Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Infection with hepatitis A virus (HAV) occurs worldwide and is the most common cause of acute viral hepatitis. The highest prevalence of this infection is seen where low standards of sanitation are adopted. Acquired primarily by the fecaloral route, HAV infection is easily disseminated, either by person-to-person contact or by ingestion of contaminated food or water. The HAV is a extremely steady non-enveloped particle, which comprises a single stranded plus-sense RNA with approximately 7,5 kb. The objective of this study was to develop, based into VP1 gene sequence, a DNA probe from the HAV genome by RTPCR to detect the virus. A 783 bp amplicon of HAV genome (Brazilian standard strain HAF-203), amplified by RT-PCR was labelled by coupling of alkaline phosphatase enzyme from Gene ImagesTM AlkPhos DirectTM System (Amersham). Specificity was determined by dot blot hybridization of RNA from standard strain HAF-203 and DNA of the own amplicon. To asses the sensitivity of detection hybridization was done in serially diluted up to 1 pg of purified viral RNA. Sewage water samples were artificially ontaminated with dilutions up to 10 PFU/mL of the virus. The particles were precipitated with amonium sulfate and the total RNA obtained by treatment with proteinase k and phenol:chloroform. Samples were transferred to a nylon membrane by dot blot and hybridized according to labelling system manufacturer s instructions. Dots were detected in spots containing 1 pg of purified RNA, just like the amplicon. The DNA probe did not hybridize to total DNA prepared from BoHV-1, XL2B DNA and Human total RNA. The probe hybridized to samples containing up to 100 PFU/mL of the virus. This results showed the probe specificity and sensitivity level is sufficient to detect the virus in environmental samples, making this technique able to be used in environmental molecular monitoring of the virus. / Infecção pelo vírus da hepatite A (HAV) acontece mundialmente e é a causa mais comum de hepatites virais agudas. A prevalência mais alta desta infecção é observada onde baixos padrões de serviço de saúde pública são adotados. Adquirida principalmente pela rota fecal-oral, a infecção por HAV é disseminada facilmente, através de contato de pessoa-para-pessoa ou por ingestão de comida ou água contaminada. O HAV é uma partícula não envelopada, extremamente estável que possui RNA fita simples de sentido positivo com aproximadamente 7,5 kb. O objetivo deste estudo foi desenvolver, baseado na seqüência do gene VP1, uma sonda de DNA a partir do genoma de HAV por RT-PCR para detectar o vírus. Um amplicon de 783 bp do genoma de HAV (amostra padrão HAF-203), amplificado por RT-PCR foi marcado pela enzima fosfatase alcalina do sistema Gene ImagesTM AlkPhos DirectTM (Amersham). A especificidade foi determinada por hibridização em dot blot com RNA da amostra padrão brasileira HAF-203 e DNA do próprio amplicon. Para testar a sensibilidade de detecção por hibridização foi realizada diluição seriada até 1 pg de RNA viral purificado. Foram contaminadas artificialmente amostras de água de esgoto com diluições até 10 PFU/mL do vírus. As partículas foram precipitadas com sulfato de amônia e RNA total obtido através de tratamento com proteinase k e fenol:clorofórmio. Amostras foram transferidas para uma membrana de nylon por dot blot e hibridizadas de acordo com as instruções do fabricante do sistema. A sonda detectou amostras nos poços contendo até 1 pg de RNA purificado, da mesma forma que o amplicon. A sonda de DNA não hibridizou com DNA preparado de BoHV-1, XL2B DNA e RNA total Humano. A sonda hibridizou com amostras que contêm até 100 PFU/mL do vírus. Estes resultados mostram que a os níveis de especificidade e sensibilidade da sonda é suficiente para detectar o vírus em amostras ambientais, tornando esta técnica aplicável no monitorando molecular ambiental do vírus.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O vÃrus da hepatite C à considerado como o principal agente causador de hepatite nÃo-A, nÃo-B e, desde sua descoberta em 1989, tem sido reconhecido como a principal causa de doenÃa crÃnica do fÃgado em todo o mundo. O VHC possui um genoma de RNA de sentido positivo e à classificado como um flavivÃrus. O vÃrus apresenta considerÃvel variabilidade em sua seqÃÃncia e as variantes do VHC podem ser divididas em seis genÃtipos principais (numerados de 1 a 6) e diversos subtipos. A distribuiÃÃo dos genÃtipos varia tanto geograficamente, quanto entre os grupos de risco. Este estudo teve como objetivo analisar a distribuiÃÃo dos genÃtipos do VHC entre pacientes atendidos em um hospital de referÃncia em Fortaleza, CearÃ. Cento e vinte pacientes com sorologia positiva para anti-VHC ou histÃria clÃnica sugestiva de infecÃÃo pelo vÃrus foram estudados. O RNA viral foi extraÃdo a partir do soro dos pacientes e a detecÃÃo molecular do vÃrus foi feita por PCR, utilizando iniciadores especÃficos para a regiÃo 5 (linha) nÃo-codificadora do genoma viral. A genotipagem foi baseada em tÃcnica de RFLP utilizando as enzimas de restriÃÃo HaeIII, RsaI, MvaI e HinfI. Noventa e seis pacientes (80%) foram positivos no teste qualitativo para VHC. A mÃdia de idade desses pacientes foi de 44 anos. HistÃria clÃnica de cirurgia foi o fator de risco mais presente, seguido por transfusÃo sangÃÃnea, DST, uso de drogas, tatuagem, diÃlise e exposiÃÃo ocupacional. A relaÃÃo entre o resultado do teste qualitativo e o uso de drogas apresentou significÃncia estatÃstica, com 96% dos usuÃrios de drogas positivos para VHC. NÃo houve diferenÃa significativa no resultado do teste qualitativo quando transfusÃes sangÃÃneas feitas antes ou depois de 1993 foram analisadas. ManifestaÃÃes clÃnicas ou Ãndices de ALT alterados tambÃm nÃo foram preditivos de positividade para VHC. O genÃtipo 1 foi o mais prevalente na populaÃÃo estudada (46,9%), seguido pelos genÃtipos 3 (34,4%) e 2 (8,3%). O genÃtipo 4 viral foi detectado em um paciente. Em nove amostras nÃo foi possÃvel a genotipagem do VHC. Esses casos foram denominados indeterminados. CaracterÃsticas epidemiolÃgicas como idade, sexo, fatores de risco, Ãndices de ALT e manifestaÃÃes clÃnicas foram relacionadas com os diversos genÃtipos virais. A distribuiÃÃo dos genÃtipos virais entre as categorias estudadas ocorreu de forma homogÃnea na maioria dos casos. Foi observada significÃncia estatÃstica apenas na relaÃÃo entre genÃtipo e exposiÃÃo ocupacional ao VHC, com predominÃncia do genÃtipo 1 neste grupo de risco. NÃo foram observadas co-infecÃÃes com mais de um genÃtipo viral. / Hepatitis C virus is considered as the main causative agent of non-A non-B hepatitis and has been recognised as the major cause of chronic liver disease worldwide, since its discovery in 1989. It has a positive-sense RNA and belongs to the flavivirus family. The HCV shows considerable sequence variability and its variants can be divided into six main genotypes (numbered from 1 to 6) and several subtypes. The genotypes distribution varies from the geographic area and among risk groups. This study had the main aim to study HCV genotypes distribution in patients from a reference hospital in Fortaleza, Ceara. It was investigated a hundred and twenty patients with anti-HCV positive or clinical history suggesting virus infection. Viral RNA was extracted from patients serum and the virus molecular detection was made by PCR, using specific primers to the 5â non-coding region of viral genome. Genotyping was based in a RFLP technique, using the restriction enzymes HaeIII, RsaI, MvaI and HinfI. Ninety six patients (80%) were positive in the qualitative test for HCV. The mean age of these patients was 44 years old. Surgery clinical history was the most frequent risk factor, followed by blood transfusion, sexual transmitted disease, drugs use, tattoos, dialysis, and occupacional exposure. The relation between qualitative test result and drug users showed statistical significance, with 96% of drugs users being positive for HCV. There was no significant difference in qualitative test result when we analysed blood transfusions done after or before the year 1993. Clinical symptoms and ALT levels also were not predictive of HCV positive result. Genotype 1 was the most prevalent in the studied population (46,9%), followed by genotype 3 (34,4%) and 2 (8,3%). There was also a molecular characterization of one patient with genotype 4 whereas nine samples were not HCV genotyped and were called as undetermined. The epidemiological characteristics such as age, gender, risk factors, ALT levels and clinical manifestations were related with viral genotypes. The HCV genotypes had a homogeneous distribution, in the majority of cases, among the different categories. A statistical significance was observed only when genotype 1 was related to HCV occupational exposure. Co-infections with more than one viral genotype were not observed.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-11-27T19:29:54Z No. of bitstreams: 2 Dissertação - Ágabo Macêdo da Costa e Silva- 2014.pdf: 1890977 bytes, checksum: 8a4d7bcee9ab084556c93c3c72e6a9f8 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Cláudia Bueno (claudiamoura18@gmail.com) on 2014-12-01T14:46:26Z (GMT) No. of bitstreams: 2 Dissertação - Ágabo Macêdo da Costa e Silva- 2014.pdf: 1890977 bytes, checksum: 8a4d7bcee9ab084556c93c3c72e6a9f8 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-12-01T14:46:26Z (GMT). No. of bitstreams: 2 Dissertação - Ágabo Macêdo da Costa e Silva- 2014.pdf: 1890977 bytes, checksum: 8a4d7bcee9ab084556c93c3c72e6a9f8 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-02-28 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Hepatitis C represents a public health problem worldwide. Hepatitis C virus (HCV) is classified into seven genotypes and many subtypes. Besides their epidemiological importance, these genotypes have influence on the response to hepatitis C treatment, as well as other factors related to the virus and its host, such as polymorphisms upstream of interleukin (IL) 28B locus. Despite the importance of these factors in the response to treatment of hepatitis C, there are no data regarding the subject in the Midwest Region of Brazil. The present study aimed to identify the genotypes of HCV among patients attended at a reference laboratory in Goiânia-GO, and also to assess response to treatment of patients infected with genotype 1 with pegylated interferon (PEG-IFN) and ribavirin (RBV), with emphasis on the polymorphism upstream of IL28B gene (rs 12979860). For HCV genotyping, a cross-sectional study was conduct in an anti-HCV positive patients in a reference laboratory in Goiânia-GO, during in a period of 10 years (2003 to 2012) (n = 1300). From January/2012 to December/2013 (n = 101), a cohort was conduct among patients infected with HCV genotype 1 treated with PEG-IFN and RBV in order to evaluate treatment response. Patients were interviewed and blood samples collected for detection of viral RNA by polymerase chain reaction (PCR) with primers complementary to the region 5’ noncoding (NC) of HCV. All HCV RNA positive samples were genotyped by line probe assay – LiPA, and the samples typed as 1a/1b, 2 and 4 were sequenced in the NS5B region of the viral genome. The analysis of single nucleotide polymorphism (SNP) rs12979860 C/T was performed by restriction fragment length polymorphism (RFLP). Of the 1300 samples tested for HCV RNA, 894 were positive. Among these, genotypes 1 (76.3%), 2 (1.9%), 3 (21.6%) and 4 (0.2%) were found. These data show the predominance of genotype 1, followed by 3 and 2, and support the results of other studies in Goiânia. In addition, genotype 4 was found in the studied population. Regarding to SNP rs12979860 C/T, the TC genotype was the most common (57.4%), followed by CC (23.8%) and TT (18.8%). The rate of sustained virologic response (SVR) was 28.7%, being higher in individuals with the CC genotype (54.2%) when compared to those with CT and TT genotypes (22.4% and 15.8%, respectively). These results indicate that analysis of SNP rs12979860 is an important predictor of SVR following PEG-IFN and RBV treatment in patients infected with HCV genotype 1 in our region. / A hepatite C representa um problema de saúde pública mundial. O vírus da hepatite C (HCV) é classificado em sete genótipos e vários subtipos. Além da importância epidemiológica, os genótipos desse vírus influenciam a resposta ao tratamento da hepatite C, assim como outros fatores relacionados ao vírus e ao hospedeiro, como o polimorfismo relacionado ao gene da interleucina (IL) 28B. Apesar da importância desses fatores na resposta ao tratamento da hepatite C, não existem dados sobre o tema na Região Centro-Oeste. O presente estudo teve como objetivos identificar os genótipos do HCV em pacientes atendidos em laboratório de referência em Goiânia-GO e avaliar a resposta ao tratamento com interferon peguilado (PEG IFN) e ribavirina (RBV) em portadores do genótipo 1, com ênfase no polimorfismo relacionado ao gene IL28B (rs 12979860). Foi realizado um estudo de corte transversal para genotipagem do HCV em pacientes anti-HCV reagentes atendidos em laboratório de referência em Goiânia-GO, em um período de 10 anos (2003 a 2012) (n = 1300). Entre janeiro/2012 e dezembro/2013 (n = 101), foi formada uma coorte de portadores do genótipo 1 do HCV submetidos ao tratamento da hepatite C com PEG IFN e RBV, para avaliação de sua resposta. Os pacientes foram entrevistados e amostras sanguíneas coletadas para detecção do RNA viral por reação em cadeia da polimerase (PCR) com iniciadores complementares a região 5’ não codificante (NC) do HCV. Todas as amostras RNAHCV positivas foram genotipadas por line probe assay – LiPA, e as tipadas como 1a/1b, 2 e 4 foram sequenciadas para a região NS5B do genoma viral. A análise do single nucleotide polymorphism (SNP) rs12979860 C/T foi realizada por restriction fragment length polymorphism (RFLP). Das 1300 amostras testadas para RNA-HCV, 894 foram positivas, sendo identificados os genótipos 1 (76,3%), 2 (1,9%), 3 (21,6%) e 4 (0,2%). Estes dados mostram o predomínio do genótipo 1, seguido do 3 e 2, e corroboram os de outros estudos realizados em Goiânia, com a adição do genótipo 4 na população estudada. Em relação ao SNP rs12979860 C/T, o genótipo TC foi o mais frequente (57,4%), seguido por CC (23,8%) e TT (18,8%). A taxa de resposta virológica sustentada (RVS) foi de 28,7%, sendo superior nos portadores do genótipo CC (54,2%) em relação aos genótipos TC e TT (22,4% e 15,8%, respectivamente). Estes resultados indicam que a análise do SNP rs12979860 é importante preditor de RVS ao tratamento com PEG IFN e RBV em pacientes infectados com o genótipo 1 do HCV em nossa região.

Made available in DSpace on 2015-04-11T13:54:59Z (GMT). No. of bitstreams: 1 soriane.pdf: 3202394 bytes, checksum: 55f87924522756721f3a0349e37b2bac (MD5) Previous issue date: 2013-09-17 / Fundação de Amparo à Pesquisa do Estado do Amazonas / Introduction: Hepatitis C virus (HCV) infection is the major cause of liver disease worldwide. Approximately 170 million more people are infected with the virus . The immune response against HCV leads to secretion of cytokines and activation and proliferation of effector T cells to act in infected hepatocytes and thus initiate the inflammatory process in the liver. Objective: This study aimed to characterize the immune response and to estimate the frequency of polymorphism of TNF - α -308 G / A in patients with chronic hepatitis C treated at the Tropical Medicine Foundation Dr. Heitor Vieira Dourado. Material and Methods: In this study we used samples from 30 patients with hepatitis C virus infection and 28 samples from individuals not infected with HCV . The analysis of T lymphocyte and adhesion molecules were performed by flow cytometry. The cytokine levels were assessed by flow cytometry using the CBA kit. The characterization of TNF- α -308 polymorphism was obtained by PCR-RFLP. The fibrosis was determined after histological analysis of liver biopsy according to the METAVIR score. Results: Our data revealed that percentage of regulatory T lymphocytes (T regs) and cytokines are increased in patients with hepatitis C when compared to the control group. Patients with fibrosis ≥F3 (N = 10) was characterized relevant frequency ( 100%) of individuals with high production of IL-2 and low frequency (50%) of individuals with high production of IL-10 and patients with fibrosis ≤F2 was characterized relevant frequency ( 85%) of individuals with high production of IL-4 and frequency ( 55%) of individuals with high production of IL-10. Conclusion: The effector T cells are regulated by T lymphocytes regs. In the fibrosis ≥F3 was characterized Th1 cytokine profile while in fibrosis ≤F2 ws characterized TH2 cytokine profile. The low IL-10 production was associated an increased fibrogenic activity in patients with hepatitis C. / Introdução: A infecção pelo vírus da hepatite C (VHC) é a principal causa de doença hepática em todo mundo. Aproximadamente 170 milhões de pessoas estão infectadas com o vírus. A resposta imune contra o VHC conduz a secreção de citocinas e ativação e proliferação de células T efetoras para atuarem nos hepatócitos infectados e assim iniciam o processo inflamatório no fígado. Objetivo: o estudo teve como objetivo caracterizar a resposta imune e estimar a frequência do polimorfismo do TNF-α -308 G/A em pacientes com hepatite C crônica atendidos na Fundação de Medicina Tropical Doutor Heitor Vieira Dourado. Material e Métodos: Foram utilizadas amostras de 30 de pacientes com hepatite C e 28 amostras de indivíduos não infectados com o VHC. As análises dos linfócitos T e moléculas de adesão foram realizadas por citometria de fluxo. As citocinas séricas foram analisadas por citometria de fluxo utilizando o kit CBA. A caracterização do polimorfismo de TNF-α- 308 foi obtida por PCR-RFLP. A fibrose foi determinada após análise histológica de biópsia hepática de acordo com o escore METAVIR. Resultados: Os pacientes com hepatite C apresentaram porcentagem de linfócitos T regulatórios (T regs) e citocinas aumentadas quando comparados ao grupo controle. Na fibrose ≥F3 (N=10) foi caracterizada relevante frequência (100%) de indivíduos com alta produção de IL-2 e baixa frequência (50%) de indivíduos com alta produção de IL-10 e na fibrose ≤F2 foi caracterizada relevante frequência (85%) de indivíduos com alta produção de IL-4 e frequência (55%) de indivíduos com alta produção de IL-10. Conclusão: Os linfócitos T efetores são regulados pelos linfócitos T regs. Na fibrose ≥F3 foi caracterizado um perfil de citocinas Th1 enquanto na fibrose ≤F2 um perfil de citocinas Th2. A baixa produção de IL-10 foi associada com uma atividade fibrogênica aumentada em pacientes com hepatite C.

Orientadores: Clarice Weis Arns, Luciana Konecny Kohn / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T21:48:22Z (GMT). No. of bitstreams: 1 Bastos_JulianaCristinaSantiago_M.pdf: 1673040 bytes, checksum: 1234828d961406573d21b2476f1f1800 (MD5) Previous issue date: 2013 / Resumo: O vírus da Hepatite C (família Flaviviridae, gênero Hepacivirus) é causador de infecções crônicas em humanos, que podem evoluir para quadros de cirrose hepática e carcinoma hepatocelular. Até o momento, não há vacina disponível contra essa infecção e o tratamento disponível é caro, tem eficácia limitada e gera uma vasta gama de efeitos secundários, o que dificulta a continuidade do tratamento. Como esse vírus não replica eficientemente em cultura de células e em animais, o vírus da diarréia viral bovina é utilizado como modelo substituto para ensaios de avaliação de atividade antiviral e em ensaios de mecanismo de ação. A partir de invertebrados e micro-organismos marinhos, foram preparados extratos e frações, e algumas substâncias foram isoladas para a avaliação da sua possível atividade antiviral. Dos 422 testados, 5% foram considerados promissores e, destes, 20% mostraram-se ativos apresentando uma proteção de mais de 97% às células frente ao vírus. Os melhores resultados foram obtidos dos extratos produzidos a partir das amostras de esponjas Hyrtios sp. (BA07ES-56: PI=99%, IS=25), Aaptos sp. (BA07ES-59: PI=99%, IS=8,25) e de bactérias Bacillus sp. (555: PI=98%, IS>18; 584: PI=98%, IS=27) isoladas da esponja Petromica citrina. Os extratos e compostos promissores foram capazes de atuar em diversas etapas do ciclo replicativo viral (adsorção, penetração, etapas intracelulares do ciclo replicativo e também inativação da partícula viral), levando à sua interrupção quase completa nas condições analisadas. Desse modo, diversas substâncias presentes nesses organismos estudados são ativas e podem levar ao desenvolvimento de fármacos que garantam uma terapia alternativa para o tratamento da hepatite C / Abstract: The Hepatitis C virus (family Flaviviridae, genus Hepacivirus) causes chronic infections in humans, which can develop to liver cirrhosis and hepatocellular carcinoma. This represents a major public health problem worldwide. To this moment, there is no vaccine available against this infection and the treatment available is expensive, has limited efficacy and generates a wide range of side effects, making it difficult to continue the treatment. All this reflects the need to seek new agents with antiviral action against this virus. As this virus does not replicate efficiently in cell culture and in animals, bovine viral diarrhea virus is used as a surrogate model for screening assays of antiviral activity, and mechanism of action assays. From marine invertebrates and micro-organisms isolated from them, extracts and fractions were prepared, and substances were isolated for assessment of their possible antiviral activity. Of the 422 tested, 5% were considered promising, and of these, 20% were active presenting a protection percentage of more than 97%. The best results were obtained from the extracts produced from the samples of sponge Hyrtios sp. (BA07ES-56: IP=99%, SI=25), Aaptos sp. (BA07ES-59: IP=99%, SI=8,25) and bacteria Bacillus sp. (555: IP=98%, SI>18; 584: IP=98%, SI=27) isolated from the sponge Petromica citrina. The promising extracts and compounds acted in several stages of viral replicative cycle (adsorption, penetration, intracellular steps of the replicative cycle and also inactivation of the viral particle). Thus, various substances are active and may lead to the development of drugs which ensure an alternative therapy for the treatment of hepatitis C / Mestrado / Microbiologia / Mestra em Genética e Biologia Molecular

La polymérase NS5B du virus de l’hépatite C (VHC) porte une activité ARN polymérase ARN-dépendante essentielle pour la réplication de l'ARN génomique viral. Cette réplication implique la synthèse d'un intermédiaire de réplication de polarité négative. In vitro et probablement in vivo, la NS5B initie la synthèse d'ARN par un mécanisme de novo qui nécessite des interactions spécifiques entre la polymérase virale et des éléments des ARN viraux. Dans une première partie nous avons étudié le rôle du GTP et d’un domaine C-terminal nommé linker de la polymérase. Nos résultats démontrent que des concentrations élevées de GTP sont nécessaires pour la transition de l'initiation à l'élongation de la synthèse de l'ARN. Des mutations dans le linker à la position 556 ne modifient pas la concentration de GTP nécessaire pour la transition. Toutefois, l'initiation de la synthèse d'ARN est augmentée par la mutation S556K. Une analyse structurale menée en parallèle suggère une implication directe du linker dans l'initiation de novo de la synthèse de l'ARN. Dans les deuxièmes et troisièmes parties, nous avons étudié le rôle de motifs ARN dans la traduction et la synthèse de l’'ARN du VHC. Nous avons démontré que la tige boucle SL-E1 formée par la région entre les nt 177 et 222 de l'extrémité 3' de l’ARN (-) est importante pour la synthèse d'ARN in vitro par la NS5B recombinante et dans les cellules Huh7 exprimant le complexe de réplication (RC) du VHC. SL-E1 est impliquée dans l’initiation de la synthèse d’ARN, au moins in vitro. Nous avons également étudié le rôle des tiges boucles SLV et SLVI du gène core. Nos données n'ont pas montré de rôle évident de ces séquences ou de leur complément dans la synthèse de l'ARN in vitro par la NS5B recombinante et en culture cellulaire par le RC du VHC. Nous avons confirmé leur effet négatif sur la traduction IRES dépendante par interaction ARN-ARN longue distance entre SL-VI et le 5'UTR et démontré que le miR122 ne peut pas empêcher cet interaction. Par contre, la présence de SL-VI prévient l’inhibition de la traduction induite par l’interaction entre le domaine III de l’IRES et la tige boucle 5BSL3.2 en 3’ du génome. Ces résultats démontrent la complexité des interactions ARN/ARN et ARN/protéines dans la régulation de la réplication virale. / The hepatitis C virus (HCV) NS5B protein displays a RNA-dependent RNA polymerase activity essential for replication of the viral RNA genome. This replication involves the synthesis of a replication intermediate of negative polarity. In vitro and likely in vivo, the NS5B initiates RNA synthesis by a de novo mechanism which requires specific interactions between the polymerase and viral RNA elements. In the first part of results, we described a combined structural and functional analysis of HCV-NS5B to study the role of a C-terminal segment (termed linker) and of GTP in RNA synthesis. Our results demonstrated that high GTP concentrations are necessary for the transition from the initiation to the elongation of RNA synthesis, and that linker mutations at position S556 did not modify the GTP requirement of NS5B for this transition. However, the initiation of RNA synthesis was greatly enhanced by a S556K mutation. These results together with a structural analysis point to the direct involvement of the linker in the de novo initiation of RNA synthesis. In the second and third parts of results, we studied the role of RNA elements in RNA synthesis. We demonstrated that the SL-E1 stem–loop formed by nucleotides 177–222 from the 3’-end of the HCV (-) RNA is important for RNA synthesis both in vitro by the recombinant NS5B and in Huh7 cells by HCV replication complex (RC). We also showed that SL-E1 is involved in initiation of RNA synthesis, at least in vitro. Then we studied the role of other viral RNA elements in core coding sequences (SLV and SLVI stem loops) and the involvement of the microRNA miR122 in RNA translation and RNA synthesis. For SLV and SLVI, our data did not show any clear role of these core-coding sequences or of their complement in the (-) RNA in RNA synthesis both in vitro by the recombinant NS5B and in cell culture by HCV-RC. We confirmed their negative effect on HCV-IRES translation through long range RNA-RNA interaction between SL-VI sequences and the 5’UTR and demonstrated that miR122 cannot disrupted this interaction and switches the region to an open conformation. Conversely, our data indicated that the SL-VI domain can counteract the negative effect of the interaction between the domain III of IRES and the 5BSL3.2 stem loop localized at the 3’end of the genome. These results point to the complexity of RNA/RNA and RNA/proteins interactions in the HCV replication cycle.

Avec près de 350 millions de personnes chroniquement infectées, et malgré l’existence de vaccins efficaces, le virus de l’Hépatite B (VHB) reste un problème majeur de santé publique. Parmi les protéines virales, la protéine régulatrice HBx possèdent des activités qui pourraient être particulièrement impliquées dans le développement de CHC. Au cours de ce travail, nous nous sommes intéressés aux différences biologiques entre la protéine HBx issue d’une région non tumorale (HBx-NT) et la protéine HBx issue d’une région tumorale (HBx-T) d’un même patient. En particulier, nous nous sommes intéressés à la régénération hépatique après hépatectomie partielle et à la carcinogenèse hépatique dans un modèle murin transgénique. Nous avons démontré l’absence d’impact de la forme tronquée de la protéine HBx sur la régénération hépatique. Nous avons démontré que la protéine HBx entière avait la capacité d’activer la sécrétion d’IL-6 dans la phase d’initiation de la régénération hépatique, conduisant à l’hyperactivation de STAT3, l’accumulation de SOCS3 et la diminution de phosphorylation de ERK. Au final, la protéine HBx entière induit un retard de régénération hépatique. Nous avons démontré une cinétique d’apparition de tumeurs plus rapide chez les souris HBx-T que chez les souris HBx-NT après injection d’un carcinogène chimique. Nous avons aussi pu observer que les deux formes HBx-T et HBx-NT sensibilisaient les hépatocytes à l’apoptose, au cours d’un dommage hépatique aigue, et que cette sensibilisation à l’apoptose pouvait en partie rendre compte de l’effet co-carcinogène observé chez les souris HBx-T et HBx-NT. L’ensemble de mes résultats a permis de mieux comprendre les mécanismes par lesquels la protéine HBx participe au développement de CHC. / Hepatitis B virus (HBV) is a worldwide health issue, as it is estimated that 350 millions people are chronically infected. Among the viral proteins, HBx is thought to be involved in hepatocellular carcinoma (HCC) development. In this work, we were interested in biological differences between HBx sequence from non tumoral region (HBx-NT) compared to HBx from tumoral region (HBx-T) from a single patient. In particular, we studied liver regeneration after partial hepatectomy et hepatocarcinogenesis in a transgenic mice model. We demonstrated that HBx-T did not modulate liver regenereation. We also showed that HBx-NT induced IL-6 overexpression during priming phase of liver regeneration, and that IL-6 overexpression was involved in STAT3 hyperactivation, SOCS3 accumulation and inhibition of ERK. Overall, HBx-NT induced IL-6 overexpression was responsible for a delay in liver regeneration. Moreover, we showed that HBx-T induced a faster development of hepatic tumor after DEN initiation, compared to HBx-NT. Both HBx forms were involved in an apoptosis sensibilization during acute liver injury, that could be involved in co-carcinogenic effect of HBx-T and HBx-NT. Overall, my results participate to the comprehension of HBx impact on liver carcinogenesis

Hepatitis B is one of the most common infectious diseases, which leads to public health problems in the world, especially in Asian counties. In recent years, extensive human genetic association studies have been carried out to identify susceptible genes and genetic polymorphisms to understand the genetic contributions to the disease progression of HBV infection. HLA-DQ gene variations have been reported to be associated with HBV infection/clearance, disease progression and the development of hepatitis B-related complications, including liver cirrhosis (LC) and hepatocellular carcinoma (HCC). However, the results are either inconclusive or controversial. Therefore, to derive a more precise estimation of the association, a meta-analysis was performed. Our data revealed that the HLA-DQ alleles rs2856718-G, rs7453920-A and rs9275319-G were significantly associated with decreased risk of HBV infection and HBV natural clearance. Logistic regression analyses showed that HLA-DQ alleles rs9275572-A significantly increased HBV infection clearance, and decreased HBV natural clearance. However, rs2856718-G and rs9275572-A were not associated with development of cirrhosis. The HLA-DQ polymorphisms (rs2856718 and rs9275572) were associated with a decreased HBV-related HCC risk in all genetic models, but rs9272105-A increased the risk of HBV-related HCC. In addition, no significant association was observed between HLA-DQ rs9275319-G polymorphism and HBVrelated HCC. These stratified analyses were limited due to relatively modest size of correlational studies. In future, further investigation on a large population and different ethnicities are warranted. Our findings contribute to the personalized care and prognosis in hepatitis B.

Since the discovery in 1989 of hepatitis C virus (HCV) as the infectious agent responsible for the vast majority of post-transfusion non-A non-B hepatitis, blood transfusions are no longer a source for HCV transmission in Sweden. Anti-HCV testing was implemented for all blood donations in 1992. Since then intravenous drug use (IDU) has become the major route of transmission in the western world. Six genotypes and more than 80 subtypes of HCV have now been identified world-wide. These genotypes and subtypes are determined by genetic divergences between the HCV strains. Subtypes 1a, 1b, 2b, 2c, and 3a have global spread, while the other subtypes have a more limited geographical distribution. Little was known on the prevalence of HCV among blood donors and on which genotypes and subtypes of HCV were circulating in Sweden before the testing of all blood donations was implemented. The prevalence of anti-HCV was therefore investigated in sera sent to the Swedish Institute for Infectious Disease Control (SMI) from 412 patients; 241 were sampled between 1970 and 1991 before the universal screening in 1992, while 171 were sampled between 1992 and 2002. The samples derived from 193 (47%) blood donors, (104 sampled before, and 89 after 1992), and from seven other groups of patients. Two groups had suspected known routes of infection, intravenous drug use (IDU) 33 patients and hemodialysis, 16 patients, while it was unknown for the other patients. Anti-HCV was detected in 120 (29%) samples. The highest frequency was found among IDUs, (91%). Before general screening was implemented, 2.8% of the blood donors were positive for hepatitis C, whereas 28% of those sampled after 1992 were anti-HCV positive. Those latter samples were sent to SMI due to anti-HCV reactivity in a primary test at the blood centre. HCV RNA could be detected by PCR in 56 (47%) of the anti-HCV positive samples, the subtype could be determined by sequencing in 45 (80%) of those. The subtypes found were 1a in 31 %, 1b in 18%, 2b in 22%, and 3a in 27%. One sample was of subtype 2c. There was a tendency of increase of genotype 2 and a decrease in subtype 1a with time. 1a was found in 38% of the samples collected before 1992, while it was only found in 19% of the samples from 1992 or later. On the other hand genotype 2 was found in 17% sera sampled before 1992 and in 37% of the samples collected 1992 or later. It is not known if this genotype has recently been introduced into Sweden. Further analysis on larger series of samples is needed to confirm these preliminary results. / AcknowledgmentsI would like to express my gratitude to several people who have been supportive in different ways throughout this project.First of all, I want to thank my supervisor Helene Norder, for giving me the possibility to do my diploma thesis at the Department of Virology, Swedish Institute for Infectious Disease control (SMI) and for helping me during this study and for the many insightful conversations during the design and development stages of the application, and also for the many helpful comments and suggestions on the text of the thesis.I want to express my appreciation to my laboratory supervisor Regina Wallin, Camilla Jern and Josefine Ederth for helping me during the procedure for this study. Then, I want to thank my examiner Magnus Johansson from the Södertörns university collegefor his advice on writing this paper. Finally, I would like to thank my family and specially my mother Bahar Hamid for always supporting me during my whole life.Last, but not least, I would like to thank my friends Annika Andersson and Yourdons Yemane for being encouraging, understanding and always supportive.

Philosophiae Doctor - PhD / Hepatitis C was first recognized as a transfusion-associated liver disease not caused by hepatitis A or hepatitis B virus after serological tests were developed to screen for their presence in the blood. The infectious agent was finally identified with the cloning of the cDNA of hepatitis C virus (HCV) using random polymerase chain reaction (PCR) screening of nucleic acids extracted from plasma of a large pool of chimpanzee infected with non-A non-B hepatitis. NS5B, a membrane-associated RNA-dependent RNA polymerase essential in the replication of HCV, initiates the synthesis of a complementary negative-strand RNA from the genomic positive-strand RNA so that more positive-strand HCV RNA can then be generated from the newly synthesised negative-strand template. The crystal structure of NS5B presented typical fingers, palm and thumb sub-domains encircling the GDD active site, which is also seen in other RNA-dependent RNA polymerases, and is similar to the structure of reverse transcriptase of HIV-1 and murine Moloney leukaemia virus. The last 21 amino acids in the C-terminus of NS5B anchor the protein to the endoplasmic reticulum (ER)-derived membranous web. NS5B has been shown to interact with the core, NS3/NS4A, NS4B and NS5A proteins, either directly or indirectly. Numerous interactions with cellular proteins have also been reported. These proteins are mainly associated with genome replication, vesicular transport, protein kinase C-related kinase 2, P68 (DDX5), α-actinin, nucleolin, human eukaryotic initiation factor 4AII, and human VAMP-associated protein. Previous studies have confirmed that NS5B binds to full-length DDX5. By constructing deletion mutants of DDX5, we proceeded to characterize this interaction between DDX5 and HCV NS5B. We report here the identification of two exclusive HCV NS5B binding sites in DDX5, one in the N-terminal region of amino acids 1 to 384 and the other in the C-terminal region of amino acids 387 to 614. Proteins spanning different regions of DDX5 were expressed and purified for crystallization trials. The N-terminal region of DDX5 from amino acids 1 to 305 which contains the conserved domain I of the DEAD-box helicase was also cloned and expressed in Escherichia coli. The cloning, expression, purification and crystallization conditions are presented in this work. Subsequently, the crystal structure of DDX5 1-305 was solved and the high resolution three-dimensional structure shows that in front of domain I is the highly variable and disordered N terminal region (NTR) of which amino acids 51-78 is observable, but whose function is unknown. This region forms an extensive loop and supplements the core with an additional α-helix. Co-immunoprecipitation experiments demonstrated that the NTR of DDX5 1-305 auto-inhibit its interaction with NS5B. Interestingly, the α-helix in NTR is essential for this auto-inhibition and seems to mediate the interaction between the highly flexible 1-60 residues in NTR and NS5B binding site in DDX5 1-305, presumably located within residues 79-305. Furthermore, co-immunoprecipitation experiments revealed that DDX5 can also interact with other HCV proteins, besides NS5B.