ABSTRACT Chronic wasting disease (CWD) affecting various species of cervids in North American and Northern Europe represents a serious problem, because it continues to propagate uncontrollably among wild and captive cervids. CWD appears to be very heterogeneous with multiple different strains and can be transmitted to other animal species. The risk of CWD transmission to humans is unknown which is a major concern because the number of sick animals and their geographical distribution is rapidly increasing. The mechanism by which CWD propagates so efficiently among cervids is also unknown. The main goal of this project is to utilize a set of highly innovative techniques to study the cellular, molecular and structural features of naturally occurring CWD strains and their potential for inter- species transmission, particularly focusing on the possibility that certain CWD strains may infect humans. We will also attempt to elucidate the atomic resolution structure of CWD prions using cryo-electron microscopy. The overarching hypothesis is that CWD exists as multiple strains in distinct individuals and even within the same individual in different brain cells and that inter- species transmission and zoonotic potential depend on the specific strain characteristics. The project is divided in the following specific aims: (1) Study the structural and molecular diversity of natural CWD strains and the high resolution three-dimensional structure of CWD prions. (2) Understand CWD prion strain diversity in single brain cells isolated by laser capture microdissection and subsequently amplified by PMCA. (3) Evaluate CWD inter-species transmission spillover potential and its effect on zoonotic potential. (4) Analyze the deer-human prion species barrier in vivo using chimeric mice harboring human and cervid neuronal cells. The studies included in this projects will address some of the most pressing questions regarding CWD, including (i) the CWD prion strain variability, (ii) the zoonotic potential of different CWD prion strains, (iii) the atomic resolution structure of infectious prions and the structural basis of prion strains, (iv) the cellular distribution of CWD prion strains in the brain and its gene expression consequences, (v) the spillover potential of CWD to other animal species, (vi) the potential role of intermediate species in the transmission of CWD prions to humans. The findings generated in this project will be essential to design measures to prevent further propagation of CWD, and to avoid the emergence of new diseases with potentially disastrous consequences.