Supplementary Material for: NS3 Variability in Hepatitis C Virus Genotype 1A Isolates from Liver Tissue and Serum Samples of Treatment-Naïve Patients with Chronic Hepatitis C

<b><i>Background:</i></b> Hepatitis C virus (HCV) NS3 resistance-associated substitutions (RASs) reduce HCV susceptibility to protease inhibitors. Little is known about NS3 RASs in viral isolates from the liver of chronic hepatitis C (CHC) patients infected with HCV genotype-1a (G1a). <b><i>Aim:</i></b> The objective of this work was to study NS3 variability in isolates from the serum and liver of HCV-G1a-infected patients naïve to direct-acting antivirals (DAAs). <b><i>Methods:</i></b> NS3 variability of HCV-G1a isolates from the serum and liver of 11 naïve CHC patients, and from sera of an additional 20 naïve CHC patients, was investigated by next-generation sequencing. <b><i>Results:</i></b> At a cutoff of 1%, NS3 RASs were detected in all the samples examined. At a cutoff of 15%, they were found in 54.5% (6/11) and 27.3% (3/11) of the paired liver and serum samples, respectively, and in 22.5% (7/31) of the overall serum samples examined. Twenty-six out of thirty-one (84%) patients showed NS3 variants with multiple RASs. Phylogenetic analysis showed that NS3 sequences clustered within 2 clades, with 10/31 (32.2%) patients infected by clade I, 15/31 (48.8%) by clade II, and 6/31 (19.3%) by both clades. <b><i>Conclusions:</i></b> Though the number of patients examined was limited, NS3 variants with RASs appear to be major components of both intrahepatic and circulating viral quasispecies populations in DAA-naïve patients.