无偿献血者隐匿性乙型肝炎病毒Pre-S/S区变异的研究

Study on Pre-S/S gene mutation of occult hepatitis B virus from blood donors

目的调查无偿献血人群的隐匿性乙型肝炎病毒感染（OB I）流行率情况与分析Pre-S/S区变异特征。方法使用EIA/NAT方法筛查深圳地区15 338人份无偿献血者血标本,经跟踪检测,获得1例HBV窗口期感染毒株和5例OB I毒株,采用荧光定量聚合酶链反应（QPCR）测定OB I病毒载量,应用巢式聚合酶链反应（Nested-PCR）技术从5例OB I样品中获得3例Pre-S/S基因片段并测定序列,进行基因分型,与对应基因型HBsAg阳性野毒株序列比对。结果深圳市无偿献血者隐匿性乙型肝炎病毒感染（OB I）的流行率为1∶3 068。5例OB I样品其病毒载量为〈1 IU/m l（不能定量）~121.8 IU/m l（中位数为54.6 IU/m l）。3例OB I样本为基因C型,在Pre-S/S蛋白区出现随机变异,OB I样品Pre-S/S区氨基酸置换率显著高于对应基因型野毒株（P〈0.01）,其中在Pre-S/S区的免疫表位区氨基酸置换率亦显著高于野毒株（P〈0.01）,但在高频变异的主要亲水区（MHR）却是高度保守,只有1例OB I出现改变基因亚型的I126S变异。结论乙肝病毒的Pre-S/S蛋白区变异,特别是Pre-S/S蛋白免疫表位区的变异,可能是OB I发生机制之一。

Objective To investigate prevalence of occult HBV infection among blood donors and analyze the mutation of Pre-S/S region.Methods A total of 15 338 blood samples were screened for HbsAg,anti-HCV and HIV antibodies by EIAs and also tested for HBV,HCV and HIV-1 nucleic acids by NAT（nucleic acid test）.Through follow-up tests,5 plasma samples were confirmed as OBI,1 sample was classified as window period infection（WP）.Viral loads of OBIs were quantified by QPCR.Pre-S/S fragment from 5 samples were amplified by nested PCR and sequenced,and 3 Pre-S/S fragmengs were obtained.HBV genotype was determined,amino acid sequences of OBI were compared to the corresponding sequences of HBV strains of similar genotype obtained from the GenBank database to analyze the mutation characterristics.Results Prevalence of occult HBV infection in Shenzhen was 1∶3 068.The viral loads of all 5 OBIs were very low,ranging between un-quantified（1 IU/ml） and 121.8 IU/ml with a median of 54.6 IU/ml.Three OBI samples were genotype C,showing random mutations in Pre-S/S proteins,with more frequent substitution of amino acids in Pre-S/S of OBIc than that of wild type HBV genotype C（P0.01）,but MHR（Major Hydrophilic Region）region was found to be conserved.Only 1 OBI sample showed I126S mutation,presumably related to subtype.Conclusion The relatively high frequency of mutations,especially the mutations of immune epitope in the Pre-S/S region could be one of the main mechanisms of OBI.