Background: Mouse bioassays remain the gold standard for the proof of infectivity, strain typing and titre estimation in ruminant TSE research. An alternative approach employing ex vivo cell-based assays is now however a real possibility. The first tissue culture to support the propagation of PrPSc directly from ovine scrapie was developed by modifying a rabbit kidney cell line to express ovine PrP of a susceptible genotype (Rov cell line, Vilette et al 2001). Aims / Objectives: The primary aim was to identify cell lines highly permissive to ovine scrapie by further investigation of existing cell lines with proven ability to propagate sheep scrapie. Methods: Rov9 cells were subcloned by limiting dilution and permissiveness to infection with SSBP/1, CH1641 and 127S scrapie isolates assessed using the Elispot assay (Klöhn et al; 2003) optimised for Rov cells. Subclones found to be highly permissive to infection with experimental strains of scrapie were further challenged using field case samples. Results: After two rounds of subcloning and screening using Elispot, we have identified subclones of the Rov9 cell line that are more permissive to infection with the SSBP/1 strain of scrapie than the parental line. PrPSc expressing cells could be detected in the parental cell line when infected with a 0.1% solution of SSBP/1 infected brain homogenate, whereas with the most permissive subclones PrPSc infected cells could be detected after exposure to scrapie brain homogenate at least 100 x more dilute. Furthermore, the most permissive subclones have been challenged with field case samples of scrapie and found to be permissive to infection with 2 out of 7 cases tested so far. To date, no cell lines have propagated the CH1641 isolate. Discussion: The 96 well Elispot platform is a useful tool for screening multiple subclones in parallel for de novo scrapie infection. By subcloning the Rov9 cell line we have generated subclones that are over 100x more sensitive to infection with the SSBP/1 strain of scrapie using this methodology. At present these cells are only permissive to a small number of scrapie field cases, further development and characterisation is required to assess a wider range of natural scrapie cases and subclones. This work was funded by DEFRA