Efficacy of the carbocyclic 2'-deoxyguanosine nucleoside BMS-200475 in the woodchuck model of hepatitis B virus infection

Daily oral treatment with the cyclopentyl 2'-deoxyguanosine nucleoside BMS-200475 at doses ranging from 0.02 to 0.5 mg/kg of body weight for 1 to 3 months effectively reduced the level of woodchuck hepatitis virus (WHV) viremia in chronically infected woodchucks as measured by reductions in serum WHV DNA levels and endogenous hepadnaviral polymerase activity. Within 4 weeks of daily therapy with 0.5 or 0.1 mg of BMS-200475 per kg, endogenous viral polymerase levels in serum were reduced about 1,000-fold compared to pretreatment levels. Serum WHV DNA levels determined by a dot blot hybridization technique were comparably decreased in these treated animals. In the 3-month study, the sera of animals that had undetectable levels of WHV DNA by the dot blot technique were further analyzed by a highly sensitive semiquantitative PCR assay. The results indicate that BMS-200475 therapy reduced mean WHV titers by 10(7)- to 10(8)-fold, down to levels as low as 10(2) to 10(3) virions/ml of serum. Southern blot hybridization analysis of liver biopsy samples taken from animals during and after BMS-200475 treatment showed remarkable reductions in the levels of WHV DNA replicative intermediates and in the levels of covalently closed circular viral DNA. WHV viremia in BMS-200475-treated WHV carriers eventually returned to pretreatment levels after therapy was stopped. These results indicate that BMS-200475 should be evaluated in clinical trials for the therapy of chronic human hepatitis B virus infections.     

Systemic distribution of talc after intrapleural administration in rats

BMS-200475 was recently shown to have potent antiviral activity against hepatitis B virus (50% effective concentration = 3.7 nM; 50% cytotoxic concentration = 30 microM). In metabolic studies in both HepG2 and hepatitis B virus-transfected 2.2.15 human hepatoma cell lines, the metabolism was similar, the primary products being the di- and triphosphates. The accumulation of triphosphate was rapid and detectable down to a 5 nM concentration of added drug. When cells were labeled at 25 microM, the intracellular triphosphate concentration attained 30 pmol/10(6) cells ( approximately 30 microM). The intracellular half-life of the triphosphate was about 15 h. Compared with five other nucleoside analogs of medical interest (lamivudine, penciclovir, ganciclovir, acyclovir, and lobucavir), BMS-200475 was most efficiently phosphorylated to the triphosphate in HepG2 cells.     

Inhibitory effect of 9-(2-phosphonylmethoxyethyl)-adenine (PMEA) on human and duck hepatitis B virus infection

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) was evaluated for its inhibitory effect on hepadnavirus replication in three different cell systems, i.e., human hepatoma cell lines HepG2 2.2.15 and HB611 (transfected with human hepatitis B virus (HBV)) and primary cultures of duck hepatocytes infected with duck hepatitis B virus (DHBV). PMEA inhibited HBV release from HepG2 2.2.15 cells and HB611 cells at a 50% inhibitory concentration (IC50) of 0.7 and 1.2 microM, respectively. Intracellular viral DNA synthesis was inhibited at concentrations equivalent to those required to inhibit virus release from the cells. DHBV secretion from duck hepatocytes was inhibited by PMEA at an IC50 of 0.2 microM. HBsAg secretion was inhibited by PMEA in a concentration-dependent manner in HB611 cells and DHBV-infected duck hepatocytes but not HepG2 2.2.15 cells. The 50% cytotoxic concentration, as measured by inhibition of [3H-methyl]deoxythymidine incorporation was 150 microM for the two human hepatoma cell lines and 40 microM for the duck hepatocyte cultures. In a pilot experiment PMEA was found to reduce the amounts of DHBV DNA in the serum of Pekin ducks.     

The GDPAL region of the pre-S1 envelope protein is important for morphogenesis of woodchuck hepatitis virus

The pre-S envelope protein of duck hepatitis B virus (DHBV) contains a region, Asp-Asp-Pro-Leu-Leu (DDPLL), that is specifically required for virus assembly and secretion (Lenhoff and Summers, J Virol 1994;68:4565-4571). We found that amino acids 201 to 205 of the pre-S envelope protein of woodchuck hepatitis virus (WHV) form a conserved amino acid cluster, Gly-Asp-Pro-Ala-Leu (GDPAL), which resembles the DDPLL sequence of DHBV. To determine whether the GDPAL region was functionally equivalent to the DDPLL region, we deleted this region from the pre-S protein of WHV or mutated individual amino acids within the region. The mutant DNA was transfected into human hepatoma cell line Huh7, and the medium was assayed for virion production by immunoprecipitation and Southern blot analysis. We found that an in-frame deletion of this small region inhibited virion formation, suggesting that the GDPAL region of the pre-S envelope protein was required for virus assembly and/or secretion of WHV. Individual replacement of alanine 204, leucine 205, or serine 206 with other amino acid residues did not affect virus production. However, substitution of either aspartic acid 202 with valine or proline 203 with leucine dramatically inhibited WHV production. Furthermore, the GDPAL mutants were individually tested for their abilities to complement a pre-S1 defective genome. The results showed that the GDPAL region functioned as part of the pre-S1 protein but was not required to function as part of the pre-S2 protein.     

Hepatitis B virus and hepatocellular carcinoma

Hepatitis B is a very important public health problem. Epidemiologic studies have shown a relationship between the hepatitis B virus (HBV) chronic carrier state and the development of hepatocellular carcinoma. HBV belongs to the Hepadna viruses family which includes the woodchuck hepatitis virus (WHV), Woodchucks infected with WHV represent a good experimental model to study the viral oncogenesis. In 85% of the studied cases, WHV acts by insertional mutagenesis in a gene of the myc family, mostly the N-myc2 gene. Expression of the myc genes is increased, suggesting the role of the viral enhancer. Study of transgenic mice have shown the liver specificity of the WHV action. In man, the liver oncogenesis is not explained. Studies are in progress to detect inactivation of tumor suppressor genes.     

Absence of mutations in the p53 tumor suppressor gene in woodchuck hepatocellular carcinomas associated with hepadnavirus infection and intake of aflatoxin B1

Infection with hepadnaviruses and exposure to aflatoxin B1 (AFB1) are considered major risk factors in the development of hepatocellular carcinoma (HCC) in humans and in animals. A high rate of mutations in the p53 tumor suppressor gene in hepatocellular carcinomas of predominantly hepatitis B virus (HBV) carrier patients has been recently related to dietary aflatoxin. Another member of the hepadnavirus family, the woodchuck hepatitis virus (WHV), infects woodchucks in a manner similar to that of HBV in humans. Therefore, it was of particular interest to determine whether the p53 gene in woodchuck HCCs associated with hepadnavirus infection and with exposure to AFB1 is affected in the same manner as in human HCCs. By direct PCR-sequencing, we analyzed exons 4-9 of the p53 gene in 13 HCCs from 12 woodchucks (two uninfected, ten WHV carriers). Six WHV carrier and two uninfected woodchucks were treated with AFB1. None of the analyzed HCC samples exhibited mutations, either in p53 gene exons 4-9, or in splicing donor-acceptor sites. The present data are consistent with our previous study that indicated a low rate of p53 mutations in HCCs of AFB1-treated ground squirrels, either infected or not infected with ground squirrel hepatitis virus, and in WHV carrier woodchucks not exposed to AFB1. Overall, our findings indicate that in woodchucks and in ground squirrels exposure to aflatoxin may affect the development of p53 mutations less than in humans.     

Structure and glycosylation patterns of surface proteins from woodchuck hepatitis virus

Woodchucks chronically infected with woodchuck hepatitis virus (WHV) are a valuable model for human hepatitis B virus (HBV) in studies of pathogenesis, immunity, and antiviral therapy. For this reason, substantial efforts to characterize both the similarities and the differences between HBV and WHV are being made. The structure of the WHV surface proteins (WHs proteins) has not yet been adequately elucidated. The bands that would be expected for glycosylated and nonglycosylated small (S) WHs protein are found by sodium dodecyl sulfate gel electrophoresis of purified WHs protein, but the bands corresponding to the middle (M) and large (L) WHs proteins of HBV are not seen at the expected sizes, even though the sequences of the WHV and HBV surface protein genes are 60% homologous. By amino-terminal sequencing we have identified two bands at 41 and 45 kDa as the MWHs proteins, 8 kDa larger than expected. We have also confirmed that two bands at 24 and 27 kDa are SWHs proteins. A protein of 49 kDa was blocked at the N terminus, which using immunoblotting with an antiserum against WHV pre-S1 (positions 126 to 146) was identified, together with a part of the 45-kDa protein, as glycosylated and nonglycosylated LWHs protein of the expected size. Sialidase and O-glycosidase digestion showed that the larger size of MWHs protein results from the presence of O glycoside groups which are probably in the pre-S2 domain of MWHs protein. Since the pre-S2 domains of HBV and WHV have similar numbers of potential O glycosylation sites, it appears to be likely that the glycosyltransferases act differently on the viral proteins in woodchucks and humans.     

Woodchuck hepatitis virus enhancer I and enhancer II are both involved in N-myc2 activation in woodchuck liver tumors

Direct activation of the N-myc2 oncogene by insertion of woodchuck hepatitis virus (WHV) DNA is a major oncogenic step in woodchuck hepatocarcinogenesis. We previously reported that WHV enhancer II (We2), which controls expression of the core/pregenome RNA, can also activate the N-myc2 promoter in hepatoma cell lines. To better define the integrated WHV regulatory sequences responsible for N-myc2 promoter activation in woodchuck liver tumors, we analyzed the structure and enhancer activity of a single viral integrant found at the win locus in tumor 2260T1 and mapping approximately 175 kb 3' of N-myc2. This viral insert was made of 11 concatemerized WHV fragments, 5 of which overlapped with We2 sequences and 1 with WHV sequence homologous to that of hepatitis B virus enhancer I (We1). In transient transfection assays in hepatoma-derived cells, the We2 activator was found to be fully effective only when inserted in close proximity to the N-myc2 promoter whereas the We1 element by itself was apparently devoid of activity. In contrast, the 2260T1 viral insert exhibited a potent enhancer capacity that depended both on multimerized We2 and on We1 sequences. In a survey of different woodchuck hepatomas, both elements were commonly found within integrated viral sequences involved in long-range N-myc2 activation.     

Role of surgical interventions in the treatment of patients with trophoblastic tumor resistant to chemotherapy

A novel strategy for anti-viral intervention of hepatitis B virus (HBV) through the disruption of the proper folding and transport of the hepadnavirus glycoproteins is described. Laboratory reared woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated with N-nonyl-deoxynojirimycin (N-nonyl-DNJ), an inhibitor of the endoplasmic reticulum (ER) alpha-glucosidases. The woodchucks experienced significant dose dependent decreases in enveloped WHV, resulting in undetectable amounts in some cases. The reduction in viremia correlated with the levels of hyperglucosylated glycan in the serum of treated animals. This correlation supports the mechanism of action associated with the drug and highlights the extreme sensitivity of the virus to this type of glycan inhibitor. At N-nonyl-DNJ concentrations that prevented WHV secretion, the glycosylation of most serum glycoproteins appeared unaffected, suggesting great selectivity for this class of therapeutics. Indeed, this may account for the low toxicity of the compound over the treatment period. We provide the first evidence that glucosidase inhibitors can be used in vivo to alter specific steps in the N-linked glycosylation pathway and that this inhibition has anti-viral effects.     

Color mixing in dental porcelain

One hundred and two apparently healthy Indian domestic ducks from the Poultry Research Station, Madras were screened for duck hepatitis B virus (DHBV) infection by; 1. screening for the duck hepatitis B virus surface antigen (DHBsAg) in their sera using hepatitis B virus (HBV) reagents, 2. screening for DHBsAg using specific duck hepatitis B virus (DHBV) reagents and 3. demonstration of DHBV DNA using DHBV DNA probe by dot blot hybridisation. While 5 ducks (4.9%) were consistently positive with HBV reagents, use of DHBV reagents showed a total of 4 ducks (including 3 of the above 5) to be positive for DHBsAg. DNA hybridisation showed 6 ducks to be positive for DHBV DNA. On clinical examination, 5 out of these 6 ducks did not reveal abnormalities, the other one showed hepatomegaly and ascites. Post-mortem studies showed the presence of nodules on the surface of the liver in all 5 which were positive with HBV reagents including the one with hepatomegaly. On histopathological evaluation, they were found to be hepatocellular carcinoma with or without bile duct carcinoma. The present study is a pilot report on the occurrence of DHBV infection in Indian domestic ducks and the possibility of antigenic cross reactivity between human HBV and duck hepatitis B virus antigens.     

Inhibition of human hepatitis B virus replication by AT-61, a phenylpropenamide derivative, alone and in combination with (-)beta-L-2',3'-dideoxy-3'-thiacytidine

AT-61, a member of a novel class of phenylpropenamide derivatives, was found to be a highly selective and potent inhibitor of human hepatitis B virus (HBV) replication in four different human hepatoblastoma cell lines which support the replication of HBV (i.e., HepAD38, HepAD79, 2.2.15, and transiently transfected HepG2 cells). This compound was equally effective at inhibiting both the formation of intracellular immature core particles and the release of extracellular virions, with 50% effective concentrations ranging from 0.6 to 5.7 microM. AT-61 (27 microM) was able to reduce the amount of HBV covalently closed circular DNA found in the nuclei of HepAD38 cells by >99%. AT-61 at concentrations of >27 microM had little effect on the amount of viral RNA found within the cytoplasms of induced HepAD38 cells but reduced the number of immature virions which contained pregenomic RNA by >99%. The potency of AT-61 was not affected by one of the mutations responsible for (-)-beta-L-2', 3'-dideoxy-3' thiacytidine (3TC) resistance in HBV, and AT-61 acted synergistic with 3TC to inhibit HBV replication. AT-61 (81 microM) was not cytotoxic or antiproliferative to several cell lines and had no antiviral effect on woodchuck or duck HBV, human immunodeficiency virus type 1, herpes simplex virus type 1, vesicular stomatitis virus, or Newcastle disease virus. Therefore, we concluded that the antiviral activity of AT-61 is specific for HBV replication and most likely occurs at one of the steps between the synthesis of viral RNA and the packaging of pregenomic RNA into immature core particles.     

Urea denaturation of staphylococcal nuclease monitored by Fourier transform infrared spectroscopy

Hepadnavirus invasion in woodchucks has been identified as a potent inducer of autoantibodies against asialoglycoprotein receptor (anti-ASGPR), a molecule essentially unique to hepatocytes that mediate clearance of desialylated serum proteins. We evaluated the possible pathogenetic importance of anti-ASGPR triggered by woodchuck hepatitis virus (WHV), using anti-ASGPR-reactive serum immunoglobulins (Igs) from five animals with different stages of WHV hepatitis or self-limited WHV infection and isolated woodchuck hepatocytes or HepG2 cells as targets. The results revealed that WHV-induced anti-ASGPR can specifically inhibit asialoglycoprotein recognition by both homologous and heterologous liver cells, as tested in an asialofetuin (ASFN)-binding radioassay. However, the extent of the interference significantly varied (from 85% inhibition to none) for anti-ASGPR with similar titer from different animals, indicating a high degree of heterogeneity in the ASGPR epitope specificity and in the potential biological effects of these autoantibodies. The WHV-triggered anti-ASGPR also induced complement-mediated hepatocytolysis in a microculture tetrazolium (MTT) assay, which ranged from 8.9% +/- 0.3% to 33.6% +/- 3.6% (mean +/- SD) for different animals and target cell numbers. This cytopathic effect was strictly ASGPR-specific, complement-dependent, and was not related to the anti-ASGPR ability to inhibit ligand-hepatocyte binding. Our findings indicate that among pathways by which anti-ASGPR autoimmunity could cause liver damage, hepadnavirus-induced anti-ASGPR might impair hepatocytes by both disrupting clearance of desialylated proteins and activation of the complement-mediated cytolysis. These cytopathic effects might contribute to the pathogenesis, aggravate severity, and prolong recovery from liver injury in viral hepatitis.     

Metabolic labeling of woodchuck hepatitis B virus X protein in naturally infected hepatocytes reveals a bimodal half-life and association with the nuclear framework

In 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.     

Covalently closed circular viral DNA formed from two types of linear DNA in woodchuck hepatitis virus-infected liver

We found that livers from woodchucks chronically infected with woodchuck hepatitis virus (WHV) contained covalently closed circular DNA (cccDNA) molecules with deletions and insertions indicative of their formation from linear viral DNA by nonhomologous recombination, as we previously described for the duck hepatitis B virus (W. Yang and J. Summers, J. Virol. 69:4029-4036, 1995). However, evidence for two different types of linear precursors was obtained by analysis of the recombination joints in WHV cccDNA. Type 1 linear precursors possessed the structural properties that correspond to those of in situ-primed linear DNA molecules, which constitute between 7 and 20% of all viral DNA replicative intermediates synthesized in the liver. Type 2 linear precursors are hypothetical species of linear DNAs with a terminal duplication of the cohesive-end region, between DR1 and DR2. This type of linear DNA has not been previously described and was not detected among the DNA species present in nucleocapsids. A fraction of cccDNAs formed from both type 1 and type 2 linear DNAs are predicted to be functional for further DNA synthesis, and some evidence for the formation of two or more generations of cccDNA from linear DNA was observed.     

T-Cell response to woodchuck hepatitis virus (WHV) antigens during acute self-limited WHV infection and convalescence and after viral challenge

The infection of woodchucks with woodchuck hepatitis virus (WHV) provides an experimental model to study early immune responses during hepadnavirus infection that cannot be tested in patients. The T-cell response of experimentally WHV-infected woodchucks to WHsAg, rWHcAg, and WHcAg peptides was monitored by observing 5-bromo-2'-deoxyuridine and [2-3H]adenine incorporation. The first T-cell responses were directed against WHsAg 3 weeks after infection; these were followed by responses to rWHcAg including the immunodominant T-cell epitope of WHcAg (amino acids 97 to 110). Maximal proliferative responses were detected when the animals seroconvered to anti-WHs and anti-WHc (week 6). A decrease in the T-cell response to viral antigens coincided with clearance of viral DNA. Polyclonal rWHcAg-specific T-cell lines were established 6, 12, 18, and 24 weeks postinfection, and their responses to WHcAg peptides were assessed. Five to seven peptides including the immunodominant epitope were recognized throughout the observation period (6 months). At 12 months after infection, T-cell responses to antigens and peptides were not detected. Reactivation of T-cell responses to viral antigens and peptides occurred within 7 days after challenge of animals with WHV. These results demonstrate that a fast and vigorous T-cell response to WHsAg, rWHcAg, and amino acids 97 to 110 of the WHcAg occurs within 3 weeks after WHV infection. The peak of this response was associated with viral clearance and may be crucial for recovery from infection. One year after infection, no proliferation of T cells in response to antigens was observed; however, the WHV-specific T-cell response was reactivated after challenge of woodchucks with WHV and may be responsible for protection against WHV reinfection.     

Biodistribution, stability, and antiviral efficacy of liposome-entrapped phosphorothioate antisense oligodeoxynucleotides in ducks for the treatment of chronic duck hepatitis B virus infection

This study investigated the feasibility of using liposomes to increase the hepatic delivery and antiviral efficacy of phosphorothioate antisense oligodeoxynucleotides (PS-ODN) for the in vivo treatment of hepatitis B virus (HBV) infection. Ducks infected with duck hepatitis B virus (DHBV) were used as the model. We studied the stability of an antisense PS-ODN in duck plasma, its integrity during the process of liposome entrapment, its in vivo biodistribution, plasma clearance, and excretion. In addition, the intrahepatic distribution of a labeled free and liposome-entrapped ODN was also investigated. The results of our studies show that: 1) phosphorothioate ODN remain stable during the process of liposome entrapment; 2) are stable in duck plasma for many hours; 3) are rapidly cleared from the plasma when injected intravenously; 4) intravenous injection of antisense ODNs entrapped within liposomes enhances delivery of the ODN to the liver; and 5) inhibit DHBV replication. Serum DHBV DNA levels fell rapidly, with a corresponding decrease in intrahepatic viral replicative intermediates at the end of the 5-day study period. Although inhibition of viral replication and a fall in the target protein was observed, a marked inhibition of viral replication was also observed with high doses of a random-sequence ODN. Thus, it is not certain that inhibition of viral replication was entirely through an antisense mechanism. Therefore, liposomes may be effective vehicles to improve the delivery of antisense oligonucleotides to the liver for the therapy of hepatotropic viruses.