BACKGROUND: Bovine viral diarrhea virus (BVDV) causes significant economic and health effects in cattle farms. Because of the relative inefficiency of BVDV eradication programs, in recent years, numerous strategies such as anti-viral gene therapy has been used extensively to fight it. One of the ways to evaluate the quality of these anti-viral strategies is the study of their efficacy in the specific cell lines expressing those viral genes through the induction of gene expression by transfection of plasmids or infection of viral vectors. OBJECTIVES: This study was performed for preparation of a cell line expressing some portions of BVDV genome to evaluate the efficacy of preventive and therapeutic strategies against this virus. METHODS: After the culture of BVDV NADL, vRNA was extracted and proliferation of 5'UTR and NS3-coding genes and cloning of these gene segments was performed in the upstream of GFP gene in pWPI-linker B lentivirus plasmid. After confirmation of cloning, lentiviral vector containing BVDV-NS3 and BVDV-5'UTR were generated in 293T cells using third-generation packaging system. Then MDBK cells persistently express 5'UTR and NS3 were generated by the infection with lentiviral vectors containing BVDV-NS3 and BVDV-5'UTR. RESULTS: The efficacy of infection with lentiviral vectors carrying transgenes, examined by fluorescence microscopy, Western blotting and RT-PCR tests, were compared with control groups and the presence and expression of the above mentioned genes were confirmed. CONCLUSIONS: The results indicate that the successful production of a MDBK cell line expressing both genes of BVDV and lentiviral vectors are preferred to the others for their persistent and long-term gene expression in several cell generations and the ability to infect many different cell lines.