Project Number: 5438-32000-029-03
Project Type: Specific Cooperative Agreement

Start Date: Sep 15, 2005
End Date: Mar 14, 2009

Objective:
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.

Approach:
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.