| AIMHepatitis B (HB) is one of the most severe infectious diseases, which remains a serious global public health problem. In China, one of the major reasons for the high prevalence of hepatitis B virus (HBV) infection is that mothers transmit the HBV to their neonates during the intrauterine period. It is estimated that HBV infection occurs in approximately 5% to 15% of neonates born to mothers positive for hepatitis B surface antigen (HBsAg), which is an important cause for a large number of persistent HBsAg positive carriers in areas with a high HBV prevalence, too. At present, studies have shown that some factors including environmental factors, viral factors, host immune factors and genetic factors may be associated with HBV infection. Based on our previous studies on risk factors and mechanisms associated with HBV intrauterine infection, in the present study, HBsAg positive mothers with their neonates infected with HBV via intrauterine transmission were recruited as cases, and HBsAg positive mothers with their neonates, who had no HBV transmission, were selected as controls. The possible association between HLA-DRB1 polymorphism and disease susceptibility as well as resistance was investigated by comparing the frequencies of HLA genotypes between two groups of neonates. And the possible association between the HBV genomic heterogeneity and intrauterine infection were investigated by comparing the quasi species isolated from two groups of hosts. These investigations might be helpful to comprehensively understand the detailed mechanisms of HBV intrauterine infection and provide powerful support for the valid preventive measures.METHODS1. Subjects of this study had been recruited consecutively for our previous retrospective studies carried out in the Maternal and Neonatal Health Hospital of ShaanXi Province and the Infectious Disease Hospital of Taiyuan, Shanxi Province. HBV intrauterine infection was defined as the situation where the neonate was positive of HBsAg in serum specimens within 24 hours after birth and before the immunoprophylaxis, whose mother was HBsAg carrier but father was not. Twelve neonates (Shaanxi) as well as eleven neonates (Shanxi) who met this criterion with their mothers were classified into intrauterine infection group. Twenty-four (Shaanxi) and twenty-two (Shanxi) neonates who were not infected with HBV were collected from the same cohort and classified as controls (control group). Each case was compared with two controls based on factors which were associated with the pregnant women as well as the neonates. These factors included the HBV marker levels in the maternal serum after delivery immediately, similar age and same gender of the neonates.2. HLA-DRB1 alleles of neonates were typed using sequence specific prime-polymerase chain reaction technique (SSP-PCR). The frequencies of HLA-DRB1 alleles were calculated by direct counting and compared between two groups by the Fisher's exact test.3. In the further study, eight HBV infected neonates with HBV-DNA positive and their mothers from Shanxi province were collected, as well as eight mothers from control group based on same factors. Quasi species of HBV were isolated from the maternal and neonatal serum specimens by using high fidelity PCR and cloning-sequencing techniques.4. Based on the reference sequences of genotypes A to H, the genotypes and the serotypes of clones were identified by analysis of the viral sequences using MegAlign software. And the difference in the proportion of clones with specific mutation was analyzed among the three groups by Fisher's exact test.5. For the phylogenetic analysis, sixteen reference strains of genotypes A to F, which were reported to be representative of human HBV genotypes, were retrieved from Genbank database. All clone sequences with reference sequences were screened for recombination by the recombination detection methods with default parameters implemented in RDP3beta software. After the clones with recombination, deletion and premature stop codons having been excluded, viral sequences were aligned with reference strains by using the ClusatlX1.81 program and edited by MEGA3.1 software package. Phylogenetic tree was reconstructed by the neighbor-joining method (NJ method) using the programs in the Phylip software package. The selective pressure was analyzed by codon-based maximum likelihood method (CODEML) included in the PAML4 software package.RESULTS1. Thirteen alleles of HLA-DRB1 were identified in this study. There were no significant differences of allele frequencies between two groups. While the mothers were HBsAg carriers with HBeAg negative, the allele frequencies of DRB1*08 and 09 were higher in infection group (21.43% and 35.71%) than control group (3.57% and 7.14%). Especially, there was a significant difference of DRB1*09 frequency between two groups (P=0.031, OR=7.22, 95%CI 1.19-43.98). It was also observed that the allele frequencies of DRB1*08 and 09 in infection group of HBeAg-negative neonates (22.22% and 27.78%) were higher than those in infection group of HBeAg-positive neonates, and frequency of DRB1*09 was significantly different (P=0.028, OR=10.39, 95%CI 1.10-98.20). The allele frequencies of DRB1*08 and 09 in infection group of HBeAg-negative neonates were also significantly higher than those in non-infection group (P=0.038 and 0.026, OR=4.97 and 4.67, 95%CI 1.19-20.79 and 1.29-16.93).2. A total of 89 HBV clones were obtained from 24 hosts. The 2.5 kb nucleotide sequences of all clones were most similar to the genome of genotype C (reference strain M12906) with the mean similarity rate of 97.88%, which was highest among those of genotypes A to H (P<0.001). This result indicated that all clones belonged to genotype C. All clones belonged to serotype adr because their aa122 of HBsAg was K and aa160 was R.3. There were seven single nucleotide substitutions in the core promoter with significant difference in the proportions of mutations among three groups (i.e. C1637G, G1658A, G1719T, A1727G, G1742T, A1762T and G1764A). Except G1742T, the others were located in binding sites of transcription factors with significantly higher proportions in control group than infection group (P<0.05). Thirteen mutations in pre-C/C were detected with significantly different proportions among three groups, but all of them were synonymous and resulted in no amino acid substitutions in HBeAg/HBcAg. Nineteen point-mutations were detected in pre-S/S region with significant difference in the proportions of mutations among three groups, six of which were non-synonymous. Mutations of C3116T and C705T, which were detected in all clones isolated from group C, emerged with lower proportions in infection group. Mutations of C3175T, C96T and T473C were only detected in clones isolated from infection group and emerged more frequently in infected neonates. Except G162A, amino acid residues coded by the other five mutations were located in T and/or B cell epitopes of HBsAg (i.e. A90V and P110S in preS1, P36L in preS2, C107R and A184V in S). Among these point-mutations with statistic significances in the pre-S/S, only T473C and T499A were non-synonymous and resulted in substitutions of residues L461S and Y470N in the polymerase domain, respectively.4. Based on the analysis results, there were no recombination strains in all clones and reference strains. The CODEML analysis of pre-C/C and pre-S reading frames coding HBeAg/HBcAg and pre-S domain showed that there were no significant evidences for positive selection in both regions in this study. But in major S protein and polymerase protein, the selection models of M2, M3 and M8 significantly favored over the neutral models of M0, M1 and M7 (P<0.05). Furtherly, the positively selected sites were detected in the major S protein by M8 model with the Bayes Empirical Bayes (BEB) analysis and mainly distributed in the two major hydrophilic regions withωvalues 4.13 of group C, 3.27 of group M and 2.52 of group N. Compared with the analysis result of group C, more positively selected sites were detected in groups M and N. Particularly, residues 68T and 126I were detected only in group N. The positively selected sites were also identified in P gene reading frame overlapping with S gene among three groups, but showed a different pattern in the S antigenCONCLUSIONS1. While the mothers were HBsAg carriers with HBeAg negative, the allele frequencies of DRB1*08 and 09 were significantly higher in infection group than control group. It was also observed that the allele frequencies of DRB1*08 and 09 in HBeAg-negative neonates of infection group were higher than those in HBeAg-positive neonates of infection group, as well as significantly higher than those in non-infection group. These results indicated that HLA-DRB1*08 and 09 might be risk factors for intrauterine infection while the hosts were HBeAg negative.2. Including A1762T/G1764A, there were six point-mutations located in binding sites of transcription factors with significantly higher proportions in control group than infection group. It indicated a disadvantage for virus with specific mutations, such as A1762T/G1764A, to transmit from the mother to the fetus. It also revealed that conservation of the major functional regions in CP might be propitious to HBV intrauterine infection. To the best of our knowledge, this was the first report involving the definite association between the heterogeneity of CP and HBV intrauterine infection. Our results also indicated the conservation of HBeAg/HBcAg in intrauterine HBV transmission.3. The analysis results suggested that epitope variants of viral surface protein might contribute to viral intrauterine transmission by affecting immunogenicity of HBsAg. C3175T/C96T/T473C were detected only in the infection groups and emerged more frequently in infected neonates, which indicated that these mutations in epitopes might be tightly associated with HBV intrauterine infection with advantage for virus to transmit from the mother to the fetus. Our findings also indicated that two mutations of C3116T and C705T might be disadvantageous to intrauterine HBV transmission, which provided the supplement to our previous studies.4. The result of phylogenetic analysis also indicated that the conservation of HBeAg/HBcAg might be beneficial to HBV maternal-fetal transmission. The positively selected sites of major surface protein were located in hydrophilic regions which suggested that the selective pressure coming from host's immune reaction might act dominantly on the surface protein during the HBV transmission from the mother to the fetus. It also supported the opinion that varieties in immune epitopes of HBsAg might contribute to intrauterine HBV infection. |
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