Viral Hepatitis
Copyright ©2008 The WJG Press and Baishideng. All rights reserved.
World J Gastroenterol. Jun 14, 2008; 14(22): 3490-3496
Published online Jun 14, 2008. doi: 10.3748/wjg.14.3490
Replication of clinical hepatitis B virus isolate and its application for selecting antiviral agents for chronic hepatitis B patients
Yin-Ping Lu, Tao Guo, Bao-Ju Wang, Ji-Hua Dong, Jian-Fang Zhu, Zhao Liu, Meng-Ji Lu, Dong-Liang Yang
Yin-Ping Lu, Tao Guo, Ji-Hua Dong, Jian-Fang Zhu, Zhao Liu, Department of Virology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China
Yin-Ping Lu, Bao-Ju Wang, Dong-Liang Yang, Division of Clinical Immunology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
Meng-Ji Lu, Institute of Virology, Duisburg-Essen University, Essen 45122, Germany
Author contributions: Lu YP and Guo T contributed equally to this work; Lu YP, Guo T and Yang DL designed the research; Lu YP, Guo T, Wang BJ, Dong JH and Zhu JF performed the research; Lu YP and Guo T analyzed the data; Lu MJ provided reagents/analytic tools; and Yang DL offered financial support.
Correspondence to: Dr. Yin-Ping Lu, Department of Virology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022, Hubei Province, China. yinpinglu@163.com
Telephone: +86-27-85726121
Fax: +86-27-85776343
Received: February 4, 2008
Revised: April 10, 2008
Accepted: April 17, 2008
Published online: June 14, 2008
Abstract

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.

Keywords: Hepatitis B virus; Chronic hepatitis B; Hepatitis B virus isolate; Antiviral agents