Clinical and virological properties of hepatitis C virus genotype 4 infection in patients treated with different direct-acting antiviral agents

Claudia Minosse,1,* Marina Selleri,1,* Emanuela Giombini,1 Barbara Bartolini,1 Maria Rosaria Capobianchi,1 Stefano Cerilli,2 Laura Loiacono,2 Chiara Taibi,2 Gianpiero D’Offizi,2 Fiona McPhee,3 AnnaRosa Garbuglia1 1Department of Pre-clinical Research Epidemiology and Advanced Diagnostics, Laboratory of Virology, National Institute for Infectious Diseases “Lazzaro Spallanzani”– IRCCS, Rome, Italy; 2Clinical Department, Infectious Disease-Hepatology Unit, National Institute for Infectious Diseases, “Lazzaro Spallanzani” – IRCCS, Rome, Italy; 3Bristol-Myers Squibb Research and Development, Wallingford, CT, USA *These authors contributed equally to this work Background: 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.Patients and methods: 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.Results: 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.Conclusion: 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. Keywords: hepatitis C virus genotype 4, virological failure, population sequencing, deep sequencing, resistance-associated substitution, RAS, identification