深度测序用于家庭内传播HIV-1耐药及准种特征分析

The application of deep sequencing in analyzing drug resistance and characterizing HIV-1 quasispecies in families

目的通过深度测序获得家庭内成员HIV-1准种信息,分析相互之间的传播进化关系及耐药情况。方法收集1对阳性夫妻与其阳性新生儿产时、产后48小时、42天及2岁龄时的血液样本,对母亲产时血浆和新生儿产时干血斑样本进行HIV-1病毒载量检测;提取干血斑中HIV-1 DNA,经巢式PCR扩增HIV-1 gag、pol、env基因区后进行亚型鉴定、耐药分析和Hiseq高通量测序。计算各样本内/样本间基因离散率,选择最大似然法构建准种进化树。结果母婴产时病毒载量分别为5600拷贝/mL和1000拷贝/mL;3人均感染CRF07_BC亚型;新生儿2年后K65R低频耐药株占比较出生时增高;母子间基因离散率低于父子间;pol区进化树显示三者之间为“父-母-子”的传播关系;新生儿4个时期样本内基因离散率在0.47%~0.68%间波动,与基线样本相比基因离散率在0.49%~0.82%间波动;pol、env进化树表明新生儿优势准种是由基线准种进化而来。结论通过深度测序可以明确家庭内成员HIV-1准种传播规律、遗传特征、不同时期的准种动态以及耐药变化,指导后续的cART。

Objective To utilize deep sequencing to investigate HIV-1 quasispecies among family members,and to analyze their transmission dynamics,evolutionary relationships,and drug resistance profiles.Methods Blood samples were collected from a couple and their newborns,all testing positive for HIV-1 at delivery,48 h post-delivery,42 days post-delivery,and at 2 years old.Plasma viral loads were determined in maternal plasma and neonatal dried blood spots.HIV-1 DNA was extracted from dried blood spots,and gag,pol,and env gene regions were amplified by nested PCR for subtype identification,drug resistance analysis,and HiSeq high-throughput sequencing.Genetic dispersion rate within and between samples were calculated,and phylogenetic trees of quasispecies were constructed using the maximum likelihood method.Results Maternal and infant viral loads were 5600 copies/mL and 1000 copies/mL,respectively.All family members were infected with the CRF07_BC subtype.The proportion of the K65R minority drug-resistant variant in the infant at 2 years old was higher than at birth.The genetic dispersion rate of infected viruses between the mother and infant was smaller than between the father and infant.The phylogenetic tree of the pol region indicated virus transmission in the order of"father-mother-infant".Genetic dispersion rate of infant samples at different timepoints varied between 0.47%to 0.68%,and 0.49%to 0.82%compared to baseline samples.Phylogenetic trees of the pol and env regions showed that dominant quasispecies in the infant evolved from baseline quasispecies.Conclusions Deep sequencing allows for determination of transmission patterns,genetic characteristics,dynamic changes,and drug resistance profiles of HIV-1 quasispecies over time,informing follow-up combination antiretroviral therapy(cART).