Evaluation of Phyllanthus amarus and Phyllanthus maderaspatensis as agents for postexposure prophylaxis in neonatal duck hepatitis B virus infection

The therapeutic potential of plant extracts of Phyllanthus amarus and Phyllanthus maderaspatensis for postexposure prophylaxis against infection by Hepadnaviruses was studied in ducklings infected by the duck hepatitis B virus (DHBV). Forty-four Pekin ducklings were inoculated intraperitoneally with DHBV at 24 hr posthatch. They were treated by intraperitoneal injection of Phyllanthus amarus (aqueous extract) (100 mg/kg body weight) or Phyllanthus mad eraspatensis (alcoholic extract) (100 mg/kg body weight) for a period of 4 weeks. Infected ducklings treated with saline served as controls. Weekly serum samples obtained before, during, and after treatment were analysed for the presence of DHBV DNA in serum by dot blot hybridisation using α ³²P-labelled probes. Liver tissue was collected after killing the ducks at various time intervals and was studied for replicative status of the viral DNA and liver histopathology; 17 of 21 ducks were viraemic on completion of treatment with Phyllanthus amarus. At 16 week posttreatment follow-up four of seven animals remained viraemic. Similar results were obtained with Phyllanthus maderaspatensis. There was no alteration in DHBV replication in the liver. No toxicity was observed with this treatment. These observations suggest that Phyllanthus amarus and Phyllanthus maderaspatensis are not useful as therapeutic agents for postexposure prophylaxis against DHBV infection. © 1993 Wiley-Liss, 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.     

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     

Antiviral strategies in chronic hepatitis B virus infection: I. Establishment of an in vitro system using the duck hepatitis B virus model

Primary duck hepatocyte (PDH) cultures were established from ducklings congenitally infected with the duck hepatitis B virus (DHBV), plated onto feeder cell layers of irradiated human embryonic lung fibroblasts, and observed for 2 to 3 weeks. This system permitted the survival of the PDH in a differentiated form free of fibroblastic overgrowth for at least 3 weeks. The hepatocytes were shown to contain all the replicative DNA intermediates found during DHBV replication as well as the DHBV structural proteins PRE-S1, PRE-S2, and S of duck hepatitis B surface antigen (DHBsAg). The pool of supercoiled (SC) DHBV DNA increased dramatically from days 10 to 14 postplating. This PDH-feeder cell layer cell culture model provides a convenient system to study the effects of conventional inhibitors of DHBV replication and compounds targeted at the supercoiled form of DHBV DNA. This approach should allow the evaluation of a variety of strategies for treating chronic carriers of hepadnaviruses.     

鸭乙型肝炎病毒的免疫学检测方法及区域分布差异

由于乙型肝炎病毒(HBV)只感染人和黑猩猩,缺少实验动物模型,所以乙型肝炎的发病机理和HBV在肝癌发生中的作用尚未完全清楚。而鸭乙型肝炎病毒(DHBV)与HBV同属于嗜肝DNA病毒家族的成员。这些病毒为直径42～47nm的球形颗粒,由相当于表面抗原成分的外壳和含核心抗原、DNA、DNA聚合酶的核心组成,对宿主有种属特异性,易引起宿主的慢性感     

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.     

Serological analysis of duck hepatitis B virus infection

A radioimmunoassay was developed to detect duck hepatitis B virus surface antigen and antibody; viraemia (DHBV DNA or DHBsAg) was detected in all ducks inoculated within 3 weeks post-hatch, and persistent infection developed in 93% of birds in this group. In contrast, only 80% and 60% of ducks inoculated 4- and 6-weeks post-hatch respectively developed viraemia, and approximately 70% of the viraemic ducks became carriers. Markers of viraemia were undetected in ducks inoculated 8 weeks post-hatch and in uninfected controls. A typical anti-DHBs seroconversion developed subsequently in 2 of 4 birds that showed transient viraemia, and antibody also developed in 3 of 7 ducks inoculated 4-8 weeks post-hatch that showed no viraemia. However, gene amplification by the polymerase chain reaction demonstrated DHBV DNA in ducks from the latter group suggesting that the antibody did not result from passive vaccination. Thus, increased resistance to infection develops with increasing age that may be related to several factors including host immunity. This model may help elucidate similar age-related features of human hepatitis B virus infections.     

磷甲酸钠在鸭体内对鸭乙型肝炎病毒的抑制作用

以鸭乙型肝炎病毒（ＤＨＢＶ）静脉感染雏鸭为模型,分组腹腔注射磷甲酸钠（ＰＦＡ）２５０ｍｇ、１２５ｍｇ、６２．５ｍｇ／ｋｇ及生理盐水,观察治疗后鸭血清中ＤＨＢＶＤＮＡ及ＤＨＢｓＡｇ的动态变化,并检测肝、肾、脾及胰中ＤＨＢＶＤＮＡ的分布;提取肝脏中超螺旋ＤＮＡ（ＳＣＤＮＡ）,检测ＰＦＡ对ＤＨＢＶＤＮＡ复制的影响。结果表明：ＰＦＡ治疗第７天到第２１天,１２５ｍｇ和２５０ｍｇ／ｋｇ剂量组对ＤＨＢｓＡｇ有显著的抑制作用;１２５ｍｇ和２５０ｍｇ／ｋｇ剂量组治疗第１４、２１天对血清中ＤＨＢＶＤＮＡ有显著的抑制作用;１２５ｍｇ和２５０ｍｇ／ｋｇ剂量组治疗第２１天肝及肾中ＤＨＢＶＤＮＡ明显下降;２５０ｍｇ／ｋｇ剂量组治疗２１天对ＤＨＢＶ感染鸭肝细胞内ＤＨＢＶＲＣＤＮＡ、ＬＤＮＡ及ＳＣＤＮＡ的合成有明显抑制作用。可见,最大剂量２５０ｍｇ／ｋｇＰＦＡ每天２次,治疗２１天,对ＤＨＢＶ感染鸭血清中ＤＨＢｓＡｇ、血清及肝、肾中ＤＨＢＶＤＮＡ都有抑制作用,对脾、胰中ＤＨＢＶＤＮＡ抑制不明显。提示该药能抑制ＤＨＢＶ感染,但未能清除病毒,故停药后可出现反跳现象。     

Natural infection of duck embryos with duck hepatitis B virus: time and tissue sequence of infection

There have been no studies addressing the detailed sequence of embryonic infection with duck hepatitis B virus (DHBV). Therefore, duck embryos from flocks infected with DHBV were examined to study the sequence of infection by DHBV in various embryonic tissues. Embryos from flocks infected with DHBV were harvested in duplicates from 7 to 25 days of incubation. Whole embryos (to 12 days) or dissected embryonic tissues were fixed, paraffin embedded, and stained for DHBV surface antigen (DHBsAg) using a peroxidase-antiperoxidase technique. Isolated hepatic cells were infected in 7-day-old embryos, and these increased in number until 11 days, when most cells were positive for DHBsAg. Endocrine pancreatic cells were positive from day 10, but only an occasional exocrine pancreatic cell was infected after day 20. Renal tubule cells were positive for DHBV by day 11, increasing in number until about day 18, after which a decline in numbers of infected cells occurred. Renal glomeruli became positive for DHBsAg from day 24. When present in the developing embryo, thymus, bursa of Fabricius, spleen, bone marrow, lung, and duodenum remained negative for DHBsAg. It was concluded that the timing of infection of specific tissues was not necessarily related to cellular maturity but may reflect a need for specific metabolic functions that permit viral replication.     

Epitope-specific antibody response to the surface antigen of duck hepatitis B virus in infected ducks.

In order to investigate the immune response to duck hepatitis B virus (DHBV) infection, newly hatched DHBV DNA negative ducklings were injected with infectious serum of sufficiently low DHBV-DNA titer to allow clearance of viremia. Of 20 injected ducklings, 13 (65%) became viremic. Of these, 6 (46%) cleared virus from the serum 3 to 22 weeks postinjection. The convalescent sera of these 6 animals were tested for an epitope-specific antibody response in a highly specific competitive inhibition assay using a panel of monoclonal antibodies against duck hepatitis B surface antigen (DHBsAg) that had been well-characterized. All 6 animals recovering from DHBV infection developed antibodies to epitopes on the preS and S proteins of DHBV. Antibody responses were highly variable with marked differences between animals in the extent and specificity of the antibody response. The humoral response to DHBsAg was prolonged in some animals but transient in others. No antibody to preS or S was detected in either preimmune sera or sera of control animals from an uninfected flock. Infected animals that did not clear viremia also remained antibody negative. The humoral responses to neutralizing preS epitopes III and V were weak but antibodies to two immunodominant epitopes on the preS region (II and B) were present in all 6 animals. The humoral response to the two epitopes in the S region was transient and of lower titer when compared to the two immunodominant preS epitopes. The two immunodominant preS epitopes may play an important role in clearance of DHBV infection in ducks.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.