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

• Published in: PloS one Vol. 10; no. 3; p. e0119145
• Main Authors: Ogishi, Masato, Yotsuyanagi, Hiroshi, Tsutsumi, Takeya, Gatanaga, Hiroyuki, Ode, Hirotaka, Sugiura, Wataru, Moriya, Kyoji, Oka, Shinichi, Kimura, Satoshi, Koike, Kazuhiko
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 06.03.2015; Public Library of Science (PLoS)
• Subjects: Acquired immune deficiency syndrome; AIDS; Antiviral agents; Bioinformatics; Blood; Blood transfusion; Feasibility studies; Female; Genetic aspects; Genetic Variation; Genotypes; Genotyping; Haplotypes; Health aspects; Hemophilia; Hemophilia A - complications; Hemophilia A - epidemiology; Hemophilia A - genetics; Hemophiliacs; Hepacivirus - genetics; Hepatitis; Hepatitis C; Hepatitis C - complications; Hepatitis C - epidemiology; Hepatitis C - genetics; Hepatitis C virus; HIV; HIV Infections - complications; HIV Infections - epidemiology; HIV Infections - genetics; Human immunodeficiency virus; Humans; Infections; Japan; Male; Mutation; Patients; Prevalence; Screening; Subgroups; Transfusion; Viral Nonstructural Proteins - genetics; Viruses; Japan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0119145

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.