Validation of a Genotype-Independent Hepatitis C Virus Near-Whole Genome Sequencing Assay.

Hope R Lapointe,Weiyan Dong,Art F Y Poon,Conan Woods,P Richard Harrigan,Winnie W Y Dong,Anita Y M Howe,Chanson J Brumme,Don Kirkby

doi:10.3390/v13091721

Abstract

Despite the effectiveness of direct-acting antiviral agents in treating hepatitis C virus (HCV), cases of treatment failure have been associated with the emergence of resistance-associated substitutions. To better guide clinical decision-making, we developed and validated a near-whole-genome HCV genotype-independent next-generation sequencing strategy. HCV genotype 1–6 samples from direct-acting antiviral agent treatment-naïve and -treated HCV-infected individuals were included. Viral RNA was extracted using a NucliSens easyMAG and amplified using nested reverse transcription-polymerase chain reaction. Libraries were prepared using Nextera XT and sequenced on the Illumina MiSeq sequencing platform. Data were processed by an in-house pipeline (MiCall). Nucleotide consensus sequences were aligned to reference strain sequences for resistance-associated substitution identification and compared to NS3, NS5a, and NS5b sequence data obtained from a validated in-house assay optimized for HCV genotype 1. Sequencing success rates (defined as achieving >100-fold read coverage) approaching 90% were observed for most genotypes in samples with a viral load >5 log10 IU/mL. This genotype-independent sequencing method resulted in >99.8% nucleotide concordance with the genotype 1-optimized method, and 100% agreement in genotype assignment with paired line probe assay-based genotypes. The assay demonstrated high intra-run repeatability and inter-run reproducibility at detecting substitutions above 2% prevalence. This study highlights the performance of a freely available laboratory and bioinformatic approach for reliable HCV genotyping and resistance-associated substitution detection regardless of genotype.

Highlights

Hepatitis C virus (HCV) infection is a major public health concern in Canada and worldwide
HCVpositive samples used to develop and validate this near whole genome, hepatitis C virus (HCV) genotypeindependent assay were sourced from Merck from the C-WORTHY trial (NCT01717326/ Protocol PN035) and the Vancouver Injection Drug Users Study (VIDUS) [31,32]
To assess the ability of the genotype-independent assay to reliably amplify and sequence the HCV virus of different genotypes, we evaluated the PCR and sequencing success rate 146 samples consisting of GT 1 to 6 samples

Summary

Introduction

Hepatitis C virus (HCV) infection is a major public health concern in Canada and worldwide. In 2015, the World Health Organization (WHO) estimated that 71 million people globally had chronic HCV infection [1]. The therapeutic approach towards HCV chronic infection has shifted in recent years with the advent of oral direct-acting antiviral (DAA) agents. Interferon- and ribavirin-based therapies were non-specific to HCV and caused moderate to severe side effects universally, while firstgeneration protease inhibitors had moderate to high potential for drug–drug interactions [3]. Newer therapies against specific viral targets use direct-acting antiviral agents (DAA), small-molecule inhibitors that can be categorized on the basis of their viral targets. These drug targets are important for viral replication, notably non-structural proteins NS3/4a, NS5a, and NS5b

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Results

Discussion

Conclusion