Identification of rare polymerase variants of hepatitis B virus using a two-stage PCR with peptide nucleic acid clamping.
Emergence of lamivudine-resistant variants, with amino acid substitutions in the Tyr-Met-Asp-Asp (YMDD) motif of hepatitis B virus (HBV) reverse transcriptase, is a serious problem in antiviral therapy. Presence of YMDD motif variants in patients who had never been treated with lamivudine has been reported recently. However, no analysis of nucleotide and amino acid sequences of these variants has been performed. In the present study, using polymerase chain reaction (PCR) with peptide nucleic acid (PNA) clamping, we detected many new variants, such as Tyr-Arg-Asp-Asp (YRDD), Tyr-Met-Asp-Asn (YMDN). Many of them had stop codon(s) in overlapping HBs gene. Although the biological activity of these HBV polymerase variants remains to be determined, our results showed that numerous quasispecies are created during virus replication. A typical lamivudine-resistant Tyr-Val-Asp-Asp (YVDD) variant was detected in only one of 62 (1.6%) anti-HBe patients with HBV infection before administration of lamivudine. This variant did not have the L528M mutation, which is often associated with YVDD variants, and lamivudine therapy in this patient suppressed HBV replication. Thus, care should be taken when interpreting the results of detection of YMDD variants, especially when the sensitivity of the assay is very high. Amplification of rare variants by PCR with PNA seems a useful tool to examine the emergence of drug-resistant variants as well as naturally occurring mutants, such as the hepatitis B e antigen (HBeAg) stop codon and vaccine escape mutants. Examination of rare variants should enhance the understanding of the mechanism for emergence of drug-resistant HBV variants and help in developing strategies for new antiviral drugs.
Ohishi
Waka
W
Department of Medicine and Molecular Science, Division of Frontier Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
Shirakawa
Hiroo
H
Kawakami
Yoshiiku
Y
Kimura
Sonoko
S
Kamiyasu
Masaya
M
Tazuma
Susumu
S
Nakanishi
Toshio
T
Chayama
Kazuaki
K
D000328:Adult; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000097:blood / Q000302:isolation & purification; D024882:Drug Resistance, Viral / Q000235:genetics; D005260:Female; D015684:Gene Products, pol / Q000235:genetics*; D006510:Hepatitis B Antibodies / Q000097:blood; D006514:Hepatitis B Surface Antigens / Q000097:blood; D006513:Hepatitis B e Antigens / Q000097:blood; D006515:Hepatitis B virus / Q000235:genetics* / Q000302:isolation & purification*; D019694:Hepatitis B, Chronic / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology / Q000627:therapeutic use; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D020135:Peptide Nucleic Acids; D016133:Polymerase Chain Reaction / Q000379:methods*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D017422:Sequence Analysis, DNA
D004279:DNA, Viral; D015684:Gene Products, pol; D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; C066027:P protein, Hepatitis B virus; D020135:Peptide Nucleic Acids; D018894:Reverse Transcriptase Inhibitors; D019259:Lamivudine