Founder effect of NS3 variant Q80K in HCV1a infected patients in Italy

Abstract

Background: Italy is characterized by a high HCV burden, with higher prevalence rates in Southern and Insular areas compared to Central and Northern regions. Despite 95% success rates reported for all HCV genotypes with direct-acting antivirals (DAAs), eradication is hampered by the impact of natural resistance-associated variants (RAVs), especially in case of retreatment after DAA failure. This study aimed to investigate HCV1a variability within Italy, as well as the association of clusters with epidemiological factors and NS3 RAVs. Materials and methods: Between 2011 and 2015, 183 baseline NS3 sequences from serum samples of HCV1a infected patients were obtained with Sanger sequencing. Individuals were sampled in seven Italian regions (Abruzzo, Apulia, Emilia-Romagna, Lazio, Liguria, Lombardy and Sardinia). HCV1a control strains coding for protein NS3 and sequenced worldwide, were gathered from Genbank. A total dataset of 1084 sequences was obtained after subtyping, quality control and alignment. Sequences were annotated with RAVs using Geno2pheno, with year and country, and when available with gender, age, therapy status, transmission route and viral co-infection. Phylogenetic reconstruction was performed using RAxML, with GTR model and supported by 1000 bootstrap replicates. Clustering of sequences was identified with Cluster Picker, using 70% bootstrap support as threshold. Phylogenetic and temporal signal were determined with TreePuzzle and Path-O-Gen, after which clusters were confirmed using Bayesian phylogeny with BEAST. Results: Median age of the 183 HCV1a positive patients was 55 years (IQR: 50-66), with the majority being male (80.9%) and DAA naïve (89.1%). Transmission route was available for 61/183 patients (33%) and only 12/183 patients (7%) were co-infected with HIV. Based on a maximum-likelihood and Bayesian phylogenetic tree of the whole dataset, we identified two clusters, which included 3 patients from our cohort in total. The first cluster consisted of two DAA-naïve HCV mono-infected patients from Abruzzo (PP = 1). Both patients were male with one infected through intravenous drug use. For the second cluster (PP > 0.97), an HIV/HCV co-infected patient from Lazio clustered with an Italian control sequence, with for the latter lacking information for viral co-infections. Both sequences were derived from DAA-naïve patients with unknown transmission route. Remarkably, both clusters included patients all harbouring RAV Q80K. In depth-analysis showed a dispersion of the Italian sequences across the whole phylogenetic tree and a distinction of the tree into two major clades, with one consisting of all, except for one, sequences harbouring variant Q80K (n=96). Due to low support for the two separate clades, downsampling to 20% of the tree was performed multiple times, and this confirmed each time the existence of a founder effect for RAV Q80K. Conclusions: Phylogeny of HCV1a NS3 sequences identified two clusters among DAA-naïve Italian patients each harbouring RAV Q80K. All HCV1a sequences could be assigned to one of two major clusters, one with and one without Q80K. This founder effect of Q80K has implications for treatment of HCV1a cirrhotic patients with simeprevir and sofosbuvir, since Q80K is associated with lower success rates. Phylogenetic cluster analysis may aid in predicting treatment response and assessing viral evolution under DAA selective pressure.status: accepte