儿童乙型肝炎病毒携带者分子流行病学特征

Molecular epidemiology of hepatitis B virus in child carriers

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.