2',3'-dideoxy-3'-fluoroguanosine inhibits duck hepatitis B virus in vivo

SUMMARY. Duck hepatitis B virus (DHBV) belongs to the same virus family as the human hepatitis B virus (HBV). Domestic ducks infected with DHBV can be used as an animal model for chronic hepatitis B virus infection in therapeutic trials. In this study the antiviral effect of the guanosine analogue 2',3'-dideoxy-3'-fluoroguanosine (FLG) was tried in vivo on chronically DHBV-infected ducks. The ducks were either congenitally infected, or inoculated with DHBV immediately post-hatch. FLG was given as intraperitoneal injections twice daily, at different dosages. Serum DHBV levels were determined by DNA dot-blot hybridization. A strong inhibition of serum DHBV DNA was observed with FLG doses down to 1 mg kg^-1 day^-1. given for 7 to 10 days. With the corresponding thymidine analogue, 2',3'-dideoxy-3'-fluorothymidine; however, no inhibition was obtained. This difference may be due to different phosphorylation mechanisms. Independently of FLG dose, serum DHBV DNA returned to pretreatment levels within a few days after cessation of therapy. After a long-term trial (FLG, 5mg kg^-1 day^-1 for 33 days), the same relapse of DHBV production was seen. Thus, FLG is an efficient inhibitor of DHBV replication, and is a candidate for treatment of HBV infections. However, the effect is transient, and therefore combination with other types of anti-HBV drugs should be considered.     

Virological efficacy of combination therapy with corticosteroid and nucleoside analogue for severe acute exacerbation of chronic hepatitis B

The short‐term prognosis of patients with severe acute exacerbation of chronic hepatitis B (CHB) leading to acute liver failure is extremely poor. We have reported the efficacy of corticosteroid in combination with nucleoside analogue in the early stages, but virological efficacy has not been documented. Our aim was to elucidate the virological efficacy of this approach. Thirteen patients defined as severe acute exacerbation of CHB by our uniform criteria were prospectively examined for virological responses to treatment. Nucleoside analogue and sufficient dose of corticosteroids were introduced as soon as possible after the diagnosis of severe disease. Of the 13 patients, 7 (54%) survived, 5 (38%) died and 1 (8%) received liver transplantation. The decline of HBV DNA was significant between the first 2 weeks (P = 0.02) and 4 weeks (P < 0.01). Mean reduction in HBV DNA during the first 2 weeks was 1.7 ± 0.9 log copies per mL in overall patients, 2.1 ± 0.8 in survived patients and 1.2 ± 0.9 in dead/transplanted patients. The decline of HBV DNA was significant between the first 2 weeks (P = 0.03) and 4 weeks (P = 0.02) in survived patients, but not in dead/transplanted patients. Our study shows that corticosteroid treatment in combination with nucleotide analogue has sufficient virological effect against severe acute exacerbation of CHB, and a rapid decline of HBV DNA is conspicuous in survived patients.     

A double blind, placebo-controlled study to assess the effect of famciclovir on virus replication in patients with chronic hepatitis B virus infection

SUMMARY. This is the first double-blind controlled study of famciclovir, an oral antiviral agent, as potential therapy for chronic hepatitis B virus (HBV) carriers. A fall of more than 90% in HBV DNA levels was noted in six of 11 evaluable patients treated with a 10 day course of oral famciclovir. Further studies with more prolonged therapy are ongoing.     

Replication of hepatitis B virus in primary duck hepatocytes transfected with linear viral DNA

AIM: To explore the expression and replication of hepatitis B virus (HBV) DNA in primary duck hepatocytes (PDHs). METHODS: Complete HBV genome was transfected into PDHs by electroporation (transfected group, 1.19×1012 copies of linear HBV DNA/1×107 PDHs). After 1-5 d of transfection, HBsAg and HBeAg in the supernatant and lysate of PDHs were measured with the IMX System. Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PDHs electroporated were used as control group. RESULTS: HBsAg in the hepatocyte lysates of transfected group was 15.24 (1 d), 14.55 (3 d) and 5.13 (5 d; P/N values, positive≥2.1) respectively. HBeAg was negative (<2.1). Both HBsAg and HBeAg were negative in the supernatant of transfected group. Dot blotting revealed that HBV DNA was strongly positive in the transfected group and negative in the control group. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (rc DNA), covalently closed circular (ccc DNA), and single-stranded (ss DNA) HBV DNA replicative intermediates in the transfected group, there was no integrated HBV DNA in the cellular genome. These parameters were negative in control group. CONCLUSION: Expression and replication of HBV genes can occur in hepatocytes from non-mammalian species. HBV replication has no critical species-specificity, and yet hepatic-specific regulating factors in hepatocytes may be essential for viral replication.     

拉米夫定联合泛昔洛韦抗鸭乙型肝炎病毒的实验研究

观察核苷酸类似物拉米夫定联合泛昔洛韦体内抗鸭乙型肝炎病毒（DHBV）的作用。方法 采用重庆麻鸭乙型肝炎动物模型,用拉米夫定联合泛昔洛韦口服治疗4周,停药观察1周,检测用药前后血清中的DHBV DNA、DHBsAg及血清转氨酶（ALT、AST）、肝组织HE染色病理。并以单用拉米夫定、泛昔洛韦、阿昔洛韦作对照。结果 拉米夫定联合泛昔洛韦用药后能使血清中DHBV DNA含量总体水平显著降低（P＜0．01）,停药1周后DHBV DNA较用药4周时DHBV DNA含量回升现象不明显。用药前后清血DHBsAg的吸光度值（490nm)的变化与DNA含量改变相似;此外,肝脏病理检查及治疗4周、停药1周后血清转氨酶检测未发现联合用药对鸭肝组织有明显的毒性损害。结论 拉米夫定联合泛昔洛韦连续用药4周在鸭体内有抗鸭乙型肝炎病毒的作用,且停药后DHBV DNA无明显“反跳”,二者用药有协同作用。     

载脂蛋白质B mRNA编辑酶催化多肽3G抑制乙型肝炎病毒和鸭乙型肝炎病毒复制

目的了解载脂蛋白质BmRNA编辑酶催化多肽3G（APOBEC3G）对乙型肝炎病毒（HBV）和鸭乙型肝炎炎病毒（DHBV）复制的抑制作用。方法从健康人外周血单个核细胞提取RNA，逆转录聚合酶链反应扩增APOBEC3G，将产物克隆到pXF3H载体的EcoRⅠ和Hind Ⅲ酶切位点以构建真核表达质粒；以ayw亚型HBV全长质粒构建具有复制能力的1．3倍HBV质粒（pHBV1．3）。不同剂量的APOBEC3G真核表达质粒与pHBV1．3共转染HepG2细胞；酶联免疫吸附法检测细胞培养上清液的乙型肝炎表面抗原和e抗原水平，Southernblot和Northernblot分析HBV核衣壳相关DNA和RNA的水平变化。不同剂量APOBEC3G真核表达质粒与头尾相接的2倍DHBV质粒共转染LMH鸡肝癌细胞，Southernblot分析DHBV核衣壳相关DNA水平变化。结果成功构建APOBEC3G真核表达质粒和具有复制能力的1．3倍HBV质粒。APOBEC3G抑制乙型肝炎表面抗原和e抗原的分泌，转染细胞内HBV核衣壳相关RNA表达水平下降，而对核心蛋白质的表达没有影响；APOBEC3G对转染细胞内HBV和DHBV核衣壳相关DNA水平具有剂量依赖的抑制效应。结论APOBEC3G对HBV和DHBV复制具有抑制作用。     

HBV DNA转染原代鸭肝细胞初步观察

目的：通过观测人HBV DNA在非哺乳动物－－鸭肝细胞中的复制和表达水平，探讨人HBV感染与复制的跨种属特异性，为建立人HBV DNA转染跨种属肝细胞模型奠定基础。方法：获取线性HBV DNA并电转染原代鸭肝细胞，电转后48h采用IMX系统检测鸭肝细胞中HBsAg表达水平，用Southern blot-ting和dot blotting检测HBV DNA复制情况。以单纯电击肝细胞为对照。结果：转染组原代鸭肝细胞裂解液中HBsAg为9．10（P／N值≥2．1为阳性），HBeAg为阴性；上清液中二者均为阴性。转染组原代鸭肝细胞裂解液dot blotting呈强阳性；转染组肝细胞总DNA Southern blotting显示约4.0kb以下分子涂抹带，为游离复制型HBV DNA，包括rcDNA,cccDNA与ssDNA等复制中间体，未见整合型HBV DNA－－高分子区（4.0-24.0kb)涂抹带，对照上述指标均为阴性。结论：人HBV DNA能在原代鸭肝细胞中复制和表达，可能为肝细胞内环境依赖性，无严格种属特异性限制。     

肝康栓抗鸭乙型肝炎病毒的实验研究

目的：用鸭乙肝模型研究肝康栓对鸭乙型肝炎病毒（DHBV）的体内抗病毒作用。方法：用DHBV阳性血清感染1日龄的樱桃谷鸭，制备鸭乙型肝炎模型。随机分成5组：肝康栓大、中、小3个剂量治疗组，生理盐水模型组和阿昔洛韦（ACV）对照组，每组12只，均连续给药4周。分别于给药前，给药14天、28天，停药7天时取血清，用real-timePCR法检测鸭血清中DHBV DNA拷贝数的变化情况。结果：肝康栓大剂量组给药14天、28天及停药7天，中剂量组给药14天、28天，小剂量组给药28天，鸭血清中DHBV DNA拷贝数的对数值均较给药前降低（〉2个对数级），差异有统计学意义（P〈0．01）。结论：肝康栓具有有效抑制鸭体内DHBV DNA复制的作用。     

Alterations in intrahepatic expression of duck hepatitis B viral markers with ganciclovir chemotherapy

Ducks congenitally infected with duck hepatitis B virus (DHBV) were treated with the guanosine analogue, ganciclovir, and the effect on serum and intrahepatic expression of DHBV DNA and viral proteins was examined. After 21 days of ganciclovir treatment, a substantial reduction in viraemia occurred; in contrast, the level of circulating DHBV surface antigen was unchanged. Ganciclovir therapy also substantially reduced the level of DHBV DNA replicative intermediates and the expression of viral core and surface antigen in hepatocytes. However, despite the antiviral treatment some liver cells, including the bile duct epithelial cells and putative oval cells, maintained their intense staining for the viral proteins. Furthermore, DHBV-infected cells in extrahepatic sites such as the pancreas, kidney and spleen were also unaffected by ganciclovir treatment. These results suggest that monotherapy with nucleoside analogues is unlikely to eliminate chronic hepadnaviral infection, and antiviral programs should be designed to target all cell populations infected by the virus.     

联合核苷类药物治疗干扰素应答不佳的慢性乙型肝炎时机选择及疗效研究

目的：探讨干扰素治疗HBeAg阳性慢性乙型肝炎时,联合核苷（酸）类药物的不同时机对治疗应答的影响。方法：观察干扰素治疗患者分别在治疗起始时联合阿德福韦酯、12周应答不佳者及24周应答不佳者联合拉米夫定最终各组疗效。结果：48周时及停药后24周时,起始时联合阿德福韦酯治疗组及12周应答不佳者联合拉米夫定组患者的病毒转阴率、 ALT复常率、 HBeAg转阴率均高于对照组（ P＜0.05）,而HBeAg转换率并未有明显提高（ P＞0.05）。结论：治疗起始干扰素联合阿德福韦酯或根据12周应答情况加用拉米夫定治疗均能一定程度提高治疗应答率。     

核苷类似物干预下乙型肝炎病毒前S抗原消长与DNA复制水平的关系

目的 监测核苷类似物干预下HBV前S1抗原(PreS1)、前S2抗原(PreS2)的动态变化,探讨PreS1、PreS2与HBV DNA复制水平的相关性.方法 选取拉米夫定治疗有效应答后出现病毒学反跳的慢性乙型肝炎患者12例,以及持续5年阿德福韦酯抗病毒治疗的慢性乙型肝炎患者20例,动态监测PreS1、PreS2及其他HBV标志物.根据不同资料分别选择t检验、X2检验、线性相关与回归分析或方差分析.结果 12例拉米夫定治疗的患者,在HBV DNA发生剧烈变化的4个时间点,均未观察到PreS1、PreS2滴度随HBV DNA复制水平的一致变化;20例阿德福韦酯治疗的患者中,仅1例患者PreS1、PreS2随HBV DNA和HBeAg一起阴转,其余均无显著变化;采用不等距重复测量设计方差分析绘制变化趋势轮廓图,未观察到PreS1、PreS2滴度随HBV DNA水平一致的变化趋势.结论 在核苷类似物干预下PreS1、PreS2滴度不能反映HBV DNA复制水平以及HBeAg的血清学转换.     

Characterization of rat hyperplastic bile ductular epithelial cells in culture andin vivo

A novel intrahepatic biliary cell culture/in vivo transplantation system has been developed with an essentially pure population of bile ductular epithelial cells isolated from rat liver 6–12 weeks after bile duct ligation. In primary culture, these cells retain staining strongly for -glutamyltranspeptidase and glutathione S-transferase P. The cytoplasm of cultured bile ductular cells reacts with an anti-laminin antibody, but loses immunoreactivity with a monoclonal anti-cytokeratin 19 antibody. Semiconservative DNA synthesis in the cultured cells was dependent upon the continued presence of 10% fetal calf serum in the medium. Replicating bile ductular cells could be subcultured for a finite number of passages. In addition, freshly isolated bile ductular epithelial cells gave rise to well differentiated bile ductular structures when transplanted into the interscapular fat pads of syngeneic recipient rats.Presented at the Proceedings of the International Meeting on Normal and Neoplastic Growth in Hepatology, Bari, Italy, June 1989.This work was supported by USPHS Grant RO1 CA39225 to Dr. Sirica by the National Cancer Institute, Department of Health and Human Services.     

干扰素联合核苷（酸）类似物治疗慢性乙型肝炎的疗效观察

目的观察干扰素（IFN）与核苷（酸）类似物联合治疗慢性乙型肝炎的疗效。方法选择慢性乙型肝炎病例207例,分别予聚乙二醇干扰素（PEG-IFNα-2a或IFNα-2b）治疗52周,随访24周,其中146例（A组）初始24周联合核苷（酸）类似物治疗（59例联合拉米夫定,56例联合阿德福韦酯,31例联合恩替卡韦）,另61例单用IFN治疗（B组）。结果 A、B组治疗结束时HBV DNA阴转率分别为86.3%（126/146）、65.6%（40/61）;ALT复常率87.7%（128/146）、76.5%（39/61）;HBeAg阴转率分别为69.3%（70/101）、40%（10/25）;HBsAg阴转率分别为30.1%（44/146）、16.4%（10/61）;抗-HBs阳转率分别为26.7%（39/146）、11.5%（7/61）,差异均有统计学意义（P〈0.05）。结论 IFN联合核苷（酸）类似物治疗慢性乙型肝炎的疗效优于单用IFN组。     

Corticosteroid and nucleoside analogue for hepatitis B virus-related acute liver failure

The early introduction of combination therapy of high-dose corticosteroid and nucleoside analogue is beneficial for the rescue of severe acute exacerbation of chronic hepatitis B.     

Inflammation of the liver causes mutations in duck hepatitis B virus genome

To investigate whether hepatitis causes mutation in the viral genome, DNA sequences in the pre-core region of duck hepatitis B virus (DHBV) DNA were analyzed in both ducks with hepatitis and without hepatitis. Five DHBV carrier ducks were injected with DHBV particle proteins purified from duck serum with Freund’s complete adjuvant (FCA) intrahepatically from 14 day posthatch for 9 weeks (immunized group). Serum was drawn at the end of the 1st and 4th week after the 1st injection of DHBV particle protein and ducks were killed at the end of the 9th week to obtain the liver. Another five ducks without treatment were used as controls. All ducks of the immunized group showed moderate to severe hepatitis at the 9th week. All ducks in the immunized group showed one mutation except one duck that showed two mutations only at the 9th week. Mutations were observed in the 5th, 13th, 21st, 22nd, and 28th codon of the precore region. All of them were point mutation at the 3rd base in the triplets. The frequency of mutation was different in each duck from 20% to 60% but not 100%. There was no mutations in ducks in control group. These results suggest that hepatitis causes mutation in the pre-core lesion genome of duck hepatitis B virus.     

Correlation of hepatitis B core antigen and beta-catenin expression on hepatocytes in chronic hepatitis B virus infection: relevance to the severity of liver damage and viral replication

BACKGROUND AND AIMS: The topographical distribution of hepatitis B core antigen (HBcAg) is related to the pathogenesis of liver damage caused by hepatitis B virus (HBV) infection. beta-catenin plays an important role in both intracellular adhesion and Wnt signaling transduction pathways. This study investigated the intrahepatic expression of HBcAg and beta-catenin in chronic HBV infection, and correlated the results with the degree of liver damage and viral replication. METHOD: Liver sections from 73 patients with chronic HBV infection were examined immunohistochemically for HBcAg and beta-catenin. RESULTS: The distribution of HBcAg could be classified into four types: only nucleus (C-1), both nucleus and cytoplasm (C-2), only cytoplasm (C-3) and all negative for nucleus and cytoplasm (C-4). Significant differences in serum aminotransferase level, HBV DNA and necroinflammatory score were observed among the different distribution types, and as the distribution of HBcAg changed from C-1 to C-4, fibrosis stage and hepatitis B e antigen (HBeAg) negative/anti-HBe positive rate increased concurrently. The distribution of beta-catenin could be classified into two types: only membrane (B-1) and membrane with nucleus or cytoplasm (B-2). B-2 showed higher serum aminotransferase level and necroinflammatory score than B-1. Between B-1 and B-2, there was no significant difference in serum HBV DNA level or fibrosis stage. CONCLUSIONS: In chronic HBV infection, HBcAg distribution may change from C-1 to C-4 gradually, and in correlation with serum aminotransferase, and HBV DNA and HBeAg negative/anti-HBe positive rate. Nuclear or cytoplasmic distribution of beta-catenin, compared with exclusive membranous distribution of beta-catenin, is related to active hepatitis, but not viral replication.     

Studies on duck hepatitis B virus: Identification and epidemiological survey in Taiwan

The presence of duck hepatitis B virus (DHBV) in domestic ducks in Taiwan was confirmed by DNA polymerase assay, Southern blot analysis and electron microscopy. To investigate the epidemiology of this virus, a total of 1274 serum samples were collected from 30 duck farms from different areas of Taiwan and studied by spot hybridization and/or DNA polymerase assay. The positive rates varied among different strains of ducks: 16% in 243 Pekin ducks, 12% in 392 Chinese common domestic ducks, 4% in 196 Muscovy, 25% in 292 Taiwan Kaiyas and 13% in 151 mule ducks. The positive rate was much higher in the younger ducks; it was highest (30.7%) in ducklings under 1 month of age, followed by ducks aged 1–12 months (11.8%), and lowest in those ducks older than 1 year (7.7%). It was concluded that the prevalence of DHBV infection in domestic ducks in Taiwan is generally high, and that the infected ducks may serve as an animal model for human hepatitis B virus infection which is also prevalent in Taiwan.     

乙型肝炎病毒基因型与病毒复制的关系

检测慢性乙型肝炎患者血清乙型肝炎病毒(HBV)基因型和病毒载量(HBV-DNA定量),探讨它们之间的关系。用微板核酸杂交-ELISA方法对HBV进行基因分型,用PCR荧光定量检测血清HBV-DNA水平,共318例。检测到HBV B型111例(35％);C型128(40％);混合型(B+C,C+D,B+C+D)45例(14％);D型2例;F型2例;未分型30例(9.4％);没有发现A、E型。结果发现,C型和混合型HBV的血清DNA水平高于B型(1.14×107vs2.2×107vsl.60×106拷贝/ml,P<0.05);而混合型HBV的血清DNA水平与C型无差别(P=0.127)。研究提示,我国HBV以B,C两基因型为主,HBV基因型可能是影响HBV复制的重要因素之一。