conditions. Pour les deux virus, un attachement maximal a été observé après un temps de contact de 10 minutes. L'évaluation de la sensibilité des norovirus aux différents désinfectants chimiques a montré une plus grande sensibilité aux composés chlorés avec une réduction virale supérieure à 3 log comparativement aux alcools ou aux ammoniums quaternaires qui n'ont permis qu'une faible réduction généralement inférieure à 1 log. On a également noté que le norovirus murin est généralement plus sensible que le norovirus humain.

Les norovirus sont l'une des causes principales de gastroentérite. Depuis 2002, des variants de norovirus GII.4 successifs ont circulé dans la population par cycle de 2-3 ans, ce qui suscite des interrogations quant au rôle de leurs ligands, les antigènes tissulaires de groupes sanguins (HBGA), dans leur évolution. Nous avons analysé l'interaction entre des variants de GII.4 représentatifs et des HBGA, et déterminé le rôle d’acides aminés (aa) clés. Par mutagénèse dirigée, nous avons montré qu’une configuration stricte des aa directement impliqués dans l’accroche est indispensable. La suppression de la thréonine 395, caractéristique des variants après 2002, confère la capacité de se lier à Lex et Si-Lex, démontrant que les aa en dehors du site de liaison peuvent modifier les propriétés d’attachement. L'analyse de l'accroche de VLP de 6 variants isolés de 1987 à 2007 à des échantillons de salive phénotypés et des HBGA synthétiques montre que tous les variants sont capables de s’attacher à la salive des sécréteurs indépendamment du phénotype ABO et aux oligosaccharides propres au phénotype sécréteur. Deux variants récents ont pu également s’accrocher aux sucres présents dans la salive des nonsécréteurs Le(+). Nos données suggèrent que la capacité de se lier à Lex et Si-Lex serait une conséquence de la variation génétique des aa situés à proximité du site de liaison. L'analyse des propriétés d’attachement par résonance plasmonique de surface a montré que seuls les variants après 2002 présentent une affinité forte pour les antigènes A et B, suggérant que l’accélération évolutive des GII.4 pourrait être liée à une affinité accrue des variants pour les HBGA après 2002. / Noroviruses are one of the leading causes of gastroenteritis worldwide. Since 2002 successive GII.4 variants have circulated in the population before being replaced every 2-3 years, which raises questions about the role of their histo-blood group antigen (HBGAs) receptors in their evolution. We analyzed the interaction between representative GII.4 variants and HBGAs and determined the role of selected amino acids (aa) in the binding profiles. By mutagenesis, we showed that there was a strict structural requirement for the aa directly implicated in HBGA bindings. The ablation of the threonine 395 residue, an epidemiological feature of the post 2002 variants, allowed to gain the capacity to bind to the Lewis x and sialyl-Lewis x antigens, demonstrating that aa residues outside the HBGA binding site can modify the binding properties. The analysis of the attachment of VLPs from 6 variants isolated from 1987 to 2007 to phenotyped saliva samples and synthetic HBGAs shows that all variants could attach to saliva of secretors irrespective of the ABO phenotype and to oligosaccharides characteristic of the secretor phenotype. Interestingly, two recent variants additionally bound to carbohydrates present in the saliva of Lewis-positive non-secretors. Our data suggest that GII.4 binding to Lex and Si-Lex antigens might be a by-product of the genetic variation of the aa located in the vicinity of the binding site. Analysis of the binding properties by surface plasmon resonance showed that only post 2002 variants presented a strong affinity for A and B antigens, suggesting that the GII.4 evolution could be related to an increased affinity for HBGAs for the post 2002 variants.

Chaque année les infections nosocomiales touchent plus de 4 millions de patients en Europe, avec un impact important en termes de mortalité, de morbidité et de coût. Parmi ces infections, celles causées par des bactéries multi-résistantes aux antibiotiques (BMR) jouent un rôle majeur. La modélisation mathématique des épidémies est un outil essentiel qui permet de mieux comprendre la dynamique de diffusion des BMR et d’évaluer l’efficacité des mesures de prévention.L'objectif principal de ce projet est d'étudier la dynamique de propagation de BMR au sein d'un réseau d'hôpitaux, en prenant en compte différentes échelles : intra-service, inter-services et inter-hôpitaux. Il s'agit de mettre en place une recherche méthodologique basée sur la modélisation mathématique et informatique et validée par des données recueillies au sein du réseau de soins Paris Île de France Ouest (PIFO), afin de mieux comprendre le rôle joué par chaque hôpital dans l'émergence et la sélection de BMR, de quantifier le risque de leur dissémination (y compris dans la population générale), et d'identifier des mesures de contrôle efficaces. Ce travail s'appuiera sur des méthodes d'inférence statistiques, d'analyse de sensibilité et d'analyses d'incertitude. / Each year nosocomial infections affect more than 4 million patients in Europe, with a significant impact in terms of mortality, morbidity and cost. Of these infections, those caused by multi-resistant bacteria (BMR) play a major role. Mathematical modeling of epidemics is an important tool to better understand the dynamics of dissemination of BMR and evaluate the effectiveness of prevention measures.The main objective of this project is to study the BMR propagation dynamics within a network of hospitals, taking into account different levels: intra-ward and inter-wards and inter-hospitals. This is to establish a research methodology based on mathematical and computer modeling and supported by data collected in the Paris Île de France Ouest (PIFO), to better understand the role played by each hospital in the emergence and selection of BMR, to quantify the risk of their dissemination (including in the general population), and to identify effective control measures. This work will be based on statistical inference methods, analytical sensitivity and uncertainty analysis.

Norovirus som orsakar vinterkräksjukan är en av de vanligaste vintersjukdomarna i Sverige. Sjukdomstiden varar generellt i en till tre dagar med symptomen kräkning och/eller diarré. Till den totala sjukdomsbilden världen över gällande akut gastroenterit, bidrar norovirus med 18 %. Trots att sjukdomen är mycket vanlig är kunskapen om norovirusets förfarande till stor del okänd.Syftet med studien var att göra en tidsserieanalys, även så kallad One-Step Growth analys, av koncentrationen minus-RNA i celler som infekterats med olika koncentrationer av murint norovirus (MNV). För att detektera minus-RNA användes RT-qPCR med SYBR Green. Målet var att se om startkoncentrationerna av virus vid någon tidpunkt korrelerar med mängden minus-RNA i cellerna. Efter 4 och 8 timmar fanns ett exponentiellt samband mellan den initiala viruskoncentrationen och minus-RNA-uttrycket i cellerna. Koncentrationen minus-RNA i de infekterade cellerna ökade mellan de undersökta tiderna 4, 8 och 24 timmar. Vidare visade resultaten att det krävs 4 timmar för att minus-RNA skulle vara kvantifierbar vid en högre infektionskoncentration av viruspartiklar, medan det krävs 24 timmar för den lägre infektionskoncentrationen av viruspartiklar. / Norovirus causes winter vomiting disease and is one of the commonest cause of winter illness in Sweden. The disease period generally lasts one to three days with symptoms like vomiting and/or diarrhea. To the disease burden of acute gastroenteritis worldwide, norovirus contributes with 18 %. Even though the illness is very common, the knowledge about norovirus is poor and largely unknown.The purpose of the study was to do a time series analysis, a so-called One-Step Growth analysis, of the minus-RNA concentration in cells infected with different concentrations of murine norovirus (MNV). For the detection of minus-RNA RT-qPCR was used with SYBR Green. The goal was to correlate start concentration of virus at any time with the amount of minus-RNA in the cells. At 4 and 8 hours there was an exponential connection by the initial virus concentration and minus-RNA development in the cells. The concentration of minus-RNA in the infected cells increased between 4, 8 and 24 hours. Further, the results can be interpreted as requiring 4 hours for the higher concentrations to become quantifiable, while requiring 24 hours for the lower concentrations to become quantifiable.

Noroviren gelten neben Rotaviren, Salmonellen und Campylobacter spp. derzeit als Hauptursache infektiöser meldepflichtiger Gastroenteritiden des Menschen in Deutschland. Im Jahr 2008 wurden 212.692 Fälle norovirusbedingter Erkrankungen gemeldet (RKI 2009b). Dabei spielen lebensmittelassozierte Infektionen eine bedeutende Rolle. Besonders rohe und unerhitzt verzehrte Produkte bergen ein Risikopotential. Neben Muscheln, Salaten und Früchten konnten die Erreger auch aus Rohwurstprodukten isoliert werden. Aus mikrobiologischer Sicht sind Rohwürste bereits als Risikoprodukte bekannt. Bisher existieren jedoch nur unzureichende wissenschaftliche Ergebnisse über die Infektionsgefahr durch Viren in Lebensmitteln. In der vorliegenden Arbeit wurde die Tenazität und Inaktivierungskinetik von Noroviren anhand des Norovirus-Surrogates Felines Calicivirus untersucht. Zunächst wurde in Suspensionsversuchen der Einfluss verschiedener Konzentrationen von D/L-Milchsäure (0,1; 0,15; 0,2; 0,3 und 0,4 %), Natriumchlorid (2; 6; 12 und 20 %) und Natriumnitrit (100; 150 und 200 ppm) über einen Zeitraum von 7 Tagen bei 4 und 20 °C auf das Virus geprüft. Anschließend wurden Versuchsreihen mit artifiziell kontaminierten Rohwürsten durchgeführt. Der Infektiositätsnachweis erfolgte im Crandell-Reese-Feline-Kidney-Zellkultursystem. In den Suspensionsversuchen zeigte sich eine konzentrations-, zeit- und temperaturabhängige Wirkung der geprüften Parameter. Signifikante Infektiositätsreduktionen ergaben sich bei 20 °C- Lagerung für D/L-Milchsäure-Konzentrationen ab 0,15 % und bei 4 °C -Lagerung ab 0,3 %. Natriumchlorid bewirkte signifikante Titerreduktionen bei 20 °C-Lagerung ab einer NaCl-Konzentration von 2 %, jedoch nicht unter Kühlbedingungen. Die Reduktion der Virusinfektiosität nahm mit steigender Natriumchlorid- bzw. D/L-Milchsäurekonzentration zu. Für praxisübliche Konzentrationen von Natriumnitrit konnte keine zusätzliche Reduktion der Infektiosität nachgewiesen werden. In den Versuchsreihen mit kurz- und langgereiften Rohwürsten erwies sich FCV als sehr stabil. In beiden Produkten konnte bis zum Ende des Versuchszeitraumes nach 21 bzw. 56 Tagen infektiöses Virus nachgewiesen werden. Somit könnten Rohwurstprodukte bei einer möglichen Kontamination mit Noroviren zum Zeitpunkt des Verzehrs ein Gesundheitsrisiko für den Verbraucher darstellen. Jedoch zeigten sich auch deutliche temperatur- und zeitabhängige Wirkungen. Ein positiver Einfluss auf die Inaktivierung ist durch die Anwendung von Reife- und Lagerungstemperaturen um 22 °C zu erwarten. Es kam zu einer kontinuierlichen Virustiterreduktion von insgesamt 1,6 log10 TCID50 / g (für kurzgereifte Produkte) bzw. 3,1 log10 TCID50 / g (für langgereifte Produkte). Eine Beeinflussung der Tenazität des Norovirus-Surrogates FCV durch spezifische Herstellungs- und Lagerungsbedingungen ist somit möglich. Insbesondere die gezielte Kombination einzelner Faktoren miteinander, wie NaCl- und Milchsäuregehalt der Rohwurst, in Verbindung mit spezifischer Lagerungstemperatur und –dauer führt zu effektiver Infektiositätsminderung potentiell enthaltener Viren. Die so erzielte Risikominimierung durch Kombination ausgewählter Parameter bedeutet für die Rohwurstproduktion eine sowohl effiziente, als auch praktikable Möglichkeit, die Lebensmittelsicherheit in Bezug auf virale Infektionserreger deutlich zu erhöhen.

Norovirus (NoV) is a leading causative agent of non-bacterial gastroenteritis in humans worldwide. NoV is genetically classified into five distinct genogroups in which genogroups I (GI), II, and rarely IV infect humans. Each genogroup is further subdivided into different genotypes. Previous local surveillance studies demonstrated that NoV GII, in particular the genotype 4 (GII/4) strain, is the predominant genogroup circulating in Hong Kong since 2001. Similar epidemiologic observations were also reported in the US, Europe, UK, Australia, and Japan, highlighting the enormous pandemic and epidemic potential of this genogroup. However, explanation for its predominance has been lacking. In this study, we demonstrated that NoV GII, comprised mostly of the GII/4 strain, showed an increased median viral RNA level in fecal specimens which was at least 100-fold higher than that of GI. The high level of viral shedding may confer greater opportunity for transmission of GII strains through the fecal-oral route. We also demonstrated that fecal viral RNA level correlated positively and independently with diarrhea duration in NoV GII/4 infections. The median fecal viral level in patients with protracted (last for &ge;4 days) diarrhea was 100-fold higher than that in patients with only limited diarrhea. Longer infectivity period may also confer greater opportunity for virus transmission through the fecal-oral route. Higher chance of transmission may result in more efficient person-to-person transmission and rapid dissemination, maintaining a high level of NoV GII persistence in the community. In summer 2006, a territory-wide gastroenteritis outbreak attributed to NoV has occurred with more than 3,000 cases of laboratory-confirmed NoV infections in Hong Kong. Phylogenetic analysis showed that the virus causing this unprecedented outbreak was a novel NoV GII/4 variant distinct from all previously reported global pandemic and local epidemic strains. In this 2006 variant, we identified two hypervariable regions when compared with previous local epidemic strains in 2005: protruding domain 2 (P2 domain) of viral protein 1 (VP1) and VP1-binding domain of VP2. We mapped frequent amino acid substitutions to the modeled antigenic loop regions of P2 domain. We also identified in carboxyl-terminus of VP1 an epidemiologically important, putative conformational epitope that alternates between two 3-amino acid signatures during pandemic NoV GII/4 strains evolution since 1995. Our findings reflect the rapid evolution of NoV GII/4 under immunological pressure and suggest that immune evasion might be a potential mechanism for pandemic NoV GII/4 strains emergence. Taken together, high level of fecal viral shedding, longer infectivity period, and periodic emergence of novel variant may underlie the global predominance of NoV GII. Further investigations are warranted to better understand the public health and biological importance of NoV GII. / Chan, Chi Wai. / Adviser: Wai K. Leung. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0818. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 124-143). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / School code: 1307.

Challenged by a huge and diverse antigenic stimulus, the intestinal mucosa has developed a unique immune system that mainly functions to maintain tolerance to innocuous antigens while retaining the ability to respond swiftly to pathogenic threats. Central to this specialised immune system are the Intraepithelial Lymphocytes (IELs). These cells are uniquely located between Intestinal Epithelial Cells (IECs) ready to respond to exogenous antigens in the intestinal lumen. The intestinal immune system is constantly influenced, not only by the commensal microbiota, but also by the nutritional status of the host and the availability of certain essential micronutrients that are derived from a healthy-balanced diet. Additionally, age has a significant impact on the efficiency of gut immunity in responding to infectious pathogens, as reflected by the increased burden of gastrointestinal infections at the extremes of age. In this thesis, using the Murine Norovirus (MNV) oral infection model, I aimed to characterize intestinal mucosal antiviral-responses with specific focus on the role of IELs, the impact of aging and the influence of certain micronutrients whose effects are mediated through the Aryl Hydrocarbon Receptor (AhR). Employing different knock-out and adoptive transfer experiments, I concluded that, at least in our experimental conditions and in a viral strain-specific manner, the activated IELs are not essential and may play a minor role in the protective response against MNV infection. This work also demonstrated that various MNV virus strains activate IELs differentially and for the first time (to our knowledge) revealed distinct abilities of these different Norovirus variants to infect IECs. Recognising an impaired response in old (2-year) mice, we were also able to identify a specific defect in the IFN-Lambda response of aged IECs. Furthermore, using the model of MNV infection to investigate the role of AhR signalling, the data I generated suggested a direct link between constitutive AhR signalling and innate interferon-mediated responses. These findings have uncovered a potential preventive/therapeutic targets for enhancing anti-viral responses.

Human norovirus (HuNoVs) are the most common enteric pathogen around the world that cause ~50% of foodborne illness of disease outbreaks annually. HuNoVs are the member of the Caliciviridae family, which consist of small (38 nm), unenveloped, single stranded RNA (ssRNA) viruses. Norovirus are divided into 5 genogroup (GI, GII, GIII, GIV, GV, GVI and GVII). The GI, GII, and GIV cause human illness, in addition, GII.4 genotype cause the most human disease. Due to HuNoVs are difficult cultured in vitro, the cultivable HuNoVs surrogates have been widely studied. Recently, some studies have been conducted with HuNoVs surrogates, for example bacteriophage MS2. MS2 is conservative surrogate for nonenveloped viruses which there is a close relationship to the behavior of HuNoVs, thus we can examine the infection control measures for HuNoVs. Despite plenty of treatment method been done on testing antiviral effect on bacteriophage MS2, for example UV inactivation, steam ultrasound and antimicrobial etc., plant-based nanoemulsion treatment has yet to be explored. Carvacrol is a major component of oregano essential oil and is responsible for their antimicrobial activity on the growth of various microorganism. In this study, carvacrol nanoemulsions were formed by using the spontaneous emulsification for testing the nanoemulsion stability (14 days shelf life study on its droplet size and particle charge) and antimicrobial activity. In carvacrol nanoemulsion 14 days shelf life test, the droplet size and particle charge stay stable at three different treatment environments (4°C, 20°C and 37°C). The results proved that nanoemulsion (was formed with surfactant agents and medium-chain triglycerides) is stable system that gives consistent droplet size and charge. Although, the low antimicrobial activity was investigated at carvacrol nanoemulsion, the strong antimicrobial effects have been found when carvacrol or carvacrol combined with ionic surfactant of treatment on MS2 and Escherichia coli. Taken together, in the wake of growing consumer demand for different “natural” products in a number of industries, our study broadly informs the development and study of functionalized carvacrol active compound that can not only provide beneficial health for human but can also examine antimicrobial efficacy of control measures for public health.