Development of Sensitive In Vitro Techniques for Prion Detection

APPROACH: Uninfected stable ovine and cervid cell lines, from genetically susceptible animals, will be developed and then assayed for the ability to accumulate PrPd after inoculation with the TSE agent. Cells will originate from tissues that accumulate PrPd in natural disease, namely reproductive tissue (placental trophoblasts) and brain (microglia and astrocytes). Preliminary work shows successful immortalization and culture of ovine microglia and demonstration of prion accumulation in the cell cultures following exposure to scrapie agent. The PrPd accumulating cells lines will be used for comparative transcriptomic experiments toward identifying accessory molecules for prion conversion and mechanisms of genetic resistance to prion conversion. Unique gene expression profiles in PrPd-accumulating cells will be identified using bovine gene chip expression arrays and specific prion conversion associated genes identified by RNA interference. The findings of the proposed study have applications to both human and veterinary medicine and will increase understanding of the pathogenesis of prion diseases by defining the genetic response to prion accumulation. Identification of cofactors involved in the accumulation of PrPSc will identify putative targets for therapeutic intervention and potential biomarkers of infection. Finally, the development of PrPSc-accumulating sheep cell lines could provide an in vitro infectivity assay for prion disease diagnosis and a model to further study pathogenesis using cells from a natural host species. Documents SCA with WSU.