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

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.

Figure 1

The schematic map of HBV expression vector of pHY106.

Figure 2

Construction of replicons of clinical HBV isolates using pHY106. Full-length HBV genomes from PCR amplication can be digested with Sap I and inserted into Sap I-digested pHY106 to produce HBV replicon. HBV open reading frames (ORFs) for the pre-core (PC), core, preS1 (PS1), preS2 (2) surface (S), polymerase, and X genes are indicated by square frame. Promoter sequences for the preS (PS), surface (S) and core (C) genes are indicated by arrows.

Figure 3

Identification of recombinant expression plasmids by restriction endonuclease digestion analysis. M: 1 kb DNA ladder marker; Lane 1-8: Recombinant plasmids digested with Hind III and Nsi I.

Figure 4

Analysis of intracellular replication of clinical HBV isolates. HBV isolates were cloned into the vector pHY106 and transfected into Huh2 cells. Seventy-two hours after transfection, intracellular replicative intermediates were isolated and analyzed by Southern blot. Double-stranded (DS) and single-stranded (SS) intermediates are indicated. Lane 1-8: eight replicons of clinical HBV isolates; Lane 9: pHY106 negative control.

Figure 5

Antiviral susceptibility of an HBV isolate cloned from a patient with lamivudine resistance. An HBV isolate amplified from the sera of patients failing in lamivudine therapy was cloned into the pHY106 vector and analyzed in vitro. Lane 1-4: transfected cells treated with 0.01, 0.1, 1 and 10 μmol/L of adefovir; Lane 5-8: transfected cells treated with 0.01, 0.1, 1 and 10 μmol/L of lamivudine.