Rapid bioassay of cervid prions in PrP transgenic Drosophila: addressing the threat to animal and human health from Chronic Wasting Disease

Pron病是人类和其他动物的致命性神经退行性疾病。这些情况在物种内部和物种之间都是可以传播的。导致这些传染性脑部疾病的病原体是在神经元中发现的一种正常蛋白质的聚集形式，被称为普恩。检测普恩病毒感染性的唯一可靠方法是在实验动物中进行生物测定。由于具有人畜共患病的潜在性，动物病毒病对人类健康构成了威胁，牛的疯牛病和随后在人类中出现的vCJD突出了这一点。慢性消耗性疾病(CWD)是一种高度传染性和日益流行的致命的普里恩疾病的鹿及其近缘物种。CWD是一种潜在的人畜共患病，因为人类消耗的鹿骨肌中存在传染性普恩病毒。这一观点得到了一些研究的支持，这些研究表明，非人类灵长类动物可以通过实验感染CWD感染的宫颈脑组织。CWD曾被认为仅限于美国西北部，但现在已在欧洲发现，挪威和芬兰也发现了病例。为了控制这种疾病，挪威开始大规模扑杀野生驯鹿，而芬兰禁止出口活鹿，这两项行动对各自的鹿业产生了重大影响。这些CWD新疫源地的出现对包括英国在内的整个欧洲的Cervid和人类健康构成了重大的新的未满足的挑战。英国的疯牛病流行病突出表明，迫切需要及早开发一种易于处理的普里恩生物测定方法，以备在评估新的动物普恩病毒疾病对动物和人类健康的风险时，或在CWD的情况下，这是一种现有普恩病毒疾病的新宿主。为了制定基于风险的控制措施，遏制疾病在动物之间的传播，重要的是开发一种易于处理的Prion生物测定方法，该方法可以测量CWD感染宫颈组织和血液中的Prion感染性。此外，为了帮助预测这种疾病感染人类的能力，有必要在实验动物中进行比灵长类动物更道德上更可接受的CWD Prion生物测试。这两个问题都曾尝试使用转Cervid或人类PrP的小鼠，这为CWD生物检测提供了鹿和人的小鼠模型。这些小鼠的Prion生物检测包括将感染CWD的物质注射到实验小鼠中，并等待观察这些动物是否会发展成Prion疾病。小鼠生物化验可能需要数年时间才能完成，而且非常昂贵，因为长期饲养大量小鼠的成本很高。因此，有必要开发一种更快、更便宜的动物生物测定法来评估CWD普恩病毒的感染性，这与小鼠普恩生物测定法一样灵敏。在我们的实验室里，我们在果蝇黑腹果蝇身上建立了Pron疾病的模型，因为与大型实验动物和小鼠相比，它们相对容易操作，能够更快速地收集数据，并且饲养成本更低。在这里，我们将利用果蝇来开发一种灵敏的生物检测方法来检测CWD普恩病毒。为此，我们将Cervid或人类PrP基因引入果蝇，使它们能够产生分别在患有CWD的鹿或患有vCJD的人的大脑中聚集的蛋白质。从我们之前发表的研究中我们已经知道，当转基因果蝇被喂食具有感染性的普恩时，这种哺乳动物蛋白质将聚集在一起，并导致普恩疾病。我们将确定我们基于苍蝇的CWD Prion生物检测的灵敏度，包括它对宫颈血液进行生物检测的能力，并研究它模拟人类对CWD Prion的易感性的能力。我们估计，PrP转基因果蝇可以用小鼠Prion生物检测的10%(或更少)的成本来对接种物进行PrP生物检测。此外，我们基于苍蝇的普恩生物测试在6周内完成，而小鼠生物测试可能需要一年或更多年来评估相同的普恩接种。

Prion diseases are fatal neurodegenerative disorders of humans and other animal species. These conditions are transmissible both within and between species. The pathogen that causes these transmissible brain diseases is an aggregated form of a normal protein found in neurons and is referred to as a prion. The only reliable method to detect prion infectivity is by bioassay in experimental animals. Animal prion diseases are a threat to human health because of their zoonotic potential, highlighted by the BSE epizootic in cattle and subsequent emergence of vCJD in humans. Chronic wasting disease (CWD) is a highly contagious and increasingly prevalent fatal prion disease of deer, and related species. CWD is a potential zoonosis since infectious prions are present in cervid skeletal muscle, which is consumed by humans. This view is supported by studies that show non-human primates can be experimentally infected with CWD-infected cervid brain tissue. CWD was considered to be restricted to N. West America but has now been found in Europe with cases detected in Norway and Finland. In attempts to control the disease Norway initiated the large-scale cull of wild reindeer, while Finland banned the export of live deer, two acts that had a significant impact upon their respective cervid industries. Emergence of these new foci of CWD infection poses significant new unmet challenges to cervid and human health across Europe, including the UK. The BSE epizootic in the UK highlighted the critical need for early development of a tractable prion bioassay for preparedness in assessment of the risk to animal and human health from new animal prion diseases, or as in the case of CWD, new reservoirs of an existing prion disease. It is important to develop a tractable prion bioassay that measures CWD prion infectivity in tissues and blood from affected cervids in order to formulate risk-based control measures that curb the spread of the disease amongst animals. In addition, it is necessary to carry out further CWD prion bioassays in experimental animals that are more ethically acceptable than primates in order to help predict the ability of this disease to infect humans. Both of these issues have been attempted using mice transgenic for cervid or human PrP, which provided murine models of deer and humans for CWD bioassays. These mouse prion bioassays involve injecting CWD-infected material into experimental mice and waiting to see if these animals develop prion disease. The mouse bioassay may take years to complete and is very expensive because of the cost of keeping large numbers of mice for a long period of time. Consequently, there is a need to develop a faster, cheaper, animal bioassay to assess CWD prion infectivity, that is as sensitive as the mouse prion bioassay. In our lab, we model prion disease in the fruit fly Drosophila melanogaster because they are relatively easy to work with, enable more rapid data collection and are less expensive to keep compared to large experimental animals and mice. Here, we will use Drosophila to develop a sensitive bioassay to detect CWD prions. To do so, we have introduced the cervid or human PrP gene into the flies that allows them to produce the protein that aggregates in the brain of deer with CWD or humans with vCJD, respectively. We already know, from our previously published studies, that this mammalian protein will aggregate and cause prion disease in transgenic Drosophila when flies are fed infectious prions. We will determine the sensitivity of our fly-based CWD prion bioassay, including its ability to bioassay cervid blood, and investigate its ability to model human susceptibility to CWD prions. We estimate that PrP transgenic Drosophila can be used to bioassay prion inocula at 10% (or less) of the cost of the mouse prion bioassay. Furthermore, our fly-based prion bioassay is completed within 6 weeks, whereas the mouse bioassay may require one or more years to assess the same prion inocula.