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首页> 外文期刊>Journal of Clinical Microbiology >Novel Recombinant Virus Assay for Measuring Susceptibility of Human Immunodeficiency Virus Type 1 Group M Subtypes To Clinically Approved Drugs
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Novel Recombinant Virus Assay for Measuring Susceptibility of Human Immunodeficiency Virus Type 1 Group M Subtypes To Clinically Approved Drugs

机译：用于测量人类免疫缺陷病毒1型M组亚型对临床批准药物的敏感性的新型重组病毒分析

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Combination therapy can successfully suppress human immunodeficiency virus (HIV) replication in patients but selects for drug resistance, requiring subsequent resistance-guided therapeutic changes. This report describes the development and validation of a novel assay that offers a uniform method to measure susceptibility to all clinically approved HIV type 1 (HIV-1) drugs targeting reverse transcriptase (RT), protease (PR), integrase (IN), and viral entry. It is an assay in which the antiviral effect on infection within a single replication cycle is measured in triply transfected U87.CD4.CXCR4.CCR5 cells, based on homologous recombination between patient-derived amplicons and molecular proviral clones tagged with the enhanced green fluorescent protein (EGFP) reporter gene and from which certain viral genomic regions are removed. The deletions stretch from p17 codon 7 to PR codon 98 in pNL4.3-ΔgagPR-EGFP, from PR codons 1 to 99 in pNL4.3-ΔPR-EGFP, from RT codons 1 to 560 in pNL4.3-ΔRT-EGFP, from IN codons 1 to 288 in pNL4.3-ΔIN-EGFP, and from gp120 codon 34 to gp41 codon 237 in pNL4.3-Δenv-EGFP. The optimized experimental conditions enable the investigation of patient samples regardless of viral subtype or coreceptor use. The extraction and amplification success rate for a set of clinical samples belonging to a broad range of HIV-1 group M genetic forms (A-J, CRF01-03, CRF05, and CRF12-13) and displaying a viral load range of 200 to >500,000 RNA copies/ml was 97%. The drug susceptibility measurements, based on discrimination between infected and noninfected cells on a single-cell level by flow cytometry, were reproducible, with coefficients of variation for resistance ranging from 7% to 31%, and were consistent with scientific literature in terms of magnitude and specificity.
机译：联合疗法可成功抑制患者体内的人类免疫缺陷病毒（HIV）复制，但选择耐药性，需要随后进行耐药性指导的治疗性改变。本报告介绍了一种新型测定方法的开发和验证，该测定方法提供了一种统一的方法来测量对所有临床批准的靶向逆转录酶（RT），蛋白酶（PR），整合酶（IN）和病毒进入。它是一种分析，其中基于患者衍生的扩增子与标记有增强的绿色荧光蛋白的分子前病毒克隆之间的同源重组，在三倍转染的U87.CD4.CXCR4.CCR5细胞中测量了单个复制周期内对感染的抗病毒作用（EGFP）报告基因，并从中删除了某些病毒基因组区域。缺失从pNL4.3-ΔgagPR-EGFP中的p17密码子7延伸到PR密码子98，从pNL4.3-ΔPR-EGFP中的PR密码子延伸到1至99，从pNL4.3-ΔRT-EGFP中的RT密码子延伸到1至560， pNL4.3-ΔIN-EGFP中的IN密码子1至288，和pNL4.3-Δenv-EGFP中的gp120密码子34至gp41密码子237。优化的实验条件使得无论病毒亚型或共受体使用情况如何，都可以对患者样品进行调查。一组广泛的HIV-1 M组遗传形式（AJ，CRF01-03，CRF05和CRF12-13）的临床样品的提取和扩增成功率，其病毒载量范围为200至> 500,000 RNA拷贝/毫升为97％。基于流式细胞术在单细胞水平上区分感染细胞和未感染细胞的药物敏感性测定，具有可重复性，耐药性的变异系数范围为7％至31％，并且与科学文献的数量级一致和特异性。

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