Use of Illumina Deep Sequencing Technology To Differentiate Hepatitis C Virus Variants

Abstract

Hepatitis C virus (HCV) is a positive-strand enveloped RNA virus that shows diverse viral populations even in one individual. Though Sanger sequencing has been used to determine viral sequences, deep sequencing technologies are much faster and can perform large-scale sequencing. We demonstrate the successful use of Illumina deep sequencing technology and subsequent analyses to determine the genetic variants and amino acid substitutions in both treatment-naïve (patient 1) and treatmentexperienced (patient 7) isolates from HCV-infected patients. As a result, almost the full nucleotide sequence of HCV was detectable for patients 1 and 7. The reads were mapped to the HCV reference sequence. The coverage was 99.8% and the average depth was 69.5؋ for patient 7, with values of 99.4% (coverage) and 51.1؋ (average depth) for patient 1. In patient 7, amino acid (aa) 70 in the core region showed arginine, with methionine at aa 91, by Sanger sequencing. Major variants showed the same amino acid sequence, but minor variants were detectable in 18% ( . The single open reading frame encodes a polyprotein of 3,011 amino acids (aa) that is cleaved by viral and cellular proteases into 10 different proteins. The three structural proteins, which constitute the viral particle, include the core protein and the envelope glycoproteins E1 and E2. Two regions in E2, known as hypervariable regions 1 and 2, are reported to have extreme sequence variability. The seven nonstructural components include the p7 polypeptide, the NS2-3 protease, the NS3 serine protease and RNA helicase, the NS4A polypeptide, the NA4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase (RdRp) (29). At both ends of the open reading frame lie the 5=-and 3=-untranslated regions (5=-UTR and 3=-UTR). The nucleotide sequence of the 5=-UTR is relatively well conserved among different HCV genotypes. The HCV 5=-UTR contains an internal ribosome entry site (IRES) that directs the cap-independent initiation of virus translation and forms on four characteristic stem-loop structures (17, 18). HCV displays very high genetic variability both in populations and within infected individuals, where it exists as a cluster of closely related but distinct variants, termed "quasispecies," as occurs in many other RNA viruses with a polymerase enzyme lacking proofreading ability (6,8,26).Current standard treatment of chronic HCV infection is based on the combination of pegylated alpha interferon (peg-IFN-␣) and ribavirin (RBV). However, patients with a high load of genotype 1b virus (Ͼ1 ϫ 10 5 log IU/ml) do not achieve high sustained virological response (SVR) rates (Ͻ50%), even when the most effective combination treatment (IFN plus RBV) is administered for 48 weeks (14,25). Some investigations concerning therapeutic prediction based on virological features revealed that substitutions of arginine for glutamine at amino acid (aa) 70 and/or leucine for methionine at aa 91 in the core region are independent and significant factors associated with SVR or that patients whose viruses ...

Hepatitis C virus genetic variability and evolution

“…This method provides a good idea of the major sequences present, but unfortunately it cannot detect minor variants that are present at a frequency below 20%-25% [106] . With the development of deep sequencing technologies, detection of drug-resistant variants became more sensitive allowing the identification of variants present at very low frequencies (about 0.1%-1%) [107][108][109] . For HCV, the deep sequencing method was first used to detect emergence of NS3 mutants.…”

Prediction of Treatment Efficacy and Telaprevir-Resistant Variants after Triple Therapy in Patients Infected with Hepatitis C Virus Genotype 1

“…Recent reports have described the advantages of ultradeep sequencing technology, including faster processing and large-scale sequencing, in addition to providing a better understanding of the dynamics of variants in HCV quasispecies (8)(9)(10)(11). However, it is not clear at this stage whether such technology is useful for the prediction of the emergence of telaprevir-resistant variants during or after the administration of triple therapy.…”

Abundant Drug-Resistant NS3 Mutants Detected by Deep Sequencing in Hepatitis C Virus-Infected Patients Undergoing NS3 Protease Inhibitor Monotherapy

“…The method provides a composite of the major sequences present but is limited in the detection of minor viral mutant subpopulations that present at Ͻ20% to 25% (7). With the development of allele-specific PCR, single-genome sequencing, and deep-sequencing technologies, the detection of drug-resistant variants became more sensitive with the current assay cutoff at about 0.1% to 1% (3,6,9). Further improvement of the assay sensitivity is constrained by uniform amplification of viral quasispecies and background noise from mutations generated during PCR amplification and sequencing.…”

“…Further improvement of the assay sensitivity is constrained by uniform amplification of viral quasispecies and background noise from mutations generated during PCR amplification and sequencing. Several recent studies clearly demonstrated the utility of deep sequencing and other sensitive technologies to detect HCV quasispecies (3,6,9). The deep-sequencing method was first used to detect the emergence of NS3 mutants in an elegant infectious model of HCV using human hepatocyte chimeric mice.…”