Abstract: Objective To clarify the long-term evolution of hepatitis B virus (HBV) quasi-species in HBsAg asymptomatic carriers in Long'an county, Guangxi. Methods ELISA was used to detect serological markers of HBV. Viral loads were measured by real time PCR. HBV DNA was extracted from serum by kits. The whole HBV genome was amplified using nested PCR and amplicons were sequenced by next-generation sequencing (NGS). These sequences from NGS were analyzed by the software like Mega. Results Serum samples were collected from 9 HBsAg asymptomatic carriers in Longan County，Guangxi at 4 different time points in 2004, 2007, 2013, 2019 or 2020. A total of 23 serum samples and 309 full-length gene quasi-species sequences were obtained, with an average amount of (0.18±0.07) G sequencing data for each sample. Genotype of 55.54%(5/9) the studied subjects underwent genotype conversion during the long-term evolution process of HBV quasi-species, and the genotyping results of the phylogenetic tree in the PreS/S region are in perfect agreement with the results of the whole genome analysis; recombinant B/C, I/C were found; the Sn ranged from 0 to 0.37 and the genetic diversity ranged from 0 to 0.11, respectively. A total of 21 special single nucleotide/amino acid mutations (7 in the S region, 2 in the X region, 3 in the PreC region and 9 in the BCP region) and 6 deletion mutations were detected, multiple mutations were found and no drug resistant mutations were found; 77.8%(7/9) of the HBV strains carried by the subjects in 2004 had double mutations at nt1 762(A→T) and 1 764(G→A) and a stop mutation at nt1 896(G→A); HBV mutations can be restored from the mutant type to the wild type and (or) vice versa without antiviral drug pressure, and The evolution rate of HBV genome was 2.03×10-5~3.50×10-3.Conclusion HBV genotype, recombinants, genetic complexity and diversity of HBV quasi-species can change over time during in natural infection. The transformation between HBV mutation type and wild type reduces the value of predicting clinical outcomes by genetic types and related mutations to some extent. The HBV genome evolution rate of asymptomatic carriers of HBsAg in Long'an County is very high.

OBJECTIVEIn order to understand the prevalence of hepatitis B virus (HBV) precore mutants isolated from asymtomatic carriers in Guangxi.

METHODSNested polymerase chain reaction (nPCR) was used for amplification of HBV DNA precore in 77 carrier sera, followed by HBV DNA nPCR products sequencing using direct sequencing.

RESULTS50.7% of 77 carriers was positive for HBV DNA with a prevalence of mutants 22.1% (17/77). HBV DNA positive rate in the southern part of the autonomous region was 55.6% (20/36). Six of them were mutants, counting for 30%. The common mutation in the southern part was seen T-->C at nt1858 while nt1896 stop mutation was discovered in one sample only, which was accompanied by point mutation at nt1837 (A-->G). HBV DNA positive rate in the northern part was 46.3% (19/41) with 11 of them were mutants, counting for 57.9%. The common mutation in that area stopped at nt1896. Among samples with stopped mutation, 4 samples had mutation at nt1846 (A-->T), 2 samples at nt1862 (G-->T). Both mutation at nt1856 (C-->T) and nt1858 (T-->C) could be seen in sample 734.

CONCLUSIONSThe prevalence of HBV precore mutant in asymptomatic carriers in Guangxi was at the average level in China. Further study is needed to determine the difference between the southern and the northern part of the region in the common type of mutation exists.

Base Sequence ; Carrier State ; virology ; DNA, Viral ; chemistry ; Hepatitis B virus ; genetics ; Humans ; Molecular Sequence Data ; Mutation ; Polymerase Chain Reaction