A Study On The Heterogeneity Of HBV Genes And Its Relationship With Intrauterine Infection

ObjectiveHBV intrauterine infection is associated with HBV itself, maternal and fetal immune status and placental factors. Different HBV strains exist in the body of Patients who have HBV, showing "similar strains "phenomenon, called a heterogeneous group. Domestic scholars tend to explore the mechanism of intrauterine transmission by researching how the HBV viruses break through the placental barrier. But it is not systematic for the research of how a virus encoding the product of the structure of the hepatitis B virus and its mutations affect the intrauterine transmission. Exact mechanisms of HBV intrauterine infection is not very clear, so it is necessary to start from the HBV genome structure and focus on the heterogeneity of groups. A theoretical basis shall be provided for the interruption of HBV intrauterine transmission and possible relationship between HBV genetic heterogeneity and intrauterine infection through use of molecular biology techniques, combined with clinical epidemiological data to analyze of the variability of the HBV genome, and to screen possible specific gene structure related to the intrauterine infection.Methods①Peripheral blood of1622puerpera and their new born babies were collected from the third People’s Hospital of Taiyuan from2001.11to2009.7.22pairs of mothers and babies were left for research by choosing the pregnant women and their babies who have the positive HBsAg, HBVDNA, while other cases were removed because of the loss of serum.8pairs of mothers and babies were selected as the intrauterine infection group where gel electrophoresis bands of HBVDNA is clear when HBVDNA copy number≥105.8mothers are belonged to uninfected group as comparison when mothers are HBVDNA positive and babies are HBV DNA negative in the collected simples.②We pick up the HBV DNA in serum and amplify gene segments of Pre S/S1.2kb and Pre C/C909bp in HBV DNA of8pairs of mothers and infants which is belonged to infected group and8cases mothers in uninfected group, respectively. After purification PCR products cloning into the pUCm-T carrier, and enzymolysis of PCR, we obtain386sequences of HBV Pre S/S zone of6pairs of mothers and babies,3cases of infected mothers and67sequences of Pre C/C zone in HBV of6pairs of mothers and babies,3cases of infected mothers. We analysis the whole82gene sequences of Pre S/S zone and67gene sequences of Pre C/C zone by using Megalin of Lasergene software. We define the genotype and build the phylogenetic tree. At the same time, the differences between every genotype of Pre S/S zone nucleotide sequence and standard sequence are compared.③Compare the differences of gene locus of Pre C/C and Pre S/S region6pairs mothers and infants which have intrauterine HBV infection, compare the level of gene differences and relationship of evolution, compare the similarity of each clone of mothers and infants and define the degree of similarity.④Compare the differences of gene sequences between mothers in infected group and uninfected group, the differences between new born in infected group and mothers in uninfected group.⑤Compare rate of intrauterine infection between different gene styles and judge the relationship between intrauterine infection and gene styles.Results①We obtain1.2kb and909bp DNA segments from serum of6neonates (NA-NF) by using amplification. Simultaneously, we obtain the1.2kb and909bp DNA segments from HBsAg positive serum of6neonates and HBV DNA positive mothers (MA-MF). And we also obtain the1.2kb and909bp DNA segments from serum of3cases HBV DNA negative mothers (I, J, K).②We build the phylogenetic tree by using79sequences of Pre S/S region of HBV and gene subtypes standard sequence of A-F in GeneBank. Simultaneously, we build the phylogenetic tree by using82clone plants and gene subtypes standard sequence of A-F in GeneBank. The evolutionary distances of74clones are close to standard sequences. Expect the17clones of mothers in uninfected group, all nucleotide sequences of Pre S/S region of65clones have the highest similarity with M12906, the value is96.25%. The average level to the A, B, C, D, E, F gene subtypes standard sequences are91.46%、88.45%、87.22%、85.43%, respectively. There are differences between similarity of six gene subtypes (F=98.944,P=0.00). The average similarity between82clones and C gene subtypes standard sequences is96.50%.③We compare the82nucleotide sequences of Pre S/S region and nucleotide sequences of Pre C/C region to the C gene-type standard sequences, and we obtain similarity of every clone, finally we obtain the average similarity rate are96.5%,98.32%, respectively. There is the difference between nucleotide sequences in gene region by using t test analysis and inspection (t=-3.096, p=0.007). The nucleotide similarity of Pre C/C is higher than Pre S/S.④In the HBV Pre S/S region of6pairs of mothers and infants which are infected, the average difference ratios of every neonate are99.83%,99.83%,99.10%,99.51%,99.61%,96.52%, respectively. The average difference ratios of each mother are99.05%,99.04%,99.39%,99.39%,99.47%,94.69%, respectively. The similarity of HBV DNA of neonates is higher than mothers (t=2.119, p=0.088). The similarity of every pair of mother and baby are98.81%,99.29%, 99.28%,99.45%,99.52%,95.61%, respectively. The sequences of mothers and infants have high similarity. We build the evolutionary tree by using nucleotide sequences of every mother and baby in Pre S/S region. It can be seen that the evolutionary distance of each mother-baby sequence is small. There are0.9、2.9、1.2、0.7、0.7、4.9, respectively. It is suggested that the evolutionary distance between mothers and babies is small, genetic relationship is closer. In the sequences of mothers and babies which have intrauterine infections, Advantage strains in mothers’body is not same as in babies’. The advantage strain in MA is not found in NA. The weak strains in ME become the advantage strains in NE, and the advantage strains of MB and MD occur frequently in neonates. And we find that two genes (nt529and nt531) mutated in "a" antigen determinant (nt524-nt595).⑤By intrauterine infection of pregnant women carry a variety of HBV genotypes, the infection rate of pregnant women which is carrying the HBV C intrauterine is64.6%, infection rate of pregnant women which is carrying of HBV B intrauterine is100%, For the intrauterine infection rate of the B and C type statistical test still can not believe the difference between the two (χ2=0.427, p=0.513).⑥By comparing the HBV DNA positive sequences between neonates in intrauterine infection group, mother in intrauterine infection group and uninfected group, we can see the difference rate from the clone rate mothers which has never been infected, the mother in the intrauterine infection group to newborns in intrauterine infection group is increasing, and the difference has statics significant (Fs=26.389,p<0.000; Fc=5.680, p=0.004). The rate of nucleotide differences of each group Pre S/S region Pre C/C gene differences in rates of difference.Conclusion①86clone plants of Pre S/S aera obtained in our experiment. When we define genotype and study the similarity and difference,7plants are eliminated for their bad properties, which may influent the analysized results. Finally, we obtain82plants include74plants of C sub-genotype and8plants of B sub-genotype. It is suggested that pregnant women and newborn who is HBsAg positive and HBV DNA positive mainly carry the HBV C genotype in Taiyuan distinct. The B genotype is less than C. Such a distribution is concident with HBV genotype spatial distribution in our country.②The clone sequences between6babies and their mothers who suffer the intrauterin infection are analyzed phylogeneticly. The similarity, difference and virable site are compared. It is shown that sequences between mother and babies are homologous, and this result support that the direction of HBV spearding is from mother to babies. Maternal HBV heterogeneity groups, both of the advantages and disadvantaged groups can infect newborns through intrauterine infections. Neonatal, monoclonal infections and mixed infections have occurred, and the advantages plants of maternal and neonatal body strain not the same. It is prompted that HBV intrauterine infection may have selectivity.③We analyze the stuiation of3pairs of B sub-genotype and51C sub-genotype pregnant women who suffer from intrauterin infection. And we find that B and C genotype which genotype has the advantage of intrauterine infection in infection process.④The difference of sequences of pre S/S aera and pre C/C aera between mother in intrauterin infection group and uninfected mother is compared. It is shown that difference is increased from uninfected mother to infected mother to infected newborns. The difference is significant (Fs=26.389,p＜0.000; Fc=5.680, p=0.004). The difference of nucleotide of Pre S/S aera in each group is higher than pre C/C aera, the difference of gene have the same characterics. The difference have statistical significance (p<0.01).⑤We compare the sequences between mother and newborns who are in intrauterin infected group, mothers who are in intrauterin infected group and uninfected group, and babies who are in intrauterin infected group and unfected mothers. Variable sites are choosen preliminarily. These mutations are mainly mutated; few of them are loss or insertion. But it can still be seen the mutations rate of pre S/S region is higher than pre C/C area, and found that two genes of "a" antigenic determinant (nt524～nt595) in S region are mutated, which are nt529and nt531.