A Study On HBV Heterogeneity And Its Relationship With Intrauterine Infection

HBV is a worldwide infection which incidence rate is very high in China. Intrauterine infection is a main pathway of HBV, and it can cause many people to be chronic carriers. Because of using of HB vaccine and anti-hepatis B immunoglobulin, the incidence rate of intrauterine infection in newborns has decreased obviously, but there are still 5%-15% children infected in uterus by HBV. The accurate mechanism is still unclear, which is thought being related to the factors of viral structure, mothers and fetus. Our department has researched HBV the intrauterine infection for several years, and proposed the"cellular transfer"pathway which might cause the newborn infected, but the mechanism is still being researched. Su in our group has found that the mutation rate of the HBV preS/S gene in the group whose children were infected in uterus by HBV was lower than that of the control group. In order to explore the mechanism of HBV intrauterine infection, we could use the technique of molecular biology to study the HBV gene structure, then analyzed the mutation of HBV genes with clinical epidemiologic data, and screened the characteristic gene structures which were probably related to intrauterine infection. It might have a theoretical and practical significance for preventing the HBV intrauterine infection. Methods1. The venous blood of HBsAg-positive pregnant women with full-term delivery and their neonates were collected in Shannxi Mother and Child Health Care Hospital from August 2004 to June 2006. According to whether their neonates were infected in uterus or not, the HBsAg-positive mothers were divided into two groups: intrauterine infection group and non-infection group.2. The HBV markers were detected by the ELISA method. HBV DNA was extracted to be the template of PCR from serum by phenol- chloroform extraction.3. The primers were designed, and 2.5 kb HBV gene fragment which included pre-S/S and pre-C/C gene of HBV was amplified by PCR. After purified, the PCR product was cloned into pGM-T vector. The recombinant plasmids were transformed into competent cell of DH5α. The positive colony was identified by Ampicillin, blue/white selection, restriction endonuclease and sequencing.4. The recombinant plasmids with 2.5kb HBV gene were sequenced. With Megalign software, the pre-C/C and pre-S/S gene sequences were compared with HBV standard strains in Genbank, and then phylogenetic trees were constructed.5. The differences of nucleotide sequences and amino acid sequences were compared among mothers and neonates in intrauterine infection group and women in non-infection group. The different distribution of nucleotide mutation and the change of amino acid sequences were analyzed, which might have relationship with the HBV intrauterine infection.Results2.1 12 objects including 2 objects of the intrauterine infected mothers (groupⅠ)and their infants (groupⅡ), 8 objects of the non-infection group (group Ⅲ) were obtained. According to the ELISA results, the HBsAg, HBeAg and anti-HBc of objects were positive. The HBsAg results had no statistically significant difference between groupⅠand groupⅢ(t-test, t=0.667, P=0.518), and the HBeAg also had no statistically significant difference between the two group (t=1.636, P=0.140).2.2 The 2.5Kb DNA fragment was obtained from all the 12 samples by PCR, and 4-6 clones were selected random from the every mother and infant of infection group, and 2-3 clones from non-infection group, which were sequenced. Then the sequences of 41 clones were obtained including 10 clones in groupⅠ, 10 clones in groupⅡ, and 21 clones in groupⅢ.2.3 A phylogenetic tree of HBV strains was constructed from the pre-S/S regions of 41 HBV isolates and standard sequences of A-H genotypes in GenBank. The phylogenetic tree showed that all HBV isolates were closely related to standard sequence of genotype C (M12906). Moreover, it was M12906 that had a highest similarity rates with all isolates, which were 96.5%-98.8%. All the 41 HBV isolates belonged to serotype adr because their aa122 amino acid of HBsAg is K and aa160 is R.2.4 The types of all the mutations in the HBV preS/S gene and preC/C gene were classified, which showed that the substitution was the main type of mutation, insertion and deletion were occured individually.2.5 The nucleotide sequences of mothers and neonates in intrauterine infection group were compared with sequences of HBsAg-positive pregnant women in non-infection group, and the mutation positions that possibly related to HBV intrauterine infection were screened. The results indicated that the frequencies of 19 point mutations had a significant difference among these three groups (Fisher's exact test, P<0.05). These positions were only found in the non-infection group except nt3026, and there were 11 missense mutations, 2 nonsense mutations and the others were silence mutation. The missense mutations K10Q and V60A in the preS1 protein were caused by the mutations of nt2875 and nt3026 respectively. Nt3212 had caused the preS2 protein aa3 W nonsense mutation. Nt287, nt339, nt435, nt531, nt810 had caused S protein T45A, L62P, P94L, T126I, S219F missense mutations. Only T126I mutation was in the"a"determinant. Nt1913, nt2149, nt2159 and nt2189 had caused T5P, D83E, G87S, L97I mutations in the C protein. Nt2444 caused aa182Q nonsense mutation. In the above mutations, nt3212, nt287 and nt435 were in the CTL cell epitope. Nt1913 and nt2149 were in the CD4+Th epitope. Nt2159 and nt2189 were in the B cell epitope.2.6 The mutations in the nt2875 and nt3026 in the preS1 protein might cause that placenta cell could not identify the HBV or HBV failed in infecting the placenta cell. The mutation in the nt3212 in the preS2 might cause HBV low secretion in the non-infection group, which would depress the viral pressure to host cell. The mutations in the S protein and C protein might decrease the immunoreactivity of the strains, which might influence the HBsAg-anti-HBs formation, and then the HBsAg-anti-HBs compounds would not combine the IgG-F receptor or the complement components C3c, so the"cellular transfer"pathway would fail and the baby would not be suffered.Conclusions1. The sequences of the HBV strains between the intrauterine infection group and non-infection group were different. 19 mutation positions which only occurred in the non-infection group except nt3026 might possibly relate to HBV intrauterine infection. It indicated that the strains without the above mutations might have the selective advantage in the intrauterine infection.2. The 13 positions including missense mutations and nonsense mutations were distributed in different area, which suggested that the different area would have the different effect in the infection course. Whether the intrauterine infection occurred or not might relate to change of some positions or some amino acid areas.3. The HBV intrauterine infection might have relationship with the infectious ability of the virus, the secrection of the viral protein and the complete antigenicity and the immunogenicity.