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Investigate the usefulness of an in vitro prion protein (PrP) conversion assay as a means to assess in vivo interspecies transmissibility of transmissible spongiform encepahlopathy (TSE) among various livestock and wildlife species. This assay estimates the efficiency with which the abnormal isoform of this protein, PrP**Sc, can subvert conformation of the isoform found in normal brain (PrP**C).
Preliminary research in the cooperator's laboratory has shown that proteins produced in recombinant bacteria, even though nongycosylated and refolded, can be successfully used in this conversion assay. Comparative results showed that the assay accurately reflects differences in interspecies transmissibility and incubation times in mice and hamsters experimentally exposed to different transmissible spongiform encephalopathy (TSE) strains. To validate the assay for species naturally affected by TSEs, sheep PrP**C containing gene polymorphisms important for scrapie susceptibility/resistance will be used to assess conversion by PrP**Sc extracted from bovine spongiform encephalopathy (BSE) brains. Results of these assays can then be compared to experimental transmissions of BSE into the sheep of the same genotypes, work that is in progress at the cooperator's laboratory. The second phase of work will concentrate on similar evaluations of PrP polymorphisms that occur in the cervid species affeced by a TSE known as chronic wasting disease (CWD). The assay will be used to investigate potential transmissibility of BSE, scrapie, and CWD between the various natural host species. These results will then be compared to results from a variety of in vivo transmission experiments in progress or planned by the cooperator or the ARS.
What were the most significant accomplishments this past year? D. Progress Report. This report serves to document research conducted under a specific cooperative agreement between ARS and the Institutes for Animal Health (IAH), Edinburgh, UK. Additional details of research can be found in the report for the parent project 3625-32000-066-00D, Bovine Spongiform Encephalopathy and Other Transmissible Spongiform Encephalopathies. Bacterial recombinant PrP (rPrP) of the three most frequently occurring sheep PrP variants, ARR, ARQ and VRQ has been cloned, radiolabelled during expression, purified, refolded into a normal PrPc-like form and used as substrate in our novel, non-guanidine cell-free conversion assay. Initial experiments established that sheep rPrP could be converted, by sheep PrPSc isolated from sheep of the ARQ and VRQ genotypes, to PrPres. They showed clear differences in their convertibility, mimicking the pattern of transmissibility observed in vivo. This data demonstrates the suitability of the assay to reveal the association of sheep PrP variants to transmissible spongiform encephalopathies (TSEs) from other species. It is essential to investigate the potential transmission of TSEs between cattle, sheep and cervids. To this end, two cervid PrP genotypes have been cloned, expressed and characterised. These will be used, alongside sheep and bovine PrPSc and further cervid material in the cell-free assay.