798 CELL CULTURE REPLICATION OF A GENOTYPE 1B HEPATITIS C VIRUS ISOLATE CLONED FROM A PATIENT WHO UNDERWENT LIVER TRANSPLANTATION

POSTERS miR-122 expression and viral load in vivo. In addition, decreased pretreatment level of miR-122 is associated with no response during IFN therapy. In the present study, the miR-122 and miR221 expression was analysed in chronic HCV infection with and without steatosis compared to non-infected, normal liver tissues. Methods: The microRNA expression was determined in 67 biopsy (12 HCV [genotype 1/b] without steatosis, 36 HCV with steatosis, 19 steatosis) and 6 normal formalin-fixed paraffin-embedded liver samples. TaqMan MicroRNA Assays (ID: 002245 and 000524, Life Technologies) were employed for the analysis of miR-122 and miR221 expression from isolated total RNA (modified RNeasy FFPE kit, Qiagen). Results: The expression of both microRNAs were decreased in the three sample groups when compared to normal liver tissue. In average, the least miR-122 expression was observed in HCV without steatosis followed by HCV with steatosis and steatosis, while miR221 expression was equally decreased in HCV without steatosis and HCV with steatosis but moderately decreased in steatosis. Conclusions: The results indicate that steatosis causes a less pronounced, however not significant, decrease in miR-122 expression even when associated with HCV infection, while no increase – observed during hepatocarcinogenesis – but decrease of miR-221 expression is present in chronic hepatitis C. Acknowledgement: This study was supported by OTKA grant #75468. 797 NITAZOXANIDE IS AN INDIRECT INHIBITOR OF HCV REPLICATION BY MODULATION OF HCV NS5A HYPER-PHOSPHORYLATION THROUGH UP-REGULATION OF CKI ALPHA A. Montero, P. Viswanathan, C. Yon, B. Korba. Microbiology & Immunology, Georgetown University Medical Center, Washington, DC, USA E-mail: [email protected] Background and Aims: Nitazoxanide (NTZ, Alinia® , Romark Laboratories, LC) is a licensed thiazolide anti-infective currently in advanced stage clinical development for treatment of chronic hepatitis C. Studies by ourselves and others previously demonstrated that NTZ and its active metabolite, tizoxanide (TIZ), exhibit potent antiviral activity against HCV replication in cell culture, but the antiviral mechanism has remained undetermined. The current investigations sought to determine the effect of TIZ on intracellular HCV proteins to reveal a mechanism of action. Methods: The status of HCV and specific cellular proteins in NTZtreated HCV replicon cultures were followed by Western blot, immunoprecipitation, and assays for enzymatic activities. Results: TIZ was inactive against HCV polymerase, protease, and helicase in enzymatic assays. The rate of reduction of HCV proteins in NTZ-treated HCV replicon cells was consistent with loss of viral RNA template. NTZ-treatment induced a 4- to 6-fold enhancement of hyper-phosphorylated HCV NS5A (p58), and a similar reduction of basally phosphorylated NS5A (p56) in intracellular membrane preparations (where HCV replication is localized). The phosphorylation state of NS5A is a regulator of the switch from active viral genome replication to packaging, and overproduction of p58 is known to reduce HCV replication. Evaluations of the effect of TIZ on casein kinase I-alpha (CKIa), the cellular kinase reported to be responsible for conversion of HCV NS5A p56 to p58 were performed. CKI activity in intracellular membrane preparations from NTZ-treated HCV replicon cells was 3- to 4-fold higher than those from untreated cells in enzymatic assays. TIZ had no direct effect on purified CKIa activity in enzymatic assays, including auto-phosphorylation. Examination of the effect of NTZ and TIZ on cellular factors involved in the regulation of casein kinases is in progress. Conclusions: Over-production of hyper-phosphorylated HCV NS5A appears to be the primary antiviral mode of action of NTZ against S320

HCV. A drug-associated intracellular enhancement of the cellular enzyme activity responsible for hyper-phosphorylation of NS5A provides a cell-based mechanism. Since TIZ appears to have no direct effect on CK1a in enzymatic assays, we hypothesize the primary cellular target for TIZ is one of several proteins involved in the regulation of CK1a. 798 CELL CULTURE REPLICATION OF A GENOTYPE 1B HEPATITIS C VIRUS ISOLATE CLONED FROM A PATIENT WHO UNDERWENT LIVER TRANSPLANTATION G. Koutsoudakis, S. Perez-del-Pulgar, M. Coto-Llerena, P. Gonzalez, J. Dragun, L. Mensa, G. Crespo, M. Navasa, X. Forns. Liver Unit. Institut de Malalties Digestives, Hospital Clinic, CIBERehd, and IDIBAPS, Barcelona, Spain E-mail: [email protected] Introduction and Aim: Hepatitis C virus (HCV) replicates poorly in cell culture. Up to date, only the genotype 2a isolate JFH1 replicates in vitro to levels that allow the study of the full life cycle. Since hepatitis C patients’ serum is an abundant natural source of the virus, we have chosen a serum with an exceptional high viral load, derived from a patient who underwent liver transplantation, in order to clone a new full-length replication-competent genotype 1b HCV isolate Methods: Huh7.5 cells were inoculated with the patient’s serum. In Huh7.5 cells, the HCV proteins were analyzed by immunofluorescence (IFM) and the RNA by quantitative reversetranscription real-time PCR. In order to clone the full-length isolate from the serum, we used overlapping PCRs to amplify the full genome, which was named Barcelona Hepatitis C Virus 1 – BHCV1. Subgenomic replicons of BHCV1 were constructed and subjected also to replication studies. Results: Initial inoculations of Huh7.5 cells by the patient serum led to the detection of HCV RNA and proteins in the cells. The full-length isolate BHCV1 belonged to the genotype 1b and it was composed of 9591 nt. Subgenomic replicons of BHCV1 with luciferase reporters, replicated poorly in cell culture. Full-length clone also replicated poorly in a transient fashion after electroporation of Huh7.5 cells with in vitro transcribed RNA. Nevertheless, approximately 3 weeks after electroporation, HCV core protein positive cells were detected by IFM. Interestingly, small amounts of core protein, in comparison to the core secreted by the JFH1 isolate, were also measured in the supernatant of the cells, suggesting that HCV particles might be assembled and released. Six weeks post electroporation and after several cell passages, the BHCV1 isolate was extracted from the cells, sequenced and one adaptive mutation was mapped. For further analysis, we created an in vitro infectious BHCV1/JFH1 intergenotypic chimeric virus. After several cell passages the chimeric virus increased its titers and accumulated few adaptive mutations mostly at the glycoproteins region. Conclusion: HCV patients’ sera can be used for inoculation of Huh7.5 cells. Cloning of recombinant viruses from selected sera could reveal new in vitro replication-competent isolates. 799 KINETICS OF HEPATIC AND CIRCULATING MICRORNA-122 DURING EXPERIMENTAL ACUTE HCV INFECTION K. Krawczynski1 , Y. Choi1 , M. Odenthal2 , M. Ruhrlander2 , M. Muller2 , H.P. Dienes2 . 1 Division of Viral Hepatitis, Centers for Disease Control and Prevention, Atlanta, GA, USA; 2 Institute of Pathology, University of Cologne, Cologne, Germany E-mail: [email protected] Background and Aims: MicroRNA 122 (miR-122) is involved in hepatic gene regulation and, as shown by in vitro studies and in chronically HCV-infected chimpanzees, may be also essential for hepatitis C virus (HCV) replication. We analyzed miR-122

Journal of Hepatology 2011 vol. 54 | S209–S361