L'une des principales cibles pour la thérapie visant le virus de l'hépatite C (VHC) est l'ARN polymérase dépendante de l'ARN NS5B indispensable à la réplication du génome virale. NS5B est l'une des enzymes clefs du cycle virale de VHC et son activation met en jeu aussi bien des interactions intramoléculaires que des interactions avec des cofacteurs viraux et cellulaires au sein du complexe de réplication. Nous avons développé une nouvelle stratégie d'inhibition de NS5B basée sur l'élaboration de peptides courts dérivés de motifs exposés à la surface de l'enzyme dans le but de cibler les nombreuses interactions impliquées dans l'activation de cette protéine. En associant une analyse fine de la structure cristallographique de NS5B avec de la modélisation moléculaire, nous avons élaboré des peptides courts mimant les motifs « hotspot » de la protéine. Ces peptides ont été évalués sur système réplicon de génotype 1b et nous avons ainsi identifié un peptide leader Moon1 de 15 résidus correspondant à un motif hautement conservé du domaine "thumb". Dans ce travail, nous avons étudié en détail la structure et le mécanisme moléculaire de ce nouvel inhibiteur de NS5B. Moon1 inhibe l'activité polymérase de la forme sauvage de NS5B ainsi que celle de mutants résistants au inhibiteurs nucléosidiques et non nucléosidiques. Nous avons démontré que la fixation de Moon1 entraine un changement de conformation de NS5B et se fait préférentiellement avec NS5B dans une conformation fermée. Ce peptide inhibe spécifiquement l'interaction entre NS5B et l'ARN double brin, indépendamment de la présence d'ions métalliques et de manière dose-dépendante. Moon1 bloque la transition entre l'étape d'initiation de novo de la synthèse d'ARN et l'extension du primer. Nous avons démontré que les résidus essentiels à l'activité de Moon1 sont hautement conservés à travers les différents génotypes et sous-types de VHC. De plus, nous avons établi une séquence minimale pour l'activité de Moon1. Nos travaux permettent de valider l'intérêt d'une stratégie interfaciale ciblant une enzyme clef du cycle du VHC et les interactions intra et intermoléculaires nécessaires à son activation. / The non-structural protein RNA-dependent RNA polymerase (RdRp) NS5B plays a key role in hepatitis C virus (HCV) replication and is currently considered as one of the most relevant target to develop safe anti-HCV agents. Although many small molecules have been identified as inhibitors of NS5B, very few are active in clinic. The structure and function of NS5B have been well characterized and as other polymerases, NS5B adopts a typical “right-hand” conformation containing the characteristic fingers, palm and thumb subdomains. The activation of NS5B requires conformational changes involving intramolecular contacts as well interactions with viral proteins and host factors in the replication complex. We developed a new strategy for NS5B inhibition based on short interfacial peptides derived from NS5B surface accessible motifs that target protein-protein interfaces or essential motifs involved in NS5B-activation. Combining the NS5B crystallogaphic structure and molecular modelling, we have designed short peptides derived from NS5B surface “hotspots” that were screened using HCV genotype 1b replicon cell system. We have identified Moon1, a short 15-residu peptide, derived from a well-conserved motif located in the NS5B thumb domain that inhibits HCV replication in the low nanomolar range. Moon1 tightly binds NS5B in a conformational-dependent manner and induces NS5B conformational changes. This peptide specifically inhibits double-stranded RNA/NS5B interactions in a dose-dependent and metal ions-independent manner. Moon1 blocks the transition between RNA de novo initiation and primer-extension. We showed that residues required for Moon-1 anti-polymerase activity are well-conserved among HCV genotypes and subtypes and a minimal Moon1 active motif was established. Taken together, these results demonstrate that NS5B structural dynamics constitute an attractive target for HCV chemotherapeutics and for the design of more specific new antiviral drugs.

NS5B est l’ARN polymérase du virus de l’hépatite C (VHC). Sa structure a beaucoup été étudiée par radiocristallographie, elle contient trois sous-domaines appelés doigts, paume et pouce. Cependant, les études structurales de cette protéine en solution sont très limitées. La résonance magnétique nucléaire (RMN) a été utilisée pour étudier NS5B en solution ainsi que son interaction avec différents partenaires. L’emploi d’un échantillon de NS5B (65kDa) perdeuterée et sélectivement enrichie au niveau des méthyles 1 des résidus d’isoleucines a permis d’obtenir un spectre simplifié et de bonne qualité. Cette étude a confirmé la présence d’une dynamique particulière dans le pouce et a permis de mettre en évidence des effets à longues distances que se transmettent aux autres sous-domaines. Cette approche a alors été utilisée pour étudier l’interaction entre NS5B et le domaine 2 de la protéine NS5A (NS5A-D2) du VHC. Celui-ci est un domaine intrinsèquement désordonné qui interagit directement avec NS5B in vitro. Nous avons identifié que NS5A-D2 se lie via deux sites d’interaction sur le sous-domaine du pouce. Puisqu’un de ces sites est le site de liaison de l’inhibiteur allostérique filibuvir, nous avons étudié la liaison de cette molécule à la polymérase. Sa liaison cause des effets à longues distances tout au long de NS5B. Enfin, nous avons caractérisé la liaison d’un ARN simple brin à NS5B et nous avons identifié que NS5A-D2 et filibuvir réduisent mais ne suppriment pas l’interaction de NS5B avec l’ARN. L’analyse de NS5B par RMN en solution a permis d’étudier des interactions et d’accéder à des paramètres dynamiques très complémentaires des études cristallographiques. / NS5B is the hepatitis C virus (HCV) RNA-dependent RNA polymerase. This protein has been extensively studied by X-ray crystallography and shows an organization in three subdomains called fingers, palm and thumb. Whereas static crystallographic data are abundant, structural studies of this protein in solution are limited. Nuclear magnetic resonance (NMR) spectroscopy was used to study the 65 kDa NS5B in solution as well as its interaction with binding partners. It was characterized using selective isotopic labeling of isoleucine side-chain methyl groups, which gives rise to a simplified NMR spectrum with an improved signal-to-noise ratio. This characterization confirmed the presence of particular dynamics in the subdomains, especially in the thumb, as well as long-range effects that are transmitted through to other subdomains. Furthermore, this system was used to investigate the binding of the domain 2 of NS5A (NS5A-D2), a disordered domain of another HCV protein that has been shown to directly interact with NS5B in vitro. With paramagnetic relaxation enhancement experiments we showed that NS5A-D2 binds to NS5B via, at least, two binding sites on the thumb subdomain. As one of these sites was the binding site of allosteric inhibitor filibuvir, we characterized the binding of this small molecule to NS5B by NMR and found long-range effects of its binding throughout the polymerase. Finally, we studied the binding of a small RNA template strand to NS5B and found that both NS5A-D2 and filibuvir reduce but do not abolish the interaction between the polymerase and RNA. In sum, NMR spectroscopy was used to study dynamic properties of NS5B and its interactions with binding partners.

Hepatitis C virus (HCV) replication is directed by NS5b, the viral RNA dependent RNA polymerase (RdRp). To date, our understanding of the HCV polymerase has come almost entirely from genotype 1. The aim of this study was to examine the influence of sequence variation in the polymerase region by characterising a polymerase derived from genotype 3a. The genotype 3a CB strain polymerase was cloned into the bacterial expression vector pTrcHis2C incorporating a hexahistidine tag to facilitate purification. An optimised process produced 2.5 mg of highly purified recombinant protein per litre of bacterial culture. The 3a preparation possessed an RdRp activity and could utilise both homopolymeric and heteropolymeric RNA templates. Optimal activity was seen at 30oC at pH 8 in reactions containing 160nM enzyme, 10??g/ml RNA template and 2.5mM MnCl2. Subsequently, three genotype 1b polymerases including the HCV-A, Con1 and JK1 strains were cloned for the comparison of activity under identical conditions. Steady state kinetic parameters for GMP incorporation revealed the 3a polymerase exhibited the highest activity, with an almost two fold higher catalytic efficiency (Kcat/Km) than HCVA-1b, primarily due to differences in Km for GTP (2.984??M vs 5.134??M). Furthermore, the 3a polymerase was 3.5 fold and 15 fold more active than JK1-1b and Con1-1b respectively. Improving our understanding of the influence of sequence difference on polymerase activity, particularly in the context of replication will be crucial to developing effective antiviral therapies.

Hepatitis C virus (HCV) replication is directed by NS5b, the viral RNA dependent RNA polymerase (RdRp). To date, our understanding of the HCV polymerase has come almost entirely from genotype 1. The aim of this study was to examine the influence of sequence variation in the polymerase region by characterising a polymerase derived from genotype 3a. The genotype 3a CB strain polymerase was cloned into the bacterial expression vector pTrcHis2C incorporating a hexahistidine tag to facilitate purification. An optimised process produced 2.5 mg of highly purified recombinant protein per litre of bacterial culture. The 3a preparation possessed an RdRp activity and could utilise both homopolymeric and heteropolymeric RNA templates. Optimal activity was seen at 30oC at pH 8 in reactions containing 160nM enzyme, 10??g/ml RNA template and 2.5mM MnCl2. Subsequently, three genotype 1b polymerases including the HCV-A, Con1 and JK1 strains were cloned for the comparison of activity under identical conditions. Steady state kinetic parameters for GMP incorporation revealed the 3a polymerase exhibited the highest activity, with an almost two fold higher catalytic efficiency (Kcat/Km) than HCVA-1b, primarily due to differences in Km for GTP (2.984??M vs 5.134??M). Furthermore, the 3a polymerase was 3.5 fold and 15 fold more active than JK1-1b and Con1-1b respectively. Improving our understanding of the influence of sequence difference on polymerase activity, particularly in the context of replication will be crucial to developing effective antiviral therapies.

Le projet de thèse se focalise sur la synthèse de biomolécules et se subdivise en deux parties. La première partie concerne la conception et la synthèse de dérivés de produits naturels d'intérêt thérapeutique nommés aurones en vue de mettre au point de nouvelles molécules à activité antivirale. Récemment, les aurones ont été identifiées comme étant des inhibiteurs de l'ARN-polymérase ARN-dépendante (NS5B) du virus de l'hépatite C (VHC). Cette enzyme, présente chez le virus mais absente chez l'homme, joue un rôle central dans la réplication virale. Suite à ces résultats antérieurs, les efforts ont été poursuivis et, dans le cadre de cette thèse, nous avons entrepris,d'une part, la synthèse d'analogues originaux dont le cycle B des aurones a été remplacé par des hétérocycles et, d'autre part, la synthèse depseudodimères d'aurones dans le but d'affiner les exigences structurales pour améliorer l'effet inhibiteur.L'activité a été évaluée selon des tests enzymatiques et cellulaires et a permis d'identifier quelques candidats doués d'une bonne activité inhibitrice et d'une faible toxicité. La deuxième partie du projet de thèse, sans lien avec la première partie,concerne des aspects plus fondamentaux et porte sur la synthèse de nouveaux surfactants agissant comme agents stabilisants lors des procédures d'extraction et de cristallisation des protéines membranaires. Les surfactants sont des composants clés dans le domaine de la biologie structurale et de la biochimie des protéines membranaire. Ils sont nécessaires pour maintenir les protéines membranaires dans leur état fonctionnel après extraction. La grande majorité des protéines membranaires est riche en résidus basiques à l'interface. Sur la base de cette caractéristique, une nouvelle famille de surfactants est développée et testée sur des protéines membranaires appartenant aux pompes d'efflux ABC multi-résistantes. / The PhD project focuses on biomolecules and is divided into two parts. The first part concerns the design and synthesis of natural product derivatives with therapeutic interest in order to develop new molecules with antiviral activity. Recently, aurones were identified as new inhibitors of hepatitis C virus (HCV) NS5B polymerase. Following these results, efforts were continuedand we undertook, on the one hand,the synthesis of original analogues in which the aurone B-ring was replaced by a heterocyclic rings and, on the other hand, the synthesis of aurone pseudodimers in order to refine the structural requirements to improve the inhibitory effect. The potent NS5B inhibitory activity combined with their low toxicity make aurones attractive drug candidates against HCV infection. The second part of the PhD thesis is unrelated to the first part and concerns more fundamental aspects. It focused on the synthesis of new surfactants acting as stabilizing agents during extraction of membrane proteins (PM). Surfactants are required for maintaining PM in their functional state after extraction from membrane lipid matrix. The vast majority of PM shares a net enrichment in basic residues at the interface between membrane and cytoplasm, a property known as the positive inside rule. Based on this feature, a new family of surfactants is developed and tested on membrane proteins belonging to the multidrug ABC efflux pumps family.

Le projet de thèse se focalise sur la synthèse de biomolécules et se subdivise en deux parties. La première partie concerne la conception et la synthèse de dérivés de produits naturels d'intérêt thérapeutique nommés aurones en vue de mettre au point de nouvelles molécules à activité antivirale. Récemment, les aurones ont été identifiées comme étant des inhibiteurs de l'ARN-polymérase ARN-dépendante (NS5B) du virus de l'hépatite C (VHC). Cette enzyme, présente chez le virus mais absente chez l'homme, joue un rôle central dans la réplication virale. Suite à ces résultats antérieurs, les efforts ont été poursuivis et, dans le cadre de cette thèse, nous avons entrepris,d'une part, la synthèse d'analogues originaux dont le cycle B des aurones a été remplacé par des hétérocycles et, d'autre part, la synthèse depseudodimères d'aurones dans le but d'affiner les exigences structurales pour améliorer l'effet inhibiteur.L'activité a été évaluée selon des tests enzymatiques et cellulaires et a permis d'identifier quelques candidats doués d'une bonne activité inhibitrice et d'une faible toxicité. La deuxième partie du projet de thèse, sans lien avec la première partie,concerne des aspects plus fondamentaux et porte sur la synthèse de nouveaux surfactants agissant comme agents stabilisants lors des procédures d'extraction et de cristallisation des protéines membranaires. Les surfactants sont des composants clés dans le domaine de la biologie structurale et de la biochimie des protéines membranaire. Ils sont nécessaires pour maintenir les protéines membranaires dans leur état fonctionnel après extraction. La grande majorité des protéines membranaires est riche en résidus basiques à l'interface. Sur la base de cette caractéristique, une nouvelle famille de surfactants est développée et testée sur des protéines membranaires appartenant aux pompes d'efflux ABC multi-résistantes. / The PhD project focuses on biomolecules and is divided into two parts. The first part concerns the design and synthesis of natural product derivatives with therapeutic interest in order to develop new molecules with antiviral activity. Recently, aurones were identified as new inhibitors of hepatitis C virus (HCV) NS5B polymerase. Following these results, efforts were continuedand we undertook, on the one hand,the synthesis of original analogues in which the aurone B-ring was replaced by a heterocyclic rings and, on the other hand, the synthesis of aurone pseudodimers in order to refine the structural requirements to improve the inhibitory effect. The potent NS5B inhibitory activity combined with their low toxicity make aurones attractive drug candidates against HCV infection. The second part of the PhD thesis is unrelated to the first part and concerns more fundamental aspects. It focused on the synthesis of new surfactants acting as stabilizing agents during extraction of membrane proteins (PM). Surfactants are required for maintaining PM in their functional state after extraction from membrane lipid matrix. The vast majority of PM shares a net enrichment in basic residues at the interface between membrane and cytoplasm, a property known as the positive inside rule. Based on this feature, a new family of surfactants is developed and tested on membrane proteins belonging to the multidrug ABC efflux pumps family.

Estima-se que a prevalência global da população mundial com hepatite C é de 3%. Pouco se sabe sobre a resposta ao tratamento com respeito à resistência viral. Algumas mutações no fragmento de 109 aminoácidos da NS5B são associadas com resistência ao interferon (IFN) e ribavirina (RBV). Estudos moleculares e clínicos identificaram fatores associados com o hospedeiro e vírus relacionados associada com a resposta ao tratamento, tal como o gene que codifica a IL-28B. Este estudo foi dividido em duas fases, cujos objetivos foram caracterizar a frequência de mutações que conferem resistência ao HCV e avaliar a relevância das mutações em pacientes Respondedores (R) ou Não Respondedores (NR) ao tratamento e caracterizar geneticamente as populações sobre polimorfismos genéticos nos SNPs da IL-28B em relação ao prognóstico da resposta ao tratamento. As amostras dos pacientes foram submetidas a testes de genotipagem e carga viral. As sequências geradas foram comparadas no BLAST e no banco de dados Los Alamos HCV. Realizamos o alinhamento das sequências homólogas e as mutações identificadas. Com base no genótipo e carga viral determinamos a classificação dos pacientes de acordo com a resposta à terapia. O DNA genômico foi isolado a partir de sangue periférico para a realização da tipagem de SNPs de IL-28B. A metodologia utilizada foi de PCR em tempo real utilizando sondas TaqMan SNP específico. A análise dos dados foi realizada utilizando GraphPad Prism com qui-quadrado, risco relativo (RR), Odds Ratio (OR) e intervalo de confiança de 95%, com um nível de significância de P <0,05. Foi encontrado na primeira fase deste estudo uma taxa significativa mutações associadas ao tratamento nas amostras estudadas. A prevalência de mutações associadas à resistência ao IFN e RBV bem como a novos medicamentos antivirais localizados no fragmento de 109 aminoácidos da NS5B foi examinado em 69 indivíduos infectados naïve no Rio de Janeiro, Brasil. Na segunda fase, as mutações foram clinicamente relevantes. Desde então, procuramos observar as diferenças entre melhor ou pior prognóstico de acordo com a imunogenética que mostrou diferenciação entre os grupos R e NR ao tratamento em relação ao prognóstico da resposta terapêutica. Quando as diferenças entre as sequências da NS5B e a resposta ao tratamento foram consideradas verificou-se que associada a mutação R254K, estava a C316N que poderia conduzir a uma não resposta à terapia no genótipo 1b. Os nossos dados também suportaram forte associação de IL-28B rs12979860, com elevada probabilidade de resposta à terapia de IFN + RBV. Nossos dados evidenciam a presença de pacientes virgens de tratamento que abrigam mutações de resistência previamente descritas na literatura. A análise dos fatores preditores de resposta virológica mostrou que a predição de boa resposta ou não ao tratamento e ainda da progressão da doença é dependente de uma importante interação entre a genética viral e a do hospedeiro. Fato este importante para que no momento de avaliação de diagnóstico e conduta terapêutica, o médico possa tomar medidas apropriadas para o tratamento de cada paciente individualmente independentemente do genótipo do HCV em questão. / It is estimated that the overall prevalence of the average world population with hepatitis C is 3%. Little is known about the treatment response with respect to viral resistance. Some mutations in the 109-aminoacid fragment of NS5B are associated to Interferon (IFN) and Ribavirin (RBV) resistance. Molecular and clinical studies have identified factors associated with the host and related viruses associated with response to treatment, as the gene encoding IL-28B. This study was divided into two phases whose objectives were to characterize the frequency of mutations conferring resistance to HCV viral evaluating the relevance of these in Responders (R) or Non-Responders (NR) patients to treatment and to characterize genetically the populations regarding genetic polymorphisms SNPs IL-28B in relation to prognosis of response to treatment for HCV. Patient samples were subjected to tests for genotyping and viral load. The sequences generated were compared in the BLAST and the Los Alamos database HCV. We conducted the alignment of homologous sequences and mutations identified. Based on virological parameters genotype and viral load determined the classification of patients according to response to therapy. Genomic DNA was isolated from peripheral blood for carrying out the typing of SNPs of IL-28B. The methodology used was real-time PCR using TaqMan probes specific SNPs. Data analysis was performed using GraphPad Prism with chi-square, relative risk (RR), Odds Ratio (OR) and confidence interval of 95% with a significance level of P <0.05. To study these biological parameters we associated the responsive patients, non-responders, the viral load, genotype, and IL-28B polymorphism to treatment outcome. We found in the first phase of this study a significant rate of treatment-associated mutations in the samples studied. The prevalence of mutations associated to resistance to interferon and ribavirin (IFN/RBV) as well new antiviral drugs located in the 109 aminoacid fragment of NS5B was examined in 69 Hepatitis C Virus drug naïve (HCV)-infected individuals in Rio de Janeiro, Brazil. In the second phase, the mutations revealed clinically relevant from the gene in question. Since then, we seek to observe the differences between better or worse prognosis according to immunogenetic showed that differentiation between the immunogenetics of the groups R and NR to treatment in relation to prognosis of therapeutic response. When the differences between the NS5B sequences at baseline and the treatment response were considered we found that R254K associated with C316N mutations could lead to a non-response to IFN-RBV therapy in genotype 1b. Our data also strong support the association of rs12979860 IL-28B polymorphism with high probability of response to IFN + RBV therapy. Our data highlight the presence of HCV genotypes from drug naïve patients harboring resistance mutations previously described in literature. The analysis of predictors virologic response demonstrated that the prediction of better or worse therapy response and further the disease progression is dependent of a significant interaction between viral and host genetics. This fact is important for diagnosis evaluation and clinical therapeutic, the medico can take appropriate measures to treat each individual patient irrespective of the genotype of HCV in question.

Estima-se que a prevalência global da população mundial com hepatite C é de 3%. Pouco se sabe sobre a resposta ao tratamento com respeito à resistência viral. Algumas mutações no fragmento de 109 aminoácidos da NS5B são associadas com resistência ao interferon (IFN) e ribavirina (RBV). Estudos moleculares e clínicos identificaram fatores associados com o hospedeiro e vírus relacionados associada com a resposta ao tratamento, tal como o gene que codifica a IL-28B. Este estudo foi dividido em duas fases, cujos objetivos foram caracterizar a frequência de mutações que conferem resistência ao HCV e avaliar a relevância das mutações em pacientes Respondedores (R) ou Não Respondedores (NR) ao tratamento e caracterizar geneticamente as populações sobre polimorfismos genéticos nos SNPs da IL-28B em relação ao prognóstico da resposta ao tratamento. As amostras dos pacientes foram submetidas a testes de genotipagem e carga viral. As sequências geradas foram comparadas no BLAST e no banco de dados Los Alamos HCV. Realizamos o alinhamento das sequências homólogas e as mutações identificadas. Com base no genótipo e carga viral determinamos a classificação dos pacientes de acordo com a resposta à terapia. O DNA genômico foi isolado a partir de sangue periférico para a realização da tipagem de SNPs de IL-28B. A metodologia utilizada foi de PCR em tempo real utilizando sondas TaqMan SNP específico. A análise dos dados foi realizada utilizando GraphPad Prism com qui-quadrado, risco relativo (RR), Odds Ratio (OR) e intervalo de confiança de 95%, com um nível de significância de P <0,05. Foi encontrado na primeira fase deste estudo uma taxa significativa mutações associadas ao tratamento nas amostras estudadas. A prevalência de mutações associadas à resistência ao IFN e RBV bem como a novos medicamentos antivirais localizados no fragmento de 109 aminoácidos da NS5B foi examinado em 69 indivíduos infectados naïve no Rio de Janeiro, Brasil. Na segunda fase, as mutações foram clinicamente relevantes. Desde então, procuramos observar as diferenças entre melhor ou pior prognóstico de acordo com a imunogenética que mostrou diferenciação entre os grupos R e NR ao tratamento em relação ao prognóstico da resposta terapêutica. Quando as diferenças entre as sequências da NS5B e a resposta ao tratamento foram consideradas verificou-se que associada a mutação R254K, estava a C316N que poderia conduzir a uma não resposta à terapia no genótipo 1b. Os nossos dados também suportaram forte associação de IL-28B rs12979860, com elevada probabilidade de resposta à terapia de IFN + RBV. Nossos dados evidenciam a presença de pacientes virgens de tratamento que abrigam mutações de resistência previamente descritas na literatura. A análise dos fatores preditores de resposta virológica mostrou que a predição de boa resposta ou não ao tratamento e ainda da progressão da doença é dependente de uma importante interação entre a genética viral e a do hospedeiro. Fato este importante para que no momento de avaliação de diagnóstico e conduta terapêutica, o médico possa tomar medidas apropriadas para o tratamento de cada paciente individualmente independentemente do genótipo do HCV em questão. / It is estimated that the overall prevalence of the average world population with hepatitis C is 3%. Little is known about the treatment response with respect to viral resistance. Some mutations in the 109-aminoacid fragment of NS5B are associated to Interferon (IFN) and Ribavirin (RBV) resistance. Molecular and clinical studies have identified factors associated with the host and related viruses associated with response to treatment, as the gene encoding IL-28B. This study was divided into two phases whose objectives were to characterize the frequency of mutations conferring resistance to HCV viral evaluating the relevance of these in Responders (R) or Non-Responders (NR) patients to treatment and to characterize genetically the populations regarding genetic polymorphisms SNPs IL-28B in relation to prognosis of response to treatment for HCV. Patient samples were subjected to tests for genotyping and viral load. The sequences generated were compared in the BLAST and the Los Alamos database HCV. We conducted the alignment of homologous sequences and mutations identified. Based on virological parameters genotype and viral load determined the classification of patients according to response to therapy. Genomic DNA was isolated from peripheral blood for carrying out the typing of SNPs of IL-28B. The methodology used was real-time PCR using TaqMan probes specific SNPs. Data analysis was performed using GraphPad Prism with chi-square, relative risk (RR), Odds Ratio (OR) and confidence interval of 95% with a significance level of P <0.05. To study these biological parameters we associated the responsive patients, non-responders, the viral load, genotype, and IL-28B polymorphism to treatment outcome. We found in the first phase of this study a significant rate of treatment-associated mutations in the samples studied. The prevalence of mutations associated to resistance to interferon and ribavirin (IFN/RBV) as well new antiviral drugs located in the 109 aminoacid fragment of NS5B was examined in 69 Hepatitis C Virus drug naïve (HCV)-infected individuals in Rio de Janeiro, Brazil. In the second phase, the mutations revealed clinically relevant from the gene in question. Since then, we seek to observe the differences between better or worse prognosis according to immunogenetic showed that differentiation between the immunogenetics of the groups R and NR to treatment in relation to prognosis of therapeutic response. When the differences between the NS5B sequences at baseline and the treatment response were considered we found that R254K associated with C316N mutations could lead to a non-response to IFN-RBV therapy in genotype 1b. Our data also strong support the association of rs12979860 IL-28B polymorphism with high probability of response to IFN + RBV therapy. Our data highlight the presence of HCV genotypes from drug naïve patients harboring resistance mutations previously described in literature. The analysis of predictors virologic response demonstrated that the prediction of better or worse therapy response and further the disease progression is dependent of a significant interaction between viral and host genetics. This fact is important for diagnosis evaluation and clinical therapeutic, the medico can take appropriate measures to treat each individual patient irrespective of the genotype of HCV in question.

<p>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.</p> / 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.

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.