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ObjectiveHepatitis delta virus (HDV) was shown to persist for weeks in the absence of HBV and for months after liver transplantation, demonstrating the ability of HDV to persevere in quiescent hepatocytes. The aim of the study was to evaluate the impact of cell proliferation on HDV persistence in vitro and in vivo.DesignGenetically labelled human sodium taurocholate cotransporting polypeptide (hNTCP)-transduced human hepatoma(HepG2) cells were infected with HBV/HDV and passaged every 7 days for 100 days in the presence of the entry inhibitor Myrcludex-B. In vivo, cell proliferation was triggered by transplanting primary human hepatocytes (PHHs) isolated from HBV/HDV-infected humanised mice into nai¨ve recipients. Virological parameters were measured by quantitative real time polymerase chain reaction (qRT-PCR). Hepatitis delta antigen (HDAg), hepatitis B core antigen (HBcAg) and cell proliferation were determined by immunofluorescence.ResultsDespite 15 in vitro cell passages and block of viral spreading by Myrcludex-B, clonal cell expansion permitted amplification of HDV infection. In vivo, expansion of PHHs isolated from HBV/HDV-infected humanised mice was confirmed 3 days, 2, 4 and 8 weeks after transplantation. While HBV markers rapidly dropped in proliferating PHHs, HDAg-positive hepatocytes were observed among dividing cells at all time points. Notably, HDAg-positive cells appeared in clusters, indicating that HDV was transmitted to daughter cells during liver regeneration even in the absence of de novo infection.ConclusionThis study demonstrates that HDV persists during liver regeneration by transmitting HDV RNA to dividing cells even in the absence of HBV coinfection. The strong persistence capacities of HDV may also explain why HDV clearance is difficult to achieve in HBV/HDV chronically infected patients.

ObjectiveThe stability of the covalently closed circular DNA (cccDNA) in nuclei of non-dividing hepatocytes represents a key determinant of HBV persistence. Contrarily, studies with animal hepadnaviruses indicated that hepatocyte turnover can reduce cccDNA loads but knowledge on the proliferative capacity of HBV-infected primary human hepatocytes (PHHs) in vivo and the fate of cccDNA in dividing PHHs is still lacking. This study aimed to determine the impact of human hepatocyte division on cccDNA stability in vivo.MethodsPHH proliferation was triggered by serially transplanting hepatocytes from HBV-infected humanised mice into naive recipients. Cell proliferation and virological changes were assessed by quantitative PCR, immunofluorescence and RNA in situ hybridisation. Viral integrations were analysed by gel separation and deep sequencing.ResultsPHH proliferation strongly reduced all infection markers, including cccDNA (median 2.4 log/PHH). Remarkably, cell division appeared to cause cccDNA dilution among daughter cells and intrahepatic cccDNA loss. Nevertheless, HBV survived in sporadic non-proliferating human hepatocytes, so that virological markers rebounded as hepatocyte expansion relented. This was due to reinfection of quiescent PHHs since treatment with the entry inhibitor myrcludex-B or nucleoside analogues blocked viral spread and intrahepatic cccDNA accumulation. Viral integrations were detected both in donors and recipient mice but did not appear to contribute to antigen production.ConclusionsWe demonstrate that human hepatocyte division even without involvement of cytolytic mechanisms triggers substantial cccDNA loss. This process may be fundamental to resolve self-limiting acute infection and should be considered in future therapeutic interventions along with entry inhibition strategies.

The mechanisms determining hepatitis B virus (HBV) persistence and pathogenesis are not fully elucidated, but appear to be multi-factorial. Current medication to repress viral replication is available; however, the unique replication strategies employed by HBV enable the virus to persist within the infected hepatocytes. Consequently, cure is rarely achieved. Progresses in HBV research and preclinical testing of antiviral agents have been limited by the narrow species- and tissue-tropism of the virus, the paucity of infection models available and the restrictions imposed by the use of chimpanzees, the only animals fully susceptible to HBV infection. Mice are not HBV permissive but major efforts have focused on the development of mouse models of HBV replication and infection, such as the generation of humanized mice. By presenting the different animal models available, this review will highlight the most important and clinically relevant findings that have been retrieved from the respective systems.

Background Suppression of viral replication with nucleoside/nucleotide inhibitors has been shown to greatly improve the outcome of chronic HBV infection. (ß-l-nucleoside analogues, especially ß-l-deoxycytidine derivatives represent one of the most efficient groups of antiretroviral compounds. We recently described that hydroxylation of the amino group of these ß-l-deoxycytidine derivatives preserved their strong HBV inhibitory activity in vitro, but strongly reduced their cytotoxicity. From this new group of compounds we selected ß-l-2',3'-didehydro-2',3'-dideoxy-N4-hydroxy-5-fluorocytidine (l-Hyd4FC) for a first in vivoinvestigation. The aim of this study was to determine the antiviral activity of l-Hyd4FC in HBV-infected human liver chimeric urokinase plasminogen activator (uPA)/SCID mice.Methods Stably infected animals (median 6x107HBV DNA/ ml) were injected daily with either l-Hyd4FC (50 mg/kg) or saline as controls. Mice treated with lamivudine served to compare the in vivoantiviral potency of l-Hyd4FC. Virological changes were determined by quantitative PCR.Results Treatment with l-Hyd4FC for 4 weeks induced a 2-log reduction of viraemia, while a median 1.5-log decline was achieved with lamivudine. Intrahepatically, l-Hyd4FC induced a median eightfold decline of viral activity (relaxed circular DNA/covalently closed circular DNA), and threefold reduction of pregenomic RNA/GAPDH levels. No significant decline of subgenomic HBV transcripts, as well as of circulating hepatitis B e antigen and hepatitis B surface antigen was detected. Maintenance of human serum albumin concentrations throughout the study, negative TUNEL staining and occurrence of viral rebound after drug withdrawal indicated that l-Hyd4FC was not toxic in human hepatocytes.Conclusions Administration of l-Hyd4FC in uPA/SCID mice harbouring HBV-infected human hepatocytes demonstrated the high antiviral potency of this drug in vivo. Such characteristics make l-Hyd4FC a good candidate for further investigations a as potential HBV therapeutic agent.

Background We previously reported that 48 weeks of combination therapy with pegylated interferon-a2b (PEG-IFN-a2b) and adefovir dipivoxil (ADV) in patients with chronic hepatitis B led to marked decreases of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) (-2.4 log10copies/ml). Combination therapy was followed by 96 weeks of ADV monotherapy.Methods Here, we report on the final outcome after 144 weeks of sequential antiviral treatment. Twenty-four patients were analysed, triplet liver biopsies (taken at baseline, week 48 and week 144) were available from 16 patients.Results At week 144, 12/15 patients positive for hepatitis B virus e antigen (HBeAg) had lost HBeAg, alanine transaminase (ALT) levels were normal in 23 patients (96%), median serum HBV DNA had decreased by -4.9 log10copies/ml and was undetectable (<100 copies/ml) in 11/24 individuals (46%). Median total intrahepatic HBV DNA had decreased by -2.2 log. Although no further significant cccDNA changes occurred between week 48 and week 144, two years of ADV monotherapy proved capable of controlling cccDNA levels in most patients. Analysis of intrahepatic HBV DNA species demonstrated that combination therapy with PEG-IFN-a2b and ADV inhibited viral productivity by 99% and subsequent ADV monotherapy by 76%, respectively. Virus suppression to undetectability within the first 12 weeks of treatment was strongly associated with long-term virological response and HBeAg and hepatitis B virus surface antigen HBsAg seroconversion. Histological improvement was determined in 11/16 patients at week 144. Two patients developed ADV resistance during the third year of treatment.Conclusions Reduction of intrahepatic viral load achieved after 48 weeks of combination therapy with PEG-IFN-a2b and ADV was maintained in the following 96 weeks of ADV monotherapy and translated into long-term clinical benefit for most of the treated patients.

Liver neo-tissue suitable for transplantation has not been established. Primary rat hepatocytes were cultured on three-dimensional biodegradable polymer matrices in a pulsatile flow bioreactor with the intention of inducing tissue formation and improving cell survival. Functional and structural analysis of the hepatocytes forming liver neo-tissue was performed. Biodegradable poly(L-lactic acid) (PLLA) polymer discs were seeded with 4×106 primary rat hepatocytes each, were exposed to a pulsatile medium flow of 24 mL/min for 1, 2, 4, or 6 days and were investigated for monoethylglycinexylidine (MEGX) formation, ammonia detoxification, Cytokeratin 18 (CK18) expression, and preserved glycogen storage. Fine structural details were obtained using scanning and transmission electron microscopy. Spheroids of viable hepatocytes were formed. MEGX-specific production was maintained and ammonia removal capacity remained high during the entire flow-culture period of 6 days. CK18 distribution was normal. Periodicacid- Schiff reaction demonstrated homogenous glycogen storage. The hepatocytes reassembled to form intercellular junctions and bile canaliculi. Functional and morphological analysis of rat hepatocytes forming spheroids in a pulsatile flow bioreactor indicated preserved and intact hepatocyte morphology and specific function. Pulsatile flow culture on PLLA scaffolds is a promising new method of hepatic tissue engineering leading to liver neo-tissue formation.

ABSTRACTIn order to identify potential sites of hepadnavirus X protein action, we have investigated the subcellular distribution and the stability of woodchuck hepatitis virus (WHV) X protein (WHx) in primary hepatocytes isolated from woodchucks with persistent WHV infection. In vivo cell labeling and cell fractionation studies showed that the majority of WHx is a soluble cytoplasmic protein while a minor part of newly synthesized WHx is associated with a nuclear framework fraction (20%) and with cytoskeletal components (5 to 10%). Pulse-chase experiments revealed that cytoplasmic WHx has a short half-life and decays with bimodal kinetics (approximately 20 min and 3 h). The rates of association and turnover of nucleus-associated WHx suggest that compartmentalization may be responsible for the bimodal turnover observed in the cytoplasm.