Molecular Epidemiology Of Hepatitis B Virus S Gene Among HBsAg Carriers After HepB Vaccination

Objective:To describe the composition of the HBV genotype, serotype and mutation in three-dimension distribution among people who developed chronic hepatitis B virus infection after universal HepB vaccination. To investigate the possible reason for infection after universal HepB vaccination. To explore the relationship between mutant and immune failure and escape. To evaluate the escape mutant threat on the vaccines and immunization strategy. To provide the baseline data for escape mutant monitor platform, vaccines and immunization strategy optimizations.Methods:168blood samples were collected from six areas with clearly immunization history, including Hebei, Henan, Qinghai, Hunan, Guangxi, and Shanghai. All samples were identified as HBsAg positive by abbott chemiluminescence immunoassay (CLIA). Total DNA was extracted by QIAamp DNA Blood Kit from serum. The S gene fragment was amplified by nested PCR. All fragment were sequenced directly or cloned into pMD18-T vector then sequenced. The sequencing results were assembled by DNAStar5.0. Genotype and serotype were identified by local blast software and blast interpreter software. All of the amino acid sites of S gene fragment ware analyzed by statistical software SAS9.0with epidemiological survey information.Results:168S gene sequence were amplified from the patient of HBV infection after universal HB vaccination. The genotype of these people were B, C, D, I. The serotype of these people were adr, adw, ayw. There was statistically significant difference in genotype and serotype between six areas. In the northern china and southeast china, genotype C is dominant. Contrarily, in the southwest china, the genotype B is majority.234amino acid mutation sites were detected from the168sequence. The key mutation sites were aa126, aa127, aa129, aa131, aa133, aa140, aa143and aa145.The Type and quantity of mutations were genotype-related and independent on whether the timely vaccination. There were13%sequence contained ’α’ determinant mutants. In quasispecies analysis, the genotype of HBV in first sample was B2and other samples were C5. Two kinds of predominant quasispecies were observed in sample one base on genetic distances and quasispecies distribution. One predominant quasispecies were observed in other samples. Entropy values of three samples has no significant difference. The ratios of Ka/Ks of sample one were lower than other samples with ω=0.58(Ka<Ks). The quasispecies in sample one were under negative (purifying) selection pressure. The point mutations of aaF134S and aaS143P at ’α’ determinant were observed in the sample two. The point mutation aaT125A and nonsense mutations ntG494A and ntC500A at ’α’ determinant were observed in the sample three.Conclusions:’α’ determinant mutants may not the main reason of immune failure. There is no need to adjust the current vaccines and immune strategies. The mutations we detected may change antigenically. The function of mutation need to be verified in vitro experiment. It is necessary to establish epidemiology monitoring system against acute hepatitis B infection. The complexity of hepatitis B virus S-gene quasispecies was existed in neonatal HepB vaccination patients with concurrent HBsAg and HBsAb in serum.’α’ determinant mutation was observed by quasispecies analysis. The experimental results indicated that quasispecies analysis can describe HBV status more accurately and practically.