Prion diseases are inevitably fatal neurodegenerative zoonotic disorders of animals, including humans, with noknown treatment or cure. Prions are comprised largely, if not entirely, of PrPSc, a misfolded form of the normalnon-infectious prion protein PrPC. Prion strains are operationally defined by differences in neuropathology thatbreed true under controlled conditions that are encoded by strain-specific conformations of PrPSc. The long-term goal of this work is to understand the dynamics of prion strains. The objective of this application is todetermine if prion strains exist as a mixture of substrains or as a homogeneous population. We hypothesizethat prions strains are a dynamic mixture of prion substrains (i.e. quasispecies). This would fundamentallyimpact our understanding of the mechanism of interspecies transmission, adaptation of prions in response toanti-prion drugs, the ecology of prion transmission in natural settings and may be relevant to other proteinmisfolding diseases that share prion strain-like features (e.g. Alzheimer?s). Since the structure of PrPSc ispoorly defined and technologies do not exist to measure the structure of an individual protein in a mixture, wewill test the hypothesis based on the predicted properties of quasispecies that are experimentally feasible. Firstwe will determine the effect of repeated limiting dilutions on prion fitness. Repeated bottleneck passage of aquasispecies leads to a reduction in fitness (i.e. Muller?s ratchet) since, on average, the fitness of an individualinfectious unit is lower compared to the fitness of the overall population. We anticipate that repeatedtransmission of prions at limiting dilutions in PMCA or in cell culture will result in a reduction in the fitness of theprion agent compared to agent passaged at high titer. Second we will establish if biologically cloned prionstrains contain substrains. Quasispecies hypothesis predicts that any given prion strain is comprised of adynamic population of substrains. We will test this by amplifying PrPSc in vitro or passaging in cells underconditions that favor the selection of a minor substrain. In a second series of experiments, we will select forsubpopulations of PrPSc that have common shared biochemical features to seed PMCA reactions or prionsusceptible RK13 cells. Serial repeated rounds of selection followed by transmission in animals will determineif biologically cloned prion strains contain additional strains and if biochemical selective pressure results in theemergence of strains with the selected properties. The results of these experiments will directly test thehypothesis that prions are quasispecies.