There is no comprehensive study available on the natural hepatitis C virus (HCV) polymorphism in sites associated with resistance including all viral genotypes which may present variable susceptibilities to particular direct-acting antivirals (DAAs). This study aimed to analyze the frequencies, genetic barriers, and evolutionary histories of naturally occurring resistance-associated variants (RAVs) in the six main HCV genotypes. A comprehensive analysis of up to 103 RAVs was performed in 2,901, 2,216, and 1,344 HCV isolates for the NS3, NS5A, and NS5B genes, respectively. We report significant intergenotypic differences in the frequencies of natural RAVs for these three HCV genes. In addition, we found a low genetic barrier for the generation of new RAVs, irrespective of the viral genotype. Furthermore, in 1,126 HCV genomes, including sequences spanning the three genes, haplotype analysis revealed a remarkably high frequency of viruses carrying more than one natural RAV to DAAs (53% of HCV-1a, 28.5% of HCV-1b, 67.1% of HCV-6, and 100% of genotype 2, 3, 4, and 5 haplotypes). With the exception of HCV-1a, the most prevalent haplotypes showed RAVs in at least two different viral genes. Finally, evolutionary analyses revealed that, while most natural RAVs appeared recently, others have been efficiently transmitted over time and cluster in well-supported clades. In summary, and despite the observed high efficacy of DAA-based regimens, we show that naturally occurring RAVs are common in all HCV genotypes and that there is an overall low genetic barrier for the selection of resistance mutations. There is a need for natural DAA resistance profiling specific for each HCV genotype.
Hepatitis C virus (HCV) infection is considered a major public health problem. More than 170 million people are chronically infected worldwide, with the consequent risk of developing liver diseases such as cirrhosis and liver cancer, which can eventually cause death (1, 2). HCV is a highly variable RNA virus which has been classified into 7 known genotypes (3). Genetic distances among genotypes reach up to 30% (4). Such diversity can be explained by an evolutionary rate of a magnitude of 10 Ϫ3 substitutions per site and year (5). These differences at the genomic level also appear to be relevant at the clinical level. Treatment of chronic HCV infection with peginterferon-ribavirin combination therapy (P/R) shows variable sustained virological response (SVR) rates depending on the infecting HCV genotype (GT), with average SVR rates of 46%, 80%, 66%, and 60% for GTs 1, 2, 3, and 4, respectively (6, 7). Even within HCV GT1, a significant difference in SVR between subtypes 1a and 1b has been reported (8).In recent years, the field of HCV therapy is blooming because of the clinical development of direct-acting antiviral drugs (DAAs) that are more effective than P/R (SVR up to Ͼ90%) and can be given in interferon (IFN)-free regimens with reduced toxicity (9, 10). DAAs that have advanced to clinical trials target three essential proteins for the HCV li...
