Rare quasispecies in the YMDD motif of hepatitis B virus detected by polymerase chain reaction with peptide nucleic acid clamping

Abstract

The emergence of drug-resistant mutants of hepatitis B virus (HBV) is a serious problem during antiviral therapy of patients with chronic hepatitis B. Lamivudine-resistant mutants with a mutation in the YMDD motif of reverse transcriptase of HBV emerge in approximately one half of the treated patients within 5 years. To date, the detection of YMDD mutants by polymerase chain reaction (PCR) with peptide nucleic acid (PNA) clamping is most sensitive. In this study, the performance of this method was evaluated in various clinical settings. The PCR-PNA method was able to detect the emergence of YMDD mutants 2-3 months earlier than the previously developed method involving restriction fragment length polymorphism. Further, rare quasispecies were detected by PCR-PNA in patients with chronic hepatitis B who were positive for hepatitis B e antigen (HBeAg). Many previously unrecognized mutants, such as those with YLDD and YMED, were found in them. Although precise sequence analyses of 10 patients identified YVDD and YIDD sequences in 6 of them, only 1 patient had a typical YVDD sequence that was identical with that in the reported lamivudine-resistant strain. All HBV mutants with the YIDD sequence accompanied stop codon(s) in the overlapping envelope (S) gene, suggesting that these strains would have no relevance as regards the emergence of lamivudine resistance. These results suggest that it would be difficult to detect lamivudine-resistant mutants before the therapy and that they would have a greater ability to evade the attack of antiviral drugs by frequent nucleotide substitutions than previously expected.