Antiviral therapy with entecavir combined with post-exposure "prime-boost" vaccination eliminates duck hepatitis B virus-infected hepatocytes and prevents the development of persistent infection

Short-term antiviral therapy with the nucleoside analogue entecavir (ETV), given at an early stage of duck hepatitis B virus (DHBV) infection, restricts virus spread and leads to clearance of DHBV-infected hepatocytes in approximately 50% of ETV-treated ducks, whereas widespread and persistent DHBV infection develops in 100% of untreated ducks. To increase the treatment response rate, ETV treatment was combined in the current study with a post-exposure "prime-boost" vaccination protocol. Four groups of 14-day-old ducks were inoculated intravenously with a dose of DHBV previously shown to induce persistent DHBV infection. One hour post-infection (p.i.), ducks were primed with DNA vaccines that expressed DHBV core (DHBc) and surface (pre-S/S and S) antigens (Groups A, B) or the DNA vector alone (Groups C, D). ETV (Groups A, C) or water (Groups B, D) was simultaneously administered by gavage and continued for 14 days. Ducks were boosted 7 days p.i. with recombinant fowlpoxvirus (rFPV) strains also expressing DHBc and pre-S/S antigens (Groups A, B) or the FPV-M3 vector (Groups C, D). DHBV-infected hepatocytes were observed in the liver of all ducks at day 4 p.i. with reduced numbers in the ETV-treated ducks. Ducks treated with ETV plus the control vectors showed restricted spread of DHBV infection during ETV treatment, but in 60% of cases, infection became widespread after ETV was stopped. In contrast, at 14 and 67 days p.i., 100% of ducks treated with ETV and "prime-boost" vaccination had no detectable DHBV-infected hepatocytes and had cleared the DHBV infection. These findings suggest that ETV treatment combined with post-exposure "prime-boost" vaccination induced immune responses that eliminated DHBV-infected hepatocytes and prevented the development of persistent DHBV infection.     

Association of intrahepatic cccDNA reduction with the improvement of liver histology in chronic hepatitis B patients receiving oral antiviral agents

Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is difficult to eradicate using current antiviral therapy. This study compares cccDNA reduction with relation to liver histology in nucleoside/nucleotide‐naïve chronic hepatitis B patients receiving oral antiviral monotherapy (n = 35), including entecavir (ETV, n = 13), adefovir dipivoxil (ADV, n = 22) or placebo (n = 14). Serum HBV DNA, intrahepatic total HBV DNA and cccDNA are quantified. Histological hepatic examination is performed at baseline and at 48 weeks of treatment. Treatment with ETV or ADV shows significant median reduction in serum HBV DNA (?6.21 and ?4.27 log₁₀ copies/mL) and intrahepatic total HBV DNA (?1.69 and ?1.23 log₁₀ copies/cell). Intrahepatic cccDNA levels are reduced slightly in the ETV and the ADV groups, but do not differ statistically from the placebo group (?0.17 vs. ?0.01 vs. 0.02 copies/cell). Only the level of intrahepatic cccDNA correlates with Knodell necroinflammation activity (r = 0.527, P < 0.001) and Ishak fibrosis severity (r = 0.348, P = 0.015) before treatment. Multivariate logistic regression analysis indicates that treatment‐induced cccDNA reduction is associated with improved necroinflammation (P = 0.041) and fibrosis (P = 0.026). In conclusion, baseline intrahepatic cccDNA loads correlate with histologic activity. Although one‐year ETV or ADV treatment is insufficient for cccDNA eradication, oral antiviral therapies may improve liver histology, probably by suppressing intrahepatic cccDNA. J. Med. Virol. 83:602–607, 2011. © 2011 Wiley‐Liss, Inc.     

Vaccination of ducks with a whole-cell vaccine expressing duck hepatitis B virus core antigen elicits antiviral immune responses that enable rapid resolution of de novo infection

As a first step in developing immuno-therapeutic vaccines for patients with chronic hepatitis B virus infection, we examined the ability of a whole-cell vaccine, expressing the duck hepatitis B virus (DHBV) core antigen (DHBcAg), to target infected cells leading to the resolution of de novo DHBV infections. Three separate experiments were performed. In each experiment, ducks were vaccinated at 7 and 14 days of age with primary duck embryonic fibroblasts (PDEF) that had been transfected 48 h earlier with plasmid DNA expressing DHBcAg with and without the addition of anti-DHBcAg (anti-DHBc) antibodies. Control ducks were injected with either 0.7% NaCl or non-transfected PDEF. The ducks were then challenged at 18 days of age by intravenous inoculation with DHBV (5 x 10(8) viral genome equivalents). Liver biopsies obtained on day 4 post-challenge demonstrated that vaccination did not prevent infection of the liver as similar numbers of infected hepatocytes were detected in all vaccinated and control ducks. However, analysis of liver tissue obtained 9 or more days post-challenge revealed that 9 out of 11 of the PDEF-DHBcAg vaccinated ducks and 8 out of 11 ducks vaccinated with PDEF-DHBcAg plus anti-DHBc antibodies had rapidly resolved the DHBV infection with clearance of infected cells. In contrast, 10 out of 11 of the control unvaccinated ducks developed chronic DHBV infection. In conclusion, vaccination of ducks with a whole-cell PDEF vaccine expressing DHBcAg elicited immune responses that induced a rapid resolution of DHBV infection. The results establish that chronic infection can be prevented via the vaccine-mediated induction of a core-antigen-specific immune response.     

Effect of Phyllanthus amarus on duck hepatitis B virus replication in vivo

Nine ducks congenitally infected with the duck hepatitis B virus (DHBV) were treated either orally (four ducks for 10 weeks) or intraperitoneally (five ducks for 12 weeks) with the Indian traditional herbal remedy Phyllanthus amarus. Compared to placebo-treated control ducks, these treatments did not result in a reduction of circulating viral DNA in the serum or in the level of viral DNA replication in the liver. In two of the five intraperitoneal-treated ducks, a reduction in the levels of duck hepatitis B surface antigenaemia (DHBsAg) was observed. The data strongly suggest that Phyllanthus amarus has no significant inhibitory effect on DHBV DNA replication and only a minor effect on DHBsAg production.     

A sequential study of viral DNA in serum in experimental transmission of duck hepatitis B virus

To understand the relationship among the time of infection, infection patterns, and liver diseases, experimental transmission of duck hepatitis B virus (DHBV) utilizing 165 Japanese white domestic ducklings was performed. Twenty to 25 ducklings were each inoculated with DHBV-positive serum intravenously at day one, 3, 5, 7, 10, and 14 posthatch and were sacrificed during the 1st, 2nd, 3rd, and 4th (and 24th in those inoculated on day one and day 3 posthatch) week after inoculation to obtain sera and the liver. The sera served for the measurement of DHBV DNA by spot hybridization test and DNA polymerase activity, and the liver was subjected to morphological examination including immunostaining for DHBV. The ducklings inoculated with DHBV on 1 day and 3 days posthatch always revealed persistent viremia, whereas those on and after 5 days posthatch showed persistent or transient viremia. The hepatitis activity in the liver was seen in ducklings inoculated with DHBV on and after 3 days posthatch and was very weak consistent with the diagnosis of mild acute hepatitis of humans. The serum transaminase activity was not significantly elevated at the time of occurrence of histological hepatitis activity. Since host immune mechanism establish at 3 to 5 days posthatch in birds, the host immune response seemed to determine whether DHBV infection was persistent or transient and the occurrence of hepatitis activity as seen in human hepatitis B virus (HBV) infection.     

Serum HBV RNA composition dynamics as a marker for intrahepatic HBV cccDNA turnover

Current therapies can suppress the replication of hepatitis B virus (HBV) but cannot clear chronic HBV infection, which afflicts hundreds of millions worldwide. HBV persistence is sustained by the viral covalently closed circular DNA (cccDNA), an episome in the nucleus of infected hepatocytes. cccDNA is refractory to current therapies and its clearance is the holy grail for HBV cure. However, it has been difficult to monitor the fate of cccDNA in the liver directly. The current study takes a novel approach to this critical issue by monitoring the dynamic change in the genetic composition of the serum HBV RNA, which appears to reflect the dynamics of intrahepatic cccDNA turnover.     

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.     

Alteration of infection pattern of duck hepatitis B virus by immunomodulatory drugs

The relationship between host immune state and hepatic inflammation and infection pattern of the Duck hepatitis B virus (DHBV) was investigated by experimental transmission of DHBV into 98 Japanese 7-day-old ducklings that had been pretreated with immunoregulatory drugs including cyclophosphamide, OK 432, and a steroid hormone. Immunosuppressive treatment with cyclophosphamide revealed an extension of the viremic period associated with an absence of inflammatory changes in the liver. Although immunostimulating treatment with OK 432 showed a remarkable accumulation of inflammatory cells in the liver, the viremic period was not shortened. Treatment with a steroid used as a immunosuppressant did not suppress the hepatitis; moreover, it increased viral DNA replication and extended the viremic period. This phenomenon of viral replication seemed to be caused by the direct effects of the steroid. Alteration of DHBV infection by modifying the host immune state is quite similar to that of hepatitis B virus (HBV) in humans. In DHBV infection, the host immune state seemed to have a considerable role in determining the infection pattern and degree of hepatitis activity. DHBV may be a helpful model of HBV for studying host-viral interaction and the immunological mechanism of viral hepatitis.     

Hepatitis B virus DNA in liver, serum, and peripheral blood mononuclear cells after the clearance of serum hepatitis B virus surface antigen

The integration of hepatitis B virus (HBV) DNA in the liver of chronic HBV carriers has been documented extensively. However, the status of the viral genome during acute infection has not been assessed conclusively. While HBV DNA sequences are detected often in serum, liver, and peripheral blood mononuclear cells (PBMCs) after the clearance of serum the hepatitis B virus surface antigen (HBsAg), the precise status of the viral genome, and in particular the possible persistence of integrated genomes in PBMCs, has not been established. A highly sensitive PCR-derived assay (Alu-PCR) was employed to re-examine liver and PBMC specimens obtained from patients with acute (n = 19) and chronic (n = 22) hepatitis in whom serum HBsAg was present (n = 12) (HBV-related chronic active hepatitis) or absent with anti-HCV (n = 10) (HCV-related chronic active hepatitis). Viral integration was demonstrated in 3 out of 19 liver specimens from patients with acute hepatitis and 12 out of 12 specimens from patients with chronic hepatitis. Viral integration was also observed in 4 out of 7 PBMC samples from HBV-related chronic active hepatitis patients and 2 out of 10 liver and PBMC samples from HCV-related chronic active hepatitis patients. In one liver specimen from an acute hepatitis patient, HBV DNA was found integrated in the intronic sequence of the tumour necrosis factor (TNF)-induced protein gene; viral integration into cellular sequences was also found in the PBMCs of four HBV-related chronic active hepatitis and two HCV-related chronic active hepatitis. The results demonstrate the early integration of HBV genome during acute viral infections and the persistence of the viral genome in an integrated form in PBMCs.     

Detection of duck hepatitis B virus DNA on filter paper by PCR and SYBR green dye-based quantitative PCR

Duck hepatitis B virus (DHBV) belongs to the Hepadnaviridae family, which includes human Hepatitis B virus (HBV) and Woodchuck hepatitis virus. It is widely distributed in wild and domestic ducks due to congenital transmission. HBV is a worldwide health problem, with carriers at risk of developing cirrhosis and liver cancer. Medical staff and scientists working with HBV must be vaccinated because of its contagious nature. DHBV is a safe surrogate for HBV because of their similarities. Collection of serum and blood samples on filter paper has been used to screen for metabolic disorders, genetic diseases, and viral infection and for evolutionary studies of the genome. In this study, DHBV from serum and blood dried on filters was detected by PCR. A 0.1-microl sample was sufficient for detection. The immobilization potential of filter papers for DHBV was examined, and the highest yield of PCR products was observed with Whatman paper. Dried serum was stable under different storage temperatures for 4 weeks, but the yields of PCR products decreased when the temperature was >or=4 degrees C. The optimal condition for storage was -70 degrees C. A newly developed quantitative PCR based on monitoring the amplification by measuring the increase in fluorescence caused by the binding of SYBR green dye to double-stranded products was applied herein. DHBV genomic DNA cloned in a plasmid was used for the generation of standard DHBV DNA for quantitative PCR. It validated results from PCR in terms of the copy number of DHBV particles. The specificity of PCR was demonstrated by melting curve analysis, and the differentiation of two DHBV isolates amplified from dried serum was demonstrated based on their melting temperatures determined by GC contents and sequence. It was easier and simpler than other PCR-based DNA techniques. The use of serum dried on filters allows samples from distant field for which cold storage and transportation are a problem to be mailed to the diagnostic laboratory. Samples can be archived for comparison and used as a source of DNA for cloning and sequencing.     

Delipidation of a hepadnavirus: Viral inactivation and vaccine development

Many viruses including HIV, hepatitis C and hepatitis B, have an outer lipid envelope which maintains inserted viral peptides in the "correct" functional conformation and orientation. Disruption of the lipid envelope by most solvents destroys infectivity and often results in a loss of antigenicity. This communication outlines a novel approach to viral inactivation by specific solvent delipidation which modifies the whole virion rendering it non-infective, but antigenic. Duck hepatitis B virus (DHBV) was delipidated using a diisopropylether (DIPE) and butanol mixture and residual infectivity tested by inoculation into day-old ducks. Delipidation completely inactivated the DHBV (p < 0.001). Delipidated DHBV was then used to vaccinate ducks. Three doses of delipidated DHBV induced anti-DHBs antibody production and prevented high dose challenge infection in five out of six ducks. In comparison, five of six ducks vaccinated with undelipidated DHBV and four of four ducks vaccinated with glutaraldehyde inactivated DHBV were unprotected (p < 0.05). Although this solvent system completely inactivated DHBV, viral antigens were retained in an appropriate form to induce immunity. Delipidation of enveloped viruses with specific organic solvents has potential as the basis for development of vaccines.     

Impact of peginterferon alpha‐2b and entecavir hydrate combination therapy on persistent viral suppression in patients with chronic hepatitis B

The ideal approach to treat chronic hepatitis B remains controversial. This pilot study aimed to evaluate the effectiveness of peginterferon (PEG‐IFN) α‐2b and entecavir hydrate (ETV) as a combination therapy for patients with chronic hepatitis B, particularly in the context of virological response and the reduction of intrahepatic covalently closed circular DNA (cccDNA). A total of 17 patients with hepatitis B virus (HBV) genotype C were enrolled in this study. All subjects were treated with this combination therapy for 48 weeks and observed for an additional 24 weeks. All patients underwent liver biopsy before and after the therapy period. Changes in cccDNA levels and liver histology were monitored between biopsies. Among the 11 patients who exhibited pre‐therapy hepatitis B e antigen (HBeAg), 8 (73%) showed evidence of HBeAg seroconversion by the end of the follow‐up period. Serum HBV DNA levels decreased by 5.2 and 3.3 log copies/ml (mean) by the end of the therapy and follow‐up periods, respectively. In addition, intrahepatic cccDNA decreased significantly to 1.4 log copies/µg (mean) by the end of the therapy period. Among the 11 patients who did not experience viral relapse, only 2 (18%) exhibited high levels of cccDNA (>4.5 log copies/µg) by the end of the treatment period. In contrast, all relapsed subjects exhibited significantly higher levels of cccDNA than subjects who did not relapse (P = 0.027). The combination regimen is a promising approach to treat chronic hepatitis B and may achieve significant reduction in serum HBV DNA and intrahepatic cccDNA. J. Med. Virol. 85: 987–995, 2013. © 2013 Wiley Periodicals, Inc.     

Evaluation of anti-hepadnavirus activity of Phyllanthus amarus and Phyllanthus maderaspatensis in duck hepatitis b virus carrier Pekin ducks

Extracts of the two traditional Indian herbs, Phyl lanthus amarus (P. amarus) and Phyllanthus maderaspatensis (P. maderaspatensis), described by others as useful in the treatment of chronic hepatitis B virus infection were studied for antiviral properties on duck hepatitis B virus infection. One hundred and fourteen ducks infected posthatch with the duck hepatitis B virus (DHBV) were divided into groups at three months of age and treated intraperitoneally with the aqueous, butanol, and alcoholic extracts of these two plants at doses of 25, 50, or 200 mg/kg body weight. Saline-treated animals served as controls. In the ducks negative for DHBV in serum after treatment, we observed replicative intermediates in the liver. There was no definite antiviral property observed in the treated ducks.     

DNA vaccines expressing the duck hepatitis B virus surface proteins lead to reduced numbers of infected hepatocytes and protect ducks against the development of chronic infection in a virus dose-dependent manner

We tested the efficacy of DNA vaccines expressing the duck hepatitis B virus (DHBV) pre-surface (pre-S/S) and surface (S) proteins in modifying the outcome of infection in 14-day-old ducks. In two experiments, Pekin Aylesbury ducks were vaccinated on days 4 and 14 of age with plasmid DNA vaccines expressing either the DHBV pre-S/S or S proteins, or the control plasmid vector, pcDNA1.1Amp. All ducks were then challenged intravenously on day 14 of age with 5 x 10(7) or 5 x 10(8) DHBV genomes. Levels of initial DHBV infection were assessed using liver biopsy tissue collected at day 4 post-challenge (p.c.) followed and immunostained for DHBV surface antigen to determine the percentage of infected hepatocytes. All vector vaccinated ducks challenged with 5 x 10(7) and 5 x 10(8) DHBV genomes had an average of 3.21% and 20.1% of DHBV-positive hepatocytes respectively at day 4 p.c. and 16 out of 16 ducks developed chronic DHBV infection. In contrast, pre-S/S and S vaccinated ducks challenged with 5 x 10(7) DHBV genomes had reduced levels of initial infection with an average of 1.38% and 1.93% of DHBV-positive hepatocytes at day 4 p.c. respectively and 10 of 18 ducks were protected against chronic infection. The pre-S/S and the S DNA vaccinated ducks challenged with 5 x 10(8) DHBV genomes had an average of 31.5% and 9.2% of DHBV-positive hepatocytes on day 4 p.c. respectively and only 4 of the 18 vaccinated ducks were protected against chronic infection. There was no statistically significant difference in the efficacy of the DHBV pre-S/S or S DNA vaccines. In conclusion, vaccination of young ducks with DNA vaccines expressing the DHBV pre-S/S and S proteins induced rapid immune responses that reduced the extent of initial DHBV infection in the liver and prevented the development of chronic infection in a virus dose-dependent manner.     

G1 phase dependent nuclear localization of relaxed-circular hepatitis B virus DNA and aphidicolin-induced accumulation of covalently closed circular DNA

During chronic hepatitis B virus (HBV) infection, virus persistence relies on the maintenance of a pool of covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes. To achieve this, HBV DNA has to be transported from the cytoplasm to the nucleus. By carrying out subcellular fractionation experiment, both of the relaxed-circular (RC) and single-stranded (SS) HBV DNA were found in the cytoplasm whereas only RC form could be detected in the nucleus of a hepatoblastoma cell line (HepG2) stably producing HBV. This fraction of nuclear RC viral DNA was clearly demonstrated in the G₁ but not S phase of synchronized HepG2 cells. Conversely, the relative amount of cytoplasmic RC viral DNA in the S phase was larger than that in the G1 phase. Although no cccDNA could be detected in HepG2 cells without synchronization, an increasing amount of cccDNA in the nucleus was demonstrated after prolonged incubation of the cells in aphidicolin. Finally, by undertaking in situ hybridization using a probe specific to plus-strand HBV DNA, nuclear viral DNA was detected predominantly in the G1 phase of HepG2 cells. In summary, the results indicated that only RC but not SS form of HBV DNA was localized to the nuclei of HepG2 cells. The nuclear localization occurred preferentially in the G₁ but not S phase and prolonged treatment with aphidicolin resulted in accumulation of nuclear cccDNA. J. Med. Virol. 55:42–50, 1998. © 1998 Wiley-Liss, Inc.