NS5A Gene Analysis by Next Generation Sequencing in HCV Nosocomial Transmission Clusters of HCV Genotype 1b Infected Patients

background: the aim of the study was to investigate the intra-host variability through next-generation-sequencing (NGS) of the NS5A-gene in nosocomial transmission-clusters observed in two Italian hospitals among hepatitis C virus (HCV)-genotype-1b infected patients. methods: HCV-sequencing was performed by sanger-sequencing (NS3 + NS5A + NS5B) and by NGS (NS5A, MiSeq-Illumina) in 15 HCV-1b infected patients [five acute with onco-hematologic-disease and 10 (4/6 acute/chronic) with beta-thalassemial. Resistance-associated-substitutions (RAS) were analysed by geno2pheno-algorithm. nucleotide-sequence-variability (NSV, at 1%, 2%, 5%, 10% and 15% NGS-cutoffs) and shannon entropy were estimated. phylogenetic analysis was performed by mega6-software and bayesian-analysis. results: phylogenetic analysis showed five transmission-clusters: one involving four HCV-acute onco-hematologic-patients; one involving three HCV-chronic beta-thalassemia-patients and three involving both HCV-acute and chronic beta-thalassemia-patients. the NS5A-RAS Y93H was found in seven patients, distributed differently among chronic/acute patients involved in the same transmission-clusters, independently from the host-genetic IL-28-polymorphism. the intra-host NSV was higher in chronic-patients versus acute-patients, at all cutoffs analyzed (p < 0.05). even though shannon-entropy was higher in chronic-patients, significantly higher values were observed only in chronic beta-thalassemia-patients versus acute beta-thalassemia-patients (p = 0.01). conclusions: In nosocomial HCV transmission-clusters, the intra-host HCV quasispecies divergence in patients with acute-infection was very low in comparison to that in chronic-infection. the NS5A-RAS Y93H was often transmitted and distributed differently within the same transmission-clusters, independently from the IL-28-polymorphism.