S214 Poster Session − Friday, April 24 581 QUANTITATIVE LONGITUDINAL EVALUATION OF HEPATITIS DELTA VIRUS RNA AND HEPATITIS B VIRUS DNA SHOWS A DYNAMIC COMPLEX REPLICATIVE PROFILE IN CHRONIC B/D COINFECTION D. Tabernero 1 , M. Schaper 1,2 , F. Rodr´ıguez-Fr´ıas 1,2,3 , R. Jardi 1,2,3 , M. Homs 1 , G. Ruiz 3 , M. Buti-Ferret 1,2,4 , R. Esteban 1,2,4 . 1 Centro Investigacion Biomedica en Red de Enfermedades Hep ´ aticas y Digestivas (CIBEREHD), 2 ViRgil, European Network of Excellence on Antiviral Drug Resistance (ViRgil LSHM-CT-2004–503359), 3 Biochemistry, 4 Hepatology, Universitary Hospital Vall d’Hebron, Barcelona, Spain E-mail:david.tabernero@ciberehd.org Summary: Coinfection with hepatitis B virus (HBV) and hepatitis delta virus (HDV) is associated with severe liver disease; nonetheless our understanding of the clinical and virological implications of these viruses in coinfection is limited. Aim: Develop and assess a real-time reverse-transcription PCR (rt-RT- PCR) method to quantify HDV-RNA levels. This method and HBV-DNA quantification have been used to determine virological profiles in untreated patients with chronic HBV and HDV coinfection. Patients and Methods: A total of 42 patients with chronic HBV and HDV infection were included in a cross-sectional study. In 25 of these patients, a retrospective longitudinal study of HBV-DNA and HDV-RNA levels was performed (298 samples). HDV-RNA was quantified by a single-step rt-RT- PCR reaction in the LightCycler™ analyzer using fluorescently labelled probes corresponding to a conserved autocatalytic HDV region. Results: The linearity range for HDV-RNA was 10 3 to 10 8 equivalent/mL. In the reproducibility analysis, intra-assay coefficients of variation (CVs) at two known HDV-RNA concentrations were 5.2% and 2.7%, and inter- assay CVs were 6.7% and 4.8%. The cross-sectional study detected active infection by both viruses in 19 cases (45.2%), inactive HBV/active HDV infection in 12 (28.6%), active HBV/inactive HDV in 7 (17%), and inactive infection by both viruses in 4 cases (10%). These findings question the majorsuppressor effectof HDV overHBV and disclose an important role of HBV in coinfection, confirmed by longitudinal analysis. Moreover, evaluation of HBV and HDV activitiesshowed 7 different replication profiles,with a high frequency of fluctuating activity of one or both viruses, including alternating predominance. In 24% of cases, longitudinal HBV-DNA and HDV-RNA valuesdiffered from cross-sectional data, indicating a high risk of misinterpreting HBV/HDV interactions when assessing viral load in a single determination. Conclusions: The LightCycler™ real-time PCR is a practical sensitive reproductible assay with a wide dynamic range of detection. Both,longi- tudinaland cross-sectional, studies question the major suppressor effect of HDV overHBV, disclosing an important role of HBV. Longitudinal evaluation of viremia has evidenced a highly complex interaction between HBV and HDV.Thus,longitudinal evaluation of viremia is essential to understand the pathophysiology of HBV and HDV coinfection. 582 MODULATION OF AKT, NFKB ACTIVITY, APOPTOSIS AND CELL CYCLE IN HEPATOCYTES BY THE LAMIVUDINE RESISTANT RTM204I HBV MUTANT R. Chin 1 , U. Nachbur 2 , L. Earnest-Silveira 3 , A. Bankovacki 2 , H. Zentgraf 4 , C.-T. Bock 5 , J. Silke 2 , J. Torresi 6 . 1 Department of Medicine, The University of Melbourne, Heidelberg, 2 Biochemistry, La Trobe University, Bundoora, 3 Department of Medicine, Austin Hospital, The University of Melbourne, Heidelberg, Victoria,Australia; 4 Applied Tumor Virology, German Cancer Research Center (DKFZ), Heidelberg, 5 Department Molecular Pathology, University Hospital of Tuebingen, Tuebingen, Germany; 6 Department of Infectious Diseases Austin Hospital, The University of Melbourne, Heidelberg, Victoria,Australia E-mail:josepht@unimelb.edu.au Introduction: The mechanisms by which HBV causes HCC invariably involve a complex interplay of dysregulated signals thatcontrolcell cycle progression, apoptosisand the inflammatory response. We have investigated the alterations in Akt signalling, apoptosisand cellcycle induced by infection with wild-type (wt) and rtM204I HBV. Methods: Recombinant adenoviruses expressing a full-length infectious genome of wt and rtM204I HBV together with an internal GFP reporter or a controladeno-GFP virus were used to infect primary mouse hep- atocytes (PMoH) and Huh7 cells. Western immunoblot analyseswere performed for MAPK intermediates, cIAP, cleaved caspase 3 and cell cycle regulatory proteins. NFkB activity was assessed using a Lentiviral NFkB-Luciferase reporter. Cell viability wasanalysed after incubation of cells with TNF-alpha, FasL and TRAIL.Cell cycle was examined in synchronised hepatocytes and analysed by flow cytometry. Results: Infection of PMoH and Huh7 cells with rAd-HBV-M204I resulted in a 93%±SD50 increase in pERK and a 68%±SD33 increase pAkt. The p53 and p21cip1 proteins wereboth increased by 130%±SD95 and 65%±SD4.5 respectively. Infection ofHuh7 cellswith rAdHBV- M204Iand rAdHBV-wtresulted in comparable increases in the levels of cyclin A (120%±SD42 vs161%±SD8, respectively) and cyclin B (225% ±SD132 vs 210%±SD93). Cell cycle analysis in synchronized infected Huh7 cells showed a greater proportion of cells in the G2 phase (28% for rAdHBV-M204I and 30% for the and rAdHBV). In rAdHBV- wt and rAdHBV-M204I infected cells, levels of pcdc2 were increased by 128%±SD67 and 158%±SD52 respectively, consistent with G2 arrest. Cell viability of HBV infected hepatocytes was significantly reduced after treatment with TNF-alpha, FasL and TRAIL.NFkB and cIAP were increased in the first 24 hours post-infection but this was followed by a reduction in NFkB activity and cIAP levels that were accompanied by a r in cleaved caspase 3 (53%±SD 21), consistent with increased apoptosis in HBV infected hepatocytes. Conclusion: We have shown here that the lamivudine resistant rtM204I mutant is able to upregulate hepatocyte Akt and NFkB activity and prod cell cycle dysregulation and apoptosis in a manner analogous to wild- type HBV.The combination of these effects may contribute to HCC development. 583 EVALUATION OF AAV-MEDIATED IFNa-GENE THERAPY EFFICACY IN HBV TRANSGENIC MICE: CONSTITUTIVE VERSUS INDUCIBLE EXPRESSION L. Vanrell 1 , C. Olague 1 , A. Vales 1 , A. Pa˜ neda 1 , L. Tenembaum 2 , G. Gonzalez-Aseguinolaza 1 . 1 Center for Applied Medical Research (CIMA). University of Navarra,Pamplona, Spain; 2 Universit ´ e Libre de Bruxelles, Laboratory for Experimental Neurosurgery, Brussels, Belgium E-mail:lvanrell@unav.es Background: HepatitisB virus (HBV) and hepatitis C virus (HCV) constitute the main etiologic factors for the chronic viral hepatitis affect more than 550 million people worldwide. Interferon alpha (IFNa) has demonstrated therapeutic efficacy in both chronic hepatitis B and C. However, the monotherapy response rate (25−35%) can be increased, and side effects limited, by improving the pharmacokinetic of IFNalpha therapy with stabilising ligands. Gene therapy allows a continuous in vivo expression of the transgene at the desired site. AAV vectors lack pathogenicity in humans and have demonstrated prolonged expression of numerous transgenes, in several tissues and immunocompetent animal models. Aims: Therapeutic efficacy evaluation of a recombinant AAV expressing murine IFN alpha (AAVIFN) underthe control of a constitutive or an inducible promoter in HBV transgenic mice. Methods: We have produced AAV vectors expressing luciferase or mur IFN-alpha1 into two different expression cassettes (AAVIFN). The first one containsthepromoter for the human Elongation Factor-1 alpha (hEF1alpfa), the transgene, and the SV40 polyadenilation signal sequen The second one contains a CMV-TetON promoter, the transgene, and the SV40 polyadenilation signal. AAV serotype 8 virus production was performed by cotransfection into 293 T cells of the plasmid containing t expression cassette flanked by the AAV ITRs, and the plasmids containin the necessary AAV and adenoviral proteins, using linear polyethylenimin (25 kDa). Viruses were purified by iodixanol centrifugation gradient and