GFP-Tagged E^rns in Bungowannah Pestivirus: A Tool for Viral Tracking and Functional Studies

Pestiviruses, such as bovine viral diarrhea virus (BVDV) or classical swine fever virus (CSFV), are members of the family Flaviviridae and infect a broad range of species, causing significant economic losses in livestock. A unique feature of pestiviruses is the E^rns protein, which is part of the glycoprotein complex at the surface of the virion, but it is also secreted as an RNase that functions as an interferon (IFN) antagonist. This dual nature makes E^rns a particularly complex and multifunctional protein, highlighting its importance for understanding pestivirus biology. Bungowannah pestivirus (BuPV) was reported to exhibit high genetic plasticity, making it suitable for engineering recombinant tools. In this study, we generated a recombinant BuPV expressing green fluorescent protein (GFP) fused to the N-terminus of the E^rns protein from BVDV. The GFP-E^rns fusion was detected by fluorescence microscopy and remained stable across five serial passages. The recombinant virus infected all tested mammalian cell lines but replicated more slowly than the parental BuPV stock. RNase activity assays confirmed retention of enzymatic function. These results demonstrate stable expression, broad infectivity, and preserved activity of GFP-E^rns in the recombinant BuPV, indicating that this might be a useful tool for further investigations on pestivirus pathogenesis.