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首页> 外文期刊>Journal of Clinical Microbiology >In Vitro Selection of Clinically Relevant Bevirimat Resistance Mutations Revealed by “Deep” Sequencing of Serially Passaged, Quasispecies-Containing Recombinant HIV-1
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In Vitro Selection of Clinically Relevant Bevirimat Resistance Mutations Revealed by “Deep” Sequencing of Serially Passaged, Quasispecies-Containing Recombinant HIV-1

机译：体外选择临床相关的抗细菌性耐药突变，通过“深”测序对连续传代的准物种重组HIV-1进行揭示

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Initial in vitro studies of bevirimat resistance failed to observe mutations in the clinically significant QVT motif in SP1 of HIV-1 gag. This study presents a novel screening method involving mixed, clinically derived gag-protease recombinant HIV-1 samples to more accurately mimic the selection of resistance seen in vivo. Bevirimat resistance was investigated via population-based sequencing performed with a large, initially antiretroviral-na?ve cohort before (n = 805) and after (n = 355) standard HIV therapy (without bevirimat). The prevalence of any polymorphism in the motif comprising Q, V, and T was ～6%, 29%, and 12%, respectively, and did not change appreciably over the course of therapy. From these samples, three groups of 10 samples whose bulk sequences were wild type at the QVT motif were used to generate gag-protease recombinant viruses that captured the existing diversity. Groups were mixed and passaged with various bevirimat concentrations for 9 weeks. gag variations were assessed by amplicon-based “deep” sequencing using a GS FLX sequencer (Roche). Unscreened mutations were present in all groups, and a V370A minority not originally detected by bulk sequencing was present in one group. V370A, occurring together with another preexisting, unscreened resistance mutation, was selected in all groups in the presence of a bevirimat concentration above 0.1 μM. For the two groups with V370A levels below consistent detectability by deep sequencing, the initial selection of V370A required 3 to 4 weeks of exposure to a narrow range of bevirimat concentrations, whereas for the group with the V370A minority, selection occurred immediately. This approach provides quasispecies diversity that facilitates the selection of mutations observed in clinical trials and, coupled with deep sequencing, could represent an efficient in vitro screening method for detecting resistance mutations.
机译：Bevirimat耐药性的初步体外研究未能观察到HIV-1 gag SP1中具有临床意义的QVT基序的突变。这项研究提出了一种新颖的筛选方法，该方法涉及临床上混合的 gag-蛋白酶重组HIV-1样品，以更准确地模拟体内 的抗性选择。通过对大规模的，最初抗逆转录病毒初治的队列在（ n = 805）和之后（ n = 355）标准HIV进行的基于人群的测序研究了Bevirimat耐药性治疗（无抗病毒治疗）。在包含Q，V和T的基序中，任何多态性的患病率分别为〜6％，29％和12％，并且在治疗过程中没有明显变化。从这些样本中，使用10个样本的三组，这些样本的QVT基序为野生型，以生成捕获现有多样性的 gag-蛋白酶重组病毒。将各组混合，并以各种浓度的紫杉醇传代9周。使用GS FLX测序仪（Roche），通过基于扩增子的“深度”测序来评估 gag 变异。在所有组中都存在未筛选的突变，并且在一组中最初没有通过批量测序检测到的V370A少数族群也存在。在存在高于0.1μM的bevirimat浓度的情况下，在所有组中选择了V370A与另一个先前存在的未筛选的抗性突变一起出现。对于通过深度测序检测到的V370A水平低于一致可检测性的两组，V370A的初始选择需要在狭窄范围的bevirimat浓度下暴露3至4周，而对于V370A少数的组，则立即进行选择。这种方法提供了准种多样性，有助于选择在临床试验中观察到的突变，并且结合深度测序，可以代表一种有效的体外筛选方法来检测抗性突变。

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