Laboratoire de Virologie, CHU Hôtel Dieu Nantes, UNAM, EA4271, Nantes, France.
J Virol Methods. 2012 Oct;185(1):94-100. doi: 10.1016/j.jviromet.2012.06.011. Epub 2012 Jun 21.
The objective was to develop a method of NS3 gene sequencing that allowed simultaneous genotyping and protease inhibitor (PI) resistance profiling of HCV genotypes 1-6. To validate the use of a unique RT-PCR for genotypes 1-6 and evaluate its sensitivity, the NS3 protease region was amplified from 140 plasma samples from patients infected with HCV without previous PI therapy. In parallel, NS5b sequences were obtained. Amplification of NS3 was successful in 139/140 samples (99%). For the 135 samples with both NS5b and NS3 sequencing results, phylogenetic analysis showed concordance of genotypes with a bootstrap >90% for each cluster. PI resistance mutations were analyzed using the Geno2pheno [hcv] v1.0 tool. For the 63 genotype 1 (G1) Nantes clinical strains, 12 (19%) presented a natural resistance mutation. This proportion was higher (p<0.05) than that observed in a sample of 374 G1 reference sequences. This significant difference was observed only in subtype 1b (n=7; 25% against n=19; 8%). In conclusion, this tool allows determination of both HCV genotype and identification of PI-resistance mutations. It can be used to detect pre-existing resistance mutations in NS3 before treatment and follow the emergence of resistant viruses during therapy.
目的是开发一种能够同时对 HCV 基因型 1-6 进行基因分型和蛋白酶抑制剂（PI）耐药性分析的 NS3 基因测序方法。为了验证用于基因型 1-6 的独特 RT-PCR 的使用及其敏感性，从未接受过 PI 治疗的 HCV 感染患者的 140 份血浆样本中扩增 NS3 蛋白酶区域。同时，获得 NS5b 序列。在 140 个样本中的 139 个（99%）成功扩增了 NS3。对于同时具有 NS5b 和 NS3 测序结果的 135 个样本，系统发生分析显示每个聚类的基因型与 bootstrap >90%的一致性。使用 Geno2pheno [hcv] v1.0 工具分析 PI 耐药突变。对于 63 个 G1（Nantes 临床株），有 12 个（19%）存在天然耐药突变。这一比例高于在 374 个 G1 参考序列样本中观察到的比例（p<0.05）。这种显著差异仅在 1b 亚型中观察到（n=7；25%对 n=19；8%）。总之，该工具可用于确定 HCV 基因型并鉴定 PI 耐药性突变。它可用于在治疗前检测 NS3 中预先存在的耐药突变，并在治疗期间检测耐药病毒的出现。
