2006 Annual Report 4d.Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and the University of Illinois. Additional details of research can be found in the report for the parent project 5438-32000-023-00D. Using an expressed sequence tag (EST)-library-based antisense method of random gene inactivation and a phenotypic screen for limitation of African Swine Fever Virus (ASFV) replication in cultured human cells, six host genes whose cellular functions are required by ASFV were identified. These included three loci, BAT3 (HLA-B associated transcript 3), C1qTNF (C1q and tumor necrosis factor related protein 6), and TOM40 (translocase of outer mitochondrial membrane 40), for which antisense expression from a tetracycline (Tet)-regulated promoter resulted in reversible inhibition of ASFV production by >99%. The effects of antisense transcription of the BAT3 EST and also of expression in the sense orientation of this EST, which encodes amino acid residues 450 to 518 of the mature BAT3 protein, were investigated more extensively. Sense expression of the BAT3 peptide, which appears to reversibly interfere with BAT3 function by a dominant negative mechanism, resulted in decreased synthesis of viral DNA and proteins early after ASFV infection, altered transcription of apoptosis-related genes as determined by cDNA microarray analysis, and increased cellular sensitivity to staurosporine-induced apoptosis. Antisense transcription of BAT3 reduced ASFV production without affecting abundance of the virus macromolecules we assayed. Overall results reveal a novel collection of cellular genes previously not known to be required for ASFV replication. To further define the molecular functions of BAT3 in viral replication, monoclonal antibodies to detect protein levels and an RT-PCR to detect transcript levels are being developed for use with BAT3 sense and antisense expressing cell lines 1.3-3s and 1.3-4as, respectively.