Duck Hepatitis B Virus DNA Research Articles

Glucagon treatment interferes with an early step of duck hepatitis B virus infection.

The effect of glucagon on the establishment of hepadnavirus infection was studied in vitro with the duck hepatitis B virus (DHBV) model. The presence of the peptide hormone throughout infection or starting up to 8 h after virus uptake resulted in a dose-dependent reduction in the levels of intra- and extracellular viral gene products and of secreted virions. Treatment with forskolin or dibutyryl-cyclic AMP, two drugs that also stimulate the cyclic AMP (cAMP) signal transduction pathway, resulted in comparable inhibition, suggesting that the inhibitor effect is related to changes in the activity of protein kinase A. In persistently infected hepatocytes, only a slight, but continuous, decrease in viral replication was observed upon prolonged drug treatment. Time course analysis, including detection of DHBV covalently closed circular (ccc) DNA templates, revealed that glucagon acts late during the establishment of infection, at a time when the virus is already internalized, but before detectable ccc DNA accumulation in the nucleus. These data suggest that nuclear import (and reimport) of DHBV DNA genomes from cytosolic capsids is subject to cAMP-mediated regulation by cellular factors responding to changes in the state of the host cell.

Inhibition of human and duck hepatitis B virus by 2′,3′-dideoxy-3′-fluoroguanosine in vitro

The fluorinated guanosine analog 2′,3′-dideoxy-3′-fluoroguanosine (FLG) has been shown to have an effect on duck hepatitis B virus (DHBV) in vivo and in vitro. In this study the inhibitory effect of FLG on DHBV and human hepatitis B virus (HBV) was evaluated in vitro. Cell lines transfected either with DHBV or HBV DNA and primary duck hepatocyte cell cultures were used. Virus production was analysed by PCR and a quantitative PCR was established for DHBV for determination of the inhibitory concentrations of the drug. 50% inhibition was achieved with an FLG concentration of 0.2 μg/ml (0.7 μM) and 90% inhibition was observed with an FLG concentration of 1.0 μg/ml (3.7 μM) using the DHBV transfected cell line. FLG showed an effect on DHBV production in primary duck hepatocyte cell cultures at concentrations down to 0.1 μg/ml (0.4 μM). However, the DHBV production returned to pre-treatment levels within a few days after cessation of treatment. HBV production in transfected cell lines was also inhibited by FLG. Both DHBV and HBV DNA-polymerases were inhibited by FLG triphosphate and 50% inhibition was observed at a concentration of 0.05 μg/ml (0.1 μM) for DHBV and 0.03 μg/ml (0.05 μM) for HBV. FLG is an efficient inhibitor of DHBV replication both in vivo and in vitro and of HBV in vitro which makes it a good candidate for treatment of HBV infections. However, it does not completely eliminate the virus since a relapse in virus production was observed when treatment was withdrawn. Therefore it would be interesting to evaluate FLG in combination with other types of anti-HBV drugs.

Comparison of Antiviral Effects of Mismatched Double-Stranded RNA and 1 -(2′-Deoxy-2′,-Fluoro-β-D-Arabinofuranosyl)-5-Methyluracil (D-FMAU) against Duck Hepatitis B virus in Vitro

The effects of mismatched double-stranded RNA (m-dsRNA) and 1-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)-5-methyl-uracil (D-FMAU) on the replication of duck hepatitis B virus (DHBV) were examined in duck primary hepatocytes infected with DHBV. The 50% inhibitory doses of m-dsRNA and D-FMAU for the replication of DHBV DNA were 0.34±0.06 and 0.007±0.001 μg mL -1 , respectively. Mismatched dsRNA slightly inhibited the intermediate DHBV DNA at a concentration as high as 1.0 μg mL -1 , whereas the DHBV replicative form was markedly suppressed in the presence of 0.1 μg mL -1 of D-FMAU. On the other hand, m-dsRNA inhibited DHBV DNA replication even after the compound was removed from the culture medium, while the efficacy of D-FMAU did not persist after the cessation of treatment. The analysis of DHBV RNA revealed that m-dsRNA markedly inhibited DHBV RNA transcription, while D-FMAU only marginally suppressed the transcription of viral RNA. These results indicate that m-dsRNA inhibits DHBV RNA transcription rather than DHBV DNA replication and that this suppression of DHBV RNA transcription may produce persistent inhibition of DHBV replication.

A novel transcriptional element in circular DNA monomers of the duck hepatitis B virus.

We report the presence of two elements, pet and net, that are required for proper transcription of the duck hepatitis B virus (DHBV). These regions were previously identified by using plasmid clones of the virus in transient expression assays (M. Huang and J. Summers, J. Virol. 68:1564-1572, 1994). In this study, we further analyzed these regions by using in vitro-synthesized circular DHBV DNA monomers to mimic the authentic transcriptional template. We observed that pet was required for pregenome transcription from circular viral monomers, and in the absence of pet-dependent transcription, expression of the viral envelope genes was increased. We found that deletion of net in circularized DNA monomers led to the production of abnormally long transcripts due to a failure to form 3' ends during transcription. In addition, we report the presence of a net-like region in the mammalian hepadnavirus woodchuck hepatitis virus. These results are consistent with a model that net is a region involved in transcription termination and that in DHBV, pet is required for transcription complexes to read through this region during the first pass through net.

Increase in the frequency of hepadnavirus DNA integrations by oxidative DNA damage and inhibition of DNA repair.

Persistent hepadnavirus infection leads to oxidative stress and DNA damage through increased production of toxic oxygen radicals. In addition, hepadnaviral DNA integrations into chromosomal DNA can promote the process of hepatocarcinogenesis (M. Feitelson, Clin. Microbiol. Rev. 5:275-301, 1992). While previous studies have identified preferred integration sites in hepadnaviral genomes and suggested integration mechanisms (M. A. Buendia, Adv. Cancer Res. 59:167-226, 1992; C. E. Rogler, Curr. Top. Microbiol. Immunol. 168:103-141, 1991; C. Shih et al., J. Virol. 61:3491-3498, 1987), very little is known about the effects of agents which damage chromosomal DNA on the frequency of hepadnaviral DNA integrations. Using a recently developed subcloning approach to detect stable new integrations of duck hepatitis B virus (DHBV) (S. S. Gong, A. D. Jensen, and C. E. Rogler, J. Virol. 70:2000-2007, 1996), we tested the effects of increased chromosomal DNA damage induced by H2O2, or of the disturbance in DNA repair due to the inhibition of poly(ADP-ribose) polymerase (PARP), on the frequency of DHBV DNA integrations. Subclones of LMH-D21-6 cells, which replicate DHBV, were grown in the presence of various H2O2 concentrations and exhibited up to a threefold increase in viral DNA integration frequency in a dose-dependent manner. Moreover, inhibition of PARP, which plays a role in cellular responses to DNA breakage, by 3-aminobenzamide (3-AB) resulted in a sevenfold increase in the total number of new DHBV DNA integrations into host chromosomal DNA. Removal of either H2O2 or 3-AB from the culture medium in a subsequent cycle of subcloning was accompanied by a reversion back towards the original lower frequency of stable DHBV DNA integrations for LMH-D21-6 cells. These data support the hypothesis that DNA damage sites can serve as sites for hepadnaviral DNA integration, and that increasing the number of DNA damage sites dramatically increases viral integration frequency.

Potential antiviral effects ofTerminalis chebula, Sanguisorba officinalis, Rubus coreanus and Rheum palmatum against duck hepatitis B virus (DHBV)

The potential antiviral effects against duck hepatitis B virus were investigated using aqueous extracts of Terminalis chebula, Sanguisorba officinalis, Rubus coreanus and Rheum palmatum. Previously, these herbal extracts were shown to inhibit hepatitis B virus (HBV) DNA polymerase activity and to bind hepatitis B virus surface antigen (HBsAg). Antiviral effects of herbal extracts were determined using Peking ducks (Anas domesticus) infected with duck hepatitis B virus (DHBV). During a 4-week treatment period with selected plant extracts, serum DHBV DNA levels and DHBV DNA polymerase activity were measured as antiviral indicators. Although all extracts demonstrated antiviral activity against DHBV, Terminalis chebula appeared to exhibit the highest antiviral activity. © 1997 John Wiley & Sons, Ltd.

Potential antiviral effects of Terminalis chebula, Sanguisorba officinalis, Rubus coreanus and Rheum palmatum against duck hepatitis B virus (DHBV)

The potential antiviral effects against duck hepatitis B virus were investigated using aqueous extracts of Terminalis chebula, Sanguisorba officinalis, Rubus coreanus and Rheum palmatum. Previously, these herbal extracts were shown to inhibit hepatitis B virus (HBV) DNA polymerase activity and to bind hepatitis B virus surface antigen (HBsAg). Antiviral effects of herbal extracts were determined using Peking ducks (Anas domesticus) infected with duck hepatitis B virus (DHBV). During a 4-week treatment period with selected plant extracts, serum DHBV DNA levels and DHBV DNA polymerase activity were measured as antiviral indicators. Although all extracts demonstrated antiviral activity against DHBV, Terminalis chebula appeared to exhibit the highest antiviral activity. © 1997 John Wiley & Sons, Ltd.

Demonstration of duck hepatitis B virus in bile duct epithelial cells: implications for pathogenesis and persistent infection.

Hepatitis B virus (HBV) has been demonstrated in bile duct epithelial cells (BDEC) during chronic infection. The persistence of virus in BDEC may play an important role in disease pathogenesis, and may be at least partly responsible for the relapse phenomenon observed in antiviral treatments using nucleoside analogues. The aims of this study were to examine the morphological changes within the liver in the duck hepatitis B model following bile duct ligation (BDL), and to assess the effect of biliary hyperplasia upon viral DNA and proteins. Seven-day-old ducklings, congenitally infected with the duck hepatitis B virus (DHBV), were subject to BDL. The pathological and virological changes were then followed at 5, 10, 15, and 20 days after ligation. All results were compared with age-matched unligated control birds congenitally infected with DHBV. To assess the early morphological changes, additional animals were sacrificed at 1, 2, 3, and 4 days post-BDL. The proportion of DHBV-infected BDEC, was examined by immunohistochemistry and in situ hybridization. BDL induced rapid biliary hyperplasia, with a doubling time for BDEC of 1.3 days. The proliferated BDEC displayed immunohistochemical features identical to resting BDEC. More than 50% of BDEC in unligated controls, and more than 46% of proliferated BDEC in ligated animals were positive for DHBV DNA and structural proteins. The intensity of immunohistochemical staining and in situ hybridization signal in the BDEC was consistently greater than that of the hepatocytes, both before and after BDL. BDL induces biliary hyperplasia in the duck model, and BDEC division does not reduce the viral burden in infected cells.

Demonstration of duck hepatitis B virus in bile duct epithelial cells: Implications for pathogenesis and persistent infection

Hepatitis B virus (HBV) has been demonstrated in bile duct epithelial cells (BDEC) during chronic infection. The persistence of virus in BDEC may play an important role in disease pathogenesis, and may be at least partly responsible for the relapse phenomenon observed in antiviral treatments using nucleoside analogues. The aims of this study were to examine the morphological changes within the liver in the duck hepatitis B model following bile duct ligation (BDL), and to assess the effect of biliary hyperplasia upon viral DNA and proteins. Seven-day-old ducklings, congenitally infected with the duck hepatitis B virus (DHBV), were subject to BDL. The pathological and virological changes were then followed at 5, 10, 15, and 20 days after ligation. All results were compared with age-matched unligated control birds congenitally infected with DHBV. To assess the early morphological changes, additional animals were sacrificed at 1, 2, 3, and 4 days post-BDL. The proportion of DHBV-infected BDEC, was examined by immunohistochemistry and in situ hybridization. BDL induced rapid biliary hyperplasia, with a doubling time for BDEC of 1.3 days. The proliferated BDEC displayed immunohistochemical features identical to resting BDEC. More than 50% of BDEC in unligated controls, and more than 46% of proliferated BDEC in ligated animals were positive for DHBV DNA and structural proteins. The intensity of immunohistochemical staining and in situ hybridization signal in the BDEC was consistently greater than that of the hepatocytes, both before and after BDL. BDL induces biliary hyperplasia in the duck model, and BDEC division does not reduce the viral burden in infected cells.(Hepatology 1997 Feb;25(2):463-9)

DHBV manipulation and prediction of the outcome of infection

Background/Aims: The immunological response of ducks to acute infection with duck hepatitis B virus (DHBV) has not been fully characterised. In this study the relationship between viral dose and the outcome of infection in immune competent 26-day-old ducks was examined. Methods: Indirect ELISA assays were deployed to detect the presence of antibody to DHB surface antigen and DHB core antigen. A DHBV serum pool was titrated in 1-day-old and 26-day-old ducklings. Results: The ID 50 dose of the ducks injected at 26 days of age was found to be 100 times that of the ducks injected on day of hatch. The antibody responses and serum DHBV DNA were followed in eight ducks inoculated with DHBV positive serum when 26 days of age and in three ducks infected with DHBV on day of hatch. The three ducks infected on day of hatch were viraemic by day 7 and remained highly viraemic throughout the experimental period. In the older ducks, inoculation with 1000ID 50 resulted in the development of chronic carriage, while inoculation with either 100 or 10ID 50 doses resulted in acute infection with or without viraemia. These ducks were able to clear the infection from their circulation, but only 50% cleared DHBV from the liver within the experimental period. All infected ducks developed anti-core activity. Only non-viraemic ducks developed anti-surface activity. Conclusions: DHBV infection can be established in immune competent adolescent ducks, with variable disease outcomes comparable to HBV infection in humans.

Relationship between viral DNA synthesis and virion envelopment in hepatitis B viruses

While the intracellular pool of encapsidated hepatitis B viral DNA contains genomes in all stages of DNA replication, serum-derived virions contain predominantly mature, partially duplex, circular DNA genomes. To account for this finding, Summers and Mason proposed in 1982 that virion envelopment is somehow linked to the state of genomic maturation (J. Summers and W.S. Mason, Cell 29:403-415, 1982). Core gene mutations with phenotypes consistent with this concept have previously been identified in the duck hepatitis B virus (DHBV). Here we show that DHBV polymerase mutants with altered DNA synthesis also display defects in envelopment, and we provide quantitative estimates of the magnitude of the preference for the envelopment of mature DNA. In cells transfected with wild-type DHBV DNA, immature minus-strand DNA represents 18% of the intracellular pool but only 4% of extracellular virion DNA. A point mutation in the C-terminal domain of the polymerase strongly and selectively impairs plus-strand synthesis; in this mutant, the ratio of immature to mature DNA in the intracellular pool rises to 6:1 but is reduced to 1.5:1 in released virions. A missense mutation in the polymerase active site inactivates all viral DNA synthesis but still allows efficient RNA encapsidation; in this mutant, no detectable viral nucleic acid is enveloped and released. Thus, viral DNA synthesis is absolutely required for envelopment and export, and a strong further bias exists in favor of the export of genomes that have completed minus-strand synthesis and at least initiated plus-strand synthesis. These results imply that events within the interior of the nucleocapsid can powerfully influence its interactions with external viral envelope glycoproteins.

Antiviral Activities and Pharmacokinetics of Penciclovir and Famciclovir in Pekin Ducks Chronically Infected with Duck Hepatitis B Virus

Famciclovir (FCV) is the oral form of the potent and selective antiherpesvirus agent penciclovir (PCV). In order to provide more information on the spectrum of activity of PCV, the activities of FCV and PCV against duck hepatitis B virus (DHBV) in chronically infected ducks were examined. As part of this investigation, the oral pharmacokinetics of FCV and PCV were determined in ducks. Oral treatment of DHBV-infected ducks (twice daily for 21 days) with either PCV (20 mg kg−1 or 100 mg kg−1) or FCV (5 mg kg−1 or 25 mg kg−1) suppressed virus replication, as measured by both plasma viral DNA and DNA polymerase levels. Both markers were reduced to undetectable levels within 4 days of the start of treatment, and after the cessation of treatment there was a delay of 2 to 8 days before plasma DHBV DNA and DNA polymerase levels began to increase, indicating that virus replication had resumed. The demonstration of efficacy of PCV and its oral form FCV against DHBV suggested that the two compounds may have clinical benefits. against human hepatitis B virus. Clinical trials are currently ongoing.

2',3'-dideoxy-3'-fluoroguanosine inhibits duck hepatitis B virus in vivo.

Duck hepatitis B virus (DHBV) belongs to the same virus family as the human hepatitis B virus (HBV). Domestic ducks infected with DHBV can be used as an animal model for chronic hepatitis B virus infection in therapeutic trials. In this study the antiviral effect of the guanosine analogue 2',3'-dideoxy-3'-fluoroguanosine (FLG) was tried in vivo on chronically DHBV-infected ducks. The ducks were either congenitally infected, or inoculated with DHBV immediately post-hatch. FLG was given as intraperitoneal injections twice daily, at different dosages. Serum DHBV levels were determined by DNA dot-blot hybridization. A strong inhibition of serum DHBV DNA was observed with FLG doses down to 1 mg kg-1 day-1, given for 7 to 10 days. With the corresponding thymidine analogue, 2',3'-dideoxy-3'-fluorothymidine; however, no inhibition was obtained. This difference may be due to different phosphorylation mechanisms. Independently of FLG dose, serum DHBV DNA returned to pretreatment levels within a few days after cessation of therapy. After a long-term trial (FLG, 5 mg kg-1 day-1 for 33 days), the same relapse of DHBV production was seen. Thus, FLG is an efficient inhibitor of DHBV replication, and is a candidate for treatment of HBV infections. However, the effect is transient, and therefore combination with other types of anti-HBV drugs should be considered.

Inhibition of duck hepatitis B virus replication by 2',3'-dideoxy-3'-fluoroguanosine in vitro and in vivo.

The antiviral activity of 2',3'-dideoxy-3'-fluoroguanosine (FdG) or its triphosphate was evaluated in the duck hepatitis B virus (DHBV) system in vitro and in vivo. In primary DHBV-infected hepatocytes FdG results in a dose-dependent inhibition of viral replication with a nearly complete inhibition at a concentration of 1 microM. Also in vivo, FdG treatment of DHBV-infected ducklings reduces DHBV DNA replication by more than 90%. These data demonstrate that FdG is a strong inhibitor of DHBV replication in vitro and in vivo.

2',3'-dideoxy-beta-L-5-fluorocytidine inhibits duck hepatitis B virus reverse transcription and suppresses viral DNA synthesis in hepatocytes, both in vitro and in vivo

beta-L-Nucleoside analogs represent a new class of potent antiviral agents with low cytotoxicity which provide new hope in the therapy of chronic hepatitis B virus (HBV) infections. We evaluated the anti-HBV activity of 2',3'-dideoxy-beta-L-5-fluorocytidine (beta-L-F-ddC), a beta-L-nucleoside analog derived from 2',3'-dideoxycytidine (ddC), in the duck HBV (DHBV) model. This compound was previously shown to inhibit HBV DNA synthesis in a stably transfected hepatoma cell line (F2215). Using a cell-free system for the expression of an enzymatically active DHBV polymerase, we could demonstrate that the triphosphate form of beta-L-F-ddC does inhibit hepadnavirus reverse transcription. In primary duck hepatocyte culture, beta-L-F-ddC showed a potent inhibitory effect on DHBV DNA synthesis which was concentration dependent. Although beta-L-F-ddC was shown to be less active than ddC against the DHBV reverse transcriptase in vitro, beta-L-F-ddC was a stronger inhibitor in hepatocytes. The oral administration of beta-L-F-ddC in experimentally infected ducklings showed that beta-L-F-ddC is a potent inhibitor of viral replication in vivo. Short-term therapy could not prevent a rebound of viral replication after the drug was withdrawn. Preventive therapy with beta-L-F-ddC could delay the onset of viremia by only 1 day compared with the time to the onset of viremia in the control group. The in vivo inhibitory effect of beta-L-F-ddC was much stronger than that of ddC and was not associated with signs of toxicity. Our data show that beta-L-F-ddC inhibits hepadnavirus reverse transcription and is a strong inhibitor of viral replication both in vitro and in vivo.

Selective inhibition of the reverse transcription of duck hepatitis B virus by binding of 2',3'-dideoxyguanosine 5'-triphosphate to the viral polymerase

Hepatitis B virus (HBV) replication is mediated by the viral polymerase that possesses three functional domains: primer, DNA polymerase/reverse transcriptase, and RNase H. Using the Pekin duck as an animal model, we demonstrate a novel mechanism of inhibition of duck hepatitis B virus (DHBV) by 2,6-diaminopurine 2',3'-dideoxyriboside (ddDAPR), a prodrug of 2',3'-dideoxyguanosine (ddG). A selective and irreversible inhibition of DHBV DNA replication is found in ducklings treated with high doses of ddDAPR (20 to 50 mg/kg), but not with similar doses of 2',3'-dideoxycytidine (ddC). The inhibition mediated by ddDAPR occurs at a very early stage of the reverse transcription. Despite the inhibition of DHBV DNA replication by ddDAPR, the DNA polymerase and reverse transcriptase activities of the polymerase are found to remain active when tested on exogenous templates in activity gels. We have demonstrated direct binding of [alpha-32P]ddGTP to the DHBV polymerase expressed in an in vitro transcription and translation system. These results suggest that the binding of ddGTP to the polymerase blocks the initial DNA replication. (Hepatology 1996 Jan;23(1):87-96)

Selective inhibition of the reverse transcription of duck hepatitis B virus by binding of 2',3'-dideoxyguanosine 5'-triphosphate to the viral polymerase.

Hepatitis B virus (HBV) replication is mediated by the viral polymerase that possesses three functional domains: primer, DNA polymerase/reverse transcriptase, and RNase H. Using the Pekin duck as an animal model, we demonstrate a novel mechanism of inhibition of duck hepatitis B virus (DHBV) by 2,6-diaminopurine 2',3'-dideoxyriboside (ddDAPR), a prodrug of 2',3'-dideoxyguanosine (ddG). A selective and irreversible inhibition of DHBV DNA replication is found in ducklings treated with high doses of ddDAPR (20 to 50 mg/kg), but not with similar doses of 2',3'-dideoxycytidine (ddC). The inhibition mediated by ddDAPR occurs at a very early stage of the reverse transcription. Despite the inhibition of DHBV DNA replication by ddDAPR, the DNA polymerase and reverse transcriptase activities of the polymerase are found to remain active when tested on exogenous templates in activity gels. We have demonstrated direct binding of [alpha-32P]ddGTP to the DHBV polymerase expressed in an in vitro transcription and translation system. These results suggest that the binding of ddGTP to the polymerase blocks the initial DNA replication.

Spectrum of liver disease and duck hepatitis B virus infection in a large series of chinese ducks with hepatocellular carcinoma

The striking difference in the geographical distribution of liver cancer in ducks raised the question of whether duck hepatitis B virus (DHBV), like mammalian hepadnaviruses, could be an oncogenic agent. Hepatocellular carcinomas (HCCs) have been found only in domestic ducks in Qidong, China, where hepatitis B virus infection and aflatoxin B 1 (AFB 1) are both risk factors and where a high frequency of human HCCs has been reported. To date, the study of liver pathology occurring in Chinese ducks has been hampered by the small number of samples available. We describe here a series of 59 Chinese brown duck livers that were collected in Qidong more than 20 years ago and formalin fixed. Thirty-six HCCs, which ranged from well-differentiated trabecular to highly anaplastic type, were identified in relatively young ducks (average age, 3.3 years). Several unique features not previously reported, such as tumor giant cells, tumor necrosis, tumor thrombi in blood vessels, and inactive cirrhosis, were observed. Bile ductule proliferation, known to be a prominent feature of AFB 1 exposure in ducks, was present in 86% of livers. Using polymerase chain reaction (PCR) and two primer pairs, located within conserved portions of the DHBV S and C genes, we demonstrated the presence of DHBV DNA in 23 of 34 HCCs analyzed (68%). The spectrum of liver pathology that we report in brown ducks from Qidong was never observed in Pekin ducks of the same age chronically infected with DHBV and followed under controlled conditions outside China, suggesting that causative factors other than virus infection may be involved in duck liver carcinogenesis observed in this area. However, because DHBV DNA was present in most HCCs, and given the lower sensitivity of PCR in formalin-fixed tissue, we cannot rule out the importance of DHBV infection in the liver disease of ducks from this study. The specific role of Chinese DHBV isolates and brown duck breed in liver pathology and oncogenesis observed in Qidong ducks should be further investigated in controlled experimental transmission studies with long-term follow-up in the absence of external carcinogens.

Spectrum of liver disease and duck hepatitis B virus infection in a large series of chinese ducks with hepatocellular carcinoma

The striking difference in the geographical distribution of liver cancer in ducks raised the question of whether duck hepatitis B virus (DHBV), like mammalian hepadnaviruses, could be an oncogenic agent. Hepatocellular carcinomas (HCCs) have been found only in domestic ducks in Qidong, China, where hepatitis B virus infection and aflatoxin B1 (AFB1) are both risk factors and where a high frequency of human HCCs has been reported. To date, the study of liver pathology occurring in Chinese ducks has been hampered by the small number of samples available. We describe here a series of 59 Chinese brown duck livers that were collected in Qidong more than 20 years ago and formalin fixed. Thirty-six HCCs, which ranged from well-differentiated trabecular to highly anaplastic type, were identified in relatively young ducks (average age, 3.3 years). Several unique features not previously reported, such as tumor giant cells, tumor necrosis, tumor thrombi in blood vessels, and inactive cirrhosis, were observed. Bile ductule proliferation, known to be a prominent feature of AFB1 exposure in ducks, was present in 86% of livers. Using polymerase chain reaction (PCR) and two primer pairs, located within conserved portions of the DHBV S and C genes, we demonstrated the presence of DHBV DNA in 23 of 34 HCCs analyzed (68%). The spectrum of liver pathology that we report in brown ducks from Qidong was never observed in Pekin ducks of the same age chronically infected with DHBV and followed under controlled conditions outside China, suggesting that causative factors other than virus infection may be involved in duck liver carcinogenesis observed in this area.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversion of duck hepatitis B virus DNA replication in vivo following cessation of treatment with the nucleoside analogue ganciclovir

In order to define, in more detail, the virological events which occur after completion of antiviral chemotherapy, ducks congenitally infected with the duck hepatitis B virus (DHBV) were treated for 4 weeks with the nucleoside analogue ganciclovir and followed up over a 7-day period. Specimens of serum and liver were collected daily during follow-up for virological analysis. Treatment resulted in a substantial reduction in both viraemia and liver DHBV DNA replicative intermediates. However, after cessation of treatment, viraemia returned to detectable levels within 4 days. In the liver, the viral supercoiled DNA (SC DNA) was the form least affected by therapy and returned to near control levels by day 2 post-treatment. The other hepatic replicative intermediates reached pretreatment levels within 4 days of cessation of therapy. This study has defined the kinetics of relapse of viral replication after completion of antiviral therapy in the duck hepatitis B model. Of all viral replicative forms, the SC DNA appears to be the one which is most resistant to nucleoside analogue therapy and is presumably responsible for the relapse phenomenon observed post-treatment.