Development of a rapid antiviral screening assay based on GFP reporter virus of bovine enterovirus

In recent years, bovine enterovirus (BEV) has been increasingly associated with diarrhea in cattle in China, posing new challenges for disease control in the cattle industry. However, the mechanisms underlying BEV pathogenesis and virulence remain poorly understood. Infectious cDNA clones provide a powerful tool for dissecting viral replication and pathogenic mechanisms. In this study, we generated a full-length infectious cDNA clone of the BEV-F isolate HB19-1. Three overlapping fragments spanning the complete viral genome were amplified by RT-PCR and assembled downstream of a cytomegalovirus (CMV) promoter placed immediately upstream of the 5' untranslated region (5'UTR). To establish a reporter virus system, the green fluorescent protein (GFP) gene was inserted between the 5'UTR and the N terminus of VP4, followed by a 2A cleavage sequence (IKTAG) at the C terminus of GFP. The recombinant rHB19-GFP virus was successfully rescued. Growth curve analysis demonstrated that rHB19-GFP exhibited slower replication kinetics at early time points relative to the parental HB19-1 virus, with no significant difference in their peak viral titers. This GFP-expressing reporter virus enables convenient monitoring of BEV replication and provides a useful platform for antiviral screening. Using this system, we found that 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) inhibited BEV replication, suggesting its potential as an antiviral candidate. Overall, the rHB19-GFP infectious clone developed here offers a practical tool for studying BEV biology and for identifying antiviral compounds against BEV.