A BOVINE CELL-LINE STABLY EXPRESSING PORCINE TUMOR-NECROSIS-FACTOR-ALPHA (TNF-ALPHA) - GROWTH-PROPERTIES AND PERMISSIVITY FOR PSEUDORABIES VIRUS-REPLICATION

To explore the feasibility of modifying the pathogenicity of pseudorab ies virus (PrV), we have undertaken a program to develop recombinant P rV carrying the porcine tumor necrosis factor alpha (TNF-alpha) gene. We have used a cloned genomic DNA fragment containing the entire porci ne 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 containin g a TATA-box and binding sites for other transcription factors. The re sulting TNF-alpha cassette has been inserted into a neomycin-selectabl e shuttle vector. This construct has been used to select MDBK cell lin es harbouring the TNF-alpha-carrying plasmids in order to verify the p roduction of biologically active TNF-alpha. One cell line thus obtaine d 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, indic ating that TNF-alpha expression in MDBK cells does neat abrogate their permissivity for virus replication. Our results show that constructio n of recombinant PrV expressing TNF-alpha should be possible using the MDBK cell line as host.