京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20989号 / 医博第4335号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 朝長 啓造, 教授 松田 文彦, 教授 小柳 義夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Despite our growing knowledge of virus biology they continue to present a problem to global public health. This problem arises from their high mutation rates that allow them to evade antiviral therapies that we have developed to date. An alternative solution for developing antiviral therapies could be to target host cell factors that are hijacked by the virus. The basis of this hypothesis is that if we can stop the virus from using host cell machinery or from evading host immune mechanisms we could treat the infection more efficiently. With the major research focus being on viral proteins and how we can prevent their functions, there is a lot of work to be done in finding host factors that could be the key to treating an infection. The three themes presented in this thesis broadly focus on this goal. The first theme looks at miRNAs, their interacting partners, and their dysregulation during HCV infection. A microRNA is identified from a small molecule screen of miRNAs that are dysregulated during HCV infection and its role in liver immunometabolism is examined to determine its antiviral potential and identify host factors that could be of interest to target with antiviral therapeutics. The second theme examines the potential of activity-based protein profiling techniques for complementing existing antiviral therapies. An azauracil probe is characterized to examine its ability to interact with viral polymerases and its suitability as a building block for antiviral research or therapies. The final theme uses activity-based protein profiling techniques to study a novel carbamate-hydrazone chemotype and establish its suitability as a chemical probe. The hydrazone probe’s reactivity with the mammalian proteome was determined and its interacting partners were identified using chemoproteomic techniques with an overall goal of examining its suitability for antiviral research. Overall, this thesis uses chemical and molecular biology techniques to present three differing perspectives on how to approach the discovery of host factors and develop novel antiviral therapies.

As novel or variant strains of HEV continue to evolve rapidly both in humans and other animals, it is important to develop a rapid pre-sequencing screening method to select field isolates for further molecular characterization. Two heteroduplex mobility assays (HMA) were developed to genetically differentiate field strains of swine HEV and avian HEV from known reference strains. It was shown that the HMA profiles generally correlate well with nucleotide sequence identities and with phylogenetic clustering between field strains and the reference swine HEV or avian HEV strains. Therefore, by using different HEV isolates as references, the HMA developed in this study can be used as a pre-sequencing screening tool to identify variant HEV isolates for further molecular epidemiological studies. Our previous study showed that avian HEV antibody is prevalent in apparently healthy chickens. A prospective study was conducted on a known seropositive but healthy chicken farm. Avian HEV was identified from the healthy chicken flock. Avian HEV isolates recovered from the healthy chicken share 70-97% nucleotide sequence identities with those isolates which cause hepatitis-splenomegaly (HS) syndrome based on partial helicase and capsid gene regions. Recovery of identical viruses from the experimentally inoculated chickens in the subsequent transmission study further confirmed our field results. The capsid gene of avian HEV isolates from chickens with HS syndrome were also characterized and found to be heterogeneic, with 76-100% nucleotide sequence identities to each other. The study indicates that avian HEV is enzootic in chicken flocks and spread subclinically among chicken populations, and that the virus is heterogeneic. As HEV can not be propagated <i>in vitro</i>, in order to further characterize avian HEV, an infectious viral stock with a known infectious titer must be generated. Bile and feces collected from specific-pathogen-free (SPF) chickens experimentally infected with avian HEV were used to prepare an avian HEV infectious stock. The infectivity titer of this infectious stock was determined, by intravenously inoculating one-week old SPF chickens, to be 5 x 10^4.5 50% chicken infectious doses (CID₅₀) per ml. Seroconversion, viremia as well as fecal virus shedding were observed in the inoculated chickens. Contact control chickens also became infected via direct contact with inoculated ones. Avian HEV infection in chickens was found to be dose-dependent. To determine if avian HEV can infect across species, one-week old SPF turkeys were intravenously inoculated each with 10^4.5(CID₅₀) of avian HEV. The inoculated turkeys seroconverted to avian HEV antibodies at 4-8 weeks postinoculation (WPI). Viremia was detected at 2-6 WPI, and fecal virus shedding at 4-7 WPI in inoculated turkeys. This is the first demonstration of cross-species infection by avian HEV. Little is known regarding the characteristics of the small ORF3 protein largely due to the lack of a cell culture system for HEV. To characterize the small protein, the ORF3 proteins of avian HEV and swine HEV were expressed in <i>Escherchia coli</i>, and purified by BugBuster His-Bind Purification System. Western blot analysis showed that avian HEV ORF3 protein is unique and does not share common antigenic epitopes with those of swine HEV and human HEV. However, swine HEV (genotype 3) and human HEV (genotype 1) ORF3 proteins cross-react with each other antigenically. To determine if the ORF3 protein is a virion protein, infectious stocks of avian HEV and swine HEV were first generated in SPF chickens and pigs, respectively. Virions were subsequently purified by sucrose density gradient centrifugation and virion proteins were characterized by SDS-PAGE and Western blot analysis. Two major forms of ORF2 proteins of avian HEV were identified: a 56 kDa and an 80 kDa proteins. Multiple immunoreactive forms of ORF2 proteins of swine HEV were also observed: 40 kDa, 53 kDa, 56 kDa and 72 kDa. However, the ORF3 protein was not detected from the native virions of avian HEV or swine HEV. These findings provide direct evidence that ORF2 indeed encodes a structural protein of HEV, whereas ORF3 does not. To search for other potential animal reservoirs for HEV, the prevalence of IgG anti-HEV antibody was determined in field mice caught in chicken farms to assess the possibility of mice as a potential reservoir for HEV infection in chickens. Three different recombinant HEV antigens derived from avian HEV, swine HEV, and human HEV were used in the ELISA assays. The anti-HEV seropositive rates in wild field mice (<i>Mus musculus</i>), depending upon the antigen used, are 15/76 (20%), 39/74 (53%), and 43/74 (58%), respectively. HEV RNA was also detected from 29 fecal and/or serum samples of mice. The HEV sequences recovered from field mice shared 72-100% nucleotide sequence identities with each other, 73-99% sequence identities with avian HEV isolates, and 51-60% sequence identities with representative strains of swine and human HEVs. However, attempts to experimentally infect laboratory mice (Mus musculus) with the PCR-positive fecal materials recovered from the wild field mice were unsuccessful. We also attempted to experimentally infect 10 Wistar rats each with avian HEV, swine HEV, and an US-2 strain of human HEV, respectively. However, the inoculated rats did not become infected as evidenced by the lack of viremia, virus shedding in feces or seroconversion. These data suggest that mice caught in chicken farms are infected by a HEV-like virus, but additional work is needed to determine the origin of the mouse virus as well as the potential role of rodents in HEV transmission. In summary, we developed two HMAs which are useful for differentiation and identification of variant strains of swine and avian HEVs. We genetically identified and characterized an avian HEV strain from apparently healthy chickens in seropositive flocks. We showed that avian HEV can cross species barriers and infect turkeys. Our data indicated that avian and swine HEV ORF2 genes encode structural proteins, whereas ORF3 genes do not. Evidence in this study also showed that HEV or HEV-like agent exists in field mice on a chicken farm. / Ph. D.

Hepatitis E Virus (HEV), the causative agent of hepatitis E, is a zoonotic pathogen of worldwide significance. The genus Orthohepevirus A of the family Hepeviridae includes all mammalian strains of HEV and consists of 8 recognized genotypes. Genotypes 1 and 2 HEVs only infect humans and genotypes 3 and 4 infect humans and several other animal species including pigs and rabbits. An ever-expanding host range of genetically-diversified strains of HEV now include bat, fish, rat, ferret, moose, wild boar, mongoose, deer, and camel. Additionally, the ruminant species goats, sheep, and cattle have been implicated as potential reservoirs as well. My dissertation research investigates a novel animal model for HEV, examines the immune dynamics during acute infection, and evaluates the possibility of additional animal reservoirs of HEV. The first project established an immunoglobulin (Ig) heavy chain knock-out JH (-/-) gnotobiotic piglet model that mimics the course of acute HEV infection observed in humans and evaluated the pathogenesis of HEV infection in this novel animal model. The dynamics of acute HEV infection in gnotobiotic pigs were systematically determined with a genotype 3 human strain of HEV. We also investigated the potential role of immunoglobulin heavy-chain JH in HEV pathogenesis and immune dynamics during the acute stage of virus infection. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems. The objective of the second project for my PhD dissertation was to determine if cattle in the United States are infected with a bovine strain of HEV. We demonstrated serological evidence of an HEV-related agent in cattle populations with a high level of IgG anti-HEV prevalence. We demonstrated that calves from a seropositive cattle herd seroconverted to IgG binding HEV during a prospective study. We also showed that the IgG anti-HEV present in cattle has an ability to neutralize genotype 3 human HEV in vitro. However, our exhaustive attempts to detect HEVrelated sequence from cattle in the United States failed, suggesting that one should be cautious in interpreting the IgG anti-HEV serological results in bovine and other species. Collectively, the work from my PhD dissertation delineated important mechanisms in HEV pathogenesis and established a novel animal model for future HEV research. / Ph. D. / Hepatitis E Virus (HEV), the causative agent of hepatitis E, is a zoonotic pathogen of worldwide significance. According to the World Health Organization, there are approximately 20 million HEV infections annually, which result in 3.3 million cases of acute hepatitis E and >44,000 HEV-related deaths. Hepatitis E is a self-limiting acute disease in general, but carries the ability to cause high mortality in pregnant women and chronic hepatitis in immunocompromised individuals. The underlying mechanisms of HEV host tropism and progression of disease to chronicity are unknown. My dissertation work investigates a novel animal model for HEV, evaluates the possibility of additional animal reservoirs of HEV, and examines the immune dynamics during acute infection. The first project established an immunoglobulin (Ig) heavy chain knock-out JH (-/-) gnotobiotic piglet model that mimics the course of acute HEV infection observed in humans. The dynamics of acute HEV infection were determined in both the knock-out and wild-type piglets with a genotype 3 strain of human HEV. We also investigated the potential role of immunoglobulin heavy-chain JH in HEV pathogenesis and virus infection. In the second project, we determined if cattle in the United States are infected with a bovine strain of HEV. We showed serological evidence of an HEV-related agent in cattle as well as calves born in a seropositive herd. Despite the detection of specific antibodies recognizing HEV in cattle, definitive evidence of virus infection could not be demonstrated. Our exhaustive attempts to detect HEV-related sequence from cattle in the United States failed, suggesting that one should be cautious in interpreting the IgG anti-HEV serological results in bovine and other species. Collectively, the work from my PhD dissertation research delineated important mechanisms in HEV pathogenesis and established a novel animal model for future HEV research.

Malgré l’existence d’un vaccin préventif efficace, l’infection chronique par le virus de l’hépatite B (HBV) demeure un problème majeur de santé publique. La persistance de l’infection par HBV étant clairement associée à des réponses immunitaires insuffisantes, l’immunothérapie par le vaccin à base d’ADN nu, visant à stimuler les réponses humorales et cellulaires, apparaît comme particulièrement pertinente pour la thérapie des hépatites B chroniques. Toutefois, l’efficacité thérapeutique d’une telle stratégie reste limitée chez l’homme, d’où la nécessité d’optimiser cette approche vaccinale pour une utilisation ultérieure en clinique. Ainsi, l’objectif général de ce travail de thèse était d’explorer, avec le modèle du DHBV (« Duck Hepatitis B Virus »), étroitement apparenté au HBV humain, si l’administration du vaccin à ADN par électroporation (EP) pouvait davantage améliorer son efficacité prophylactique et thérapeutique. Nous avons montré, dans un 1er temps chez des canards naïfs, que l’administration du vaccin à ADN par EP permet de potentialiser le pouvoir neutralisant et d’élargir le répertoire épitopique de la réponse humorale dirigée contre la protéine d’enveloppe du DHBV, même avec des doses d’ADN relativement faibles. Dans un 2ème temps, nous avons montré chez des animaux chroniquement infectés par le DHBV, que l’administration par EP du vaccin à ADN ciblant les protéines structurales du DHBV et le DuIFN-γ améliore considérablement l’efficacité thérapeutique du vaccin, notamment au regard de la séroconversion et de la clairance virale. Les résultats ainsi obtenus confirment l’intérêt majeur de cette approche vaccinale pour la thérapie des hépatites B chroniques / Despite the existence of an effective prophylactic vaccine, chronic hepatitis B virus (HBV) infection remains a major public health problem. Since persistence of HBV infection is mostly associated with insufficient immune responses, therefore DNA vaccination capable of activating both humoral and cellular immune responses appears as a pertinent strategy for chronic hepatitis B therapy. However, the efficacy of such therapeutic approach remains limited in humans. Improvement of DNA vaccine efficacy is therefore needed for future therapeutic applications in clinic. The main objective of this thesis was to investigate in the duck hepatitis B virus (DHBV) model, whether the protective and therapeutic efficacy of DNA vaccine can be enhanced using EP-based delivery system. Firstly, we showed in naïve ducks that EP-based delivery was able to improve the dose efficiency of DNA vaccine and to maintain a highly neutralizing, multi-specific B-cell response even with relatively low DNA doses, suggesting that it may be an effective approach for chronic hepatitis B therapy at clinically feasible DNA dose. Secondly, we showed in chronic DHBV-carriers that in vivo EP is able to dramatically enhance the therapeutic potency of DNA vaccine targeting hepadnaviral proteins. Indeed, this approach was able to consistently restore humoral immune response and to sustainably decrease and even clear viral infection. Thus, these data strongly support the use of this approach for chronic hepatitis B therapy in humans

Thesis (MScMedSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Despite the decreased rate of HBV horizontal transmission in South Africa (SA) due to the HB vaccine, the risk of perinatal transmission remains of concern, especially in HIV/HBV co-infected women. Loss of HBV immune control, resulting in higher HBV replication and thus increasing the risk of transmission is described in HIV/HBV co-infected women. Chronic hepatitis is a well-recognized risk factor for hepatocellular carcinoma (HCC). The presence of specific HBV mutations has been reported in chronic and HCC patients and is used in algorithms for the prediction of HCC in CHB patients in Asia. While these mutations are extensively described in male patients, little is known regarding the antenatal and paediatric populations. This study aimed to determine the prevalence of HBV infection in HIV-exposed infants and to investigate the presence of HCC-related mutations in pregnant women and HIV-exposed children in SA. Residual samples of infants born to HIV-infected mothers were collected from the P1041 study previously conducted in SA. HBV markers (HBsAg, anti-HBs and anti-HBc) were tested on the Architect (Abbott). HBsAg positive samples were tested for HBV DNA to determine HBV viral loads. HBV strains were characterised by sequencing of the HBsAg gene and genotypes were determined by phylogenetic analysis using HepSEQ (www.hepseq.org.uk). For the HCC-related mutations investigation, samples and data were collected from three HBV-related studies: the NHLS Paediatric Study, an Antenatal Study and the current study. Pre-S, basal core promoter (BCP) and pre-core data was collected from all samples. Multiple alignments were formed and the nucleotide sequences of these extracts were translated into protein sequences. These protein sequences were compared manually to the HBV reference genes to identify HCC-related mutations. Of 850 HIV-exposed infants tested, three infants were positive for both HBsAg and HBV DNA. Two samples show evidence of past, but cleared HBV infection. Sequence analysis showed that the infants were infected with a subgenotype A1. At follow up, only one infant and mother were able to be traced and contacted. The infant was HIV-infected and had been on an ART regimen, including lamivudine for two years. HBV testing showed that the infant was HBsAg positive and had an undetectable viral load. Core sequence analysis showed clustering between mother and infant sequences. Transmission of mutant HBV previously associated with HCC prompted the question of what the prevalence of mutations in the antenatal and paediatric population is. In this investigation of HCC-related mutations study, a higher prevalence of combined pre-S, BCP and pre-core mutations was found in HIV-infected as compared to HIV-uninfected women. This study shows that vertical transmission is occurring in HIV-exposed infants in SA despite HB vaccination. Data described in this study suggests the importance of HB vaccination closer to the time of birth in SA. Moreover, data on the higher prevalence of HCC-related mutations in HIV-infected pregnant women provide a background for further longitudinal studies to confirm these findings and their implications in SA. / AFRIKAANSE OPSOMMING: As gevolg van die beskikbaarheid van die Hepatitis B virus (HBV) entstof , het horisontale transmissie van die virus drasties in Suid-Afrika (SA) verminder. Ten spyte hiervan, is daar steeds ‘n hoë risiko van perinatale transmissie van swanger vroue na hulle babas, dit word veral gesien met MIV/HBV positiewe vroue. Dit is wyd beskryf dat vroue wat mede-besmet is met MIV/HBV gewoonlik beheer verloor oor hulle immuunstelsel, wat lei tot ‘n hoër mate van HBV replikasie en dus ‘n hoër risiko van virus oordrag. Kroniese hepatitis is wel bekend as ‘n hoë risiko faktor vir HCC. Die teenwoordigheid van spesifieke HBV mutasies in kroniese en HCC pasiënte word alreeds in Asië gebruik in sekere algoritmes en formules om infeksie aan te dui en te voorspel. Hierdie mutasies is omvattend beskryf in manlike pasiënte, maar baie min is bekend in voorgeboorte en pediatriese gevalle. In hierdie studie het ons die teenwoordigheid van HCC-verwante mutasies in swanger vroue en MIV-blootgestelde kinders in Suid-Afrika ondersoek. Monsters is verkry van babas gebore van MIV-positiewe moeders van die P1041 studie wat voorheen in SA gedoen is. Die HBV merkers (HbsAg, teen-HBs en teen-HBc) was op die Architect (Abbott) getoets. HBsAg positiewe monsters was getoets vir HBV DNA om die virale lading te bepaal. Die verskeidenheid HBV stamme was gekarakteriseer deur die virus se nukleïensuur volgordes te bepaal. Die verskillende genotipes is bepaal deur filogenetiese analises te doen met behulp van die HepSEQ (www.hepseq.org.uk) program. Vir die HCC-verwante mutasie studie is monsters en data vergelyk met 3 HBV-verwante studies: die NHLS pediatriese studie, ‘n voorgeboorte studie en hierdie spesifieke studie. Voor-S, basale kern promoter en voor-kern data was van alle monsters bekom. ‘n Veelvoudige belyning was gedoen met die nukleïensuur volgordes van die verskeie DNA ekstrakte, wat daarna vertaal is in proteïen volgordes. Hierdie proteïenvolgordes translasie was by hand vergelyk met verwysings gene om die relatiewe HCC mutasies te probeer identifiseer. Van die 850 blootgestelde MIV babas wat getoets is, het 3 positief getoets vir beide HbsAg en HBV DNA. Twee monsters het bewys van verlede , maar vrygestelde HBV infeksie. Data analise bewys dat die babas met subtipe A1 besmet was. Ons kon slegs een moeder en baba paar opvolg en kontak vir verdere toetse. Die baba was MIV-positief en was op antiretrovirale behandeling , insluitend lamivudine, vir ten minste 2 jaar. HBV toetse het gewys dat die baba HbsAg positief is en ‘n onopspoorbare virale lading gehad het. Kern nukleïensuur volgorde analise het groepering getoon tussen die ma en baba se virus monsters . Die transmissie van die mutante HBV wat geassosieer is met HCC het gelei tot die vraag wat die voorkomssyfer is van hierdie spesifieke mutasies in die voorgeboorte en pediatriese populasies in SA. In hierdie studie het ons ‘n hoër gekombineerde voorkomssyfer gevind van die voor-S, basale kern promoter en voor-kern mutasies in MIV-positiewe vroue, in vergelyking met MIV-negatiewe vroue. Hierdie studie bewys dus dat vertikale transmissie van HBV in blootgestelde MIV babas steeds plaasvind, ten spyte van HBV inenting. Die data wat in hierdie studie beskryf was dui daarop dat die belangrikheid van HBV inenting nader aan die tyd van die geboorte in SA gegee moet word.As gevolg van die hoë voorkomssyfer van HCC-verwante mutasies in swanger vroue, is daar verdere longitudinale studies nodig om hierdie bevindinge en hul implikasies in SA te bevestig.

Understanding the transmission dynamics of infectious diseases is important to well-informed public health policy, responsive infection control and individual patient management. The on-going revolution in whole-genome sequencing provides unprecedented resolution for detecting evidence of recent transmission and characterising population-level transmission dynamics. In this thesis, I develop and apply evolutionary approaches to investigating transmission, focusing on three globally important pathogens. Hepatitis C virus (HCV) is a major cause of liver disease affecting 150 million people and killing 350,000 annually. I conducted a meta-analysis of twentieth-century HCV epidemics, finding that the age of the epidemic can be predicted by genetic diversity. Using the coalescent, I fitted classic susceptible-infected (SI), susceptible-infected-susceptible (SIS) and susceptible-infected-recovered (SIR) epidemiological models. Most epidemics showed signatures of SI dynamics, but three, from Argentina, Hong Kong and Thailand, revealed complex SIR dynamics. Norovirus is the leading viral cause of diarrhoea, estimated to cost the NHS around £115 million annually. I analysed whole norovirus genomes via a stochastic transmission model, finding that up to 86% of hospital infection was attributable to transmission from another patient in the hospital. In contrast, the rate of new introductions to hospital by infected patients was extremely low (<0.0001%), underlining the importance of ward management during outbreaks. Campylobacter is the most commonly identified cause of bacterial gastroenteritis worldwide. I developed a zoonotic transmission model based on phylogeography approaches to test whether three strains previously associated with multiple host species were in fact aggregates of strongly host-restricted sub-strains, or genuine generalists. Members of the same strain isolated from different host species were often more closely related than those isolated from the same host species. I estimated 419, 389 and 31 zoonotic transmissions in ST-21, ST-45 and ST-828 respectively, strongly supporting the hypothesis that these strains are adapted to a generalist lifestyle.

Le virus de l’hépatite C (VHC) est une des principales causes d’hépatite chronique. La protéine F du VHC est codée par un cadre de lecture alternatif du gène de la capside, Core. La protéine F a été découverte après que l’on ait associé Core à plusieurs des fonctions pathogènes du VHC. Nous proposons donc que certaines fonctions biologiques et pathogènes attribuées à la protéine Core résultent de l’activité de la protéine F. Nous avons choisi de développer trois lignées de poissons zébrés (Danio rerio) qui expriment différentes versions de la protéine F afin d’étudier les effets de la protéine F et leur incidence dans la pathogenèse du VHC. Deux versions de la séquence codant pour la protéine F (AF11 et AUG26) et une version mutante du gène core (CoremutI) ont été introduites sur les vecteurs d’un système d’expression répressible spécifique au foie. Ces vecteurs ont été co-injectés dans des embryons unicellulaires de poissons zébrés pour générer les poissons fondateurs des lignées transgéniques. 19, 21 et 36 poissons ont été choisis comme fondateurs pour les lignées AF11, AUG26 et CoremutI respectivement. De ce nombre, 9, 11 et 11 poissons ont atteint la maturité, dans l’ordre pour les mêmes lignées, et seront croisés pour donner naissance à des lignées transgéniques stables. Les résultats de ces expériences nous permettront de mieux cerner les propriétés biologiques de la protéine F et de définir son rôle dans la pathogenèse du VHC. / Hepatitis C virus (HCV) is a major cause of liver steatosis, fibrosis and hepatocellular carcinoma. HCV F protein is expressed from an alternative reading frame within the Core sequence. F protein was discovered after many of the pathogenic determinants of HCV had been associated with the effects of Core. Hence, we propose that a part of the functions attributed to Core result from the activity of the F protein. We produced and selected 19, 21 and 36 transgenic zebrafish (Danio rerio) to give rise to 3 independent lines expressing different versions of the F protein. Of these founders, 9, 11 and 11 were raised to maturity and will be bred to generate stable transgenic lines. Characterizing the phenotype of these transgenic fish will help determine the precise role of the F protein in the pathogenesis of hepatitis C.

L'hépatite C pose un problème de santé publique majeur, dans la mesure où le risque de développer une infection chronique est relativement élevé (40 à 60%) et où la résistance au traitement de choix - l’interféron alpha pégylé et la ribavirine - touche près de la moitié des patients. Cette persistence virale repose avant tout sur de puissantes stratégies d’évasion du système immunitaire inné de l’hôte par le virus. Dans ce projet, nous nous sommes intéressés à la caractérisation de la réponse antivirale dans des hépatocytes primaires humains normaux et chroniquement infectés avec le VHC, un domaine encore largement inconnu dû à la difficulté d’obtenir ce type de matériel primaire. Nous avons étudié la fonctionnalité de deux voies majeures de détection des pathogènes viraux suite à l’exposition d’hépatocytes primaires humains à de l’ARNdb intracellulaire, via le récepteur et adaptateur RIG-I/MDA5-CARDIF, et extracellulaire via TLR3-TRIF, mimant ainsi les étapes précoces de la détection d’un virus par la cellule hôte. Nous avons établi par RT-PCR quantitatif et analyse transcriptomique par microarray, que ces deux voies de stimulation sont fonctionnelles dans des hépatocytes primaires normaux et que leur activation entraîne à la fois l’expression de gènes antiviraux communs (ISG56, ISG15, CXCL10, …) mais aussi spécifiques avec les gènes IL28A, IL28B et IL29 qui sont une signature de l’activation de la voie de détection de l’ARNdb intracellulaire. La protéine virale NS3/4A joue un rôle majeur à la fois dans le clivage de la polyprotéine virale initiale, mais aussi en interférant avec les cascades de signalisation engagées suite à la détection par la cellule hôte de l’ARN du VHC. Plus particulièrement, nous avons démontré que l’expression ectopique de NS3/4A dans des hépatocytes primaires humains normaux entraîne une diminution significative de l’induction des gènes antiviraux dûe au clivage de CARDIF au cours de l’activation de la voie de signalisation médiée par RIG-I. Nous avons également démontré que l’expression de la NS3/4A entraîne des modifications de l’expression de gènes-clé impliqués dans la régulation de l’apoptose et du programme de mort cellulaire, en particulier lorsque la voie TLR3 est induite. L’ensemble de ces effets sont abolis en présence de BILN2061, inhibiteur spécifique de NS3/4A. Malgré les stratégies de subversion de l’immunité innée par le VHC, nous avons démontré l’induction significative de plusieurs ISGs et chemokines dans des hepatocytes primaires provenant de patients chroniquement infectés avec le VHC, sans toutefois détecter d’interférons de type I, III ou certains gènes antiviraux précoces comme CCL5. Ces observations, concomitantes avec une diminution de l’expression de CARDIF et une correlation inverse entre les niveaux d’ARNm des ISGs et l’ARN viral révèlent une réponse antivirale partielle dûe à des mécanismes interférents sous-jacents. Cette réponse antivirale détectable mais inefficace est à mettre en lien avec l’échec du traitement classique PEG-IFN-ribavirine chez la moitié des patients traités, mais aussi en lien avec l’inflammation chronique et les dommages hépatiques qui mènent ultimement au développement d’une fibrose puis d’une cirrhose chez une grande proportion de patients chroniquement infectés. / Hepatitis C infection is a worldwide health problem since the risk to develop a persistent infection is relatively elevated (40 to 60%) and nearly half of the infected patients do not respond to the classical anti-HCV therapy based on a combination of PEG-IFNα and ribavirin. Viral persistence is based on powerful evasion strategies of the host’s innate immune system. In our study, we characterized antiviral response in primary human normal and chronically HCV-infected hepatocytes, a cutting-edge in our field due to the difficulty to isolate this particular cell type. In order to better define the antiviral response in freshly isolated human primary hepatocytes, we stimulated these cells with extracellular and intracellular dsRNA to trigger TLR3/TRIF and RIG-I-MDA5/CARDIF-mediated antiviral signaling pathways. By using qRT-PCR and microarray analysis, we report that both detection pathways are functional in normal human hepatocytes, their activation leading to the expression of both common (IFIT1, OASL, ISG15 and CXCL10) and specific genes (IL28A, IL28B and IL29), these last ones being a signature of the intracellular dsRNA-mediated pathway. HCV NS3/4A plays a key role in the viral polyprotein processing and upon viral RNA detection by interfering with the host’s antiviral signalling cascades. We report that major antiviral genes induction following activation of RIG-I mediated pathway are severely impaired in ectopically NS3/4A expressing normal hepatocytes due to CARDIF cleavage, but can be restored by specific NS3/4A inhibitor BILN2061. Our microarray analysis also revealed a role for NS3/4A following TRL3-mediated pathway activation on regulation of apoptosis and programmed cell death, which could be linked to strategies for the virus to persist in its host. Despite HCV strategies to circumvent the host’s immune defense system, we observed significant upregulation of ISGs and chemokines in liver biopsies and corresponding isolated hepatocytes from chronically HCV-infected patients. However, no type I and III interferon, neither key-antiviral genes (e.g., CCL5) were detected, underlying an ongoing –but inefficient- antiviral response unable to eradicate the virus. Moreover, we obtained significant inverse correlations between ISGs mRNAs and viral RNA in addition to CARDIF decrease, clearly unravelling efficient viral interfering strategies in a context of chronic HCV infection. This sustained -albeit incomplete- hepatic innate immune response is certainly associated to the failure of the classical IFN-based therapy in half of the infected patients and to the chronic inflammation causing liver damages and eventually leading to hepatocarcinoma which is often observed at late stage of the disease.