Lamivudine Therapy And Hepatitis B Virus Polymerase Gene Mutation

Objective To study quasispecies and mutations of hepatitis B virus polymerase gene in pre- and post-therapy of lamivudine in chronic hepatitis B patients. Lamivudine, a nucleoside analogue, suppresses hepatitis B virus (HBV) replication in most patients during short-term therapy. However, prolonged treatment with lamivudine results in emergence of resistant viruses in 24% of patients following 1 year of therapy and 70% of patients following 4 years of therapy. Lamivudine resistance may be associated with loss of clinical benefit. Viral resistance to lamivudine has been mapped to the specific mutations in the tyrosine-methionine-aspartate-aspartate (YMDD) motif in the C domain of the HBV polymerase.Methods Six patients infected with hepatitis B virus were chosen in this study, so as to investigate the mutations of HBV polymerase gene in lamivudine therapy. All of the patients broke out when they received lamivudine 36 weeks to 48 weeks. Before and after lamivudine therapy serum specimens were collected for the study. 1) SSCP/HDA was applied to analyse quasispecies complexity and character of mutation after PCR, TA-clone and transfected into Colibacillus INVaF'. 2) Melting curve analysed the YMDD mutation roughly. 3) One patient was researched by RFLP.Results 1) Pre-therapy quasispecies composing were more complicated than post-therapy. The number of quasispecies was 7~14(average 9.8) and 4~8(average 5.7), respectively(t=3.98, P<0.05). 6 patients had one or two predominant quasispecies before therapy, but percentage of pre-therapy (33.3%~81.8%) was lower than post-therapy samples(78.8%~90.9%), t=3.24, P<0.05. 2) By sequence analyzing predominant quasispecies, experiment found that, 2 patients with M550V/L526M, 3 patients with M550I and 1 patient with wild type after lamivudine therapy. Additionally, there were individuation mutations which had no obvious current. The nucleotide mutation ratios ofpatients A~F were 6.5, 6.5, 4.3, 10.9, 13.0, 8.7bp/lkb, respectively. 3) RFLP shown that, in the 33 clones of patient D ,there were 32 clones were YMDD of pre-therapy and 29 clones were YIDD of post-therapy sample, except land 4 synonymous mutations, respectively.Conclusion 1) After lamivudine therapy, 5 patients emerged YI/VDD mutation and 1 patient were wild type all along. Suggested that there were other resistant factors except YMDD mutation.2) Under the selective pressure of lamivudine, oquasispecies number of post-therapy was less than pre-therapy. 3) All patients with higher HBV DNA titer and most people had elevatory ALT when YMDD mutation emergence.