Our broad, long-term objectives are to assess and manage the risk posed to humans from continually evolving prions, specifically those causing chronic wasting disease (CWD), an uncontrollable contagious epidemic of cervids of uncertain zoonotic potential. Understanding the properties of emergent CWD strains, their origins, how they adapt and evolve, and their zoonotic threats are important goals. We propose four Specific Aims to address the hypothesis that conformational conversion of host prion protein (PrPC) and adaptive selection of the resulting infectious conformers (PrPSc) is influenced by variation at PrP residue 226, the singular primary structural difference in PrP among susceptible cervid species. Since we further propose that strain selection in non-CNS compartments influences CWD transmission, our expanded use of gene targeting (Gt) strategies for accurate physiological PrP expression is a key component of this proposal. Aim I will characterize the properties of emergent CWD strains. Our strategy builds upon evidence indicating that prion strains driving the current North American CWD epidemic evolved from unstable emergent strains. Aim II will explore the origins of CWD by addressing the hypothesis that atypical strains originated either from exposure to prions in sympatric species, or from stochastic spontaneous conversion of cervid PrPC. We expect that iterative passaging and adaptation will result in strains with properties consistent with established CWD. Aim III will explore the proposal that accurate physiological PrP expression in Gt mice makes them uniquely suited to study strain adaptation. We expect that different inoculation routes will select different strains, and that the properties of CWD prions in peripheral compartments are different to those in the CNS. Our demonstration of novel emergent strains and their demonstrated potential for adaptation increases uncertainties about CWD zoonosis. In Aim IV we employ newly optimized Gt mice expressing human PrP that we expect will provide an improved application to explore human prion disease pathogenesis, and an accurate means to assess the potential for zoonotic CWD transmission. Our findings will aid in combating the incurable lethality and unpredictable epidemic and zoonotic potential of CWD prions by understanding the means by which they propagate and exist as heritable strains with protean host range properties that adapt and evolve under selective pressure.