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。     

乙型肝炎病毒在异种动物原代肝细胞中复制与表达的研究

目的 探讨乙型肝炎病毒(HBV)DNA复制和表达的跨种属特异性。 方法 分离培养原代大鼠肝细胞(PRH)与原代鸭肝细胞(PDH),电转HBV线性裸DNA(转染组每1×107PRH或PDH 1.19×1012拷贝),分别于转染后1～15d各时点,收集PRH或PDH培养上清液与细胞裂解液,分别以Southern杂交分析和斑点杂交法分析HBV DNA的复制中间体与复制型式;以全自动免疫荧光检测系统检测乙型肝炎表面抗原(HBsAg)、乙型肝炎e抗原(HBeAg),以western免疫印迹、免疫斑点印迹和免疫细胞化学法检测乙型肝炎核心抗原(HBcAg);用逆转录聚合酶链反应检测HBV S/X mRNA。以单纯电击PRH/PDH为对照组。 结果 Southern杂交分析显示:PRH/PDH转染组HBV DNA均为游离复制型,可见4.0 kb以下分子区带,包括松弛环状DNA、共价闭环形DNA和单链线形DNA等复制中间体;未见整合型HBVDNA-高分子(4.0～24.0 kb)区带。HBV DNA在PRH中表达蛋白产物水平,HBsAg于转染后各时点PRH裂解液中均可检测到(P/N值2.17～93.41,平均值为14.74±31.82,阳性≥2.1),峰值于1～3 d;仅转染后1d PRH培养上清液中检测到HBsAg,P/N值为6.66;HBcAg和HBeAg仅于转染后1～3 d时点内检测到低度表达;HBV S mRNA为阳性,而X mRNA为阴性。HBV DNA转染PDH组,转染后1、3、5 d各时点PDH裂解液中HBsAg分别为15.24、     

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。     

乙型肝炎病毒裸DNA转染原代大鼠肝细胞模型的建立

目的建立乙型肝炎病毒(HBV)环化裸DNA转染原代大鼠肝细胞瞬时表达模型,为进一步研究肝细胞与HBV之间的互动关系奠定基础. 方法采用原代大鼠肝细胞(PRH)作为靶细胞,电转环化HBV DNA,于转染后1～10d各时点分别以Southern杂交和斑点杂交分析HBV DNA的复制中间体与复制形式,以IMX系统检测HBsAg、HBeAg,以western免疫印迹、免疫斑点印迹和免疫细胞化学法检测HBcAg,用RT-PCR检测HBV S/X mRNA,并采用电镜观察有无Dane颗粒合成.以单纯电击PRH为对照组. 结果 HBV环化裸DNA在PRH中的复制为游离型,可见rcDNA、cccDNA和ssDNA等复制中间体;表达的蛋白质产物、HBsAg于转染后各时点PRH裂解液中均可检测到(P/N值4.83～85.69,阳性≥2.1),峰值于1～3d,转染后1～10d平均P/N值为18.239±27.459;PRH培养上清液中未检测到HBsAg;HBeAg于PRH培养上清液和细胞裂解液中均呈阴性(P/N值＜2.1);HBcAg于转染后1～3d时点内检测到低度表达;HBV S mRNA为阳性,而X mRNA为阴性;电镜未观察到Dane颗粒. 结论 HBV环化裸DNA转染原代大鼠肝细胞瞬时表达模型稳定性、可重复性均良好.     

厦门市乙型肝炎病毒亚基因型分布的初步研究

目的探讨厦门地区乙型肝炎患者血清乙型肝炎病毒（HBV）基因型的分布情况。方法应用混合性型特异性引物聚合酶链反应法结合琼脂糖电泳，根据PCR产物片段大小直接判定HBV基因型；应用PCR扩增全S基因序列-DNA测序-生物信息学分析方法确定部分样本的亚基因型，并探讨前前S区编码的流行情况。结果在217例可以通过混合性型特异性引物聚合酶链反应法确定基因型的感染者中，B型83例（38．2％）、C型71例（32．7％），B／C混合型63例（29．0％）。HBeAg阳性患者中感染B基因型占50％，B／C型混合感染占22％；抗-HBe阳性患者中B／C型混合感染36％，B型38％；在慢性乙型肝炎患者中，以B基因型多见（46％），在肝硬化和肝癌患者中，以C（41％和42％）或B／C混合基因型（38％和33％）感染为主；20例患者血清经过DNA测序-生物信息学分析方法确定了亚基因型，其中B2亚型2例，C2亚型18例，其中混合性型特异性引物聚合酶链反应判断为B型的4例经过测序分析为C2亚型。B2亚型不编码前前S区，18例C2亚型均编码前前S区。结论本研究提示厦门地区乙型肝炎患者群的HBV基因型B、C型的感染率大致相同，亚基因型分析提示C2亚基因型占90％，可能是B／C基因型重组的结果。C2亚基因型病毒株均编码前前S区，提示前前S区的编码可能具有亚基因型特异性。     

原发性肝癌患者血清HBV标志物和HBV DNA测定的临床意义

目的观察原发性肝癌患者血清HBV标志物模式及HBV DNA水平。方法采用化学发光法检测151例PHC、132例慢性乙型肝炎和140例乙型肝炎肝硬化患者血清乙型肝炎病毒标记物;采用聚合酶链反应检测血清HBV DNA水平。结果在151例原发性肝癌患者中,HBV感染率达97.4%（147/151）,HBV DNA阳性率为74.8%（113/151）,平均水平为5.6±1.1 lgcopies/ml;HBsAg（＋）、HBeAb（＋）、HBcAb（＋）90例（59.6%）,其血清HBVDNA检出率为76.7%,平均水平为5.1±0.9lgcopies/ml;HBsAg（＋）、HBeAg（＋）、HBcAb（＋）38例（25.2%）,血清HBVDNA检出率100.0%,平均水平为6.4±0.9 lgcopies/ml。结论 PHC患者HBV感染率高,HBV与PHC发生有十分密切的关系。随着慢性乙型肝炎和乙型肝炎肝硬化病情的进展,血清HBeAg自发性发生血清学转换和HBV DNA载量下降,要警防原发性肝癌的发生。     

鸭乙型肝炎病毒体液免疫血清学指标的系统建立与应用

目的：建立简便,特异的鸭乙型肝炎病毒（DHBV）感染及免疫血清学检测系统。方法：制备、纯化检测所需同的抗DHVB前S区单克隆抗体腹水,DHBsAg和rDHBcAg , 对随机筛选的80份感染血清及实验感染1日龄重庆麻鸭系列血清分别进行DHBsAg 和抗－DHBc,抗－DHBs检测。结果：PCR方法检测出阳性66份,阴性14例,选用PCR阳性的66份标本,经HDBsAg ELISA检测出58份阳性,斑点杂交检出阳性份数为62份。与DHBVRCR相比较,灵敏度分别为87．9％和93．9％,而8只实验感染鸭仅2只在感染后第3周血清抗－DHBc阳性,抗体效价为1：10,至第4周一已为阴性,抗－DHBs则在所有标本均为阴性,结论：DHBV感染及免疫血清学酶联免疫方法的建立,为进一步研究DHBV感染后腹制规律及体风免疫应答状况奠定了基础。     

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。     

RNA干扰抑制乙型肝炎病毒复制的实验研究

目的 以乙型肝炎病毒(HBV)核心区为靶位，构建表达小干扰RNA(siRNA)的质粒载体pSilencer3．1-Hlhygro，体外观察siRNA抗HBV的效果。方法 以HepG2 2．2．15细胞为靶细胞，利用脂质体Metafectene与表达siRNA的质粒载体pSilencer3．1-Hlhygro共转染，用定量聚合酶链反应检测细胞上清液中DNA，用逆转录聚合酶链反应检测HBV C-mRNA。结果 成功构建了表达siRNA的转录质粒载体，两条siRNA均可抑制HBV的复制，而且与siRNA浓度成正相关。结论 靶向HBV核心区的siRNA能抑制HBV的复制。     

慢性乙型肝炎患者血清乙型肝炎病毒表面大蛋白水平检测意义探讨

目的探讨慢性乙型肝炎、乙型肝炎肝硬化和原发性肝癌患者血清乙型肝炎病毒表面大蛋白（HBV-LP） 水平差异及意义。方法选取2014年8月~2015 年12月我院诊治的慢性乙型肝炎患者508例,乙型肝炎肝硬化患者74例,原发性肝癌患者29例,采用ELISA 法检测血清HBV-LP水平,采用FQ-PCR法检测血清HBV DNA水平。结果慢性乙型肝炎、乙型肝炎肝硬化和原发性肝癌患者血清HBV-LP阳性率分别为77.2%、82.4%和89.7%,血清HBV DNA阳性率分别为78.9%、83.8%和93.1%;3组血清HBV-LP水平分别为（9.78±4.25）μg/L、（17.24±8.63）μg/L和（38.65±19.38）μg/L,差异有统计学意义（P<0.05）。结论HBV-LP是乙型肝炎病毒感染者血清重要的标记物,与血清HBV DNA存在某种相关性,其检测的意义值得探讨。     

Relationship between hepatitis B virus DNA levels and liver histology in patients with chronic hepatitis B

AIM： To study the relationship between hepatitis B virus （HBV） DNA levels and liver histology in patients with chronic hepatitis B （CHB） and to determine the prevalence and characteristics of hepatitis B e antigen （HBeAg） negative patients.
METHODS： A total of 213 patients with CHB were studied, and serum HBV DNA levels were measured by the COBAS Amplicor HBV Monitor test. All patients were divided into two groups according to the HBeAg status.The correlation between serum HBV DNA levels and liver damage （liver histology and biochemistry） was explored.
RESULTS： Of the 213 patients with serum HBV DNA levels higher than 10^5 copies/mL, 178 （83.6%） were HBeAg positive, 35 （16.4%） were HBeAg negative. The serum HBV DNA levels were not correlated to the age,history of CHB, histological grade and stage of liver disease in either HBeAg negative or HBeAg positive patients. There was no correlation between serum levels of HBV DNA and alanine aminotransferanse （ALT）,aspartate aminotrans-ferase （AST） in HBeAg positive patients. In HBeAg negative patients, there was no correlation between serum levels of HBV DNA and AST,while serum DNA levels correlated with ALT （r = 0.351, P = 0.042）. The grade （G） of liver disease correlated with ALT and AST （P 〈 0.05, r = 0.205, 0.327 respectively）in HBeAg positive patients. In HBeAg negative patients,correlations were shown between ALT, AST and the G （P 〈 0.01, and r = 0.862, 0.802 respectively）. HBeAg negative patients were older （35 ± 9 years vs 30 ±9 years, P 〈 0.05 ） and had a longer history of HBV infection （8 ± 4 years vs 6 ± 4 years, P 〈 0.05） and a lower HBV DNA level than HBeAg positive patients （8.4± 1.7 Log HBV DNA vs 9.8 ± 1.3 Log HBV DNA, P 〈0.001）. There were no significant differences in sex ratio,ALT and AST levels and liver histology between the two groups.
CONCLUSION： Serum HBV DNA level is not correlated to histological grade or stage of liver disease in CHB patients with HBV DNA mor     

乙型肝炎病毒Ⅹ蛋白对原代小鼠肝细胞周期的影响

目的 探讨HBV复制过程中HBx蛋白对原代小鼠肝细胞周期的影响. 方法 采用原代小鼠肝细胞为研究系统,用野生型HBV质粒(pHBV)和HBx蛋白表达缺失的HBV质粒(pHBV△X)分别转染细胞.Western blot检测细胞周期蛋白cyclin D1、cyclin E、cyclin A及细胞周期蛋白依赖性激酶抑制因子p16 (p16)、p21的表达水平;实时荧光定量PCR和Southern blot检测HBVDNA复制水平.两组之间比较用两样本均数t检验,多样本均数间的比较用方差分析及q检验. 结果 新分离肝细胞和原代培养24、48、72 h肝细胞的细胞周期蛋白表达水平无明显差异;转染48 h后,pHBV组细胞与pHBV△X组细胞周期蛋白条带灰度值相比,前者cyclin D1、p21、cyclin E表达量较高,p16的表达量较低,统计量t值分别为15.713,22.897,14.680,-19.584,P值均＜0.05.培养72 h后提取HBV DNA,Southem blot检测HBV DNA复制中间体松散环状DNA、双链线性DNA、单链DNA的水平,经条带灰度扫描,pHBV组的水平(分别为3288.336±448.011、6458.318±182.163、2760.613±393.561)明显高于pHBV△X组(分别为515.721±62.530、2122.228±28.347、1632.013±207.021)及空白对照组,P值均＜0.05;实时荧光定量PCR检测HBV DNA复制水平,pHBV组[每个细胞(987.50±47.80)拷贝]也明显高于pHBV△X组[每个细胞(303.67±33.94)拷贝],t=20.203,P＜0.05.结论 HBx蛋白能通过调节细胞周期中各蛋白的表达,使细胞由静止期(G0)进入DNA合成前期(G1)并停滞于G1期,从而促进HBV的复制.     

HDV感染后乙型肝炎患者血清肝炎病毒标志物的变化

目的　分析HDV感染患者血清病毒性肝炎标志物的变化和意义 ,探讨HDV致病机理。方法　对 469例HDV阳性乙型肝炎患者常见各类型病毒性肝炎血清标志物的变化等作统计分析 ,以 2 13例HDV( -)乙型肝炎患者作对照。结果　HDV感染后血清HBeAg检出率降低 (P <0 .0 1)。在HDV ( +)HBVDNA( -)组 ,HBeAg( -)的机会大 (P <0 .0 1)。在急性肝炎、重型肝炎和肝硬化患者HDAg( +)HBeAg( -)为主要血清病毒表现形式 (P <0 .0 1或 0 .0 5 )。HDV感染后合并其它肝炎病毒感染率高于乙型肝炎组。结论　HDV感染可抑制HBV复制或HBeAg表达 ,混合感染HDV的乙型肝炎中HDV的直接细胞毒性作用可能起主要致病作用。重叠感染HDV的乙型肝炎患者其病情重、病死率高和容易慢性化。     

Replication of clinical hepatitis B virus isolate and its application for selecting antiviral agents for chronic hepatitis B patients

AIM： To establish a cell model harboring replicative clinical hepatitis B virus （HBV） isolates and evaluate its application in individualized selection of anti-HBV agents for chronic hepatitis B （CHB） patients.
METHODS： The full-length HBV genomic DNA from 8 CHB patients was amplified by polymerase chain reaction （PCR）. All the patients were treated with lamivudine for at least seven months and finally became resistant to lamivudine. The amplified HBV DNA fragments were inserted into pHY106 vectors by Sap Ⅰ digestion. The recombinant plasmids containing 1.1 copies of HBV genome were transiently transfected into Huh7 cell line, and the levels of HBsAg, HBeAg and intercellular HBV replicative intermediates were determined by ELISA and Southern blot analysis, respectively, with or without lamivudine and adefovir treatment. The antiviral treatment with adefovir was administered to the patients and analyzed in parallel.
RESULTS： A total of 25 independent HBV isolates were obtained from the sera of 8 patients, each patient had at least two isolates. One isolate from each individual was selected and subcloned into pHY106 vector, including 5 isolates with YVDD mutation and 3 isolates with YIDD mutation. All recombinant plasmids harboring HBV isolates were transfected into Huh7 cells. The results indicated that HBV genome carried in HBV replicons of clinical HBV isolates could effectively replicate and express in Huh7 cells. Adefovir, but not lamivudine, inhibited HBV replication both in vitro and in vivo, and in vitro inhibition was dose-dependent.
CONCLUSION： The novel method described herein enables individualized selection of anti-HBV agents in clinic and is useful in future studies of antiviral therapy for CHB.     

DHBV感染鸭特异免疫应答比较

目的 研究嗜肝病毒感染病毒清除向慢性化转变的免疫机理。方法 通过接种不同龄鸭、不同感染方案建立DHBV急性、慢性感染组及免疫组模型,并检测各组病毒复制及特异体液和细胞介导免疫反应。结果 成年鸭接种DHBV导致一过性病毒血症的形成,感染后抗－DHBs和抗-DHBc的水平比慢性感染组高（P＜0．05）,而免疫组抗－DHBs、抗－DHBc的水平又比急感染组高（P＜0．01）。特异细胞介导免疫反应（CMI）的分析提示急性感染组感染后10d外周血单个核细胞（PBMC）对特异抗原的体外增殖反应强于慢性感染组,但在感染5周内快速下降。而免疫组CMI与急性感染组比差异无显著意义。结论 感染宿主免疫应答特别是特异免疫反应是决定感染转归的重要因素。     

乙型肝炎病毒对人肝癌细胞脂类代谢相关蛋白表达的影响

目的 从mRNA水平和蛋白水平检测乙型肝炎病毒(Hepatitis B Virus,HBV)对人肝癌细胞脂类代谢相关蛋白表达的影响。方法 以HBV细胞株或以1.2倍体HBV基因组瞬时转染人肝癌细胞株,采用实时定量PCR和Western blot方法验证人肝癌细胞载脂蛋白AI(apolipoprotein A-I,ApoAI)、载脂蛋白E(apolipoprotein E, ApoE)、超氧化物歧化酶2(superoxide dismutase 2,SOD 2)、NADPH脱氢酶(醌)1(NAD(P)H dehydrogenase, quinone 1,NQO 1),以及肝型脂肪酸结合蛋白(fatty acid binding protein 1,FABP 1)和乙酰辅酶A乙酰转移酶2(acetyl-Coenzyme A acetyltransferase 2,ACAT 2)等脂类代谢相关蛋白表达水平的改变。结果 HBV细胞株或瞬时转染HBV均使人肝癌细胞SOD 2、NQO 1基因的mRNA和蛋白表达量下调,FABP 1基因的mRNA和蛋白表达量上调。结论 HBV可影响人肝癌细胞脂类代谢相关蛋白表达,为HBV致肝细胞脂肪变机制的深入研究打下良好基础。     

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.     

Hepatitis B virus infection and replication in primarily cultured human fetal hepatocytes

AIMTo investigate the infection and replication of hepatitis B virus(HBV)in primarily cultured human fetal hepatocytes(HFHs).METHODSThe human fetal hepatocytes were cultured in serum-free medium,HBV-positive serum was added into the medium to study the susceptibility of hepatocytes to HBV infection.The supernatant was collected for ELISA assay of HBsAg and HBeAg,and quantitative fluorescence PCR for HBV-DNA assay daily.Albumin and HBcAg,CK8 and CK18 expressions were detected by immunohistochemistry in cultured hepatocytes.Content of lactate dehydrogenate(LDH)was measured to find out the integrity of the cell membrane.RESULTSA stable hepatocyte culture system was established.HBV could infect the hepatocytes and replicate,and HBcAg expression could be detected by immunohistochemistry in hepatocyte-like cells.HBV-DNA in the supernatant could be detected from d 2 to d 18 and HBsAg and HBeAg were positive on d 3-d 18 after HBV infection.HBV in medium increased from d 0 to d 6 and subsequently decreased as the cells were progressively loosing their hepatocyte phenotypes.CONCLUSIONHBV could infect human fetal hepatocytes and replicate.This in vitro model allowed a detailed Study on early events associated with human HBV entry into cells and subsequent replication.     

Gene expression analysis of primary normal human hepatocytes infected with human hepatitis B virus

AIM: To find the relationship between hepatitis B virus (HBV) and hepatocytes during the initial state of infection by cDNA microarray. METHODS: Primary normal human hepatocytes (PNHHs) were isolated and infected with HBV. From the PNHHs, RNA was isolated and inverted into complement DNA (cDNA) with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-κB luciferase reporter assay. RESULTS: From the cDNA microarray, we obtained 98 differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes: LT-α, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, Huh7, and Chang liver cells, which were trans-fected with pHBV1.2x, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBV-mediated NF-κB activation. CONCLUSION: During the initial state of HBV infection, hepatocytes facilitate the activation of NF-κB through up regulation of LT-α, TRAF2, and NIK.     

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.