Mutation Analysis Of Hepatitis B Virus Pre-s Region Before And After Adefovir Dipivoxil Treatment

Hepatitis B virus (HBV) infection is a major public health problemworldwide. HBV reverse transcriptase lacks a proofreading function whichleads to the emergence of a great deal of genetic variants undermulti-selective pressures. Pre-S region is the most variable part of the HBVgenome. HBV isolates with Pre-S deletion mutations are often found inhepatitis B carriers. Hot spots of mutation include the3’ half of the Pre-S1and the5’ half of the pre-S2, and Pre-S2start codon deletion/pointmutation. The prevalence of Pre-S deletions is higher in genotype B and C.Pre-S deletions, as an independent factor, have been documented to play apotential role in hepatocarcinogenesis. Therefore, effectively suppressingHBV replication, especially the isolates with Pre-S deletions, may help toreduce the incidence of HCC. Nucleot(s)ide analogues (NAs) used to treatHBV infection have been demonstrated clinically to significantly suppressHBV replication, decrease serum ALT level and improve liver histology.Recent studies showed that Pre-S deletions were found during the treatment of NAs. The effects of NAs therapy on Pre-S deletion strains are largelyunknown. These questions related to drug safety require to be resolved.Objective. To investigate HBV Pre-S mutants in patients with chronichepatitis B infection (CHB) before and after adefovir dipivoxil (ADV)treatment.Methods. HBV DNA was extracted from the sera of61CHB patientsat baseline and at week48of ADV treatment. Semi-nested PCR wasperformed to amplify HBV Pre-S region. PCR products were purified andligated with pMD19-T Simple Vector at16℃overnight, then transformedinto an E. coli strain DH5α. PCR amplifications of insert-DNA werepurified and sequenced. Quantification of HBV DNA was performed byreal-time genotyping and quantitative PCR. Serological markers includingHBsAg、 HBeAg and HBeAb were determined using commercial kits(Abbott and Kehua). ALT levels were tested using Roche Modular DDPanalyzer.Results.1. Pre-S mutants were detected in55.7%(34/61) of CHBpatients at baseline, and37.7(23/61) of CHB patients at week48of ADVtreatment. In most patients with Pre-S mutants infection, pre-S mutantscoexisted with the wild-type HBV strain except patient X39. Based on thepresence of Pre-S mutants at baseline, Patients were divided2groups: W-0(wild-type only) and M-0(Pre-S mutants existing). No significance wasdetermined in gender, age, genotype, HBV Viral load, and ALT levels between the2groups. At week48of ADV treatment, HBV Viral load andALT levels were decreased markedly in group M-0compared with groupW-0. Also, HBeAg loss were significantly elevated in group M-0.2. A45bp insertion mutation was investigated in one colony producenamed No.7from patient X37at week48. The inserted site was between nt2978and2979. The inserted segment was same as the nt2934-2978in thisPre-S sequence.3.61CHB patients were infected with genotype B and/or C. Genotypes inCHB patients which infected genotype C and B/C mixture were changed atweek48of ADV treatment. Of20patients infected genotype C,5patientspresented genotype B infection,6patients presented B/C mixture attreatment week48. Of3patients infected B/C mixture, one was changed togenotype C infection; the other was changed to genotype B infection; theremainder presented B/C mixture infection with increased genotype B attreatment week48.4. Recombinations based on Pre-S between genotypes B and C weredetermined in5patients with B/C mixture infection. One patient was foundrecombinations at baseline; the other4were found recombinations attreatment week48.Conclusion. The prevalence of Pre-S mutants was high in CHBpatients. ADV can effectively suppress Pre-S mutants replication. Pre-Smutation strains may be more sensitive for ADV therapy compared with wild-type strain. Mixed genotype infections were common in CHB patients.Genotype C may be more sensitive for ADV therapy than genotype B.