Hepatitis E virus (HEV), the causative agent of hepatitis E, is an emerging virus of global distribution. At least four distinct genotypes of HEV exist worldwide: genotype 1 and 2 HEV strains are responsible for waterborne epidemics; genotype 3 and 4 HEV strains are responsible for sporadic occurrences of acute hepatitis E. Genotype 3 and 4 HEVs are zoonotic and have a more expanded host range than genotypes 1 and 2 which are restricted to humans. Genotype 3 and 4 HEV isolates obtained from animal tissues are genetically very similar, or identical in some cases, to human HEV recovered from hepatitis E patients. The objectives of this dissertation research were to assess the zoonotic foodborne transmission of HEV and elucidate the viral determinants of HEV host range. To determine the risk of HEV foodborne transmission, 127 packages of commercial pig liver were tested for HEV RNA. Eleven percent of them were positive for HEV RNA and the contaminating virus remained infectious. We also demonstrated that medium-to-rare cooking condition (56°C) does not completely inactivate HEV, although frying and boiling of the contaminated livers inactivated the virus. To reduce the risk of foodborne HEV transmission, commercial pig livers must be thoroughly cooked for consumption. To determine the host range of genotype 4 HEVs, pigs were inoculated with a genotype 4 human HEV. All pigs developed an active HEV infection indicating that genotype 4 human HEVs can cross species barriers and infect pigs. To identify viral determinant(s) of species tropism, ORF2 alone or in combination with its adjacent 5′ junction region (JR) and 3′ non-coding region (NCR), were swapped between genotypes 1 and 4, 3 and 4, and 1 and 3 to produce 5 chimeric viruses. Chimeric viruses containing ORF2 or JR+ORF2+3' NCR from genotype 4 human HEV in the backbone of genotype 3 swine HEV were viable in vitro and infectious in vivo. Chimeric viruses containing the JR+ORF2+3'NCR of genotypes 3 or 4 HEV in the backbone of genotype 1 human HEV were viable in vitro but non-infectious in pigs, suggesting that ORF1 may also be important for host range. / Ph. D.

HEV is an important public health concern due largely to water-borne outbreak. Recent research confirms individual cases of zoonotic transmission due to human exposure to contaminated animal meats. At least four recognized and two putative genotypes of mammalian HEV have been reported: genotypes 1 and 2 are restricted to humans whereas genotypes 3 and 4 are zoonotic. In addition to humans, strains of HEV have been genetically identified from pigs, chickens, rats, mongoose, deer, rabbits and fish. The current experimental vaccines are all based on a single strain of HEV, even though multiple genotypes of HEV are co-circulating in some countries and thus an individual may be exposed to more than one genotype. Therefore, it is important to know if prior infection with a genotype 3 swine HEV will confer protective immunity against subsequent exposure to genotypes 3 and 4 human and swine HEV. In the first study, specific-pathogen-free pigs were divided into 4 groups of 6 each. Pigs in the three treatment groups were each inoculated with a genotype 3 swine HEV, and 12 weeks later, challenged with the same genotype 3 swine HEV, a genotype 3 human HEV, and a genotype 4 human HEV, respectively. Sera from all pigs were tested for HEV RNA and IgG anti-HEV, and fecal samples were also tested for HEV RNA each week. The pigs inoculated with swine HEV became infected as evidenced by fecal virus shedding and viremia, and the majority of pigs also developed IgG anti-HEV prior to challenge at 12 weeks post-inoculation. After challenge, viremia and fecal virus shedding of challenge viruses were not detected, suggesting that prior infection with a genotype 3 swine HEV prevented pigs from developing viremia and fecal virus shedding after challenge with homologous and heterologous genotypes 3 and 4 HEV, respectively. Immunogenic epitopes are located within the open reading frame 2 (ORF 2) capsid protein and recombinant ORF 2 antigens are capable of preventing HEV infection in non-human primates and chickens. In the second study we expressed and purified N-truncated ORF 2 antigens based on swine, rat, and avian HEV strains. Thirty pigs were randomly divided into groups of 6 pigs each and initially vaccinated with 200µg swine ORF 2 antigen, rat ORF 2 antigen, avian ORF 2 antigen, or PBS buffer (positive and negative control groups) and booster with the same vaccine 2 weeks later. At 4 wks, after confirming seroconversion to IgG anti-HEV antibody with ELISA, all groups except the negative control were challenged with swine genotype 3 HEV (administered intravenously). The protective and cross-protective abilities of these antigens were determined following swine genotype 3 challenge by evaluating both serum and fecal samples for HEV RNA using nested RT-PCR and IgG anti-HEV using ELISA. The results from these two studies have important implications for future development of an effective HEV vaccine. As a part of our ongoing efforts to search for potential animal reservoirs for HEV, we tested goats from Virginia for evidence of HEV infection and showed that 16% (13/80) of goat sera from Virginia herds were positive for IgG anti-HEV. Importantly, we demonstrated that selected goat sera were capable of neutralizing HEV in cell culture. Subsequently, in an attempt to genetically identify the HEV-related agent from goats, we conducted a prospective study in a closed goat herd with known anti-HEV seropositivity and monitored a total of 11 kids from the time of birth until 14 weeks of age for evidence of HEV infection. Seroconversion to IgG anti-HEV was detected in 7 out of the 11 kids, although repeated attempts to detect HEV RNA by a broad-spectrum nested RT-PCR from the fecal and serum samples of the goats that had seroconverted were unsuccessful. In addition, we also attempted to experimentally infect laboratory goats with three well-characterized mammalian strains of HEV but with no success. The results indicate that a HEV-related agent is circulating and maintained in the goat population in Virginia and that the goat HEV is likely genetically very divergent from the known HEV strains. / Ph. D.

Hepatitis E Virus (HEV), the causative agent of hepatitis E, is a zoonotic pathogen of worldwide significance. The genus Orthohepevirus A of the family Hepeviridae includes all mammalian strains of HEV and consists of 8 recognized genotypes. Genotypes 1 and 2 HEVs only infect humans and genotypes 3 and 4 infect humans and several other animal species including pigs and rabbits. An ever-expanding host range of genetically-diversified strains of HEV now include bat, fish, rat, ferret, moose, wild boar, mongoose, deer, and camel. Additionally, the ruminant species goats, sheep, and cattle have been implicated as potential reservoirs as well. My dissertation research investigates a novel animal model for HEV, examines the immune dynamics during acute infection, and evaluates the possibility of additional animal reservoirs of HEV. The first project established an immunoglobulin (Ig) heavy chain knock-out JH (-/-) gnotobiotic piglet model that mimics the course of acute HEV infection observed in humans and evaluated the pathogenesis of HEV infection in this novel animal model. The dynamics of acute HEV infection in gnotobiotic pigs were systematically determined with a genotype 3 human strain of HEV. We also investigated the potential role of immunoglobulin heavy-chain JH in HEV pathogenesis and immune dynamics during the acute stage of virus infection. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems. The objective of the second project for my PhD dissertation was to determine if cattle in the United States are infected with a bovine strain of HEV. We demonstrated serological evidence of an HEV-related agent in cattle populations with a high level of IgG anti-HEV prevalence. We demonstrated that calves from a seropositive cattle herd seroconverted to IgG binding HEV during a prospective study. We also showed that the IgG anti-HEV present in cattle has an ability to neutralize genotype 3 human HEV in vitro. However, our exhaustive attempts to detect HEVrelated sequence from cattle in the United States failed, suggesting that one should be cautious in interpreting the IgG anti-HEV serological results in bovine and other species. Collectively, the work from my PhD dissertation delineated important mechanisms in HEV pathogenesis and established a novel animal model for future HEV research. / Ph. D. / Hepatitis E Virus (HEV), the causative agent of hepatitis E, is a zoonotic pathogen of worldwide significance. According to the World Health Organization, there are approximately 20 million HEV infections annually, which result in 3.3 million cases of acute hepatitis E and >44,000 HEV-related deaths. Hepatitis E is a self-limiting acute disease in general, but carries the ability to cause high mortality in pregnant women and chronic hepatitis in immunocompromised individuals. The underlying mechanisms of HEV host tropism and progression of disease to chronicity are unknown. My dissertation work investigates a novel animal model for HEV, evaluates the possibility of additional animal reservoirs of HEV, and examines the immune dynamics during acute infection. The first project established an immunoglobulin (Ig) heavy chain knock-out JH (-/-) gnotobiotic piglet model that mimics the course of acute HEV infection observed in humans. The dynamics of acute HEV infection were determined in both the knock-out and wild-type piglets with a genotype 3 strain of human HEV. We also investigated the potential role of immunoglobulin heavy-chain JH in HEV pathogenesis and virus infection. In the second project, we determined if cattle in the United States are infected with a bovine strain of HEV. We showed serological evidence of an HEV-related agent in cattle as well as calves born in a seropositive herd. Despite the detection of specific antibodies recognizing HEV in cattle, definitive evidence of virus infection could not be demonstrated. Our exhaustive attempts to detect HEV-related sequence from cattle in the United States failed, suggesting that one should be cautious in interpreting the IgG anti-HEV serological results in bovine and other species. Collectively, the work from my PhD dissertation research delineated important mechanisms in HEV pathogenesis and established a novel animal model for future HEV research.

Dans cette étude, la bile d’un porc canadien naturellement infecté par une souche du virus de l’hépatite E (VHE) a été utilisée afin d’inoculer deux groupes de porcelets. Dans l’étude précoce (E), 4 porcelets âgés de 4 semaines et exempts de pathogènes spécifiques (SPF), ont été suivis jusqu’à 14 jours post-inoculation (pi). Dans l’étude tardive (L), 9 porcelets ont été suivis à chaque semaine jusqu’à l’abattage, soit 120 jours pi. À la nécropsie, la présence du VHE a été évaluée dans différents organes à 7, 14 et 120 jours pi. Des porcelets témoins (E=2 et L=3) ont été inoculés par de la bile exempte de VHE. Le virus a persisté chez certains animaux jusqu’à 84 à 105 jours pi dans le sérum malgré la présence d’anticorps IgG anti-VHE dans le sang, suggérant une virémie prolongée. L’excrétion virale dans les fèces s’est étalée également sur une période de 105 jours pi chez certains animaux. De plus, la détection de l’ARN viral dans les organes évalués s’est révélée presque nulle à l’âge d’abattage à l’exception de quelques vésicules biliaires, alors qu’on retrouvait l’ARN viral dans plusieurs organes à 7 et 14 jours pi. Pour évaluer la distribution du VHE chez les porcs commerciaux du Québec, un échantillonnage de porcs de trois abattoirs a été réalisé. Environ 100 échantillons de sang, fèces, foies et bile provenant des mêmes animaux en processus d’abattage ont été prélevés dans chacun des abattoirs, sur des porcs destinés à la consommation humaine. La détection de l’ARN viral et des anticorps du VHE a été réalisée à l’aide d’une RT-PCR nichée et d’un test ELISA adapté pour déceler les anticorps porcins anti-VHE. Chez les porcs d’abattoir, 12,9 % des échantillons de bile contenaient de l’ARN viral du VHE, alors que la détection virale était moindre dans les autres organes. Une séroprévalence en IgG de 26,0 % a été obtenue pour les sérums porcins analysés. Une analyse phylogénétique des différentes souches isolées pendant l’étude a démontré qu’elles sont du génotype 3. Ces données indiquent une exposition potentielle des travailleurs de l’industrie porcine au VHE porcin, notamment par les fèces, le sang et les organes et également pour les consommateurs par le biais des foies. / In this study, a strain of porcine hepatitis E virus (HEV) isolated from the bile of a naturally-infected Canadian pig was used to inoculate two groups of piglets. In the early-phase experiment (E), 4 one month-old piglets, specific pathogen free (SPF), were monitored for 14 days. In the late-phase experiment (L) 9 piglets were monitored up to slaughter (120 days post-inoculation (pi)). Controls piglets (E=2 and L=3) were inoculated with free HEV bile. The presence of HEV was monitored routinely in their blood and feces. At necropsy, viral occurence was evaluated in organs at 7, 14 and 120 days pi. Interestingly, HEV was found to persist in the serum of some animals up to 84-105 days pi, despite the presence of IgG HEV antibodies in their blood. Fecal shedding was detected until 105 days pi for a portion of pigs. In organs, HEV RNA was detected in low amount of gallbladders at killing time, while it was detected in a large number of organs at 7 and 14 days pi. To assess the distribution of HEV in commercial finishing pig in Quebec, a sampling was realised in pigs from three slaughterhouses from Quebec. Approximately a hundred samples of feces, blood, bile and liver were collected in each slaughterhouse, on pigs intended for human consumption. A sample of each type was collected on each of the chosen pigs. Detection of HEV RNA was carried out using a nested RT-PCR on each sample and a human ELISA test was adapted for the detection of swine antibodies against HEV in swine serum samples. For pigs at slaughter, 12,9 % of the bile samples were positive to HEV RNA and a seroprevalence of IgG of 26,0 % was detected in swine. A phylogenetic analysis demonstrated that all strains of the study were in the genotype 3. All results demonstrate that porcine industry workers are potentially exposed to swine HEV by feces, blood and organs.

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