- Email: [email protected]
380 Genotype inclusivity of HBV DNA quantitation with the cobas taqman HBV test
Category 5a: Viral Hepatitis: Hepatitis B – Basic ITOR, (Roche; sensitivity 2x102 HBV DNA copies/ml) were quantified with HBV DNA Assay v3.0 (bDNA) (Bayer Healthcare-Diagnostics; sensitivity 2x103 HBV DNA copies/ml). HBV genotype and presence of mutations in Basal Core Promotor (BCP) or PreCore were available for 461 serum samples. Results: In 909 samples tested 60% and 72% had HBV DNA detectable with bDNA v3.0 and COBAS, respectively. Correlation between the two assays was r2= 0.657. Median viral loads were: 4.35 log 10 copies/ml (3.03 - 7.38 log 10 copies/ml) vs 4.07 log 10 copies/ml (2.3 -7.21 log 10 copies/ml) with bDNA v3.0 vs COBAS, respectively (p=0.001). Six dilutions of HBV WHO ranging from 5x105 to 2.5 x102 IU/ml were tested with both assays; 4 dilutions were detectable with bDNA v3.0 (3.4x105 to 5.1x102 IU/ml) (r2= 0.999); all were detectable with COBAS (5.1x104 to 1.4x102 IU/ml)(r2= 0.911). Correlation (r2) between the two assays was r2= 0.475 for wild type HBV; r2= 0.588 for BCP or PreCore mutations; r2= 0.616 for genotype A; r2 = 0.566 for genotype B; r2 = 0.534 for genotype D and r2= 0.661 for genotype E. Conclusion: The increased sensitivity of bDNA v3.0 will avoid retesting, with quantitative PCR, 60% of samples not detectable with bDNA v1.0. 377 EVALUATION OF THE REALQUANT HBV-DNA ASSAY (REALTIME PCR)
M. Martinot-Peignoux 1 , V. Esnault 2 , C. Kumas 1 , B.N. Pham 3 , P. Marcellin 2 . 1 INSERM U481 et Unité Claude Bernard Hôpital Beaujon, Clichy, France; 2 Service D’Hépatologie,Hôpital Beaujon, Clichy, France; 3 Service D’Hématologie-Immunologie Biologiques Hôpital Beaujon, Clichy, France The aim of our study was to assess the relevance of the with REALQUANT HBV-DNA assay for a routine laboratory. Methods: 180 serum samples tested with Cobas Amplicor HBV MONITOR, Roche (sensitivity 2x102 HBV DNA copies/ml) were quantified with REALQUANT HBV-DNA, (DiaTech, Italy) on the Rotor-Gene system (Corbett Research, Australia). REALQUANT allows the amplification of a short length of the gene coding for the Surface Antigen and utilizes a Taqman probe for the detection. Twenty samples HBsAg negative/antiHCV negative and 20 samples HBsAg negative/HCV RNA positive were tested for the specificity. Reproducibility was calculated with 3 serum samples tested 10 times. Sensitivity was determined with 76 samples HBV DNA undetectable with COBAS (30 patients under therapy and 46 untreated) and 35 samples HBV DNA detectable with COBAS. Results: All the samples HBsAg negative/anti-HCV negative were found negative; 3/20 samples HBsAg negative/HCV RNA positive were found positive.(7.4x103; 181; and 70 copies/ml) with REALQUANT . The 3 samples tested 10 times showed CV ranging from 13% to 24%. HBV DNA was detectable with REALQUANT in 58% and 63% of the samples from under therapy and untreated patients, respectively. Among the 35 samples COBAS positive 94% had HBV DNA detectable with RealQuant HBVDNA. The correlation between the two assays was good (r2 =0.695). The HBV DNA levels were higher with REALQUANT than with COBAS (ratio=9). Conclusion: REALQUANT HBV-DNA assay showed a good specificity 93%, a high sensitivity (mainly for patients under therapy) suggesting that monitoring patients with a sensitive assay could be clinically relevant.
378 VIRAL, HOST AND PATHOLOGICAL PROFILE OF WHV/HDV INFECTION IN WOODCHUCK MODEL
E. D’Ugo 1 , S. Orobello 1 , A. Canitano 1 , C. Argentini 1 , R. Giuseppetti 1 , G. Palmieri 2 , M. Rapicetta 1 . 1 Laboratory of Virology, Istituto Superiore Di Sanità, Rome, Italy; 2 Pathological Anatomy Chair, Tor Vergata University, Rome, Italy Introduction: HDV superinfection in WHV carrier woodchucks has shown similar biological and pathological features of HDV in humans.
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Aim: Our aim was to characterise WHV/HDV superinfection with particular regard to viral determinants possibly involved in pathogenesis. Materials and Methods: Chronically WHV infected woodchucks were infected with HDV genotype 1. Serum and liver samples were monitored for virological and antibody markers (RT-PCR, dot-blot hybridisation, ELISA). Sequence and phylogenetic analyses of WHV Pre-S/S and HDAg were performed by Neighbour-Joining method. AST levels were determined and histological scoring performed according Ishak. Results: HDV-RNA and HDAg were detected in follow-up sera from all animals at 2 to 4 and 6 to 8 weeks post-infection. High HDV-RNA titers in livers and elevated AST values were present in infected animals. Phylogenetic analysis showed relatedness of WHV sequence to HBV genotype F. All HDV isolates from woodchucks clustered in a unique group separate from human isolates. Different amino acid usage, but conservation of the total positive charge of HDAg was found. Liver histological analysis performed on all animals showed normal liver tissue histology before infection, despite massive panacinar liver necrosis on autoptic tissues (Ishak score B6). A short survival was observed in all HDV infected animals that died before the 30th week after infection. Conclusions: A severe course of HDV infection has been observed in woodchucks possibly associated to the particular HDV and helper virus strains. A detailed molecular characterisation of HDV and helper virus is reported as well as a fine histological scoring of infected livers.
379 EXPRESSION OF HEPATITIS C VIRUS CORE PROTEIN IMPAIRS DNA-REPAIR IN HUMAN HEPATOMA CELLS
T. Severi 1 , T. Crabbe 1 , A. Van Eetveldt 1 , C. Verslype 1 , T. Roskams 2 , J. Fevery 1 , J.F. van Pelt 1 . 1 Labo Hepatology, 2 Dept Morph&Molec. Pathol., Univ. Hospital Gasthuisberg, Leuven, Belgium Several studies have documented the important association between HCV infection and hepatocellular carcinoma. The mechanisms involved are still unknown and could involve viral proteins. We investigated the effect of HCV core protein on DNA repair after UV-induced DNA damage. Therefore we developed and characterized stably transfected HepG2 cell lines that express HCV core protein (genotype 1b) or truncated HCV core as shown by immunohistochemistry. To study the DNA repair, cells were seeded at 5,000 cells/well in 96-well culture plates. The next day, the cells were UV radiated (14 J/cm2 ) and cultured for 3 additional days after which time an XTT-assay was performed. We calculated the reduction in cell number per well with or without UV-exposure for the different cell lines. Core transfected cells were significantly less capable to repair the DNA damage than control cells or cells expressing truncated core. Interestingly, expression of the full length HCV core did increase the cell doubling time in one of the cell lines. Therefore we investigated apoptosis and telomerase activity in these cells. Conclusion: This suppression of DNA repair by HCV core protein renders the cells more sensitive to acquire mutations that in combination with enhanced in vivo cell turnover in the infected liver might increase the likelihood of malignant transformation of HCV-infected cells by other viral factors or upon exposure to environmental factors (food, drugs, smoking, alcohol etc). Further studies are required to determine for which phase of the developmental process of HCV-induced HCC our cells can be used as model.
380 GENOTYPE INCLUSIVITY OF HBV DNA QUANTITATION WITH THE COBAS TAQMAN HBV TEST
J. Weiss, G. Song, Y. Alemayehu, S. Austin, S. Shah, H. Wu, B. Farrenkopf. Roche Molecular Systems, Pleasanton CA, USA The COBAS TaqMan Hepatitis B Virus Test For Use With The High Pure System was developed for the quantitation of HBV DNA from EDTAplasma and serum specimens. COBAS TaqMan technology provides a rapid method for real-time amplification and quantitation of nucleic acid
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from HBV. An internal quantitation standard is included in each assay to compensate for effects of inhibition and DNA recovery during sample preparation and amplification/detection. The COBAS TaqMan HBV Test has a 7-log dynamic range using a single aliquot of specimen without requiring dilutions and sensitivity of 6 IU/mL (∼40 copies/mL). Analysis of 65 genotyped HBV clinical specimens by the COBAS TaqMan HBV Test showed that genotypes A through G are detected. Genotype F was substantially underquantitated by the COBAS AMPLICOR HBV MONITOR Test, while other genotypes were detected similarly. Analysis of 57 HBsAg-positive specimens with the COBAS TaqMan HBV, COBAS AMPLICOR HBV MONITOR and AMPLICOR HBV MONITOR Tests showed a good correlation in HBV DNA titers between the three tests over the linear range of the COBAS TaqMan HBV Test, with the exception of four samples that were underquantitated by the COBAS AMPLICOR and AMPLICOR HBV MONITOR Tests. These samples were typed as genotype F. The limit of detection and linearity of the COBAS TaqMan HBV Test with genotypes A through G was equivalent. The COBAS TaqMan HBV Test detects genotypes A through G equivalently and has a large dynamic range and good sensitivity, and is well-suited to monitoring patients being treated with efficacious therapies for HBV.
the intrahepatic total HBV DNA and covalently closed circular (ccc) DNA in the tumorous and non-tumorous tissues of HCC patients. Methods: Paired liver biopsy samples from tumorous and non-tumorous tissues were obtained from 19 patients with HBV-related HCC. Total HBV DNA, cccDNA and human genomic DNA were measured by the Invader assay. Results: There was no significant difference in the median total HBV DNA levels between the tumorous and non-tumorous paired tissues (0.354 vs. 0.220 copies/cell respectively, P = 0.904). The tumorous tissues tended to have a higher median cccDNA level than their non-tumorous counterparts (0.350 vs. 0.160 copies/cell respectively, P = 0.053). The proportion of cccDNA over total HBV DNA in the tumorous tissues was significantly higher than that in the non-tumorous counterparts (median values: 100% vs. 27.6% respectively, P = 0.005). In 15 out of 19 (79%) HCC tissues, HBV DNA were 100% in the form of cccDNA. Conclusions: HBV DNA was present in both the tumor and non-tumor tissues in HCC patients. The proportion of cccDNA/total HBV DNA was significantly higher in the tumor tissue, with 79% of tumors having HBV DNA solely in the form of cccDNA. The role of HBV cccDNA in carcinogenesis remains to be elucidated. Whether the depletion of HBV cccDNA with nucleoside analogues can prevent the formation of HCC remains to be studied.
381 HEPATITIS DELTA VIRUS PROTEINS P24 AND P27 SUPPRESS HEPATITIS B VIRUS (HBV) REPLICATION BY TRANS REPRESSING HBV ENHANCERS 1 AND 2, AND BY ACTIVATING THE ALPHA IFN-INDUCIBLE MXA GENE 1
2
3
1
1
V. Williams , C. Peltekian , C. Sureau , P. Deny , E. Gordien . 1 Laboratoire De Bacteriologie – Virologie – Hygiene; EA 3406; Bobigny France, Laboratoire Associe Au Centre National De Reference Des Hepatites B Et C, Pour Lehepatite Delta, Bobigny, France; 2 INSERM U370, Hopital Necker, Paris, France; 3 INSERM U76; Institut National De Transfusion Sanguine (INTS, Paris, France Background: Epidemiological and in vitro studies have shown that hepatitis B virus (HBV) coinfection or superinfection with hepatitis delta virus (HDV) frequently leads to suppression of HBV replication. The mechanisms involved in this phenomenon are still unknown. Methods: As HDV encodes a phosphoprotein under two isoforms, p24 and p27, we reasoned that p24 and/or p27 could alter the activities of HBV enhancers 1 and 2 and/or Pre S-, S-, X- and C- HBV promoters. We previously demonstrated that MxA protein could inhibit HBV replication, and we questioned whether delta proteins could activate MxA gene. Vectors encoding the luciferase reporter gene under the control of HBV regulatory sequences, and of a minimal MxA promoter, were cotransfected with p24 or p27 encoding plasmids in HuH7 cell line. Results: Both p24 and p27 inhibit enhancer 1 and 2 activity by 60% to more than 80%. In contrast, p24 could selectively activate Pre S promoter by 3fold while p27 activated all HBV promoters by 2- to 4-fold. Moreover, p27 but not p24, was able to activate the MxA promoter by 3-fold, and could also increase the effect of alpha interferon (alpha IFN) on MxA promoter up to 20-fold. Conclusions: p24 and p27 HDV proteins might be responsible for modulation of the helper HBV replication at least by (i) directly trans-repressing HBV enhancers 1 and 2, and (ii) by indirectly trans-activating the MxA promoter and enhancing αIFN effects on MxA promoter. 382 QUANTITATION OF HBV COVALENTLY CLOSED CIRCULAR DNA (HBV CCC DNA) IN HCC PATIENTS
D.K.H. Wong 1 , M.F. Yuen 1 , R.T.P. Poon 2 , S.S.M. Sum 1 , C.L. Lai 1 . 1 Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, Hong Kong; 2 Department of Surgery, The University of Hong Kong, Queen Mary Hospital, Hong Kong, Hong Kong Objective: Hepatitis B virus (HBV) is a major causative agent of hepatocellular carcinoma (HCC). We aimed to use the Invader assay to measure
383 IN VITRO CHARACTERIZATION AND MOLECULAR MODELING ANALYSIS OF A NOVEL ADEFOVIR RESISTANCE MUTATION RTN236T IN THE HBV POLYMERASE
H. Yang 1 , X. Qi 1 , K. Das 2 , E. Arnold 2 , C.E. Westland 1 , W.E. Delaney IV 1 , C.L. Brosgart 1 , C.S. Gibbs 1 , M.D. Miller 1 , S. Xiong 1 . 1 Gilead Sciences, Foster City CA, USA; 2 Center For Advanced Biotechnology and Medicine, Dept of Chemistry, Rutgers University, Piscataway NJ, USA Background: The rtN236T mutation in the HBV reverse transcriptase (RT) confers in vitro and clinical resistance to adefovir. This mutation emerges in <2% of adefovir dipivoxil (ADV)-treated patients after 2 years of ADV therapy. We evaluated the cross-resistance profile and viral replication fitness of the rtN236T mutant in vitro and proposed a hypothesis for the mechanism of rtN236T induced resistance. Methods: In vitro drug susceptibility and viral replication fitness were determined by transient transfection of a patient-derived full-length HBV clone with the rtN236T mutation into HepG2 cells. Intracellular HBV replication signals were determined by Southern blotting. A computer model of HBV RT based on HIV-1 and murine leukemia virus RT structures was used to study the possible role of rtN236T. Results: The rtN236T mutant studied showed a 9.6-fold decrease in adefovir susceptibility in vitro. Susceptibilities to acyclic phosphonates tenofovir and MCC-478 were also reduced by 4.2- and 8.6-fold, respectively. The rtN236T mutant remained sensitive to lamivudine, L-dT and entecavir with IC50 changes <2.4-fold. Cross resistance to FTC and L-FMAU is being investigated. The rtN236T mutation resulted in >60% reduction in replication capacity. Modeled HBV RT structure suggests that the side chain of the mutated rtN236T may have a more favorable interaction with the gamma-phosphate of dATP compared to adefovir diphosphate thus providing for selectivity against adefovir diphosphate and other acyclic phosphonate analogs versus the natural substrate. Conclusion: The in vitro cross-resistance profile of the adefovir resistance mutation rtN236T shows full susceptibility to lamivudine and investigational agents entecavir and L-dT.
M. Martinot-Peignoux 1 , V. Esnault 2 , C. Kumas 1 , B.N. Pham 3 , P. Marcellin 2 . 1 INSERM U481 et Unité Claude Bernard Hôpital Beaujon, Clichy, France; 2 Service D’Hépatologie,Hôpital Beaujon, Clichy, France; 3 Service D’Hématologie-Immunologie Biologiques Hôpital Beaujon, Clichy, France The aim of our study was to assess the relevance of the with REALQUANT HBV-DNA assay for a routine laboratory. Methods: 180 serum samples tested with Cobas Amplicor HBV MONITOR, Roche (sensitivity 2x102 HBV DNA copies/ml) were quantified with REALQUANT HBV-DNA, (DiaTech, Italy) on the Rotor-Gene system (Corbett Research, Australia). REALQUANT allows the amplification of a short length of the gene coding for the Surface Antigen and utilizes a Taqman probe for the detection. Twenty samples HBsAg negative/antiHCV negative and 20 samples HBsAg negative/HCV RNA positive were tested for the specificity. Reproducibility was calculated with 3 serum samples tested 10 times. Sensitivity was determined with 76 samples HBV DNA undetectable with COBAS (30 patients under therapy and 46 untreated) and 35 samples HBV DNA detectable with COBAS. Results: All the samples HBsAg negative/anti-HCV negative were found negative; 3/20 samples HBsAg negative/HCV RNA positive were found positive.(7.4x103; 181; and 70 copies/ml) with REALQUANT . The 3 samples tested 10 times showed CV ranging from 13% to 24%. HBV DNA was detectable with REALQUANT in 58% and 63% of the samples from under therapy and untreated patients, respectively. Among the 35 samples COBAS positive 94% had HBV DNA detectable with RealQuant HBVDNA. The correlation between the two assays was good (r2 =0.695). The HBV DNA levels were higher with REALQUANT than with COBAS (ratio=9). Conclusion: REALQUANT HBV-DNA assay showed a good specificity 93%, a high sensitivity (mainly for patients under therapy) suggesting that monitoring patients with a sensitive assay could be clinically relevant.
378 VIRAL, HOST AND PATHOLOGICAL PROFILE OF WHV/HDV INFECTION IN WOODCHUCK MODEL
E. D’Ugo 1 , S. Orobello 1 , A. Canitano 1 , C. Argentini 1 , R. Giuseppetti 1 , G. Palmieri 2 , M. Rapicetta 1 . 1 Laboratory of Virology, Istituto Superiore Di Sanità, Rome, Italy; 2 Pathological Anatomy Chair, Tor Vergata University, Rome, Italy Introduction: HDV superinfection in WHV carrier woodchucks has shown similar biological and pathological features of HDV in humans.
113
Aim: Our aim was to characterise WHV/HDV superinfection with particular regard to viral determinants possibly involved in pathogenesis. Materials and Methods: Chronically WHV infected woodchucks were infected with HDV genotype 1. Serum and liver samples were monitored for virological and antibody markers (RT-PCR, dot-blot hybridisation, ELISA). Sequence and phylogenetic analyses of WHV Pre-S/S and HDAg were performed by Neighbour-Joining method. AST levels were determined and histological scoring performed according Ishak. Results: HDV-RNA and HDAg were detected in follow-up sera from all animals at 2 to 4 and 6 to 8 weeks post-infection. High HDV-RNA titers in livers and elevated AST values were present in infected animals. Phylogenetic analysis showed relatedness of WHV sequence to HBV genotype F. All HDV isolates from woodchucks clustered in a unique group separate from human isolates. Different amino acid usage, but conservation of the total positive charge of HDAg was found. Liver histological analysis performed on all animals showed normal liver tissue histology before infection, despite massive panacinar liver necrosis on autoptic tissues (Ishak score B6). A short survival was observed in all HDV infected animals that died before the 30th week after infection. Conclusions: A severe course of HDV infection has been observed in woodchucks possibly associated to the particular HDV and helper virus strains. A detailed molecular characterisation of HDV and helper virus is reported as well as a fine histological scoring of infected livers.
379 EXPRESSION OF HEPATITIS C VIRUS CORE PROTEIN IMPAIRS DNA-REPAIR IN HUMAN HEPATOMA CELLS
T. Severi 1 , T. Crabbe 1 , A. Van Eetveldt 1 , C. Verslype 1 , T. Roskams 2 , J. Fevery 1 , J.F. van Pelt 1 . 1 Labo Hepatology, 2 Dept Morph&Molec. Pathol., Univ. Hospital Gasthuisberg, Leuven, Belgium Several studies have documented the important association between HCV infection and hepatocellular carcinoma. The mechanisms involved are still unknown and could involve viral proteins. We investigated the effect of HCV core protein on DNA repair after UV-induced DNA damage. Therefore we developed and characterized stably transfected HepG2 cell lines that express HCV core protein (genotype 1b) or truncated HCV core as shown by immunohistochemistry. To study the DNA repair, cells were seeded at 5,000 cells/well in 96-well culture plates. The next day, the cells were UV radiated (14 J/cm2 ) and cultured for 3 additional days after which time an XTT-assay was performed. We calculated the reduction in cell number per well with or without UV-exposure for the different cell lines. Core transfected cells were significantly less capable to repair the DNA damage than control cells or cells expressing truncated core. Interestingly, expression of the full length HCV core did increase the cell doubling time in one of the cell lines. Therefore we investigated apoptosis and telomerase activity in these cells. Conclusion: This suppression of DNA repair by HCV core protein renders the cells more sensitive to acquire mutations that in combination with enhanced in vivo cell turnover in the infected liver might increase the likelihood of malignant transformation of HCV-infected cells by other viral factors or upon exposure to environmental factors (food, drugs, smoking, alcohol etc). Further studies are required to determine for which phase of the developmental process of HCV-induced HCC our cells can be used as model.
380 GENOTYPE INCLUSIVITY OF HBV DNA QUANTITATION WITH THE COBAS TAQMAN HBV TEST
J. Weiss, G. Song, Y. Alemayehu, S. Austin, S. Shah, H. Wu, B. Farrenkopf. Roche Molecular Systems, Pleasanton CA, USA The COBAS TaqMan Hepatitis B Virus Test For Use With The High Pure System was developed for the quantitation of HBV DNA from EDTAplasma and serum specimens. COBAS TaqMan technology provides a rapid method for real-time amplification and quantitation of nucleic acid
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from HBV. An internal quantitation standard is included in each assay to compensate for effects of inhibition and DNA recovery during sample preparation and amplification/detection. The COBAS TaqMan HBV Test has a 7-log dynamic range using a single aliquot of specimen without requiring dilutions and sensitivity of 6 IU/mL (∼40 copies/mL). Analysis of 65 genotyped HBV clinical specimens by the COBAS TaqMan HBV Test showed that genotypes A through G are detected. Genotype F was substantially underquantitated by the COBAS AMPLICOR HBV MONITOR Test, while other genotypes were detected similarly. Analysis of 57 HBsAg-positive specimens with the COBAS TaqMan HBV, COBAS AMPLICOR HBV MONITOR and AMPLICOR HBV MONITOR Tests showed a good correlation in HBV DNA titers between the three tests over the linear range of the COBAS TaqMan HBV Test, with the exception of four samples that were underquantitated by the COBAS AMPLICOR and AMPLICOR HBV MONITOR Tests. These samples were typed as genotype F. The limit of detection and linearity of the COBAS TaqMan HBV Test with genotypes A through G was equivalent. The COBAS TaqMan HBV Test detects genotypes A through G equivalently and has a large dynamic range and good sensitivity, and is well-suited to monitoring patients being treated with efficacious therapies for HBV.
the intrahepatic total HBV DNA and covalently closed circular (ccc) DNA in the tumorous and non-tumorous tissues of HCC patients. Methods: Paired liver biopsy samples from tumorous and non-tumorous tissues were obtained from 19 patients with HBV-related HCC. Total HBV DNA, cccDNA and human genomic DNA were measured by the Invader assay. Results: There was no significant difference in the median total HBV DNA levels between the tumorous and non-tumorous paired tissues (0.354 vs. 0.220 copies/cell respectively, P = 0.904). The tumorous tissues tended to have a higher median cccDNA level than their non-tumorous counterparts (0.350 vs. 0.160 copies/cell respectively, P = 0.053). The proportion of cccDNA over total HBV DNA in the tumorous tissues was significantly higher than that in the non-tumorous counterparts (median values: 100% vs. 27.6% respectively, P = 0.005). In 15 out of 19 (79%) HCC tissues, HBV DNA were 100% in the form of cccDNA. Conclusions: HBV DNA was present in both the tumor and non-tumor tissues in HCC patients. The proportion of cccDNA/total HBV DNA was significantly higher in the tumor tissue, with 79% of tumors having HBV DNA solely in the form of cccDNA. The role of HBV cccDNA in carcinogenesis remains to be elucidated. Whether the depletion of HBV cccDNA with nucleoside analogues can prevent the formation of HCC remains to be studied.
381 HEPATITIS DELTA VIRUS PROTEINS P24 AND P27 SUPPRESS HEPATITIS B VIRUS (HBV) REPLICATION BY TRANS REPRESSING HBV ENHANCERS 1 AND 2, AND BY ACTIVATING THE ALPHA IFN-INDUCIBLE MXA GENE 1
2
3
1
1
V. Williams , C. Peltekian , C. Sureau , P. Deny , E. Gordien . 1 Laboratoire De Bacteriologie – Virologie – Hygiene; EA 3406; Bobigny France, Laboratoire Associe Au Centre National De Reference Des Hepatites B Et C, Pour Lehepatite Delta, Bobigny, France; 2 INSERM U370, Hopital Necker, Paris, France; 3 INSERM U76; Institut National De Transfusion Sanguine (INTS, Paris, France Background: Epidemiological and in vitro studies have shown that hepatitis B virus (HBV) coinfection or superinfection with hepatitis delta virus (HDV) frequently leads to suppression of HBV replication. The mechanisms involved in this phenomenon are still unknown. Methods: As HDV encodes a phosphoprotein under two isoforms, p24 and p27, we reasoned that p24 and/or p27 could alter the activities of HBV enhancers 1 and 2 and/or Pre S-, S-, X- and C- HBV promoters. We previously demonstrated that MxA protein could inhibit HBV replication, and we questioned whether delta proteins could activate MxA gene. Vectors encoding the luciferase reporter gene under the control of HBV regulatory sequences, and of a minimal MxA promoter, were cotransfected with p24 or p27 encoding plasmids in HuH7 cell line. Results: Both p24 and p27 inhibit enhancer 1 and 2 activity by 60% to more than 80%. In contrast, p24 could selectively activate Pre S promoter by 3fold while p27 activated all HBV promoters by 2- to 4-fold. Moreover, p27 but not p24, was able to activate the MxA promoter by 3-fold, and could also increase the effect of alpha interferon (alpha IFN) on MxA promoter up to 20-fold. Conclusions: p24 and p27 HDV proteins might be responsible for modulation of the helper HBV replication at least by (i) directly trans-repressing HBV enhancers 1 and 2, and (ii) by indirectly trans-activating the MxA promoter and enhancing αIFN effects on MxA promoter. 382 QUANTITATION OF HBV COVALENTLY CLOSED CIRCULAR DNA (HBV CCC DNA) IN HCC PATIENTS
D.K.H. Wong 1 , M.F. Yuen 1 , R.T.P. Poon 2 , S.S.M. Sum 1 , C.L. Lai 1 . 1 Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, Hong Kong; 2 Department of Surgery, The University of Hong Kong, Queen Mary Hospital, Hong Kong, Hong Kong Objective: Hepatitis B virus (HBV) is a major causative agent of hepatocellular carcinoma (HCC). We aimed to use the Invader assay to measure
383 IN VITRO CHARACTERIZATION AND MOLECULAR MODELING ANALYSIS OF A NOVEL ADEFOVIR RESISTANCE MUTATION RTN236T IN THE HBV POLYMERASE
H. Yang 1 , X. Qi 1 , K. Das 2 , E. Arnold 2 , C.E. Westland 1 , W.E. Delaney IV 1 , C.L. Brosgart 1 , C.S. Gibbs 1 , M.D. Miller 1 , S. Xiong 1 . 1 Gilead Sciences, Foster City CA, USA; 2 Center For Advanced Biotechnology and Medicine, Dept of Chemistry, Rutgers University, Piscataway NJ, USA Background: The rtN236T mutation in the HBV reverse transcriptase (RT) confers in vitro and clinical resistance to adefovir. This mutation emerges in <2% of adefovir dipivoxil (ADV)-treated patients after 2 years of ADV therapy. We evaluated the cross-resistance profile and viral replication fitness of the rtN236T mutant in vitro and proposed a hypothesis for the mechanism of rtN236T induced resistance. Methods: In vitro drug susceptibility and viral replication fitness were determined by transient transfection of a patient-derived full-length HBV clone with the rtN236T mutation into HepG2 cells. Intracellular HBV replication signals were determined by Southern blotting. A computer model of HBV RT based on HIV-1 and murine leukemia virus RT structures was used to study the possible role of rtN236T. Results: The rtN236T mutant studied showed a 9.6-fold decrease in adefovir susceptibility in vitro. Susceptibilities to acyclic phosphonates tenofovir and MCC-478 were also reduced by 4.2- and 8.6-fold, respectively. The rtN236T mutant remained sensitive to lamivudine, L-dT and entecavir with IC50 changes <2.4-fold. Cross resistance to FTC and L-FMAU is being investigated. The rtN236T mutation resulted in >60% reduction in replication capacity. Modeled HBV RT structure suggests that the side chain of the mutated rtN236T may have a more favorable interaction with the gamma-phosphate of dATP compared to adefovir diphosphate thus providing for selectivity against adefovir diphosphate and other acyclic phosphonate analogs versus the natural substrate. Conclusion: The in vitro cross-resistance profile of the adefovir resistance mutation rtN236T shows full susceptibility to lamivudine and investigational agents entecavir and L-dT.