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Immune modulation for the treatment of chronic hepatitis B (CHB) has gained more traction in recent years, with an increasing number of compounds designed for targeting different host pattern recognition receptors (PRRs). These agonistic molecules activate the receptor signaling pathway and trigger an innate immune response that will eventually shape the adaptive immunity for control of chronic infection with hepatitis B virus (HBV). While definitive recognition of HBV nucleic acids by PRRs during viral infection still needs to be elucidated, several viral RNA sensing receptors, including toll-like receptors 7/8/9 and retinoic acid inducible gene-I-like receptors, are explored preclinically and clinically as possible anti-HBV targets. The antiviral potential of viral DNA sensing receptors is less investigated. In the present study, treatment of primary woodchuck hepatocytes generated from animals with CHB with HSV-60 or poly(dA:dT) agonists resulted in increased expression of interferon-gamma inducible protein 16 (IFI16) or Z-DNA-binding protein 1 (ZBP1/DAI) and absent in melanoma 2 (AIM2) receptors and their respective adaptor molecules and effector cytokines. Cytosolic DNA sensing receptor pathway activation correlated with a decline in woodchuck hepatitis virus (WHV) replication and secretion in these cells. Combination treatment with HSV-60 and poly(dA:dT) achieved a superior antiviral effect over monotreatment with either agonist that was associated with an increased expression of effector cytokines. The antiviral effect, however, could not be enhanced further by providing additional type-I interferons (IFNs) exogenously, indicating a saturated level of effector cytokines produced by these receptors following agonism. In WHV-uninfected woodchucks, a single poly(dA:dT) dose administered via liver-targeted delivery was well-tolerated and induced the intrahepatic expression of ZBP1/DAI and AIM2 receptors and their effector cytokines, IFN-β and interleukins 1β and 18. Receptor agonism also resulted in increased IFN-γ secretion of peripheral blood cells. Altogether, the effect on WHV replication and secretion following in vitro activation of IFI16, ZBP1/DAI, and AIM2 receptor pathways suggested an antiviral benefit of targeting more than one cytosolic DNA receptor. In addition, the in vivo activation of ZBP1/DAI and AIM2 receptor pathways in liver indicated the feasibility of the agonist delivery approach for future evaluation of therapeutic efficacy against HBV in woodchucks with CHB., Competing Interests: SM serves occasionally as a paid scientific consultant to Northeastern Wildlife, Inc. (Harris, ID, USA), the only commercial source for woodchucks within the United States, from which the animals of the current study were purchased. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Suresh, Li, Huang, Korolowicz, Murreddu, Gudima and Menne.)

Mechanism of initial hepatitis B virus (HBV) integrations and kinetics of DNA repair immediately after infection remain essentially unknown impairing understanding of hepadnaviral oncogenesis. WCM260 hepatocytes susceptible to HBV-compatible woodchuck hepatitis virus (WHV) were examined from 15 min to 72 h post-infection (p.i.). WHV strongly induced reactive oxygen species (ROS), transiently inducible nitric oxide (iNOS) and DNA damage from 15 min p.i. All initial WHV-host fusions had the head-to-tail format indicating their formation by non-homologous end joining (NHEJ). Transcription of poly(ADP-ribose) polymerase 1 (PARP1) and X-ray repair cross-complementing protein 1 (XRCC1), the PARP1 binding partner, were induced in 30 min p.i. and that of 8-oxyguanine DNA glycosylse (OGG1) responding to oxidative DNA damage at 12 h p.i. Nicotinamide adenine dinucleotide (NAD + ), a marker of PARP1 activation, and heme oxygenase-1 (HO1), an indicator of pro-oxidative stress, were significantly augmented from 15-30 min p.i. Additionally, PARP1 cleavage activity was evident from 30 min p.i. confirming that PARP1-mediated DNA repair became operational almost instatly after hepatocyte contact with virus. By applying complementary approaches, the study showed that initial WHV integration was due to virus-induced oxidative DNA damage and implied that the NHEJ PARP1-dependent repair pathway determined format of the first virus-host DNA junctions., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest., (Copyright © 2020. Published by Elsevier Inc.)

Hepadnaviruses are hepatotropic enveloped DNA viruses with an icosahedral capsid. Hepatitis B virus (HBV) causes chronic infection in an estimated 240 million people; woodchuck hepatitis virus (WHV), an HBV homologue, has been an important model system for drug development. The dimeric capsid protein (Cp) has multiple functions during the viral life cycle and thus has become an important target for a new generation of antivirals. Purified HBV and WHV Cp spontaneously assemble into 120-dimer capsids. Though they have 65% identity, WHV Cp has error-prone assembly with stronger protein-protein association. We have taken advantage of the differences in assemblies to investigate the basis of assembly regulation. We determined the structures of the WHV capsid to 4.5-Å resolution by cryo-electron microscopy (cryo-EM) and of the WHV Cp dimer to 2.9-Å resolution by crystallography and examined the biophysical properties of the dimer. We found, in dimer, that the subdomain that makes protein-protein interactions is partially disordered and rotated 21° from its position in capsid. This subdomain is susceptible to proteolysis, consistent with local disorder. WHV assembly shows similar susceptibility to HBV antiviral molecules, suggesting that HBV assembly follows similar transitions. These data show that there is an entropic cost for assembly that is compensated for by the energetic gain of burying hydrophobic interprotein contacts. We propose a series of stages in assembly that incorporate a disorder-to-order transition and structural shifts. We suggest that a cascade of structural changes may be a common mechanism for regulating high-fidelity capsid assembly in HBV and other viruses. IMPORTANCE Virus capsids assemble spontaneously with surprisingly high fidelity. This requires strict geometry and a narrow range of association energies for these protein-protein interactions. It was hypothesized that requiring subunits to undergo a conformational change to become assembly active could regulate assembly by creating an energetic barrier and attenuating association. We found that woodchuck hepatitis virus capsid protein undergoes structural transitions between its dimeric and its 120-dimer capsid states. It is likely that the closely related hepatitis B virus capsid protein undergoes similar structural changes, which has implications for drug design. Regulation of assembly by structural transition may be a common mechanism for many viruses., (Copyright © 2019 American Society for Microbiology.)

Mechanisms mediating clearance of hepatitis delta virus (HDV) are poorly understood. This study analyzed in detail profound down-regulation of HDV infection in the woodchuck model. Super-infection with HDV of woodchucks chronically infected with HBV-related woodchuck hepatitis virus produced two patterns. In the first, HDV viremia had a sharp peak followed by a considerable decline, and initial rise of HDV virions' infectivity followed by abrupt infectivity loss. In the second, HDV titer rose and later displayed plateau-like profile with high HDV levels; and HDV infectivity became persistently high when HDV titer reached the plateau. The infectivity loss was not due to defects in the virions' envelope, binding to anti-envelope antibodies, or mutations in HDV genome, but it correlated with profound reduction of the replication capacity of virion-associated HDV genomes. Subsequent finding that in virions with reduced infectivity most HDV RNAs were not full-length genomes suggests possible HDV clearance via RNA fragmentation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Chinese woodchucks (M. himalayana) were recently found to be susceptible to woodchuck hepatitis virus (WHV) infection. In this study, we aimed to determine the susceptibility to WHV infection of M. himalayana from different areas and their association with the animal genetic background exemplified by cytochrome B and MHC-DRB molecules., Methods: Animals from four different areas in Qinghai province were inoculated with WHV59 strains. The virological markers including WHV surface antigen (WHsAg), WHV core antibody (WHcAb), and WHV DNA in serum were measured by ELISA and Real-time PCR, respectively. The sequences of cytochrome B gene and MHC-DRB molecules were obtained and sorted with Clustalx software. The nucleotide variation sites were identified using MEGA5 software., Results: The animals from four different areas had different susceptibility to WHV infection. Animals from TR and TD areas had a high level of long-lasting viremia, while those from GD and WL areas had a low level of transient viremia after WHV inoculation. All of the animals belong to the same subspecies M. himalayana robusta identified by cytochrome B gene sequences. Based on their nucleotide variation pattern, 8 alleles of cytochrome B gene were identified, and 7 MHC-DRB alleles were identified. Allele A of cytochrome B and Allele Mamo-DRB1*02 of MHC-DRB was found to be frequent in animals from TR and TD areas, while Allele H of cytochrome B and Allele Mamo-DRB1*07 of MHC-DRB was predominant in animals from GD and WL areas., Conclusion: Chinese woodchucks from different areas differed in their susceptibility to WHV infection, though they belong to the same subspecies M. himalayana robusta. The genetic background exemplified by cytochrome B and MHC-DRB differed in Chinese woodchucks with different susceptibility to WHV infection.

Woodchuck hepatitis virus (WHV) is prone to aberrant assembly in vitro and can form a broad distribution of oversized particles. Characterizing aberrant assembly products is challenging because they are both large and heterogeneous. In this work, charge detection mass spectrometry (CDMS) is used to measure the distribution of WHV assembly products. CDMS is a single-particle technique where the masses of individual ions are determined from simultaneous measurement of each ion's charge and m/z (mass-to-charge) ratio. Under relatively aggressive, assembly promoting conditions, roughly half of the WHV assembly products are T=4 capsids composed of exactly 120 dimers while the other half are a broad distribution of larger species that extends to beyond 210 dimers. There are prominent peaks at around 132 dimers and at 150 dimers. In part, the 150 dimer complex can be attributed to elongating a T=4 capsid along its 5-fold axis by adding a ring of hexamers. However, most of the other features cannot be explained by existing models for hexameric defects. Cryo-electron microscopy provides evidence of elongated capsids. However, image analysis reveals that many of them are not closed but have "spiral-like" morphologies. The CDMS data indicate that oversized capsids have a preference for growth by addition of 3 or 4 dimers, probably by completion of hexameric vertices., (Copyright © 2015. Published by Elsevier Ltd.)

Woodchuck hepatitis virus (WHV) is often used as surrogate to study mechanism of HBV infection. Currently, most infections are conducted using strains WHV7 or WHV8 that have very high sequence identity. This study focused on natural strain WHVNY that is more genetically distant from WHV7. Three naive adult woodchucks inoculated with WHVNY developed productive acute infection with long lasting viremia. However, only one of two woodchucks infected with WHV7 at the same multiplicity demonstrated productive liver infection. Quantification of intracellular WHV RNA and DNA replication intermediates; percentages of core antigen-positive hepatocytes; and serum relaxed circular DNA showed that strains WHVNY and WHV7 displayed comparable replication levels and capacities to induce acute infection in naive adult woodchucks. Strain WHVNY was therefore validated as valuable reagent to analyze the mechanism of hepadnavirus infection, especially in co- and super-infection settings, which required discrimination between two related virus genomes replicating in the same liver., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Marmota monax and its natural infection by woodchuck hepatitis virus (WHV) could be used as a predictive model for evaluating mechanisms of viral persistence during chronic hepatitis B virus (HBV) infection. The aim of this study was to investigate the presence of viral variants in the core gene of chronically WHV-infected woodchucks that showed two different patterns of peripheral blood mononuclear cells' (PBMCs') responses after stimulation with a specific WHV core peptide. Sequences' analysis of the WHV core region from eight WHV chronically infected woodchucks have been performed after in vitro stimulation with an immunodominant epitope of the WHV core protein (amino acids [aa] 96-110). Following this stimulation, positive PBMC responses at each point of follow-up were observed for four animals (group A), and weak immune responses at one or a few points of follow-up were observed for the remaining four animals (group B). The WHV core gene sequences contained amino acid deletions (aa 84-126, aa 84-113) in three of four group A animals and in none of group B animals. In the group A animals, the same deletions were observed in liver specimens and in two of four tumor specimens. Hepatocellular carcinoma (HCC) was diagnosed in all group A animals and in one group B animal. In conclusion, internal deletions in the core region correlated with a sustained PBMC response to the immunogenic peptide (96-110) of the core protein. A possible role of this relationship in hepatocarcinogenesis could be hypothesized; however, this needs to be investigated in patients with chronic HBV infection. The evaluation of virus-specific T-cell responses and T-cell epitopes that are possibly related to the mechanisms of viral evasion should be further investigated in order to design combined antiviral and immune approaches to control chronic HBV infection.

The major histocompatibility complex (MHC)-containing genes are among the most polymorphic in vertebrates. MHC genes code for proteins that are critical in the immune system response. In this study, the polymorphism of the second exon of the MHC class II DRB gene was characterized in the Eastern woodchuck (Marmota monax). Woodchucks chronically infected with the woodchuck hepatitis virus (WHV) represent the best available animal model for the study of chronic hepatitis B infection in humans. In the genotyped animals we found fifteen alleles, which were expressed in two independent loci and that were named DRB1A and DRB1B in this work. The 15 alleles investigated showed an elevated divergence. A significant excess of non-synonymous substitutions was detected, which could indicate that a historical positive selection is acting in the woodchuck DRB1 genes. This hypothesis was confirmed in our study by the high variability in or near the antigen binding sites (ABS) and by the results obtained in sequence variability analyses. This analysis identified the presence of a microsatellite sequence that is located at the start of the second intron, which could further allow the development of a fast and cheap semiautomatic sequencing method.

Unlabelled: The determinants of the maintenance of chronic hepadnaviral infection are yet to be fully understood. A long-standing unresolved argument in the hepatitis B virus (HBV) research field suggests that during chronic hepadnaviral infection, cell-to-cell spread of hepadnavirus is at least very inefficient (if it occurs at all), virus superinfection is an unlikely event, and chronic hepadnavirus infection can be maintained exclusively via division of infected hepatocytes in the absence of virus spread. Superinfection exclusion was previously shown for duck HBV, but it was not demonstrated for HBV or HBV-related woodchuck hepatitis virus (WHV). Three woodchucks, which were chronically infected with the strain WHV7 and already developed WHV-induced hepatocellular carcinomas (HCCs), were superinfected with another WHV strain, WHVNY. Six weeks after the superinfection, the woodchucks were sacrificed and tissues of the livers and HCCs were examined. The WHVNY superinfection was demonstrated by using WHV strain-specific PCR assays and (i) finding WHVNY relaxed circular DNA in the serum samples collected from all superinfected animals during weeks one through six after the superinfection, (ii) detecting replication-derived WHVNY RNA in the tissue samples of the livers and HCCs collected from three superinfected woodchucks, and (iii) finding WHVNY DNA replication intermediates in tissues harvested after the superinfection. The results are consistent with the occurrence of continuous but inefficient hepadnavirus cell-to-cell spread and superinfection during chronic infection and suggest that the replication space occupied by the superinfecting hepadnavirus in chronically infected livers is limited. The findings are discussed in the context of the mechanism of chronic hepadnavirus infection., Importance: This study aimed to better understand the determinants of the maintenance of chronic hepadnavirus infection. The generated data suggest that in the livers chronically infected with woodchuck hepatitis virus, (i) hepadnavirus superinfection and cell-to-cell spread likely continue to occur and (ii) the virus spread is apparently inefficient, which is consistent with the interpretation that a limited number of cells in the livers facilitates the spread of hepadnavirus. The limitations of the cell-to-cell virus spread most likely are mediated at the level of the cells and do not reflect the properties of the virus. Our results further advance the understanding of the mechanism of chronic hepadnavirus infection. The significance of the continuous but limited hepadnavirus spread and superinfection for the maintenance of the chronic state of infection should be further evaluated in follow-up studies in order to determine whether blocking the virus spread would facilitate the suppression of chronic hepadnavirus infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Woodchuck hepatitis virus (WHV), a close relative of human hepatitis B virus (HBV), has been a key model for disease progression and clinical studies. Sequences of the assembly domain of WHV and HBV core proteins (wCp149 and hCp149, respectively) have 65% identity, suggesting similar assembly behaviors. We report a cryo-electron microscopy (cryo-EM) structure of the WHV capsid at nanometer resolution and characterization of wCp149 assembly. At this resolution, the T=4 capsid structures of WHV and HBV are practically identical. In contrast to their structural similarity, wCp149 demonstrates enhanced assembly kinetics and stronger dimer-dimer interactions than hCp149: at 23 °C and at 100 mM ionic strength, the pseudocritical concentrations of assembly of wCp149 and hCp149 are 1.8 μM and 43.3 μM, respectively. Transmission electron microscopy reveals that wCp149 assembles into predominantly T=4 capsids with a sizeable population of larger, nonicosahedral structures. Charge detection mass spectrometry indicates that T=3 particles are extremely rare compared to the ∼ 5% observed in hCp149 reactions. Unlike hCp149, wCp149 capsid assembly is favorable over a temperature range of 4 °C to 37 °C; van't Hoff analyses relate the differences in temperature dependence to the high positive values for heat capacity, enthalpy, and entropy of wCp149 assembly. Because the final capsids are so similar, these findings suggest that free wCp149 and hCp149 undergo different structural transitions leading to assembly. The difference in the temperature dependence of wCp149 assembly may be related to the temperature range of its hibernating host., Importance: In this paper, we present a cryo-EM structure of a WHV capsid showing its similarity to HBV. We then observe that the assembly properties of the two homologous proteins are very different. Unlike human HBV, the capsid protein of WHV has evolved to function in a nonhomeostatic environment. These studies yield insight into the interplay between core protein self-assembly and the host environment, which may be particularly relevant to plant viruses and viruses with zoonotic cycles involving insect vectors., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The woodchuck model is an informative model for studies on hepadnaviral infection. In this study, woodchuck hepatitis virus (WHV) transgenic (Tg) mouse models based on C57BL/6 mice were established to study the pathogenesis associated with hepadnaviral infection. Two lineages of WHV Tg mice, harboring the WHV wild-type genome (lineage 1217) and a mutated WHV genome lacking surface antigen (lineage 1281), were generated. WHV replication intermediates were detected by Southern blotting. DNA vaccines against WHV proteins were applied by intramuscular injection. WHV-specific immune responses were analyzed by flow cytometry and enzyme-linked immunosorbent assays (ELISAs). The presence of WHV transgenes resulted in liver-specific but sex- and age-dependent WHV replication in Tg mice. Pathological changes in the liver, including hepatocellular dysplasia, were observed in aged Tg mice, suggesting that the presence of WHV transgenes may lead to liver diseases. Interestingly, Tg mice of lineage 1281 spontaneously developed T- and B-cell responses to WHV core protein (WHcAg). DNA vaccination induced specific immune responses to WHV proteins in WHV Tg mice, indicating a tolerance break. The magnitude of the induced WHcAg-specific immune responses was dependent on the effectiveness of different DNA vaccines and was associated with a decrease in WHV loads in mice. In conclusion, sex- and age-dependent viral replication, development of autoimmune responses to viral antigens, pathological changes in the liver in WHV Tg mice, and the possibility of breaking immune tolerance to WHV transgenes will allow future studies on pathogenesis related to hepadnaviral infection and therapeutic vaccines.

Hepatitis B virus (HBV) persistence is facilitated by exhaustion of CD8 T cells that express the inhibitory receptor programmed cell death-1 (PD-1). Improvement of the HBV-specific T cell function has been obtained in vitro by inhibiting the PD-1/PD-ligand 1 (PD-L1) interaction. In this study, we examined whether in vivo blockade of the PD-1 pathway enhances virus-specific T cell immunity and leads to the resolution of chronic hepadnaviral infection in the woodchuck model. The woodchuck PD-1 was first cloned, characterized, and its expression patterns on T cells from woodchucks with acute or chronic woodchuck hepatitis virus (WHV) infection were investigated. Woodchucks chronically infected with WHV received a combination therapy with nucleoside analogue entecavir (ETV), therapeutic DNA vaccination and woodchuck PD-L1 antibody treatment. The gain of T cell function and the suppression of WHV replication by this therapy were evaluated. We could show that PD-1 expression on CD8 T cells was correlated with WHV viral loads during WHV infection. ETV treatment significantly decreased PD-1 expression on CD8 T cells in chronic carriers. In vivo blockade of PD-1/PD-L1 pathway on CD8 T cells, in combination with ETV treatment and DNA vaccination, potently enhanced the function of virus-specific T cells. Moreover, the combination therapy potently suppressed WHV replication, leading to sustained immunological control of viral infection, anti-WHs antibody development and complete viral clearance in some woodchucks. Our results provide a new approach to improve T cell function in chronic hepatitis B infection, which may be used to design new immunotherapeutic strategies in patients.

Matrix metalloproteinases (MMPs) play a central role in tumor invasion and metastasis. Increased expression of MMPs occurs during development of hepatocellular carcinoma (HCC) in humans following infection with hepatitis B virus (HBV). Woodchucks are used as an animal model for hepadnavirus-induced HCC. All woodchucks infected chronically with woodchuck hepatitis virus (WHV), a virus that is closely related to HBV, develop HCC. In the present study MMPs and related molecules were investigated in woodchucks to better understand the mechanisms of extracellular matrix remodeling in HCC. Three groups of samples were studied: liver and HCC tissues from animals infected with WHV and age- and gender-matched normal liver from animals not infected with WHV. New partial gene sequences for woodchuck MMP-2, MMP-7, and MMP-9 as well as their inhibitors NGAL, TIMP-1, and TIMP-2 were identified and used for determination of expression levels in liver and HCC by qRT-PCR. Compared to liver of WHV-naïve woodchucks, high levels of MMP-1, MMP-2, MMP-7, NGAL, and TIMP-1 were detected in liver of animals infected with WHV. However, no differences were found for TIMP-2. MMP-9 expression was higher in HCC than in liver of animals not infected with WHV. Immunohistochemical staining demonstrated that MMP-9 immunoreactivity was most intense in HCC, correlating with the progression of liver disease. Upregulation of MMP-9 in HCC was confirmed by Western blotting and zymography analysis. Furthermore, the activity of woodchuck MMPs was suppressed by BiPS, a common inhibitor of mammalian MMPs. These results suggest the use of MMP inhibitors as a potential HCC treatment strategy that could be explored in woodchucks., (Copyright © 2013 Wiley Periodicals, Inc.)

Type I interferons (IFN-α/β) serve as the first line of defense against viral infection and share the same type I IFN receptor (IFNAR) complex, which is composed of IFNAR1 and -2. The Eastern woodchuck (Marmota monax) and Chinese woodchuck (Marmota himalayana) are suitable for studying hepatitis B virus (HBV) infection. Here, the complete or partial sequences of the IFNARs of both species were obtained and analyzed. Small interference RNAs targeting wIFNAR1 and -2 specifically down-regulated the expression of wIFNAR1 and -2 and the IFN-stimulated gene MxA in a woodchuck cell line, respectively. IFNAR2 was significantly up-regulated in primary woodchuck hepatocytes stimulated with IFN-α or -γ. The expression of woodchuck IFNAR1 and -2 was decreased in woodchucks chronically infected with woodchuck hepatitis virus (WHV). These results are essential for studying type I IFN-related innate immunity and therapy in hepadnaviral infection in the woodchuck model., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: The programmed cell death 1 (PD-1)/programmed death-1 ligand 1 (PD-L1) system may play a role in the negative regulation of T cell functions in hepatitis B virus (HBV) infection. Thus, it is important to study its role in the widely used animal model for HBV infection of woodchucks with woodchuck hepatitis virus (WHV)., Methods: Woodchuck PD-L1 (wPD-L1) and -L2 (wPD-L2) were cloned and characterized. The levels of wPD-L1 expression in primary woodchuck hepatocytes (PWH), peripheral blood mononuclear cells (PBMCs), and liver tissue of naive and WHV-infected woodchucks were examined by real time reverse transcription (RT)-PCR and flow cytometry. Using antibodies against wPD-L1 and -L2, the effect of blocking PD-1/PD-L1/PD-L2 interaction on the proliferation and degranulation of woodchuck PBMCs was examined., Principal Findings: Both wPD-L1 and -L2 showed a high homology to their counterparts of other mammalian species and humans. WPD-L1 expression in PWH and PBMCs of naive animals was low but could be stimulated by Toll-like receptor (TLR) ligands and interferons (IFN). WPD-L1 expression in liver tissue was significantly higher than that measured in PWHs and was slightly elevated during acute and chronic WHV infection. However, wPD-1 and wPD-L1 expression on PBMCs was strongly up-regulated during acute and chronic infection. In vitro blockade with antibodies against wPD-L1 and -L2 partially enhanced proliferation and degranulation of PBMCs from WHV-infected woodchucks., Conclusions: Our results demonstrated that wPD-1/wPD-L1 expression in hepatocytes and PBMCs can be induced by different inflammatory stimuli and is up-regulated mainly on PBMCs during WHV infection. A blockade of the woodchuck PD-1/PD-L pathway could partially enhance T cell functions in WHV infection.

Woodchucks (Marmota monax) infected with woodchuck hepatitis virus (WHV) represent a highly valuable immunopathogenic model of hepatitis B virus (HBV) infection. Both WHV and HBV are noncytopathic hepadnaviruses which induce a strong but delayed virus-specific cellular immune response believed to be a cause of hepatitis. The reason behind this postponement is not well understood and its dissection in the woodchuck model has been hampered by the lack of appropriate research tools. In this study, we applied an assay for the simultaneous detection of cell apoptosis and proliferation to determine the fate of T lymphocytes after WHV infection leading to acute hepatitis. The results revealed that pre-acute WHV infection is associated with the significantly heightened susceptibility of T lymphocytes to activation-induced apoptotic death. This suggests that T lymphocyte function is compromised very early in the course of hepadnaviral infection and this may directly contribute to the postponement of virus-specific T cell response., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Our previous studies have shown that Toll-like receptor (TLR) ligands, Poly I:C and lipopolysaccharide (LPS), are able to activate non-parenchymal liver cells and trigger the production of interferon (IFN) to inhibit hepatitis B virus replication in vivo and in vitro. However, little is known about TLR-mediated cellular responses in primary hepatocytes. By the model of woodchuck hepatitis virus (WHV) infected primary woodchuck hepatocytes (PWHs), Poly I:C and LPS stimulation resulted in upregulation of cellular antiviral genes and relevant TLRs mRNA expression respectively. LPS stimulation led to a pronounced reduction of WHV replicative intermediates without a significant IFN induction. Poly I:C transfection resulted in the production of IFN and a highly increased expression of antiviral genes in PWHs and slight inhibitory effect on WHV replication. LPS could activate nuclear factor kappa B, MAPK and PI-3k/Akt pathways in PWHs. Further, inhibitors of MAPK-ERK and PI-3k/Akt pathways, but not that of IFN signalling pathway, were able to block the antiviral effect of LPS. These results indicate that IFN- independent pathways which activated by LPS are able to downregulate hepadnaviral replication in hepatocytes.

Small interfering RNA (siRNA) has been shown to be active to inhibit the hepatitis B virus gene expression and replication in transient and stable transfection systems. Here in primary hepatocytes prepared from naturally woodchuck hepatitis virus (WHV)-infected woodchucks, four siRNAs targeting the WHV preS1, S, C, and X region led to a depletion of WHV transcripts and replicative intermediates with different kinetics and a decreased production of viral particles. Two siRNAs targeting WHV S and X region had the highest efficacy to deplete 70% of WHV transcripts and replicative intermediates. In addition, siRNA-mediated suppression of WHV enhanced the expression of cellular genes like MxA and MHC I. Specific siRNAs are able to inhibit the hepadnaviral replication and enhance the expression of cellular genes relevant for antiviral actions. Thus, siRNAs might be useful as novel antiviral agents for the treatment of chronic HBV infection.

Adefovir dipivoxil (ADV) and tenofovir disoproxil fumarate (TDF) are nucleotide analogs that inhibit the replication of wild-type hepatitis B virus (HBV) and lamivudine (3TC)-resistant virus in HBV-infected patients, including those who are coinfected with human immunodeficiency virus. The combination of ADV or TDF with other nucleoside analogs is a proposed strategy for managing antiviral drug resistance during the treatment of chronic HBV infection. The antiviral effect of oral ADV or TDF, alone or in combination with 3TC or emtricitabine (FTC), against chronic woodchuck hepatitis virus (WHV) infection was evaluated in a placebo-controlled study in the woodchuck, an established and predictive model for antiviral therapy. Once-daily treatment for 48 weeks with ADV plus 3TC or TDF plus FTC significantly reduced serum WHV viremia levels from the pretreatment level by 6.2 log(10) and 6.1 log(10) genome equivalents/ml serum, respectively, followed by TDF plus 3TC (5.6 log(10) genome equivalents/ml), ADV alone (4.8 log(10) genome equivalents/ml), ADV plus FTC (one survivor) (4.4 log(10) genome equivalents/ml), TDF alone (2.9 log(10) genome equivalents/ml), 3TC alone (2.7 log(10) genome equivalents/ml), and FTC alone (2.0 log(10) genome equivalents/ml). Individual woodchucks across all treatment groups also demonstrated pronounced declines in serum WHV surface antigen, characteristically accompanied by declines in hepatic WHV replication and the hepatic expression of WHV antigens. Most woodchucks had prompt recrudescence of WHV replication after drug withdrawal, but individual woodchucks across treatment groups had sustained effects. No signs of toxicity were observed for any of the drugs or drug combinations administered. In conclusion, the oral administration of 3TC, FTC, ADV, and TDF alone and in combination was safe and effective in the woodchuck model of HBV infection.

The objective of this study was to evaluate, by developing one-step real-time PCR, the outcome of superinfection with hepatitis D virus (HDV) genotype I in woodchucks that were chronic carriers of woodchuck hepatitis virus (WHV) and did not show relevant signs of liver damage. Three woodchucks (Marmota monax) chronically infected with WHV were superinfected with a woodchuck HDV inoculum. The evolution of the WHV and HDV infections was monitored by quantifying HDV-RNA, WHV-DNA, and HDV-WHV antigens and antibodies. WHV and HDV sequencing was also performed and liver markers were evaluated. Liver damage was assessed using the Ishak method. All woodchucks showed a high HDV viral load, antigenemia and short survival after superinfection. Histopathological examination of autoptic liver samples showed massive liver necrosis compatible with an acute fatal course of hepatitis. The WHV sequencing showed that the virus population was not substituted by the WHV inoculum. The HDV sequencing performed during superinfection and at autopsy indicated amino acid changes in immune dominant regions of the HDV antigen. The strong correlation between acute infection with HDV genotype I and rapid and fatal liver failure indicates that HDV can be an important factor in the prognosis of HDV-WHV-superinfected woodchucks.

Experimental studies of animals with chronic hepadnavirus infection could provide valuable insight into optimal therapeutic strategies for individuals with chronic HBV infection. In this review, we focus on the contributions of the woodchuck model to our understanding of HBV biology and on its role in the development of antiviral drug. Furthermore, we consider the implications of studies focusing on the natural history of WHV infection for the management of HBV and the capacity of treatment to prevent complications of chronic hepatitis B infection.

The livers of woodchucks chronically infected with woodchuck hepatitis virus (WHV) contain foci of morphologically altered hepatocytes (FAH) with "basophilic", "amphophilic" and "clear cell" phenotypes, which are possibly pre-neoplastic in nature. Interestingly, most fail to express detectable levels of WHV proteins and nucleic acids. We studied sections of WHV-infected liver tissue to determine if all foci of hepatocytes that failed to express detectable levels of WHV, as assessed by immunoperoxidase staining for WHV core antigen, could be classified morphologically as FAH. We found that at least half of the foci of WHV core antigen-negative hepatocytes did not show clear morphological differences in either H&E or PAS (periodic acid Schiff) stained sections from surrounding hepatocytes, and were therefore not designated as FAH. In the second approach, we assayed core antigen-negative foci for the presence of fetuin B, a serum protein produced by normal hepatocytes, but not by neoplastic hepatocytes in hepatocellular carcinomas. Basophilic and amphophilic FAH had reduced levels of fetuin B compared to hepatocytes present in the surrounding liver; fetuin B staining was detected in clear cell FAH but the level could not be accurately assessed because of the displacement of fetuin B to the cell periphery by accumulated glycogen. The foci of morphologically normal WHV core antigen-negative hepatocytes had similar levels of fetuin B to that of the surrounding hepatocytes. The co-existence of at least four types of WHV core antigen-negative foci, including those with no obvious morphologic changes, raises the possibility that the different foci arise from distinct primary events. We hypothesize that a common event is loss of the ability to express WHV, allowing these hepatocytes to escape immune mediated cell death and to undergo clonal expansion to form distinct foci.

Unlabelled: Resolution of hepatitis B virus (HBV) infection is characterized by coordinated humoral and cellular immune responses. Immunity is durable over decades, protecting the host from reinfection and potential activation of residual HBV. Woodchucks infected at birth with woodchuck hepatitis virus (WHV) cleared viremia and developed antibodies to surface antigen (anti-WHs). Woodchucks became seronegative for anti-WHs 3-6 years later, but in some, WHV DNA was detected in serum, liver, and/or peripheral blood mononuclear cells (PBMCs). Those with WHV DNA had increased in vitro cellular immune responses to viral antigens, CD4 and CD8 markers, and Th1-type cytokines, suggesting active WHV-specific T lymphocytes. Immunosuppression for 12 weeks using cyclosporine A in such woodchucks resulted in transient reactivation of WHV replication. Serum of 1 woodchuck that became positive for WHV DNA during immunosuppression was inoculated into WHV-susceptible woodchucks, and a productive infection was demonstrated. The results indicate that after infection durable cellular immunity to WHV is essential for the long-term control of viral replication and is probably maintained by continuous priming from residual virus., Conclusion: These experimental observations demonstrate the potential of immunosuppression to reactivate HBV after resolution of infection.

This review describes the woodchuck and the woodchuck hepatitis virus (WHV) as an animal model for pathogenesis and therapy of chronic hepatitis B virus (HBV) infection and disease in humans. The establishment of woodchuck breeding colonies, and use of laboratory-reared woodchucks infected with defined WHV inocula, have enhanced our understanding of the virology and immunology of HBV infection and disease pathogenesis, including major sequelae like chronic hepatitis and hepatocellular carcinoma. The role of persistent WHV infection and of viral load on the natural history of infection and disease progression has been firmly established along the way. More recently, the model has shed new light on the role of host immune responses in these natural processes, and on how the immune system of the chronic carrier can be manipulated therapeutically to reduce or delay serious disease sequelae through induction of the recovery phenotype. The woodchuck is an outbred species and is not well defined immunologically due to a limitation of available host markers. However, the recent development of several key host response assays for woodchucks provides experimental opportunities for further mechanistic studies of outcome predictors in neonatal- and adult-acquired infections. Understanding the virological and immunological mechanisms responsible for resolution of self-limited infection, and for the onset and maintenance of chronic infection, will greatly facilitate the development of successful strategies for the therapeutic eradication of established chronic HBV infection. Likewise, the results of drug efficacy and toxicity studies in the chronic carrier woodchucks are predictive for responses of patients chronically infected with HBV. Therefore, chronic WHV carrier woodchucks provide a well-characterized mammalian model for preclinical evaluation of the safety and efficacy of drug candidates, experimental therapeutic vaccines, and immunomodulators for the treatment and prevention of HBV disease sequelae.

Woodchuck hepatitis virus (WHV) is a member of family Hepadnaviridae and closely related to hepatitis B virus (HBV). The WHV core protein (WHcAg) is a strongly immunogenic protein and forms virus-like particles. WHcAg may represent a suitable carrier system for B- and T-cell epitopes. However, the lack of a high expression system for WHcAg and defined antibodies to detect WHcAg prevents the use of this carrier system. In the present study, vectors expressing WHcAg with carboxyl-terminal truncations were constructed to determine the region of WHcAg required for assembly. The first 144 or 149 amino acid residues of WHcAg were able to efficiently assemble into particulate structures. Both truncated forms of WHcAg were accumulated in E. coli as uniform particles with a diameter of 34nm in large quantities and could be purified in milligram scale. As expected, the particles of truncated WHcAg retained the antigenicity of the full length WHcAg. However, denatured WHcAg remained to be reactive with specific antisera, suggesting that WHcAg may possess additional linear B-cell epitopes. Monoclonal antibodies against denatured WHcAg were generated and tested for their specificity. Five antibodies were found to direct the N-terminal region of WHcAg. Due to the conservation of the amino acid sequence in this region of WHcAg and HBcAg, these antibodies recognized recombinant HBcAg as well. Thus, this linear B-cell epitope is conserved on the core proteins of hepadnaviruses.

In liver tumors induced by chronic WHV infection in the WHV/woodchuck model of HBV infection, activation of genes of the myc family by WHV insertion has been well documented. Several studies have shown that N-myc2 is by far the most frequently involved, and in most cases, its transcriptional activation is due to WHV insertion nearby the gene. N-myc2 has been shown to be also activated by WHV insertion in two downstream loci, b3n and win. Although the extent of insertion in these latter loci in woodchuck tumors has not been investigated, their discovery has led to the notion that therein WHV insertion accounts for N-myc2 activation in the remaining tumors expressing the proto-oncogene in absence of any detectable alteration nearby the gene, a notion remained unproved and not further investigated yet. In the majority of cases, the above observations were derived from tumors developed in colony born laboratory bred woodchucks experimentally infected with standardized viral inocula, mostly of the same lineage. In the present work, we investigated a survey of liver tumors naturally developed in wild woodchucks with naturally acquired chronic WHV infection. Tumors had histological features of well to moderately differentiated HCCs. In most animals, multiple tumor nodules were observed; in the great majority of cases, they were shown to be independent tumors because their WHV integration patterns were not clonally related. 53 independent tumors were investigated for N-myc activation and WHV integration nearby N-myc genes and in the b3n and win loci. Comparison of our results with data from previous studies revealed that, in tumors from naturally infected wild woodchucks, the frequency of WHV integration nearby N-myc2 has a tendency to be lower and, in addition, N-myc2 activation is due to WHV integration nearby the gene significantly less frequently than in tumors from experimentally infected colony born animals (12/28, 43% vs. 15/20, 75%, P = 0.0397). These findings are likely related to the less uniform conditions as to infecting virus and host genetic background in naturally infected wild woodchucks with respect to experimentally infected colony born woodchucks and suggest that viral and/or host factors may influence the site of viral insertion finally detected in overt tumors. In addition, more than one third (11/28, 39%) tumors with activated N-myc2 transcription did not show rearrangement either nearby the gene, or in b3n or in win. These findings challenge the notion that integration in the downstream b3n and win loci is responsible for N-myc2 activation in tumors lacking insertion nearby N-myc2 and suggest that in a considerable fraction of liver tumors, at least from wild woodchucks, N-myc2 activation might be due either to WHV integration in further regions of the N-myc2 chromosomal domain or to other mechanisms related or unrelated to viral insertion.

Hepatitis B virus X (HBX) is essential for the productive infection of hepatitis B virus (HBV) in vivo and has a pleiotropic effect on host cells. We have previously demonstrated that the proteasome complex is a cellular target of HBX, that HBX alters the proteolytic activity of proteasome in vitro, and that inhibition of proteasome leads to enhanced viral replication, suggesting that HBX and proteasome interaction plays a crucial role in the life cycle and pathogenesis of HBV. In the present study, we tested the effect of HBX on the proteasome activities in vivo in a transgenic mouse model in which HBX expression is developmentally regulated by the mouse major urinary promoter in the liver. In addition, microarray analysis was performed to examine the effect of HBX expression on the global gene expression profile of the liver. The results showed that the peptidase activities of the proteasome were reduced in the HBX transgenic mouse liver, whereas the activity of another cellular protease was elevated, suggesting a compensatory mechanism in protein degradation. In the microarray analysis, diverse genes were altered in the HBX mouse livers and the number of genes with significant changes increased progressively with age. Functional clustering showed that a number of genes involved in transcription and cell growth were significantly affected in the HBX mice, possibly accounting for the observed pleiotropic effect of HBX. In particular, insulin-like growth factor-binding protein 1 was down-regulated in the HBX mouse liver. The down-regulation was similarly observed during acute woodchuck hepatitis virus infection. Other changes including up-regulation of proteolysis-related genes may also contribute to the profound alterations of liver functions in HBV infection.

The woodchuck together with the woodchuck hepatitis virus (WHV) is an excellent model to study the pathogenesis of hepadnaviral infections. Chronic WHV infection causes severe liver disease and hepatocellular carcinoma in woodchucks. The mechanism of viral clearance is not fully understood, interferons seem to play a major role in down-regulating viral replication prior to elimination of infected hepatocytes. We investigated on the pattern of cytokine and T-cell-marker expression in livers of woodchucks chronically infected with WHV. RNase-protection-assay (RPA) was used to determine mRNA of woodchuck specific genes (TNF-alpha, IFN-gamma, IL-15, CD3, CD4, CD8). Serial liver biopsies were performed daily or weekly in eight chronic WHV-carrier woodchucks. Cytokine/T-cell-marker expression differed significantly between the time points up to +/-50% within each woodchuck. The different expression patterns of cytokines or T-cell-markers did not correlate to the (weak) fluctuations in the viremia but may explain the observed fluctuations in the WHV/HBV-load in chronically infected individuals. Furthermore, we observed associations between cytokine and T-cell-marker expression. The marginal fluctuations in viremia during the chronic infection may indicate, that, once the chronic hepadnaviral infection is established, cytokines/interferons expressed endogenously (i.e. not vector-borne or injected) play only a minor role.

Tenofovir disoproxil fumarate (TDF) is a nucleotide analogue approved for treatment of human immunodeficiency virus (HIV) infection. TDF also has been shown in vitro to inhibit replication of wild-type hepatitis B virus (HBV) and lamivudine-resistant HBV mutants and to inhibit lamivudine-resistant HBV in patients and HBV in patients coinfected with the HIV. Data on the in vivo efficacy of TDF against wild-type virus in non-HIV-coinfected or lamivudine-naïve chronic HBV-infected patients are lacking in the published literature. The antiviral effect of oral administration of TDF against chronic woodchuck hepatitis virus (WHV) infection, an established and predictive animal model for antiviral therapy, was evaluated in a placebo-controlled, dose-ranging study (doses, 0.5 to 15.0 mg/kg of body weight/day). Four weeks of once-daily treatment with TDF doses of 0.5, 1.5, or 5.0 mg/kg/day reduced serum WHV viremia significantly (0.2 to 1.5 log reduction from pretreatment level). No effects on the levels of anti-WHV core and anti-WHV surface antibodies in serum or on the concentrations of WHV RNA or WHV antigens in the liver of treated woodchucks were observed. Individual TDF-treated woodchucks demonstrated transient declines in WHV surface antigen serum antigenemia and, characteristically, these woodchucks also had transient declines in serum WHV viremia, intrahepatic WHV replication, and hepatic expression of WHV antigens. No evidence of toxicity was observed in any of the TDF-treated woodchucks. Following drug withdrawal there was prompt recrudescence of WHV viremia to pretreatment levels. It was concluded that oral administration of TDF for 4 weeks was safe and effective in the woodchuck model of chronic HBV infection.

We examined a rational approach to therapy of chronic hepatitis B virus (HBV) infection that utilized the reduction of viral load combined with appropriately timed immune modulation/stimulation. In a placebo-controlled study, chronic woodchuck hepatitis virus (WHV) carrier woodchucks received clevudine (L-FMAU), previously shown to have especially potent and sustained antiviral activity in woodchucks, for 32 weeks followed by WHV surface antigen (WHsAg) alum-adjuvanted vaccine at 32, 36, 40 and 48 weeks. Clevudine induced significant reductions in viraemia, surface antigenaemia, hepatic WHV nucleic acids, and hepatic core and surface antigens. Viral replication markers remained markedly suppressed in 75% of the clevudine-treated woodchucks following drug withdrawal, but remained at high levels in the vaccine monotherapy and placebo groups. Combination drug and vaccine therapy had benefits based on sustained reduction of viraemia, antigenaemia, and hepatic WHV DNA and RNA; inhibition of progression of chronic hepatitis; reduced frequency of chronic liver injury; and delayed onset of hepatocellular carcinoma (HCC). Combination therapy contributed to prevention of HCC in up to 38% of treated carriers, although the growth rate of established HCC was not affected. This study demonstrates enhanced benefits of combination chemo-immunotherapy against viral load and disease progression in chronic hepadnaviral infection, and provides a platform for further development of such treatment regimens.

Antibodies against virus nucleocapsid (anticore) normally accompany hepadnaviral hepatitis but they may also occur in the absence of symptoms and other serological indicators of the infection. This situation can be encountered following a clinically and serologically unapparent exposure to hepatitis B virus (HBV) or after recovery from hepatitis B. In this study, woodchucks inoculated with woodchuck hepatitis virus (WHV) were investigated to determine the relationship between anticore detection and the molecular status of virus replication in a primary WHV surface antigen (WHsAg)-negative infection or long-after resolution of WHV hepatitis. Serial, parallel samples of sera, peripheral blood mononuclear cells (PBMC) and liver tissue, collected for more than 5 years after inoculation with virus, were examined for WHV DNA by highly sensitive polymerase chain reaction (PCR)/nucleic acid hybridization assays. Sera were also tested for WHV DNA after DNase treatment and for WHV DNA and WHsAg after concentration in sucrose. Liver and PBMC were examined for WHV covalently closed circular DNA and viral RNA transcripts by PCR-based techniques to assess virus replication status. The study showed that anticore antibodies existing in the absence of other serological markers are a reliable indicator of occult WHV infection. This state can be accompanied by traces of circulating particles behaving as intact virions and by intermittent minimal-to-mild liver inflammation. In conclusion, the long-term presence of anticore antibodies alone is a consequence of sustained restimulation of the immune system by virus nucleocapsid produced during low-level hepadnaviral assembly.

Interferon-alpha (IFN-alpha) is a potent suppressor of hepatitis B virus (HBV) replication in the HBV-transgenic mouse, depleting virus replication intermediates from infected hepatocytes via pathways mediated by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). It has also been hypothesized that cytokines induce curing of infected hepatocytes via non-cytolytic pathways during resolution of transient hepadnavirus infections. We have therefore evaluated therapy of chronic woodchuck hepatitis virus (WHV) infections using treatment with the nucleoside analog clevudine [L-FMAU; 1-(2-fluoro-5-methyl-b-L-arabinofuranosyl) uracil] and therapy with adenovirus vectors expressing INF-gamma, TNF-alpha, and beta-galactosidase. Before their use in vivo, expression of IFN-gamma and TNF-alpha from the adenovirus vectors was evaluated in vitro. Conditioned media from adenovirus-infected WC-3 cells was shown to inhibit WHV replication in baculovirus-transduced cells. Adenovirus super-infection of the liver in woodchucks led to declines in the percentage of hepatocytes with detectable core antigen and nucleic acids, and in levels of covalently closed circular DNA (cccDNA) and total WHV DNA, but a major long-term benefit of adenovirus super-infection during clevudine treatment was not demonstrated. Moreover, the effect took at least 2 weeks to develop suggesting that the declines in the percentage of WHV-infected cells, ccc, and total WHV DNA resulted from induction of the adaptive immune response by the adenovirus super-infection, and only indirectly from the expression of cytokines by the vectors.

Alpha interferon (IFN-alpha) and IFN-gamma are able to suppress hepadnavirus replication. The intrahepatic expression of high levels of IFN may enhance the antiviral activity. We investigated the effects of woodchuck-specific IFN-alpha (wIFN-alpha) and IFN-gamma(wIFN-gamma) on woodchuck hepatitis virus (WHV) replication in vivo by helper-dependent adenoviral (HD-Ad) vector-mediated gene transfer. The expression of biologically active IFNs was demonstrated in vitro after transduction of woodchuck cells with HD-Ad vectors encoding wIFN-alpha (HD-AdwIFN-alpha) or wIFN-gamma (HD-AdwIFN-gamma). The transduction efficacy of the HD-Ad vector in woodchuck liver in vivo was tested with a vector expressing green fluorescence protein (GFP). Immunohistochemical staining of liver samples on day 5 after injection showed expression of GFP in a high percentage of liver cells surrounding the central vein. The transduction of livers of WHV carriers in vivo with HD-AdwIFN-alpha or HD-AdwIFN-gamma induced levels of biologically active IFN, which could be measured in the sera of these animals. Expression of wIFN-alpha in the liver reduced intrahepatic WHV replication and WHV DNA in sera of about 1 log step in two of two woodchucks. Transduction with HD-AdwIFN-gamma, however, reduced WHV replicative intermediates only slightly in two of three animals, which was not accompanied with significant changes in the WHV DNA in sera. We demonstrated for the first time the successful HD-Ad vector-mediated transfer of genes for IFN-alpha and IFN-gamma in vivo and timely limited reduction of WHV replication by wIFN-alpha, but not by wIFN-gamma., (Copyright 2004 American Society for Microbiology)

The X protein (HBX) of the hepatitis B virus (HBV) is not essential for the HBV life cycle in vitro but is important for productive infection in vivo. Our previous study suggests that interaction of HBX with the proteasome complex may underlie the pleiotropic functions of HBX. With the woodchuck model, we demonstrated that the X-deficient mutants of woodchuck hepatitis virus (WHV) are not completely replication defective, possibly behaving like attenuated viruses. In the present study, we analyzed the effects of the proteasome inhibitors on the replication of wild-type and X-negative HBV and WHV. Recombinant adenoviruses or baculoviruses expressing replicating HBV or WHV genomes have been developed as a robust and convenient system to study viral replication in tissue culture. In cells infected with either the recombinant adenovirus-HBV or baculovirus-WHV, the replication level of the X-negative construct was about 10% of that of the wild-type virus. In the presence of proteasome inhibitors, the replication of the wild-type virus was not affected, while the replication of the X-negative virus of either HBV or WHV was enhanced and restored to the wild-type level. Our data suggest that HBX affects hepadnavirus replication through a proteasome-dependent pathway.

The middle surface antigen (M-sAg) of hepadnaviruses is one of three envelope proteins that share a common C-terminal S domain. M-sAg contains the preS2 domain in addition to the S region. The preS2 region of woodchuck hepatitis virus (WHV) contains a potential glycosylation site Asn-Gln-Thr at amino acid (aa) positions 3-5. In this study, we mutated this site by site-directed mutagenesis and confirmed that glycosylation occurs here. In in vitro translation assays, the mutation Thr to Asn at aa 5 significantly impaired glycosylation of M-sAg. The mutated M-sAg formed abnormal clustered structures in transfected cells as determined by immunofluorescent staining. Confocal microscopic analysis showed that an enrichment of this glycosylation-deficient protein in the Golgi apparatus occurred, which is not typical for the wild-type protein. These results are consistent with earlier findings that incorrect glycosylation of viral proteins may interfere with virus assembly.

Background: Our aim was to evaluate the anti-HBV activity of a novel L-nucleoside analog, 2',3'-dideoxy-2',3'-didehydro-beta-L-5-fluorocytidine (beta-L-Fd4C), in study models of HBV infection., Method: Its mechanism of action was evaluated on the in vitro expressed duck HBV (DHBV) reverse transcriptase and in primary hepatocyte cultures of duck and human origin. The capacity of antiviral therapy to clear viral infection was analyzed in vivo in the duck and woodchuck models., Results: beta-L-Fd4C-TP exhibited a more potent inhibitory effect on the RT activity of the DHBV polymerase than other cytidine analogs (lamivudine-TP, ddC-TP, beta-L-FddC-TP). In primary duck hepatocyte cultures, beta-L-Fd4C exhibited a long-lasting inhibitory effect on viral DNA synthesis but could not clear viral cccDNA. In vivo treatment with beta-L-Fd4C in infected ducklings and woodchucks, induced a greater suppression of viremia and intrahepatic viral DNA synthesis than with lamivudine. However, covalently closed circular DNA persistence explained the relapse of viral replication after treatment withdrawal. Viral spread was strongly reduced in the case of early therapeutical intervention, but the number of infected cells did not decline when therapy was started during chronic infection. Liver histology analysis showed a decrease in the inflammatory activity of chronic hepatitis while no ultrastructural modification of liver cells was observed in electron microscopy studies. Furthermore, in human primary hepatocyte cultures, beta-L-Fd4C induced a significant inhibition of HBV DNA synthesis., Conclusion: beta-L-Fd4C is a potent inhibitor of hepadnavirus RT and inhibits viral DNA synthesis in hepatocytes both in vitro and in vivo. These experimental studies allowed as to show that beta-L-Fd4C is a promising anti-HBV agent. Combination therapy should be evaluated to eradicate viral infection., (Copyright 2002 Blackwell Publishing Asia Pty Ltd)

The presence of Deleted Genomes has been shown in a number of viral models including Hepadnaviridae. The analysis of woodchuck hepatitis B virus (WHV) population after experimental infection of woodchuck 197 (W197) with WHV7-PI inoculum revealed the presence of two Deleted Genomes: DG600 lacking a 1330 bp region (Core/Polymerase/PreS1) and DG900 showing a deletion of 869 nts (Pol/PreS/S). These mutants were also present in WHV7-PI. The successive WHV experimental infections in adult animals were performed using W197-w7 inoculum containing DG600 and DG900. Infections were divided into three groups presenting different patterns of viral replication, different presence of markers, occurrence of variants and persistence of infection. The first group displayed 2-3 weeks viremic phase and WHV-DNA titres of 10-30 ng/ml; the second a longer viremic phase (8-9 weeks) and higher WHV-DNA titres (up to 78 ng/ml). In contrast, the third group exhibited lifetime presence of WHV-DNA and WHVeAg in serum and viral replication in liver. The Deleted Genomes were transmitted in the newly infected animals with the same genomic organization. DG600 was persistently found only in chronically infected woodchuck, whereas a different pattern of presence was described for DG900. The characterization of these classes of deleted mutants in woodchuck-WHV model raises new questions on the link between DGs and persistent infections.

Administration of either lamivudine (2'-deoxy-3'-thiacytidine) or L-FMAU (2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil) to woodchucks chronically infected with woodchuck hepatitis virus (WHV) induces a transient decline in virus titers. However, within 6 to 12 months, virus titers begin to increase towards pretreatment levels. This is associated with the emergence of virus strains with mutations of the B and C regions of the viral DNA polymerase (T. Zhou et al., Antimicrob. Agents Chemother. 43:1947-1954, 1999; Y. Zhu et al., J. Virol. 75:311-322, 2001). The present study was carried out to determine which of the mutants that we have identified conferred resistance to lamivudine and/or to L-FMAU. When inserted into a laboratory strain of WHV, each of the mutations, or combinations of mutations, of regions B and C produced a DNA replication-competent virus and typically conferred resistance to both nucleoside analogs in cell culture. Sequencing of the polymerase active site also occasionally revealed other mutations, but these did not appear to contribute to drug resistance. Moreover, in transfected cells, most of the mutants synthesized viral DNA nearly as efficiently as wild-type WHV. Computational models suggested that persistence of several of the WHV mutants as prevalent species in the serum and, by inference, liver for up to 6 months following drug withdrawal required a replication efficiency of at least 10 to 30% of that of the wild type. However, their delayed emergence during therapy suggested replication efficiency in the presence of the drug that was still well below that of wild-type WHV in the absence of the drug.

Human hepatitis B virus (HBV) variants containing in-frame core internal deletion (CID) have been demonstrated to contain all the functional features of defective interfering (DI) particles (Yuan, T. T.-T., M.-H. Lin, D. S. Chen, and C. Shih, 1998, J. Virol. 72, 578-584). Here, we report that out-of-frame HBV CID variants exhibit defective interfering property similar to in-frame CID variants characterized previously. This result raises the possibility that it may be the deleted pregenomic RNA product, rather than the deleted core protein product, that is responsible for interference. Furthermore, a genomic deletion elsewhere does not cause interference since preS2 deletion variants exhibit no influence on wild-type HBV replication. Consistent with the natural occurrence of HBV CID variants, we recently identified CID variants of woodchuck hepatitis virus (WHV) in natural infection. However, unlike HBV CID variants, functional characterization of WHV CID variants using a human hepatoma cell line has not revealed any interference in tissue culture. In summary, defective interference is a general phenomenon for both in-frame and out-of-frame HBV CID variants.

The X protein (HBX) of the hepatitis B virus (HBV) has been shown to be important for the establishment of HBV infection in vivo. Our previous studies suggested that interaction of HBX with the proteasome complex may underlie the pleiotropic functions of HBX. In this study, we generated a series of woodchuck hepatitis virus (WHV) X mutants, including mutants of the domain interacting with the proteasome, and studied their infectivity in woodchucks. Many of the mutants were defective in transactivation but none of them were completely replication defective in vitro. In vivo, all the wild-type and some X mutant-transfected animals demonstrated evidence of infection with anti-WHc and/or anti-WHs seroconversion. Most of the wild-type- and X mutant-transfected animals had transient viremia. Some animals were later challenged with infectious WHV. Animals inoculated with X mutants, including those with no serologic evidence of infection, were protected from the challenge, suggesting previous infection with resulting protective immunity. Our study demonstrates that the previously described functional domains of HBX are biologically important and the X-defective mutants, possibly as attenuated viruses, are not completely replication defective in vivo.

Isolated hepatocytes represent a relevant model of the liver and are highly required both for research and therapeutic applications. However, sources of primary liver cells from human beings and from some animal species are limited. Therefore, cryopreservation of hepatocytes could greatly facilitate advances in various research areas. The aim of this study was to evaluate whether cryopreserved primary woodchuck hepatocytes could be used for woodchuck hepatitis B virus (WHV) infection studies, and whether they could maintain their regenerative potential in vivo after thawing. Critical steps for good quality of cryopreserved hepatocytes included the use of University of Wisconsin (UW) solution as a main component of the freezing medium, stepwise reduction of dimethylsulfoxide (DMSO) to avoid osmotic shock, and maintenance of low concentrations of DMSO in the culture medium. After cryopreservation, cell viability was still high (70% to 80%), and 50% to 60% of thawed cells attached to the plates. The appearance of covalently closed circular (ccc)DNA and of WHV-replicative forms a few days after in vitro infection demonstrated that thawed woodchuck hepatocytes were still susceptible to viral infection, thus proving maintenance of a very high hepatocyte-specific differentiation status. Furthermore, transplantation of woodchuck hepatocytes into the liver of urokinase-type plasminogen activator (uPA)/recombination activation gene-2 (RAG-2) mice, a model of liver regeneration, demonstrated that cryopreserved cells retained the ability to divide and to extensively repopulate a xenogenic liver. Notably, in vivo susceptibility to infection with WHV and proliferative capacity of frozen/thawed woodchuck hepatocytes in recipient mice were identical to those observed by transplanting fresh hepatocytes.

A unique series of simple unnatural L-nucleosides that specifically inhibit hepatitis B virus (HBV) replication has been discovered. These molecules have in common a hydroxyl group in the 3'-position (3'-OH) of the beta-L-2'-deoxyribose sugar that confers antiviral activity specifically against hepadnaviruses. Replacement of the 3'-OH broadens activity to other viruses. Substitution in the base decreases antiviral potency and selectivity. Human DNA polymerases and mitochondrial function are not effected. Plasma viremia is reduced up to 8 logs in a woodchuck model of chronic HBV infection. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.

Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNA-enriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.

Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (approximately 10(3) virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses.

Emtricitabine [(-)FTC] [(-)-beta-2', 3'-dideoxy-5-fluoro-3'-thiacytidine] has been shown to be an effective inhibitor of hepatitis B virus (HBV) in cell culture, with a potency and selectivity that are essentially identical to those of lamivudine. The antiviral activity of oral administration of (-)FTC against WHV replication in chronically infected woodchucks, an established and predictive model for antiviral therapy against HBV, was examined in a placebo-controlled study. (-)FTC significantly reduced viremia and intrahepatic WHV replication in a dose-dependent manner that was comparable to the antiviral activity of lamivudine observed in previous studies conducted by our laboratories. No effect on the levels of hepatic WHV RNA or the levels of woodchuck hepatitis surface antigen or anti-woodchuck hepatitis surface and core antibodies in the serum of the treated animals was observed. No evidence of drug-related toxicity was observed in any of the animals treated.

Woodchuck hepatitis virus (WHV), similar to human hepatitis B virus, causes acute liver inflammation that can progress to chronic hepatitis and hepatocellular carcinoma. WHV also invades cells of the host lymphatic system, where it persists for life. We report here that acute and chronic hepadnavirus hepatitis is characterized by a profound difference in the expression of class I major histocompatibility complex (MHC) molecules on the surface of infected hepatocytes and, notably, lymphoid cells. While acute WHV infection is accompanied by the enhanced hepatocyte surface presentation of class I MHC antigen and upregulated transcription of the relevant hepatic genes, inhibition of class I antigen display on liver cells is a uniform hallmark of chronic WHV infection. This inhibition in chronic hepatitis occurs despite augmented (as in acute infection) expression of hepatic genes for class I MHC heavy chain, beta(2)-microglobulin, and transporters associated with antigen processing (TAP1 and TAP2). Further, the class I antigen inhibition is not related to the histological severity of hepatocellular injury, the extent of lymphocytic infiltrations, the level of intrahepatic gamma interferon induction, or the hepatic WHV load. Importantly, the antigen expression is also inhibited on organ lymphoid cells of chronically infected hosts. The results obtained in this study demonstrate that the defective presentation of class I MHC molecules on cells supporting persistent WHV replication is due to viral posttranscriptional interference. This event may diminish the susceptibility of infected hepatocytes to virus-specific T-cell-mediated elimination, hinder virus clearance, and deregulate the class I MHC-dependent functions of the host immune system. This multifarious effect could be critical for perpetuation of liver damage and evasion of the antiviral immunological surveillance in chronic infection and therefore could be supportive of hepadnavirus persistence.

Hepatitis B virus (HBV) is a major human pathogen that causes chronic infection and life-threatening liver diseases in millions of individuals. While pathological and epidemiological consequences of clinically evident HBV infections are well recognized, there is no similar knowledge on an asymptomatic, silently progressing virus persistence. Contrary to previous opinion, current evidence indicates that a serologically undetectable (occult) HBV carriage is a common outcome of recovery from symptomatic illness and that scanty amounts of the virus are carried by apparently healthy individuals for years after resolution of hepatitis B despite the presence of presumably protective antiviral antibodies. Recent studies on this silent form of hepadnavirus carriage in an experimental woodchuck hepatitis virus (WHV) infection, which is considered to be the closest natural model of HBV disease, revealed that the life-long occult persistence of traces of pathogenic virus is an invariable consequence of recovery after hepadnaviral invasion and that this state always co-exists with a steady low-rate virus replication in both the liver and the lymphatic system. Importantly, this serologically concealed infection can be accompanied by development of hepatocellular carcinoma in convalescent animals and is transmittable from mothers to offspring as an asymptomatic, indefinitely long infection which involves the lymphatic system but not always the liver. This review focuses on the features of hepadnavirus occult persistence and its lymphotropism, and on what is currently understood about the contribution of the lymphatic system in maintaining hepadnavirus carriage based on insights provided by analysis of the woodchuck-WHV experimental system.

Chronic infection by hepatitis B virus is a leading cause of human liver cancer and liver disease. The hepatitis B virus HBx protein is a regulatory factor that is essential for virus infection in mammals and is implicated in development of liver cancer and liver disease. Among the reported activities of HBx is the ability to stimulate Src tyrosine kinases, Ras-GTPases and transcriptional activation. We now demonstrate that HBx activation of Src tyrosine kinases, but not Ras, promotes a high level of viral replication in cell culture. HBx is shown to stimulate reverse transcription of the viral pregenomic mRNA into genomic DNA through a Src-mediated pathway in tissue culture cells. Targeted inhibition of Src tyrosine kinase activity, mutational inactivation of the HBx gene or retargeting of HBx to the nucleus to abolish cytoplasmic signal transduction activity, are shown to impair viral reverse transcription strongly. These studies implicate HBx stimulation of the Src family of tyrosine kinases in stimulation of viral polymerase activity.