肝细胞刺激物质对感染鸭乙型肝炎病毒的雏鸭体内抗病毒作用的实验研究

以感染鸭乙型肝炎病毒（ＤＨＢＶ）的北京雏鸭为体内实验动物模型，观察了肝细胞刺激物质（ＨＳＳ）对鸭乙型肝炎病毒的抗病毒作用。１日龄北京鸭实验感染ＤＨＢＶ，７天后血清ＤＨＢＶＤＮＡ阳性，给予药物治疗１０天，用斑点杂交方法检测用药前后血清ＤＨＢＶＤＮＡ，分析药物对血清ＤＨＢＶＤＮＡ是否有抑制作用，结果显示：ＨＳＳ大、小两个剂量组（分别为５０ｍｇ／ｋｇ／ｄ和２００ｍｇ／ｋｇ／ｄ）均对ＤＨＢＶＤＮＡ有抑制作用，大剂量组出现ＤＨＢＶＤＮＡ抑制作用时间早于小剂量组，用药５天、１０天及停药３天血清ＤＨＢＶＤＮＡ中位抑制率显著高于生理盐水对照组（Ｐ＜０．０５）；两个剂量组均无停药反跳；生理盐水组用药前后ＤＨＢＶＤＮＡ的Ａ值比较差异无显著性意义（Ｐ＞０．０５）。停药后３天肝组织病理光镜及电镜均显示ＨＳＳ组病变较生理盐水组轻。结论：肝细胞刺激物质有一定的保肝和抑制ＤＨＢＶＤＮＡ复制的作用     

HBV感染与IgA肾病肾小管间质病变的关系

目的探讨ＩｇＡ肾病ＨＢＶ感染与肾小管间质病变的关系。方法利用原位分子杂交（ＨＢＶＤＮＡ）、免疫组化（ＨＢＡｇ、ＣＤ３、ＣＤ８）以及ＨＢＶＤＮＡＨＢＡｇ和ＨＢＡｇＣＤ４３双标记技术，对９１例ＩｇＡ肾病肾穿刺标本进行研究。结果肾组织内ＨＢＡｇ阳性率为６９．２％。ＨＢＶＤＮＡ原位杂交阳性率为４２９％。ＨＢＶＤＮＡ阳性的病例，双重标记染色发现ＨＢＶＤＮＡ阳性的肾小管上皮细胞可表达ＨＢｃＡｇ或／和ＨＢｓＡｇ。ＨＢＶ感染标记（ＨＢＶＤＮＡ、ＨＢｃＡｇ、ＨＢｓＡｇ）阳性组ＣＤ３阳性细胞和ＣＤ８阳性细胞数明显高于阴性组（Ｐ＜００１），并可见数量不等的Ｔ淋巴细胞入侵ＨＢｃＡｇ及ＨＢｓＡｇ阳性肾小管管壁或围绕其周围。结论感染ＨＢＶ的肾组织细胞能够表达ＨＢＡｇ，并诱导ＣＤ３阳性细胞和ＣＤ８阳性细胞浸润，从而加重肾小管、间质损害。ＨＢＶ感染对ＩｇＡ肾病的发生发展可能起着重要作用     

反义寡脱氧核苷酸在鸭体内抑制鸭乙型肝炎病毒复制与表达的研究

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ＤＨＢＶＤＮＡ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ＤＨＢＶＤＮＡ阳性未见明显改变，肝组织ＤＮＡＳｏｕｔｈｅｒｎ杂交在３０与２３ｋｂ左右杂交信号明显。注射ＡＳＯＤＮ鸭在１０天后，２／３鸭血清ＤＨＢｓＡｇ及ＤＨＢＶＤＮＡ量显著降低，３／３鸭肝组织中３０ｋｂ左右的杂交信号显著减弱，２３ｋｂ左右未见杂交信号。结论说明该段ＡＳＯＤＮ在鸭体内能部分抑制ＤＨＢＶ的复制与抗原表达。     

鸭乙型肝炎病毒DNA体内转染的研究

利用两种鸭乙型肝炎病毒（ＤＨＢＶ）ＤＮＡ双体质粒及环化的ＤＨＢＶＤＮＡ体内转染２ｄ龄鸭及４～２０ｄ龄鸭，大多数鸭产生了短暂病毒血症，少数鸭产持续病毒血症，转染鸭血清中检测得ＤＨＢｓ／ｐｒｅＳＡｇ，ＤＨＢＶＤＮＡ及ＤＨＢＶ病毒颗粒，转染鸭血清腹腔注射１ｄ龄鸭可感染成功，转染鸭肝组织检测到复制型ＤＨＢＶＤＮＡ，提示转染后既有抗原有的表达，又有病毒的复制。另外，用两种ＤＨＢＶＤＮＡ单体质粒体内转染２ｄｘ     

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     

肝细胞刺激物质对感染鸭乙型肝炎病毒的争论鸭体…

以感染鸭乙型肝炎病毒（ＤＨＢＶ）的北京雏鸭为体内实验动物模型，观察了肝细胞刺激物质（ＨＳＳ）对鸭乙型肝炎病毒的抗病毒作用，１日龄北京鸭实验感染ＤＨＢＶ，７天后血清ＤＨＢＶＤＮＡ阳性，给予药物治疗１０天，用斑点杂交方法检测用药前后血清ＤＨＢＶＤＮＡ，分析药物对血清ＤＨＢＶＤＮＡ有抑制作用，大剂量量组出现ＤＨＢＶＤＮＡ抑制作用时间早小剂量组，用药５天，１０天及停药３天血清ＤＨＢＶＤＮＡ中位抑制率显著高     

慢性乙型肝炎肝内HBVDNA和HBsAg的双标记定位研究

应用原位杂交和免疫组化ＰＡＰ法双标记技术，结合病人乙型肝炎（乙肝）病毒血清学标志物检测结果，研究了３１例慢性乙肝病人肝穿刺组织中乙型肝炎病毒ＤＡＮ和ＨＢｓＡｇ的分布及意义。结果显示，肝辔内检ＨＶＤＮＡ２３例，ＨＢｓＡｇ２６例，二者同时检出者２１例。从同组病人肝组织的ＨＢＶＤＮＡ和ＨＢｓＡｇ双标检测结果与其乙肝病毒血清学标志物检测结果的比较来看，肝组织内ＨＢＶＤＮＡ和ＨＢｓＡｇ同时很可能表明ＨＢＶ正     

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％。     

乙肝张黄曲霉毒素B1诱发树Qu肝癌的病理变化

目的：对人乙型肝炎病毒（ＨＢＶ）和黄曲霉毒素Ｂ１（ＡＦＢ１）引起的树Ｑｕ肝脏病变进行动态比较观察。方法：成年树Ｑｕ按不同处理分为Ａ（ＨＢＶ＋ＡＦＢ１）、Ｂ（ＨＢＶ）、Ｃ（ＡＦＢ１）、Ｄ（空白对照）４组。全部动物定期肝活检,并于１６０周结束实验时处理所有存活者。各次活检及尸检肝组织均作常规病理组织学检查,部分同时作ＨＢｓＡｇ及ＨＢｓＡｇ免疫组织化学、ＨＢＶＤＮＡ原位杂交,以及ＰＡＳＤ、网状纤维染色等     

HBV感染与IgA肾病肾小管-间质病变的关系

目的 探讨ＩｇＡ肾病ＨＢＶ感染与肾小管间质病变的关系。方法 利用原位分子杂交（ＧＨＢＶ ＤＮＡ）、免疫组化（ＨＢＡｇ、ＣＤ３、ＣＤ８）以及ＨＢＶ ＤＮＡ－ＨＢＡｇ和ＨＢＡｇ－ＣＤ４３双标记技术,对９１例ＩｇＡ肾病肾穿刺标本进行研究。结果 肾组织内ＨＢＡｇ阳性率为６９．２％。ＨＢＶ ＤＮＡ原位杂交阳性率为４２．９％。ＨＢＶ ＤＮＡ阳性的病例,双重标记染色发现ＨＢＶ ＤＮＡ阳性肾小管上皮细胞可表达ＨＢ     

Oral famciclovir against duck hepatitis B virus replication in hepatic and nonhepatic tissues of ducklings infected in ovo

Detection of hepadnaviral DMA in extrahepatic tissues of human and animal models of hepatitis B virus (HBV) has raised the question of whether virus replication in organs other than the liver could be targeted for the treatment of chronic hepatitis B. Since duck hepatitis B virus (DHBV) replication is dynamic in the liver, kidney, pancreas, and spleen of newly hatched ducklings infected in ovo, we used the duck model and the new antiherpesvirus agent, famciclovir (FCV), to determine whether antiviral effect of nucleoside analogues on DHBV replication is pluripotential. Day-old ducklings hatched from eggs laid by a DHBV-carrier duck were bled and administered FCV (25 mg/kg/bd) orally for periods of 1, 2, 3, 6, 9, and 12 days. Seventeen (17) hours after the last dose of each regimen the duckling(s) was bled and postmortem samples of liver, kidney, pancreas, and spleen were snap-frozen and stored at ?70°. Analysis of plasma samples of ducklings treated for 2 days and longer by dot-blot hybridisation showed that levels of DHBV DNA were reduced significantly compared to levels in samples collected before treatment begun. Southern blot hybridisation of tissue DNA corroborated these results and showed that DHBV DNA replicative intermediates in all the tissues examined were reduced to levels that reflected the amount of virus released into the blood of each treated duckling. It is concluded from these results that if antiviral agents could be transformed to active metabolites in any infected tissues including the liver, replication of hepadnaviruses would be inhibited. We also note that the ability of young ducklings to metabolise FCV to the parent compound, penciclovir, suggests that hatchlings could be used for screening antiviral compounds under development. © 1994 Wiley-Liss, Inc.     

Effects of purine nucleoside analogues with a cyclobutane ring and erythromycin A oxime derivatives on duck hepatitis B virus replication in vivo and in cell culture and HIV-1 in cell culture.

The effects on duck hepatitis B virus (DHBV) replication of specific analogues of two classes of chemical compounds not previously tested against hepadnaviruses are described. One is erythromycin A-9-methyloxime (EMO) and other oxime derivatives of erythromycin A, and the other is purine nucleoside analogues (cyclobut A and cyclobut G) with cyclobutane rings. Viral replication was assessed by measuring serum levels of DHBV DNA in infected ducklings and DHBV DNA in infected primary duck hepatocyte cultures. Administration of EMO 15 mg/kg of body weight IM to infected ducklings resulted in a rapid fall in DHBV DNA levels during therapy and a return to pretreatment levels after EMO administration was stopped. There was local toxicity at injection sites with muscle necrosis in some animals. When 100 mg/kg EMO was administered by gastric tube no such viral response was observed. The difference in virus response to EMO 15mg/kg IM and 100 mg/kg by gastric tube was not due to failure to achieve comparable blood and tissue levels of EMO administered by the different routes. The results suggest an indirect effect dependent on IM injection of EMO rather than a direct antiviral effect of the compound. Administration of cyclobut G or cyclobut A at 70 mg/kg IM led to a rapid reduction of DHBV DNA to undetectable levels in serum, and in only 1 of 4 animals did DHBV DNA became detectable again within 10 days after stopping the drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

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).     

湖北麻鸭乙型肝炎病毒体内感染模型的建立

目的 建立标准化的湖北麻鸭乙型肝炎病毒(DHBV)动物模型,并初步观察拉米呋啶(3TC)体内抗DHBV的作用.方法将收集的转染细胞培养上清液纯化后作为体内实验感染的标准毒种,经腹腔接种2日龄雏鸭,连续观察50 d,用Real-Time PCR检测血清中DHBV DNA出现和持续时间.药物组则于接种3 d后给予3TC,检测观察期间感染鸭血清中病毒含量的变化情况.结果雏鸭实验感染率达到87.5%;雏鸭在接种后第7天出现病毒血症,血清病毒含量于第11天达到高峰值,后逐步降低,但在观察期内仍维持较高水平.在3TC治疗期间,感染鸭体内病毒血症处于较低水平,被感染肝细胞的数量也明显减少,且无明显的毒性作用;在停药3 d后,病毒血症即出现反跳现象,此后又有上升趋势.结论通过接种转染细胞上清液成功建立湖北麻鸭乙型肝炎病毒动物模型,药物评估实验证明其良好的稳定性和实用性;该模型也为研究DHBV生物学特性提供了有力的手段.     

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.     

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.     

Reduced duck hepatitis B virus viraemia in ducklings coinfected with the immunodepressive reticuloendotheliosis virus

Coinfection of avian hosts by duck hepatitis B virus (DHBV) and reticuloendotheliosis virus (REV) was studied to assess the effect of immunodepression by REV on the replication of DHBV. One-day-old ducklings, domestic chickens, and turkey poults were inoculated either with DHBV or DHBV and REV and were bled and weighed at regular intervals. DHBV infection as manifested by viraemia and DHBV DNA in liver was established only in ducklings. All chickens and turkeys were negative for DHBV DNA in serum and liver. However, ducklings coinfected with REV showed a delayed onset and reduced level of viraemia compared to ducklings infected only with DHBV. The narrow host range of DHBV was confirmed even in immunodepressed species. It is suggested that the reduction in DHBV viraemia in ducklings was due to factors not involving the specific immune system.     

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.     

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.