Original ResearchFull Report: Basic and Translational—LiverHCV Genotype 6a Escape From and Resistance to Velpatasvir, Pibrentasvir, and Sofosbuvir in Robust Infectious Cell Culture Models
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HCV Genotype 6a Consensus Sequences and Plasmid Construction
To obtain full-length clones of genotype 6a, the complete consensus open reading frame (ORF) sequences of prototype strains HK6a (MG717928) and HK2 (MG717925) were determined from serum samples4, 19 by using direct sequence analysis of reverse-transcription polymerase chain reaction (RT-PCR) products. These strains were originally recovered from patients with chronic HCV.4, 19 The HK6a 5ʹ untranslated region (UTR) was determined previously using the 5ʹRACE system.8, 14 A partial HK6a 3ʹUTR
Development of Highly Efficient HCV Genotype 6a Full-Length Infectious Cell Culture Systems
We initially developed full-length clones of prototype strains HK6a (MG717928)8, 19 and HK2 (MG717925)4, 5 with strain-specific ORF differing by 4% and 3% at nt and aa level, respectively, and encoding polyproteins of 3020 and 3019 aa, respectively. They had authentic HK6a 5ʹUTR8 and adapted chimeric HK6a/H77 3ʹUTR comprising the H77 3ʹX-stem-loop I (SLI)20 (Supplementary Results; Supplementary Figures 1A and B and 2). However, the HK2(H77_3ʹXSLI) (MG717926) and HK6a(H77_3ʹXSLI) (MG717929)
Discussion
We developed efficient HCV in vitro models for genotype 6a prototype strains, HK2 and HK6a. These are the first infectious full-length culture systems for major genotype 6, that poses an important disease burden in Asia. They permitted efficacy studies of NS5A- and NS5B-inhibitors, including the combination treatment velpatasvir/sofosbuvir. For 6a, combination treatment is essential to overcome the relatively low resistance barrier to velpatasvir or sofosbuvir. In vitro, 6a viruses readily
Acknowledgments
We thank L. Ghanem and L. Mikkelsen (Copenhagen University Hospital, Hvidovre) for laboratory assistance, and B. Ørskov Lindhardt and O. Andersen (Copenhagen University Hospital, Hvidovre), as well as Carsten Geisler (University of Copenhagen) for valuable support. We thank C.M. Rice (Rockefeller University, New York, NY) and R. Purcell (National Institutes of Health, Bethesda, MD) for providing reagents.
The present address for Yi-Ping Li is: Institute of Human Virology and Key Laboratory of
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Conflicts of interest The authors disclose no conflicts.
Funding This study was supported by a PhD stipend from Faculty of Health and Medical Sciences, University of Copenhagen (L.V.P.) and by grants from The Region H Foundation (S.R., J.M.G., J.B.), The Lundbeck Foundation (S.R., J.B.), The Novo Nordisk Foundation (J.M.G., J.B.), The Danish Council for Independent Research (DFF), Medical Sciences (J.M.G., S.R., J.B.), and Innovation Fund Denmark (Infect-ERA EU, J.B.). J.B. is the 2014 recipient of an advanced top researcher grant from DFF and the 2015 recipient of the Novo Nordisk Prize.
GenBank accession numbers: MG717925- MG717930.
Author names in bold designate shared co-first authorship.