Wu Shouli, Yan Pingping, Yan Yansheng, Qiu Lijun, Xie Meirong
Fujian Provincial Center for Disease Control and Prevention, No. 76 Jintai Road, Fuzhou, Fujian Province, 350001, China.
Arch Virol. 2015 Jun;160(6):1385-95. doi: 10.1007/s00705-015-2386-2. Epub 2015 Mar 22.
HIV/AIDS is a leading public health concern throughout the world. Currently, treatment of HIV/AIDS still depends on highly active antiretroviral therapy (HAART); however, there is increasing evidence showing the emergence of resistance to antiretroviral drugs in HIV-1 strains, making ART less effective over time. Intensive monitoring of HIV-1 drug resistance is therefore of great importance to evaluate the current sensitivity of antiretroviral agents and is urgently needed. The aim of this study was to develop a single-loop recombinant pseudotyped-virus-based assay to detect phenotypic resistance in clinical HIV-1 strains. HIV-1 RNA was extracted from HIV-1-infected human plasma samples, and an approximately 3-kb fragment containing p7/p1/p6 cleavage sites and full-length protease (PR), reverse transcriptase (RT), thermonuclease (TNase), and integrase (1-280 aa) genes was amplified by nested RT-PCR. A retroviral vector was constructed using the HIV-1 infectious molecular clone pLWJ to test antiretroviral drug susceptibility. pLWJ-SV40-Luc contained a luciferase expression cassette inserted within a deleted region of the envelope (env) gene as an indicator gene. Resistance test vectors (RTVs) were constructed by incorporating amplified target genes into pLWJ-SV40-Luc by using ApaI or AgeI and AarI restriction sites and conventional cloning methods. The virus stocks used for drug susceptibility test were produced by co-transfecting 293T cells with RTVs and a plasmid that provided vesicular stomatitis virus glycoprotein (VSV-G). Viral replication was monitored by measuring luciferase activity in infected target cells at approximately 48 h postinfection. A total of 35 clinical plasma samples from HIV-1-infected humans were tested, and target fragments were successfully amplified from 34 samples (97.1 %) and 33 RTVs were successfully constructed by directional cloning, with an overall success rate of 94.3 %. A clear-cut dose-dependent relationship was detected between virus production and luciferase activity in the constructed phenotypic resistance testing system. The highest coefficient of determination (R(2)) was found between luciferase activity and drug concentration and viral inhibition at 293T cell concentrations of 5 × 10(4) cells per well. The phenotypic profiles of the viruses from 29 clinical samples almost completely matched the observed genotypes. The results demonstrate that a single-loop recombinant pseudotyped-virus-based assay was successfully developed, and this testing system has high stability and appears to be applicable for testing phenotypic resistance of clinical HIV-1 strains to commonly used antiretroviral agents.
人类免疫缺陷病毒/获得性免疫综合征（HIV/AIDS）是全球主要的公共卫生问题。目前，HIV/AIDS的治疗仍依赖于高效抗逆转录病毒疗法（HAART）；然而，越来越多的证据表明，HIV-1毒株对抗逆转录病毒药物产生了耐药性，使得抗逆转录病毒疗法随着时间的推移效果越来越差。因此，对HIV-1耐药性进行强化监测对于评估抗逆转录病毒药物目前的敏感性至关重要，且迫切需要。本研究的目的是开发一种基于单环重组假型病毒的检测方法，以检测临床HIV-1毒株的表型耐药性。从感染HIV-1的人类血浆样本中提取HIV-1 RNA，通过巢式逆转录聚合酶链反应（RT-PCR）扩增出一个约3 kb的片段，该片段包含p7/p1/p6切割位点以及全长蛋白酶（PR）、逆转录酶（RT）、核酸酶（TNase）和整合酶（1 - 280个氨基酸）基因。使用HIV-1感染性分子克隆pLWJ构建逆转录病毒载体，以测试抗逆转录病毒药物的敏感性。pLWJ-SV40-Luc包含一个插入包膜（env）基因缺失区域内的荧光素酶表达盒作为指示基因。通过使用ApaI或AgeI以及AarI限制性酶切位点和常规克隆方法，将扩增的靶基因整合到pLWJ-SV40-Luc中，构建耐药性测试载体（RTV）。用于药物敏感性测试的病毒储备液是通过将RTV与提供水疱性口炎病毒糖蛋白（VSV-G）的质粒共转染293T细胞产生的。在感染后约48小时，通过测量感染靶细胞中的荧光素酶活性来监测病毒复制。共检测了35份来自感染HIV-1的人类的临床血浆样本，34份样本（97.1%）成功扩增出靶片段，通过定向克隆成功构建了33个RTV，总体成功率为94.3%。在构建的表型耐药性测试系统中，检测到病毒产生与荧光素酶活性之间存在明确的剂量依赖关系。在每孔5×10⁴个细胞的浓度下，293T细胞中荧光素酶活性与药物浓度和病毒抑制之间的决定系数（R²）最高。来自29份临床样本的病毒表型谱几乎与观察到的基因型完全匹配。结果表明，成功开发了一种基于单环重组假型病毒的检测方法，该测试系统具有高稳定性，似乎适用于检测临床HIV-1毒株对常用抗逆转录病毒药物的表型耐药性。
