RECOMBINANT VIRUS ASSAY - A RAPID, PHENOTYPIC ASSAY FOR ASSESSMENT OF DRUG SUSCEPTIBILITY OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 ISOLATES
RECOMBINANT VIRUS ASSAY - A RAPID, PHENOTYPIC ASSAY FOR ASSESSMENT OF DRUG SUSCEPTIBILITY OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 ISOLATES
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DOI：
10.1128/aac.38.1.23
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发表时间：
1994-01-01
影响因子：
4.9
通讯作者：
LARDER, BA
中科院分区：
文献类型：
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作者：
KELLAM, P;LARDER, BA
需要对临床人类免疫缺陷病毒1型（HIV-1）分离株进行抗病毒药物敏感性试验，以监测临床试验和抗逆转录病毒药物治疗期间耐药性的发展。第一代表型测定具有许多缺点，尤其是在共培养期间选择不具有代表性的病毒群体。在这里，我们描述了一种快速的表型测定的临床分离株逆转录酶（RT）抑制剂的敏感性评估。该程序称为重组病毒试验，允许通过将PCR衍生的RT编码序列库同源重组至RT缺失的非感染性前病毒克隆pHIV DELTABstEII中来生成活病毒。巢式PCR程序已被优化，以允许从未培养和共培养的感染患者外周血淋巴细胞（PBL）DNA扩增RT池，用于随后的重组病毒的创建。在齐多夫定治疗过程中对两名患者的分析表明，这种方法产生的病毒准确地表现出与原始感染PBL DNA相同的基因型和表型。重组病毒检测可在约3周内进行，无需使用供体PBL，因此代表了用于临床分离株检测的快速、非选择性程序。
Antiviral drug susceptibility assays for clinical human immunodeficiency virus type 1 (HIV-1) isolates are required to monitor the development of drug resistance during clinical trials and antiretroviral drug therapy. First-generation phenotypic assays possess a number of drawbacks, not least the selection of unrepresentative virus populations during cocultivation. Here we describe a rapid phenotypic assay for the assessment of the susceptibility of clinical isolates to reverse transcriptase (RT) inhibitors. This procedure, called the recombinant virus assay, allows the generation of viable virus by homologous recombination of a PCR-derived pool of RT coding sequences into an RT-deleted, noninfectious proviral clone, pHIVDELTABstEII. A nested PCR procedure has been optimized to allow the amplification of an RT pool from both uncultured and cocultured infected patient peripheral blood lymphocyte (PBL) DNA for subsequent use in the creation of recombinant viruses. Analysis of two patients during the course of zidovudine therapy showed that this approach produced viruses which accurately exhibited the same genotype and phenotype as that of the original infected PBL DNA. The recombinant virus assay can be performed in approximately 3 weeks without the use of donor PBLs and therefore represents a rapid, nonselective procedure for the assay of clinical isolates.