Detection And Sequence Analysis Of HBV DNA From Immuned Childern And Their Mothers

Objective:Investigating the possible reason account for the phemonenon that HBsAg negative cases with HBV DNA positive and establishing the standard method to evaluate the effect of the interruption for mother to children transmission. To describe the distribution of the amino acid substitutions in the successful and unsuccessful interruption cases.To find the differences of amino acid in every site of HBV S and C gene fragment between successfully interrupted group and unsuccessful group.Methods:The blood samples were collected from three cities,including Beijing,Lanzhou and Ningbo,which were used for the research of interrupting mother to children transmission of HBV.All serological indexes of the samples were detected by EIA.The chidren who are HBsAg positive and anti-HBs negative were considered interrupted unsuccessfully and their mothers were unsuccessful mother.HBV DNA was extracted by QIAamp DNA Blood Mini Kit.The S gene fragment was amplified by nested PCR and the C gene fragment was amplified by half nested PCR.Both of the fragments were purificated and sequenced.Choosing the reference sequences from GenBank which were included 8 genotypes.All of the S gene fragments were aligned with the reference sequences to distinguish the genotype used the soft of DNASTAR.All of the amino acid sites of S gene and C gene fragments were analyzed by the soft of DNA STAR and statistical soft of SAS9.0.Results:1,273 cases of successfully interrupted children's mothers and 72 unsuccessfully children's mothers.HBV DNA detected positive were 123 and 60 respectively,and the positive rate were 45.05%and 83.33%;the successfully and unsuccessfully interrupted children were 149 and 79 respectively.HBV DNA detected positive were 18 and 69 of the two groups,and the positive rate were 12.08%and 87.34%.2,221 samples of S gene fragment were analyzed.The genotypes of 72 unsuccessfully interrupted children were A,B,C,D.The successfully interrupted children's mothers were 87 and the unsuccessfully were 62. The genotypes of them were B,C and D.No differences between the genotype B and C mothers in the effect of interruption mother to children transmission,P=0.8682.3,179 samples of the S gene fragments were sequenced.63 samples of children,14 samples successfully and 49 samples unsuccessfully.116 samples of mothers,69 samples successfully and 47 samples unsuccessfully.138 samples of the C gene fragment were sequenced.63 samples of children,10 samples successfully and 32 samples unsuccessfully.96 samples of mothers,62 samples successfully and 34 samples unsuccessfully.The analysis of the amino acid of S gene fragment showed the substitution rate were 60.3%(38/63) in children,72.5%(50/69) in successfully interrupted children's mothers and 74.5%(35/47) in unsuccessfully children's mothers.The data shows no differences among them.4,The analysis of the single amino acid in successfully and unsuccessfully children shows the important sites were 27^th,38^th,65^th,87^th,88^th,114^th site of pre S gene fragment,46^th,122^nd,133^rd, 159^th site of S gene fragment,15^th,30^th,33^rd,86^th,109^th site of C gene fragment;In the mothers,the meaningful amino acid site were 35th,39th,45th,48th,51st,54th,57th, 115^th,121^st,138^th,148^th,152^nd,154^th of pre S,5^th,8^th,24^th,56^th,57^th,59^th,64^th,85^th, 143^rd,161^st of S gene fragment,61^st,80^th site of C gene fragment.Conclusions:Some children with HBsAg negative and HBV DNA positive indicates that serological index couldn't completely reflect the infected condition. HBV DNA detection should be included;No differences between the mothers infected by HBV of genotype B and infected by HBV of genotype C.Some of the amino acid substitution of HBV pre S and S gene fragment may have relationship with the effect of mother to child transmission.