HIV-1临床毒株的分离培养及生物学表型和基因型鉴定

目的从晚期艾滋病（AIDS）病人中分离培养艾滋病病毒Ⅰ型（HIV-1）毒株,对其生物学表型和基因型进行鉴定,了解中国临床HIV-1毒株的生物学特征,以期探讨HIV-1生物学特征与疾病进展的关系。方法收集6例AIDS晚期病人的静脉全血,分离血浆和外周血单个核淋巴细胞（PBMCs）;流式细胞仪检测CD4细胞,bDNA法检测病毒载量;PBMCs共培养法分离HIV-1毒株;终点稀释法滴定分离株的感染力;逆转录-聚合酶链反应（RT-PCR）扩增分离株的env区;以MT-4细胞检测毒株的融合诱导性。结果6例病例中分离出3株能在PBMCs中连续稳定传代的HIV-1毒株,50%组织培养感染剂量（TCID50/ml）分别为6.3×10^3、10.2×10^3、2.51×10^3;3株毒株均为B亚型,均不能引起MT-4细胞病变。结论共分离出3株感染力相对较高、呈慢/高型复制动力学、M-嗜性、非融合诱导性的HIV-1 B亚型临床分离株。

Isolation and cultivation of clinical HIV-1 strains and identification of their biological phenotype and subtype

Objective To isolate and cultivate clinical HIV-1 strains from advanced AIDS patients and identify their biological phenotype and subtype in order to comprehend their biological feature in China and explore the correlation with progression of disease. Methods Venous blood was taken from six advanced AIDS patients, from which plasma and PBMCs were separated. CD4 cell count and viral load were detected by flow cytometer and bDNA respectively. Isolation of HIV-1 strains and their infectivity titer were performed by co-culture of PBMCs and end-point dilution, respectively. Env gene of the isolates was amplified using RT-PCR. Capacity of inducing syncytium of the isolates was tested in MT-4 cells. Results Three strains which could be passaged stably in PBMCs were isolated from the six AIDS patients, and the 50 % tissue culture infectious doses （TCID50/ml） were 6.3 × 10^3 . 10. 2 × 10^3 and 2.51 × 10^3 respectively. All the three strains were B subtype based on env gene and neither of them was able to cause cyto-pathic effect in MT-4 cells. Conclusions Three strains with relatively high infeetivity titer were isolated, which were M-tropic, non-syncytia inducing B-subtype, showing slow/high growth dynamics.