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目的对HBsAg阴性和阳性献血者血样HBV DNA存在的确认并分析隐匿性乙型肝炎病毒S区变异特征。方法使用EIA/NAT方法筛查深圳地区19 397份无偿献血者血样,把109例乙肝不合格样品分成3类(HBs Ag+/NAT+、HBs Ag+/NAT-、HBs Ag-/NAT+),通过跟踪检测,确认为OBI毒株10例、HBV窗口期感染期3例和5例缺失追踪的HBs Ag-/HBV DNA+样品,采用荧光定量聚合酶链反应(QPCR)测定HBV病毒载量,应用NestedPCR技术扩增S基因片段并测定序列,与B/C基因型HBs Ag+/HBV DNA+阳性野毒株序列比对。结果深圳市无偿献血者经乙肝表面抗原胶体金快速试纸筛查后的HBs Ag阳性检出率为0.34%(66/19 397);隐匿性乙型肝炎病毒感染(OBI)的流行率范围为1∶1 939-1∶1 293,HBV窗口期感染流行率范围为1∶6 465-1∶2 424;10例OBI样品其病毒载量介于不能定量至112.0 IU/m L(中位数98.5 IU/m L)。10例OBI样本在S蛋白区(nt215-710)出现随机变异,OBI样品S区氨基酸置换率显著高于野毒株(P<0.000 1),有4、2、3个OBI样品分别在CTL表位21-29、86-96、172-180出现L21S(2)、K/R24E(1)、I25M(1)、L88P(2)、S172F/L(2)、V178T(1)变异;OBI非CTL表位免疫区的氨基酸置换率亦显著高于野毒株(P<0.05);其中1个OBI样品在nt636发生缺失变异。结论深圳献血者OBI流行率有增高趋势,OBI发生机制与乙型肝炎病毒的S蛋白区变异,特别是免疫活性区的变异密切相关。
Objective To confirm the presence of HBV DNA in blood samples from HBsAg-negative and positive blood donors and analyze the characteristics of S-zone mutation of occult hepatitis B virus. Methods EIA / NAT was used to screen blood samples from 19 397 blood donors in Shenzhen. 109 unhealthy hepatitis B patients were divided into three groups (HBsAg + / NAT +, HBsAg + / NAT-, HBsAg- / NAT +) by tracing , Confirmed as OBI strain in 10 cases, HBV window infection period in 3 cases and 5 cases missing tracking of HBsAg- / HBV DNA + samples, using quantitative PCR (QPCR) determination of HBV viral load, the application of NestedPCR technology The S gene fragment was amplified and sequenced, and the sequence was aligned with that of HBs Ag + / HBV DNA + positive wild type strain of B / C genotype. Results The positive detection rate of HBsAg in unpaid blood donors in Shenzhen was 0.34% (66/19 397) after HBsAg positive test strips. The prevalence of occult hepatitis B virus (OBI) ranged from 1 : 1 939-1: 1 293, the prevalence of HBV window phase infection ranged from 1: 465-1: 424; 10 cases of OBI samples were unable to quantify the viral load to 112.0 IU / m L (median 98.5 IU / m L). 10 cases of OBI samples showed random variation in the S protein region (nt215-710). The amino acid substitution rate in the S region of OBI samples was significantly higher than that in the wild-type strains (P <0.0001). There were 4, 2 and 3 OBI samples in the CTL table The mutations of L21S (2), K / R24E (1), I25M (1), L88P (2), S172F / L (2) and V178T (1) The amino acid substitution rate of CTL epitope immunized region was also significantly higher than that of wild-type strain (P <0.05). One OBI sample had a deletion mutation at nt636. Conclusion The prevalence of OBI in blood donors in Shenzhen has an increasing trend. The mechanism of OBI is closely related to the variation of S protein region of hepatitis B virus, especially the immunoreactive region.