The Studies On The Quasispecies Sequence Of PreS/S And The Mutations Of EhnⅡ/CP/PreC In Children Infected HBV Through Mother-to-Infant Transmission And Their Mother

ObjectiveTo investigate that there are quasispecies populations of HBV and that the characteristics of mutations in EnhII/CP/PreC gene of HBV in AsC children infected through mother-to-infant transmission and their AsC mothers with different degree of viremia . Methods1 Establishment of the consensus sequence of PreS/S and Enhll/CP/PreC gene of hepatitis B virus in Chongqing of China: There were 10 AsC children in this study. Mothers of all children were AsC before pregnancy. They were 3~6 years old, and HBV vaccine and anti-HBV medicine were never administrated. The serological markers of hepatitisA,B,C,D and E virus were tested by ELISA. The titers of serum HBV DNA were measured by the quantitative polymerase chain reaction(Q-PCR) method. DNA in serum was extracted with phenol and chloroform. PreS/S and EnhII/CP/PreC genes were amplified by PCR and the PCR products were sequenced directly. Genotype was determined on the basis that the homology of S gene sequence was less than 4% comparing with that of different genotype of HBV in GenBank. Serotype of HBV was determined by special ammo acids in HBsAg codoned by S gene. Under the same genotype and serotype of HBV, the consensus sequence of PreS/S and EnhII/CP/PreC gene were established by dominant nuleotide at every position.2 The study on the quasispecies of HBV in children infected HBV through mother-to-infant transmission and their mothers: There were three pairs of child and mother in this study. Mothers of all children were AsC before pregnancy and the children did not inoculated against HBV after birth. Anti-HBV medicine was never administrated to all subjects. The serological markers of hepatitis A,B,C,D and E virus were tested and the titers of serum HBV DNA were quantitated. PreS/S gene was amplified by PCR and cloned into pGEM-T vector with A-T cloning technique. The recombinant plasmid pGEM-PreS/S was confirmed by digestionwith restriction enzyme Apa I and Sac I . Six clones were selected to be sequenced each patient. Phylogenetic tree was established based on the 36 clones of HBV PreS/S gene. The sequences were analyzed comparing with consensus sequence of Chongqing.3 The study on the mutations in EnhII/CP/PreC gene in children infected HBV through mother-to-infant transmission and their mothers: There were 15 pairs of child and mother in this study. Mothers of all children were AsC before pregnancy and the children did not inoculated against HBV after birth. Anti-HBV medicine was never administrated to all subjects. The serological markers of hepatitis A,B,C,D and E virus were tested and the titers of serum HBV DNA were quantitated. S gene was amplified by PCR and the PCR product was sequenced directly to determine the genotype and serotype of HBV in all patients. EnhII/CP/PreC gene was amplified by PCR and cloned into pGEM-T vector with A-T cloning technique. The recombinant plasmid pGEM-EnhII/CP/PreC was confirmed by digestion with restriction enzyme Apa I and Sac I .Tow clones were selected to be sequenced each patient. The mutations of EnhII/CP/PreC were analyzed comparing with consensus sequence of Chongqing.Results1 Establishment of the consensus sequence of PreS/S and EnhII/CP/PreC gene of hepatitis B virus in Chongqing: All ten AsC children were high viremia with HBsAg HBeAg and anti-HBc positive. HBV strains of 7/10 were genotype B/serotype adw2 and 3/10 were genotype C/serotype adrq+. Comparing with the consensus sequence of PreS/S and EnhII/CP/PreC gene of HBV with those of Southeast of China, there were 6 and 1 nucleotide variants and the homology were 99.5% and 99.6% respectively in HBV with genotype B/serotype adw2,and there were 13 and 2 nucleotide variants and the homology were 98.9% and 99.3% respectively in HBV with genotype C/serotype adrq+. Comparing between the two consensus sequence of PreS/S and EnhII/CP/PreC gene of different subtype HBV in Chongqing, there were 95 and 9 nucleotide variants and the homology were 92.1% and 96.8% respectively.2 The study on the quasispecies...