Construction of enterovirus G expressing reporter genes for antiviral drug screening assays.

Abstract

The emergence of Enterovirus G strains harboring recombinant papain-like protease (EV-G-PLP) poses a significant threat to the global swine health with zoonotic potential. Addressing the critical lack of targeted treatments, we developed a reverse genetics system for the CH/20GXNN/PLP2020 strain and generated three isogenic reporter recombinants (GFP/RFP/iLOV) through precise PLP gene substitution. Viral progeny exhibited parental-like replication kinetics, with r20GXNN-iLOV demonstrating genetic stability (> 10 passages) compared to GFP/RFP variants. Plaque phenotyping revealed an inverse correlation between plaque size and insert length at the 2C/3A junction. Leveraging this system, we engineered a high-throughput screening platform identifying four anti-EV-G compounds: niclosamide (0.02 µM), salinomycin (2 µM), chloroquine phosphate (12 µM), and ribavirin (400 µM). This study establishes a reverse genetics platform enabling picornavirus research advancement and reveals structural plasticity in the 2C/3A junction that facilitates antiviral screening. Furthermore, it identifies EV-G-specific drug candidates while providing a framework for pan-picornaviral (including EV-A71) compound discovery.