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.     

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.     

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.     

核酶对鸭乙型肝炎病毒感染体内抗病毒作用效果的观察

目的观察核酶在体内抗病毒作用效果。方法选择鸭乙型肝炎（ＤＨＢＶＬＪ－７６）及其鸭动物模型作为检测系统，将针对ＤＨＢＶＰｒｅ－Ｓ７３６位点的核酶（ＲｚＤＳ）插入ｐＪ１２０质粒构建成ｐＪ－ＲｚＤＳ。与痘苗病毒天坛７６１株（ＶＶ）同源重组后获得含ＲｚＤＳ的重组痘苗病毒（Ｖ－ＲｚＤＳ）。用ＤＨＢＶ感染１日龄北京鸭，分别在感染的同时、感染后２４小时和７２小时注射Ｖ－ＲｚＤＳ和ＶＶ，１０天后检测血清中ＤＨＢＶＤＮＡ和ＤＨＢｓＡｇ的含量。结果北京鸭在感染ＤＨＢＶ的同时注射Ｖ－ＲｚＤＳ，其ＤＨＢＶＤＮＡ和ＤＨＢｓＡｇ的含量均低于ＶＶ对照组，两组之间有显著性差异。结论提示ＲｚＤＳ对ＤＨＢＶ基因组复制和表达均有抑制作用，其抑制率分别为４５％和６３％。     

Experimental in ovo transmission of duck hepatitis B virus

Inoculation of fertile Pekin duck eggs with diluted serum containing DHBV into eggs incubated for 24 h and into the extra-embryonic cavities of 14-day-old embryos resulted in a high proportion of viraemic ducklings irrespective of the route of inoculation. Long-term observation of som of the ducks established that the viraemia induced experimentally is long-lasting and has persisted for periods up to 16 mth post-hatch. Separation of DHBV from the plasma of carrier ducks by rate zonal centrifugation was examined by DNA polymerase (DNAP) activity. Particles in the fraction with peak DNAP activity had a buoyant density of 1.16 g X cm-3 in sucrose and an estimated sedimentation coefficient, S20.w of 77. DHBV particles, the morphology of which could be resolved under the electron microscope, consisted of a coat (about 10 nm in thickness) surrounding a core with a diameter measuring 40 nm but not 27 nm as previously reported. Spike-like projections were found on the surface of the core as described previously by W.S. Mason, G. Seal and J. Summers, 1980, J. Virol. 36, 829-836.     

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.     

反义寡脱氧核苷酸在鸭体内抑制鸭乙型肝炎病毒复制与 …

目的 研究反义核酸的抗病毒作用。方法 设计合成了针对鸭乙型肝炎病毒（ＤＨＢＶ）前Ｓ（ＰｒｅＳ）基因区第９５１－９６８位核苷酸的硫代反义寡脱氧核苷酸（ＡＳ－ＯＤＮ），以２０μｇ／ｇ体重／日剂量对３只腹腔感染ＤＨＢＶ５．２毒株后，血清ＤＨＢｓＡｇ及ＤＨＢＶ ＤＮＡ阳性鸭连续静脉注射１０天，同时以等体积生理盐水注射另３只感染鸭作为对照。结果 对照鸭注射生理盐水后，血清ＤＨＢｓＡｇ及ＤＨＢＶ ＤＮＡ阳性未     

Characterization of infectious and defective cloned avian hepadnavirus genomes

The infectivity in vivo, replication competence in vitro, and expression of viral genes of several molecularly cloned duck hepatitis B virus (DHBV) genomes were investigated. In addition, replication competence, core protein expression, and secretion of viral proteins were investigated for a grey heron hepatitis B virus genome. Except two, all DHBV isolates tested induced a systemic infection in Pekin ducks when injected as cloned viral DNA into the liver. After transfection of chicken hepatoma cells, both defective DHBV genomes expressed intracellular nucleocapsid and pre-S envelope proteins and secreted DHBs/pre-S particles into the medium. One of the defective DHBV genomes and HHBV produced within the cells replicative intermediates encapsidated in core particles and secreted virions, whereas the other defective DHBV genome did not and was unable to efficiently encapsidate the RNA pregenome. Comparative sequence analysis was performed to identify potential amino acid changes in viral proteins of both defective DHBV genomes. The data obtained demonstrate that most cloned avian hepadnaviruses are infectious or replication competent and suggest defects in envelope, polymerase or encapsidation function, respectively, in two cloned DHBV genomes.     

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.     

The effect of surgical immunomodulation on liver inflammation and clearance of DHBV infection

The key to developing a therapeutic vaccine for chronic hepadnavirus infection lies in the characteristics of the host-immune response which leads to clearance of acute infection. Groups of 28-day-old ducks which had been surgically bursectomized (n = 10) or thymectomized (n = 13) on the day of hatch or were untreated (n = 21) were inoculated with 10(9) viral genome equivalents (vge) DHBV, then bled twice a week, and euthanased 40 days later. Serum and liver were tested for DHBV DNA and total leukocytes and peripheral blood mononuclear cells (PBMCs) counted. Liver and spleen sections were either stained with hematoxylin and eosin, and graded for inflammation or stained with peroxidase-labeled anti-human CD3 antibody and examined for T lymphocyte distribution. PBMC counts were similar in all groups. DHBV infection combined with bursectomy increased significantly, while thymectomy decreased significantly the total leukocyte count. The spleen and liver bursectomy increased T lymphocyte number while B cells were decreased. Converse changes were observed in thymectomized ducks. Histological evidence of hepatitis was present in infected control and bursectomized ducks but not in the uninfected control or infected thymectomized ducks. In control animals, DHBV challenge caused viremia in 17 and persistent infection in 11 (56%). Fewer thymectomized ducks (3/13, 23%) and significantly more (100%) bursectomized ducks remained persistently infected (P < 0.001). Unexpectedly, bursectomy led to persistence of infection while clearance of infection occurred normally in thymectomized ducks despite decreased T lymphocyte numbers. This suggests that clearance requires T and B lymphocyte collaboration.     

Dissociated antibody responses to the S and pre-S2 regions of the hepatitis B virus after vaccination in hemophiliacs

The membranes of hepatocytes and the pre-S2 envelope protein of the hepatitis B virus (HBV) contain binding sites for polymerized human albumin, which is thought to act as a link between HBV and hepatocytes. Hence, anti-pre-S2 antibodies should prevent HBV uptake by the liver, and there is indeed preliminary evidence that they protect chimpanzees from HBV infection. To evaluate whether a plasma-derived vaccine containing the pre-S2 sequence induced an anti-pre-S2 response in 105 vaccinated hemophiliacs, anti-pre-S2 was measured in parallel with antibody to hepatitis B surface antigen (anti-HBs). Eighty-five percent of the hemophiliacs had both anti-pre-S2 and anti-HBs when vaccination was completed, 13% had anti-HBs alone, and 2% (two cases) had anti-pre-S2 alone. Eighty-seven percent of anti-pre-S2-positive hemophiliacs compared with only 50% of anti-pre-S2-negative hemophiliacs (P less than 0.001) developed high anti-HBs titers (greater than or equal to 1,000 mlU/ml). This study demonstrates, therefore, that the antibody responses to the S and pre-S2 regions of HBV may be dissociated after vaccination in hemophiliacs and that higher anti-HBs titers are attained in anti-pre-S2-positive hemophiliacs.     

Molecular cloning and sequence analysis of duck hepatitis B virus genomes of a new variant isolated from Shanghai ducks

The genomes of duck hepatitis B virus (DHBV) from a brown duck (S5) and a white duck (S31) kept independently in Shanghai, China, were cloned and the complete nucleotide sequence of each virion DNA (DHBV-S5 and DHBV-S31) was determined. DHBV-S5 and DHBV-S31 were both 3027 bp in length and 6 bp longer than the other two DHBVs analyzed previously, DHBV16 and DHBV3. The genomes of DHBV-S5 and DHBV-S31 encoded three long overlapping open reading frames designated as P, S, and C. A possible new open reading frame was found in a complementary strand of each viral genome, as 336 bp for DHBV-S5 and 306 bp for DHBV-S31, respectively. A pair of 3-bp insertions were found in the overlapping region of pre-S2 and P and so two amino acids were inserted in this region in DHBV-S5 and DHBV-S31. The nucleotide sequence variation between DHBV-S5 and DHBV-S31 (4.9%) was similar to that between DHBV16 and DHBV3 (5.6%), and less than the variations between either of these Shanghai clones and DHBV16 or DHBV3 (9.5-10.4%). The amino acid sequence was also conserved in the two Shanghai clones but showed group difference from DHBV16 or DHBV3. Thus these two independent Shanghai clones of DHBV showed geographical characteristics of genomic structure.     

A study of duck hepatitis B virus infection in Chinese ducklings]

Intraperitoneal inoculation of duck hepatitis B virus in three different dosages (9 x 10(7), 1.8 x 10(8), 9 x 10(8) DHBV particles) into 3 to 21 day-old Chinese ducklings provided from a DHBV free flock produced a persistent infection up to 93.3% in 60 animals. The serum and liver specimens of these ducklings were examined by DNA dot blot hybridization on the 30th day after inoculation. The results showed that: (1) examination of viral DNA in liver was more sensitive and reliable than estimation of the DNA in serum for detecting DHBV infection in inoculated ducklings; (2) the liver DHBV DNA level did not coincide with the degree of liver hepatitis induced; (3) 21-day-old Chinese ducklings were also susceptible to DHBV infection, the infection rate of this group was 100% (10/10).     

Intracellular factors, but not virus receptor levels, influence the age-related outcome of DHBV infection of ducks.

Previous serological studies of experimental infection with duck hepatitis B virus (DHBV) have shown that the outcome of infection depends largely on the age of the duck at the time of inoculation. To examine the hypothesis that decreased susceptibility with increased age might be due to the loss of the virus receptor on hepatocyte membranes in adult ducks, we performed receptor binding studies using intact serum-derived DHBV virions and purified liver plasma membranes from both young ducklings and adult ducks. These studies showed that (1) DHBV was able to bind specifically to duck liver plasma membranes but not to internal membranes; (2) this binding could be inhibited by a monoclonal antibody to DHBV preS, a corresponding region in hepatitis B virus that binds to human hepatocytes; and (3) there was no significant difference in the receptor binding ability between plasma membranes from ducklings and from adult ducks. Since hepatocytes in the neonatal ducks are actively dividing, in contrast to the situation in adult ducks, we examined the effect of partial hepatectomy on DHBV-carrier ducks. A sharp increase was noted in the level of DHBV in the serum after partial hepatectomy suggesting that DHBV replication was enhanced in dividing hepatocytes. Thus the age-related difference in susceptibility of ducks to DHBV infection is not due to loss of the receptor but may be related to an intracellular event associated with cell division.     

The insertion domain of the duck hepatitis B virus core protein plays a role in nucleocapsid assembly

Synthesis of hepadnaviral DNA is dependent upon both the viral DNA polymerase and the viral core protein, the subunit of the nucleocapsids in which viral DNA synthesis takes place. In a study of natural isolates of duck hepatitis B virus (DHBV), we cloned full-length viral genomes from a puna teal. One of the clones failed to direct viral DNA replication in transfected cells, apparently as a result of a 3 nt inframe deletion of histidine 107 in the core protein. Histidine 107 is located in the center of a predicted helical region of the "insertion domain", a stretch of 45 amino acids which appears to be at the tip of a spike on the surface of the nucleocapsid. The mutation was introduced into a well-characterized strain of DHBV for further analysis. Core protein accumulated in cells transfected with the mutant DHBV but was partially degraded, suggesting that it was unstable. Assembled nucleocapsids were not detected by capsid gel electrophoresis. Interestingly, the mutant protein appeared to form chimeric nucleocapsids with wild-type core protein. The chimeric nucleocapsids supported viral DNA replication. These results suggest that the insertion domain of the spike may play a role either in assembly of stable nucleocapsids, possibly in formation of the dimer subunits, or in triggering nucleocapsid disintegration, required during initiation of new rounds of infection.     

Alternative methods for validation of cell culture infection with duck hepatitis B virus

Hepatitis B virus (HBV) is an important virus used in disinfection procedures for blood spillage. However, validation of HBV inactivation is difficult, since there are no feasible infectivity assays. In some countries, the duck HBV (DHBV) is recognized as a suitable model for testing antiviral activity of chemical biocides against HBV. Currently, DHBV-infected ducks are required for preparation of the test virus as well as eggs from DHBV-free flocks for testing DHBV infectivity. To improve the practicality of the system, we suggested to use commercially available embryonated duck eggs for preparation of DHBV-susceptible hepatocyte cultures and to exclude infected hepatocytes by pre-screening with qualitative detection of DHBV DNA using polymerase chain reaction (PCR). A standardized DHBV test virus was prepared from the DHBV DNA-transfected hepatoma cell line D2, which contained 10(11)DHBV DNA molecules per mL detected by light cycler real-time PCR. Infection of cell cultures was most efficient 4 days after plating. The best identification of infected cultures was possible 6 days after infection with immunofluorescence using an antiserum against DHBV surface antigen.     

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.     

Comparison of cell culture systems for duck hepatitis B virus using SyBr green quantitative PCR

The Hepadnaviridae family contains DNA viruses such as human hepatitis B virus (HBV), woodchuck hepatitis B virus (WHV), and duck hepatitis B virus (DHBV). DHBV is distributed in both wild and domestic ducks. HBV is a worldwide health problem with carriers at risk of developing cirrhosis and liver cancer. All medical staff and scientists working with HBV must be vaccinated, because of its highly contagious nature. DHBV is a safe surrogate for HBV because of their similarities. Several cell culture systems have been developed to study anti-DHBV drugs and disinfectants. However, differences in their capabilities to support DHBV propagation have not been reported. Therefore, a sensitive and reproducible quantitative PCR based on SyBr green dye was developed. This system does not need electrophoresis for analysis of PCR products, thus reducing processing time and potential for cross-contamination. It allowed precise quantification of DHBV over 8-logarithm dynamic range with a good correlation (R(2) = 0.9689) and showed minimal run-to-run deviation. Sensitivity was 820 copies of DHBV genome and specificity was confirmed by melting curve analysis. It demonstrated good repeatability in quantification of DHBV loads from serum of infected ducks. This assay compared DHBV yields from different cultured cells. All cells had similar kinetic curves for DHBV replication and replication peaks appeared 4 days post-infection. Duck embryonic hepatocytes showed the highest (P > 0.05) replication peak for DHBV. Therefore, duck embryonic hepatocytes and quantitative PCR based on SyBr green dye are a good choice for anti-DHBV drug and disinfectant testing.