Abstract
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.
Highlights
Hepatitis C virus (HCV) infects over 180 million individuals worldwide and is the leading cause of liver transplantation due to cirrhosis and hepatocellular carcinoma [1]
We show that naturally occurring, low frequency resistance-associated variants (RAVs) are common in chronic HCV, and can persist long term following liver transplantation
To minimize re-sampling of low viral load RNA templates, all samples were quantified by quantitative RT-PCR and a median 2.56105 copies of RNA per reaction were used in the amplification step
Summary
Hepatitis C virus (HCV) infects over 180 million individuals worldwide and is the leading cause of liver transplantation due to cirrhosis and hepatocellular carcinoma [1]. In treatment-naıve patients, naturally occurring dominant RAVs are common [12]. The quasispecies nature of HCV raises the concern that viral swarms may harbor preexisting mutations at low frequency not readily detectable by conventional genotyping methods, which may influence treatment outcome. The presence of preexisting RAVs is further supported by the observation that RAVs are selected rapidly in subjects receiving protease inhibitor monotherapy [10,11], typically within days of initiating DAAs. Importantly, lessons from the HIV field indicate that preexisting drug resistant variants at low frequencies could contribute to treatment failure [15], and genotypic resistance testing is the standard of care prior to initiating antiretroviral therapy. A detailed examination of resistance profiles is important for identifying clinically relevant drug resistance variants and optimizing strategy to improve treatment outcome
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