- Email: [email protected]
HDV co-infected mice
POSTERS cccDNA in induced AD38 cells. Importantly, when CaMPs treatment was started during infection, cccDNA formation/accumulation was completely inhibited (>95%) and viral replication was blunted. Conclusions: Anti-capsid compounds (CpAMs) have an impact on Cp nuclear functions at multiple levels: block of new cccDNA formation / accumulation, reduction of an established cccDNA pool and inhibition of HBc occupancy and histone acetylation on the cccDNA that translate into a reduced pgRNA transcription. P0530 MYRCLUDEX-B INHIBITS ESTABLISHMENT OF HDV SUPER-INFECTION IN HBV INFECTED MICE AND REDUCES HDV VIREMIA IN STABLY HBV/HDV CO-INFECTED MICE T. Volz1 , K. Giersch1 , L. Allweiss1 , O.D. Bhadra1 , J. Petersen2 , 1,4 A.W. Lohse1,3 , M. Lutgehetmann ¨ , S. Urban5,6 , M. Dandri1,3 . 1 I. Department of Internal Medicine, University Medical Center Hamburg Eppendorf, 2 IFI Institute for Interdisciplinary Medicine, Asklepios Clinic St. Georg, 3 Hamburg and Heidelberg Sites, German Center for Infection Research, 4 Institute of Microbiology, Virology and Hygiene, University Medical Center Hamburg Eppendorf, Hamburg, 5 Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, 6 Hamburg and Heidelberg Sites, German Center for Infection Research, Heidelberg, Germany E-mail: [email protected] Background and Aims: 15 million people are infected with the hepatitis Delta virus (HDV) worldwide. Therapeutic strategies specifically targeting HDV infection are not available but urgently needed. We previously demonstrated prevention of de novo HBV/HDV co-infection using the entry inhibitor Myrcludex-B in naïve human liver-chimeric uPA/SCID mice (Lutgehetmann, ¨ Hepatology 2012). Aim of the study was to assess (I) whether treatment with Myrcludex-B can hinder infection establishment and spreading of HDV also in humanized mice already infected with HBV, and (II) whether long-term entry inhibition can affect viremia levels in stably HBV/HDV co-infected mice. Methods: Humanized uPA/SCID mice were first stably infected with HBV, then super-infected with HDV and treated with Myrcludex-B (2 mg/kg; daily) 2 days before until 5 weeks after HDV inoculation (I), while stably HBV/HDV co-infected mice received Myrcludex-B daily for 9 weeks (II). Viral loads were quantified in serum and liver by qRT-PCR and visualized by immunohistochemistry. Results: (I) In HBV-infected mice, which received Myrcludex-B during the first 5 weeks of HDV super-infection, HDV viremia, intrahepatic HDV RNA and HDAg remained below detection levels. However, one mouse showed development of HDV viremia 3 weeks after treatment cessation, demonstrating that Myrcludex-B strongly hindered but did not completely abrogate establishment of HDV infection in humanized mice. (II) In stably HBV/HDV co-infected mice, 9 weeks of Myrcludex-B treatment induced 1-log reduction of HDV, while HBV viremia was similar in untreated and treated mice. However, no significant intrahepatic viral changes could be determined by comparing treated and untreated animals. The ratio of HDV RNA quasi-species encoding for the small and large HDAg was also maintained after 9 weeks of Myrcludex-B administration. Conclusions: Myrcludex-B significantly inhibited HDV infection establishment also in the presence of a productive HBV infection. However, continuous drug administration was necessary to prevent HDV spreading from the very few initially infected human hepatocytes. Long-term treatment of HBV/HDV co-infected mice with the entry inhibitor was needed to detect HDV viremia decrease. The high infection efficiency and great survival capacities of HDV shown in this study highlight the difficulties encountered in treating HBV/HDV co-infected patients.
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P0531 TRANSGENIC MICE CARRYING HEPATITIS B VIRUS PRE-S/S GENE CONTAINING THE rtA181T/sW172* MUTATION DEVELOP HEPATOCELLULAR CARCINOMA C.-T. Yeh1 , M.-W. Lai1 , K.-H. Liang1 , Y.-H. Huang1 . 1 Liver Research Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan E-mail: [email protected] Background and Aims: Hepatitis B virus carrying the rtA181T/sW172* mutation conferred cross-resistance to adefovir and lamivudine. Cell-based and epidemiological studies indicated that hepatitis B virus pre-S/S proteins carrying this mutation had an increased oncogenic potential. Here we created transgenic mice models to study this issue. Methods: Transgenic mice were generated by transfer of hepatitis B virus pre-S/S gene (genotype A) together with its own promoter into C57B6 mice. Results: Four lines of transgenic mice were created. Two of them carried wild type pre-S/S gene and produced high and low levels of HBsAg (TgWT-H and L). The other two carried mutant type pre-S/S gene containing the rtA181T/sW172* mutation (TgSW172-H and L). Western blot/IHC analysis detected high and low levels of intrahepatic truncated pre-S/S proteins respectively in TgSW172-H and L mice. The former had detectable low level of HBsAg in the serum, while the latter was negative for serum HBsAg (secretion failure). All mice were sacrificed 18 months after birth. None of the TgWT mice developed hepatocellular carcinoma (HCC), whereas 6/26 (23.1%) TgSW172-H and 2/24 (8.3%) TgSW172-L mice developed HCC. Molecular analysis of liver tissues revealed significantly increased expression of GPR78 and phosphorylated ERK1 in TgSW172 mice versus TgWT mice; and decreased expression of Bcl-XL in TgSW172-H mice versus TgSW172-L and TgWT mice. Higher proportion of apoptotic cells assessed by TUNEL assay was found in TgSW172-H than in TgWT mice, but both showed increased cyclin E levels, suggesting increased hepatocyte turnover and regeneration. Combined analysis of cDNA microarray and microRNA array showed reduced expression of the CUB and Sushi multiple domains 3 (CSMD3) protein, a putative tumor suppressor, in TgSW172 but not TgWT mice. Conclusions: Transgenic mice experiments confirmed that the pre-S/S gene carrying rtA181T/sW172* mutation had an increased oncogenic potential in development of liver cancer. Increased ER stress with more rapid hepatocyte turnover as well as decreased expression of CSMD3 contributed in part to hepatocarginogenesis. P0532 HBx–DLEU2 lncRNA COMPLEX AFFECTS TRANSCRIPTION OF NEW TARGET PROMOTERS F. Guerrieri1 , L. Chiodo2 , S. Jeddari3 , D. D’Andrea4 , G. Ruocco1 , A. Tramontano4 , M. Levrero3 . 1 CLNS@SAPIENZA, Istituto Italiano di Tecnologia (IIT), Rome, 2 CLNS@SAPIENZA, Istituto Italiano di Tecnologia (IIT), IIT, 3 Dept of Internal Medicine, Sapienza University, 4 Biocomputing Lab, Department of Physics, Sapienza University of Rome, Rome, Italy E-mail: [email protected] Background and Aims: HBx affects HBV mini-chromosome transcription, by preventing HDACs and PMRT1 recruitment on the cccDNA, and cellular genes expression, by favoring the recruitment of positive and negative chromatin modifying enzymes. A ChIPSeq analysis of HBx genome wide recruitment identified 39 long non coding RNAs (lncRNAs), including DLEU2, as direct HBx transcriptional targets in HBV replicating cells. DLEU2 lncRNA overlaps the first exon of the TRIM13 gene in the opposite orientation and the pri-Mir of the miR-15a/16–1 cluster. Up-regulation of specific DLEU2 splicing variants correlates with tumors. TRIM13 induces autophagy and increase ectopic levels of p53.
Journal of Hepatology 2015 vol. 62 | S263–S864
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P0531 TRANSGENIC MICE CARRYING HEPATITIS B VIRUS PRE-S/S GENE CONTAINING THE rtA181T/sW172* MUTATION DEVELOP HEPATOCELLULAR CARCINOMA C.-T. Yeh1 , M.-W. Lai1 , K.-H. Liang1 , Y.-H. Huang1 . 1 Liver Research Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan E-mail: [email protected] Background and Aims: Hepatitis B virus carrying the rtA181T/sW172* mutation conferred cross-resistance to adefovir and lamivudine. Cell-based and epidemiological studies indicated that hepatitis B virus pre-S/S proteins carrying this mutation had an increased oncogenic potential. Here we created transgenic mice models to study this issue. Methods: Transgenic mice were generated by transfer of hepatitis B virus pre-S/S gene (genotype A) together with its own promoter into C57B6 mice. Results: Four lines of transgenic mice were created. Two of them carried wild type pre-S/S gene and produced high and low levels of HBsAg (TgWT-H and L). The other two carried mutant type pre-S/S gene containing the rtA181T/sW172* mutation (TgSW172-H and L). Western blot/IHC analysis detected high and low levels of intrahepatic truncated pre-S/S proteins respectively in TgSW172-H and L mice. The former had detectable low level of HBsAg in the serum, while the latter was negative for serum HBsAg (secretion failure). All mice were sacrificed 18 months after birth. None of the TgWT mice developed hepatocellular carcinoma (HCC), whereas 6/26 (23.1%) TgSW172-H and 2/24 (8.3%) TgSW172-L mice developed HCC. Molecular analysis of liver tissues revealed significantly increased expression of GPR78 and phosphorylated ERK1 in TgSW172 mice versus TgWT mice; and decreased expression of Bcl-XL in TgSW172-H mice versus TgSW172-L and TgWT mice. Higher proportion of apoptotic cells assessed by TUNEL assay was found in TgSW172-H than in TgWT mice, but both showed increased cyclin E levels, suggesting increased hepatocyte turnover and regeneration. Combined analysis of cDNA microarray and microRNA array showed reduced expression of the CUB and Sushi multiple domains 3 (CSMD3) protein, a putative tumor suppressor, in TgSW172 but not TgWT mice. Conclusions: Transgenic mice experiments confirmed that the pre-S/S gene carrying rtA181T/sW172* mutation had an increased oncogenic potential in development of liver cancer. Increased ER stress with more rapid hepatocyte turnover as well as decreased expression of CSMD3 contributed in part to hepatocarginogenesis. P0532 HBx–DLEU2 lncRNA COMPLEX AFFECTS TRANSCRIPTION OF NEW TARGET PROMOTERS F. Guerrieri1 , L. Chiodo2 , S. Jeddari3 , D. D’Andrea4 , G. Ruocco1 , A. Tramontano4 , M. Levrero3 . 1 CLNS@SAPIENZA, Istituto Italiano di Tecnologia (IIT), Rome, 2 CLNS@SAPIENZA, Istituto Italiano di Tecnologia (IIT), IIT, 3 Dept of Internal Medicine, Sapienza University, 4 Biocomputing Lab, Department of Physics, Sapienza University of Rome, Rome, Italy E-mail: [email protected] Background and Aims: HBx affects HBV mini-chromosome transcription, by preventing HDACs and PMRT1 recruitment on the cccDNA, and cellular genes expression, by favoring the recruitment of positive and negative chromatin modifying enzymes. A ChIPSeq analysis of HBx genome wide recruitment identified 39 long non coding RNAs (lncRNAs), including DLEU2, as direct HBx transcriptional targets in HBV replicating cells. DLEU2 lncRNA overlaps the first exon of the TRIM13 gene in the opposite orientation and the pri-Mir of the miR-15a/16–1 cluster. Up-regulation of specific DLEU2 splicing variants correlates with tumors. TRIM13 induces autophagy and increase ectopic levels of p53.
Journal of Hepatology 2015 vol. 62 | S263–S864