Since the first approved hepatitis C virus (HCV) NS3 protease inhibitors in 2011, numerous direct acting antivirals (DAAs) have reached late stages of clinical trials. Today, several combination therapies, based on different DAAs, with or without the need of pegylated interferon-α injection, are available for chronic HCV infections. The chemical foundation of the approved and late-stage HCV NS3 protease inhibitors is markedly similar. This could partly explain the cross-resistance that have emerged under the pressure of NS3 protease inhibitors. The first-generation NS3 protease inhibitors were developed to efficiently inhibit genotype 1 of the virus and were less potent against other genotypes. The main focus in this thesis was to design and synthesize a new class of 2(1H)-pyrazinone based HCV NS3 protease inhibitors, structurally dissimilar to the inhibitors evaluated in clinical trials or approved, potentially with a unique resistance profile and with a broad genotypic coverage. Successive modifications were performed around the pyrazinone core structure to clarify the structure-activity relationship; a P3 urea capping group was found valuable for inhibitory potency, as were elongated R6 residues possibly directed towards the S2 pocket. Dissimilar to previously developed inhibitors, the P1’ aryl acyl sulfonamide was not essential for inhibition as shown by equally good inhibitory potency for P1’ truncated inhibitors. In vitro pharmacokinetic (PK) evaluations disclosed a marked influence from the R6 moiety on the overall drug-properties and biochemical evaluation of the inhibitors against drug resistant enzyme variants showed retained inhibitory potency as compared to the wild-type enzyme. Initial evaluation against genotype 3a displayed micro-molar potencies. Lead optimization, with respect to improved PK properties, were also performed on an advanced class of HCV NS3 protease inhibitors, containing a P2 quinazoline substituent in combination with a macro-cyclic proline urea scaffold with nano-molar cell based activities. Moreover, an efficient Pd-catalyzed C-N urea arylation protocol, enabling high yielding introductions of advanced urea substituents to the C3 position of the pyrazinone, and a Pd-catalyzed carbonylation procedure, to obtain acyl sulfinamides, were developed. These methods can be generally applicable in the synthesis of bioactive compounds containing peptidomimetic scaffolds and carboxylic acid bioisosteres.

Hepatitis C Virus (HCV) infects more than 170 million people worldwide but no vaccine is available yet. HCV entry may represent a promising target for therapies and is mediated by two envelope glycoproteins, E1 and E2, assembled as heterodimer onto the virus surface. However, how E1 and E2 dialog, structurally rearrange and act together during these steps remain poorly defined. In this work, we aimed to clarify the interrelation of E1E2 during virus entry, thus opening ways to potential new therapeutic strategies. We first investigated whether a strong genetic divergence between E1E2 heterodimers may highlight distinct functions. We observed that B-cell derived E1E2 were specialized for B-cell infection, suggesting that new functions can emerge from the E1E2 conformational plasticity. In a second approach, we identified a conserved dialog between E1 and the domain III of E2 that was critical for virus binding and fusion. Moreover, a computational model predicted a strong co-evolution between E1 and E2 as well as potential structural rearrangements, suggesting that HCV E2 is likely a fusion protein able to fold over via its domain III through the mediation of E1. Altogether, these different works highlight that E1 and E2 are involved in complex dialogs that regulate the heterodimer folding and functions, suggesting that E1E2 heterodimer is more likely a single functional protein entity than an association of two proteins with specific functions.

Thesis (Ph.D.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor in Philosophy in Experimental Surgery, Department of Surgery. Title from pdf file main screen (viewed on October 13, 2009). Includes bibliographical references.

Chez un individu infecté, le VHC circule sous la forme d’une population de variants viraux appelés quasi-espèce. Lors d’un évènement de transmission, certains variants viraux sont préférentiellement transmis et un effondrement de la diversité virale chez l’individu nouvellement infecté est souvent observé. Les propriétés électives de ces variants ainsi que leur rôle dans l’évolution clinique sont méconnus. L’objectif de cette étude est d’identifier si des déterminants moléculaires situés au niveau des glycoprotéines d’enveloppe du VHC sont associés à une plus grande capacité de transmission. Les propriétés fonctionnelles des variants transmis et non transmis seront étudiées, en particulier la sensibilité à la neutralisation autologue. Les échantillons étudiés proviennent de couples mère-enfant infectés chroniquement par le VHC issus de d’un essai clinique réalisé en Thaïlande. La composition des populations virales au sein de 3 paires a été étudiée à l’aide d’une technique d’amplification après dilution limite (SGA) suivie d’un séquençage profond (Illumina). Le variant majoritaire chez la mère était retrouvé majoritaire chez l’enfant pour les paires 1 et 3. Pour 2 paires (2 et 3), une moindre diversité génétique a été observée chez l’enfant par rapport à la mère témoignant d’un goulot d’étranglement génétique lors de la transmission. Après clonage des gènes E1E2, des tests d’infectivité sur cellules hépatocytaires ainsi que des tests de neutralisation par le sérum maternel sont réalisés avec le modèle de rétrovirus pseudotypés (VHCpp). Pour la 1ère paire, le variant majoritaire chez la mère (variant transmis à l’enfant) est infectieux et résistant au sérum autologue. Pour la deuxième paire, le variant minoritaire (transmis) est légèrement résistant à la neutralisation autologue. Un variant majoritaire non transmis apparait sensible à la neutralisation autologue. Des études complémentaires en système de virus réplicatifs issus de la culture cellulaire (VHCcc) sont en cours. Au final, les résultats de cette étude contribuent à comprendre les étapes précoces de l’infection par le VHC, afin de mieux appréhender de futures approches immunoprophylactiques ou vaccinales. / In infected individuals, HCV circulates as a complex mixture of genetically different, but closely related viral variants named quasispecies. In a transmission event, some viral variants are preferentially transmitted. The genetic and functional properties of these variants are still unknown. The aim of our work was to identify molecular determinants of E1E2 associates with a greater capacity of transmission. We also intend to study the functional properties of transmitted and no transmitted variants, as for example sensibility to autologous neutralization. Studied sera samples were obtained from three women and their child infected by the HCV, who were participating in HIV prevention clinical trial for the prevention of perinatal transmission of HIV in Thailand. Quasispecies were studied with single genome amplification (SGA) followed by deep sequencing (Illumina). A decrease in intra-host diversity (genetic bottleneck) was observed in the viral population of child near birth (week 6) compared with that observed in the mother (just before delivery). For 2 pairs, the major variant observed in the mother was the same as the major one identified in the child. Retroviral pseudotypes (HCVpp), bearing each transmitted and non-transmitted envelope glycoproteins were produced. For each one, the level of infectivity on HuH7 cells was measured as well as the neutralizing activity of the autologous sera. For the first pair, the major variant (transmitted) appears resistant to autologous neutralization. For the second pair, the transmitted minor variant appears slightly resistant to autologous neutralization. A non-transmitted major variant is sensitive to autologous neutralization. Complementary studies with HCV derived from cell culture (HCVcc) are in progress We hope that the results of this study may be helpful to better understand early steps of HCV infection, which is of great interest for the development of immunoprophylaxis and vaccine strategies.

L'infection par le virus de l'hépatite C implique des processus d'interaction complexe entre l'hôte et le virus. Plusieurs facteurs de l'hôte incluant des polymorphismes génétiques et les réponses immunitaires influent sur l'infection et les réponses au traitement. Aussi, il est important d'identifier en amont les facteurs pour prédire la réponse au traitement. L'objectif de la thèse est d'étudier l'influence de certains polymorphismes génétiques de l'hôte sur la réponse à la bithérapie et sur le statut immunitaire du foie dans l'infection chronique par le VHC. L'étude a porté sur les polymorphismes des gènes de l'interféron lambda 3 et 4, l'interféron gamma, l'interleukine 10, et l'interleukine 17, conjointement à la réponse au traitement avec le peg-IFNα et la RBV et aux réponses immunitaires du foie chez les patients. Nous avons établi une méthode PCR-RFLP simple et fiable pour le typage de deux polymorphismes de l'IFNL3. En utilisant les enzymes de restriction BstUI et BrsDI permet le génotypage de deux variantes de IFNL3 (rs12979860 C/T et rs8099917 T/G, respectivement). Les résultats indiquent que cette méthode PCR-RFLP donne des résultats similaires à ceux des méthodes standard et présente un intérêt pour des analyses de routine en laboratoire clinique car elle est peu coûteuse. Nous avons analysé l'association des polymorphismes avec la réponse au traitement antiviral sur une cohorte de 108 patients infectés par le VHC de génotype 1 traités par la bithérapie. Nous avons ainsi démontré que le génotype de l'IFNL4 TT/TT de ss469415590 et une réponse virologique rapide sont des facteurs prédictifs indépendants pour atteindre un taux de réponse virologique soutenue (OR = 3,93, p = 0,004 et OR = 6,74, p = 0,021). D'autre part, une charge virale initiale haute est associée à l'échec au traitement (OR = 0,34, p = 0,023). Ainsi, ces paramètres sont utiles pour la définition d'un traitement personnalisé. Pour expliquer l'influence de ces polymorphismes dans l'infection chronique par le VHC, nous avons étudié l'association des polymorphismes IFNL3 et 4 avec la réponse immunitaire du foie chez les patients atteints d'une infection chronique par le VHC. En utilisant l'expression de CD107a comme marqueur de l'activité sécrétoires des lymphocytes, nous avons observé une activité de dégranulation des lymphocytes du foie plus importante les patients porteurs des génotypes de IFNL4 favorables en comparaison avec les patients porteurs de l'allèle défavorable. En utilisant des analyses de régression, les taux d'ALT sont en corrélation avec la fréquence des cellules NKT CD107a+ dans le foie. Enfin, chez les patients traités par la bithérapie, une forte activité de dégranulation est observée chez les patients avec les génotypes favorables IFNL3 et 4. Nous suggérons que les polymorphismes des gènes de l'interféron lambda sont associés à l'activité de la dégranulation des lymphocytes intra-hépatiques et contribuent à un mécanisme de clairance du VHC sous la bithérapie. Nous avons également étudié l'impact de plusieurs polymorphismes génétiques sur la gravité de l'hépatite C chronique. Les résultats montrent une association significative observée entre le polymorphisme de l'IFN-γ et la gravité de l'hépatite C chronique. Pour l'analyse de régression logistique, l'allèle T et la présence d'une stéatose sont des facteurs prédictifs indépendants de la sévérité de la maladie hépatique chronique associée au VHC. L'utilisation du génotypage de l'IFN-γ pourrait être utile dans la prise en charge des patients. En conclusion, nous avons montré que les polymorphismes des gènes des IFNL3 et 4 et de l'IFN-γ de l'hôte jouent un rôle important dans l'efficacité du traitement et les réponses immunitaires hépatiques. Ces résultats aident à définir un traitement personnalisé pour le contrôle de l'infection chronique par le VHC, en particulier dans les régions aux ressources limitées où les nouveaux traitements ne sont pas accessibles. / Hepatitis C virus (HCV) infection is a complex interaction process between the host and viral factors. The host immune responses and genetic polymorphisms have been shown to be associated with the outcome of HCV infections and the responses to treatments. Thus, it is very important to identify pre-treament factors to predict treatment outcomes. The overall aim of the thesis study is to investigate the role of host genetic polymorphisms on response to combination therapy and immune response in the liver in chronic HCV infection. The study has focused on polymorphisms in the interferon lambda (IFNL) genes, interferon gamma, interleukin 10, and interleukin 17 in relation to response to therapy with peg-IFNα and Ribavirin (RBV) and liver immune responses in patients with chronic HCV infection.First, we have established a simple and reliable method for genotyping of the IFNL3 polymorphisms. We designed primers and selected restriction enzymes BstUI and BrsDI for genotyping 2 variants rs12979860 C/T and rs8099917 T/G, respectively. The results indicate that this PCR-RFLP method yields to identical data than standard sequencing method and commercial kit. We suggest that PCR-RFLP method could be used routinely in conventionally clinical laboratory for genotyping of IFNL3 polymorphisms. Next, we analysed the association of these variants with response in combination therapy of peg-IFNα and RBV. Among 108 treated patients infected with HCV genotype 1, by using logistic regression model analyses, we showed that patients who had favorable IFNL4 genotype (genotype TT/TT of ss469415590) and presented a rapid virological response (RVR) were independent predictors of achieving sustained virological response rate (OR = 3.93, CI = 1.53 -10.08, p = 0.004 and OR = 6.74, CI = 1.33 - 34.06, p= 0.021), whereas patients with high baseline viral load level were associated with failure to treatment (OR = 0.34, CI = 0.13 - 0.87, p = 0.023). We suggest that patients had favorable IFNL4 genotype and achieved RVR should benefit an individualized treatment of combination therapy of peg-IFNα and RBV. To explain the influence of these polymorphisms in chronic HCV infection, we investigated the association of IFNL4 polymorphisms with immune response in the liver in patients with chronic HCV infection. By using marker CD107a, a marker expressing degranulation activity of cytotoxic lymphocytes, we indicated that degranulation process was found in liver lymphocytes in patients carrying favourable IFNL4 genotypes compared with patients with unfavourable genotypes. By using multiple regression analyses, we demonstrated that ALT levels correlate with frequency of CD107a+ NKT cells in the liver. Finally, in patients treated by peg-IFNα and RBV, high degranulation activity observed in patients with favourable genotypes of IFNL3 and IFNL4 (CC of rs12979860 and TT/TT of ss469415590). We suggest that polymorphisms in the interferon lambda genes associated with intrahepatic lymphocyte degranulation activity and contribute to clearance mechanism of HCV under combination treatment of peg-IFNα and RBV.We investigated the impact of several genetic polymorphisms on the severity of chronic hepatitis C. We showed a significant association observed between polymorphism of IFN-γ and the severity of chronic hepatitis C. By using logistic regression analysis, T allele of IFN-γ and the presence of steatosis are independent predictive factors of severity of HCV-1 - related liver disease. This suggests we can use genetic variant of IFN-γ in classification and management of chronic hepatitis C. In conclusion, we indicated that host genetic polymorphisms play critical roles both in responses to treatment and in the immunopathogenesis of chronic HCV infection. This study can help to reach a closer step to individualized medicine for the control of chronic HCV infection in resource-limited regions when new treatment regimens are not available.

Made available in DSpace on 2014-06-11T19:27:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-14Bitstream added on 2014-06-13T18:31:21Z : No. of bitstreams: 1 yamasaki_lht_me_sjrp.pdf: 6194517 bytes, checksum: 350d37acd1e46ef2885eecc1354a317c (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A infecção pelo vírus da Hepatite C (HCV) é considerada um grande problema de saúde pública, desde a sua descoberta em 1989. Entretanto a terapia mais utilizada atualmente, baseada no uso de Peginterferon, tem sucesso em aproximadamente 50% dos pacientes com o genótipo 1. Embora os mecanismos envolvidos nesta resistência viral ainda não sejam esclarecidos, sugere-se que fatores virais e do hospedeiro participam deste. A proteína não-estrutural 5A (NS5A) está envolvida em diversos processos celulares e é um componente essencial para o HCV. Entretanto, sua estrutura e função ainda não foram bem elucidadas. A partir destes fatos, os objetivos do presente estudo foram elaborar um modelo teórico da NS5A e investigar as propriedades estruturais e funcionais in silico. Foram analisadas 345 sequências da proteína NS5A do HCV de 23 pacientes infectados com o genótipo 1 ou 3. As composições de aminoácidos e de estrutura secundária demonstraram que há diferença entre os genótipos, podendo indicar que há diferenças nas interações proteína-proteína entre os genótipos, o que pode estar relacionado com a diferença da taxa de resistência ao tratamento. A análise funcional foi realizada com o ProtFun, que sugeriu que a NS5A estaria envolvida nas funções celulares de metabolismo intermediário central, tradução, crescimento, tranporte, ligação e hormônio. Estas funções variaram entre os domínios, suportando a hipótese de que a NS5A é uma proteína multifuncional. A análise pelo PROSITE indicou vários sítios de glicosilação, fosforilação e miristoilação, que são altamente conservados e podem ter função importante na estabilização da estrutura e função, sendo assim possíveis alvos de novos antivirais. Alguns deles estão em regiões relacionadas com a resposta ao tratamento. Outro... / Hepatitis C virus (HCV) infects almost 3% of people worldwide and it is considered the main cause of liver chronic diseases and transplants. Until today, there is no effective vaccine and the current most used therapy, based on Peginterferon, is successful only in 50% of patients infected by genotype 1. Although the outcomes of this treatment resistance are unclear, it is suggested host and virus factors may participate in this mechanism. Non-structural 5A (NS5A) protein is involved in several cellular and virus processes and it is a critical component of HCV. However, its structure and function are still uncertain. Regarding these facts, the present study attachments were to elaborate a model of the NS5A protein and to investigate NS5A structural and functional features, using in silico tools. It was analyzed 345 sequences of HCV NS5A protein from 23 patients infected by genotypes 1 or 3. Residues and secondary structure composition of all sequences demonstrated that there are differences between genotypes. It may indicate that there are differences in interactions between genotypes, which could be related with the distinct average of treatment resistance. In addition, among those that varied between genotypes, there were amino acids in regions that studies suggested as related with virus persistence. Functional analysis was performed with ProtFun. It suggested that NS5A is involved with central intermediary metabolism, translation, growth, transport, ligation and hormone functions in the cell. These functions vary between the domains, strengthening the hypothesis that NS5A is a multifunctional protein. Prosite motif search indicated that there are many glicosilation, fosforilation and myristoilation sites, which are highly conserved and may play an important role in structural stabilization and... (Complete abstract click electronic access below)

L’infection par le virus de l’hépatite C (HCV) est une cause majeure de maladies hépatites sévères et constitue un problème majeur de santé public. Le HCV est associé aux lipopoprotéines formant une lipoviroparticule (LVP) qui est la forme infectieuse du virus. L’apolipoprotéine E (apoE), associée aux lipoprotéines, est impliquée dans les étapes précoces et tardives de l’infection. Elle interagit avec de récepteurs impliqués dans le métabolisme lipidique tels les héparanes sulfates protéoglycanes (HSPG). Durant ma thèse, j’ai démontré que les acides aminés chargés positivement du domaine de liaison aux HSPG d’apoE sont impliqués dans l’entrée du HCV dans l’hépatocyte. J’ai également démontré que la production et/ou l’infectiosité des particules virales est corrélée au taux d’expression d’apoE dans les cellules sans avoir d’impact sur la traduction ou la réplication virales. Enfin, j’ai identifié syndecan-4, un membre de la famille des HSPG, comme l’HSPG principal impliqué dans l’entrée du HCV dans les lignées Huh7.5.1. L’ensemble de ces résultats démontre qu’HCV utilise l’interaction apoE-SDC4 pour établir une infection virale efficace. / Hepatitis C virus (HCV) infection is a major cause of liver disease worldwide and represents a major health problem. HCV associates with host lipoproteins forming host/viral hybrid complexes termed lipoviral particles. Apolipoprotein E (apoE) is a lipoprotein component that interacts with heparan sulfate proteoglycans (HSPG) to mediate hepatic lipoprotein uptake, and may likewise mediate HCV entry. I sought to define the functional regions of apoE with an aim to identify critical apoE binding partners involved in HCV infection. I demonstrated a direct correlation of apoE expression and HCV infectivity, whereas no correlation exists with viral protein translation or replication. Mutating the HSPG binding domain (HSPG-BD) of apoE revealed key residues that are critical for mediating HCV infection. Finally, I identified Syndecan-4 (SDC4), an HSPG family member, as the principal HSPG mediating HCV entry. Our data demonstrate that HCV uses apoE-SDC4 interactions to enter hepatocytes and establish efficient viral infection.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Schistosomiasis is a parasitic disease, severe, chronic waterborne and development, whose etiologic agent is Schistosoma mansoni. It occurs in 74 countries with 207 million people infected and 700 million in risky areas. In Brazil, data indicate a prevalence of eight million. The Schistosomiasis can be exacerbated when patients are carriers of the Hepatitis B Virus (HBV) and C (HCV), resulting in simultaneous evolution of both pathologies. In Brazil, the prevalence of co-infection with HBV/HCV and S. mansoni found in studies ranged from 13.6% to 40% for HBV and 0.5% to 19.66% for the HCV. On this basis, this research aimed to identify the prevalence of serological markers of HBV and HCV and risky factors in individuals carrying the S. mansoni in the Santa Maria neighborhood, Aracaju, state of Sergipe. We conducted a cross-sectional epidemiological study. Data were collected for each patient by means of a questionnaire investigation. This questionnaire included variables identifying the subject of the research participants and variables that included the epidemiological risky factors for infection with HBV or HCV. We collected serum samples of research participants and forwarded to the laboratory testing to identify serological markers of hepatitis B and C: Total Anti-HBc IgG, Anti-HBs, HBsAg and Anti-HCV. All procedures performed using the technique of chemiluminescence immunoassay using the ARCHITECT assay for each serological marker following the protocols established by the laboratory equipment manufacturer SYSTEM ARCHITECT. In addition, we carried out the analysis of spatial distribution of co-infection in the district through the program using the TerraView Kernel intensity estimation. It was found that 16 individuals had contact with HBV (9.41%), one of these was positive for HBsAg. Only Thirty-two samples (18.82%) were positive for the marker Anti-HBs. Three samples were positive for anti-HCV (1.76%), and also a positive for Anti-HBc. The main risks of HBV and HCV infection were related to parenteral interventions of health services, as well as sexual activity without condom use in the case of HBV. Spatial analysis of cases of co-infection (Schistosomiasis and Hepatitis) allowed the visualization of areas of higher concentration of these infections, as well as those that are exposed to different degrees of risk of transmission. The survey results allow to offer, the municipal health services, a tool to facilitate the understanding of the spatial distribution of schistosomiasis and hepatitis (B and C) in Santa Maria neighborhood. Although our values are above the estimated prevalence for the Brazilian population and the Northeast, we can t infer that the individuals with Schistosomiasis are more susceptible to infection with HBV or HCV, since the risky factors were the means of risky transmission of causative agents of hepatitis and not infected with S. mansoni. / A Esquistossomose Mansônica é uma doença parasitária grave, de veiculação hídrica e evolução crônica, cujo agente etiológico é o Schistosoma mansoni. Ocorre em 74 países, com 207 milhões de pessoas infectadas e 700 milhões em áreas de risco. No Brasil, dados indicam uma prevalência de oito milhões. A Esquistossomose pode ser agravada quando os pacientes são portadores dos Vírus das Hepatites B (VHB) e C (VHC), resultando na evolução simultânea de ambas as patologias. No Brasil, As prevalências da co-infecção VHB/VHC e S. mansoni encontradas nos estudos realizados variaram de 13,6% a 40% para o HBV e de 0,5% a 19,66% para o HCV. Com base nisso, esta pesquisa objetivou identificar a prevalência de marcadores sorológicos do VHB e VHC e os fatores de risco em indivíduos portadores do S. mansoni no Bairro Santa Maria, Aracaju/SE. Foi realizado um estudo epidemiológico do tipo transversal. Coletaram-se dados de cada paciente por meio de um questionário investigativo. Este questionário contemplou variáveis de identificação do sujeito participante da pesquisa e variáveis epidemiológicas que incluíram os fatores de risco para infecção pelo HBV ou HCV. Foram coletadas alíquotas de soro dos participantes da pesquisa e encaminhadas para a realização das análises laboratoriais para identificação de marcadores sorológicos dos Vírus das Hepatites B e C: Anti-HBc Total IgG, Anti-HBs, HBsAg e Anti-HCV. Todos os procedimentos realizados utilizaram a técnica de Imunoensaio Quimioluminescente através do Ensaio ARCHITECT para cada marcador sorológico seguindo os protocolos de análises laboratoriais estabelecidos pelo fabricante do equipamento ARCHITECT SYSTEM. Além disso, realizou-se a análise espacial da distribuição da co-infecção no bairro através do programa TerraView utilizando o estimador de intensidade Kernel. Constatou-se que 16 indivíduos tiveram contato com o HBV (9,41%), desses um foi positivo para HBsAg. Apenas Trinta e duas amostras (18,82%) foram positivas para o marcador Anti-HBs. Três amostras foram positivas para Anti-HCV (1,76%), sendo uma também positiva para Anti-HBc. Os principais riscos de infecção pelo HBV e HCV foram relacionados às intervenções parenterais dos serviços de saúde, assim como à atividade sexual sem uso de preservativo, no caso do HBV. A análise espacial dos casos de co-infecção (Esquistossomose e Hepatite) permitiu a visualização de áreas de maior concentração dessas infecções, assim como as que são expostas a diferentes graus de risco de transmissão. Os resultados da pesquisa possibilitam oferecer, aos serviços municipais de saúde, um instrumento que facilite a compreensão da distribuição espacial da Esquistossomose e Hepatites (B e C) no bairro Santa Maria. Embora nossos valores sejam acima da prevalência estimada para a população brasileira e da região nordeste, não podemos inferir que os indivíduos portadores da Esquistossomose são mais susceptíveis à infecção pelo HBV ou HCV, uma vez que, os fatores de risco foram as vias de risco de transmissão de agentes causadores de hepatite e não a infecção pelo S. mansoni.

O presente estudo realizou o sequenciamento de nova geração do vírus da hepatite C genótipo 1, incluindo os subtipos 1a (n=51) e 1b (n=49), e identificou variantes associadas com resistência (RAV) aos antivirais de ação direta em pacientes sem tratamento prévio. No subtipo 1a, foram encontradas RAV para as regiões NS3-4A, NS5A e NS5B em 10%, 22% e 8% dos pacientes, respectivamente. RAV detectadas foram: T54S (2%), V55A (2%), Q80K (4%) e R155K (2%) na protease NS3-4A; Q30H (4%), H58P (10%) e Q30H/R+Y93C/H/N (8%) na região NS5A; e A421V (8%) na polimerase NS5B. As frequências das RAV para o subtipo 1b foram 12%, 53% e 31% para as regiões NS3-4A, NS5A e NS5B, respectivamente. Foram encontradas as RAV F43I (2%), T54S (4%), Q80H (2%), D168E (2%) e M175L (2%) na região NS3-4A; L28M (2%), R30Q (2%), L31M (2%), Q54H (27%), A92T (2%), Y93H (4%), Q54H+A92T (6%), Q54H+Y93H (6%) e A92T+Y93H (2%) na região NS5A e, L159F (2%), C316N (4%), A421V (7%), L159F+C316N (9%) e S556G (9%) na polimerase. Utilizando esta metodologia, um recombinante inter-subtipo 1a/1b foi identificado. / This study performed the next-generation sequencing of the hepatitis C virus genotype 1, including subtypes 1a (n = 51) and 1b (n = 49), and identified resistance-associated variants (RAVs) to direct-acting antivirals in previously untreated patients. In subtype 1a, RAVs were found for NS3-4A, NS5A, and NS5B regions in 10%, 22% and 8% of patients, respectively. RAVs detected were: T54S (2%), V55A (2%), Q80K (4%) and R155K (2%) in NS3-4A protease; Q30H (4%), H58P (10%) and Q30H/R+Y93C/H/N (8%) in NS5A region; and A421V (8%) in NS5B polymerase. Frequencies of RAV for subtype 1b were 12%, 53% and 31% for NS3-4A, NS5A and NS5B regions, respectively. RAVs F43I (2%), T54S (4%), Q80H (2%), D168E (2%) and M175L (2%) were found in NS3-4a region; L28M (2%), R30Q (2%), L31M (2%), Q54H (27%), A92T (2%), Y93H (4%), Q54H+A92T (6%), Q54H+Y93H (6%) and A92T+Y93H (2%) in NS5A region and, L159F (2%), C316N (4%), A421V (7%), L159F+C316N (9%) and S556G (9%) in polymerase. By using this methodology, a recombinant inter-subtype 1a/1b was identified.

A Síndrome de Gilbert é caracterizada por uma hiperbilirrubinemia indireta benigna que ocorre na ausência de hemólise ou doença estrutural do fígado. Manifesta-se por episódios intermitentes de icterícia, desencadeados por exposição a estressores físicos, baixa ingesta calórica, entre outros. A base genética da redução da atividade da enzima UDP - Glucoroniltransferase foi descoberta em 1995: em uma população caucasiana. Todos os pacientes estudados apresentaram uma adição dos nucleotídeos Timina-Adenina (TA) na região TATA box presente no promotor do gene UGT1A1, em ambos os alelos. Embora considerada uma condição benigna, a síndrome de Gilbert tem sido recentemente associada à hiperbilirrubinemia e a outros efeitos colaterais na utilização de algumas drogas como o Indinavir e Irinotecan. Outro ponto importante diz respeito ao nível de bilirrubina sérica como um indicador da severidade do acometimento de hepatopatas. A presença de mutação no gene UGT1A1 em pacientes hepatopatas pode levar ao aumento da bilirrubina sérica, supervalorizando o acometimento hepático da condição patológica. O objetivo deste estudo foi verificar a frequência do alelo UGT1A1*28 em doadores de sangue da Fundação Pró-sangue Hemocentro de São Paulo HC-FMUSP e em pacientes portadores de hepatite crônica C atendidos no ambulatório de Gastroenterologia Clínica da FMUSP. Relacionar o genótipo TA7/7 com o aumento de bilirrubina sérica nos pacientes com hepatite crônica C e avaliar a técnica de análise de fragmento no rastreamento e genotipagem da Síndrome de Gilbert. A frequência encontrada para o genótipo TA7/7 no grupo doador foi de 9% (30/313) e no grupo de pacientes VHC, de 10% (51/494). O genótipo TA7/7 parece estar relacionado com o aumento de bilirrubina. A técnica de análise de fragmentos mostrou-se rápida, sendo possível para fazer uma análise em grande escala. A herança genética da população brasileira é muito heterogênea. É constituída de caucasianos, africanos, indios, orientais e outros. Os dados sugerem que a variação genética da região promotora do gene UGT1A1 é alta entre pacientes com bilirrubina maior que 1mg/dL, e que a genotipagem para UGT1A1*28 deve ser considerada na avaliação dos pacientes com hepatite C crônica com hiperbilirrubinemia. / Gilberts syndrome is a benign condition characterized by unconjugated hiperbilurubinemia that occurs in the absence of hemolysis or liver chronic disease. It is clinically manifested by intermittently jaundice, triggered by exposition to physical stress, low calory diet, among others. The genetic base is the reduction of the activity of UDP-glucuronosyltransferase enzyme described in 1995: in a Caucasian population, all patients studied presented a Thymine Adenine (TA) addition in the TATA box region in both alleles of the UGT1A1 gene promoter. Although, Gilberts syndrome has been considered a benign condition, recently it has been associated to hiperbilirrubinemia and other adverse events during the utilization of some drugs such as Indinavir and Irinotecan. Another important issue to consider is that bilirubin is used to evaluate the severity of liver dysfunction in chronic liver diseases. The presence of this mutation in those patients could increase bilirubin levels, overestimating liver damage. The aim of this study were: 1) to verify the frequency of the genotype UGT1A1*28 (TA7/7) in blood donors and in chronic hepatitis C patients from the Gastroenterology outpatients clinics of the University of São Paulo School of Medicine; 2) to establish a relationship with TA7/7 genotype and bilirubin elevation in chronic hepatitis C patients and 3) to evaluate the fragment analysis technique to screening and genotyping the Gilbert syndrome. The frequencies of TA7/7 genotype found in blood donors group were 9.6% (30/313) and in the chronic hepatitis C group were 10% (51/494). The TA7/7 genotype seems to be related with increase of bilirubin. The fragment analysis technique is fast and able to a large scale screening approach. The genetic background of Brazilian population is highly heterogeneous. It is comprised of Caucasians, Africans, Indians, Orientals and others. The data suggests that genetic variation of promoter region of UGT1A1 gene is high among patients with bilirubin levels greater than 1 mg/dl, and UGT1A1*28 genotypes should be considered when evaluating chronic hepatitis C patients with hiperbilirubinemia.