HIV-1 CRF07_BC病毒适应性的体外生长竞争试验的建立  

Establishment of an in vitro growth competition assay for HIV-1 CRF07_BC viral fitness

作　　者：何林[1] 焦洋[2] 丁奕博 张媛媛 林倩茹 汪立杰 李丹[1] 洪坤学[1] 马丽英[1] He Lin;Jiao Yang;Ding Yibo;Zhang Yuanyuan;Lin Qianru;Wang Lijie;Li Dan;Hong Kunxue;Ma Liying(National Center for AIDS/STD Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Beijing Chaoyang Center for Disease Control and Prevention,Beijing 100021,China)

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]北京市朝阳区疾病预防控制中心,100021

出　　处：《中华实验和临床病毒学杂志》2021年第4期457-462,共6页Chinese Journal of Experimental and Clinical Virology

基　　金：十三五国家科技重大专项(2018ZX10101002-004-003);国家自然科学基金(81871694)。

摘　　要：目的:通过突变的方式构建含有鼠Thy1.1基因或鼠Thy1.2基因的HIV-1 CRF07_BC感染性克隆,建立CRF07_BC亚型体外竞争试验。方法:用鼠Thy1.1基因和鼠Thy1.2基因替换CRF07_BC亚型感染性克隆pXJDC6291-13 Nef区前218个碱基,构建成CRF07_BC感染性克隆BCEA1和BCEA2质粒。BCEA1和BCEA2进行293T细胞转染后获得病毒,测定病毒滴度,以等比例病毒含量共感染PM1细胞,在感染的第3、4、5、6天收集细胞并用Thy1.1和Thy1.2特异性抗体标记细胞,并进行流式细胞检测病毒适应性。通过"TFitness"程序(http://bis.urmc.rochester.edu/vFitness)计算病毒相对适应性。并观察耐药位点K103 N、多态性位点K166R相对于CRF07_BC亚型相对适应性的影响。结果:CRF07_BC亚型BCEA1和BCEA2两病毒的相对适应性(1+s)结果为1.06±0.23693,无统计学差异,建立了CRF07_BC亚型体外生长竞争性试验。利用该体外生长竞争试验验证K103 N和K166R突变株与野生株的相对适应性,BCEA2-K103 N/BCEA1相对适应性是0.728282±0.16608、BCEA2-K166R/BCEA1是0.883385±0.19023、BCEA2-K103 N+K166R/BCEA1是0.804604±0.06164。K103耐药位点突变株与野生株相对适应性存在统计学差异。结论:建立HIV-1 CRF07_BC病毒适应性的体外生长竞争试验,基于该体外竞争试验,HIV-1 K103 N的耐药毒株的病毒适应性降低。Objective To establish an in vitro growth competition assay using HIV-1 CRF07_BC infectious clone containing the mouse Thy1.1 gene or Thy1.2 gene.Methods The mouse Thy1.1 gene and Thy1.2 gene were used to replace the 218 bases of Nef region of the CRF07_BC infectious clone(pXJDC6291-13)and construct the CRF07_BC infectious clones BCEA1 and BCEA2 plasmid.BCEA1 and BCEA2 plasmids were transfected into 293T to obtain the virus and the viral titration was measured.PM1 cells were co-infected with an equal virus.On the 3^(rd) to 6^(th) day of infection the cells were collected and labeled with specific antibodies Thy1.1 and Thy1.2.The viral fitness was detected by flow cytometry.Through"TFitness"(http://bis.urmc.rochester.edu/vFitness)the relative viral fitness was calculated.The influence K103 N and K166R on the relative fitness of HIV-1 CRF07_BC were observed.Results The relative fitness(1+s)of CRF07_BC subtypes BCEA1 and BCEA2 viruses were 1.06±0.23693,which was not statistically significant.An in vitro growth competition assay for CRF07_BC was established.The in vitro growth competition assay was used to verify the relative fitness of the K103 N and K166R mutant strains to the wild strain.The relative fitness of BCEA2-K103 N/BCEA1 was 0.728282±0.16608,BCEA2-K166R/BCEA1 was 0.883385±0.19023,BCEA2-K103 N+K166R/BCEA1 was 0.804604±0.06164.The relative fitness of K103 N resistant mutant strain and the wild strain was significantly different.Conclusions An in vitro growth competition assay for the fitness of HIV-1 CRF07_BC virus was established.Based on the in vitro growth competition assay,the HIV-1 K103 N resistant mutant strain has reduced viral fitness.

关 键 词：HIV-1 CRF07_BC 体外竞争试验 病毒适应性 

分 类 号：R73[医药卫生—肿瘤]