Infection/transfection scheme for generation of recombinant ORFV. A. Construction of the recombinant transfer vector pSPV-EGFP. A cassette of selectable markers of E.coli neo and gusA genes in pZIPPY-neo/gus vector was replace by the EGFP reporter gene amplified from pEGFP-N1 vector (Clontech, CA) to generate the recombinant vector pSPV-EGFP. B. Generation of recombinant ORFV. OFTU Cells are infected with OV-IA82 and transfected with the transfer vector pSPV-113LF-EGFP-113RF. The resultant virus mixture is then plated on OFTu cells to eliminate OV-IA82 and the desired viruses were isolated. MCS: Multiple cloning sites. B: BglII; E: EcoRI; H: HindIII; N: NotI; and X: XhoI. V: vaccinia virus (strain WR) VV early/late protomer VVp7.5. L: Up stream of ORFV113 un-transcription region; R: Down stream of ORFV113 un-transcription region. DHR: double homologous recombination.