Construction of a reverse genetics and fluorescent reporter system for a bovine enterovirus isolated from cattle with diarrhea

Bovine enteroviruses are widely detected in cattle, but molecular tools for functional studies and antiviral screening remain limited. In this study, a bovine enterovirus strain, GXHC2317, was isolated from diarrheic cattle in Hechi, Guangxi, China and propagated in BHK-21 cells. Full-genome sequencing and phylogenetic analyses classified GXHC2317 as Enterovirus E genotype 3, and recombination analyses indicated a mosaic genome with regions related to both northern and local Chinese lineages. To enable genetic manipulation, we constructed a full-length infectious cDNA clone of GXHC2317 under a CMV promoter and successfully rescued a recombinant virus, rGXHC2317, which showed replication kinetics comparable to the parental isolate in BHK-21 cells. Using an oral infection model in 4-week-old ICR mice, rGXHC2317 and GXHC2317 displayed similar tissue distribution and broadly comparable histopathology. Furthermore, we generated a fluorescent reporter virus by inserting UnaG at the 5′UTR–VP4 junction. The reporter virus produced clear cytopathic effects and strong fluorescence, although its peak titer was reduced compared with the parental strain. Finally, a fluorescence-based assay using rGXHC2317-UnaG enabled antiviral evaluation and distinguished inhibitory effects of ribavirin and pleconaril from the limited activity of chloroquine phosphate under our conditions. Together, these results provide a reverse genetics and fluorescent reporter platform for BEV that supports studies of viral biology and practical antiviral screening.