Carboxypeptidase D Is an Avian Hepatitis B Virus Receptor

The receptor molecules for human and animal hepatitis B viruses have not been defined. Previous studies have described a 170 to 180 kDa molecule (p170 or gp180) that binds in vitro to the pre-S domain of the large envelope protein of duck hepatitis B virus (DHBV); cDNA cloning revealed the binding protein to be duck carboxypeptidase D (DCPD). In the present study, the DCPD cDNA was transfected into several nonpermissive human-, monkey-, and avian species-derived cell lines. Cells transfected with a plasmid encoding the full-length DCPD protein bound DHBV particles, whereas cells expressing truncated versions of DCPD protein that fail to bind the pre-S protein did not. The DHBV binding to DCPD-reconstituted cells was blocked by a monoclonal antibody that neutralizes DHBV infection of primary duck hepatocytes (PDH) and also by a pre-S peptide previously shown to inhibit DHBV infection of PDH. In addition to promoting virus binding, DCPD expression was associated with internalization of viral particles. The entry process was prevented by incubation of reconstituted cells with DHBV at 4 degrees C and by the addition of energy-depleting agents known to block DHBV entry into PDH. These results demonstrated that DCPD is a DHBV receptor. However, the lack of complete viral replication in DCPD-reconstituted cells suggested that additional factors are required for postentry events in immortalized cell lines.     

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.     

A hydrophobic domain in the large envelope protein is essential for fusion of duck hepatitis B virus at the late endosome

The duck hepatitis B virus (DHBV) envelope is comprised of two transmembrane (TM) proteins, the large (L) and the small (S), that assemble into virions and subviral particles. Secondary-structure predictions indicate that L and S have three alpha-helical, membrane-spanning domains, with TM1 predicted to act as the fusion peptide following endocytosis of DHBV into the hepatocyte. We used bafilomycin A1 during infection of primary duck hepatocytes to show that DHBV must be trafficked from the early to the late endosome for fusion to occur. Alanine substitution mutations in TM1 of L and S, which lowered TM1 hydrophobicity, were used to examine the role of TM1 in infectivity. The high hydrophobicity of the TM1 domain of L, but not of S, was shown to be essential for virus infection at a step downstream of receptor binding and virus internalization. Using wild-type and mutant synthetic peptides, we demonstrate that the hydrophobicity of this domain is required for the aggregation and the lipid mixing of phospholipid vesicles, supporting the role of TM1 as the fusion peptide. While lipid mixing occurred at pH 7, the kinetics of insertion of the fusion peptide was increased at pH 5, consistent with the location of DHBV in the late-endosome compartment and previous studies of the nonessential role of low pH for infectivity. Exchange of the TM1 of DHBV with that of hepatitis B virus yielded functional, infectious DHBV particles, suggesting that TM1 of all of the hepadnaviruses act similarly in the fusion mechanism.     

Central role of a serine phosphorylation site within duck hepatitis B virus core protein for capsid trafficking and genome release

Viral nucleocapsids compartmentalize and protect viral genomes during assembly while they mediate targeted genome release during viral infection. This dual role of the capsid in the viral life cycle must be tightly regulated to ensure efficient virus spread. Here, we used the duck hepatitis B virus (DHBV) infection model to analyze the effects of capsid phosphorylation and hydrogen bond formation. The potential key phosphorylation site at serine 245 within the core protein, the building block of DHBV capsids, was substituted by alanine (S245A), aspartic acid (S245D) and asparagine (S245N), respectively. Mutant capsids were analyzed for replication competence, stability, nuclear transport, and infectivity. All mutants formed DHBV DNA-containing nucleocapsids. Wild-type and S245N but not S245A and S245D fully protected capsid-associated mature viral DNA from nuclease action. A negative ionic charge as contributed by phosphorylated serine or aspartic acid-supported nuclear localization of the viral capsid and generation of nuclear superhelical DNA. Finally, wild-type and S245D but not S245N virions were infectious in primary duck hepatocytes. These results suggest that hydrogen bonds formed by non-phosphorylated serine 245 stabilize the quarterny structure of DHBV nucleocapsids during viral assembly, while serine phosphorylation plays an important role in nuclear targeting and DNA release from capsids during viral infection.     

A short sequence within domain C of duck carboxypeptidase D is critical for duck hepatitis B virus binding and determines host specificity

Virus-cell surface receptor interactions are of major interest. Hepadnaviruses are a family of partially double-stranded DNA viruses with liver tropism and a narrow host range of susceptibility to infection. At least in the case of duck hepatitis B virus (DHBV), host specificity seems controlled partly at the receptor level. The middle portion in the pre-S region of the viral large envelope protein binds specifically to duck carboxypeptidase D (DCPD) but not to its human or chicken homologue. Although domain C of DCPD is implicated in ligand binding, the exact pre-S contact site remains to be determined. We prepared and tested a panel of chimeric constructs consisting of DCPD and human carboxypeptidase D (HCPD). Our results indicate that a short region at the N terminus of domain C (residues 920 to 949) is critical to DHBV binding and is a major determinant for the host specificity of DHBV infection. Replacing this region of the DCPD molecule with its human homologue abolished the DHBV interaction, whereas introducing this DCPD sequence into HCPD conferred efficient DHBV binding. Extensive analysis of site-directed mutants revealed that both conserved and nonconserved residues were important for the pre-S interaction. There were primary sequence variations and secondary structural differences that contributed to the inability of HCPD to bind the DHBV pre-S domain.     

Superinfection exclusion in duck hepatitis B virus infection is mediated by the large surface antigen

Superinfection exclusion is the phenomenon whereby a virus prevents the subsequent infection of an already infected host cell. The Pekin duck hepatitis B virus (DHBV) model was used to investigate superinfection exclusion in hepadnavirus infections. Superinfection exclusion was shown to occur both in vivo and in vitro with a genetically marked DHBV, DHBV-ClaI, which was unable to establish an infection in either DHBV-infected ducklings or DHBV-infected primary duck hepatocytes (PDHs). In addition, exclusion occurred in vivo even when the second virus had a replicative advantage. Superinfection exclusion appears to be restricted to DHBV, as adenovirus, herpes simplex virus type 1, and vesicular stomatitis virus were all capable of efficiently infecting DHBV-infected PDHs. Exclusion was dependent on gene expression by the original infecting virus, since UV-irradiated DHBV was unable to mediate the exclusion of DHBV-ClaI. Using recombinant adenoviruses expressing DHBV proteins, we determined that the large surface antigen mediated exclusion. The large surface antigen is known to cause down-regulation of a DHBV receptor, carboxypeptidase D (CPD). Receptor down-regulation is a mechanism of superinfection exclusion seen in other viral infections, and so it was investigated as a possible mechanism of DHBV-mediated exclusion. However, a mutant large surface antigen which did not down-regulate CPD was still capable of inhibiting DHBV infection of PDHs. In addition, exclusion of DHBV-ClaI did not correlate with a decrease in CPD levels. Finally, virus binding assays and confocal microscopy analysis of infected PDHs indicated that the block in infection occurs after internalization of the second virus. We suggest that superinfection exclusion may result from the role of the L surface antigen as a regulator of intracellular trafficking.     

Therapeutic Antiviral Effect of the Nucleic Acid Polymer REP 2055 against Persistent Duck Hepatitis B Virus Infection

Previous studies have demonstrated that nucleic acid polymers (NAPs) have both entry and post-entry inhibitory activity against duck hepatitis B virus (DHBV) infection. The inhibitory activity exhibited by NAPs prevented DHBV infection of primary duck hepatocytes in vitro and protected ducks from DHBV infection in vivo and did not result from direct activation of the immune response. In the current study treatment of primary human hepatocytes with NAP REP 2055 did not induce expression of the TNF, IL6, IL10, IFNA4 or IFNB1 genes, confirming the lack of direct immunostimulation by REP 2055. Ducks with persistent DHBV infection were treated with NAP 2055 to determine if the post-entry inhibitory activity exhibited by NAPs could provide a therapeutic effect against established DHBV infection in vivo. In all REP 2055-treated ducks, 28 days of treatment lead to initial rapid reductions in serum DHBsAg and DHBV DNA and increases in anti-DHBs antibodies. After treatment, 6/11 ducks experienced a sustained virologic response: DHBsAg and DHBV DNA remained at low or undetectable levels in the serum and no DHBsAg or DHBV core antigen positive hepatocytes and only trace amounts of DHBV total and covalently closed circular DNA (cccDNA) were detected in the liver at 9 or 16 weeks of follow-up. In the remaining 5/11 REP 2055-treated ducks, all markers of DHBV infection rapidly rebounded after treatment withdrawal: At 9 and 16 weeks of follow-up, levels of DHBsAg and DHBcAg and DHBV total and cccDNA in the liver had rebounded and matched levels observed in the control ducks treated with normal saline which remained persistently infected with DHBV. These data demonstrate that treatment with the NAP REP 2055 can lead to sustained control of persistent DHBV infection. These effects may be related to the unique ability of REP 2055 to block release of DHBsAg from infected hepatocytes.     

Interaction between duck hepatitis B virus and a 170-kilodalton cellular protein is mediated through a neutralizing epitope of the pre-S region and occurs during viral infection.

Identification of cell surface viral binding proteins is important for understanding viral attachment and internalization. We have fused the pre-S domain of the duck hepatitis B virus (DHBV) large envelope protein to glutathione S-transferase and demonstrated a 170-kDa binding protein (p170) in [35S]methionine-labeled duck hepatocyte lysates. This glycoprotein was found abundantly in all extrahepatic tissues infectible with DHBV and in some noninfectible tissues, though it is not secreted into the blood. The interaction of pre-S fusion protein with p170 was competitively inhibited by wild-type DHBV in a dose-dependent manner. In addition, infection of hepatocytes with DHBV blocked the binding of pre-S fusion protein to p170, which suggests a biological role for p170 during natural infection. The p170 binding site was mapped to a conserved sequence of 16 amino acid residues (positions 87 to 102) by using 24 pre-S deletion mutants; this binding domain coincides with a major virus-neutralizing antibody epitope. Furthermore, site-directed mutagenesis revealed that an arginine residue at position 97 is critical for p170 binding. p170 was purified by a combination of ion-exchange and affinity chromatographies, and four peptide sequences were obtained. Two peptides showed significant similarities to human and animal carboxypeptides H, M, and N. Taken together, these results raise the possibility that the p170 binding protein is important during the replication cycle of DHBV.     

Isolation and characterization of a hepatitis B virus endemic in herons.

A new hepadnavirus (designated heron hepatitis B virus [HHBV]) has been isolated; this virus is endemic in grey herons (Ardea cinerea) in Germany and closely related to duck hepatitis B virus (DHBV) by morphology of viral particles and size of the genome and of the major viral envelope and core proteins. Despite its striking similarities to DHBV, HHBV cannot be transmitted to ducks by infection or by transfection with cloned viral DNA. After the viral genome was cloned and sequenced, a comparative sequence analysis revealed an identical genome organization of HHBV and DHBV (pre-C/C-, pre-S/S-, and pol-ORFs). An open reading frame, designated X in mammalian hepadnaviruses, is not present in DHBV. DHBV and HHBV differ by 21.6% base exchanges, and thus they are less closely related than the two known rodent hepatitis B viruses (16.4%). The nucleocapsid protein and the 17-kilodalton envelope protein sequences of DHBV and HHBV are well conserved. In contrast, the pre-S part of the 34-kilodalton envelope protein which is believed to mediate virus attachment to the cell is highly divergent (less than 50% homology). The availability of two closely related avian hepadnaviruses will now allow us to test recombinant viruses in vivo and in vitro for host specificity-determining sequences.     

Preparation of monoclonal antibodies against duck hepatitis B core antigen and analysis of the monoclonal antibodies

Mice were immunized against duck hepatitis B virus core (DHBc) particles isolated from the liver of asymptomatic carrier ducks of duck hepatitis B virus (DHBV) by ultracentrifugation. Their spleen cells were fused with mouse myeloma (NS-1) cells, and 12 clones of hybridoma cells secreting antibodies against DHBc (anti-DHBc) were isolated. According to the reactivity to core particles and core peptide obtained from DHBc particles treated with SDS-2ME, the 12 antibodies were classified into two groups. Two monoclonal antibodies reacted against both core particles and core peptide (B-type), the other ten monoclonal antibodies reacted against core particles but did not react against core peptide obtained from DHBc particles treated with SDS-2 ME. (A-type). Solid phase enzyme immuno assay (EIA) using these two types of antibodies could detect core antigenisity not only in the liver homogenate but also in the DHBV infected serum. Sucrose gradient analysis and gel filtration analysis revealed this DHBc antigenisity in the serum is not carried by core particles but carried by core peptide, equivalent to HBe antigen in the serum of Hepatitis B virus (HBV) carrier. This EIA may provide sensitive test monitoring both serum DHBe antigen levels and DHBc antigen levels in the liver during DHBV infection.     

Cellular receptor traffic is essential for productive duck hepatitis B virus infection

We have investigated the mechanism of duck hepatitis B virus (DHBV) entry into susceptible primary duck hepatocytes (PDHs), using mutants of carboxypeptidase D (gp180), a transmembrane protein shown to act as the primary cellular receptor for avian hepatitis B virus uptake. The variant proteins were abundantly produced from recombinant adenoviruses and tested for the potential to functionally outcompete the endogenous wild-type receptor. Overexpression of wild-type gp180 significantly enhanced the efficiency of DHBV infection in PDHs but did not affect ongoing DHBV replication, an observation further supporting gp180 receptor function. A gp180 mutant deficient for endocytosis abolished DHBV infection, indicating endocytosis to be the route of hepadnaviral entry. With further gp180 variants, carrying mutations in the cytoplasmic domain and characterized by an accelerated turnover, the ability of gp180 to function as a DHBV receptor was found to depend on a wild-type-like sorting phenotype which largely avoids transport toward the endolysosomal compartment. Based on these data, we propose a model in which a distinct intracellular DHBV traffic to the endosome, but not beyond, is a prerequisite for completion of viral entry, i.e., for fusion and capsid release. Furthermore, the deletion of the two enzymatically active carboxypeptidase domains of gp180 did not lead to a loss of receptor function.     

Glycine decarboxylase mediates a postbinding step in duck hepatitis B virus infection

Envelope protein precursors of many viruses are processed by a basic endopeptidase to generate two molecules, one for receptor binding and the other for membrane fusion. Such a cleavage event has not been demonstrated for the hepatitis B virus family. Two binding partners for duck hepatitis B virus (DHBV) pre-S envelope protein have been identified. Duck carboxypeptidase D (DCPD) interacts with the full-length pre-S protein and is the DHBV docking receptor, while duck glycine decarboxylase (DGD) has the potential to bind several deletion constructs of the pre-S protein in vitro. Interestingly, DGD but not DCPD expression was diminished following prolonged culture of primary duck hepatocytes (PDH), which impaired productive DHBV infection. Introduction of exogenous DGD promoted formation of protein-free viral genome, suggesting restoration of several early events in viral life cycle. Conversely, blocking DGD expression in fresh PDH by antisense RNA abolished DHBV infection. Moreover, addition of DGD antibodies soon after virus binding reduced endogenous DGD protein levels and impaired production of covalently closed circular DNA, the template for DHBV gene expression and genome replication. Our findings implicate this second pre-S binding protein as a critical cellular factor for productive DHBV infection. We hypothesize that DCPD, a molecule cycling between the cell surface and the trans-Golgi network, targets DHBV particles to the secretary pathway for proteolytic cleavage of viral envelope protein. DGD represents the functional equivalent of other virus receptors in its interaction with processed viral particles.     

Susceptibility to duck hepatitis B virus infection is associated with the presence of cell surface receptor sites that efficiently bind viral particles.

To test the hypothesis that susceptibility of hepatocytes to duck hepatitis B virus (DHBV) infection requires cell surface receptors that bind the virus in a specific manner, we developed an assay for the binding of DHBV particles to monolayers of intact cells, using radiolabeled immunoglobulin G specific for DHBV envelope protein. Both noninfectious DHBV surface antigen particles and infectious virions bound to a susceptible fraction (approximately 60%) of Pekin duck hepatocytes. In contrast, binding did not occur to cells that were not susceptible to DHBV infection, including Pekin duck fibroblasts and chicken hepatocytes, and binding to Muscovy duck hepatocytes, which are only weakly susceptible (approximately 1% of cells) to DHBV infection, was virtually undetectable. Within a monolayer, individual Pekin duck hepatocytes appeared to differ markedly in the capacity to bind DHBV, which may explain difficulties that have been encountered in infecting 100% of cells in culture. We have also found that the loss of susceptibility to infection with DHBV that occurs when Pekin duck hepatocytes are maintained for more than a few days in culture correlates with a decline in the number of cells that bind virus particles efficiently. All of these results support the interpretation that the binding event detected by our assay is associated with the interaction between DHBV and specific cell surface receptors that are required for initiation of infection. Our assay may facilitate isolation and identification of hepatocyte receptors for this virus.     

Duck hepatitis B virus infection of hepatocytes is not dependent on low pH.

The pH dependency for initiation of infection by the hepadnavirus duck hepatitis B virus (DHBV) was investigated in primary duck hepatocytes. First, an infection assay was developed using a radioimmunoblot to measure DHBV e antigen secreted into tissue culture fluid from infected hepatocytes. The quantity of this viral marker was proportional to the duration of inoculation and the amount of DHBV used as inoculum. The role of pH in initiation of DHBV infection was investigated by using this assay, but no dependence on low pH was found. DHBV was able to infect hepatocytes in the presence of NH4Cl and monensin, agents that raise the pH in intracellular vesicles and prevent penetration of viruses dependent on low pH in endosomes. In control experiments, infection by Semliki Forest virus, which is low pH dependent, was inhibited, whereas herpes simplex virus type 1 infection, which is pH independent, occurred. Attempts to trigger DHBV-cell fusion by exposure of DHBV prebound to hepatocytes to mildly acidic pH were unsuccessful. In these experiments, it was also observed that internalization of DHBV occurred only between pH 6.8 and 8.0. Additionally, in the absence of cells, infectivity of DHBV was stable at pH 4.6 to 4.8, which is lower than the pH encountered in endosomes (pH 5 to 6.6). Thus, no evidence for a role for mildly acidic pH in the initiation of DHBV infection was found. Therefore, we propose that the infection route followed by DHBV resembles that of the group of enveloped viruses, including herpesviruses, that fuse with their host cells at neutral pH.     

Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

Persistence of hepatitis B virus (HBV) infection requires covalently closed circular (ccc)DNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc) DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV) in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process.     

Identification of antigenic regions of duck hepatitis B virus core protein with antibodies elicited by DNA immunization and chronic infection

The induction of humoral response in ducks by DNA-based immunization against duck hepatitis B virus (DHBV) core protein (DHBc) was investigated. In addition, the amino acid specificity of the induced response was compared by using peptide scanning to that elicited either by protein immunization or during chronic DHBV infection. Immunization of ducks with a plasmid expressing DHBc protein led to the induction of a long-lasting antibody response able to specifically recognize viral protein in chronically infected duck livers. Peptide scanning analysis of anti-DHBc response induced during chronic DHBV infection allowed us to identify six major antigenic regions (AR1 to AR6). The reactivity spectrum of duck sera elicited by protein immunization appeared narrower and was restricted to only four of these antigenic regions in spite of higher anti-DHBc antibody titers. Interestingly, anti-DHBc antibodies induced by DNA-based immunization recognized five of six antigenic regions, and the epitope pattern was broader and more closely related to that observed in chronic viral infections. To gain more insight into the location of antigenic regions, we built a three-dimensional (3-D) model of DHBc protein based on human and duck core sequence alignment data and the HBc 3-D crystal structure. The results suggest that two identified antigenic regions (AR2, amino acids [aa] (64)T-P(84), and AR5, aa (183)A-R(210)) are located at positions on the protein surface equivalent to those of the two HBc major epitopes. Moreover, we identified another antigenic region (AR3, aa (99)I-I(112)) that was recognized by all sera from chronically infected, DNA- or protein-immunized ducks within the large 45-aa insertion in DHBc protein, suggesting that this region, which lacks HBc, is externally exposed.     

Avian Hepatitis B Virus Infection Is Initiated by the Interaction of a Distinct Pre-S Subdomain with the Cellular Receptor gp180

Functionally relevant hepadnavirus-cell surface interactions were investigated with the duck hepatitis B virus (DHBV) animal model by using an in vitro infection competition assay. Recombinant DHBV pre-S polypeptides, produced in Escherichia coli, were shown to inhibit DHBV infection in a dose-dependent manner, indicating that monomeric pre-S chains were capable of interfering with virus-receptor interaction. Particle-associated pre-S was, however, 30-fold more active, suggesting that cooperative interactions enhance particle binding. An 85-amino-acid pre-S sequence, spanning about half of the DHBV pre-S chain, was characterized by deletion analysis as essential for maximal inhibition. Pre-S polypeptides from heron hepatitis B virus (HHBV) competed DHBV infection equally well despite a 50% difference in amino acid sequence and a much-reduced infectivity of HHBV for duck hepatocytes. These observations are taken to indicate (i) that the functionality of the DHBV pre-S subdomain, which interacts with the cellular receptor, is determined predominantly by a defined three-dimensional structure rather than by primary sequence elements; (ii) that cellular uptake of hepadnaviruses is a multistep process involving more than a single cellular receptor component; and (iii) that gp180, a cellular receptor candidate unable to discriminate between DHBV and HHBV, is a common component of the cellular receptor complex for avian hepadnaviruses.     

New hepatitis B virus of cranes that has an unexpected broad host range

All hepadnaviruses known so far have a very limited host range, restricted to their natural hosts and a few closely related species. This is thought to be due mainly to sequence divergence in the large envelope protein and species-specific differences in host components essential for virus propagation. Here we report an infection of cranes with a novel hepadnavirus, designated CHBV, that has an unexpectedly broad host range and is only distantly evolutionarily related to avihepadnaviruses of related hosts. Direct DNA sequencing of amplified CHBV DNA as well a sequencing of cloned viral genomes revealed that CHBV is most closely related to, although distinct from, Ross' goose hepatitis B virus (RGHBV) and slightly less closely related to duck hepatitis B virus (DHBV). Phylogenetically, cranes are very distant from geese and ducks and are most closely related to herons and storks. Naturally occurring hepadnaviruses in the last two species are highly divergent in sequence from RGHBV and DHBV and do not infect ducks or do so only marginally. In contrast, CHBV from crane sera and recombinant CHBV produced from LMH cells infected primary duck hepatocytes almost as efficiently as DHBV did. This is the first report of a rather broad host range of an avihepadnavirus. Our data imply either usage of similar or identical entry pathways and receptors by DHBV and CHBV, unusual host and virus adaptation mechanisms, or divergent evolution of the host genomes and cellular components required for virus propagation.     

Production of infectious duck hepatitis B virus in a human hepatoma cell line.

The differentiated human hepatoma cell line Hep-G2 was transfected with cloned duck hepatitis B virus (DHBV) DNA. Introduction of closed circular DNA into the human liver cells resulted in the production of viral proteins: core antigen was detected in the cytoplasm, and e antigen, a related product, was secreted into the medium. Moreover, viral particles were released into the tissue culture medium which were indistinguishable from authentic DHBV by density, antigenicity, DNA polymerase activity, and morphology. Intravenous injection of tissue culture-derived DHBV particles into Pekin ducks established DHBV infection. In conclusion, transfection of human hepatoma cells with cloned DHBV DNA results in the production of infectious virus, as occurs with cloned human hepatitis B virus DNA. Human liver cells are therefore competent to support production of the avian and mammalian hepadnaviruses, indicating that liver-specific viral gene expression is controlled by evolutionarily conserved mechanisms. This new DHBV transfection system offers the opportunity to rapidly produce mutated DHBV which then can be further investigated in Pekin ducks.