A hepatite C afeta cerca de 150 milhões de pessoas no mundo e é a razão mais comum de transplantes de fígado. A erradicação viral, por meio de tratamento medicamentoso, é a única intervenção que pode deter a progressão da doença, reduzir a mortalidade e melhorar a qualidade de vida dos pacientes. Em 2011, foi aprovado o boceprevir, um inibidor de protease, que passou a ser adicionado à terapia padrão dupla (interferon peguilado e ribavirina) pelo Protocolo Clínico brasileiro para tratamento de Hepatite C genótipo 1 em pacientes com grau de fibrose maior que F2. Devido ao alto custo de aquisição deste medicamento e à produção cada vez maior de novas tecnologias para essa área terapêutica, foi proposta essa pesquisa que tem como objetivo analisar o custo-efetividade da terapia tripla em relação à terapia dupla, no tratamento da hepatite C crônica genótipo 1 em pacientes virgens de tratamento para todos os graus de fibrose. Para tanto, foi construído um modelo de Markov com 15 estados de saúde representando a história natural da Hepatite C crônica. O modelo seguiu uma coorte hipotética pela vida toda, em que os custos foram expressos em Reais e os desfechos em anos de vida ganhos. A perspectiva adotada foi a do SUS. A RCEI calculada, com taxa de desconto de 5% para custos e desfechos, foi R$ 201.504,92 por ano de vida ganho. Considerando um limiar de custo efetividade de 3 vezes o valor do PIB per capita, segundo recomendação da OMS, a adição do boceprevir não foi custo-efetiva no tratamento de pacientes virgens em todos os graus de fibrose. Pela análise de sensibilidade, nenhuma variável teve grande impacto na RCEI, exceto quando a taxa de desconto aplicada em desfechos foi zerada, em que a terapia tripla passou a ser custo-efetiva. / The Hepatitis C virus affects around 150 million of people worldwide and it is the most common reason for liver transplantation. Viral eradication, by drug treatment, is the only therapeutic intervention that may halt the disease progression, reduce HCV-related mortality and improve the quality of life of infected patients. Boceprevir, a protease inhibitor, was approved in 2011, being to be added to standard of care (peguilated interferon-α and ribavirin) by the Brazilian Protocol of treatment of genotype 1 Hepatitis C, in patients with degree of fibrosis greater than F2. Due to the high cost of acquisition of this drug and the increasing production of new technologies in this therapeutic area, the aim of this work was develop a cost-effectiveness analysis, comparing the triple therapy with the standard of care (double therapy) for treatment of genotype 1 chronic hepatitis C in treatment-naïve patients of all degrees of fibrosis. It was constructed a Markov Model with 15 health states representing the natural history of chronic Hepatitis C. The model followed a hypothetic cohort by lifetime, where costs were expressed in Reais and outcomes in life-years gained, under the perspective of Brazilian public health system. The calculated ICER, with discount rate of 5% to costs and outcomes, was R$201.504, 92 by life-years gained. Considering three times GDP per capita for cost-effectiveness threshold, according WHO recommendation, boceprevir was not cost-effective, when considered treatment-naïve patients of all degrees of fibrosis. By sensitivity analysis, none of the variables had a big impact in the ICER, except when it was stopped applying the discount rate in outcomes, in which the triple therapy became cost-effective.

As hepatites virais estão entre as mais importantes pandemias mundiais da atualidade. Existem várias causas de hepatite, entre elas, o vírus da hepatite B (HBV), o vírus da hepatite C (HCV) e o vírus da Hepatite Delta (HDV). Da mesma forma, o vírus GB-C (GBV-C) é importante na coinfecção com outros vírus, como o HIV. Nesse estudo, várias regiões da América do Sul foram analisadas. Na Colômbia, os estados do Amazonas e Magdalena foram encontradas como regiões hiperendêmicas para HBV. O genótipo F3 (75%) foi o mais prevalente. Determinou-se que o subgenótipo F3 é o mais antigo dos subgenótipos F. No estado de Chocó, encontrou-se o subgenótipo A1 (52,1%) como o mais prevalente. Surpreendentemente, nesse mesmo estado foram encontrados nove casos autóctones de infecção pelo genótipo E (39,1%). Para o HCV, em Bogotá, encontrou-se o subtipo 1b (82,8%) como o mais prevalente. Da mesma forma, estimou-se que esse subtipo foi introduzido por volta de 1950 e se propagou exponencialmente entre 1970 a 1990. O HDV foi identificado em casos de hepatite fulminante do estado de Amazonas, todos classificados como genótipo 3. Se determinou que o HDV/3 se espalhou exponencialmente a partir de 1950 a 1970 na América do Sul e depois desta época, esta infecção deixou de aumentar, provavelmente devido a introdução de vacinação contra o HBV. GBV-C foi procurado em doadores de sangue colombianos infectados com HCV e/ou HBV de Bogotá e em povos indígenas com infecção pelo HBV no Amazonas. A análise filogenética revelou a presença do genótipo 2a como o mais prevalente entre os doadores de sangue e o 3 nos povos indígenas estudados. A presença do genótipo 3 na população indígena foi previamente relatada na região de Santa Marta, na Colômbia e nos povos indígenas da Venezuela e da Bolívia. No Chile, foi realizado um estudo com 21 pacientes cronicamente infectados pelo HBV sem tratamento antiviral prévio. Todas as sequências obtidas eram do subgenótipo F1b e se agrupavam em quatro diferentes grupos, sugerindo que diferentes linhagens desse subgenótipo estão circulando no Chile. No Brasil, no estado de Rondônia, para o HCV, encontramos o subtipo 1b (50,0%) como o mais frequente. Esse foi o primeiro relato sobre os genótipos do HCV neste estado. Para o HBV, o subgenótipo A1 (37,0%) foi o mais frequente. Os resultados do estado de Rondônia são consistentes com outros estudos no Brasil, mostrando a presença de vários genótipos do HBV, refletindo a origem mista da população Brasileira. Estudando o estado do Maranhão, avaliamos a frequência da infecção pelo HBV e seus genótipos. Foram encontradas 4 sequencias genótipo A1 que agruparam com outras sequências reportadas do Brasil. Em outro estudo, caracterizamos os subgenótipos do HBV em 68 pacientes com hepatite crônica B em Pernambuco, encontrando 78,7% de presença do subgenótipo A1. Finalmente, em um estudo realizado com amostras da cidade de São Paulo, encontramos um caso de HBV genótipo C em um brasileiro nativo, sendo essa a primeira sequência completa do genoma de HBV/C2 notificados no Brasil / Viral hepatitis are among the major pandemics in the world nowadays. There are many causes of hepatitis, including hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis delta virus (HDV). Similarly, GB virus C (GBV-C) is a relevant agent in co-infection with HIV. In this study, several regions of South America were studied. In Colombia, the states of Amazonas and Magdalena were identified as highly endemic areas for HBV. Genotype F3 (75%) was the most prevalent. It was determined that subgenotype F3 is the oldest among all F subgenotypes. In the state of Chocó, subgenotype A1 (52.1%) was the most prevalent. Surprisingly, nine indigenous cases of infection by genotype E (39.1%) were found in this state. For HCV, in Bogotá, subtype 1b (82.8%) was the most frequent. Likewise, it was estimated that this subtype was introduced around 1950 and spread exponentially from 1970 to 1990. HDV has been identified in cases of fulminant hepatitis in the state of Amazonas, all of them classified as genotype 3. It was determined that the HDV/3 spread exponentially from 1950 to 1970 in South America and after this time, this infection stopped to increase, probably due to introduction of vaccination against HBV. GBV-C was sought in Colombian blood donors infected with HCV and/or HBV in Bogotá and indigenous peoples with HBV infection in the Amazon. The phylogenetic analysis revealed the presence of genotype 3 as the most prevalent among blood donors and in three studied indigenous people. The presence of genotype 3 in the indigenous population has been previously reported in the region of Santa Marta, Colombia, and in the indigenous peoples of Venezuela and Bolivia. In Chile, a study was carried out with 21 patients chronically infected with HBV without any prior antiviral treatment. All sequences obtained belonged to subgenotype F1b and clustered into four different groups, suggesting that different strains that are circulating in Chile. In Brazil, the state of Rondônia, we found HCV subtype 1b (50.0%) as the most frequent. This was the first report on HCV genotypes in this state. For HBV, subgenotype A1 (37.0%) was the most frequent. The results of the state of Rondônia are consistent with other studies carried out in Brazil, showing the presence of several HBV genotypes, reflecting the mixed origin of the Brazilian population. Studying the state of Maranhão, we evaluated the frequency of HBV infection and its genotypes and we found 4 genotype A1 sequences that grouped with other sequences reported in Brazil. In another study, we characterized HBV subgenotypes in 68 patients with chronic hepatitis B in Pernambuco and we found subgenotype A1 in 78.7% cases. Finally, in a study of samples from São Paulo, we found a case of HBV genotype C in a native Brazilian patient and this is the first complete genome sequence of HBV/C2 reported in Brazil

A evolução da recorrência da hepatie C pós-transplante hepático pode ter um curso bastante variável. Raramente a doença pode progredir para uma forma conhecida como hepatite recorrente colestática grave, cuja patogenia ainda não é bem conhecida. Nós estudamos nesse trabalho alguns aspectos virológicos, histológicos e imunohistoquímicos de seis pacientes com essa forma rara de recorrência da doença, tendo como comparação um grupo pareado de seis pacientes transplantados com a forma leve de hepatite C recorrente, e como controle imunocompetente, cinco pacientes não transplantados com hepatite crônica pelo vírus C. Foram avaliados como possíveis fatores preditivos de gravidade da progressão da recorrência: viremia do VHC, evolução de quasispécies, parâmetros histopatológicos, e imunoreatividade para o antígeno core do VHC. / Following liver transplantation (OLT) HCV-related disease severity is highly variable, with a minority of cases progressing to an extremely severe form of cholestatic hepatitis, in which the pathogenesis is not yet understood. We aim to compare virological, histological and immunohistological changes in patients developing mild and severe post-OLT HCV recurrence. Twelve patients with recurrent HCV infection were studied (6 with severe and 6 with mild disease). Five HCV-infected immunocompetent patients were used as controls. We looked at viral load, quasispecies evolution of HCV, several histological parameters and immuno-reactivity of core antigens at three time-points (pre-OLT, early post-OLT and late post-OLT) as predictors of severity of recurrence post-OLT.

A hepatite C afeta cerca de 150 milhões de pessoas no mundo e é a razão mais comum de transplantes de fígado. A erradicação viral, por meio de tratamento medicamentoso, é a única intervenção que pode deter a progressão da doença, reduzir a mortalidade e melhorar a qualidade de vida dos pacientes. Em 2011, foi aprovado o boceprevir, um inibidor de protease, que passou a ser adicionado à terapia padrão dupla (interferon peguilado e ribavirina) pelo Protocolo Clínico brasileiro para tratamento de Hepatite C genótipo 1 em pacientes com grau de fibrose maior que F2. Devido ao alto custo de aquisição deste medicamento e à produção cada vez maior de novas tecnologias para essa área terapêutica, foi proposta essa pesquisa que tem como objetivo analisar o custo-efetividade da terapia tripla em relação à terapia dupla, no tratamento da hepatite C crônica genótipo 1 em pacientes virgens de tratamento para todos os graus de fibrose. Para tanto, foi construído um modelo de Markov com 15 estados de saúde representando a história natural da Hepatite C crônica. O modelo seguiu uma coorte hipotética pela vida toda, em que os custos foram expressos em Reais e os desfechos em anos de vida ganhos. A perspectiva adotada foi a do SUS. A RCEI calculada, com taxa de desconto de 5% para custos e desfechos, foi R$ 201.504,92 por ano de vida ganho. Considerando um limiar de custo efetividade de 3 vezes o valor do PIB per capita, segundo recomendação da OMS, a adição do boceprevir não foi custo-efetiva no tratamento de pacientes virgens em todos os graus de fibrose. Pela análise de sensibilidade, nenhuma variável teve grande impacto na RCEI, exceto quando a taxa de desconto aplicada em desfechos foi zerada, em que a terapia tripla passou a ser custo-efetiva. / The Hepatitis C virus affects around 150 million of people worldwide and it is the most common reason for liver transplantation. Viral eradication, by drug treatment, is the only therapeutic intervention that may halt the disease progression, reduce HCV-related mortality and improve the quality of life of infected patients. Boceprevir, a protease inhibitor, was approved in 2011, being to be added to standard of care (peguilated interferon-α and ribavirin) by the Brazilian Protocol of treatment of genotype 1 Hepatitis C, in patients with degree of fibrosis greater than F2. Due to the high cost of acquisition of this drug and the increasing production of new technologies in this therapeutic area, the aim of this work was develop a cost-effectiveness analysis, comparing the triple therapy with the standard of care (double therapy) for treatment of genotype 1 chronic hepatitis C in treatment-naïve patients of all degrees of fibrosis. It was constructed a Markov Model with 15 health states representing the natural history of chronic Hepatitis C. The model followed a hypothetic cohort by lifetime, where costs were expressed in Reais and outcomes in life-years gained, under the perspective of Brazilian public health system. The calculated ICER, with discount rate of 5% to costs and outcomes, was R$201.504, 92 by life-years gained. Considering three times GDP per capita for cost-effectiveness threshold, according WHO recommendation, boceprevir was not cost-effective, when considered treatment-naïve patients of all degrees of fibrosis. By sensitivity analysis, none of the variables had a big impact in the ICER, except when it was stopped applying the discount rate in outcomes, in which the triple therapy became cost-effective.

As hepatites virais estão entre as mais importantes pandemias mundiais da atualidade. Existem várias causas de hepatite, entre elas, o vírus da hepatite B (HBV), o vírus da hepatite C (HCV) e o vírus da Hepatite Delta (HDV). Da mesma forma, o vírus GB-C (GBV-C) é importante na coinfecção com outros vírus, como o HIV. Nesse estudo, várias regiões da América do Sul foram analisadas. Na Colômbia, os estados do Amazonas e Magdalena foram encontradas como regiões hiperendêmicas para HBV. O genótipo F3 (75%) foi o mais prevalente. Determinou-se que o subgenótipo F3 é o mais antigo dos subgenótipos F. No estado de Chocó, encontrou-se o subgenótipo A1 (52,1%) como o mais prevalente. Surpreendentemente, nesse mesmo estado foram encontrados nove casos autóctones de infecção pelo genótipo E (39,1%). Para o HCV, em Bogotá, encontrou-se o subtipo 1b (82,8%) como o mais prevalente. Da mesma forma, estimou-se que esse subtipo foi introduzido por volta de 1950 e se propagou exponencialmente entre 1970 a 1990. O HDV foi identificado em casos de hepatite fulminante do estado de Amazonas, todos classificados como genótipo 3. Se determinou que o HDV/3 se espalhou exponencialmente a partir de 1950 a 1970 na América do Sul e depois desta época, esta infecção deixou de aumentar, provavelmente devido a introdução de vacinação contra o HBV. GBV-C foi procurado em doadores de sangue colombianos infectados com HCV e/ou HBV de Bogotá e em povos indígenas com infecção pelo HBV no Amazonas. A análise filogenética revelou a presença do genótipo 2a como o mais prevalente entre os doadores de sangue e o 3 nos povos indígenas estudados. A presença do genótipo 3 na população indígena foi previamente relatada na região de Santa Marta, na Colômbia e nos povos indígenas da Venezuela e da Bolívia. No Chile, foi realizado um estudo com 21 pacientes cronicamente infectados pelo HBV sem tratamento antiviral prévio. Todas as sequências obtidas eram do subgenótipo F1b e se agrupavam em quatro diferentes grupos, sugerindo que diferentes linhagens desse subgenótipo estão circulando no Chile. No Brasil, no estado de Rondônia, para o HCV, encontramos o subtipo 1b (50,0%) como o mais frequente. Esse foi o primeiro relato sobre os genótipos do HCV neste estado. Para o HBV, o subgenótipo A1 (37,0%) foi o mais frequente. Os resultados do estado de Rondônia são consistentes com outros estudos no Brasil, mostrando a presença de vários genótipos do HBV, refletindo a origem mista da população Brasileira. Estudando o estado do Maranhão, avaliamos a frequência da infecção pelo HBV e seus genótipos. Foram encontradas 4 sequencias genótipo A1 que agruparam com outras sequências reportadas do Brasil. Em outro estudo, caracterizamos os subgenótipos do HBV em 68 pacientes com hepatite crônica B em Pernambuco, encontrando 78,7% de presença do subgenótipo A1. Finalmente, em um estudo realizado com amostras da cidade de São Paulo, encontramos um caso de HBV genótipo C em um brasileiro nativo, sendo essa a primeira sequência completa do genoma de HBV/C2 notificados no Brasil / Viral hepatitis are among the major pandemics in the world nowadays. There are many causes of hepatitis, including hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis delta virus (HDV). Similarly, GB virus C (GBV-C) is a relevant agent in co-infection with HIV. In this study, several regions of South America were studied. In Colombia, the states of Amazonas and Magdalena were identified as highly endemic areas for HBV. Genotype F3 (75%) was the most prevalent. It was determined that subgenotype F3 is the oldest among all F subgenotypes. In the state of Chocó, subgenotype A1 (52.1%) was the most prevalent. Surprisingly, nine indigenous cases of infection by genotype E (39.1%) were found in this state. For HCV, in Bogotá, subtype 1b (82.8%) was the most frequent. Likewise, it was estimated that this subtype was introduced around 1950 and spread exponentially from 1970 to 1990. HDV has been identified in cases of fulminant hepatitis in the state of Amazonas, all of them classified as genotype 3. It was determined that the HDV/3 spread exponentially from 1950 to 1970 in South America and after this time, this infection stopped to increase, probably due to introduction of vaccination against HBV. GBV-C was sought in Colombian blood donors infected with HCV and/or HBV in Bogotá and indigenous peoples with HBV infection in the Amazon. The phylogenetic analysis revealed the presence of genotype 3 as the most prevalent among blood donors and in three studied indigenous people. The presence of genotype 3 in the indigenous population has been previously reported in the region of Santa Marta, Colombia, and in the indigenous peoples of Venezuela and Bolivia. In Chile, a study was carried out with 21 patients chronically infected with HBV without any prior antiviral treatment. All sequences obtained belonged to subgenotype F1b and clustered into four different groups, suggesting that different strains that are circulating in Chile. In Brazil, the state of Rondônia, we found HCV subtype 1b (50.0%) as the most frequent. This was the first report on HCV genotypes in this state. For HBV, subgenotype A1 (37.0%) was the most frequent. The results of the state of Rondônia are consistent with other studies carried out in Brazil, showing the presence of several HBV genotypes, reflecting the mixed origin of the Brazilian population. Studying the state of Maranhão, we evaluated the frequency of HBV infection and its genotypes and we found 4 genotype A1 sequences that grouped with other sequences reported in Brazil. In another study, we characterized HBV subgenotypes in 68 patients with chronic hepatitis B in Pernambuco and we found subgenotype A1 in 78.7% cases. Finally, in a study of samples from São Paulo, we found a case of HBV genotype C in a native Brazilian patient and this is the first complete genome sequence of HBV/C2 reported in Brazil

A evolução da recorrência da hepatie C pós-transplante hepático pode ter um curso bastante variável. Raramente a doença pode progredir para uma forma conhecida como hepatite recorrente colestática grave, cuja patogenia ainda não é bem conhecida. Nós estudamos nesse trabalho alguns aspectos virológicos, histológicos e imunohistoquímicos de seis pacientes com essa forma rara de recorrência da doença, tendo como comparação um grupo pareado de seis pacientes transplantados com a forma leve de hepatite C recorrente, e como controle imunocompetente, cinco pacientes não transplantados com hepatite crônica pelo vírus C. Foram avaliados como possíveis fatores preditivos de gravidade da progressão da recorrência: viremia do VHC, evolução de quasispécies, parâmetros histopatológicos, e imunoreatividade para o antígeno core do VHC. / Following liver transplantation (OLT) HCV-related disease severity is highly variable, with a minority of cases progressing to an extremely severe form of cholestatic hepatitis, in which the pathogenesis is not yet understood. We aim to compare virological, histological and immunohistological changes in patients developing mild and severe post-OLT HCV recurrence. Twelve patients with recurrent HCV infection were studied (6 with severe and 6 with mild disease). Five HCV-infected immunocompetent patients were used as controls. We looked at viral load, quasispecies evolution of HCV, several histological parameters and immuno-reactivity of core antigens at three time-points (pre-OLT, early post-OLT and late post-OLT) as predictors of severity of recurrence post-OLT.

Unravelling the regulators of translation and replication of Hepatitis C virus Hepatitis C virus (HCV) is a positive sense, single stranded RNA virus belonging to the genus Hepacivirus and the family Flaviviridae. It infects human liver cells predominantly. Although, the treatment with α interferon and ribavirin can control HCV in some cases, they fail to achieve sustained virological response in others, thus emphasizing the need of novel therapeutic targets. The viral genome is 9.6 kb long consisting of a 5’ untranslated region (5’UTR), a long open reading frame (ORF) that encodes the viral proteins and the 3’ untranslated region (3’UTR). The 5’UTR contains a cis acting element, the internal ribosome entry site (IRES) that mediates the internal initiation of translation. The HCV 5’UTR is highly structured and consists of four major stem-loops (SL) and a pseudoknot structure. HCV proteins are synthesized by the IRES mediated translation of the viral RNA, which is the initial obligatory step after infection. The viral proteins are synthesized in the form of a long continuous chain of proteins, the polyprotein, which is then processed by the host cell and the viral proteases. Once viral proteins are synthesized sufficiently, the viral RNA is replicated. However the mechanism of switch from translation to viral RNA replication is not well understood. Several host proteins as well as the viral proteins help in the completion of various steps in the HCV life cycle. In this thesis, the role of two such factors in HCV RNA translation and replication has been characterized and exploited to develop anti-HCV peptides. The HCV proteins are categorized into two major classes based on the functions broadly: the non structural and the structural proteins. HCV NS3 protein (one of the viral non structural proteins) plays a central role in viral polyprotein processing and RNA replication. In the first part of the thesis, it has been demonstrated that the NS3 protease (NS3pro) domain alone can specifically bind to HCV-IRES RNA, predominantly in the SLIV region. The cleavage activity of the NS3 protease domain is reduced upon HCV-RNA binding owing to the participation of the catalytic triad residue (Ser 139) in this RNA protein interaction. More importantly, NS3pro binding to the SLIV region hinders the interaction of La protein, a cellular IRES-trans acting factor required for HCV IRES-mediated translation, thus resulting in the inhibition of HCV-IRES activity. Moreover excess La protein could rescue the inhibition caused by the NS3 protease. Additionally it was observed that the NS3 protease and human La protein could out-compete each other for binding to the HCV SL IV region indicating that these two proteins share the binding region near the initiator AUG which was further confirmed using RNase T1 foot printing assay. Although an over expression of NS3pro as well as the full length NS3 protein decreased the level of HCV IRES mediated translation in the cells, replication of HCV RNA was enhanced significantly. These observations suggested that the NS3pro binding to HCV IRES reduces translation in favour of RNA replication. The competition between the host factor (La) and the viral protein (NS3) for binding to HCV IRES might contribute in the regulation of the molecular switch from translation to replication of HCV. In the second part the interaction of NS3 protease and HCV IRES has been elucidated in detail and the insights obtained were used to target HCV RNA function. Computational approach was used to predict the putative amino acid residues within the protease that might be involved in the interaction with the HCV IRES. Based on the predictions a 30-mer peptide (NS3proC-30) was designed from the RNA binding region. This peptide retained the RNA binding ability and also inhibited IRES mediated translation. The NS3proC-30 peptide was further shortened to 15-mer length (NS3proC-C15) and demonstrated ex vivo its ability to inhibit translation as well as replication. Additionally, its activity was tested in vivo in a mice model by encapsulating the peptide in Sendai virus based virosome followed by preferential delivery in mice liver. This virosome derived from Sendai virus F protein has terminal galactose moiety that interacts with the asialoglycoprotein receptor on the hepatocytes leading to membrane fusion and release of contents inside the cell. Results suggested that this peptide can be used as a potent anti-HCV agent. It has been shown earlier from our laboratory, that La protein interacts with HCVIRES near initiator AUG at GCAC motif by its central RNA recognition motif, the RRM2 (residues 112-184). A 24 mer peptide derived from this RRM2 of La (LaR2C) retained RNA binding ability and inhibited HCV RNA translation. NMR spectroscopy of the HCV-IRES bound peptide complex revealed putative contact points, mutations at which showed reduced RNA binding and translation inhibitory activity. The residues responsible for RNA recognition were found to form a turn in the RRM2 structure. A 7-mer peptide (LaR2C-N7) comprising this turn showed significant translation inhibitory activity. The bound structure of the peptide inferred from transferred NOE (Nuclear Overhauser Effect) experiments suggested it to be a βturn. Interestingly, addition of hexa-arginine tag enabled the peptide to enter Huh7 cells and showed inhibition HCV-IRES function. More importantly, the peptide significantly inhibited replication of HCVRNA. Smaller forms of this peptide however failed to show significant inhibition of HCV RNA functions suggesting that the 7-mer peptide as the smallest but efficient anti-HCV peptide from the second RNA recognition motif of the human La protein. Further, combinations of the LaR2C-N7 and NS3proC-C15 peptide showed better inhibitory activity. Both the peptides were found to be interacting at similar regions of SLIV around the initiator AUG. The two approaches have the potential to block the HCV RNA-directed translation by targeting the host factor and a viral protein, and thus can be tried in combination as a multi drug approach to combat HCV infection. Taken together, the study reveals important insights about the complex regulation of the HCV RNA translation and replication by the host protein La and viral NS3 protein. The interaction of the NS3 protein with the SLIV of HCV IRES leads to dislodging of the human La protein to inhibit the translation in favour of the RNA replication. These two proteins thus act as the regulators of the translation and the replication of viral RNA. The peptides derived from these regulators in turn regulate the functions of these proteins and inhibit the HCV RNA functions.

近年来，针对就业和就学中遭遇歧视的困境，中国乙肝携带者发起一场要求消除歧视、维护合法权益的抗争运动。为什么乙肝携带者就业歧视问题在中国如此显著？面对国家和市场这两大最具权势的系统，公民社会将何以对抗？本文旨在从社会学的视角对这场“将‘乙肝’去除"的运动进行解读。 / 本文采用个案研究方法，以北京益仁平中心为主要研究对象，围绕 “乙肝"在中国的建构和重构过程，将研究聚焦于三个方面：第一，乙肝污名化过程以及国家、市场中的话语权；第二，反乙肝歧视维权运动的条件和动员机制；第三，“将‘乙肝’去除"中的政治和权力话语三角。 / 本文从社会运动理论中的资源动员、政治机会结构和框架理论出发解读中国乙肝携带者的维权运动；将运动中的微观景象与宏观社会结构结合起来，考察影响乙肝携带者维权运动的主要因素以及运动的动员机制。研究伊始分别从国家和市场两个领域审视乙肝歧视问题在中国的建构过程，阐明乙肝污名化是政府权威以及市场中医药商和医学权威共谋下的合力作用，从而为乙肝携带者反歧视抗争运动的后续研究选择一个合适的立足点。笔者在案例中发现，组织在维权运动中将乙肝携带者群体动员起来，采取有效的策略，充分利用其在资源获得方面的优势、建立乙肝维权组织网络、善于把握时机营造政治机会空间、并能够吸纳律师和媒体的专业力量是维权行动能取得成功的重要因素。 / 组织在维权行动中的话语框架对运动的发展至关重要。乙肝携带者群体对组织运动框架的认同是动员成功的基础；掌握定义“乙肝"的主动权、运动领袖的可信性、框架话语表达的日常化、与媒体的良好关系等策略有助于框架在动员中与参与者、旁观者产生共鸣；抗争精英通过话语框架为抗争活动提供合法性。 / 反乙肝歧视维权运动可以看作是一个“将‘乙肝’去除"的“去污名化"运动。一方面，中央政府与地方政府有着各自自主性利益；另一方面，乙肝携带者群体内部就抗争形式也难以统一，这两种分裂情况交叉形成了一个围绕“将‘乙肝’去除"的，以规则、效益和权利为话语的权力三角，支撑反歧视行动的抗争空间。权力三角的多变性决定去除“乙肝"的行动是有策略的、冒险的，但却相对稳定。 / Hepatitis B Virus carriers (HBVers) have launched series social movements targeting at eliminating discriminations against HBVers in job market and promoting fair employment in recent decade of years. Why does nowhere match the HBV discrimination in such country like China? How is contentious politics possible when the powerless engage in struggles with power holders, like state and market in China? The thesis attempts to learn the intricacies of body politics with sociological approaches. / Yirenping, a NGO located in Beijing, is selected as research object in this case study. Concentrating on the political nature of the HBV confrontations, this thesis is comprised of three parts: first, the stigmatization of HBV in China; second, tactics and strategies that employed against the system of discrimination; third, the politics of “Removing HBV" and the power triangle among state, market and civil society. / The analysis of the HBV movement is informed by three sociological theories of social movements: resource mobilization, political opportunity structure and framing, meanwhile macro structure and micro interaction are combined. It is postulated that this disease discrimination in China occurs when confronting an entrenched stigmatization conjoined from both state and market dedicated to keeping the HBVers excluded and marginalized, which serves as a departure point for further analysis of the struggles for power against this discrimination. Resources mobilized, leaderships and organizations, networks among HBVers and outside supporters, strategies in mobilization positively facilitate the anti-discrimination movements. Utilizing the institutional advocacies as well as informal networks with officials, NPC & CPCCC delegates open more political opportunities within the preexisting political environment. / Framing is essential to the movement mobilization. Identifying closely with the visions and missions in movements, the self conceptualization of HBV-discrimination, charismatic leaders, the everyday narrative of the frame, as well as strategic media coverage help promoting resonance among movement participants and standers-by. Framing strategies provide legitimacy for HBV selves in collective movements. / Finally, the thesis came to the conclusion that anti-discrimination-against-HBVers social movements in China can be interpreted as a process of struggle to “Removing HBV" power. Central government and local ones have their autonomies and interests respectively, while weak but observed divisions in protesting strategies also exist within the HBVers, which shape a triangle of power struggles among the dominant and dominated groups. The power triangle is flexible, and the “Removing HBV" movements are strategic, risk-taking, while being routinized. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / 郭娜. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 137-156) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in Chinese and English. / Guo Nuo. / 中文摘要 --- p.i / 英文摘要 --- p.ii / 引言 --- p.1 / Chapter 第一章 --- 导论 乙肝：作为医学问题和作为社会问题 --- p.3 / Chapter 1.1 --- 作为医学问题的乙型肝炎及其全球地理分布 --- p.3 / Chapter 1.2 --- 乙型肝炎在中国 --- p.6 / Chapter 1.3 --- 作为社会问题的乙肝歧视 --- p.8 / Chapter 1.4 --- 研究方法 --- p.10 / Chapter 1.5 --- 小结 --- p.14 / Chapter 第二章 --- 研究问题与文献回顾 --- p.15 / Chapter 2.1 --- 社会运动理论的发展脉络 --- p.16 / Chapter 2.2 --- 认同与社会运动的动员 --- p.21 / Chapter 2.3 --- 中国底层社会与维权抗争研究回顾 --- p.24 / Chapter 2.4 --- 中国反乙肝歧视运动的分析框架 --- p.26 / Chapter 2.5 --- 小结 --- p.34 / Chapter 第三章 --- 政府与入职体检 --- p.35 / Chapter 3.1 --- 新中国成立之初的公共卫生政策 --- p.35 / Chapter 3.2 --- 中国人事制度改革和公务员职业声望 --- p.39 / Chapter 3.3 --- 体检标准与强制乙肝检测 --- p.43 / Chapter 3.4 --- 小结 --- p.47 / Chapter 第四章 --- 市场话语权与定义“乙肝" --- p.49 / Chapter 4.1 --- 市场中的虚假广告 --- p.50 / Chapter 4.2 --- 体检经济 --- p.54 / Chapter 4.3 --- 医药商与医学权威 --- p.56 / Chapter 4.4 --- 小结 --- p.58 / Chapter 第五章 --- 乙肝携带者的个人经验 --- p.61 / Chapter 5.1 --- 疾病的社会建构 --- p.62 / Chapter 5.2 --- 乙肝携带者的认知过程 --- p.66 / Chapter 5.3 --- 乙肝携带者的抗争选择 --- p.68 / Chapter 5.4 --- 小结 --- p.74 / Chapter 第六章 --- 从个体经验到集体行动：公民社会的回应 --- p.76 / Chapter 6.1 --- 从个人困境到集体行动 --- p.76 / Chapter 6.2 --- 反乙肝歧视运动中的资源动员 --- p.80 / Chapter 6.3 --- 反乙肝歧视组织网络 --- p.82 / Chapter 6.4 --- 反乙肝歧视运动中的机会空间 --- p.86 / Chapter 6.5 --- 小结 --- p.90 / Chapter 第七章 --- 框架策略：反乙肝歧视运动中的动员 --- p.91 / Chapter 7.1 --- 认同与社会运动 --- p.91 / Chapter 7.2 --- 反乙肝歧视运动的行动框架 --- p.95 / Chapter 7.3 --- 反乙肝歧视运动的动员策略 --- p.100 / Chapter 7.4 --- 小结 --- p.107 / Chapter 第八章 --- 身体的政治：将“乙肝"去除 --- p.108 / Chapter 8.1 --- 权力的维度 --- p.108 / Chapter 8.2 --- 反乙肝歧视行动的抗争轨迹 --- p.111 / Chapter 8.3 --- 国家、市场与公民社会：将“乙肝"去除 --- p.116 / Chapter 8.4 --- 小结 --- p.130 / Chapter 第九章 --- 总结与讨论 --- p.131 / 参考文献 --- p.137 / Chapter 附录A --- 访谈提纲 --- p.157 / Chapter 附录B --- 64名被访者基本信息概况 --- p.160 / 致谢 --- p.162

Eine Monotherapie mit Tenofovir disoproxil fumarate (TDF) stellt eine hoch effiziente Therapie-option für multipel vorbehandelte Patienten mit chronischer Hepatitis-B-Virusinfektion (HBV) dar. Eine Resistenz gegen TDF wurde bislang nicht beschrieben, jedoch wird ein möglicher negativer Einfluss von Adefovir dipivoxil (ADV)-Resistenzvarianten auf die TDF-Ansprechrate diskutiert. Diese retrospektive Kohortenstudie untersucht die Dynamik von Nukleos(t)id-Analoga (NA)-Resistenzvarianten im HBV-Polymerasegen mit Fokus auf ADV-Resistenzvarianten bei 18 chronisch HBV-infizierten Patienten mit Therapieversagen auf eine vorangegangene Lamivudin (LAM)- und ADV-Therapie, sowie nur partiellem Therapieansprechen auf eine TDF-Monotherapie. Zur Detektion von NA-Resistenzvarianten wird eine HBV-Genomsequenzierung mit Tiefenpyrosequenzierung (Genome Sequencer FLX, Roche Diagnostics, Germany) (UDPS), direkte Sequenzierung (TRUGENETM HBV Genotyping Kit, OpenGeneTM DNA Sequencing Sys-tem, Siemens Healthcare Diagnostic, USA) (TG) und Line Probe Assay (INNO-LiPa DRv2 und v3, Innogenetics, Belgium) (INNO-LiPA) durchgeführt. Unter TDF kommt es zu einer quantitati-ven Shift zugunsten der ADV-Resistenzvarianten mit konstant bleibendem Anteil und deutlich höher persistierender Virämie zu Monat 12 im Vergleich zu Patienten ohne ADV-Resistenzvarianten. Vor allem werden die Varianten rtA181V und rtN236T selektiert, jedoch nicht die Variante rtA181T. Die absolute Anzahl der LAM-Resistenzvarianten hingegen halbiert sich. Varianten mit einem initial per UDPS detektierten Anteil von >20% der patientenspezifi-schen HBV-Population werden meist selektiert und nehmen im Verlauf den Hauptanteil der Quasispezies ein. UDPS stellte ein potentes Medium der Detektion, Identifikation und Quantifi-zierung von HBV-Varianten dar und ist INNO-LiPa und TG überlegen. Es ergibt sich kein Hin-weis auf TDF-Resistenzvarianten, jedoch zeigt das Vorliegen von ADV-Resistenzvarianten ei-nen tendentiell negativen Einfluss auf die virale Kinetik. Weitere größere Langzeitstudien sind zur Bestätigung dieser Beobachtung notwendig. / Tenofovir disoproxil fumarate (TDF) is a highly efficient treatment option for nucleos(t)ide analogue (NA) pre-treated patients with chronic hepatitis B virus (HBV) infection. Little is known about the reasons for persistent virus replication in some rare cases. As of today, no TDF resistance variants have been identified, but a possible linkage to Adefovir dipivoxil (ADV) resistance associated variants negatively influencing HBV-DNA suppression by TDF has been suspected, based on the similarity of the chemical structure. In this retrospective cohort study the dynamics of NA resistance variants in the HBV polymerase gene with focus on ADV resistance variants were assessed. For this, we have chosen a cohort including patients with multiple failures to treatment with different NAs. Thus, data of 18 patients with previous treatment failure to LAM and ADV was analysed, showing a persistent viremia (HBV-DNA >35 copies/mL) despite switch to TDF monotherapy (median HBV-DNA at month 12 3,5±0,8 (2,1-4,9) log10 copies/mL). Sequencing analysis was performed with ultra-deep pyrosequencing (UDPS) (Genome Sequencer FLX, 454 Life Science, Roche Diagnostic, Branford, CT), direct sequencing (TG) (TRUGENETM HBV Genotyping Kit, OpenGeneTM DNA Sequencing System, Siemens Healthcare Diagnostic, USA) and line probe assay (INNO-LiPA) (INNO-LiPa DRv2/v3, Innogenetics, Belgium). Using TDF monotherapy, a quantitative shift in favour to ADV resistance variants was observed in this cohort. The percentage of substitutions conferring resistance to ADV at baseline (BL) and at the time of the last sequencing endpoint (EP) of the HBV genome remained constant (BL 35%, 13/37, EP 36%, 9/25). The variants rtA181V and rtN236T were mostly selected, whereas rtA181T was not selected. The total amount of substitutions conferring resistance to Lamivudin (LAM) showed a strong decline, however remained the majority part of all NA resistance variants (BL 51% (19/37), EP 40% (10/25)). The percentage of ETV resistance variants increased slightly (BL 14% (5/37), EP 24% (6/25)). Known ADV, Lam and ETV resistance variants emerged in variable abundance (1,0-99,6%) of quasispecies during TDF therapy. A homogenization of HBV quasispecies took place. Especially mutations occurring in higher abundance (>20% of viral population) were mostly selected (BL 51% (19/37), EP 80% (20/25)). No new HBV variants with possible association to resistance against TDF were identified, but patients with ADV resistance variants showed the highest HBV-DNA level at month 12 of TDF therapy (median HBV-DNA 3,57±0,72 (2,14-3,96) log10 copies/mL, not significant). A negative influence of ADV resistance variants on viral suppression with TDF monotherapy may be assumed, however more long-term studies are needed to confirm the role of ADV resistance variants in TDF therapy. UDPS is a potent medium for detection, identification and quantification of dominant to low level variants in HBV-DNA. It is superior to direct sequencing and line probe assay in the detection of variants.

La lutte pour les ressources est un phénomène qui a débuté dès l'apparition d'organismes reproductifs et dont la description a été initiée par Malthus puis remarquablement synthétisée et étendue à la biologie sous le terme d'évolution par Darwin en 1859 dans « De l'origine des espèces ». Si le concept est ancien à l'échelle des sciences biologiques, il continue à caractériser des domaines à l'époque insoupçonnés par son auteur tels que la virologie. En effet, les virus hautement variables tels que le virus de l'immunodéficience humaine (VIH), de l'hépatite B (VHB) et de l'hépatite C (VHC) sont présents sous forme de quasi espèce au sein de leur environnement réplicatif, c'est à dire qu'une multitude de virus génétiquement proche mais distincts coexistent au sein de cet espace qu'ils doivent partager selon les mêmes règles générales que les êtres vivants. Ainsi, lorsque des pressions de sélection s'exercent (immunitaires, antivirales…), une redistribution des variants majoritaires est observé au bénéfice de variants minoritaires mieux adaptés à cet environnement changeant. La modélisation mathématique et informatique de la capacité mutationnelle et la dynamique d'adaptation des variants minoritaires au travers de 6 études de cohortes de patients infectés, par la technique ultra-sensible de pyroséquençage haut débit associée à des logiciels originaux ont permis de mettre en évidence, caractériser et évaluer l'impact de marqueurs diagnostics permettant de prédire la résistance aux antiviraux mais aussi de caractériser de nouvelles cibles antivirales. / Struggle for resources is a worldwide rule which has been first described by Malthus and extended to whole world of living organisms by Darwin in 1859 in “Origin of species”. Today, this concept has been enlarged to virological field, and is particularly adapted to describe highly variable viruses like Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) which have a quasispecies distribution in infected patients characterized by the co-existence of a number of distinct but related viral populations. Selection pressure on viral replicative environment (immune, antiviral drug treatment…), generally lead to a redistribution of the viral quasispecies with an increasing of the best adapted minor viral variants at the expense of major viral populations. Mathematical and bioinformatic modelization of this phenomenon through 6 infected patients cohorts by means of ultra-deep sequencing and an original bioinformatic package allowed discovery, characterization and evaluation of new diagnostic markers that could be used to prevent resistance emergence to antiviral drugs and to characterized new therapeutics antiviral targets.