Transmissible spongiform encephalopathies, such as variant Creutzfeldt-Jakob disease in humans and 'mad cow disease' in cattle are caused by an infectious agent called 'prion'. Prions consist mainly, if not exclusively, of PrPSc, a multimer consisting of misfolded form(s) of the ubiquitous host glycoprotein PrPC. Replication of PrPSc is believed to follow the 'seeding model', whereby infecting PrPSc accrues host PrPC molecules that change their conformation as they add onto the PrPSc aggregates. Expression of PrP is essential but not sufficient for the propagation of prions; it is not known which other components are required.<P> Prions affecting any one organism can occur in the form of many distinct strains which show quite different phenotypic properties, such as incubation time between infection and appearance of clinical disease, the location of lesions and sites of PrPSc accumulation in the brain. The fact that many different prion strains can be propagated indefinitely in the same inbred line of mice homozygous for the PrP gene is explained by the proposal that the strain is encoded by conformation of PrPSc. Prions can be propagated in vitro, but only in selected cell lines. In addition, cell lines are specific in their capacity to replicate some prion strains but not others, which may reflect the specificity of brain regions for this process. <P> The aims of this project are to perfect the Scrapie Cell Assay (SCA), which allows the sensitive and rapid quantification of prions in cell culture rather than by titration in mice, to establish a panel of cell lines that allow prion strain identification by the SCA, to ascertain the fidelity of prion strain replication in cell culture and in cell-free systems by the Protein Misfolding Cyclic Amplification (PMCA). Using the tools we are developing, we intend to identify the components other than PrPC that are necessary to allow prion replication and strain recognition by cell lines (and ultimately tissues).<P> The identification of components essential for prion replication will provide additional targets, beyond PrP, for prophylactic and therapeutic interventions against prion diseases.