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  • NGS substitution linkage analysis and haplotype reconstructi

    2021-10-16

    NGS substitution linkage analysis and haplotype reconstruction was previously only used for short genome segments and not in the context of antiviral resistance. We optimized this approach to cover a complete viral protein and major DAA target, enabling us to investigate resistance associated NS3P haplotypes in unprecedented detail. Mostly for simeprevir and grazoprevir, and to a lesser extent for glecaprevir, increasing PI concentrations created an evolutionary bottleneck, driving the selection of highly resistant viruses. While low PI concentrations selected for various NS3P haplotypes with different substitutions occurring individually or in various combinations, high PI concentrations selected for few NS3P haplotypes dominated by high-level resistance RASs such as R155K and A156T. In contrast, for voxilaprevir A156T/V dominated in all escape cultures, possibly because of its increased activity against several low-level resistance RASs. RASs selected in original viruses were comparable to those selected in replicons and in patients under PIs used in this study.[11], [33], [35], [36], [3], [4], [5], [39], [40], [41], [42], [43], [44], [45], [46] In addition, selection of RASs R155K, D168E and I170T under simeprevir and of position-168 RASs under grazoprevir in genotype 1a infected patients with baseline Q80K highlights the clinical relevance of our results.[10], [40], [47], [48] Our data underline the central importance of NS3P position 156 for resistance to the clinically highly important PIs grazoprevir, glecaprevir and voxilaprevir.[3], [4], [5] The capacity to escape high PI concentrations was not directly linked to resistance exerted by Q80K. Rather, Q80K facilitated rapid co-selection of additional RASs, leading to faster escape and to escape variants with higher SNOG and/or resistance compared to escape variants selected in experiments with the original viruses. We hypothesize that upon treatment initiation, Q80K variants might have an evolutionary advantage due to increased growth capacity under treatment, allowing faster selection of variants carrying additional RASs and/or fitness compensating substitutions.
    Financial support Ph.D. stipends from Faculty of Health and Medical Sciences, Copenhagen University (L.V.P., S.B.J., S.B.N.S.), research grants from the Danish Council for Independent Research–Medical Sciences (S.R., D.H., J.B. and J.M.G.), Region Hovedstadens Forskningsfond (S.R., J.B., J.M.G.), the Lundbeck Foundation (S.R. and J.B.), the Novo Nordisk Foundation (J.B. and J.M.G.), Innovation Fund Denmark (J.B). J.B. is the recipient of an advanced-top researcher grant from the Danish Council for Independent Research in 2014 and of the 2015 Novo Nordisk Prize.
    Conflict of interest Please refer to the accompanying ICMJE disclosure forms for further details.
    Authors’ contributions
    Acknowledgements
    Introduction Hepatitis C Virus (HCV) infection is a global health threat. More than 170 million people are infected by HCV worldwide, up to 85% of whom are subjected to chronic infection with high risk of developing liver cirrhosis and ∼5% will develop hepatocellular carcinoma (HCC) [1]. HCV is a positive-strand RNA virus belonging to the Flaviviridae family (genotypes 1–6) with a single open reading frame that encodes a large polyprotein (3000 a.a.). The polyprotein is processed and cleaved by host and viral proteases into 3 structural and 7 nonstructural proteins (NSs) [[2], [3]]. Direct-acting antiviral treatments for HCV can be categorized according to their protein target. [[4], [5], [6], [7]] For example, NS3/4A inhibitors, [8] such as telaprivir, compete with the substrate NS5A/5B, and target the substrate-binding site [5]. Besides direct-acting antiviral drugs, interferons protect cells against viruses by producing interferon-stimulated genes, which generate a variety of inhibitory activities [[9], [10]]. Combinations of the various treatments have demonstrated synergistic effects or have improved the antiviral responses [11].