Diagnostic Hybrids, Inc., Cleveland, Ohio, USA.
Antimicrob Agents Chemother. 2011 Aug;55(8):3729-42. doi: 10.1128/AAC.00396-11. Epub 2011 May 31.
Twenty-six antiretroviral drugs (ARVs), targeting five different steps in the life cycle of the human immunodeficiency virus type 1 (HIV-1), have been approved for the treatment of HIV-1 infection. Accordingly, HIV-1 phenotypic assays based on common cloning technology currently employ three, or possibly four, different recombinant viruses. Here, we describe a system to assess HIV-1 resistance to all drugs targeting the three viral enzymes as well as viral assembly using a single patient-derived, chimeric virus. Patient-derived p2-INT (gag-p2/NCp7/p1/p6/pol-PR/RT/IN) products were PCR amplified as a single fragment (3,428 bp) or two overlapping fragments (1,657 bp and 2,002 bp) and then recombined into a vector containing a near-full-length HIV-1 genome with the Saccharomyces cerevisiae uracil biosynthesis gene (URA3) replacing the 3,428 bp p2-INT segment (Dudley et al., Biotechniques 46:458-467, 2009). P2-INT-recombinant viruses were employed in drug susceptibility assays to test the activity of protease (PI), nucleoside/nucleotide reverse transcriptase (NRTI), nonnucleoside reverse transcriptase (NNRTI), and integrase strand-transfer (INSTI) inhibitors. Using a single standardized test (ViralARTS HIV), this new technology permits the rapid and automated quantification of phenotypic resistance for all known and candidate antiretroviral drugs targeting all viral enzymes (PR, RT, including polymerase and RNase H activities, and IN), some of the current and potential assembly inhibitors, and any drug targeting Pol or Gag precursor cleavage sites (relevant for PI and maturation inhibitors) This novel assay may be instrumental (i) in the development and clinical assessment of novel ARV drugs and (ii) to monitor patients failing prior complex treatment regimens.
已有 26 种抗逆转录病毒药物（ARV）获批用于治疗人类免疫缺陷病毒 1 型（HIV-1）感染，这些药物针对 HIV-1 生命周期的五个不同步骤发挥作用。相应地，目前基于常见克隆技术的 HIV-1 表型检测法采用三种，甚至可能是四种不同的重组病毒。在此，我们描述了一种使用单一源自患者的嵌合病毒评估所有针对三种病毒酶以及病毒组装的抗 HIV-1 药物耐药性的系统。将源自患者的 p2-INT（gag-p2/NCp7/p1/p6/pol-PR/RT/IN）产物作为单个片段（3428bp）或两个重叠片段（1657bp 和 2002bp）进行 PCR 扩增，然后重组到含有酿酒酵母尿嘧啶生物合成基因（URA3）的载体中，取代 3428bp 的 p2-INT 片段（Dudley 等人，Biotechniques 46:458-467, 2009）。使用源自 p2-INT 的重组病毒进行药物敏感性检测，以测试蛋白酶（PI）、核苷/核苷酸逆转录酶（NRTI）、非核苷逆转录酶（NNRTI）和整合酶链转移（INSTI）抑制剂的活性。利用单一标准化检测（ViralARTS HIV），这项新技术可以快速、自动地定量检测针对所有已知和候选抗病毒药物（靶向所有病毒酶[PR、RT，包括聚合酶和 RNase H 活性，以及 IN]、一些当前和潜在的组装抑制剂以及任何靶向 Pol 或 Gag 前体切割位点的药物[与 PI 和成熟抑制剂相关]）的表型耐药性。这种新的检测方法可能对以下方面具有重要意义：（i）新型 ARV 药物的开发和临床评估，以及（ii）监测先前复杂治疗方案失败的患者。
