© 2020 American Society for Microbiology. All Rights Reserved.Soria, María EugeniaGarcía Crespo, CarlosMartínez González, BrendaVázquez Sirvent, L.Lobo Vega, RebecaÁvila, Ana Isabel deGallego, IsabelChen, QianGarcía Cehic, DamirLlorens Revull, MeritxellBriones, C.Gómez, JordiFerrer Orta, CristinaVerdaguer, NuriaGregori, JosepRodríguez Frías, FranciscoButi, MaríaIgnacio Esteban, JuanDomingo, EstebanQuer, JosepPerales, CeliaUnidad de Excelencia Científica María de Maeztu Centro de Astrobiología del Instituto Nacional de Técnica Aeroespacial y CSIC, MDM-2017-07372021-03-172021-03-172020-12Journal of Clinical Microbiology 58(12): e01985-20 (2020)0095-1137https://jcm.asm.org/content/58/12/e01985-20http://hdl.handle.net/20.500.12666/108Despite the high virological response rates achieved with current directly acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. The identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultradeep sequencing (UDS) methods, for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide resistance-associated substitutions (RAS), from 220 patients who failed therapy. They were present frequently in basal and posttreatment virus of patients who failed different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. They also have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value for treatment response, are discussed.engNext-generation sequencingViral quasispeciesViral fitnessAntiviral AgentsViral diagnosticsTreatment planninginfo:eu-repo/semantics/article10.1128/JCM.01985-201098-660Xhttp://dx.doi.org/10.13039/501100011033http://dx.doi.org/10.13039/100010784http://dx.doi.org/10.13039/501100003329http://dx.doi.org/10.13039/100008054http://dx.doi.org/10.13039/501100004587http://dx.doi.org/10.13039/501100001872info:eu-repo/semantics/restrictedAccess