A bovine cell line stably expressing porcine tumor-necrosis-factor-alpha (TNF-α) : Growth properties and permissivity for pseudorabies virus replication

To explore the feasibility of modifying the pathogenicity of pseudorabies virus (PrV), we have undertaken a program to develop recombinant PrV carrying the porcine tumor necrosis factor alpha (TNF-alpha) gene. We have used a cloned genomic DNA fragment containing the entire porcine TNF-alpha gene (Chardon et al., 1991), and have deleted the 3' non-translated region which is supposed to be a tissue-specific regulatory sequence. We have also removed the 5' non-translated region containing a TATA-box and binding sites for other transcription factors. The resulting TNF-alpha cassette has been inserted into a neomycin-selectable shuttle vector. This construct has been used to select MDBK cell lines harbouring the TNF-alpha-carrying plasmids in order to verify the production of biologically active TNF-alpha. One cell line thus obtained secreted 20-30 pg/10(6) cells of active TNF-alpha after five days in culture and exhibited normal growth kinetics. PrV titers on this cell line were the same as titers on cells not expressing TNF-alpha, indicating that TNF-alpha expression in MDBK cells does not abrogate their permissivity for virus replication. Our results show that construction of recombinant PrV expressing TNF-alpha should be possible using the MDBK cell line as host.