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首页> 外文期刊>Journal of Clinical Microbiology >Allele-Specific Real-Time PCR System for Detection of Subpopulations of Genotype 1a and 1b Hepatitis C NS5B Y448H Mutant Viruses in Clinical Samples
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Allele-Specific Real-Time PCR System for Detection of Subpopulations of Genotype 1a and 1b Hepatitis C NS5B Y448H Mutant Viruses in Clinical Samples

机译：用于检测临床样本中基因型1a和1b丙型肝炎NS5B Y448H突变病毒亚群的等位基因特异性实时PCR系统

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The Y448H mutation in NS5B has been selected by GS-9190 as well as several benzothiadiazine hepatitis C virus (HCV) polymerase inhibitors in vitro and in vivo. However, the level and the evolution kinetics of this resistance mutation prior to and during treatment are poorly understood. In this study, we developed an allele-specific real-time PCR (AS-PCR) assay capable of detecting Y448H when it was present at a level down to 0.5% within an HCV population of genotype 1a or 1b. No Y448H mutation was detected above the assay cutoff of 0.5% in genotype 1b-infected Con-1 replicons prior to in vitro treatment. However, the proportion of replicons with the Y448H mutation rapidly increased in a dose-dependent manner upon treatment with GS-9190. After 3 days of treatment, 1.2%, 6.8%, and >50% of the replicon population expressed Y448H with the use of GS-9190 at 1, 10, and 20 times its 50% effective concentration, respectively. In addition, plasma from 65 treatment-na?ve HCV-infected patients (42 and 23 with genotype 1a and 1b, respectively) was tested for the presence of Y448H by AS-PCR and population sequencing. As expected, all patient samples were wild type at NS5B Y448 by population sequencing. AS-PCR results were obtained for 62/65 samples tested, with low levels of Y448H ranging from 0.5% to 3.0% detected in 5/62 (8%) treatment-na?ve patient samples. These findings suggest the need for combination therapy with HCV-specific inhibitors to avoid viral rebound of preexisting mutant HCV.
机译：GS-9190和几种苯并噻二嗪丙型肝炎病毒（HCV）聚合酶抑制剂 和 in vivo 已选择了NS5B中的Y448H突变。然而，对这种抗性突变在治疗之前和治疗过程中的水平和进化动力学了解甚少。在这项研究中，我们开发了一种等位基因特异性实时PCR（AS-PCR）分析法，能够检测到在基因型1a或1b的HCV人群中Y448H的水平低至0.5％。在进行体外处理之前，在基因型1b感染的Con-1复制子中，未检测到超过0.5％的分析阈值的Y448H突变。然而，在用GS-9190治疗后，具有Y448H突变的复制子的比例以剂量依赖的方式迅速增加。治疗3天后，分别使用有效浓度50倍，10倍和20倍的GS-9190，分别有1.2％，6.8％和> 50％的复制子群体表达Y448H。另外，通过AS-PCR和群体测序测试了来自65名初治HCV感染患者（分别为42和23个基因型为1a和1b）患者的血浆中是否存在Y448H。如预期的那样，通过群体测序，所有患者样品均为NS5B Y448的野生型。对于测试的62/65样品获得了AS-PCR结果，在5/62（8％）初治患者样品中检测到的低水平Y448H范围为0.5％至3.0％。这些发现表明需要与HCV特异性抑制剂联合治疗，以避免先前存在的突变HCV病毒反弹。

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