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目的 探索江苏省和浙江省部分地区儿童乙型肝炎病毒(HBV)携带者的病毒分子流行病学特征.方法 采用酶免疫法(EIA)和Abbott公司微粒子酶免疫(MEIA)法筛选乙型肝炎表面抗原(HBsAg)阳性儿童;采崩荧光定量PCR法检测HBV DNA载量;采用巢式-聚合酶链反应扩增乙型肝炎病毒表面抗原基因(HBV S基因),采用DNASTAR软件进行序列分析,用VMD1.8.6软件展示HBV表面蛋白的立体结构,用SPSS 12.0软件进行数据处理.结果 共筛选出64例HBsAg阳性患儿,扩增后获得41株HBV S基因全序列.64例HBsAg阳性患儿的血清HBV DNA平均载量为(4.15±0.79)×10~7拷贝/mL.41株HBV S基因全序列中,C基因型34株(82.93%),B基因型5株(12.19%),B+C型2株(4.88%);血清型主要为adr型(34/39,87.18%)、adw型(4/39,10.24%)和ayr型(1/39,2.56%),2株未分型.HBV表面抗原"a"决定簇常见的变异位点是第129位点的Q→F(谷氨酰胺→苯丙氨酸)、第145位点的G→R(甘氨酸→精氨酸)、第131位点的S→N(丝氨酸→天冬酰胺)和第144位点的C→A(半胱氨酸→丙氨酸),变异频率分别为12.20%(5/41)、4.88%(2/41)、2.27%(1/41)和2.27%(1/41),总变异频率为21.95%(9/41).常见变异株第129位点"Q→F"和第145位点"G→R"S蛋白的"a"抗原决定簇空间结构与野生株完全不同.结论 江苏省和浙江省部分地区儿童HBV携带者体内的HBV呈高复制状态.感染以C/adr型的野生株为主,目前的乙型肝炎疫苗仍然有效.

Objective To investigate the molecular epidemiology of hepatitis B virus(HBV) in child carriers. Methods Blood samples were collected from children under 15 in Jiangsu and Zhejiang provinces. Enzyme immunoassay(EIA) and microparticle enzyme immunoassay(MEIA) were applied to screen hepatitis B surface antigen(HBsAg) positive children. Nested-PCR and real time PCR were used to amplify the HBV S gene and detected HBV DNA loads. S gene sequence and three-dimensional structure were analyzed by the DNASTAR and VMD1.8.6, respectively. SPSS 12.0 software was applied for data processing. Results A total of 64 HBsAg-positive cases were found in the screened children, from which 41 HBV S gene sequences were obtained. The average HBV DNA loads were(4.15±0.79)×10~7 copies/mL in 64 HBV carriers. Among 41 sequences. genotype C, B and B+C accounted for 82.93%(34/41), 12.19%(5/41)and 4.88%(2/41), respectively; and the serotypes were adr(34/39,87.18%), adw(4/39,10.24%) and ayr (1/39, 2.56%) with 2 strains unable to be sub-typed. The most common variants of "a" determinant in HBV S gene were 129 site Q→F(glutamine→phenylalanine), 145 site G→R(glycine→lysine), 131 site S→N(serine→asparagine)and 144 site C→A(cysteine→alanine), and the mutation frequencies were 12.20%(5/41), 4.88%(2/41), 2.27%(1/41)and 2.27%(1/41), respectively . The total mutation frequency was 21.95%(9/41). The S protein spatial structures of 129 site "Q→F" and 145 site "G→R" were entirely different from that of the wild strain. Conclusion Wild strain of HBV(C/adr) is predominant in the children HBV carriers, exhibiting a high replication, and the HBV vaccine should be still effective.