No description available.

Background: Hepatitis C virus (HCV) is a widespread problem in the United States, but the disease's low screening rates mean that reported cases account for only a fraction of the population's antibody prevalence. In reality, chronic HCV is the most common chronic blood-borne infection in the country. While newer infections may be completely asymptomatic, the virus can lead to serious complications in the liver down the line, including hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Because HCV is a reportable disease, the Philadelphia Department of Public Health (PDPH) hepatitis surveillance registry has records of all of the positive tests performed in the city. Negative test results are not readily available, however, making it difficult to create an accurate picture of who is being tested for HCV. Our study used negative results collected from reference laboratories throughout the city to fill in many of the gaps and determine which neighborhoods were not sufficiently screening at-risk populations. Methods: Our dataset included approximately 100,000 individuals, a little over 90% of whom were HCV negative. Negative test results were obtained from Quest and Lab Corp, two of the major reference laboratories in the area that account for approximately 80% of all of the results of tests performed in Philadelphia. The screening data were then combined with records from the PDPH hepatitis surveillance registry. ArcGIS geographic information software was used to create maps out of neighborhood and census tract data, providing a visual representation of HCV screening distribution in Philadelphia. We also explored differences in demographic characteristics and testing facility information by test result. The data included anyone in the past 2 years who had undergone an antibody (Ab) or RNA test for HCV. Results: We found that census tract poverty rate was positively associated with HCV screening rate. Also, the majority of testing occurred in either hospital networks or private practices. There were significantly more males testing positive for HCV, despite the fact that many more females were tested overall. Similarly, the "baby boomer" age range (50-69) had the highest proportion of HCV positive test results, yet those aged 30-49 had the highest HCV testing rates. Conclusions: Negative test results for reportable diseases are rarely utilized, but can be extremely useful in identifying problem areas and focusing testing resources. Because so many people with HCV go undiagnosed, it is especially important that populations requiring additional attention be recognized and screened. / Epidemiology

CD4 T cell homeostasis and competency are critical for the effectiveness of antiviral immunity. However, CD4 T cells derived from people living with HIV (PLWH) and individuals with chronic HCV infection often exhibit an inflammaging phenotype, evidenced by persistent inflammation, immune activation, exhaustion, senescence, and cellular apoptosis. Despite intensive investigations, the molecular mechanisms underlying CD4 T cell dysfunction in antiretroviral therapy (ART)-controlled PLWH and HCV-infected patients remain poorly understood. By investigating the roles of non-coding (nc)RNA transcripts in regulating the functions of CD4 T cells derived from PLWH and HCV-infected patients, we demonstrated that long non-coding (lnc)RNA - growth arrest-specific transcript 5 (GAS5) - is downregulated and plays a crucial role in regulating CD4 T cell functions through and beyond the microRNA (miR)-21-mediated signaling network. Our data suggest that disrupting the GAS5-miR21 axis may restore CD4 T cell homeostasis and competency during latent HIV infection and prevent premature CD4 T cell aging or immune senescence. Moreover, our results also showed that TRF2, a component of the shelterin complex maintaining the integrity of telomeres, is post-transcriptionally inhibited, which is one of the major forces driving cellular dysregulation in CD4 T cells from PLWH and HCV patients. Importantly, our study identified miR-23a as the key regulator of TRF2 translational expression by targeting its 3’UTR in CD4 T cells and that targeting miR-23a may restore the TRF2 protein level, and thereby reconstitute CD4 T cell homeostasis and competency to rescue CD4 T cells from premature aging and immunosenescence during latent HIV infection. The findings from these studies improved our understanding and knowledge of how ncRNA-mediated networks regulate the functions of CD4 T cells during chronic viral (HIV and HCV) infections. Understanding such mechanisms is important for developing therapeutic approaches to reverse the inflammaging phenotype observed in CD4 T cells from ART-controlled PLWH and chronically HCV-infected patients to improve their immunological functions and quality of life.

INTRODUÇÃO: Infecções pelo HIV, HBV, HCV e Treponema pallidum encontram no sistema prisional um ambiente favorável para suas propagações e representam grave problema de saúde pública. Neste estudo buscamos conhecer o perfil epidemiológico dos reeducandos de uma unidade prisional e a dinâmica do seu comportamento associado às infecções estudadas. Foram medidas as prevalências dessas infecções e suas coinfecções, investigados os potenciais fatores de risco, assim como medido o padrão de uso de drogas no presídio com ênfase no crack. MÉTODOS: Estudo epidemiológico transversal realizado de fevereiro a dezembro de 2007, na Penitenciária I de São Vicente. Realizada uma análise descritiva utilizando medidas de frequência, médias e desvio padrão. Foram considerados apenas os diagnósticos sorológicos para definição das infecções. Utilizado o OR (odds ratio) como medida de associação com nível de significância de 5% (?). Utilizada a regressão logística para estimar OR ajustado por algumas variáveis. RESULTADOS: Participaram das entrevistas 546 (84,1%) reeducandos e 514 (94,1%) realizaram as sorologias. Apresentaram um perfil jovem com idade média de 29,8 anos. 52,0% (283) referiram relacionamento conjugal com média de dois filhos. Declararam ser de cor parda 51,5% (280). O tempo médio de prisão foi de 10,5 anos. As prevalências identificadas foram: HIV 1,8% [IC95% = 0,1- 3,3], HBV 21,0% [IC95% = 17,8-25,1], HCV 5,3% [IC95% = 3,5-7,6], e 5,3% [IC95% =3,5-7,6] para a infecção pelo Treponema pallidum. Os fatores de risco associados com a infecção pelo HIV foram: uso de droga injetável (OR=15,38), > 30 anos (OR=13,3), uso de cocaína na vida (OR= 5,36) e uso de crack na vida (OR= 5,21). Nas análises multivariadas as variáveis que se mantiveram associadas com o HBV foram: uso de droga injetável (OR=3,36), ter referido DST (OR= 2,28), > de 30 anos (OR=1,86) e mais de cinco anos de prisão (OR= 2,17); com o HCV foram: uso de droga injetável (OR=9,65), uso de maconha na prisão (OR=2,91) e idade >30 anos (OR=8,41); com o Treponema pallidum foram: relação homossexual (OR=11,92) e ter referido sífilis (OR=10,88). As prevalências das coinfecções foram: 0,8% [IC95%= 0,2-2,0] para HIV/HBV, 1,4% [IC95%=0,5-2,8], para HBV/Treponema pallidum, 1,8% [IC95%= 0,8-3,3] para HBV/HCV, e para a tripla infecção 0,4% [IC95%= 0,5-1,4]. Confirmaram o uso de crack na vida 25,0% (136) dos entrevistados. Idade média de início de uso de drogas ilícitas foi de 15,4 anos. Na prisão, o uso de álcool foi relatado por 8,4% (45), tabaco por 62,0% (318), maconha por 36,5 % (194), cocaína por 9,0% (48), e crack por 11,7% (15), sendo seu uso diário referido por 2,3% (3). Uso de drogas injetáveis na vida foi referido por 5,9% (32) e nenhum uso nos últimos seis meses. As situações de violência relacionadas com drogas para 14,3% (28) foram de ameaças de morte, 16,7% (57) agressões físicas e 27,3% (3) sofreram agressão sexual. Para 15,8% (30) o crack esteve relacionado com estas situações. O seu uso com outras drogas foi referido por 41,5% (54). CONCLUSÃO: As prevalências encontradas na população confinada continuam maiores que as observadas na população geral. O consumo de drogas e praticas sexuais desprotegidas foram mantidas no período de confinamento. O tempo de confinamento mostrou-se fator importante na análise de risco. Os fatores de riscos identificados aparecem como importantes indicadores para a estruturação de estratégias de controle dessas infecções junto à população confinada. / INTRODUCTION: Infection by HIV, HBV, HCV and Treponema pallidum are common in the prison system due to an environment favorable to their propagation and represent a serious public health problem. This study aims to understand the epidemiological profile of the inmates in a prison unit and the behaviors associated with the infectious diseases studied. We estimated the prevalence of these infections and their co-infections, investigated potential risk factors, as well as identified the pattern of drug use in prison, especially crack use. METHODS: A cross-sectional epidemiological study conducted from February to December 2007 at the São Vicente Penitentiary . A descriptive analysis using frequency measures, means and standard deviations was conducted. We considered only the setting for serological diagnosis of infections. Odds ratio (OR) with a significance level of 5% (?) was used as a measure of association and a logistic regression was applied to estimate adjusted OR for some variables. RESULTS: A total of 546 prisoners were interviewed (84.1%). Of these, 514 (94.1%) underwent serological analysis. Results showed a profile of young men with an average age of 29.8 years. Fifty-two percent (283) reported marital relationship with an average of two children, and 51.5% (280) reported being of mixed ethnicity. The average time of arrest was 10.5 years. The prevalences were identified: HIV 1.8% [95% CI = 0.1 - 3.3], HBV 21.0% [95% CI 17.8 to 25.1], HCV 5.3% [95% CI = 3.5 to 7.6] and 5.3% [95% CI = 3.5 to 7.6] for infection with Treponema pallidum. The risk factors associated with HIV infection were injected-drug use (OR = 15.38), > 30 years (OR = 13.3), cocaine use (OR = 5.36) and use of crack during lifetime (OR = 5.21). According to multivariate analyzes, variables associated with HBV were: injected-drug use (OR = 3.36), reported any STD (OR = 2.28), > 30 years (OR = 1.86) and more than five years in prison (OR = 2.17); the variables associated with HCV were: injected-drug use (OR = 9.65), marijuana use in prison (OR = 2.91) and age> 30 years (OR = 8.41); the variables associated with Treponema pallidum were: homosexual intercourse (OR = 11.92) and have referred syphilis (OR = 10.88). The prevalence of co-infections were 0.8% [95% CI = 0.2 to 2.0] for HIV / HBV, 1.4% [95% CI = 0.5 to 2.8] for HBV / Treponema pallidum, 1.8% [95% CI = 0.8 to 3.3] for HBV / HCV infection and for the triple infection 0.4% [95% CI = 0.5 to 1.4]. Average age of onset of illicit drug use was 15.4 years. The use of crack during lifetime was reported by 25.0% (136) of respondents. In prison, alcohol use was reported by 8.4% (45), tobacco by 62.0% (318), marijuana by 36.5% (194), 9.0% for cocaine (48), and crack by 11.7% (15), with the daily use of crack reported by 2.3% (3). Injected-drug use during lifetime was reported by 5.9% (32), but no use in the last six months was reported. Drug-related violence episodes reported were death threats 14.3% (28), 16.7% (57) assaults and 27.3% (3) suffered sexual assault. To 15.8% (30) of the respondents, crack use was associated with violence episodes. The use of crack-cocaine in association with other drugs was reported by 41.5% (54). CONCLUSION: The prevalence found in the confined population is still larger than those observed in the general population. Drug use and unprotected sexual practices were maintained during the period of confinement. The confinement time proved to be an important factor in the analysis of risk of infection. The identified risk factors appear to be important indicators for developing strategies to control these infections in the prison environment.

No description available.

Le virus de l'hépatite C (HCV) est une cause majeure de maladie chronique du foie et de carcinome hépatocellulaire. Les options thérapeutiques pour traiter l'hépatite chronique sont limitées par des coûts élevés, des effets secondaires et une résistance virale. L'entrée du HCV est la première étape d'interaction entre le virus et la cellule hôte. Elle est requise pour l'initiation, la propagation et le maintien de l'infection, ce qui en fait une cible prometteuse pour les traitements antiviraux. L’entrée du HCV nécessite l'interaction coopérative de plusieurs facteurs cellulaires, y compris CD81 et claudine-1 (CLDN1). Nous avons récemment identifié un rôle pour le récepteur à l’EGF (EGFR) et le récepteur à l’ephrine A2 (EphA2) dans l'entrée du HCV par la régulation de la formation du complexe de co-récepteurs CD81-CLDN1, ce qui suggère que la signalisation de ces récepteurs joue un rôle dans l'entrée du virus. Nous avons voulu identifier les mécanismes moléculaires de signalisation de l’EGFR requis pour l'entrée du HCV et avons identifié HRas comme un transducteur de signalisation clé de l'hôte. Des études d'imagerie ont révélées que la signalisation de HRas peut moduler la diffusion et le trafic membranaire de CD81, ce qui permet l’assemblage du complexe de récepteurs. De plus, HRas s’associe avec les récepteurs de l'hôte CD81 et CLDN1 et des facteurs d’entrée du HCV inconnus jusque là: l’intégrine beta1 et Rap2B. Le HCV profite donc de la signalisation de HRas pour l'entrée cellulaire. Ces données améliorent notre compréhension des mécanismes moléculaires de l'entrée du HCV induite par l’EGFR et ouvrent de nouvelles perspectives pour le développement d'antiviraux. / Hepatitis C virus (HCV) is a major cause of chronic liver disease and hepatocellular carcinoma worldwide. Therapeutic options to treat chronic viral hepatitis are limited by high costs, side effects and viral resistance in most patients. HCV entry is the first step of interaction between the virus and the host cell. It is required for the initiation, propagation and maintenance of infection, making it a promising target for antiviral therapy. HCV entry requires the cooperative interaction of several cellular factors, including CD81 and claudin-1 (CLDN1). We have recently identified a role for EGF receptor (EGFR) and ephrin receptor A2 (EphA2) in HCV entry by regulating the formation of the co-receptor complex CD81-CLDN1, suggesting that the signaling of these receptors might play a role in viral entry. However, the precise mechanisms of regulation are unknown. We wanted to identify the molecular mechanisms of EGFR signaling required for the HCV entry process. We identify HRas as key host signaling transducer for HCV entry. Advanced imaging studies have revealed that HRas signaling can modulate the lateral diffusion and membrane trafficking of CD81. A modified diffusion of CD81 allows the assembly of the receptors complex. In addition, HRas associates with tetraspanin microdomains containing the host receptors CD81 and CLDN1 and HCV entry factors previously unknown: the integrin beta1 and Rap2B. HCV therefore exploits HRas signaling for cellular entry. These data improve our understanding of the molecular mechanisms of HCV entry induced by EGFR and open new perspectives for the development of antivirals targeting signaling pathways.

INTRODUÇÃO: Infecções pelo HIV, HBV, HCV e Treponema pallidum encontram no sistema prisional um ambiente favorável para suas propagações e representam grave problema de saúde pública. Neste estudo buscamos conhecer o perfil epidemiológico dos reeducandos de uma unidade prisional e a dinâmica do seu comportamento associado às infecções estudadas. Foram medidas as prevalências dessas infecções e suas coinfecções, investigados os potenciais fatores de risco, assim como medido o padrão de uso de drogas no presídio com ênfase no crack. MÉTODOS: Estudo epidemiológico transversal realizado de fevereiro a dezembro de 2007, na Penitenciária I de São Vicente. Realizada uma análise descritiva utilizando medidas de frequência, médias e desvio padrão. Foram considerados apenas os diagnósticos sorológicos para definição das infecções. Utilizado o OR (odds ratio) como medida de associação com nível de significância de 5% (?). Utilizada a regressão logística para estimar OR ajustado por algumas variáveis. RESULTADOS: Participaram das entrevistas 546 (84,1%) reeducandos e 514 (94,1%) realizaram as sorologias. Apresentaram um perfil jovem com idade média de 29,8 anos. 52,0% (283) referiram relacionamento conjugal com média de dois filhos. Declararam ser de cor parda 51,5% (280). O tempo médio de prisão foi de 10,5 anos. As prevalências identificadas foram: HIV 1,8% [IC95% = 0,1- 3,3], HBV 21,0% [IC95% = 17,8-25,1], HCV 5,3% [IC95% = 3,5-7,6], e 5,3% [IC95% =3,5-7,6] para a infecção pelo Treponema pallidum. Os fatores de risco associados com a infecção pelo HIV foram: uso de droga injetável (OR=15,38), > 30 anos (OR=13,3), uso de cocaína na vida (OR= 5,36) e uso de crack na vida (OR= 5,21). Nas análises multivariadas as variáveis que se mantiveram associadas com o HBV foram: uso de droga injetável (OR=3,36), ter referido DST (OR= 2,28), > de 30 anos (OR=1,86) e mais de cinco anos de prisão (OR= 2,17); com o HCV foram: uso de droga injetável (OR=9,65), uso de maconha na prisão (OR=2,91) e idade >30 anos (OR=8,41); com o Treponema pallidum foram: relação homossexual (OR=11,92) e ter referido sífilis (OR=10,88). As prevalências das coinfecções foram: 0,8% [IC95%= 0,2-2,0] para HIV/HBV, 1,4% [IC95%=0,5-2,8], para HBV/Treponema pallidum, 1,8% [IC95%= 0,8-3,3] para HBV/HCV, e para a tripla infecção 0,4% [IC95%= 0,5-1,4]. Confirmaram o uso de crack na vida 25,0% (136) dos entrevistados. Idade média de início de uso de drogas ilícitas foi de 15,4 anos. Na prisão, o uso de álcool foi relatado por 8,4% (45), tabaco por 62,0% (318), maconha por 36,5 % (194), cocaína por 9,0% (48), e crack por 11,7% (15), sendo seu uso diário referido por 2,3% (3). Uso de drogas injetáveis na vida foi referido por 5,9% (32) e nenhum uso nos últimos seis meses. As situações de violência relacionadas com drogas para 14,3% (28) foram de ameaças de morte, 16,7% (57) agressões físicas e 27,3% (3) sofreram agressão sexual. Para 15,8% (30) o crack esteve relacionado com estas situações. O seu uso com outras drogas foi referido por 41,5% (54). CONCLUSÃO: As prevalências encontradas na população confinada continuam maiores que as observadas na população geral. O consumo de drogas e praticas sexuais desprotegidas foram mantidas no período de confinamento. O tempo de confinamento mostrou-se fator importante na análise de risco. Os fatores de riscos identificados aparecem como importantes indicadores para a estruturação de estratégias de controle dessas infecções junto à população confinada. / INTRODUCTION: Infection by HIV, HBV, HCV and Treponema pallidum are common in the prison system due to an environment favorable to their propagation and represent a serious public health problem. This study aims to understand the epidemiological profile of the inmates in a prison unit and the behaviors associated with the infectious diseases studied. We estimated the prevalence of these infections and their co-infections, investigated potential risk factors, as well as identified the pattern of drug use in prison, especially crack use. METHODS: A cross-sectional epidemiological study conducted from February to December 2007 at the São Vicente Penitentiary . A descriptive analysis using frequency measures, means and standard deviations was conducted. We considered only the setting for serological diagnosis of infections. Odds ratio (OR) with a significance level of 5% (?) was used as a measure of association and a logistic regression was applied to estimate adjusted OR for some variables. RESULTS: A total of 546 prisoners were interviewed (84.1%). Of these, 514 (94.1%) underwent serological analysis. Results showed a profile of young men with an average age of 29.8 years. Fifty-two percent (283) reported marital relationship with an average of two children, and 51.5% (280) reported being of mixed ethnicity. The average time of arrest was 10.5 years. The prevalences were identified: HIV 1.8% [95% CI = 0.1 - 3.3], HBV 21.0% [95% CI 17.8 to 25.1], HCV 5.3% [95% CI = 3.5 to 7.6] and 5.3% [95% CI = 3.5 to 7.6] for infection with Treponema pallidum. The risk factors associated with HIV infection were injected-drug use (OR = 15.38), > 30 years (OR = 13.3), cocaine use (OR = 5.36) and use of crack during lifetime (OR = 5.21). According to multivariate analyzes, variables associated with HBV were: injected-drug use (OR = 3.36), reported any STD (OR = 2.28), > 30 years (OR = 1.86) and more than five years in prison (OR = 2.17); the variables associated with HCV were: injected-drug use (OR = 9.65), marijuana use in prison (OR = 2.91) and age> 30 years (OR = 8.41); the variables associated with Treponema pallidum were: homosexual intercourse (OR = 11.92) and have referred syphilis (OR = 10.88). The prevalence of co-infections were 0.8% [95% CI = 0.2 to 2.0] for HIV / HBV, 1.4% [95% CI = 0.5 to 2.8] for HBV / Treponema pallidum, 1.8% [95% CI = 0.8 to 3.3] for HBV / HCV infection and for the triple infection 0.4% [95% CI = 0.5 to 1.4]. Average age of onset of illicit drug use was 15.4 years. The use of crack during lifetime was reported by 25.0% (136) of respondents. In prison, alcohol use was reported by 8.4% (45), tobacco by 62.0% (318), marijuana by 36.5% (194), 9.0% for cocaine (48), and crack by 11.7% (15), with the daily use of crack reported by 2.3% (3). Injected-drug use during lifetime was reported by 5.9% (32), but no use in the last six months was reported. Drug-related violence episodes reported were death threats 14.3% (28), 16.7% (57) assaults and 27.3% (3) suffered sexual assault. To 15.8% (30) of the respondents, crack use was associated with violence episodes. The use of crack-cocaine in association with other drugs was reported by 41.5% (54). CONCLUSION: The prevalence found in the confined population is still larger than those observed in the general population. Drug use and unprotected sexual practices were maintained during the period of confinement. The confinement time proved to be an important factor in the analysis of risk of infection. The identified risk factors appear to be important indicators for developing strategies to control these infections in the prison environment.

Hepatitis C virus (HCV) represents a global health threat. HCV is a blood-borne positive-strand RNA virus belonging to the Flaviviridae family that infects ~160 million people worldwide. About 70% of infected individuals fail to clear the virus and subsequently develop chronic hepatitis, frequently leading to liver cirrhosis and in some cases hepatocellular carcinoma. Therapeutic options for HCV infection are still limited and a protective vaccine is not yet available. Currently available therapies include administration of pegylated alpha interferon in combination with ribavirin. The recently approved protease inhibitors Boceprevir and Telaprevir are also included in the treatment regimen. However, limitations to the treatment with direct-acting antivirals (DAAs) are associated with severe side effects and low sustained virological response (SVR) rates that vary depending on the virus and host genotype. The replication step of the viral life cycle is mostly targeted by majority of DAAs. Recent findings have suggested that a combination of entry inhibitors together with DAAs exhibit a synergistic effect in the treatment of HCV. Therefore, identification of efficient HCV entry inhibitors is of high priority In vitro studies have shown that HCV attachment and subsequent entry into the host cells is mediated by E1 and E2 viral envelope proteins. HCV entry requires interaction with a number of receptors which include CD81, scavenger receptor B1 (SR-B1) and the tight junction proteins, claudin 1 (CLDN1) and occludin (OCLN). Since the E2 glycoprotein is reported to interact directly with cellular receptors, it is an attractive target for neutralisation. The present study focuses on the establishment and characterisation of entry inhibitors as antivirals for HCV. The thesis is presented in three chapters: Chapter 1- ‘Introduction’, provides a brief overview on HCV genotypes, genome organisation, life cycle including details on the entry process and therapies used for the treatment of HCV. Chapter 2 describes the generation of monoclonal antibodies (mAbs) against HCV envelope proteins as potent anti-viral agents for the prevention of HCV infection. Data on the identification and characterization of the neutralizing epitopes of HCV envelope proteins have been presented. Chapter 3 includes isolation of entry inhibitors of HCV from natural sources and identification and characterization of the active components exhibiting antiviral property. A number of studies have reported the role of neutralizing antibodies in the course of HCV infection and emerging data suggest protective effect of antibodies against HCV infection. Most of the ongoing studies are based on HCV genotype 1a which is prevalent globally. However in India, the prevalent genotype is 3a. Therefore, we established a panel of mAbs against HCV-LPs comprising of core-E1-E2 derived from genotype 3a as described in chapter 2. HCV-LP based system has been used in this study since it mimics the biophysical conformation, morphology and antigenic properties of the native virion and represents a model system for studies on viral binding and entry. MAbs were characterised and analyzed for their ability to prevent viral binding and entry into host cells. Three mAbs namely E3D8, H6D3 and A10F2 were identified to recognize the E2 viral glycoprotein which significantly inhibited HCV-LP binding to Huh7 cells in vitro. The neutralizing epitopes corresponding to the mAbs were identified using overlapping truncated fragments and synthetic peptides of the E2 protein. Our experiments suggest that the epitopes recognised by the inhibitory mAbs are unique and different from those reported till now. The synergistic effect of a combination of mAbs on virus neutralization has shown promising results for treatment of viral infections. Since in the present study the epitopes recognised by the mAbs are non-overlapping, we went ahead to determine whether a combination of these mAbs would enhance the ability to block HCV-LP binding. Indeed, flow cytometry and fluorescence microscopy studies revealed that a combination of the antibodies efficiently blocked the binding of HCV-LP to human hepatoma cells. More importantly and of relevance is the observation that the mAbs in combination inhibited viral infection (JFH1 strain) and replication in permissive human hepatocytes as determined by real time RT-PCR. Phytochemicals present in plants have been considered as conducive for prevention of several viral infections and are found to be promising antiviral agents. Natural products which are biologically active disclose drug-like properties since they are small molecules and can be easily metabolised and absorbed by the body. In our study as described in chapter 3, we evaluated extracts from Indian medicinal plants and fruits which are known to have hepato-protective effect, for natural potent attachment and entry inhibitors for HCV. Flow cytometric analysis suggested that the root extract of the herb Boerhavia diffusa and fruit extract of Prunus domestica exhibited high antiviral activity by inhibiting the binding of Hepatitis C virus like particles (HCV-LPs) to the human hepatoma cells. We went on to isolate, identify and confirm the active principles to be Boeravinone H, a dehydrorotenoid, (from Boerhavia diffusa) and Rutin, a flavonoid, (from Prunus domestica) by LC-ESI-MS, NMR, UV and IR spectral analysis. Our study revealed that the compounds block the attachment as well as entry step probably by targeting the viral particle. We also assessed the efficiency of these small molecules (Boeravinone H and Rutin) to inhibit HCV negative strand synthesis post entry by real time RT-PCR. Results suggest significant inhibition of viral entry and infection in the HCV cell culture (ex vivo). To our knowledge it is the first report on Boeravinone H and Rutin as entry inhibitor for HCV. In conclusion, our findings support the potential of employing a cocktail of neutralizing mAbs and antiviral agents from natural source in the management of HCV infection.

Le virus de l’hépatite C (HCV) infecte les hépatocytes. Il est remarquable par le fait q’ilperturbe fortement le metabolisme lipidique de l’hôte, conduisant à des dysfonctions majeures telles qu’une stéatose ou une résistance à l’insuline. In vivo, les virions sériques présentent une densité faible et variable reflétant leur association aux lipoprotéines de faible et de tres faible densité (LDL et VLDL). Ainsi, l'existence de lipo-viro-particules (LVP), contenant à la fois les constituants viraux et les apolipoprotéines B (apoB) et E (apoE) a été suggerée. Ces LVP joueraient un rôle important dans la persistance virale. Cependant, cette association entre HCV et apoB n'a pas été retrouvée in vitro avec les modeles cellulaires disponibles.Mes travaux de thèse se sont donc concentrés sur la mise en place d'un nouveau modèle deculture cellulaire capable de produire a la fois des VLDL et des particules virales HCV, permettantainsi d’étudier l’interaction entre les deux voies de synthèse. Dans un premier temps, lacaracterisation de la production de lipoproteines par differentes lignees d'hepatocytes a permis demontrer l'existence d'un défaut de secretion de VLDL en cellules Huh7.5, classiquement utiliséespour étudier HCV in vitro, alors que les cellules HepG2 et HepaRG sont capables de produire des VLDL physiologiques. Dans un second temps, des cellules HepG2 repliquant HCV de manière persistante ont été utilisées pour caractériser les particules virales produites. Etonnamment, a l'instar des cellules Huh7.5 et malgré leur capacité a produire des VLDL, les cellules HepG2 ne secreteraient pas de LVP mais des particules virales positives pour apoE et négatives pour apoB. / Hepatitis C virus (HCV) mainly infects hepatocytes. It is unique in its ability to impair host lipidmetabolism, leading to major liver dysfunctions as, for instance, hepatic steatosis or insulinresistance. In vivo, serum virions have a low and variable density, reflecting their association withlow- and very-low-density lipoproteins (LDL and VLDL). Hence, the existence of lipo-viro-particles(LVP), containing both viral components as well as apolipoprotein B (apoB) and E (apoE), has beensuggested. These LVPs could play an important role in viral persistence. However, this associationbetween HCV and apoB has not been observed in vitro, using the currently available cell culturemodels.Therefore, during my PhD, I have worked at setting up a new cell culture model, which wascapable of producing both VLDL and HCV particles, and therefore would enable the study of theinterplay between both synthesis pathways. First of all, we characterized lipoprotein production indifferent hepatocyte cell lines and confirmed that Huh7.5 cells, usually used to study HCV in vitro,were deficient for VLDL secretion, whereas two other cell lines, HepG2 and HepaRG, were able toproduce quasi-physiological VLDLs. Therefore, in a second time, we used HepG2 cells to replicate aHCV strain containing a selection gene and to characterize viral particle production. Surprisingly,VLDL-producing HepG2 cells were also unable to secrete LVPs, but rather secreted apoE-positive andapoB-negative viral particles, which were similar to ones Huh7.5 cells produced. This suggests thatthe ability to produce LVPs does not correlate with the ability to produce VLDL.

In Anbetracht der enormen Verbesserung der HCV-Therapie durch die Entwicklung hochwirksamer und direkt wirkender Virostatika (DAA) dient diese Studie der Analyse von HCV-Koinfektionen unter den gemeldeten HIV-Neudiagnosen in Deutschland. Zunächst wurde die Eignung zweier kommerzieller HCV Ag/Ab ELISAs, dem Murex (Abbott) und dem Monolisa (Bio-Rad), zum Nachweis von HCV in getrockneten Serumspots (DSS) verglichen. Der Murex-ELISA zeigte sich sensitiver bei Antigen-positiven Plasma-HCV-Serokonversionsproben während der Monolisa eine höhere Sensitivität bei HCV-Antikörper-positiven DSS-Eluaten. Des Weiteren wurde ein Aviditätstest zur Unterscheidung von neuen und bereits länger bestehenden Infektionen bei einem Aviditätsindex Cut-off von 40% und einem Zeitraum von 364 Tagen etabliert. Von den insgesamt 6.097 untersuchten Proben der Jahre 2015-2017 waren 396 HCV-ELISA-reaktiv (6,5%). Von diesen wurden 256 (64,6%) als aktive und 140 (35,4%) als ausgeheilte Infektionen identifiziert. Ein hoher Anteil der HCV-Koinfektionen wurde bei intra-venösen Drogenkonsumenten (77,8%, n=168/216), in der Altersgruppe der 30-39 Jährigen (9%, n=179/1.978) und bei Menschen osteuropäischer Herkunft (38,3%, n=124/324) beobachtet. Im Vergleich zum Jahr 2016 hat sich der Anteil der ausgeheilten Infektionen im Jahr 2017 bei der Gesamtzahl der Patienten (p<0,01) sowie insbesondere bei Personen ausländischer (p<0,01) und osteuropäischer (p<0,05) Herkunft erhöht. Die HCV Subtypen (St)-1a, St-3a und St-1b waren mit 33,9% (n=79/233), 33,5% (n=78/233) und 23,3% (n=52/233) vorherrschend. Der Anteil der St-1a- und St-1b-Proben, deren HCV-Sequenz gegen DAAs resistent ist, war in allen untersuchten Jahren hoch (25%-42,9%). Der beobachtete Anstieg des Anteils der ausgeheilten HCV-Koinfektionen kann auf die DAA-Therapie zurückgeführt werden. Es sind jedoch weitere Anstrengungen erforderlich, damit Gruppen mit weiterhin hoher HCV-Prävalenz von dieser Behandlung profitieren. / In light of the major shift in HCV therapy resulting from the availability of highly potent direct acting antivirals (DAA), this study performed a surveillance of HCV coinfections among reported HIV new diagnoses in Germany. First the suitability of two HCV Ag/Ab ELISAs, Murex (Abbott) and Monolisa (Bio-Rad) for dried serum spots (DSS) was compared. The Murex was more sensitive in antigen positive plasma HCV seroconversion samples while the Monolisa showed a higher sensitivity in HCV antibody positive DSS eluates. Furthermore, an avidity test was established to distinguish between new and already longer existing infections at an avidity index cut-off of 40% and a period of 364 days. Of the total of 6,097 samples examined for the years 2015-2017, 396 were HCV ELISA reactive (6.5%). Of these, 256 (64.6%) were identified as active and 140 (35.4%) as resolved infections. A high proportion of HCV coinfections were observed in intravenous drug users (77.8%, n=168/216), in the age group 30-39 years (9%, n=179/1,978) and in people of Eastern European origin (38.3%, n=124/324). Compared to 2016, the proportion of resolved infections in 2017 has increased in the total number of patients (p<0.01) and especially in people of foreign (p<0.01) and Eastern European (p<0.05) origin. HCV subtypes (St)-1a, St-3a, and St-1b were predominant with 33.9% (n=79/233), 33.5% (n=78/233) and 23.3% (n=52/233), respectively. The proportion of St-1a and St-1b samples whose HCV sequence has resistance to DAAs was high in all diagnoses years (25%-42.9%). The observed increase in the proportion of resolved HCV coinfections can be attributed to DAA therapy. However, further efforts are needed to ensure that groups with continued high HCV prevalence benefit from this treatment.