深度测序用于分析抗病毒治疗急性期感染者的HIV-1准种群变化  

Next generation sequencing-based analysis of evolution of HIV-1 quasispecies in patients initiating antiretroviral therapy during acute infection

作　　者：冯凯迪 张志敏[1] 张秋月[2] 宋爱心 蒋岩[1] 于凤娇[1] 马仲慧 潘品良[1] 张彤[2] 吴昊[2] 粟斌[2] 邱茂锋[1] 

机构地区：[1]中国疾病预防控制中心性病艾滋病预防控制中心,北京102206 [2]首都医科大学附属北京佑安医院感染中心,北京100069

出　　处：《中国艾滋病性病》2017年第7期582-586,591,共6页Chinese Journal of Aids & STD

基　　金：国家"十二五"科技重大专项(2012ZX10001001-001);中美艾滋病防治合作项目(2016-2017;4.2);首都卫生发展科研专项基金项目(首发2016-1-2182);北京市科技计划资助(D141100000314002;D141100000314005)~~

摘　　要：目的建立针对艾滋病病毒I型(HIV-1)三个基因区片段的Illumina Hiseq深度测序(简称Hiseq测序)方法,并尝试用于分析抗病毒治疗急性期感染者的HIV-1准种群变化规律。方法对2例在急性期启动抗病毒治疗的HIV-1感染者进行随访检测,直至第96周。提取外周血单个核细胞(PBMC)中的脱氧核糖核酸(DNA),对基线血液样本进行HIV-1基因亚型分析。分别以gag、pol和env基因区为目的片段,对基线及随访样本进行巢式聚合酶链反应(PCR),扩增产物经一代测序确认无误后纯化、混合构建DNA文库,进行Hiseq测序。分析HIV-1准种群分布,计算基因离散率,并做系统进化树分析。结果接受抗病毒治疗后,2例感染者体内的HIV-1病毒载量下降,到第12周后无法检出;CD4+T淋巴细胞数在治疗第2、4周时有所下降,随后回升。2例感染的HIV-1毒株分别为CRF01_AE亚型和CRF07_BC亚型。核酸扩增成功的样本全部成功进行了Hiseq测序,每个目的基因区片段都获得了十万级的序列数。在基线点,频数最高的第一个准种群序列的频数占全部序列总频数的比例均超过50%。抗病毒治疗后,准种群的分布发生不同程度的变化,其中频数第一准种群频数所占比例先减小,从第12周起多数增大,第72周再减小。样本内平均基因离散率整体稳定,小范围波动。每个样本3个基因区各随访时间点与基线点之间的平均基因离散率在治疗第2周增大,随后保持稳定。系统进化树显示,不同时间的准种群序列散在分布且距离接近。结论针对HIV-1 gag、pol和env基因区的Hiseq测序方法可有效用于分析HIV-1准种群的变化规律,早期抗病毒治疗可降低病毒载量调定点且延长准种群分散时间。Objective To establish an approach of using the next generation sequencing platform（Hiseq platform）of gag,pol,env mixed DNA library and apply it to study the evolution of HIV-1 quasispecies in patients initiating antiretroviral therapy（ART） during acute infection.Methods Two patients with acute HIV-1 infection on ART were enrolled and followed up until the 96th week of the treatment.Their blood specimens were collected and DNAs were extracted from the peripheral blood mononuclear cells（PBMC）.After HIV-1 subtyping, Hiseq platform was performed to sequence the HIV-1 quasispecies targeted at gag,pol,envregion fragments.After data cleaning,the frequency of quasispecies from each specimen was counted and ranked.Intrapersonal and interpersonal genetic distance and phylogenetic tree were calculated.Results After ART,the viral load（VL）of HIV-1 in the 2 patients declined till the virus was not detectable at 12^（th） week.The number of CD4 Tcells decreased at the 2nd and 4th week then recovered.The subtypes of HIV-1 from 2 patients were CRF01_AE and CRF07_BC,respectively.Hiseq sequencing was successfully conducted and one hundred thousand level cleaned sequences were obtained.At baseline,the frequency of top 1 quasispecies sequence was counted more than 50% of the total.With time,the distribution of quasispecies changed：the frequency of top 1 quasispecies sequence decreased first,and most of them rebounded from the 12^（th） week then decreased at 72^（th） week.The average intrapersonal genetic distance rate between quasispecies was stable and fluctuated only within a narrow range.The average interpersonal genetic distance between the follow-up time and the baseline increased in the first 2 weeks and then remained stable.Phylogenetic tree indicated that the quasispecies sequences were scattered at different times and the distance wass close.Conclusion The approach of using the next generation sequencing of HIV-1 gag,pol,envgene regions has a high practical value in studying the evolution of HIV-1

关 键 词：艾滋病病毒I型 抗病毒治疗 准种 进化 深度测序 

分 类 号：R512.91[医药卫生—内科学] R373.9[医药卫生—临床医学]