Duck Hepatitis B Virus Infection Research Articles

Glucose Homeostasis Is Dysregulated in Ducks Infected with Duck Hepatitis B Virus

Introduction: The association between hepatitis B virus (HBV) infection and the development of diabetes remains controversial. This study examined the effect of HBV infection on glucose homeostasis using a duck HBV (DHBV) model. Methods: Plasma DHBV DNA was detected by quantitative polymerase chain reaction (PCR). Tissue infection of DHBV was determined by detecting DHBV covalently closed circular DNA (cccDNA) with a method of rolling circle amplification combined with cross-gap PCR, and verified by fluorescence in situ hybridization assay. An intravenous injection glucose tolerance test (GTT) was used to analyze the effect of DHBV infection on glucose tolerance. Results: Of the finally included 97 domestic ducks, 53 (54.6%) were congenitally infected by DHBV. The positive rate of DHBV cccDNA in the liver, kidney, pancreas, and skeletal muscle of the infected ducks was 100, 75.5, 67.9, and 47.2%, respectively. The DHBV-infected ducks had higher blood glucose levels at 15 and 30 min post-load glucose (p < 0.01 and p < 0.001, respectively) in the GTT, much more individuals with greater glucose area under curve (p < 0.01), and a 57% impaired glucose tolerance (IGT) rate, as compared with noninfected controls. In addition, the subgroups of the infected ducks with DHBV cccDNA positive in skeletal muscle maintained the higher blood glucose level up to 2 h post-load glucose during the GTT and had a 76% IGT rate. Conclusion: These results suggest that DHBV intrahepatic and extrahepatic infection impairs glucose tolerance, and thus evidence the association of DHBV infection with the dysregulation of glucose metabolism.

Phosphorylation and Alternative Translation on Wheat Germ Cell-Free Protein Synthesis of the DHBV Large Envelope Protein.

Wheat-germ cell-free protein synthesis (WG-CFPS) is a potent platform for the high-yield production of proteins. It is especially of interest for difficult-to-express eukaryotic proteins, such as toxic and transmembrane proteins, and presents an important tool in high-throughput protein screening. Until recently, an assumed drawback of WG-CFPS was a reduced capacity for post-translational modifications. Meanwhile, phosphorylation has been observed in WG-CFPS; yet, authenticity of the respective phosphorylation sites remained unclear. Here we show that a viral membrane protein, the duck hepatitis B virus (DHBV) large envelope protein (DHBs L), produced by WG-CFPS, is phosphorylated upon translation at the same sites as DHBs L produced during DHBV infection of primary hepatocytes. Furthermore, we show that alternative translation initiation of the L protein, previously identified in virus-producing hepatic cells, occurs on WG-CFPS as well. Together, these findings further strengthen the high potential of WG-CFPS to include the reproduction of specific modifications proteins experience in vivo.

Immunogenicity and protective efficacy of DHBV DNA vaccines expressing envelope and capsid fusion proteins in ducks delivered by attenuated Salmonella typhimurium

Duck hepatitis B virus (DHBV) shares many basic characteristics with hepatitis B virus (HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks. Attenuated Salmonella typhimurium (SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response. Based on this strategy, novel DNA vaccines (SL7207-pVAX1-LC and SL7207-pVAX1-SC) were generated. Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy. The relative transcript levels of the CD4 and CD8 T genes and the antibody levels (IgY) in ducks receiving the vaccines were higher than those in single gene delivered groups. Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.

Inhibition of hepatitis B viral entry by nucleic acid polymers in HepaRG cells and primary human hepatocytes.

Hepatitis B virus (HBV) infection remains a major public health concern worldwide with 240 million individuals chronically infected and at risk of developing cirrhosis and hepatocellular carcinoma. Current treatments rarely cure chronic hepatitis B infection, highlighting the need for new anti-HBV drugs. Nucleic acid polymers (NAPs) are phosphorothioated oligonucleotides that have demonstrated a great potential to inhibit infection with several viruses. In chronically infected human patients, NAPs administration lead to a decline of blood HBsAg and HBV DNA and to HBsAg seroconversion, the expected signs of functional cure. NAPs have also been shown to prevent infection of duck hepatocytes with the Avihepadnavirus duck hepatitis B virus (DHBV) and to exert an antiviral activity against established DHBV infection in vitro and in vivo.In this study, we investigated the specific anti-HBV antiviral activity of NAPs in the HepaRG human hepatoma cell line and primary cultures of human hepatocytes. NAPs with different chemical features (phosphorothioation, 2’O-methyl ribose, 5-methylcytidine) were assessed for antiviral activity when provided at the time of HBV inoculation or post-inoculation. NAPs dose-dependently inhibited HBV entry in a phosphorothioation-dependent, sequence-independent and size-dependent manner. This inhibition of HBV entry by NAPs was impaired by 2’O-methyl ribose modification. NAP treatment after viral inoculation did not elicit any antiviral activity.

Hepatitis Virus Infections in Poultry.

Viral hepatitis in poultry is a complex disease syndrome caused by several viruses belonging to different families including avian hepatitis E virus (HEV), duck hepatitis B virus (DHBV), duck hepatitis A virus (DHAV-1, -2, -3), duck hepatitis virus Types 2 and 3, fowl adenoviruses (FAdV), and turkey hepatitis virus (THV). While these hepatitis viruses share the same target organ, the liver, they each possess unique clinical and biological features. In this article, we aim to review the common and unique features of major poultry hepatitis viruses in an effort to identify the knowledge gaps and aid the prevention and control of poultry viral hepatitis. Avian HEV is an Orthohepevirus B in the family Hepeviridae that naturally infects chickens and consists of three distinct genotypes worldwide. Avian HEV is associated with hepatitis-splenomegaly syndrome or big liver and spleen disease in chickens, although the majority of the infected birds are subclinical. Avihepadnaviruses in the family of Hepadnaviridae have been isolated from ducks, snow geese, white storks, grey herons, cranes, and parrots. DHBV evolved with the host as a noncytopathic form without clinical signs and rarely progressed to chronicity. The outcome for DHBV infection varies by the host's ability to elicit an immune response and is dose and age dependent in ducks, thus mimicking the pathogenesis of human hepatitis B virus (HBV) infections and providing an excellent animal model for human HBV. DHAV is a picornavirus that causes a highly contagious virus infection in ducks with up to 100% flock mortality in ducklings under 6 wk of age, while older birds remain unaffected. The high morbidity and mortality has an economic impact on intensive duck production farming. Duck hepatitis virus Types 2 and 3 are astroviruses in the family of Astroviridae with similarity phylogenetically to turkey astroviruses, implicating the potential for cross-species infections between strains. Duck astrovirus (DAstV) causes acute, fatal infections in ducklings with a rapid decline within 1-2 hr and clinical and pathologic signs virtually indistinguishable from DHAV. DAstV-1 has only been recognized in the United Kingdom and recently in China, while DAstV-2 has been reported in ducks in the United States. FAdV, the causative agent of inclusion body hepatitis, is a Group I avian adenovirus in the genus Aviadenovirus. The affected birds have a swollen, friable, and discolored liver, sometimes with necrotic or hemorrhagic foci. Histologic lesions include multifocal necrosis of hepatocytes and acute hepatitis with intranuclear inclusion bodies in the nuclei of the hepatocytes. THV is a picornavirus that is likely the causative agent of turkey viral hepatitis. Currently there are more questions than answers about THV, and the pathogenesis and clinical impacts remain largely unknown. Future research in viral hepatic diseases of poultry is warranted to develop specific diagnostic assays, identify suitable cell culture systems for virus propagation, and develop effective vaccines.

Complete genome sequence of the novel duck hepatitis B virus strain SCP01 from Sichuan Cherry Valley duck.

BackgroundThe duck hepatitis B virus (DHBV) strain, designated SCP01, was isolated and identified from a Sichuan Cherry Valley duck in Southwestern China. To determine the origination and evolution of this isolated strain, we carried out complete genome sequencing of this strain.FindingsSequencing of the nucleotide sequence of DHBV strain SCP01 revealed a genome size of 3021 bp that contained three open reading frames, designated as C, S, and P, which were consistent with those of other duck hepatitis B viruses nucleotide sequences available in the GenBank of NCBI. Sequence comparisons based on the full-length genomic sequences showed that the DHBV SCP01 strain had the highest similarity (99.64 %) with the sequence of strain DHBV-XY, but had a lower similarity (90.04 %) with the sequence of strain DHBV CH5 isolated from Southwestern China. Phylogenetic analysis revealed that the DHBV-XY and DHBV SCP01 formed a branch that was clearly distinct from the other strains.ConclusionThis study show that the DHBV SCP01 strain from Sichuan belonged to “Western” isolates, while the DHBV CH5 from Sichuan belonged to “Chinese” isolates. These data will promote further research into the evolutionary biology, epidemiology and pathobiology of hepadnavirus infections. In addition, continuing duck hepatitis B virus surveillance in poultry is critical to understand the patterns of DHBV infection, and to find further animal infection models to study HBV infection.

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.

From DCPD to NTCP: the long journey towards identifying a functional hepatitis B virus receptor.

Hepatitis B virus (HBV) is the prototype of hepatotropic DNA viruses (hepadnaviruses) infecting a wide range of human and non-human hosts. Previous studies with duck hepatitis B virus (DHBV) identified duck carboxypeptidase D (dCPD) as a host specific binding partner for full-length large envelope protein, and p120 as a binding partner for several truncated versions of the large envelope protein. p120 is the P protein of duck glycine decarboxylase (dGLDC) with restricted expression in DHBV infectible tissues. Several lines of evidence suggest the importance of dCPD, and especially p120, in productive DHBV infection, although neither dCPD nor p120 cDNA could confer susceptibility to DHBV infection in any cell line. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a binding partner for the N-terminus of HBV large envelope protein. Importantly, knock down and reconstitution experiments unequivocally demonstrated that NTCP is both necessary and sufficient for in vitro infection by HBV and hepatitis delta virus (HDV), an RNA virus using HBV envelope proteins for its transmission. What remains unclear is whether NTCP is the major HBV receptor in vivo. The fact that some HBV patients are homozygous with an NTCP mutation known to abolish its receptor function suggests the existence of NTCP-independent pathways of HBV entry. Also, NTCP very likely mediates just one step of the HBV entry process, with additional co-factors for productive HBV infection still to be discovered. NTCP offers a novel therapeutic target for the control of chronic HBV infection.

Cloning, expression and purification of duck hepatitis B virus (DHBV) core protein and its use in the development of an indirect ELISA for serologic detection of DHBV infection

Infecting ducks with duck hepatitis B virus (DHBV) is widely accepted as a relevant model for studying aspects of human HBV infection. However, efficient and sensitive diagnostic methods for the various infection models are limited. In order to provide a more simple and convenient method for serologic diagnosis, we improved the production of recombinant DHBV viral capsid protein (core protein) and then used it to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detecting anti-DHBc antibodies (DHBcAg ELISA) in DHBV-infected ducks. Given the positive/negative cut-off value, the maximum dilution of duck sera in which anti-DHBc antibodies could be detected was 1:12,800. In addition, the DHBcAg ELISA displayed no cross reactivity with duck antisera against duck circovirus (DuCV), duck plague virus (DPV), duck hepatitis virus (DHV), duck swollen head septicemia virus (DSHSV), avian influenza virus (AIV), Riemerella anatipestifer, Salmonella anatum, or Escherichia coli. Furthermore, the coefficients of variation (CVs) of inter-assay and intra-assay experiments were both below than 10 %. When compared to PCR for accuracy on clinical samples from cases of suspected DHBV infection, the DHBcAg showed 95.45 % coincidence with PCR. In conclusion, recombinant DHBc was readily produced and used to establish a simple DHBcAg ELISA that provided a highly specific and sensitive method for analysis of clinical samples.

Nucleic Acid Polymers Inhibit Duck Hepatitis B Virus InfectionIn Vitro

Nucleic acid polymers (NAPs) utilize the sequence-independent properties of phosphorothioate oligonucleotides (PS-ONs) to target protein interactions involved in viral replication. NAPs are broadly active against a diverse range of enveloped viruses that use type I entry mechanisms. The antiviral activity of NAPs against hepatitis B virus (HBV) infection was assessed in vitro in duck hepatitis B virus (DHBV)-infected primary duck hepatocytes (PDH). NAPs efficiently entered PDH in the absence of any transfection agent and displayed antiviral activity at concentrations of 0.01 to 10 μM, measured by their ability to prevent the intracellular accumulation of DHBV surface antigen, which was independent of their nucleotide sequence and was specifically dependent on phosphorothioation. Higher levels of antiviral activity were observed with NAPs 40 nucleotides in length or longer. The fully degenerate NAP (REP 2006) was active during DHBV infection or when added 12 h after infection. In contrast, an acidic-pH-sensitive NAP (REP 2031) that was broadly active against other viruses displayed antiviral activity when present during DHBV infection but no activity when added 12 h after infection, suggesting that NAPs exert their postentry effect in an acidic environment unique to DHBV infection. Both REP 2006 and REP 2031 displayed negligible cytotoxicity in PDH at concentrations of up to 10 μM, as assessed using an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] cytotoxicity assay. The antiviral activity of NAPs against DHBV in vitro was strictly dependent on their amphipathic character, suggesting that NAPs interact with amphipathic target(s) that are important for DHBV entry and postentry mechanisms required for infection.

Nucleic Acid Polymers Prevent the Establishment of Duck Hepatitis B Virus Infection In Vivo

Nucleic acid polymers (NAPs) are novel, broad-spectrum antiviral compounds that use the sequence-independent properties of phosphorothioate oligonucleotides (PS-ONs) as amphipathic polymers to block amphipathic interactions involved in viral entry. Using the duck hepatitis B virus (DHBV) model of human hepatitis B virus infection, NAPs have been shown to have both entry and postentry antiviral activity against DHBV infection in vitro in primary duck hepatocytes (PDH). In the current study, various NAPs were assessed for their prophylactic activity in vivo against DHBV infection in ducks. The degenerate NAP REP 2006 prevented the development of widespread and persistent DHBV infection in 14-day-old ducks, while the acidic-pH-sensitive NAP REP 2031 had little or no prophylactic effect. REP 2006 displayed significant toxicity in ducks, which was attributed to CpG-mediated proinflammation, while REP 2031 (which has no CpG motifs) displayed no toxicity. A third NAP, REP 2055, which was designed to retain amphipathic activity at acidic pH and contained no CpG motifs, was well tolerated and displayed prophylactic activity against DHBV infection at doses as low as 1 mg/kg of body weight/day. These studies suggest that NAPs can be easily and predictably tailored to retain anti-DHBV activity and to have minimal toxic effects in vivo. Future studies are planned to establish the therapeutic efficacy of NAPs against persistent DHBV infection.

Spectroscopic Characterization and Fusogenic Properties of PreS Domains of Duck Hepatitis B Virus

In order to shed light on the hepatitis B virus fusion mechanism and to explore the fusogenic capabilities of preS regions, a recombinant duck hepatitis B virus (DHBV) preS protein (DpreS) containing six histidines at the carboxy-terminal end has been obtained. The DpreS domain, which has an open and mostly nonordered conformation as indicated by fluorescence and circular dichroism spectroscopies, has the ability to interact with negatively charged phospholipid vesicles. The observed interaction differences between neutral and acidic phospholipids can be interpreted in terms of an initial ionic interaction between the phospholipid polar headgroup and the protein followed by the insertion of probably the N-terminal region in the cellular membrane. Fluorescence polarization studies detect a decrease of the transition enthalpy together with a small modification of the transition temperature, typical effects of integral membrane proteins. The interaction of the protein with acidic phospholipid vesicles induces aggregation, lipid mixing, and leakage of internal contents, properties that have been ascribed to membrane destabilizing proteins. The fact that the preS domains of the hepadnaviruses have little similarity but share a very similar hydrophobic profile points to the importance of the overall three-dimensional structure as well as to its conformational flexibility and the distribution of polar and apolar amino acids on the expression of their destabilizing properties rather than to a particular amino acid sequence. The results presented herein argue for the involvement of DpreS in the initial steps of DHBV infection. Taken together with previously reported results, the conclusion that both S and preS regions participate in the fusion process of the hepadnaviridae family may be drawn.

Hepatocytes Traffic and Export Hepatitis B Virus Basolaterally by Polarity-Dependent Mechanisms

Viruses commonly utilize the cellular trafficking machinery of polarized cells to effect viral export. Hepatocytes are polarized in vivo, but most in vitro hepatocyte models are either nonpolarized or have morphology unsuitable for the study of viral export. Here, we investigate the mechanisms of trafficking and export for the hepadnaviruses hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) in polarized hepatocyte-derived cell lines and primary duck hepatocytes. DHBV export, but not replication, was dependent on the development of hepatocyte polarity, with export significantly abrogated over time as primary hepatocytes lost polarity. Using Transwell cultures of polarized N6 cells and adenovirus-based transduction, we observed that export of both HBV and DHBV was vectorially regulated and predominantly basolateral. Monitoring of polarized N6 cells and nonpolarized C11 cells during persistent, long-term DHBV infection demonstrated that newly synthesized sphingolipid and virus displayed significant colocalization and fluorescence resonance energy transfer, implying cotransportation from the Golgi complex to the plasma membrane. Notably, 15% of virus was released apically from polarized cells, corresponding to secretion into the bile duct in vivo, also in association with sphingolipids. We conclude that DHBV and, probably, HBV are reliant upon hepatocyte polarity to be efficiently exported and this export is in association with sphingolipid structures, possibly lipid rafts. This study provides novel insights regarding the mechanisms of hepadnavirus trafficking in hepatocytes, with potential relevance to pathogenesis and immune tolerance.

Differential gene expression analysis of in vitro duck hepatitis B virus infected primary duck hepatocyte cultures

BackgroundThe human hepatitis B virus (HBV), a member of the hepadna viridae, causes acute or chronic hepatitis B, and hepatocellular carcinoma (HCC). The duck hepatitis B virus (DHBV) infection, a dependable and reproducible model for hepadna viral studies, does not result in HCC unlike chronic HBV infection. Information on differential gene expression in DHBV infection might help to compare corresponding changes during HBV infection, and to delineate the reasons for this difference.FindingsA subtractive hybridization cDNA library screening of in vitro DHBV infected, cultured primary duck hepatocytes (PDH) identified cDNAs of 42 up-regulated and 36 down-regulated genes coding for proteins associated with signal transduction, cellular respiration, transcription, translation, ubiquitin/proteasome pathway, apoptosis, and membrane and cytoskeletal organization. Those coding for both novel as well as previously reported proteins in HBV/DHBV infection were present in the library. An inverse modulation of the cDNAs of ten proteins, reported to play role in human HCC, such as that of Y-box binding protein1, Platelet-activating factor acetylhydrolase isoform 1B, ribosomal protein L35a, Ferritin, α-enolase, Acid α-glucosidase and Caspase 3, copper-zinc superoxide dismutase (CuZnSOD), Filamin and Pyruvate dehydrogenase, was also observed in this in vitro study.ConclusionsThe present study identified cDNAs of a number of genes that are differentially modulated in in vitro DHBV infection of primary duck hepatocytes. Further correlation of this differential gene expression in in vivo infection models would be valuable to understand the little known aspects of the hepadnavirus biology.

319 THE VALUE OF TRANSIENT ELASTOGRAPHY AND SAMPLE SEROLOGICAL TEST FOR THE EVALUATION OF LIVER FIBROSIS IN CHRONIC HCV HEPATITIS

and its isoform protein (DuIL-10Dexon5) that lacks one receptor binding site for IL-10R1. The aim of this study was to identify and characterize duck IL-10R1 (DuIL-10R1) and to explore the role of IL-10 signaling in duck hepatitis B virus (DHBV) infection. Methods: The open reading frame of DuIL-10R1 was obtained by RT-PCR with primers based on chicken sequence and 5′ RACE and 3’RACE from duck splenocytes. DuIL-10, DuIL-10Dexon5 and IL-10R1 transcript levels were assessed by real-time PCR in PBMC’s and tissue of DHBV-naive animals and liver of ducks infected with DHBV. Results: The predicted DuIL-10R1 protein showed an identity of 63%, 28% and 27% with chicken, human and murine homologues, respectively. Transcripts of DuIL-10R1 and DuIL-10-isoform proteins were predominately expressed in primary and secondary immune organs and lung. DuIL-10R1 transcripts were down-regulated in PBMCs following mitogen stimulation and transcripts of DuIL-10 isoform proteins were differentially expressed in the liver of ducks with congenital or acute resolving DHBV infection and uninfected, age-matched control animals. Conclusions: DuIL-10R1 has significant homology with mammalian and chicken homologues. The interaction of DuIL-10R1 with the identified two DuIL-10 isoform proteins and their individual roles in duck immune responses and DHBV infection can now be further investigated.

The development of persistent duck hepatitis B virus infection can be prevented using antiviral therapy combined with DNA or recombinant fowlpoxvirus vaccines

We recently reported the development of a successful post-exposure combination antiviral and “prime-boost” vaccination strategy using the duck hepatitis B virus (DHBV) model of human hepatitis B virus infection. The current study aimed to simplify the vaccination strategy and to test the post-exposure efficacy of combination therapy with the Bristol-Myers Squibb antiviral drug, entecavir (ETV) and either a single dose of DHBV DNA vaccines on day 0 post-infection (p.i.) or a single dose of recombinant fowlpoxvirus (rFPV–DHBV) vaccines on day 7 p.i. Whilst untreated control ducks infected with an equal dose of DHBV all developed persistent and wide spread DHBV infection of the liver, ducks treated with ETV combined with either the DHBV DNA vaccines on day 0 p.i. or the rFPV–DHBV vaccines on day 7 p.i. had no detectable DHBV-infected hepatocytes by day 14 p.i. and were protected from the development of persistent DHBV infection.

The persistence in the liver of residual duck hepatitis B virus covalently closed circular DNA is not dependent upon new viral DNA synthesis

Residual hepatitis B virus (HBV) DNA can be detected following the resolution of acute HBV infection. Our previous work using duck hepatitis B virus (DHBV) infected ducks, indicated that ~ 80% of residual DHBV DNA in the liver is in the covalently closed circular DNA (cccDNA) form, suggesting that viral DNA synthesis is suppressed. The current study asked more directly if maintenance of residual DHBV cccDNA is dependent upon ongoing viral DNA synthesis. Ducks that recovered from acute DHBV infection were divided into 2 groups and treated with the antiviral drug, Entecavir (ETV), or placebo. No major differences in the stability of cccDNA or levels of residual cccDNA were observed in liver biopsy tissues taken 95 days apart from ETV treated and placebo control ducks. The data suggest that residual DHBV cccDNA is highly stable and present in a cell population with a rate of turnover similar to normal, uninfected hepatocytes.

Initiation of Duck Hepatitis B Virus Infection Requires Cleavage by a Furin-Like Protease

The entry mechanism of hepatitis B virus (HBV) has not been defined, and this impedes development of antiviral therapies aimed at an early step in the viral life cycle. HBV infection has both host and tissue specificities. For the related duck hepatitis B virus (DHBV), duck carboxypeptidase D (DCPD) has been proposed as the species-specific docking receptor, while glycine decarboxylase (DGD) may serve as a tissue-specific cofactor or secondary receptor. DGD binds to several truncated versions of the viral large envelope protein but not to the full-length protein, suggesting a need for proteolytic cleavage of the envelope protein by a furin-like proprotein convertase. In the present study, we found that transfected DCPD could confer DHBV binding to non-duck cell lines but that this was followed by rapid virus release from cells. Coexpression of furin led to DCPD cleavage and increased virus retention. Treatment of DHBV particles with endosome prepared from duck liver led to cleavage of the large envelope protein, and such viral preparation could generate a small amount of covalently closed circular DNA in LMH cells, a chicken hepatoma cell line resistant to DHBV infection. A furin inhibitor composed of decanoyl-RVKR-chloromethylketone blocked endosomal cleavage of the large envelope protein in vitro and suppressed DHBV infection of primary duck hepatocytes in vivo. These findings suggest that furin or a furin-like proprotein convertase facilitates DHBV infection by cleaving both the docking receptor and the viral large envelope protein.

Pre-P Is a Secreted Glycoprotein Encoded as an N-Terminal Extension of the Duck Hepatitis B Virus Polymerase Gene

The duck hepatitis B virus (DHBV) pregenomic RNA is a bicistronic mRNA encoding the core and polymerase proteins. Thirteen AUGs (C2 to C14) and 10 stop codons (S1 to S10) are located between the C1 AUG for the core protein and the P1 AUG that initiates polymerase translation. We previously found that the translation of the DHBV polymerase is initiated by ribosomal shunting. Here, we assessed the biosynthetic events after shunting. Translation of the polymerase open reading frame was found to initiate at the C13, C14, and P1 AUGs. Initiation at the C13 AUG occurred through ribosomal shunting because translation from this codon was cap dependent but was insensitive to blocking ribosomal scanning internally in the message. C13 and C14 are in frame with P1, and translation from these upstream start codons led to the production of larger isoforms of P. We named these isoforms "pre-P" by analogy to the pre-C and pre-S regions of the core and surface antigen open reading frames. Pre-P was produced in DHBV16 and AusDHBV-infected duck liver and was predicted to exist in 80% of avian hepadnavirus strains. Pre-P was not encapsidated into DHBV core particles, and the viable strain DHBV3 cannot make pre-P, so it is not essential for viral replication. Surprisingly, we found that pre-P is an N-linked glycoprotein that is secreted into the medium of cultured cells. These data indicate that DHBV produces an additional protein that has not been previously reported. Identifying the role of pre-P may improve our understanding of the biology of DHBV infection.

Duck Hepatitis B Virus Requires Cholesterol for Endosomal Escape during Virus Entry

The identity and functionality of biological membranes are determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of biological membranes favoring the formation of detergent-resistant microdomains. In the present study, we evaluated the functional role of cholesterol and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that the duck hepatitis B virus (DHBV) attaches predominantly to detergent-soluble domains on the plasma membrane. Cholesterol depletion from host membranes and thus disruption of rafts does not affect DHBV infection. In contrast, depletion of cholesterol from the envelope of both DHBV and human HBV strongly reduces virus infectivity. Cholesterol depletion increases the density of viral particles and leads to changes in the ultrastructural appearance of the virus envelope. However, the dual topology of the viral envelope protein L is not significantly impaired. Infectivity and density of viral particles are partially restored upon cholesterol replenishment. Binding and entry of cholesterol-deficient DHBV into hepatocytes are not significantly impaired, in contrast to their release from endosomes. We therefore conclude that viral but not host cholesterol is required for endosomal escape of DHBV.