Similar Prevalence of Low-Abundance Drug-Resistant Variants in Treatment-Naive Patients with Genotype 1a and 1b Hepatitis C Virus Infections as Determined by Ultradeep Pyrosequencing

Abstract

Background and ObjectivesHepatitis C virus (HCV) variants that confer resistance to direct-acting-antiviral agents (DAA) have been detected by standard sequencing technology in genotype (G) 1 viruses from DAA-naive patients. It has recently been shown that virological response rates are higher and breakthrough rates are lower in G1b infected patients than in G1a infected patients treated with certain classes of HCV DAAs. It is not known whether this corresponds to a difference in the composition of G1a and G1b HCV quasispecies in regards to the proportion of naturally occurring DAA-resistant variants before treatment.MethodsWe used ultradeep pyrosequencing to determine the prevalence of low-abundance (<25% of the sequence reads) DAA-resistant variants in 191 NS3 and 116 NS5B isolates from 208 DAA-naive G1-infected patients.ResultsA total of 3.5 million high-quality reads of ≥200 nucleotides were generated. The median coverage depth was 4150x and 4470x per NS3 and NS5B amplicon, respectively. Both G1a and G1b populations showed Shannon entropy distributions, with no difference between G1a and G1b in NS3 or NS5B region at the nucleotide level. A higher number of substitutions that confer resistance to protease inhibitors were observed in G1a isolates (mainly at amino acid 80 of the NS3 region). The prevalence of amino acid substitutions that confer resistance to NS5B non-nucleoside inhibitors was similar in G1a and G1b isolates. The NS5B S282T variant, which confers resistance to the polymerase inhibitors mericitabine and sofosbuvir, was not detected in any sample.ConclusionThe quasispecies genetic diversity and prevalence of DAA-resistant variants was similar in G1a and G1b isolates and in both NS3 and NS5B regions, suggesting that this is not a determinant for the higher level of DAA resistance observed across G1a HCV infected patients upon treatment.

Resistance-Associated Variants in HCV Subtypes 1a and 1b Detected by Ion Torrent Sequencing Platform

“…The A421V mutation, which was found in 8% (HCV-1a) and 7% (HCV-1b) of the patients in the present study, affects the Thumb 1 domain and is associated with low levels of resistance to non-nucleoside inhibitors (NNI) [37,38]. Further studies are needed to investigate the action of this variant on the NNI TMC647055, which is in Phase II trials [39].…”

Prevalence and characteristics of naturally occurring sofosbuvir resistance‐associated variants in patients with hepatitis C virus genotype 1b infection

“…Similar to C316, V321 is in close proximity to the catalytic triad of the HCV NS5B polymerase (D220, D318, and D319). In addition, NS5B‐V321A/I variants are known as active‐site variants that enhance resistance to nucleotide analogs such as PSI‐352938 and PSI‐353661 . Several previous reports have indicated that the prevalence of the V321I variant in patients with HCV genotype 1b is 2.5–3.0%, which shows a 2.0‐fold EC 50 for PSI‐352938, compared with wild‐type .…”

“…In addition, NS5B-V321A/I variants are known as active-site variants that enhance resistance to nucleotide analogs such as PSI-352938 and PSI-353661. 38,39 Several previous reports have indicated that the prevalence of the V321I variant in patients with HCV genotype 1b is 2.5-3.0%, which shows a 2.0-fold EC 50 for PSI-352938, compared with wildtype. 9,14 Moreover, Tong et al 26 reported that L320F confers a 2.7-fold increase in in vitro resistance and a 41-fold increase, compared with wild-type, when it coexists with the S282T variant in vitro.…”

Comprehensive Screening for Naturally Occurring Hepatitis C Virus Resistance to Direct-Acting Antivirals in the NS3, NS5A, and NS5B Genes in Worldwide Isolates of Viral Genotypes 1 to 6

“…Natural HCV variation is generated and transmitted over time even in the absence of DAAs. Indeed, naturally occurring DAA resistance-associated HCV variants (RAVs) have already been reported in DAA-naive patients, with some RAVs showing differential prevalence between genotypes and subtypes (13)(14)(15)(16)(17)(18)(19)(20)(21). A relevant example is the NS3-Q80K variant associated with resistance to simeprevir (SMV), which was previously found to be present in Ͼ30% of GT1a sequences but almost absent in GTs 1b, 2, 3, 4, and 5 (13,16,21).…”