Characterization of Naturally Occurring NS5A and NS5B Polymorphisms in Patients Infected with HCV Genotype 3a Treated with Direct-Acting Antiviral Agents

Abstract

Hepatitis C virus (HCV) genotype (GT)3 is associated with increased risk of steatosis, development of cirrhosis and hepatocellular carcinoma. Limited data are available regarding genetic variability and use of direct-acting antiviral agents in these patients. non-structural protein 5A (NS5A) and non-structural protein 5B (NS5B) sequencing was performed on 45 HCV GT3-infected Italian patients subsequently treated with sofosbuvir ± daclatasvir (SOF ± DCV). Novel GT3a polymorphisms were observed by Sanger sequencing in three NS5A (T79S, T107K, and T107S) and three NS5B (G166R, Q180K, and C274W) baseline sequences in patients who achieved sustained virological response (SVR). Baseline NS5A resistance-associated substitutions A30K and Y93H were detected in 9.5% of patients; one patient with A30K did not achieve SVR. Phylogenetic analyses of sequences showed no distinct clustering. Genetic heterogeneity of NS5A and NS5B was evaluated using ultra-deep pyrosequencing (UDPS) in samples longitudinally collected in patients not achieving SVR. Some novel NS5A and NS5B polymorphisms detected at baseline may not impact treatment outcome, as they were not enriched in post-failure samples. In contrast, the novel L31F NS5A variant emerged in one treatment failure, and I184T, G188D and N310S, located on the same NS5B haplotype, became predominant after failure. These findings suggest a potential impact of these novel substitutions on the treatment outcome; however, their significance requires further investigation.

Keywords: Genotype3; HCV; NS5A; NS5B; genetic variability; polymorphism; resistance-associated substitutions; ultra-deep pyrosequencing.

Figure 1

Substitutions rate at non-synonymous sites - substitutions rate at synonymous sites (dN-dS) in HCV GT3a sequences worldwide. The graphs represent dN-dS vs. each codon for: NS5A (A); and NS5B (B) of HCV GT3a sequences from the Los Alamos database.

Figure 2

Phylogenetic trees of NS5A and NS5B. Phylogenetic analyses of: NS5A (A); and NS5B (B) sequences performed using 500 bootstrap repetitions. Only bootstrap values >70% are outlined with black circles. Sequences from the present study are indicated with patient (Pt) number; HCV sequences from European countries in the Los Alamos database are shown with black triangles. The country and year of these sequences are reported after the genotype. The GT3a reference sequence (D17763, in black star) is included. Patients harboring NS5A-A30K are side marked. AU, Australia; CA, Canada; CH, Switzerland; CN, China; DE, Germany; FR, France; GB, Great Britain; IN, India; ITA, Italy; PK, Pakistan; US, United States; NZ, New Zealand.

Figure 2

Phylogenetic trees of NS5A and NS5B. Phylogenetic analyses of: NS5A (A); and NS5B (B) sequences performed using 500 bootstrap repetitions. Only bootstrap values >70% are outlined with black circles. Sequences from the present study are indicated with patient (Pt) number; HCV sequences from European countries in the Los Alamos database are shown with black triangles. The country and year of these sequences are reported after the genotype. The GT3a reference sequence (D17763, in black star) is included. Patients harboring NS5A-A30K are side marked. AU, Australia; CA, Canada; CH, Switzerland; CN, China; DE, Germany; FR, France; GB, Great Britain; IN, India; ITA, Italy; PK, Pakistan; US, United States; NZ, New Zealand.