HCV genome-wide analysis for development of efficient culture systems and unravelling of antiviral resistance in genotype 4

Abstract

ObjectiveHCV-genotype 4 infections are a major cause of liver diseases in the Middle East/Africa with certain subtypes associated with increased risk of direct-acting antiviral (DAA) treatment failures. We aimed at developing infectious genotype 4 cell culture systems to understand the evolutionary genetic landscapes of antiviral resistance, which can help preserve the future efficacy of DAA-based therapy.DesignHCV recombinants were tested in liver-derived cells. Long-term coculture with DAAs served to induce antiviral-resistance phenotypes. Next-generation sequencing (NGS) of the entire HCV-coding sequence identified mutation networks. Resistance-associated substitutions (RAS) were studied using reverse-genetics.ResultThe in-vivo infectious ED43(4a) clone was adapted in Huh7.5 cells, using substitutions identified in ED43(Core-NS5A)/JFH1-chimeric viruses combined with selected NS5B-changes. NGS, and linkage analysis, permitted identification of multiple genetic branches emerging during culture adaptation, one of which had 31 substitutions leading to robust replication/propagation. Treatment of culture-adapted ED43 with nine clinically relevant protease-DAA, NS5A-DAA and NS5B-DAA led to complex dynamics of drug-target-specific RAS with coselection of genome-wide substitutions. Approved DAA combinations were efficient against the original virus, but not against variants with RAS in corresponding drug targets. However, retreatment with glecaprevir/pibrentasvir remained efficient against NS5A inhibitor and sofosbuvir resistant variants. Recombinants with specific RAS at NS3-156, NS5A-28, 30, 31 and 93 and NS5B-282 were viable, but NS3-A156M and NS5A-L30Δ (deletion) led to attenuated phenotypes.ConclusionRapidly emerging complex evolutionary landscapes of mutations define the persistence of HCV-RASs conferring resistance levels leading to treatment failure in genotype 4. The high barrier to resistance of glecaprevir/pibrentasvir could prevent persistence and propagation of antiviral resistance.

Hepatitis C virus genotype 4: A poorly characterized endemic genotype

“…Using a chimeric JFH1-replicon in which NS5A originates from GT4r, Nguyen et al 52 reported that L31M and Y93H confer resistance to LDV. Pham et al 58…”

“…[79][80][81] Attempts to develop infectious models for other genotypes and subtypes made very limited success and unlike JFH1, all other isolates require cell-culture adaptation changes 82 as illustrated with the case of available GT4-based HCVcc. 58,83 The first model system was established by constructing a full-length ED43 and several mutants harboring ED43's replication-enhancing mutations (REM). 83 The introduction of these full-length constructs into Huh7.5.1 cells followed by a long-term passaging revealed additional mutations only for two clones releasing HCV core particles in the culture medium.…”

“…83 The latest GT4 full-length infectious clone is an ED43-based construct harboring a total of 32 mutations capable of promoting both replication and propagation mapped across the entire viral genome (except Core and P7) among which 5ʹ-UTR-G38A turned out to be paramount for substantial viral production in Huh7.5 cells. 58 Although the development of high replicative subgenomic replicon systems 71,72 has opened up avenues for the establishment of fully permissive cell culture systems; this hope was unfortunately abrogated when Pietschmann et al 84 demonstrated that the presence of REM mapped across the nonstructural region of the HCV genome are detrimental to virus production. This finding explains the advent of several JFH1-intra/intergenotypic chimeric genomes from which the J6/JFH1 system developed by a cis-fusion of the wild-type JFH1-replicase with the structural region of another GT2a strain, J6 83 turned out to be the most infectious chimeric system till today.…”

“…Using a chimeric JFH1‐replicon in which NS5A originates from GT4r, Nguyen et al 52 reported that L31M and Y93H confer resistance to LDV. Pham et al 58 used the infectious cDNA clone (ED43cc) to select RAS from a panel of DAAs. They documented that for NS3‐RAS, D168E and Y56H + D168A confer resistance to Paritaprevir; while A156T/V confer resistance to Grazoprevir and Glecaprevir.…”

“…The first infectious full‐length HCV clone (HCVcc) termed JFH1 was established in 2005 from a GT2a isolate 79–81 . Attempts to develop infectious models for other genotypes and subtypes made very limited success and unlike JFH1, all other isolates require cell‐culture adaptation changes 82 as illustrated with the case of available GT4‐based HCVcc 58,83 . The first model system was established by constructing a full‐length ED43 and several mutants harboring ED43's replication‐enhancing mutations (REM) 83 .…”