Genotyping and resistance profile of hepatitis C (HCV) genotypes 1-6 by sequencing the NS3 protease region using a single optimized sensitive method

Abstract

The objective was to develop a method of NS3 gene sequencing that allowed simultaneous genotyping and protease inhibitor (PI) resistance profiling of HCV genotypes 1-6. To validate the use of a unique RT-PCR for genotypes 1-6 and evaluate its sensitivity, the NS3 protease region was amplified from 140 plasma samples from patients infected with HCV without previous PI therapy. In parallel, NS5b sequences were obtained. Amplification of NS3 was successful in 139/140 samples (99%). For the 135 samples with both NS5b and NS3 sequencing results, phylogenetic analysis showed concordance of genotypes with a bootstrap >90% for each cluster. PI resistance mutations were analyzed using the Geno2pheno [hcv] v1.0 tool. For the 63 genotype 1 (G1) Nantes clinical strains, 12 (19%) presented a natural resistance mutation. This proportion was higher (p<0.05) than that observed in a sample of 374 G1 reference sequences. This significant difference was observed only in subtype 1b (n=7; 25% against n=19; 8%). In conclusion, this tool allows determination of both HCV genotype and identification of PI-resistance mutations. It can be used to detect pre-existing resistance mutations in NS3 before treatment and follow the emergence of resistant viruses during therapy.