Building capacity for in vitro prion amplification

慢性消耗性疾病（CWD）是一种朊病毒疾病，在美国26个州、加拿大3个省和斯堪的纳维亚半岛的野生和养殖猪中不可避免地蔓延。朊病毒是一种传染性病原体，通过将宿主编码的朊病毒蛋白（PrPC）转化为致病性的PrPSc亚型来进行复制。它在大脑中的积累诱发了发病机制，导致进行性神经变性和最终死亡。已知朊病毒疾病存在于人类和动物中。CWD人畜共患传播的风险被认为很低，但出现新的CWD菌株（PrPSc的构象，可以有不同的宿主范围）是非常令人担忧的。很可能很快就会传染给驯鹿，这将对已经在减少的驯鹿数量造成不利影响。因此，开发控制和检测CWD感染的方法以及研究CWD菌株进化的分子机制对于控制疾病和减轻传播至关重要。***本申请中所要求的设备将直接使Sabine Gilch博士和Hermann Schaetzl博士的NSERC发现计划受益。Gilch博士验证了体外朊病毒扩增检测作为一种新的工具，可以使用粪便或淋巴组织样本进行非侵入性和死前检测CWD，并研究了CWD菌株的进化。Schaetzl博士建议开发一种CWD疫苗，并分析疫苗接种对粪便和尿液中朊病毒脱落的影响。目前迫切需要建立基于PrPC底物与PrPSc种子相互作用后模板转化的体外朊病毒扩增能力。一种方法是实时振动诱导转换（RT-QuIC），这是一种高灵敏度的方法，可以将微量的PrPSc（飞图范围）扩增到可检测的水平，使用重组PrP作为底物和实时荧光读数进行检测。因此，需要一种能够运行间歇振荡和静息循环的荧光阅读器，以诱导PrPSc的指数扩增。第二种方法是蛋白质错误折叠循环扩增（PMCA）。在这里，超声诱导了PrPC的有效转换。所设置的设备包括一个声纳器、一个微孔板喇叭、一个循环水浴和一个外部温度传感器。PMCA可以重现朊病毒株特性、传播屏障并产生感染性朊病毒，而RT-QuIC是一种非常稳健和敏感的朊病毒检测方法，不会产生真正的感染性朊病毒。由于每种方法的局限性，需要为两种技术设置设备，以显着提高我们在高影响力出版物中的产出，提高我们的国际认可度并为新的合作奠定基础。我们的HQP将从这些机会中受益，并能够产生更多的相关数据，从而在加拿大的劳动力市场上更具竞争力

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.**