Serial viral passage and reverse genetics protocol for identifying the antiviral drug targets of EV-D68 antivirals

Identifying antiviral targets and mechanisms is crucial for drug discovery. Here, we present a protocol to select EV-D68 variants resistant to Jun6504 by combining serial passage and reverse genetics. We describe steps for viral serial passage, sequencing, and viral genome mutagenesis. We then detail procedures for producing and characterizing recombinant viruses. This protocol provides a robust strategy to identify targets from phenotypic screening hits.