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

Effect of Antiviral Treatment with Entecavir on Age- and Dose-Related Outcomes of Duck Hepatitis B Virus Infection

Entecavir (ETV), a potent inhibitor of the hepadnaviral polymerases, prevented the development of persistent infection when administered in the early stages of duck hepatitis B virus (DHBV) infection. In a preliminary experiment, ETV treatment commenced 24 h before infection showed no significant advantage over simultaneous ETV treatment and infection. In two further experiments 14-day-old ducks were inoculated with DHBV-positive serum containing 10(4), 10(6), 10(8), or 5 × 10(8) viral genomes (vge) and were treated orally with 1.0 mg/kg of body weight/day of ETV for 14 or 49 days. A relationship between virus dose and infection outcome was seen: non-ETV-treated ducks inoculated with 10(4) vge had transient infection, while ducks inoculated with higher doses developed persistent infection. ETV treatment for 49 days did not prevent initial infection of the liver but restricted the spread of infection more than ∼1,000-fold, a difference which persisted throughout treatment and for up to 49 days after withdrawal. Ultimately, three of seven ETV-treated ducks resolved their DHBV infection, while the remaining ducks developed viremia and persistent infection after a lag period of at least 63 days. ETV treatment for 14 days also restricted the spread of infection, leading to marked and sustained reductions in the number of DHBV-positive hepatocytes in 7 out of 10 ducks. In conclusion, short-term suppression with ETV provides opportunity for the immune response to successfully control DHBV infection. Since DHBV infection of ducks provides a good model system for HBV infection in humans, it seems likely that ETV may be useful in postexposure therapy for HBV infection aimed at preventing the development of persistent infection

Detection of Clonally Expanded Hepatocytes in Chimpanzees with Chronic Hepatitis B Virus Infection ▿ †

During a hepadnavirus infection, viral DNA integrates at a low rate into random sites in the host DNA, producing unique virus-cell junctions detectable by inverse nested PCR (invPCR). These junctions serve as genetic markers of individual hepatocytes, providing a means to detect their subsequent proliferation into clones of two or more hepatocytes. A previous study suggested that the livers of 2.4-year-old woodchucks (Marmota monax) chronically infected with woodchuck hepatitis virus contained at least 100,000 clones of >1,000 hepatocytes (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. USA 102:1139-1144, 2005). However, possible correlations between sites of viral-DNA integration and clonal expansion could not be explored because the woodchuck genome has not yet been sequenced. In order to further investigate this issue, we looked for similar clonal expansion of hepatocytes in the livers of chimpanzees chronically infected with hepatitis B virus (HBV). Liver samples for invPCR were collected from eight chimpanzees chronically infected with HBV for at least 20 years. Fifty clones ranging in size from ∼35 to 10,000 hepatocytes were detected using invPCR in 32 liver biopsy fragments (∼1 mg) containing, in total, ∼3 × 107 liver cells. Based on searching the analogous human genome, integration sites were found on all chromosomes except Y, ∼30% in known or predicted genes. However, no obvious association between the extent of clonal expansion and the integration site was apparent. This suggests that the integration site per se is not responsible for the outgrowth of large clones of hepatocytes

<i>In vivo</i> Experiment 1: Detection of DHBsAg and DHBcAg positive hepatocytes in liver tissue.

<p>^a--- = no liver tissue harvested</p><p>^bLower limit of detection of DHBsAg positive hepatocytes is 0.001%.</p><p>^cLower limit of detection of DHBcAg positive hepatocytes is 0.006%.</p><p>^dThe liver of duck 296 contained amyloid deposits</p><p>^eND = not done due to sample exhaustion</p><p><i>In vivo</i> Experiment 1: Detection of DHBsAg and DHBcAg positive hepatocytes in liver tissue.</p

<i>In vivo</i> Experiment 1.

<p><b>Response to various REP 2055 treatment regimens in Groups 1–4</b>. Individual duck data for serum DHBsAg (top row) and serum DHBV DNA (bottom row). Days of REP 2055 treatment are indicated at the top of each graph by inverted triangles (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140909#pone.0140909.g001" target="_blank">Fig 1A</a>). LLOQ for DHBsAg (0.88 μg/ml) and DHBV DNA (24000 copies/ml) are shown as dashed lines.</p

Experimental design of REP 2055 and NS treatment in <i>in vivo</i> Experiment 1 and 2.

<p>Fourteen-day-old ducks were infected with 5x10⁸ DHBV genome equivalents via the jugular vein. In Experiment 1 (a) all ducks were treated by IP injection with REP 2055. Group 1 received 10 mg/kg/day from 1 day prior to DHBV infection to 14 dpi; Group 2 received 10 mg/kg/day from 12–19 dpi and 10 mg/kg once weekly for 49 days; Group 3 received 10 mg/kg/day from 4–18 dpi and; Group 4 received 2 mg/kg/day from 4–18 dpi. After treatment, ducks in Groups 1, 3 and 4 were followed for an additional 49 days, from 19–68 dpi. Liver biopsies were performed prior to treatment in Group 2 and at the end of treatment in Groups 1, 3 and 4. Autopsies were performed at 68 dpi at the end of treatment in Group 2 and the end of follow-up in Groups 1, 3 and 4. In Experiment 2 (b), 14 DHBV-infected ducks were treated by IP injection with 10 mg/kg/day of REP 2055 from 12–40 dpi. A control group of 14 DHBV-infected ducks received daily IP injections of NS. Blood samples were collected during treatment and from 41–103 dpi during the first 9 weeks of follow-up. Based on interim analysis of serum DHBV DNA, 7 REP 2055-treated ducks that maintained control of their infection and 7 randomly selected NS-treated ducks, were followed from 103–155 dpi (total 16 weeks of follow-up). Liver biopsies were performed in all animals at 12 dpi prior to treatment. Biopsies or autopsies were performed at 103 dpi (9 weeks follow-up) and additional autopsies were performed in 7 animals per group at 155 dpi (16 weeks follow-up).</p

<i>In vivo</i> Experiment 2: Detection of DHBsAg and DHBcAg positive hepatocytes in liver tissue.

<p>^aNS or REP 2055 was administered via IP injection.</p><p>^b--- = no liver tissue harvested (ducks autopsied at 103 dpi, 9 weeks of follow-up)</p><p>^cLower limit of detection of DHBsAg positive hepatocytes is 0.001%.</p><p>^dLower limit of detection of DHBcAg positive hepatocytes is 0.006%.</p><p><i>In vivo</i> Experiment 2: Detection of DHBsAg and DHBcAg positive hepatocytes in liver tissue.</p

Lack of cytokine gene upregulation in PHH treated with REP 2055.

<p>The expression levels of <i>TNF</i> (a), <i>IL6</i> (b), <i>IL10</i> (c), <i>IFNA4</i> (d) and <i>IFNB1</i> (e) genes were assessed by quantitative RT-PCR 6 hr after treatment with the NAP REP 2055 (0.01–10 μM) or without treatment (w/o), ODN 2216 (a CpG oligonucleotide TLR-9 agonist; 2 μM), poly I:C (a double stranded RNA TLR-3 agonist; 25 μg/ml) or Pam3CK4 (a TLR-1/2 agonist; 1 μM). Values represent mean ± SEM (normalized to 100,000 copies of beta actin mRNA). Statistically significant changes compared to untreated controls are reported for p< 0.05 (*) and p< 0.01 (**).</p