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。     

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％。     

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。     

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     

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     

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     

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     

肝硬变内HBV DNA及其五种抗原的表达及意义

取２２５例人肝硬变活检组织石蜡切片，检测了ＨＢＶＤＮＡ及其５种抗原。分别用免疫组化ＡＢＣ法检测ＨＢｘＡｇ、ｐｒｅ－Ｓ＿１和ｐｒｅ－Ｓ＿２抗原；用ＰＡＰ法检测ＨＢｓＡｇ和ＨＢｃＡｇ；用原位杂交方法检测ＨＢＶＤＮＡ；用免疫组化、原位杂交双标记方法检测ＨＢＶＤＮＡ和ＨＢｓＡｇ、ＨＢｘＡｇ或ＨＢｃＡｇ。结果显示，阳性检出率ＨＢｓＡｇ为７０．０％（１２８／１８３例），ｐｒｅ－Ｓ＿１抗原为６４．４％（８５／１３２例）、ｐｒｅ－Ｓ＿２抗原为６１．４％（８１／１３２例），ＨＢｘＡｇ为７５．３％（１１３／１５０例），ＨＢｃＡｇ为２２．４％（３９／１７４例），ＨＢＶＤＮＡ为６２．４％（５８／９３例）。双标阳性检出率ＨＢＶＤＮＡ和ＨＢｓＡｇ为３７．３％（１９／５１例），ＨＢＶＤＮＡ和ＨＢｘ－Ａｇ为８６．３％（４４／５１例），ＨＢＶＤＮＡ和ＨＢｃＡｇ为３９．２％（２０／５１例）。ＨＢＶＤＮＡ和ＨＢＶ５种抗原阳性病例中８０％以上均伴有肝细胞不典型增生。这一结果表明，在我国肝硬变的发生发展与ＨＢＶ慢性感染有密切的关系。     

硫代反义寡核苷酸体外对HDV的抑制作用

观察硫代反义寡核苷酸（Ｓ－ＡＳＯＤＮ）体外对ＨＤＶ的抑制作用。方法在ＨＤＶ／ＨＢＶ感染人胎肝细胞中加入不同浓度的针对ＨＤＶ ＳｔｅｍⅠ区６８４－６９８位核苷酸的１５聚Ｓ－ＡＳＯＤＮ,分别采用ＥＬＩＳＡ和斑点杂交法检测上清液中ＨＤＡｇ和细胞中ＨＤＶ ＲＮＡ。结果ＨＢｓＡｇ、ＨＤＡｇ在感染后第２天至第１６天均可测出,以第４天至第１２天达高峰,加入Ｓ－ＡＳＯＮＤ（２、４、６μＭＯＬ／ｌ）ＲＧ ２ＧＤ ,     

成年树鼩实验感染丁型肝炎病毒的初步研究

在证实成年树可感染人乙型肝炎病毒（ＨＢＶ）的基础上，进行了丁型肝炎病毒－乙型肝炎病毒（ＨＤＶ／ＨＢＶ）实验感染的探索。ＨＤＶ／ＨＢＶ阳性人血清经同时和重叠感染方式接种于成年树后，定期留取感染树血清及肝组织，检测血清中ＨＢｓＡｇ、ＨＤＡｇ、抗－ＨＤ、ＨＢＶＤＮＡ及ＨＤＶＲＮＡ，初步探讨成年树实验感染ＨＤＶ的可能性。研究发现：①成年树对人ＨＢＶ易感，ＨＢｓＡｇ阳性率为７５％，其血清学反应及肝组织病理改变与文献报道一致；②ＨＤＶ可通过同时和重叠两种方式感染成年树，感染树血清中可出现ＨＤＡｇ及抗－ＨＤ，经分子杂交证实其血清及肝内均有ＨＤＶＲＮＡ和ＨＢＶＤＮＡ存在，且可导致明显的肝细胞损伤。结果提示：成年树既可感染人ＨＢＶ也可感染ＨＤＶ，可作为研究ＨＤＶ人工感染的实验动物；成年树感染ＨＤＶ后的特点与人及黑猩猩感染时十分相似，能较好地反映人丁型肝炎的真实情况。     

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.     

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.     

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.     

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.     

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     

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.     

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.     

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.