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

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 &mgr;mol/L of adefovir; Lane 5-8: transfected cells treated with 0.01, 0.1, 1 and 10 &mgr;mol/L of lamivudine.