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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

A genética forense tem grande importância na geração de provas em casos de violência sexual, paternidade criminal, identificação de cadáveres e investigação de evidências de locais de crime. A análise de STRs apresenta grande poder de discriminação, mas é uma metodologia multi-etapas, trabalhosa, cara e em muitos casos a análise genética é prejudicada pela baixa quantidade e qualidade das evidências coletadas. Este estudo teve como objetivo desenvolver e caracterizar uma metodologia de triagem de amostras forenses através da análise de perfis de dissociação em alta resolução (HRM) de regiões do DNA mitocondrial, o qual está presente em maior número de cópias e mais resistente a degradação. Para tanto, foram extraídos DNAs de 68 doadores. Estas amostras foram sequenciadas e analisadas por HRM para sete alvos no DNA mitocondrial. Também foram realizados ensaios para determinar a influência do método de extração, da concentração e nível de degradação do DNA no perfil de HRM obtido para uma amostra. Os resultados demonstraram a capacidade da técnica de excluir indivíduos com sequências diferentes da referência comparativa em cinco regiões amplificadas. Podem ser analisadas em conjunto, amostras de DNA com variação de concentração de até a ordem de 100 vezes e extraídas por diferentes metodologias. Condições de degradação de material genético não prejudicaram a obtenção de perfis de dissociação em alta resolução. A sensibilidade da técnica foi aprimorada com a análise de produtos de amplificação de tamanho reduzido. A fim de otimizar o ensaio foi testada a análise de HRM em reações de PCR duplex. Um dos pares de amplificação forneceu perfis de HRM compatíveis com resultados obtidos de reações com amplificação de apenas um dos alvos. Através da análise conjunta das cinco regiões, esta metodologia visa a identificação de indivíduos não relacionados com as referências comparativas, diminuindo o número de amostras a serem analisadas por STRs, reduzindo gastos e aumentando a eficiência da rotina de laboratórios de genética forense. / The forensic genetics has an important role in the generation of evidence in cases of sexual assault, criminal paternity, identification of corpses and crime scenes investigation. The analysis of STRs has great power of discrimination, but it is a multi-stage methodology, complex, expensive and in many cases the genetic analysis is hampered by the low quantity and quality of evidence collected. This study aimed to develop and characterize a forensic samples screening methodology to examine high resolution melting profiles (HRM) of regions of the mitochondrial DNA, which is present in more copies and more resistant to degradation. Thus, we extracted DNA from 68 donors. These samples were sequenced and analyzed by HRM to seven mitochondrial DNA targets. Tests were also conducted to determine the influence of extraction method, concentration and DNA degradation level of HRM profile obtained for a sample. The results demonstrated the technical ability to exclude individuals with different sequences of comparative reference amplified in five regions. Can be analyzed together samples with varying concentration to the order of 100 times and extracted by different methods. Genetic material degradation conditions did not prevent obtaining high resolution melting profiles. The sensitivity of the technique was improved with the analysis of reduced size amplification products. In order to optimize the assay HRM analysis was tested in duplex PCR reactions. A pair of amplification provided HRM profiles consistent with results from amplification in reactions with only one of the targets. Through the joint analysis of the five regions, this approach aims to identify individuals not related to comparative references, reducing the number of samples to be analyzed by STRs, reducing costs and increasing the efficiency of the routine of forensic genetics laboratories.

A genética forense tem grande importância na geração de provas em casos de violência sexual, paternidade criminal, identificação de cadáveres e investigação de evidências de locais de crime. A análise de STRs apresenta grande poder de discriminação, mas é uma metodologia multi-etapas, trabalhosa, cara e em muitos casos a análise genética é prejudicada pela baixa quantidade e qualidade das evidências coletadas. Este estudo teve como objetivo desenvolver e caracterizar uma metodologia de triagem de amostras forenses através da análise de perfis de dissociação em alta resolução (HRM) de regiões do DNA mitocondrial, o qual está presente em maior número de cópias e mais resistente a degradação. Para tanto, foram extraídos DNAs de 68 doadores. Estas amostras foram sequenciadas e analisadas por HRM para sete alvos no DNA mitocondrial. Também foram realizados ensaios para determinar a influência do método de extração, da concentração e nível de degradação do DNA no perfil de HRM obtido para uma amostra. Os resultados demonstraram a capacidade da técnica de excluir indivíduos com sequências diferentes da referência comparativa em cinco regiões amplificadas. Podem ser analisadas em conjunto, amostras de DNA com variação de concentração de até a ordem de 100 vezes e extraídas por diferentes metodologias. Condições de degradação de material genético não prejudicaram a obtenção de perfis de dissociação em alta resolução. A sensibilidade da técnica foi aprimorada com a análise de produtos de amplificação de tamanho reduzido. A fim de otimizar o ensaio foi testada a análise de HRM em reações de PCR duplex. Um dos pares de amplificação forneceu perfis de HRM compatíveis com resultados obtidos de reações com amplificação de apenas um dos alvos. Através da análise conjunta das cinco regiões, esta metodologia visa a identificação de indivíduos não relacionados com as referências comparativas, diminuindo o número de amostras a serem analisadas por STRs, reduzindo gastos e aumentando a eficiência da rotina de laboratórios de genética forense. / The forensic genetics has an important role in the generation of evidence in cases of sexual assault, criminal paternity, identification of corpses and crime scenes investigation. The analysis of STRs has great power of discrimination, but it is a multi-stage methodology, complex, expensive and in many cases the genetic analysis is hampered by the low quantity and quality of evidence collected. This study aimed to develop and characterize a forensic samples screening methodology to examine high resolution melting profiles (HRM) of regions of the mitochondrial DNA, which is present in more copies and more resistant to degradation. Thus, we extracted DNA from 68 donors. These samples were sequenced and analyzed by HRM to seven mitochondrial DNA targets. Tests were also conducted to determine the influence of extraction method, concentration and DNA degradation level of HRM profile obtained for a sample. The results demonstrated the technical ability to exclude individuals with different sequences of comparative reference amplified in five regions. Can be analyzed together samples with varying concentration to the order of 100 times and extracted by different methods. Genetic material degradation conditions did not prevent obtaining high resolution melting profiles. The sensitivity of the technique was improved with the analysis of reduced size amplification products. In order to optimize the assay HRM analysis was tested in duplex PCR reactions. A pair of amplification provided HRM profiles consistent with results from amplification in reactions with only one of the targets. Through the joint analysis of the five regions, this approach aims to identify individuals not related to comparative references, reducing the number of samples to be analyzed by STRs, reducing costs and increasing the efficiency of the routine of forensic genetics laboratories.

Hepatitis E virus (HEV) is a major cause of enterically transmitted acute viral hepatitis in developing countries that lack proper hygienic infrastructure. Hepatitis E is globally distributed and has emerged as an important public health disease in both developing and industrialized countries. HEV is a non-enveloped virus carrying a single-stranded positive-sense RNA genome of approximately 7.200 bp in length. The life cycle of HEV is poorly understood due to the lack of an efficient cell culture system. Animal model systems, including non-human primates, swine, and chickens are being used to study some fundamental aspects of the HEV biology. Recently, novel animal strains of rat and rabbit HEV have been discovered, and whose usage as animal model systems needs to be established. HEV infections in pigs and chickens provide excellent model systems to study the replication and pathogenesis aspects of HEV. Recently, we identified a hypervariable region (HVR) in the open reading frame 1 (ORF1) of HEV. The objectives of this dissertation were to utilize chicken and swine model systems to study the role of HVR in HEV infection in vivo, to determine the effects of HVR on replication of HEV in vitro, and to analyze the effect of exchange of HVR among different genotypes on the replication-competency and virion production in vitro. Extensive sequence variability in the HVR among HEV strains of different genotypes prompted us to study the dispensability of this region. Initially we constructed two partial deletion mutants of genotype 1 human HEV, hHVRd1 and hHVRd2, with in-frame deletion of amino acids (aa) 711 to 777 and 747 to 761 in the HVR of a sub-genomic GFP HEV replicon. Expression of enhanced green fluorescent protein by the mutant hHVRd2 confirmed the dispensability of amino acid residues 747-761 of the HVR. To confirm our in vitro results, specific-pathogen-free (SPF) chickens were intra-hepatically inoculated with capped RNA transcripts from three avian HEV HVR-deletion mutants: mutants aHVRd1 (Δ557-585), aHVRd2 (Δ612-641), and aHVRd3 (Δ557-641). Chickens intra-hepatically inoculated with the mutants, aHVRd1 and aHVRd2, developed active viral infection as evidenced by seroconversion, viremia, and fecal virus shedding. Mutant aHVRd3, with a larger HVR deletion, was apparently attenuated in chickens. Additionally, we used the swine model system to further verify our results from the chicken study. The infectivity of four genotype 3 swine HEV HVR-deletion mutants, sHVRd1 (Δ712-790), sHVRd2 (Δ722-781), sHVRd3 (Δ735-765), and sHVRd4 (Δ712-765) constructed using the genotype 3 swine HEV as the backbone was determined in SPF pigs. Pigs intra-hepatically inoculated with capped RNA transcripts from the mutants sHVRd2, sHVRd3, and sHVRd4 developed active viral infection, whereas mutant sHVRd1 (Δ712-790), with a nearly complete HVR deletion, exhibited an attenuation phenotype. The data from these studies indicate that deletions in HVR do not abolish HEV infectivity in vitro or in vivo, although evidence for attenuation was observed for HEV mutants with a larger or nearly complete HVR deletion. To further elucidate the role of HVR in HEV replication, we investigated the effects of serial amino acid deletions in HVR on the replication of HEV. We first constructed a genotype 1 human HEV luciferase replicon by replacing the ORF2 gene that encodes for the capsid protein with the fire fly luciferase reporter gene. Using the backbone of human HEV genotype 1 luciferase replicon, we constructed a series of HVR-deletion mutants with deletions of variable lengths in the HVR. Amino acid deletions Δ711-725, 711-740 and Δ711-750 were engineered at the N-terminus, deletions Δ729-754, Δ721-766, and Δ716-771 were engineered in the central region, and deletions Δ761-775, Δ746-775, and Δ736-775 were engineered at C-terminus of the HVR. The effects of these serial deletions on HEV RNA replication in the human liver carcinoma cell line, Huh7, were examined. Replication levels of mutants carrying these deletions were compared with that of the wild-type HEV in Huh7 cells. We observed that deletions in the HVR did not abolish viral RNA synthesis but substantially reduced the replication levels of viral RNA, as measured by the reporter luciferase activity. To further verify the effects of HVR deletions on viral RNA replication as observed with the genotype 1 human HEV replicon, we subsequently used a genetically-distinct strain of HEV, avian HEV, and constructed an avian HEV sub-genomic luciferase replicon by substituting the ORF2 gene of avian HEV with the fire fly luciferase gene. Avian HEV HVR-deletion mutants Δ557-603, Δ566-595, and Δ573-587 were then engineered using the backbone of avian HEV luciferase replicon. The replication efficiency of the three deletion mutants of avian HEV in chicken liver hepatoma cell line, LMH, was evaluated. Compared with the wild-type avian HEV, the viral RNA synthesis of the avian HEV HVR-deletion mutants was considerably reduced by the HVR deletions. To analyze the impact of the complete HVR deletion on avian HEV infectivity, we constructed an avian HEV mutant with a deletion of the entire HVR region (aaΔ557-603) using the avian HEV infectious cDNA clone as the backbone. After confirming the viability of the complete HVR-deletion mutant in LMH cells, SPF chickens were intrahepatically inoculated with capped RNA transcripts generated from the mutant. None of the chickens inoculated with the complete HVR-deletion mutant showed evidence of HEV infection, indicating that drastic reduction in replication levels due to complete HVR deletion has resulted in the loss of virus infectivity. The results indicated that HVR may have critical residues that may interact with viral/and or host factors and modulate the replication efficiency of HEV. In the final part of the dissertation research, we sought to determine if the variable sequences of HVR are genotype-specific for in vitro virus replication. By using the genotype 1 human HEV as the backbone, we swapped the HVR of genotype 1 human HEV with the HVRs of the genotype 3 swine HEV and the distantly-related avian HEV to construct two inter-genotypic chimeras, pSKHEV2-Sw and pSKHEV2-Av. Similarly, by using the genotype 3 swine HEV as the backbone, the HVR of genotype 3 swine HEV was swapped with the HVR of genotype 1 human HEV to construct the chimera, pSHEV3-Hu. The viability of these chimeras was tested in Huh7 cells that are permissive for HEV replication. Immunofluorescence assay (IFA) with anti-HEV antibodies revealed that all the three chimeras were replication-competent in Huh7 cells. The infectivity of these chimeras was subsequently evaluated in HepG2 cells. The results showed that exchange of the HVR between different genotypes of mammalian HEVs does not abolish the replication competency and infectivity of HEV. This finding suggests that HVR is not genotype-specific with respect to viral replication and infectivity. The absence of detectable viral antigen in HepG2 cells infected with chimera pSKHEV2-Av suggested a functional incompatibility of the HVR of avian HEV in the mammalian HEV genome. In summary, we identified a highly variable sequence, HVR, in the ORF1 of the HEV genome, and the sequences of the HVR vary significantly among HEV strains of different genotypes. We found that the HVR contain sequences that are dispensable for virus infectivity both in vitro and in vivo. Deletion analysis of HVR revealed that the region may play a role in modulating the replication efficiency of HEV RNA by interacting with viral and/or host factors. Finally, we demonstrated that HVR is not genotype-specific for virus replication and the region can be functionally replaced between mammalian HEV genotypes for virus replication and virion production in vitro. The results from this dissertation research have important implications for better understanding the biology and mechanism of HEV replication and may aid in our efforts to eventually develop a modified live-attenuated vaccine against HEV. / Ph. D.