| HBV intrauterine infection was thought being related to the factors of viral structure, placenta barrier and immunization condition of mother and her fetus. HBV intrauterine transmission could be caused through hematogenous transfer and cellular transfer, but its accurate mechanism was still unclear. In order to explore the mechanism of HBV intrauterine infection at gene level, we analyzed the variation of HBV genes, screened the characteristic gene structures which were probably related to intrauterine infection and investigated the relationship between heterogeneity of HBV genes and intrauterine infection, which might provide a theoretical basis for preventing intrauterine infection.METHODS: ①The venous blood of 70 pairs HBsAg-positive pregnant women with full-term delivery and their neonates were collected in Shannxi Mother and Child Health Care Hospital from August 1997 to May 1998 and from August 2001 to January 2002. According to whether their neonates were infected or not, the HBsAg-positive mothers were divided into two groups: intrauterine infection group and non-infection group. In the same time the serum of one HBsAg-positive pregnant woman undergoing induced abortion and the fetal liver were collected. ②The HBV DNA were extracted from serum. We amplified 2.5 kb or 1.2 kb HBV gene fragments of 4 pairs mother and neonate of infection group and that of 5 pregnant women of non-infection group. After purified, the PCR products were cloned into pUC19 vector. After PCR, restriction endonuclease and sequencing, 63 HBV isolates were identified. ③With Megalign software, we analyzed the pre-C/C and pre-S/S gene sequences of all HBV isolates and determined the genotype and serotype. We also constructed phylogenetic trees and compared the difference between each gene region. ④The difference of gene positions of HBV sequences were compared between mothers and neonates with intrauterine infection, then the evolutionary relation of species were determined. ⑤The difference of nucleotide sequences and amino acid sequences were compared between neonates of intrauterine infection group and pregnant women in non-infection group, they were also compared between mothers in intrauterine infection group and pregnant women in non-infection group. ⑥The 2.5kb HBV gene fragments of the pregnant women undergoing induced abortion and the 500 bp gene fragments of HBV gene S region from fetal liver were amplified. One isolate of the mother and one isolate of her fetus were chosen to analyze their homogeneity, mutation positions of them were compared with other isolates.RESULTS: ①The 2.5kb/1.2kb HBV DNA fragments were amplified from sera of neonates. The 2.5kb DNA fragments were obtained from sera of 2 neonates (NA and ND)and 1.2kb DNA fragments were obtained from sera of other 2 neonates (NB and NC). Similarly, 2.5kb DNA fragments were also obtained from sera of their HBsAg-positive mothers (MA-MD). Moreover, 2.5kb HBV fragments were also obtained from sera of 5 HBsAg-positive pregnant women (E~I) whose neonates were not infected. @2A phylogenetic tree of HBV strains was constructed from the pre-S/S regions of 63 HBV isolates and standard sequences of A-F genotypes inGenBank. The phylogenetic tree showed that all HBV isolates were closely related to standard sequence of genotype C (M12906). Moreover, the nucleotide sequence of each isolate was compared with standard sequences of 6 genotypes. It was M12906 that had a highest similarity rate with all isolates, and the mean similarity rate was 97.41%. So we concluded that these 63 HBV isolates belonged to genotype C. According to whether the rate of divergence of nucleotide< 2.0%, these HBV isolates were divided into two clusters: I and II, 36 sequences belonged to cluster I and 27 sequences to cluster II. All the 63 HBV isolates belonged to serotype adr because their aa122 amino acid of HBsAg is K and aa160 is R. ③Comparison of the similarity rates of nucleotide sequences showed a statistically significant difference among 5 gene regions (F= 146.587, P<0.0...
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