Deconvoluting the composition of low-frequency hepatitis C viral quasispecies: comparison of genotypes and NS3 resistance-associated variants between HCV/HIV coinfected hemophiliacs and HCV monoinfected patients in Japan.
Pre-existing low-frequency resistance-associated variants (RAVs) may jeopardize successful sustained virological responses (SVR) to HCV treatment with direct-acting antivirals (DAAs). However, the potential impact of low-frequency (∼0.1%) mutations, concatenated mutations (haplotypes), and their association with genotypes (Gts) on the treatment outcome has not yet been elucidated, most probably owing to the difficulty in detecting pre-existing minor haplotypes with sufficient length and accuracy. Herein, we characterize a methodological framework based on Illumina MiSeq next-generation sequencing (NGS) coupled with bioinformatics of quasispecies reconstruction (QSR) to realize highly accurate variant calling and genotype-haplotype detection. The core-to-NS3 protease coding sequences in 10 HCV monoinfected patients, 5 of whom had a history of blood transfusion, and 11 HCV/HIV coinfected patients with hemophilia, were studied. Simulation experiments showed that, for minor variants constituting more than 1%, our framework achieved a positive predictive value (PPV) of 100% and sensitivities of 91.7-100% for genotyping and 80.6% for RAV screening. Genotyping analysis indicated the prevalence of dominant Gt1a infection in coinfected patients (6/11 vs 0/10, p = 0.01). For clinical samples, minor genotype overlapping infection was prevalent in HCV/HIV coinfected hemophiliacs (10/11) and patients who experienced whole-blood transfusion (4/5) but none in patients without exposure to blood (0/5). As for RAV screening, the Q80K/R and S122K/R variants were particularly prevalent among minor RAVs observed, detected in 12/21 and 6/21 cases, respectively. Q80K was detected only in coinfected patients, whereas Q80R was predominantly detected in monoinfected patients (1/11 vs 7/10, p < 0.01). Multivariate interdependence analysis revealed the previously unrecognized prevalence of Gt1b-Q80K, in HCV/HIV coinfected hemophiliacs [Odds ratio = 13.4 (3.48-51.9), p < 0.01]. Our study revealed the distinct characteristics of viral quasispecies between the subgroups specified above and the feasibility of NGS and QSR-based genetic deconvolution of pre-existing minor Gts, RAVs, and their interrelationships.
Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, Bunkyo, Tokyo, Japan.
Masato
M
Ogishi
Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, Bunkyo, Tokyo, Japan.
Hiroshi
H
Yotsuyanagi
Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, Bunkyo, Tokyo, Japan.
Takeya
T
Tsutsumi
AIDS Clinical Center, National Center for Global Health and Medicine, Shinjuku, Tokyo, Japan.
Hiroyuki
H
Gatanaga
Department of Infectious Diseases and Immunology, Clinical Research Center, Nagoya Medical Center, Nagoya, Japan.
Hirotaka
H
Ode
Department of Infectious Diseases and Immunology, Clinical Research Center, Nagoya Medical Center, Nagoya, Japan.
Wataru
W
Sugiura
Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, Bunkyo, Tokyo, Japan.
Kyoji
K
Moriya
AIDS Clinical Center, National Center for Global Health and Medicine, Shinjuku, Tokyo, Japan.
Shinichi
S
Oka
Director, Tokyo Teishin Hospital, Tokyo, Japan; President, Tokyo Health Care University, Tokyo, Japan.
Satoshi
S
Kimura
Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, Bunkyo, Tokyo, Japan.
Kazuhiko
K
Koike
D005260:Female; D014644:Genetic Variation; D015658:HIV Infections / Q000150:complications / Q000453:epidemiology / Q000235:genetics*; D006239:Haplotypes; D006467:Hemophilia A / Q000150:complications / Q000453:epidemiology / Q000235:genetics*; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000150:complications / Q000453:epidemiology / Q000235:genetics*; D006801:Humans; D007564:Japan; D008297:Male; D015995:Prevalence; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases