Immunity in Pekin ducks experimentally and naturally infected with duck hepatitis B virus

The immune response to duck hepatitis B virus (DHBV) had not been elucidated. An assay was therefore established to detect the presence of antibody to DHB surface antigen (anti-DHBs) in serum of experimentally inoculated and naturally infected ducks. Anti-DHBs in serum was detected by indirect RIA from the percentage inhibition of binding of rabbit anti-DHBs to purified DHBsAg. Specificity was confirmed by positive and negative controls, infected and noninfected sera, and a mouse monoclonal antibody to DHB core antigen (anti-DHBc). Serum and liver samples were tested for DHBV DNA by dot-blot hybridization assay. Adult ducks repeatedly inoculated with DHBV remained non-viraemic but developed anti-DHBs. This antibody activity neutralized the infectivity of DHBV, which was experimentally inoculated into 1-day-old ducklings. In naturally infected flocks anti-DHBs was detected in a proportion of noninfected adult ducks as well as 1-day-old hatchlings. Anti-DHBs activity in hatchlings neutralized the infectivity of experimentally inoculated DHBV. Pekin ducks can therefore mount a neutralizing antibody response to DHBV, and immunity may be transferred in ovo from dam to off-spring.     

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.     

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.     

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.     

Cellular immune response of ducks to duck hepatitis B virus infection

Duck hepatitis B virus (DHBV) has been a useful model for hepadnavirus infection. There have been few studies on immunity to DHBV and none describing the cell-mediated immune response by acute and chronically infected ducks. A duck hepatitis B antigen-specific blastogenesis assay was used to measure DHBV antigen-specific responses of duck peripheral blood (PBMC) and splenic mononuclear cells (SMCs) from uninfected control ducks, ducks acutely or chronically infected with DHBV, and ducks immune to DHBV. A comparison of the group mean responses by PBMC to DHBV surface antigen (DHBsAg) found that the immune group was significantly different to the other three groups (controls or unexposed, P < 0.0001; acutely infected, P< 0.01; chronically infected, P < 0.01). The responses to DHBsAg by PBMC of the acute group (P< 0.01) were significantly different also to that of the unexposed group. For DHBV core antigen (DHBcAg), significant differences in the responses were found between immune ducks and unexposed (P < 0.0005) and acutely infected (P < 0.05) groups. The SMC showed a significant difference between unexposed ducks and immune ducks (P< 0.05) in the group mean responses to DHBsAg. The responses to DHBcAg were significantly different between the immune group and the acute (P < 0.01) and unexposed (P < 0.01) groups. The group mean of unexposed ducks was also significantly different to that of acutely infected ducks (P < 0.01). This study indicates that the cellular immune response in immune animals differs from acutely and chronically infected ducks. Further studies of these differences may provide some explanations for the differing outcomes of DHBV infection.     

鸭乙型肝炎病毒在鸭胆管上皮细胞内增殖的超微结构研究

Liver specimens from Shanghai Ma ducks infected with duck hepatitis B virus (DHBV) were examined by immunohistochemical technique and electron microscopy. The results showed that DHBV and DHBV antigen were found not only in the hepatocytes but also in the biliary epithelial cells of infected ducks. Electron microscopy also revealed that incomplete spherical particles, 40-50 nm in diameter, were present in the dilated cisternae of rough endoplasmic reticulum (RER), and complete spherical virions, 55-65 nm in diameter, existed in the cytoplasmic vesicles and cytoplasm in small amounts. The results of the present study seem to confirm the possibility of infection and replication of DHBV not only in the liver cells but also in the biliary epithelial cells of ducks.     

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.     

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.     

In vivo antiviral effects of mismatched double-stranded RNA on duck hepatitis B virus

The antiviral activity and ability of mismatched double-stranded RNA (m-dsRNA), r(I)_n′r(C₁₂-U)_n′ to induce interferon (IFN) were evaluated in ducks chronically infected with duck hepatitis B virus (DHBV). When m-dsRNA was administered intravenously at a single dose of 5 mg/kg, serum DHBV DNA concentrations decreased significantly for 3 days (P < 0.002). However, the DHBV DNA concentrations returned to the pretreatment levels 4 days after treatment. Inhibition of DHBV DNA replication in the liver was also observed 2 days after treatment. Serum IFN activity peaked 3 hours after administration of m-dsRNA, then rapidly declined. 2′-5′ Oligo-adenylate synthetase (2′-5′ AS) activity increased gradually after treatment and remained elevated for at least 48 hours. In ducks receiving m-dsRNA once daily for 7 consecutive days, serum DHBV DNA concentrations on the last day of treatment were decreased by 76 ± 12% (P < 0.05) in ducks that received 0.2 mg of m-dsRNA per kg and by 65 ± 12% (P < 0.05) in ducks that received 1 mg of m-dsRNA per kg. This decrease persisted for at least 2 weeks after the cessation of treatment in all ducks. These results suggest that m-dsRNA effectively inhibits DHBV replication in vivo, and that IFN induction and stimulation of 2′–5′AS activity contribute to the inhibition of DHBV replication by m-dsRNA. © 1994 Wiley-Liss, Inc.     

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.     

Molecular Characterization of Duck Hepatitis B Virus Isolates from South African Ducks

The objective of the study was to characterize the genome of duck hepatitis B virus (DHBV) isolates from South African Pekin ducks. Duck serum and liver samples were collected from two commercial duck farms from geographically distinct regions of South Africa. In total, 498 duck serum samples were tested for the presence of DHBV DNA using either sub-genomic or full-length polymerase chain reaction (PCR) assays. The overall prevalence of DHBV infection in South African ducks was 47%. In addition, 30% of 59 liver tissues tested were DHBV DNA-positive. Six randomly selected serum or liver samples were used to clone and sequence the genomes of the South African DHBV strains. All six isolates had DHBV genomes of 3,021 nucleotides with three characteristic overlapping reading frames encoding the polymerase, surface and core gene products. No X-like gene with a traditional start codon was found. Following phylogenetic analysis, the South African DHBV isolates clustered with DHBV isolates from other "Western" countries, including United States of America, Canada, Germany and India. On translation of the open reading frames, the South African isolates were found to share signature amino acids in the polymerase and surface genes with the "Western" country isolates as opposed to those of Chinese DHBV isolates.     

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.     

Characterization of the core promoter and enhancer of duck hepatitis B virus

The core gene promoter of duck hepatitis B virus (DHBV) has been localized and an enhancer element has been found in a region of the DHBV genome immediately upstream of the core promoter. This enhancer was able to activate expression from both the core promoter and the S promoter of DHBV, as well as from the heterologous thymidine kinase and simian virus 40 early promoters, but not from the DHBV PreS promoter, which was active both in the absence and in the presence of the enhancer. The activity of the enhancer showed a preference for cell cultures of hepatic origin, suggesting a possible tissue preference in vivo.     

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.     

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.     

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.     

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.     

Studies by electron microscopy on the assembly of duck hepatitis B virus in the liver

Liver specimens from 1-day-old ducklings infected in ovo with maternally transmitted duck hepatitis B virus (DHBV) were examined by electron microscopy. Complete and incomplete DHBV particles were located within hypertrophied cisternae of the endoplasmic reticulum of the hepatocytes. The complete viral particles found intracellularly have inner cores with a diameter ranging from 35 to 37.5 nm and an outer coat or envelope. The whole particle measures approximately 45-65 nm in diameter. Naked core particles were located in the nuclei, free in the cytoplasm, and also near or on the cisternal membrane of the endoplasmic reticulum on the cytoplasmic face. Duck hepatitis B virions appear to share morphological characteristics with the viral coat and core of human hepatitis B virus (HBV). Electron microscopy suggested that the core particles of DHBV migrate from the nucleus into the cytoplasm through the nuclear pores. The complete viral particles are probably formed by protrusion of the core particles through the endoplasmic reticulum and by simultaneous encapsulation with a coat derived from the endoplasmic reticulum.