Generation and characterization of an Npro-disrupted marker bovine viral diarrhea virus derived from a BAC cDNA

In vitro studies showed that N(pro) protein of bovine viral diarrhea virus (BVDV) interferes with cellular antiviral defense. To understand the role of N(pro) protein in successful viral invasion of the host and establishment of the lifetime persistence, an infectious N(pro)-disrupted virus with a noncytopathic (NCP) background is desired. In this study, an N(pro)-disrupted cDNA, pBSD1-N(pro)/eGFP2A, was constructed based on an infectious full-length BAC cDNA clone of NCP BVDV strain SD1, pBSD1. In this clone, whole N(pro) gene except its first 57 nucleotides (nt) was in frame substituted with an eGFP2A sequence. eGFP2A was constructed by in frame fusing a foot-and-mouth disease virus 2A protease (FMDV 2A(pro)) to C-terminus of eGFP. Intramolecular cleavage of FMDV 2A(pro) at its C-terminal glycine-proline dipeptide will release the viral nucleocapsid protein from the nascent viral polyprotein and the processed eGFP2A protein will then act as a marker protein. The resulting BAC cDNA clone was propagated stably for at least 10 passages in E. coli strain XL1-blue as determined by sequencing the progeny plasmids. The rescued virus, BSD1-N(pro)/eGFP2A, showed a peak virus titer approximately 1.2 log(10) lower and a maximum virus yield about 20 hr later than wt SD1, respectively, and was similar to wt SD1 in viral RNA replication and protein expression. FACS, fluorescent microscopy and western blotting assays confirmed that functional eGFP2A protein was expressed and processed properly in MDBK cells. In summary, the availability of BSD1-N(pro)/eGFP2A with a stable viral genome would facilitate the investigation of the role of N(pro) protein in transplacental transfer of BVDV and establishment of persistent infection in bovine fetus.