Yao Y, Tang N, Huang A
Institute for Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing 400010, China.
Zhonghua Yi Xue Za Zhi. 2001 Oct;81(19):1157-61.
By studying the replication of Hepatitis B virus (HBV) compared with that of Duck Hepatitis B virus (DHBV) in primary duck hepatocytes (PDH), we want to explore the host-specific regulating roles on the replication of HBV in hepatocytes from heterologous species.
PDH were transfected with complete HBV genome by electroporation (transfected group, 1.19 x 10(12) copies of linear HBV DNA/1 x 10(7) PDH) or infected with DHBV (infected group, 4 x 10(8) virions/1 x 10(7) PDH). 1, 3 and 5 days after transfection or infection, HBsAg, HBeAg and DHBsAg in the supernatant and lysate of PDH were measured with IMX System or ELISA. Meanwhile, replicative intermediates of HBV DNA and DHBV DNA were analyzed by Southern blotting and dot blotting. PDH electroporated only was used as control group.
HBsAg in the lysates of transfected group were 15.24 (1 day), 14.55 (3 days) and 5.13 (5 days; P/N values, positive > or = 2.1), HBeAg all was negative (< 2.1), and both were negative in the supernatants of transfected group. DHBsAg in the supernatants of infected group were 14.6 (1 day), 31.53 (3 days) and 34.73(5 days; S/N values, positive > or = 2.1). Dot blotting revealed that both the total amount of HBV DNA in the transfected group and DHBV DNA in the infected group were strongly positive, whereas that of the control group was negative. Southern blot analysis of intracellular total DNA indicated that there are relaxed circular (RC), covalently closed circular (ccc) and single-stranded (SS) HBV DNA replicative intermediates in the transfected group, there was no integrated HBV DNA in the cellular genome, as the same as that of DHBV DNA in the infected group. Control groups were negative at all.
Our results demonstrate that expression of HBV genes and production can occur in hepatocytes from nonmammalian species and strongly support the idea that HBV replication has no critical species-specificity, and yet hepatic-specific regulating factors could be essential for viral replication.
通过研究乙型肝炎病毒（HBV）与鸭乙型肝炎病毒（DHBV）在原代鸭肝细胞（PDH）中的复制情况，探讨异源物种肝细胞中宿主特异性调节因子对HBV复制的作用。
通过电穿孔法将完整的HBV基因组转染至PDH（转染组，1.19×10¹²拷贝线性HBV DNA/1×10⁷个PDH），或用DHBV感染PDH（感染组，4×10⁸个病毒粒子/1×10⁷个PDH）。转染或感染后1、3和5天，用IMX系统或ELISA检测PDH上清液和裂解物中的HBsAg、HBeAg和DHBsAg。同时，通过Southern印迹和斑点印迹分析HBV DNA和DHBV DNA的复制中间体。仅进行电穿孔的PDH作为对照组。
转染组裂解物中的HBsAg分别为15.24（1天）、14.55（3天）和5.13（5天；P/N值，阳性≥2.1），HBeAg均为阴性（<2.1），转染组上清液中的两者均为阴性。感染组上清液中的DHBsAg分别为14.6（1天）、31.53（3天）和34.73（5天；S/N值，阳性≥2.1）。斑点印迹显示，转染组中HBV DNA总量和感染组中DHBV DNA总量均呈强阳性，而对照组为阴性。细胞内总DNA的Southern印迹分析表明，转染组中有松弛环状（RC）、共价闭合环状（ccc）和单链（SS）HBV DNA复制中间体，细胞基因组中无整合的HBV DNA，感染组中DHBV DNA情况相同。对照组均为阴性。
我们的结果表明，HBV基因可在非哺乳动物物种的肝细胞中表达并产生，有力支持了HBV复制无严格物种特异性的观点，但肝脏特异性调节因子可能对病毒复制至关重要。
