Corticosterone mediates telomere length in raptor chicks exposed to chemical mixture.

Stressors experience early in life by animals may have carry over impacts on life-traits over the life cycle. Accelerated telomere attrition induced by stress during development and growth could play a role in such delayed effects. Among stressors, exposure to chemicals may modify telomere dynamic but, to date, the trends evidenced between exposure and telomere shortening remains inconsistent. Moreover, the role of corticosterone as a possible mediator of chemical impact on telomere is not yet clearly established. Here, we investigated in wild populations of Red kite whether nestling exposure to metals and pesticides was related to corticosterone concentrations in feathers and telomere length measured in 47 individuals. Lead and mercury concentrations in blood ranged from 2.3 to 59.0 µg L-1 and to 1.4 to 115.7 µg L-1, respectively, and were below the toxicity thresholds proposed for wildlife. Rodenticides were detected in 30% of the chicks. Corticosterone increased with mercury and lead in interaction, showing a synergistic effect of these 2 non-essential metals on this stress hormone. Telomere length was not linked to metals and/or rodenticide exposure while it was related negatively to corticosterone. The relationship between telomere and corticosterone was modulated by nestling's age, which suggests that the rate of telomere shortening is higher when corticosterone increases. Our findings propose an effect of low exposure of Red Kite nestlings to mercury and lead mixture to raise baseline corticosterone in feathers. The relationships established suggest the hypothesis that telomere attrition could be an indirect consequence of metal exposure mediated by corticosterone.

Intranasal dexmedetomidine in healthy beagles: An echocardiographic and pharmacokinetic/pharmacodynamic study.

The aim of this study was to compare the sedative and cardiovascular effects of dexmedetomidine (DEX) administrated via intranasal (IN) and intramuscular (IM) routes. This masked, randomised, crossover study used six male beagle dogs, receiving 0.02 mg/kg dexmedetomidine either IN (DEX-IN) or IM (DEX-IM), and an equal volume of saline by the alternative route. Dexmedetomidine plasma concentration was measured before (TB) and at time points (T) 2, 5, 10, 15, 30, 45, 60, 90 and 120 min after drug administration (T0). Physiological variables, sedation scores and sedation times were recorded until recovery. Echocardiography was performed at TB and between T20-T40. Repeated data over time were analysed using a Scheirer-Ray-Hare test. Other data were compared using a Wilcoxon or Student's t test. Times from T0 to sternal position and from lateral to sternal position were longer for DEX-IN than DEX-IM (P = 0.018 and P = 0.009, respectively). Time from sternal to standing position was shorter for DEX-IN than DEX-IM (P = 0.03). Dexmedetomidine plasma concentrations were significantly lower for DEX-IN than DEX-IM from T10 to T120. Heart rate was significantly lower for DEX-IM than DEX-IN from T5 to T20. Echocardiography showed a decrease in systolic function and calculated cardiac output in DEX-IM as compared to baseline. The DEX concentration-sedation curve for DEX-IN as compared to DEX-IM was leftward shifted, whereas the IN and IM DEX concentration-variation-in-heart-rate curves overlapped. Although reaching lower plasma concentrations, IN dexmedetomidine produced similar sedation to IM dexmedetomidine without affecting cardiovascular function.

Decreased replication capacity of a duck hepatitis B virus mutant with altered distal pre-S region.

We have observed in a previous study that insertion, deletion and partial frameshift mutation in the distal pre-S region did not abolish replication capacity of the duck hepatitis B virus (DHBV) (Li et al., 1989, J. Virol. 63, 4965-4968). To compare further the relative replication capacity between the pre-S mutant and wild type virus, ducts were infected with either the wild type DHBV strain or a pre-S mutant (FS-17) characterized by a total change of nine consecutive amino acid codons in the distal pre-S region. Compared with the wild type virus, FS-17 exhibited decreased replication capacity whether in separate or mixed infection. The decreased viral replication was correlated with delayed appearance of supercoiled DNA and viral RNA in the hepatocytes. Besides, FS-17 induced persistent viremia when inoculated into 1-day-old ducklings; hence the transient viremia which had been observed in the previous study was probably due to the time delay needed to generate compensatory deletion mutation.

Main properties of duck hepatitis B virus DNA polymerase: comparison with the human and woodchuck hepatitis B virus DNA polymerases.

The main properties of the duck hepatitis B virus (DHBV) DNA polymerase have been studied and compared with those of the human hepatitis B virus (HBV) and of the woodchuck hepatitis virus (WHV) DNA polymerases. All 3 enzymes are active under high salt conditions in the presence of high magnesium concentration. DHBV DNA polymerase was found less sensitive to ethanol and to operate at higher optimal pH than the HBV and WHV DNA polymerases. Like the other two viral endogenous DNA polymerases, the DHBV enzyme was strongly inhibited by phosphonoformic acid but not by aphidicolin, sulfhydryl group blockers or phosphonoacetic acid. Inhibition of DHBV DNA polymerase by the triphosphate derivatives of several nucleoside analogs appeared similar to that reported for HBV or WHV endogenous polymerase. FIACTP was the most, and ACVTP the least effective inhibitor; BVdUTP was of intermediary potency; araCTP and araTTP had a greater inhibitory effect on DHBV DNA polymerase than HBV or WHV DNA polymerase. The similarities in the properties of DHBV and HBV DNA polymerase justify the use of the duck hepatitis B polymerase model for screening and evaluation of potentially active drugs against HBV infection.

Histopathological assessment of tumoral destruction by alcoholization in an experimental model of human hepato-cellular carcinoma transplanted in nude mice.

This study aims to assess the efficacy of intra-tumoral alcoholization as a potential therapy against small hepatocellular carcinoma (HCC). The experimental model consisted of 20-mm diameter tumors resulting from subcutaneous abdominal injection of Hep G2 human hepatoma cell lines into nude mice (nu/nu). Alcoholization was performed using either a single centro-tumoral injection or multiple cross-shaped injections (tangential and oblique), during one sequence with a total dose of 0.1, 0.2 or 0.3 ml of 95% ethanol. Efficacy was assessed by the percentage of tumoral necrosis using a semi-quantitative method. Fifty-four tumors were alcoholized. Forty-eight hours following alcoholization, the tumor was regularly replaced by a necrotic ulcer. A single centro-tumoral injection always resulted in the persistence of a small peripheral edge of tumor cells even with the highest dose used (no dose-related effect). By contrast, the use of multiple injections restored a dose-related effect. Oblique cross-shaped injections of 0.3 ml of ethanol resulted in the highest mean tumoral necrosis rate (m +/- SD = 99 +/- 1%; range = 97-100). This study confirms that alcoholization is an efficient way to destroy hepatocarcinoma tissue and suggests that ethanol diffusion in HCC is less than 20 mm. In addition it demonstrates that increasing number and location of injections restores a dose-related effect, while efficacy is improved using oblique injections.

VKORC1 mutations detected in patients resistant to vitamin K antagonists are not all associated with a resistant VKOR activity.

BACKGROUND: The VKORC1 gene codes for the VKORC1 enzyme, which is responsible for the reduction of vitamin K epoxide into vitamin K. VKORC1 enzyme is the target of vitamin K antagonists (VKA). Twenty-eight rare single mutations in the VKORC1 coding sequence have been reported from resistant patients receiving unusually high doses of VKA to achieve therapeutic anticoagulation. OBJECTIVES: It has been suggested that these mutations are responsible for the resistant phenotype, while biochemical consequences of these mutations on the VKORC1 enzyme have not yet been evaluated. Therefore, the aim of this study was to investigate the causality of the VKORC1 mutations in the resistance phenotype. METHODS: Wild-type VKORC1 and its spontaneous mutants were expressed in Pichia pastoris and susceptibility to VKA was assessed by the in vitro determination of kinetic and inhibition constants. RESULTS AND CONCLUSIONS: The in vitro analysis revealed that six mutations only (A26P, A41S, V54L, H68Y, I123N and Y139H) were associated with increase in K(i) , suggesting their involvement in the resistance phenotype observed in patients. A41S and H68Y led to selective resistance, respectively, to indane-1,3-dione and 4-hydroxycoumarine derivatives. The other mutations did not increase the K(i). Furthermore, 10 mutations (S52L, S52W, W59L, W59R, V66M, V66G, G71A, N77S, N77T and L128R) led to an almost complete loss of activity. These results suggest the existence of other resistance mechanisms.

The carbocyclic analog of 2'-deoxyguanosine induces a prolonged inhibition of duck hepatitis B virus DNA synthesis in primary hepatocyte cultures and in the liver.

The carbocyclic analog of 2'-deoxyguanosine (2'-CDG) is a strong inhibitor of hepatitis B virus (HBV) DNA synthesis in HepG2 cells (P.M. Price, R. Banerjee, and G. Acs, Proc. Natl. Acad. USA 86:8543-8544, 1989). We now report that 2'-CDG inhibited duck hepatitis B virus (DHBV) DNA synthesis in primary cultures of duck hepatocytes and in experimentally infected ducks. Like foscarnet (phosphonoformic acid [PFA]) and 2'-,3'-dideoxycytidine (ddC), 2'-CDG blocked viral DNA replication in primary hepatocyte cultures when present during an infection but failed to inhibit the DNA repair reaction that occurs during the initiation of infection to convert virion relaxed circular DNA to covalently closed circular DNA, the template for viral mRNA transcription. Moreover, as for PFA and ddC, viral RNA synthesis was detected when infection was initiated in the presence 2'-CDG. In another respect, however, 2'-CDG exhibited antiviral activity unlike that of ddC or PFA: a single 1-day treatment of hepatocytes with 2'-CDG blocked initiation of viral DNA synthesis for at least 8 days, irrespective of whether DHBV infection was carried out at the time of drug treatment or several days later. Furthermore, orally administered 2'-CDG was long-acting against DHBV in experimentally infected ducklings. Virus replication was delayed by up to 4 days in ducklings infected after administration of 2'-CDG. These observations of long-lasting efficacy in vitro and in vivo even after oral administration suggest that this inhibitor or a nucleoside with similar pharmacological properties may be ideal for reducing virus replication in patients with chronic HBV infection.

Viral spliced RNA are produced, encapsidated and reverse transcribed during in vivo woodchuck hepatitis virus infection.

By the use of reverse transcription followed by polymerase chain reaction (RT-PCR), we have identified one shorter than full-length, pregenomic viral RNA species in liver samples of woodchucks chronically infected with the woodchuck hepatitis virus (WHV). The spliced WHV RNA of about 2.4 kb in length was cloned and partially sequenced. The splicing donor and acceptor sites of this novel RNA are located, respectively, 130 nucleotides downstream of the ATG initiation codon of the core gene and 21 nucleotides upstream of the initiation codon of the pre-S2 surface gene. The splicing event generates a new core-polymerase fusion protein and removes the terminal protein domain and the spacer region of the polymerase gene. A nucleotide probe specific for the splice junction was used following RT-PCR, to further confirm the existence of this spliced RNA in the liver of seven WHV-infected woodchucks. Deleted viral DNA molecules corresponding to the 2.4 kb spliced RNA were also detected in the liver and, to a lesser extent, in the serum of infected woodchucks, suggesting that this spliced RNA can be encapsidated and reverse-transcribed during the course of natural WHV infection.

Maintenance of woodchuck hepatitis virus activity in woodchuck hepatocyte primary culture.

Primary cultures of non-proliferating hepatocytes isolated by the two-step collagenase perfusion method from woodchuck naturally infected with hepatitis virus (WHV) were used to study WHV propagation in vitro. Hepatocytes carrying WHV DNA exhibited a very high level of survival and retained their morphological characteristics for 2 to 3 months. Over this time, they were found to produce virus-specific proteins and release viral particles with DNA polymerase activity into the medium. Using Southern blot analysis and a recombinant hepatitis B virus DNA plasmid probe, intracellular and extracellular viral DNA was consistently detected. Only extrachromosomal forms of WHV DNA were observed and no integration could be demonstrated in the DNA of the cells. The WHV DNA patterns were repeatedly identical with a characteristic smear starting from 3.3 kb associated with other smaller DNA fragments which presumably represented intermediate replicative forms of viral DNA. Furthermore, dot blot hybridization of the total RNA revealed the presence of WHV-specific transcripts in cells after 3 weeks of culture. All these results are compatible with the maintenance of active WHV replication in vitro although it was somewhat reduced after the first day of culture. This provides a mammalian model for hepadnavirus replication studies in stable primary hepatocyte cultures.

Effects of 2'-fluorinated arabinosyl-pyrimidine nucleosides on duck hepatitis B virus DNA level in serum and liver of chronically infected ducks.

The 2'-fluorinated arabinosyl-pyrimidine nucleosides, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU), are new antiviral compounds with in vitro inhibitory activity against the DNA polymerase of hepadnaviruses. Those compounds also induced permanent inhibition of viral replication in woodchucks chronically infected by woodchuck hepatitis virus. The effects of these antiviral compounds were assessed in ducks chronically infected by duck hepatitis B virus (DHBV). Following intraperitoneal administration for 5 days, FMAU (2 mg/kg/day) and FIAC (10 mg/kg/day) induced a transient decrease in DHBV replication, as shown by the decrease in both the serum and liver DHBV DNA level. After stopping therapy, DHBV replication rebounded immediately to the pretreatment level. The supercoiled form of liver viral DNA was found to be less affected by the therapy. By contrast, no obvious antiviral effect was observed with vidarabine monophosphate (ara-AMP) (80 mg/kg/day) therapy. No sign of toxicity was observed during the course of the treatment. These preliminary results confirmed in the DHBV model the higher efficacy of FIAC and FMAU as compared to ara-AMP. Pharmacokinetic studies are needed to explain the differences observed in viral replication in these 2 models of HBV infection.

Inhibitory effects of 2'-fluorinated arabinosyl-pyrimidine nucleosides on woodchuck hepatitis virus replication in chronically infected woodchucks.

The treatment of woodchuck hepatitis virus infections with 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU), given intraperitoneally, caused complete and permanent decrease of serum virus endogenous DNA polymerase and viral DNA in all treated woodchucks but was associated with severe toxicity. By contrast 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-ethyluracil (FEAU) induced a sustained, although less dramatic, decrease of viral replication without apparent toxic effect. FEAU was also effective when given orally. However, in both cases this inhibitory effect was transient.

Human hepatocellular carcinoma transplanted in nude mice: a relevant experimental model to assess tumoral destruction by alcoholization.

Intratumor ethanol injection was studied in the treatment of small hepatocellular carcinoma (HCC). One of the major drawbacks of this technique remains the lack of objective information about its efficiency and the practical conditions of injection. To ensure accurate evaluation of alcoholization, we developed a model based on the tumor obtained after subcutaneous injection of human hepatoma cell lines into nude mice. Each of three cell lines (Hep G2, Hep 3B, PLC/PRF/5) was tested on 24 mice. A total of 0.1 ml of a solution containing 5 x 10(6) cells was injected under the abdominal skin of a male nude mouse weighing 30 g. The Hep G2 cell line appeared to be the most suitable for the model. It enabled us to obtain tumors of 20 mm in diameter within a mean delay (m +/- SD) of 45 +/- 16 days (range: 29-60) with only a 25% failure rate. No visceral spreading of the carcinoma was noticed and the tumors obtained, similar to human HCC, were convenient to be measured, monitored, and treated by alcoholization. To validate this model for alcoholization, 38 tumors ranging in diameter from 10 to 20 mm were treated using either a unique centrotumoral injection (n = 27) or five cross-shaped injections (n = 11). Intratumor absolute ethanol injection resulted in tumoral necrosis which was easily quantified as a percentage of the tumor volume, using a semiquantitative method. It is concluded that the Hep G2 cell line transplanted in nude mice resulted in a relevant model to assess tumoral destruction by alcoholization.

Hepatitis B virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide.

We investigated the possibility of infecting normal adult human hepatocytes maintained in pure cultures or in cocultures with hepatitis B virus (HBV). Several assays with different infectious sera and hepatocyte populations from various donors identified only limited HBV replication, with significant variations from one cell preparation to another. The addition of 1.5% dimethyl sulfoxide to the culture medium markedly enhanced the infection process. Indeed, hepatitis B e antigen secretion, the appearance of both HBV DNA replicative forms and major HBV transcripts, and the release of complete HBV particles into the medium were demonstrated. It is possible that the significant increase in intracellular HBV DNA in dimethyl sulfoxide-treated cells was related to enhanced adsorption of the virus. When viral particles produced by a transfected HepG2 cell line were used to infect normal hepatocytes, the same results were obtained. In addition, comparative assays with hepatocytes from three different donors showed that although high amounts of intracellular viral DNA were found in all cases, viral replicative intermediates were visualized in only one case. These findings suggest that this HBV-producing cell line could serve as a reproducible source of infectious virus and that primary culturing of human hepatocytes represents a unique tool for analyzing intracellular regulating factors which, in addition to the penetration step, modulate HBV replication.

Virus-neutralizing monoclonal antibody to a conserved epitope on the duck hepatitis B virus pre-S protein.

In this study we used duck hepatitis B virus (DHBV)-infected Pekin ducks and heron hepatitis B virus (HHBV)-infected heron tissue to search for epitopes responsible for virus neutralization on pre-S proteins. Monoclonal antibodies were produced by immunizing mice with purified DHBV particles. Of 10 anti-DHBV specific hybridomas obtained, 1 was selected for this study. This monoclonal antibody recognized in both DHBV-infected livers and viremic sera a major (36-kilodalton) protein and several minor pre-S proteins in all seven virus strains used. In contrast, pre-S proteins of HHBV-infected tissue or viremic sera did not react. Thus, the monoclonal antibody recognizes a highly conserved DHBV pre-S epitope. For mapping of the epitope, polypeptides from different regions of the DHBV pre-S/S gene were expressed in Escherichia coli and used as the substrate for immunoblotting. The epitope was delimited to a sequence of approximately 23 amino acids within the pre-S region, which is highly conserved in four cloned DHBV isolates and coincides with the main antigenic domain as predicted by computer algorithms. In in vitro neutralization assays performed with primary duck hepatocyte cultures, the antibody reduced DHBV infectivity by approximately 75%. These data demonstrate a conserved epitope of the DHBV pre-S protein which is located on the surface of the viral envelope and is recognized by virus-neutralizing antibodies.

Metabolism in human cells of the D and L enantiomers of the carbocyclic analog of 2'-deoxyguanosine: substrate activity with deoxycytidine kinase, mitochondrial deoxyguanosine kinase, and 5'-nucleotidase.

The carbocyclic analog of 2'-deoxyguanosine (CdG) has broad-spectrum antiviral activity. Because of recent observations with other nucleoside analogs that biological activity may be associated the L enantiomer rather than, as expected, with the D enantiomer, we have studied the metabolism of both enantiomers of CdG to identify the enzymes responsible for the phosphorylation of CdG in noninfected and virally infected human and duck cells. We have examined the enantiomers as substrates for each of the cellular enzymes known to catalyze phosphorylation of deoxyguanosine. Both enantiomers of CdG were substrates for deoxycytidine kinase (EC 2.7.1.74) from MOLT-4 cells, 5'-nucleotidase (EC 3.1.3.5) from HEp-2 cells, and mitochondrial deoxyguanosine kinase (EC 2.7.1.113) from human platelets and CEM cells. For both deoxycytidine kinase and mitochondrial deoxyguanosine kinase, the L enantiomer was the better substrate. Even though the D enantiomer was the preferred substrate with 5'-nucleotidase, the rate of phosphorylation of the L enantiomer was substantial. The phosphorylation of D-CdG in MRC-5 cells was greatly stimulated by infection with human cytomegalovirus. The fact that the phosphorylation of D-CdG was stimulated by mycophenolic acid and was not affected by deoxycytidine suggested that 5'-nucleotidase was the enzyme primarily responsible for its metabolism in virally infected cells. D-CdG was extensively phosphorylated in duck hepatocytes, and its phosphorylation was not affected by infection with duck hepatitis B virus. These results are of importance in understanding the mode of action of D-CdG and related analogs and in the design of new biologically active analogs.

Evidence that hepatocyte turnover is required for rapid clearance of duck hepatitis B virus during antiviral therapy of chronically infected ducks.

Duck hepatitis B virus (DHBV) DNA synthesis in congenitally infected ducks is inhibited by 2'-deoxycarbocyclic guanosine (2'-CDG). Three months of therapy reduces the number of infected hepatocytes at least 10-fold (W.S. Mason, J. Cullen, J. Saputelli, T.-T. Wu, C. Liu, W.T. London, E. Lustbader, P. Schaffer, A.P. O'Connell, I. Fourel, C.E. Aldrich, and A.R. Jilbert, Hepatology 19:393-411, 1994). The present study was performed to determine the kinetics of disappearance of infected hepatocytes and to evaluate the role of hepatocyte turnover in this process. Essentially all hepatocytes were infected before drug therapy. Oral treatment with 2'-CDG resulted in a prompt reduction in the number of infected hepatocytes. After 2 weeks, only 30 to 50% appeared to still be infected, and less than 10% were detectably infected after 5 weeks of therapy. To assess the possible role of hepatocyte turnover in these changes, 5-bromo-2'-deoxyuridine (BUdR) was administered 8 h before liver biopsy to label host DNA in hepatocytes passing through S phase, and stained nuclei were detected in tissue sections by using an antibody reactive to BUdR. The extent of nuclear labeling after 5 weeks was the same as that before therapy (ca. 1%). However, biopsies taken after 2 weeks of therapy showed a ca. 10-fold elevation in the number of nuclei labeled with BUdR. This result suggested that a rapid clearance of infected hepatocytes by 2'-CDG was caused not just by the inhibition of viral replication but also by an acceleration of the rate of hepatocyte turnover. To test this possibility further, antiviral therapy was carried out with another strong inhibitor of DHBV DNA synthesis, 5-fluoro-2',3'-dideoxy-3'-thiacytidine (524W), which did not accelerate hepatocyte turnover in ducks. 524W administration led to a strong inhibition of virus production but to a slower rate of decline in the number of infected hepatocytes, so that ca. 50% (and perhaps more) were still infected after 3 months of therapy. In addition, histopathologic evaluation of 2'-CDG-treated ducks revealed liver injury, especially at the start of therapy. No liver damage was observed during 524W therapy. These results imply that clearance of infected hepatocytes from the liver is correlated with hepatocyte turnover. Thus, in the absence of immune clearance or other sources for the accelerated elimination of infected hepatocytes, inhibitors of virus replication would have to be administered for a long period to substantially reduce the burden of infected hepatocytes in the liver.

Characterization of the antiviral effects of 2' carbodeoxyguanosine in ducks chronically infected with duck hepatitis B virus.

This study was carried out to evaluate benefits and limitations of long-term therapy of hepatitis B virus infections with a nucleoside analog inhibitor of virus replication. The model we used was the domestic duck chronically infected with duck hepatitis B virus by in ovo infection. 2' Carbodeoxyguanosine was used as an inhibitor of viral DNA synthesis. In all animals examined there was a reduction in virus production during therapy. A dose of 2' carbodeoxyguanosine of 10 micrograms/kg every other day reduced the number of infected hepatocytes from greater than 95% to 25% to 50% in less than 3 mo, whereas a 10-fold higher dose produced a decline to less than 10%. Histological evaluation revealed mild to moderate liver injury in ducks receiving the higher dose of 2' carbodeoxyguanosine, suggesting that disappearance of infected hepatocytes may have been accelerated by a toxic effect of the drug. Drug treatment did not completely eliminate duck hepatitis B virus from any duck, and replication was restored in all hepatocytes within a few weeks to several months after antiviral therapy was terminated. Our results suggest that elimination of a chronic infection with a single inhibitor of replication may be difficult in a host that lacks an antiviral immune response capable of eliminating at least a portion of the infected hepatocytes and of ultimately producing antibodies capable of neutralizing residual virus.

Evidence for the presence of duck hepatitis B virus in wild migrating ducks.

A virus closely related to duck hepatitis B virus (DHBV) was isolated from serum and liver samples of wild migratory ducks (mallards) caught in two separate wildlife reserve parks in France. In the first one (Dombes region) 12% of wild mallards were positive for DHBV, and in the second (River Somme) 3% of mallards were found positive. The DHBV isolated from the serum of wild mallards was also associated with an endogenous DNA polymerase activity capable in vitro of completing a partially double-stranded viral DNA into a fully double-stranded DNA of 3 kb. The various replicative DNA forms reported for DHBV were also detected in the liver of wild viraemic mallards. The DNA restriction enzyme pattern of the wild mallard strain differed from that of American and French strains of DHBV. The wild mallard strain DHBV was experimentally transmitted to mallard and Pekin ducklings and induced a chronic viraemia in both varieties of infected birds. This strain might be the common ancestor of all DHBV strains isolated from domestic ducks world-wide. The discovery of a DHBV-related virus in the natural wild population might be an important clue in the study of the different roles of environmental, host and viral factors in the pathogenesis of DHBV infection, and their possible oncogenic action in ducks.

Prolonged duck hepatitis B virus replication in duck hepatocytes cocultivated with rat epithelial cells: a useful system for antiviral testing.

Duck cultured hepatocytes from Pekin ducks naturally infected by duck hepatitis B virus can remain functional twice longer if a coculture system with rat liver epithelial cells is used instead of ordinary primary culture. The use of a selective medium in which ornithine and lactate replaced arginine and glucose, respectively, allowed viral replication initiated in vivo to be maintained in the coculture for 2 months. Several antiviral compounds including the pyrophosphate analog (phosphonoformic acid) or nucleoside analogs (9 beta-arabinofuranosyl AMP, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine, 1,2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-ethyluracil and 1,2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl thymine) were studied in both culture systems for their ability to inhibit duck hepatitis B virus replication. Hepatocytes were treated for 7 days with 1,2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-ethyluracil (10 microM) and 1,2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl thymine (0.5 microM) or for 14 days with 9 beta-arabinofuranosyl AMP (90 microM), phosphonoformic acid (100 microM) and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (6 microM). The effects of the drugs on viral replication were monitored by testing for duck hepatitis B virus DNA in the culture supernatant and in the cells by molecular hybridization.(ABSTRACT TRUNCATED AT 250 WORDS)