The Sequence Analysis Of Complete Genome Of HBV From Vaccinated Children In Jiangxi Province

Background:With the HBV immunization covering whole newborn infants in China, theprevalence of hepatitis B has been effectively controlled，especially among thechildren. But some children with immunization of HBV vaccine are still infectedHBV. HBV infection of children can cause acute and chronic liver diseases, rangingfrom fulminant hepatitis to cirrhosis, and, eventually, hepatocellular carcinoma. Themechanism of HBV infection among immunized children is complex. The variationof HBV genome is one important mechanism. Currently there is less research aboutthe molecular characteristics of HBV infected by immunized children，and mutationanalysis of complete genome of HBV infecting children immunized in Jiangxiprovince have not been reported.Objective:This project will screen HBV positive serum among children immunized withhepatitis B vaccine in Jiangxi province. The significant mutation sites will besearched based on the PCR amplification of complete HBV genome DNA，andsequence analysis of HBV different ORFs aim to provide viral molecular informationfor prevention and treatment of hepatitis B in immunized children.Methods:1. Collection and screening of clinical samples: Blood samples of children withHBV immunization were gathered in Jiangxi Children’s Hospital between January2012to December2012. ELISA kit for detecting the HBV-M was used to screenpositive serum samples with HBV infection.2. The amplification of complete HBV genome DNA： High fidelity PCRmethod with three overlapping primers was used to amplify the whole gene of HBVDNA extracted from sera specimen and the PCR products were sent to sequencing directly.3. Comparison of nucleic acid and amino acid sequences：The sequences ofnucleotide and amino acid of complete HBV genome were divided into4ORFs andaligned with the reference sequence used software Clustal X and Bioedit to analyzethe main and significant mutation sites.4. Construction of HBV phylogenetic tree and identification genotype: DNAstar,Clustal X and Mega4software were used to construct phylogenetic tree of HBV Sgene and determined the genotype of obtained HBV isolates.5. Confirmation of subgenotype and serotype：Clustal X software and Bioeditsoftware were used to calculate the identity of nucleotide of S gene to confirm thesubgenotype. Meanwhile，ALys to Arg switch at amino acids122and160of HBsAgdefnes the d/y and w/r serotype specifcities，according to this we confirmed theserotype.Results:1. Collection of samples:11445blood samples were collected from children(7.83±3.51year) vaccinated with hepatitis B vaccine in Jiangxi Children’s Hospital.6282samples (54.89%) were collected from male children and5163samples(45.11%) from female children.117HBV-M positive serum samples were collected.2. Sequence and analysis of complete HBV genome：Complete HBV genomefrom58samples out of HBsAg positive serum samples was successfully amplifiedand sequenced. Analyze the mutations in HBV different ORFs，we found the aminoacid mutation rate of S-ORF，C-ORF，P-ORF and X-ORF is5.50%（22/400），2.36%（5/212），3.91%（33/843）and12.99%（20/154）respectively. The mutation sitesand rate in the four ORFs as follows：①The PreS region in S-ORF：G2938C→D31H（5/58,8.62%）、A2990C→H48P（11/58,18.97%）、A2999C→N51T（12/58,20.69%）、C3041T→P65L（10/58,17.24%）、C3050T／C3051T→T68I（6/58,10.34%）、G3065T→G73V（5/58,8.62%）、C3097A→L84I（20/58,34.48%）、G3109A→V88M（6/58,10.34%）and A3136G→T97A（11/58,18.97%）；S region（mutations focuson120aa-200aa）：C533A→P127T（5/58,8.62%）、A541C→Q129H（3/58,5.17%）、G588C→G145A（3/58,5.17%）、T753A→F200Y（18/58,31.03%）.②C-ORF： A1814C／T1815C→M1P（8/58,13.79%）、G1896A→W28*（1/58,1.72%）in PreCregion；C1913A→P5T（1/58,1.72%）in C region.③P-ORF：there was no YMDDmutation in RT region，and no mutation related to tolerance in P-ORF.④X-ORF:G1437T/A→G22C/S （14/58,24.14%）、 G1476A→G35R （12/58,20.69%）、G1503A/T/C／T1504G→V44I/F/L/C （31/58,53.45%）、 G1752A／T1753C→V127I/T （14/58,24.14%）、 A1762T→K130M （5/58,8.62%）、G1764A→V131I（5/58,8.62%）、T1800C→C143R（29/58,50%）、C1807G→P145R（29/58,50%）、G1809T／C1810G／A1811T→A146C/G（29/58,50%）、T1815C／G1816A／C1817T→C148R/H （29/58,50%）、 A1819G／C1820A→D149G（19/58,32.76%）.3. Determination of genotype and serotype: Analysis of phelogenetic treeshowed that58samples were found to be infected with genotype B，and calculate theidentity of nucleotide of S gene，we found58strains had highest homologue withgenotype B2with nucleotide identity range form99.0%to99.6%，suggesting allbelonged to be Genotype B2type. The alignment of amino acid sequence showed that58cases were all belonged to adw.Conclusions:1.58complete HBV genome from117HBV-M positive samples weresuccessfully sequenced and analyzed，the significant mutation sites and molecularcharacteristics of the complete genome of HBV infecting children with vaccinationwere preliminarily analyzed in Jiangxi area.2．Some mutations in S-ORF could change the biological properties such asinfectivity、antigenicity and immunogenicity, serving as a possible mechanism toescape the protection immunity and cause persistent infection of HBV in children.3. Some mutations in C-ORF could change the expression、process and secretionof HBeAg and HBcAg and also may down regulate the cellular immune responseagainst HBcAg in host as one of the molecular and immunoregulation mechanismsfor chronic infection of HBV in children.4. The mutation rate of amino acid in X gene is very high. The multiplemutation sites in HBx could transregulate the replication and gene expression of HBV genome as a potential mechanism of chronic infection of HBV in children.5. The child patients in Jiangxi area were mainly infected with subgenotype B2and adw serotype of HBV.