Ali Baghian and Knostantin Kousoulas, Pathobiological Sciences, LSUSVM

Construciton of recombinant vesicular stomatitis viruses (VSV) expressing Bovine Coronavirus E, M and S Proteins

Genes encoding BCoV M, E, and S proteins were cloned into intermediate plasmids containing specific protein epitopes used for detection purposes. The 3X FLAG epitope was designed to be located at the 5’ end of S gene while the same 3X FLAG epitope was placed at the 3’ end of the M gene and V5 epitope was located at the 3’ end of E gene. E, M, and S genes with their respective epitopes were independently inserted between the G and L genes of plasmid pVSV-XN2 containing the entire VSV genome, thus making three different large plasmids named pVSV-E, pVSV-M, pVSV-S. VSV gene containing plasmids pBS-N, pBS-P and pBS-L were all under a T7 promoter which is essential for efficient expression of viral RNA. Cells were first infected for an hour with a Vaccinia virus possessing T7 polymerase and subsequently transfected with the mix of plasmids. Vaccinia virus was removed by filtration through 0.2 µ m filter, while the much smaller recombinant VSV was recovered and purified after serial passages. Infected cells were examined and proteins expressed with their antibody epitopes were visualized using immunofluorescence microscopy.