HIV-1 CRF01-AE、CRF07/08-BC和B′构造病毒株体外适应性比较

Comparison of fitness of HIV-1 CRF01-AE,CRF07/08-BC and B′ subtype strains using chimeric provirus in vitro

目的构建NL4-3env-EGFP/DsRed2前病毒,观察中国主要流行的HIV-1 CRF01-AE?CRF07/08-BC和B′亚型构造病毒株体外适应性。方法 5份HIV-1B′,CRF07/08-BC,CRF01-AE亚型感染的干血斑样本和1份Zambia C亚型PBMC DNA样本,经单基因组扩增技术获得env全长基因,克隆、亚克隆、转染等步骤构建14个NL4-3env-EGFP/DsRed2前病毒,按顺序进行配对竞争实验,流式细胞仪定量检测被感染细胞的数量。结果 CRF01-AE亚型前病毒适应性最强,适应性值W>1,其次为B′,Zambia C,CRF07-BC/CRF08-BC,P<0.05。结论可利用NL4-3env-EGFP/DsRed2构造前病毒,通过竞争实验比较流行病毒株的体外适应性,推测env基因对适应性的影响。

Objective To construct NL4-3 env-EGFP/DsRed2 chimeric provirus and observe the fitness of HIV-1 CRF01-AE,CRF07/08-BC and B′ subtype strains in vitro.Methods Gene env was amplified from 5 dried blood samples（DBS） which were infected by Thai-B′,CRF07/08-BC,CRF01-AE subtype strains and 1 peripheral blood mononuclear cells（PBMC） sample which was infected by Zambia C subtype strain.Then 14 chimeric proviruses,NL4-3 env-EGFP/DsRed2 were constructed.Pair-wised competition assays were carried out to compare the fitness among all chimeric proviruses.Flow cytometry was used to identify the quantity of infected cells.Results Chimeric provirus from CRF01-AE subtype with relative fitness value（W） more than 1 showed greatest replicative fitness,followed by Thai-B,Zambia C,CRF07-BC/CRF08-BC（paired t-test,P0.05）.Conclusion NL4-3 env-EGFP/DsRed2 can be used to compare the fitness of prevalent HIV strains in vitro by competition assay,and infer the impact of env gene on the HIV fitness.