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.

ORF3 and partial ORF1 regions were amplified with RT-PCR f rom two patients (T1 and T11)infected with new genotype of hepatitis E Virus. Th e PCR products were cloned and sequenced. The results showed that G-C rich regi on in ORF3 was deleted when amplified with normal PCR reaction. However, PCR rea ction containing G-C melt solution can overcome this problem. The sequence anal ysis showed that T1 and T11 belong to a new genotype of HEV which differs from g enotype I,II and III reported.T1 and T11 have 79%～82%, 80%～81% and 83%～85% id entical to genotype I,II and III respectively.

OBJECTIVETo understand the prevalence of HBV core promoter mutant (T1762 A1764 mutant) isolated from asymptomatic carriers from areas with higher and lower incidence of hepatocellular carcinoma (HCC) in Guangxi.

METHODSA nested polymerase chain reaction (nPCR) was used for amplification of HBV DNA core promoter in sera, and then HBV DNA nPCR products were sequenced by direct sequencing.

RESULTSThe results show that 50.6% (39/77) of all HBV asymptomatic carriers were positive for HBV DNA HBV DNA positive rates of the samples from HCC higher incidence area, Longan County, and from lower incidence area, Guilin city were 55.6% (20/36) and 46.3% (19/41), respectively. HBV core promoter mutants could be seen in 35% in Longan positive samples and 47.4% in Guilin. The common mutations in both regions were all double mutations (nt 1,762 A-->T; nt 1,764 G-->A), accounting for 25% and 21%, respectively. The difference of the double mutant between Longan County and Guilin city was not significant (P>0.05).

CONCLUSIONSThese data implicated that the prevalence of HBV core promoter mutant isolated from asymptomatic carriers may not be correlated with the incidence of HCC in Guangxi.

Adolescent ; Adult ; Carcinoma, Hepatocellular ; virology ; Carrier State ; virology ; DNA, Viral ; genetics ; Female ; Hepatitis B ; virology ; Hepatitis B Core Antigens ; genetics ; Hepatitis B virus ; genetics ; Humans ; Liver Neoplasms ; virology ; Male ; Middle Aged ; Point Mutation ; Promoter Regions, Genetic ; genetics

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

OBJECTIVETo explore the relationship between HBV core promoter mutations and liver damage or HBeAg status.

METHODSNested polymerase chain reaction (nPCR) was used for amplification of HBV DNA core promoter in 59 sera from patients with chronic hepatitis B in Guangxi, then the HBV DNA positive products were sequenced by direct sequencing.

RESULTSThe HBV DNA positive rate of was 59.3%(35/59). All the patients were infected by mutants. The commonest mutation was the double mutation (A --> T at nt1762 and G --> A at nt1764), counting for 57.1% (20/35). The next was C --> G at nt1799, counting for 54.4% (19/35), but this was no function. A --> G at nt1752 (resulting in isoleucine to valine) was seen in 37.1% (13/35) of the HBV DNA positive patients, and T --> C at nt1753 was seen in 20% (7/35). The significant difference in the frequency of T1762A1764 mutant was found between HBeAg positive patients (31.3%) and negative patients (79.0%).

CONCLUSIONSHBV core promoter mutations are common among patients with chronic hepatitis B in Guangxi. T1762A1764 mutant is associated with HBeAg status and chronic hepatitis.

Adolescent ; Adult ; Female ; Hepatitis B Core Antigens ; genetics ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; isolation & purification ; Hepatitis B, Chronic ; virology ; Humans ; Liver ; pathology ; Male ; Middle Aged ; Point Mutation ; Promoter Regions, Genetic ; genetics