The Study Of Genes And Structure Of Hepatitis B Virus Influencing The Efficiency Of Lamivudine And Construction Of High Replication Competency, Lamivudine-resistant, Infectious Molecular Clones Of HBV

Since lamivudine was authorized to treat chronic hepatitis B virus (HBV) infection in 1998, the problem of HBV lamivudine-resistance became increasingly serious. More mutations happened in lamivudine-resistant chronic hepatitis B patients when they received new nucleoside or nucleotide analog drug treatment. So how more availably carried on anti-virus therapy become a common concern in the world. In this study, the sequences of HBV P gene in serum samples of 29 HBeAg-positive at baseline patients, who had different response to lamivudine, were analyzed to determine if specific HBV DNA sequence patterns outside YMDD motif could be correlated with lamivudine-resistance, and confirmed by evaluation of effects of lamivudine on site-mutant strains in vitro. The reverse dot hybridization was applied to detected HBV mutants. Meanwhile, we constructed high replication competency full-length lamivudine-resistant HBV infectious molecular clones. It may be the basis on pathopaiesia mechanism, resistance mechanism, and using on screening anti-lamivudine-resistant HBV drug.1. The study of genes and structure of HBV influencing the efficiency of lamivudine and its application1.1 Identification mutation outside YMDD motif of the hepatitis B virus polymerase selected during lamivudine therapyIn this study, sequences of the HBV reverse transcriptase region in 29 chronic hepatitis B patients (HBeAg-positive at baseline) treated for 48 months were analyzed to determine specific HBV DNA sequence patterns associated with lamivudine-resistance. YMDD mutants were detected before lamivudine-therapy, but not always developed into clinical resistance. We first reported two naturally-occurring DNA polymorphisms at amino acid 91 in domain A and amino acid 164 in domain B of the HBV genotype B polymerase were observed to be significantly (P<0.01), rt91L and rt164V were presented in lamivudine- sensitive strains while rt91I and rt164L in lamivudine-resistant strains. Rt91L and rt164L became the dominant circulating species in breakthroughs and non-responders under lamivudine-press. The sole genotype before therapy turned into commixed genotype B and C commixed infection after lamivudine-therapy might be a result of virus evaded immune-pressure.1.2 In vitro study of the relationship between HBV polymerase mutation rtI91L and rtL164V and lamivudine-resistanceWe constructed rt91, rt164 and rt204 single and united mutants on lamivudine- sensitive wild-type strain #56, and evaluated of the effects of lamivudine on mutant strains by transfect HepG2 cell line. RtL164V mutation decreased the YMDD wild-type HBV genotype B sensitivity to lamivudine. In despite of rtI91L mutation did not change the sensitivity of the YMDD wild-type HBV to lamivudine, it could decrease further the level of sensitivity of rtL164V-carrying HBV as an additional mutation. The addition of the rt91 and /or rtL164V mutation conferred increasing in replication efficiency to both wild-type and rtM204I-carrying HBV genotype B in vitro (P<0.05).1.3 Determination of HBV mutants correlated with lamivudine-resistance by reverse dot hybridizationHBV reverse transcriptase domain was amplified used biotin-marked reverse primers, and hybridized with oligo-nucleotide probe in nylon membranes. HBV mutants were determined by reverse dot hybridization and P gene sequencing. Complete concordance between reverse dot blot and sequencing analysis at rt91, rt164, rt204 were observed for 82.6%, 89.7% and 75.9%. The discrepant results in these three site were 1 specimen (3.4%) each. The whole rate of match was 82.8%, and total miscarriage of justice rate was 4.6%. So long-segment DNA reverse dot hybridization could be used in detected multi-mutations in lamivudine-resistant HBV.2. Construction of high replication competency, lamivudine-resistant, infectious molecular clones of HBVThe HBV full-length were amplified from chronic hepatitis B virus infected patients serum who were no response to lamivudine-treatment. We first screening a lamivudine-resistant HBV clone of genotype B with high replication competency from 7 recombinant plasmids containing full-length HBV by transfected HepG2 using calcium phosphated precipitation methods and lamivudine-resistant assay in vitro. Gene sequencing indicated that the HBV strain was YIDD mutant without rtV173L or rtL180M mutation which had been confirmed to enhance viral replication. There were not obviously changes in conserved regions of reverse transcriptase and regulated sequences. We presumed the mutation changed the structure of the reverse transcriptase, which enhanced viral replication efficiency.