A Study On Quasispecies And Genotype Prevalence Of HBV In Chengdu Area

Objective: Hepatitis B is a worldwide epidemic which is wide prevalent and extremely harmful that can not be effectively cured yet. As a most epidemic region in the world, our nation now have over one-tenth of population to be hepatitis B virus (HBV) carrier according to the report. A precise understanding of the heterogeneity of highly mutational HBV is meaning to pathology researches on clinic diagnosis and treat. Considering the quasispecies phenomenon widely exists, a long and accurate Polymerase Chain Reaction (LA-PCR) method was used to investigate the prevalence of (HBV) genotypes in patients suffering chronic HBV infection in Chengdu area of China.Methods: Specific primers were designed to amplify the homologous region containing S gene which codes hepatitis B surface protein (HBsAg) in the HBV genome. The fragments were obtained from 90 serum samples of positive patients through a long and accurate PCR procedure, and were then sequenced by a Sanger Method. The results were compared with standard sequences recorded in GeneBank for genotyping and further analysis. Meanwhile, a typespecific-primered PCR was performed on all samples for standard control. By executing this more frequently used method, a nested PCR was carried out with specific primers for each genotype. The results from the two assays were compared as to test their credibility and accuracy. Results: Genotype B was detected in 63.3 %( 57/90) of subjects by sequencing S fragment, while genotype C in 33.3 %( 30/90), and genotype D in 3.3% (3/90). None of any other genotypes in this group of patients was detected. However, by genotyping following typespecific primered PCR, genotype B was detected in 47.8 %( 43/90) of subjects, while genotype C in 23.3 %( 21/90), genotype D in 3.3% (3/90), B/C hybrid in 24.4% (22/90) and B/D hybrid in 1.1% (1/90). The research showed the highly mutational characteristic of the HBV genome, not only the wide existence of polymorphism and heterogeneity in one genotype, but also the existence of quasispecies of multi-clones in one sample.Conclusion: This genotyping result showed that HBV genotype B was the predominant genotype in Chengdu area while genotype C was secondary; genotype D was singularly. This consists well with former reports which proclaimed that genotype B was the majority genotype in southern China. The further sequence analysis indicated that LA-PCR product was not a single clone with identical sequence, but a quasispecies composed of a predominant clone and a cluster of relative mutants. The S fragment sequencing method was suggested significantly better (P<0.001) between the two genotyping assays, for it can describe diversity of HBV quasispecies, identify the genotype of predominant clone and avoid the false-positive result caused by minority clones. The phenomenon of quasispecies and characteristic of highly polymorphism was confirmed by this research, however, the influence of which on clinic diagnosis, treat and prognostic still need further investigation.