2007 Annual Report 1a.Objectives (from AD-416) The objective of this cooperative research project is to define and characterize the function of cellular genes (VRG) essential for viral replication in swine cells. Identification of genes and cellular pathways affected by VRGs will provide fundamental information about cellular resistance mechanisms to viral infection. 1b.Approach (from AD-416) Cellular genes (VRG) essential for replication of foot-and-mouth disease virus (FMDV) and African swine fever virus (ASFV) have been identified using homozygous knock-out cells. Here, cellular pathways affected by VRG function and associated with resistance to viral infection will be identified. VRG will be cloned in the appropriate orientation (sense or anti-sense) in mammalian expression vectors and transfected into cells to generate stably expressing swine cell lines. Levels of target protein will be determined by Western blot or immunoprecipitation using monospecific antibodies raised to the proteins. Anti-sense RNA levels will be determined by real time RT-PCR. Cell clones with high, moderate and low levels of target gene expression will be further evaluated. Cellular genes and pathways affected by VRG function will be defined by comparing transcriptional profiles of VRG containing cells to that of parental control cells. In these experiments, mRNA from VRG expressing cells will be compared to mRNA from control cells using swine DNA microarrays (containing 7,000 selected genes) to analyze changes in host gene expression and to identify the cellular pathways affected by individual VRG expression. Real time PCR or Northern blot analysis of selected genes will also be used to confirm the level of expression of selected critical genes. Clustering programs will be used to group the genes that are differentially co-expressed, and genes will be clustered based on functions such as inflammation, chemotaxis, transcription, apoptosis, signal transduction, extracellular matrix, cell cycle and metabolism. Biochemical databases will be searched to determine the functional relationship between these genes (http://www.genome.ad.jp/kegg/kegg4.html). All experimental designs will be compliant with standards suggested in the Minimal Information About a Microarray Experiment (MIAME) 1.1 document (www.mged.org/Workgroups/MIAME/miame_1.1.html) as a means to ensure reproducibility and standardized interpretation of microarray data. 3.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 in-house project 5438-32000-029-00D, Genetic and Biological Determinants of Respiratory Disease Susceptibility. Using an expressed sequence tag (EST)-library-based antisense method of random gene inactivation and a phenotypic screen for inhibition of FMDV replication in cultured bovine BK cells, four host genes, IRF7, SPP, ectonucleoside triphosphate diphohydrolase 6 (NTPDase. 6)and EHBP1, whose cellular functions are required by FMDV were identified and confirmed. NTPDase 6, a cellular gene not previously associated with RNA viral replication, was required for normal synthesis of FMDV RNA and viral protein synthesis. Bacterial protein expression systems were optimized for expressing identified target proteins for mono-specific antibody production. The coding regions of selected genes were amplified by PCR and cloned into the pCMV-Tag4 mammalian expression vector (Stratagene) containing a tag FLAG epitope to facilitate the detection of expression by anti-FLAG specific antibody. Conditions were optimized for expression of the tagged proteins, in transfected HeLa cells and proteins were detected by western blot using an anti-FLAG M2 antibody. Currently, proteins are being expressed/purified in sufficient quantities for animal immunization. Additionally, we are now attempting to obtain stable HeLa cell lines expressing the tagged proteins of interest by selecting for neomycin resistance. RNA extraction and purification procedures for examining transcriptional profiles in resistant (target gene-inactivated) cell lines were optimized and adapted for working with small cell numbers. Monitoring activities include telephone conversations and emails. Review Publications Chang, A.C., Zsak, L., Feng, Y., Mosseri, R., Lu, Q., Kowalski, P., Zsak, A., Burrage, T.G., Neilan, J.G., Kutish, G.F., Lu, Z., Laegreid, W.W., Rock, D.L., Cohen, S.N. 2006. Phenotype-based identification of host genes required for replication of African swine fever virus. Journal of Virology. 80(17):8705-8717.