Prion diseases are a group of infectious neurodegenerative disorders affecting humans and animals. Although rare diseases, the recent outbreak of Bovine Spongiform Encephalopathy and Chronic Wasting disease and the transmission of the disease from cattle to humans have risen a great concern about a possible epidemic of Creutzfeldt-Jakob disease. This problem is aggravated by many uncertainties surrounding the unprecedented nature of the infectious agent, its mechanism of propagation and the species barrier that seems to control prion transmission. The most accepted hypothesis proposes that the infectious agent (termed prion) is composed exclusively by a misfolded version of a normal protein and does not contain any nucleic acid. According to this hypothesis, the disease is transmitted by propagation of the misfolding from the disease associated isoform (termed PrPres) to the normal host protein (termed PrPc), which become converted into the pathological form. We have recently described a procedure to induce the conversion of PrPc into prPres in vitro starting with minute quantities of brain PrPres. This procedure, named Protein Misfolding Cyclic Amplification (PMCA) mimics the process of prion replication in vivo, but at an accelerated speed resulting in an exponential amplification of the initial amount of PrPres. The major goal of this project is to take advantage of the PMCA technology to attempt developing a highly sensitive and noninvasive diagnosis of prion diseases as well as to study diverse aspects related to the nature of the infection agent, other factors involved in prion conversion and the transmission between species. In specific aim 1 we will study the infectious and structural properties of prPres generated in vitro with the objective to attempt multiplying and producing infectivity in the test tube. This experiment is widely considered as the final pending proof for the prion hypothesis. In specific aim 2 we will attempt to identify cellular protein factors that seem to play a major role in prion replication in vivo. Specific aim 3 proposes to study the species barrier phenomenon and the influence of PrP polymorphisms in the efficiency of prion replication in vitro. In specific aim 4 we will develop a highly-sensitive diagnostic test to detect PrPres in blood of experimental animals, cattle and humans, based on amplifying minute quantities of the pathological protein present in blood to a level that enable reproducible detection. Therefore, this project offers a balanced combination between basic science studies aimed to understand the most relevant scientific problems in the prion field and applied studies to resolve the main practical problem associated to these diseases, which is the lack of a highly-sensitive pre-symptomatic blood diagnosis to limit the spreading of these incurable illnesses.