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


CONCLUSIONS
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.
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
Yin-Ping
YP
Lu
Tao
T
Guo
Bao-Ju
BJ
Wang
Ji-Hua
JH
Dong
Jian-Fang
JF
Zhu
Zhao
Z
Liu
Meng-Ji
MJ
Lu
Dong-Liang
DL
Yang
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology / Q000627:therapeutic use; D000998:Antiviral Agents / Q000494:pharmacology* / Q000627:therapeutic use*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D024882:Drug Resistance, Viral / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D019694:Hepatitis B, Chronic / Q000188:drug therapy*; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology / Q000627:therapeutic use; D021141:Nucleic Acid Amplification Techniques; D063065:Organophosphonates / Q000494:pharmacology / Q000627:therapeutic use; D010957:Plasmids / Q000235:genetics; D012093:Replicon / Q000235:genetics; D014162:Transfection; D014779:Virus Replication / Q000187:drug effects*
D000998:Antiviral Agents; D063065:Organophosphonates; D019259:Lamivudine; C053001:adefovir; D000225:Adenine