Article Figures & Data
Figures
- FIG. 1.
(A) Clinical course of a patient who developed breakthrough without emergence of YMDD mutants during lamivudine therapy. Arrows a to e indicate time points of serum sampling for direct sequencing and RFLP PCR. (B) Nucleotide sequence analysis of the reverse transcriptase/polymerase gene of hepatitis B virus by direct sequencing. Time points of serum sampling (see panel A) were as follows: (a) just before lamivudine treatment, (b) after breakthrough, (c) after cessation of lamivudine treatment, (d) just before readministration of lamivudine, and (e) during adefovir and lamivudine therapy. Note that the wild type reappeared during the cessation of therapy (c and d), but it disappeared after readministration of the drug (e).
- FIG. 2.
Comparison of nucleotide sequences and amino acid sequences of two overlapping open reading frames, reverse transcriptase/polymerase and the HBs gene of the hepatitis B virus, before and after viral breakthrough. Sequences obtained from serum samples before (a) and after (b) breakthrough were compared. See Fig. 1A for time points of serum sampling. Nucleotide sequence numbers are those of typical HBV (e.g., accession no. AB206816 [31]), which starts from a unique EcoRI site.
- FIG. 3.
Replication ability of wild-type HBV and three mutants (S331C, rtA181T, and S331C/rtA181T). Plasmids containing 1.4-genome-length HBV were transiently transfected into HepG2 cells. (A) The replicative intermediates were analyzed by Southern blot hybridization. Core-associated replicative intermediates of HBV DNA were isolated from HepG2 cells at 3 days after transfection. The positions of relaxed circular DNA (RC) and replication intermediates (RI) are indicated. (B) Quantitative analyses of core-associated intermediates of HBV. Experiments were performed in triplicate. Values are relative to those of the wild type and are expressed as means ± SD. *, not significant compared to the wild type.
- FIG. 4.
In vitro analyses of susceptibility of wild-type HBV and three mutants (S331C, rtA181T, S331C/rtA181T) to lamivudine after transient transfection into HepG2 cells. Cells were transiently transfected with plasmids containing 1.4-genome-length HBV and treated with the indicated amount of lamivudine. (A) Southern blot analysis of replicative intermediate. Representative results for the wild type (wt) and the S331C/rtA181T mutant are shown. The positions of relaxed circular (RC) and replication intermediate (RI) forms of HBV DNA are indicated. (B) Dose-response curves of the four HBV strains against lamivudine. The curves were used to estimate the lamivudine IC50s for each HBV strains. Values are relative to no-lamivudine controls for each strain. Experiments were performed in triplicate. Values are expressed as means ± SD.
- FIG. 5.
In vivo analyses of the effect of lamivudine on wild-type and S331C/rtA181T mutant HBV. Four human hepatocyte-chimeric mice were inoculated with serum samples containing wild-type or mutant HBV. One of the animals fed with lamivudine died 6 weeks after the beginning of therapy.
- FIG. 6.
Detection of the rtA181T mutant by RFLP PCR assay. PCR-amplified DNA fragments were treated with EspI, which digests only wild-type sequences, and separated in a 3.5% agarose gel. (A) Agarose gel electrophoresis of RFLP PCR products. Wild-type and rtA181T mutant plasmids were used as controls. See Fig. 1A for the time points of serum sampling (a to e) for patient 1 and see Fig. 1B for a comparison with nucleotide sequence analyses. f and g indicate the time points before and after viral breakthrough for patient 2. (B) Agarose gel electrophoresis of RFLP PCR products using HBV DNA samples obtained from 39 patients who showed lamivudine breakthrough. Of the 39 samples, 35 were wild type (lanes 1 and 2). The remaining four samples (lanes 3 to 7) showed partial digestion, suggesting a mixture of wild-type and mutant strains. (C) Nucleotide sequence analysis of a sample by RFLP PCR suggested the presence of a wild-type-mutant mixture (lane 5 of panel B).
Tables
- TABLE 1.
In vitro susceptibility of the S331/rtA181 mutant to lamivudinea
Strain S331/rtA181 mutation Lamivudine IC50 (μM) Resistance (fold) Source Type Patient WT −/− 0.19 ± 0.01 1 S331C C/− 0.23 ± 0.01 1.2* rtA181T −/T 0.58 ± 0.08 3** S331C/rtA181T C/T 0.57 ± 0.06 3** Laboratory WT −/− 0.23 ± 0.04 1 S331C C/− 0.3 ± 0.05 1.3* rtA181T −/T 0.88 ± 0.2 3.9** S331C/rtA181T C/T 0.98 ± 0.12 4.3** ↵ a Experiments were performed in triplicate. Values are expressed as means ± SD. WT, wild type. *, not significant; ** P < 0.001 compared to the wild type.
- TABLE 2.
In vitro susceptibility of the S331/rtA181 mutant to lamivudine, adefovir, and entecavira
Patient strain S331/rtA181 Lamivudine Adefovir Entecavir IC50 (μM) Resistance (fold) IC50 (μM) Resistance (fold) IC50 (nM) Resistance (fold) WT −/− 0.19 ± 0.01 1 0.37 ± 0.1 1 0.19 ± 0.02 1 S331C/rtA181T C/T 0.57 ± 0.06 3** 0.36 ± 0.08 0.98* 0.23 ± 0.05 1.2* ↵ a Experiments were performed in triplicate. Values are expressed as means ± SD. WT, wild type. *, not significant; ** P < 0.001 compared to the wild type.
