Viral variants with decreased susceptibility to HCV protease inhibitors (PIs) occur naturally and preexist at low levels within HCV populations. In patients failing PI monotherapy, single and double mutants conferring intermediate to high-level resistance to PIs have been selected in vivo. The abundance, temporal dynamics and linkage of naturally occurring resistance-associated variants (RAVs), however, have not been characterized in detail. Here, using high-density pyrosequencing, we analyzed HCV NS3 gene segments from 20 subjects with chronic HCV infection, including 12 subjects before and after liver transplantation. Bioinformatics analysis revealed that Q80 substitution was a dominant variant in 40% of the subjects, whereas other RAVs circulate at low levels within quasispecies populations. Low frequency mutation linkage was detectable by Illumina paired-end sequencing in as low as 0.5% of the mock populations constructed from in vitro RNA transcripts but were uncommon in vivo. We show that naturally occurring RAVs are common and can persist long term following liver transplant at low levels not readily detectable by conventional sequencing. Our results indicate that mutation linkage at low levels could be identified using the Illumina paired-end approach. The methods described here should facilitate the analysis of low frequency HCV drug resistance, mutation linkage and evolution, which may inform future therapeutic strategies in patients undergoing direct acting antiviral therapies
