PMCA Detection of CWD Infection in Cervid and Non-Cervid Species

描述（由申请人提供）：慢性消耗性疾病（CWD）是一种紧急的、有效传播的、致命的动物（鹿、麋鹿、驼鹿）朊病毒疾病，目前在美国15个州、加拿大2个省和韩国被发现。我们发现能够传播CWD的传染性朊病毒存在于CWD感染鹿的唾液、血液和尿液中，这可能解释了该疾病的横向传播。唾液和血液中CWD朊病毒的存在证实了朊病毒在生物体液中的感染性，并为通过更灵敏的体外检测方法进行死前检测提供了现实的希望，这构成了本应用的基础。使用现代检测方法（即免疫组织化学、western blotting、ELISA）直接检测鹿体液或排泄物中异常的CWD朊病毒蛋白（PrPRES/CWD）是不可能的。本研究拟采用序列蛋白错误折叠循环扩增技术（sPMCA）在感染鹿的唾液、血液、尿液和粪便中扩增和检测PrPCWD。直接体外检测PrPCWD将有助于症状前的死前诊断，提高对CWD的生态学理解，并有可能检测子宫颈产品中的朊病毒携带状态和污染。自然界中是否存在非宫颈型CWD感染宿主尚不清楚。同样，CWD是否对人类健康构成风险也不确定。为了开始评估CWD跨物种传播的可能性，我们建议使用sPMCA作为替代物来评估来自选定的非子宫颈物种的正常朊病毒蛋白在暴露于CWD朊病毒后是否可以转化为异常的PrPCWD亚型。这些信息将用于指导相关的体内接种和流行病学研究，以评估CWD物种屏障，确定自然界中潜在的储集宿主，并更好地估计CWD对人类或其他物种造成的潜在健康风险。

DESCRIPTION (provided by applicant): Chronic wasting disease (CWD), an emergent, efficiently transmitted, fatal prion disease of cervids (deer, elk, moose), is now identified in 15 states, 2 Canadian provinces, and Korea. We have found that infectious prions capable of transmitting CWD are present in the saliva, blood, and urine of CWD-infected deer, potentially explaining the lateral transmission of the disease. The presence of CWD prions in saliva and blood confirms prion infectivity in biological fluids and provides realistic hope for antemortem detection via a more sensitive in vitro assay, which forms the basis for this application. Direct detection of the abnormal CWD prion protein (PrPRES/CWD) in body fluids or excreta of deer has not been possible using contemporary assay methodologies (i.e. immunohistochemistry, western blotting, ELISA). Here we propose to employ serial protein misfolding cyclic amplification (sPMCA) to amplify and detect PrPCWD in saliva, blood, urine, and feces of infected deer. Direct in vitro detection of PrPCWD would enable pre-symptomatic ante-mortem diagnosis, improve ecologic understanding of CWD, and potentially enable detection of prion carrier states and contamination in cervid products. Whether a non-cervid reservoir for CWD infection exists in nature remains unknown. Likewise, whether CWD represents a risk to human health is uncertain. To begin to assess the potential for trans-species transmission of CWD, we propose to use sPMCA as a surrogate to assess whether the normal prion protein from selected non-cervid species can be converted to the abnormal PrPCWD isoform after exposure to CWD prions. This information will be used to direct correlative in vivo inoculation and epidemiologic studies needed to assess the CWD species barrier, identify potential reservoir hosts in nature, and better estimate potential human or other species health risks posed by CWD. PUBLIC HEALTH RELEVANCE: Chronic wasting disease (CWD) is a rapidly spreading fatal prion disease of deer and elk, probably spread through saliva, and now present in 15 states. Whether CWD may represent a risk to human health analogous to mad cow disease is unknown. Proposed here is a new more powerful test to detect CWD prions in saliva, blood, and excretions of infected deer-something not possible with current technology yet vital to controlling the spread of CWD among deer and potentially other species.