Minosse Claudia, Selleri Marina, Giombini Emanuela, Bartolini Barbara, Capobianchi Maria Rosaria, Cerilli Stefano, Loiacono Laura, Taibi Chiara, D'Offizi Gianpiero, McPhee Fiona, Garbuglia AnnaRosa
Department of Pre-clinical Research Epidemiology and Advanced Diagnostics, Laboratory of Virology, National Institute for Infectious Diseases "Lazzaro Spallanzani" - IRCCS, Rome, Italy.
Clinical Department, Infectious Disease-Hepatology Unit, National Institute for Infectious Diseases, "Lazzaro Spallanzani" - IRCCS, Rome, Italy,
Infect Drug Resist. 2018 Nov 2;11:2117-2127. doi: 10.2147/IDR.S179158. eCollection 2018.
The efficacy of direct-acting antivirals (DAAs) depends on the hepatitis C virus (HCV) genotype 4 (GT4) subtype which are used in the treatment of HCV. We aimed to retrospectively investigate the baseline prevalence of HCV NS5A and NS5B polymorphisms and their impact on virological outcome in GT4-infected patients treated with various DAA regimens.
Available plasma samples from HCV GT4-infected patients treated with different DAA regimens were analyzed at baseline and after treatment failure, where applicable. Sanger sequencing of patient-derived NS5A and NS5B regions was performed on all available samples, while ultradeep pyrosequencing (UDPS) of NS5A and NS5B regions was performed only on samples from treatment failures at different time points.
Sustained virological response (SVR) was achieved by 96% (48/50) of patients. Of 16 patients with baseline NS5A sequence, polymorphisms at amino acid positions associated with drug resistance were detected only at position 58: P58 (69.2%) and T58 (30.8%). Of 21 patients with baseline NS5B sequence, N142S was detected only in the two treatment failures, both with GT4d were treated with sofosbuvir (SOF)-based regimens, suggesting a potential involvement in SOF efficacy. Two patients (patient 1 [Pt1] and patient 2 [Pt2]) relapsed. In Pt1, NS5A-T56I and NS5A-Y93H/S emerged. In Pt2, NS5A-L28F emerged and a novel NS5B resistance-associated substitution (RAS), L204F, representing 1.5% of the viral population at baseline, enriched to 71% and 91.6% during and after treatment failure, respectively. UDPS of NS5B from Pt2 indicated a mixed infection of approximately 1:5, GT1a:GT4d, at baseline and GT4d during failure. Phylogenetic analysis of NS5A sequences indicated no clustering of HCV strains from patients achieving SVR vs patients who relapsed. The mean genetic distance in NS5A sequences was 5.8%, while a lower genetic distance (3.1%) was observed in NS5B sequences.
Results from these analyses confirm the importance of UDPS in the analysis of viral quasispecies variability and the identification of novel RASs potentially associated with DAA treatment failure in HCV GT4-infected patients.
直接抗病毒药物（DAA）的疗效取决于用于治疗丙型肝炎病毒（HCV）的基因4型（GT4）亚型。我们旨在回顾性研究HCV NS5A和NS5B基因多态性的基线患病率及其对接受各种DAA方案治疗的GT4感染患者病毒学结局的影响。
对接受不同DAA方案治疗的HCV GT4感染患者的可用血浆样本在基线时以及适用时在治疗失败后进行分析。对所有可用样本进行患者来源的NS5A和NS5B区域的桑格测序，而仅对不同时间点治疗失败患者的样本进行NS5A和NS5B区域的超深度焦磷酸测序（UDPS）。
96%（48/50）的患者实现了持续病毒学应答（SVR）。在16例具有基线NS5A序列的患者中，仅在第58位检测到与耐药相关的氨基酸位置的多态性：P58（69.2%）和T58（30.8%）。在21例具有基线NS5B序列的患者中，仅在两名治疗失败患者中检测到N142S，这两名患者均为GT4d，接受基于索磷布韦（SOF）的方案治疗，提示其可能影响SOF疗效。两名患者（患者1 [Pt1]和患者2 [Pt2]）复发。在Pt1中，出现了NS5A-T56I和NS5A-Y93H/S。在Pt2中，出现了NS5A-L28F以及一种新的NS5B耐药相关替代（RAS），即L204F，在基线时占病毒群体的1.5%，在治疗失败期间和之后分别富集至71%和91.6%。对Pt2的NS5B进行UDPS表明，基线时存在约1:5的GT1a:GT4d混合感染，失败期间为GT4d。对NS5A序列的系统发育分析表明，实现SVR的患者与复发患者的HCV毒株未聚类。NS5A序列的平均遗传距离为5.8%，而NS5B序列的遗传距离较低（3.1%）。
这些分析结果证实了UDPS在分析病毒准种变异性以及识别可能与HCV GT4感染患者DAA治疗失败相关的新RAS方面的重要性。
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