The Analysis Of The Mutations In S Gene Of Hepatitis B Virus Froi Vaccinated Children In Jiangxi Province

Background:With HBV vaccine adapted by programmed immunization from1992, HBVcarriers in population at low age in China are decreased significantly. Meanwhile, theemergence and epidemics of HBV strains with mutations in "a" determinant hasbecome an increasing concern worldwide, but detection of mutations in "a"determinant of HBV infecting children immunized in Jiangxi area have not beenreported.Objective:This study is to provide important evidence for prevention, diagnosis andtreatment of HBV infection in children with the investigation of HBV infection in thevaccinated children who were born after1996. Analysis of the mutations of "a"dominant in S gene of HBV with an aim to search the variation site associated withimmune escape and determination of Genotype and serotype of infected HBV.Methods:1. Collection of clinical samples: Blood samples from children immunized withhepatitis B vaccine were collected in Jiangxi Children's Hospital. HBV-M was testedusing ELISA to screen positive serum with HBV infection.2. Statistical analysis of HBV infeciton: Statitical difference of HBsAg positiverate between different age group and gender group was analyzed using x2test.3. The amplification and sequencing of HBV S gene: High fidelity PCR methodwas used to amplify S gene of HBV DNA extracted from serum and the PCRproducts were sequenced directly.4. Analysis of the variation sites in "a" dominant of HBV: The sequences ofnucleotide and amino acid of HBV S gene were alignned to determine serotype andanalyze the mutation sites in ''a'' dominant of HBV S gene.5. Identification of HBV genotype: Genotypes of infected HBV were determinedvia construction of phylogenetic tree of HBV S gene using Editseq, Clustalx (1.83) and Mega4software or by comparing each sequence of amplified HBV S gene withthe reference sequence with on-line Genotyping software.Results:1. Collection of samples: The reaserchers collected13117blood samples fromchildren (7.39±3.66year) vaccinated with hepatitis B vaccine in Jiangxi Children'sHospital from June2010to May2011.7139samples (54.42%) were collected frommale children and5978samples (45.58%) from female children.2. The analysis of HBsAg infection:230HBsAg positive serum samples werescreened from13117vaccinated children with positive rate of1.75%. HBsAgpositive rate from male children was1.78%and from female children1.72%withoutstatistically significant difference between two gender group (P>0.05). HBsAg inthree age groups set by5-year-old were1.80%,1.53%and2.00%respectivelywithout statistical differences among three age groups. No statistical differences forHBsAg positive rate were detected between two gendre group at same age group(P>0.05).3. Detection of mutations in "a" dominant: HBV S gene from118samples out of230samples was successfully amplified and sequenced.24strains with mutations in"a" dominant were detected from118samples with mutation rate of20.34%. Eighttypes of missense mutations: I126T/S,P127T,Q129H,S132T,P142T,S143T andG145A were found in seven sites in "a" determinant region. The mutation ratebetween two loops and both gender groups in HBV "a" dominant are not differentstatistically (P>0.05).4. Determination of serotype: The alignment of amino acid sequence showedthat105cases were belonged to adw2subserotype in adw serotype;7cases belongedto ayw1subtype of ayw serotype.4cases were adrq+subtype of and2were adrq-subtype of adr serotype.5. Determination of genotype: Analysis of phelogenetic tree and comparison ofon-line Genotyping showed that112samples were found to be infected withgenotype B (94.92%) with all cases of subgenotype B2and6samples were found tobe infected with genotype C (5.08%) with4cases of subgenotype C2and2cases ofsubgenotype C3. Genotype distribution in different gender has no difference (P>0.05).There was relationship and no heterogenecity between serotypes andgenotypes.Conclusions:1. Since HBV vaccination was managed by the programmed immunization in1992, the positive rate of HBsAg in children has been decreased obviously in Jiangxiprovince.2. Infectin of HBV strains with mutations in "a" dominant were detected inchildren immunized with HBV vaccines in Jiangxi province, suggesting that themutations in "a" dominant is a possible mechanism to escape the HBV vacineprotection imunity to cause infection of HBV in children.The infection with adominant mutation type of HBV strain was not found.3. Serotype of HBV infected children was mainly serotype adw with existence ofa few strains of serotype ayw and adr.4. Both of genotype B and genotype C of HBV were found in immunizedchildren. Most were subgenotype B2and a few are subgenotype C2and subgenotypeC3. It is concluded that genetic distance of HBV infected children was near in thearea.