Use of PrP transgenic Drosophila to measure mammalian prion infectivity

朊病毒疾病总是致命的脑神经退行性疾病，影响人类和其他脊椎动物物种。这些疾病包括人类的克雅氏病、牛的疯牛病和羊瘙痒病。朊病毒疾病发生时，一种蛋白质称为PrP经历了形状的变化，并聚集在大脑中，它损害神经元。虽然其他神经退行性疾病也通过其自身特定的蛋白质错误折叠事件发生，但朊病毒疾病是独特的，因为它们在同一物种的个体内以及不同物种之间都是可传播的。在朊病毒病期间，与这些病症相关的可传播感染因子（被认为是PrP的错误折叠形式）在受影响个体的大脑中积累。这种传染因子被称为朊病毒。朊病毒通过其从动物传播到人类的可能性对公共卫生构成重大风险，这可以从英国牛的BSE和随后在人类中出现的变异CJD中看出。朊病毒疾病最常见的传播途径是通过口服朊病毒感染的物质，即无意中食用朊病毒。以这种方式感染的个体在淋巴组织和血液中积累朊病毒感染性。确定朊病毒感染个体的大脑、不同组织和血液中存在多少朊病毒感染性是很重要的。这对于了解传染性朊病毒的生物学、确保人类消费的动物产品的安全以及帮助建立针对这些疾病的血液检测是必要的。朊病毒是一种非常规传染病原，检测其存在的唯一可靠方法是用朊病毒感染的样本接种实验动物，看看接受者是否会患上朊病毒疾病。这不可避免地导致大量实验动物，特别是小鼠和绵羊，被用来评估哺乳动物朊病毒的感染性，结果死于终末期朊病毒病。该提案旨在尽可能减少和取代使用小鼠和绵羊来测量朊病毒对无脊椎动物宿主果蝇的感染性。反过来，这将有助于改进朊病毒感染性实验，即使用感知能力较弱的动物，这些动物不知道是否具有疼痛系统或与哺乳动物宿主相当的意识水平。我们将在果蝇中模拟羊瘙痒症。我们已经培育出转绵羊朊蛋白基因的果蝇，并证明这些果蝇对绵羊朊蛋白的毒性作用敏感。成年苍蝇，作为幼虫喂养朊病毒感染性，显示出移动能力下降，并显示出错误折叠形式的PrP在它们的大脑中的存在。在这项提议的实验中，我们打算验证我们的新朊病毒苍蝇模型的灵敏度，以确定我们的新动物模型在多大程度上可以取代小鼠和绵羊测量朊病毒感染性。我们使用PrP转基因果蝇将产生人道的，高质量的，可重复的朊病毒为基础的科学，同时协助3R战略。使用PrP转基因果蝇将产生高质量的统计学显著性数据。可以很容易地生产和圈养大量的苍蝇，并且可以使用经济上可行的模型同时测试相关数量的复制品。我们以前使用哺乳动物宿主研究传染性朊病毒疾病的经验将使我们能够直接比较和突出使用PrP转基因果蝇所获得的好处，因此我们可以明智地告知其他科学家，并鼓励减少对小鼠和绵羊朊病毒生物测定的依赖。我们将使用先前生成的朊病毒感染样本在我们的果蝇模型中进行测试，因此无需向其他实验动物注射朊病毒材料以获得本研究中使用的样本。我们有许多这些样品的初步测试结果，因此它们可以直接与我们用PrP转基因果蝇产生的结果进行比较。

Prion diseases are invariably fatal neurodegenerative diseases of the brain that affect humans and other vertebrate species. These diseases include CJD of humans, BSE of cattle and scrapie of sheep. Prion diseases occur when a protein termed PrP undergoes a change in shape, and aggregates in the brain where it damages neurons. While other neurodegenerative diseases also occur through their own specific protein misfolding event, prion diseases are unique because they are transmissible, within individuals of the same species and also between different species. During prion disease, the transmissible infectious agent associated with these conditions, which is thought to be the misfolded form of PrP, accumulates in the brain of affected individuals. This infectious agent is termed a prion. Prions are a significant risk to public health through their potential to spread from animals to humans, seen by the appearance of BSE in UK cattle and subsequent emergence of variant CJD in humans. The most common route of transmission of prion diseases is by oral inoculation with prion-infected material i.e. by inadvertently eating prions. Individuals that become infected in such a manner accumulate prion infectivity in lymphoid tissue and blood. It is important to determine how much prion infectivity is present in the brain and different tissues, and blood from prion-infected individuals. This is necessary to understand the biology of infectious prions, to ensure the safety of animal products destined for human consumption, and to help to establish a blood test for these diseases. The prion is an unconventional infectious agent and the only reliable method to detect its presence is to inoculate experimental animals with prion-infected samples and see if the recipients develop prion disease. Inevitably, this has led to a large number of experimental animals, in particular mice and sheep, being used to assess mammalian prion infectivity, and as a consequence, dying from terminal prion disease. This proposal aims to reduce and replace, where possible, the use of mice and sheep to measure prion infectivity with an invertebrate host, Drosophila melanogaster. This in turn, will help to refine prion infectivity experiments by using less sentient animals that are not known to have a pain system or a comparable level of awareness as mammalian hosts. We will model scrapie of sheep in Drosophila. We have already generated Drosophila transgenic for sheep PrP and have shown these flies are susceptible to the toxic effects of sheep prions. Adult flies, fed prion infectivity as larvae, show a decreased ability to move and show the presence of the misfolded form of PrP in their brains. In the experiments of this proposal, we intend to validate the sensitivity of our new prion fly model to determine the extent to which our new animal model can replace mice and sheep in measuring prion infectivity. Our use of PrP transgenic Drosophila will produce humane, high-quality, reproducible prion-based science whilst assisting the 3Rs strategy. Good quality, statistically significant data will be generated using PrP transgenic Drosophila. Large numbers of flies can easily be produced and housed, and relevant numbers of replicates can be tested at the same time using a financially viable model. Our previous experience using mammalian hosts to study infectious prion disease will enable us to directly compare and highlight the benefits gained by using PrP transgenic Drosophila so we can sensibly inform other scientists and encourage less dependency on mouse and sheep prion bioassays. We will use previously generated prion-infected samples to test in our Drosophila model so that no other experimental animals need to be injected with prion material in order to obtain samples for use within this study. We have preliminary test results from many of these samples so they can be directly compared with the results we generate with our PrP transgenic Drosophila.