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Objective To analyze the sequence of Pre-S gene in asymptomatic chronic HBV carriers (ASCs) with low-level HBsAg.Methods The serum samples were collected from 654 ASCs in the First Affiliated Hospital of Zhejiang University School of Medicine , Hangzhou Sixth People’s Hospital and the 903th Hospital of PLA.According to the level of HBsAg , ASCs were divided into low-level HBsAg group (≤10 IU／mL ) and high-level HBsAg group (＞10 IU／mL ).The pre-S／S gene amplification and sequencing were performed in 138 ASCs with low-level HBsAg and 100 age-matched ASCs with high-level HBsAg.A phylogenetic tree was constructed to determine the genotype , based on the successful sequencing results of Pre-S gene in the high level HBsAg group , the Pre-S gene reference sequences of the main ASCs genotypes in Eastern China were established.The sequence of Pre-S gene in low-level HBsAg group was analyzed and compared with the reference sequences.SPSS 12.01 statistical software was used to analyze the data.Results Sixty-three cases of Pre-S／S were successfully sequenced in 138 ASCs of low-level HBsAg group, including 52 cases of B genotype and 11 cases of C genotype.Among the 100 cases of high-level HBsAg group, 94 cases of Pre-S／S were successfully sequenced , including 48 cases of B genotype and 46 cases of C genotype.The sequence analysis indicated that in the B genotype , 81 amino acid mutation sites were found in the Pre-S protein of the low-level HBsAg group, including 4 significant mutations: F56I／V, T76A／N／P in the Pre-S1 region, P15L／S／T and Y21T／F／H／N in the Pre-S2 region; while 47 amino acid mutation sites were found in Pre-S protein of high-level HBsAg group, including 3 significant mutations :L34F, V49A and P59S／L in Pre-S1 region.The total number of amino acid mutation sites in the low-level HBsAg group of B genotype was higher than that of the high-level HBsAg group (χ2 ＝14.008, P＜0.05). In the C genotype, 19 amino acid mutation sites were found in the Pre-S protein of the low-level HBsAg group, including 3 significant mutations : W66V／G and A79V in the Pre-S1 region,V32A in the Pre-S2 region; while 39 amino acid mutation sites were found in Pre-S protein of the high-level HBsAg group, including 2 significant mutations: A79V in Pre-S1 region and T49I in Pre-S2 region.The total number of amino acid mutation sites of Pre-S protein in the C genotype was significantly different between the two groups (χ2 ＝7.571, P＜0.05).Conclusion Significant mutations in Pre-S gene may be associated with the persistent expression of low-level HBsAg in ASCs.
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Objective To reveal the characteristics of S gene sequence of hepatitis B surface antigen (HBsAg) in hepatitis B virus (HBV)-infected patients with low HBsAg level.Methods From February 2016 to December 2017, 1 308 serum samples of inactive HBsAg carriers were collected from the 903rd Hospital of PLA and Hangzhou Jianggan District People′s Hospital.The cases were divided into high-level group and low-level group according to the level of serum HBsAg (10 IU／mL) expression.The HBV S gene was sequenced in patients with low-level HBsAg expression.In addition, in patients with high-level HBsAg, 100 patients were randomly selected (stratified sampling) for HBV S gene sequencing based on the matching of age and serological pattern (hepatitis B e antigen [HBeAg] negative) of low-level HBsAg group.A comparative analysis was conducted between HBV S gene sequences from inactive HBsAg carrier in low HBsAg expression group and the HBV reference S gene sequences from inactive HBsAg carrier in high HBsAg expression group .The results of normal distribution data were expressed as Mean ±SD, and analyzed using t-test.The results of non-normal distribution data were expressed by M(QR), and analyzed using Mann-Whitney U test.Chi-square test or Fisher exact test was used to compare continuous variables and classification variables between the two groups .Results There were 276 serum samples from the low level group and 1 032 serum samples from the high level group , including 257 HBsAg／HBeAg／anti-HBc-positive cases, 753 HBsAg／anti-HBe／anti-HBc-positive cases, and 22 HBsAg／anti-HBc-positive cases.Successful HBV S gene sequencing was performed on 126 out of 276 patients in the low-level HBsAg group.According to the age inthe low-level HBsAg group, 100 samples with negative HBeAg in the high-level HBsAg group were randomly selected , among which 94 patients were genotyped and hemotyped.The results showed that there were statistically significant differences in HBV serological markers , HBV DNA level and HBV genotype distribution between the high level group (94 cases) and the low level group (126 cases) (all P＜0.05).The ASC-R-B and ASC-R-C genotypes reported in this study had high homology (99.6%-100.0%) with those reported in Shanghai , Chengdu, Wuhan, Yunnan and Beijing of China , and high homology (98.2%-99.6%) with those reported in Japan and Korea of NCBI genotype B and C reference sequences, but had low homology with patients far away from China (98.2% in Canada and 98.7% in Indonesia).In genotype B of the low level group , the amino acid mutation number of SHB protein was 71, and the hot spot mutation number was 19, both higher than those in the high level group (39 and 8, respectively). The difference was statistically significant (χ2 ＝12.303 and 4.766, respectively, both P＜0.05).Amino acid mutation sites in the low HBsAg group were mainly distributed on both sides of the major hydrophilic region (MHR) (amino acid residues 40 -49 and 198 -220).There were no significant differences in amino acid mutation number and hot spot mutation number between the two groups of C genotype (χ2 ＝0.383 and 0.409, respectively, both P＞0.05).For genotype B, 12 single point mutations and 4 dual co-mutations were found in low level group.Among them, one single point mutation (S210R) and 3 dual co-mutations (G44E／V+T45P／I, G44E／V+L49P／R and N40S+I208T) were not hot spot mutations , while 2 dual co-mutations and 2 single point mutations were found in high level group.The difference between two groups was statistical significant (χ2 ＝7.533,P ＝0.006).For genotype C, 5 single point mutations ( T5A, A45T, T47A／K, Q101R and I126S／T) were found in low level group and 1 single point mutation (N3S) in high level group.The difference in mutation frequency between two groups were statistical significant (χ2 ＝47.914,P＝0.000).Conclusions Significant mutations in multiple regions and at multiple sites ( including co-mutations) on both sides of the MHR may be one of the causes of low HBsAg expression level in this population .
ABSTRACT
Objective To investigate the molecular characteristics and epidemiological signification of patients with low-level HBsAg. Methods PCR and gene sequencing were used to detect HBV DNA and Tyr-Met-Asp-Asp(YMDD) mutant in 136 serum samples with low-level HBsAg and 44 sernm samples with high-level HBsAg. Genotyping was performed in 47 cases with HBV DNA 10~5 copies/L by concentration method and 37 cases with high-level HBsAg. S gene sequences and serotypes were analyzed in 14 cases with HBV DNA 105 copies/L and 29 cases with high-level HBsAg. S gene sequences were compared with the consensus sequence of Chinese strain by BioEdit software. Results The HBV DNA-positive rate, YMDD mutation rate and HBV DNA load (logarithm) in low-level and high-level HBsAg group were 34.6% (47/136), 0% (0/136), 6.5±1.4 and 84.1% (37/44), 9.1% (4/44), 8.9±1.8, respectively. There was statistically significant differences between two groups (for concentration method,χ~2 = 30.8, P < 0.05; for direct method, χ~2 = 53.5, P < 0.05; for YMDD mutation ratio, P = 0.003, For HBV DNA (log), t = 6.5, P < 0.05). The genotypes in low-level HBsAg group included type B (16/47), type C (5/47) and non-classified ones(26/47). There were significant differences between two groups (χ~2=21.8, P <0.01). The serotypss included adw (7/14), ayw (4/14), adr (2/14) and ayr (1/14). There were significant differences in genotypes (χ~2 = 13.5, P < 0.05) but not in serotypes between two groups (χ~2 = 4.7, P >0.05). S gene sequencing results showed no S gnne variation was detected, but there were 6 single nucleotide polymorphisms in 16 cases, which would not result in the alternation of amino acid. Conclusions Low-replication phenomenon of HBV DNA was present in patients with low-level HBsAg. The major genotyps and serotype was type B and adw/ayw, respectively. Polymorphic variants have been found in the S gene. The existence of low-level HBsAg might be related with its own molecular characteristics resulting in low expression of HBsAg or immune tolerance induced by low-level HBsAg after HBV infection.