Building capacity for in vitro prion amplification

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Chronic wasting disease (CWD) is a prion disease spreading inexorably among wild and farmed cervids in 26 US states, 3 Canadian provinces and Scandinavia. Prions are infectious agents that replicate by converting the host-encoded prion protein (PrPC) into the pathogenic isoform PrPSc. Its accumulation in the brain induces the pathogenesis resulting in progressive neurodegeneration and eventually death. Prion diseases are known in humans and animals. Risk of zoonotic transmission of CWD is considered low, but emergence of new CWD strains, conformers of PrPSc that can have distinct host ranges, is of huge concern. Transmission to caribou is very likely to occur soon, and this will be detrimental to the already declining populations. Hence, developing means to control and detect CWD infection and investigating molecular mechanisms of CWD strain evolution are critical to contain the disease and mitigate transmission.***The equipment requested in this application will directly benefit Dr. Sabine Gilch's and Dr. Hermann Schaetzl's NSERC Discovery programs. Dr. Gilch validates in vitro prion amplification assays as a new tool for non-invasive and pre-mortem detection of CWD using fecal or lymphatic tissue samples, and investigates CWD strain evolution. Dr. Schaetzl proposes to develop a vaccine for CWD and analyse the effect of vaccination on prion shedding in feces and urine. There is an urgent need to build capacity for in vitro prion amplification, which is based on the templated conversion of a PrPC substrate upon interaction with PrPSc seeds. One method is real-time quaking induced conversion (RT-QuIC), a highly sensitive method which can amplify minute amounts of PrPSc (femtogram range) to detectable levels using recombinant PrP as a substrate and a real-time fluorescence read out for detection. Therefore, a fluorescence reader with capability to run cycles of intermittent shaking and resting which induces exponential amplification of PrPSc is necessary. The second method is protein misfolding cyclic amplification (PMCA). Here, sonication induces efficient conversion of PrPC. The equipment set up consists of a sonicator, a microplate horn, a circulating water bath and an external temperature sensor. PMCA allows to recapitulate prion strain properties, transmission barriers and generates infectious prions, while RT-QuIC is a very robust and sensitive method for prion detection which does not generate bona fide infectious prions. ***Because of the limitations of each method, setting up equipment for both techniques is needed to significantly enhance our output in high-impact publications, raise our international recognition and form the basis for new collaborations. Our HQP will benefit from these opportunities and be able to produce additional relevant data and become more competitive on the labor market in Canada.**