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

发布时间：2018-06-04 11:30

本文选题：艾滋病病毒I型 + 抗病毒治疗　；参考：《中国艾滋病性病》2017年07期

【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。
[Abstract]:Objective to establish a method of Illumina Hiseq deep sequencing (Hiseq sequencing) for three gene regions of HIV-1, and to analyze the changes of HIV-1 quasi population in patients with acute HIV infection treated with antiviral therapy. Methods two patients with HIV-1 were followed up for 96 weeks. DNA was extracted from peripheral blood mononuclear cells (PBMC) and the HIV-1 gene subtypes were analyzed in baseline blood samples. The genomic DNA fragments of gagapol and env were used as the target fragments. The baseline and follow-up samples were detected by nested polymerase chain reaction (NPCR). The amplified products were confirmed by a generation of sequencing and then purified. The DNA library was constructed and sequenced by Hiseq sequencing. The distribution of HIV-1 quasi population was analyzed, gene dispersion rate was calculated, and phylogenetic tree analysis was done. Results after antiviral therapy, the HIV-1 viral load in two infected patients was decreased, and the number of CD4 T lymphocytes could not be detected after 12 weeks of treatment. The number of CD 4 T lymphocytes was decreased at the 2nd week of treatment, and then rose to CRF01_AE subtype and CRF07_BC subtype in 2 cases of HIV-1 infection. All the successful nucleic acid samples were sequenced by Hiseq. At baseline, the frequency of the first quasi population sequence with the highest frequency is more than 50 percent of the total frequency of the whole sequence. After antiviral therapy, the distribution of quasipopulations changed in different degrees, in which the proportion of the first quasi population frequency decreased first, increased from the 12th week, and then decreased at the 72nd week. The average gene dispersion rate in the sample was stable and fluctuated in a small range. The average gene dispersion rate between the time points and baseline points in 3 gene regions of each sample increased at the second week of treatment and then remained stable. Phylogenetic tree shows that the quasi-population sequences are distributed and close to each other at different times. Conclusion the Hiseq sequencing method for HIV-1 gagapol and env gene regions can be used to analyze the variation of HIV-1 quasi population effectively. Early antiviral therapy can reduce the viral load regulation and prolong the time of quasi population dispersion.
【作者单位】：中国疾病预防控制中心性病艾滋病预防控制中心;首都医科大学附属北京佑安医院感染中心;
【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)~~
【分类号】：R512.91

【参考文献】