Use of PrP transgenic Drosophila to measure mammalian prion infectivity

朊病毒疾病总是致命的大脑神经退行性疾病，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、牛的疯牛病和羊的痒病。当一种被称为PrP的蛋白质发生形状变化并聚集在大脑中损害神经元时，就会发生朊病毒疾病。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具有传染性。在朊病毒疾病期间，与这些疾病相关的传染性病原体（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。朊病毒具有从动物向人类传播的潜力，对公共卫生构成重大风险，英国牛中出现疯牛病以及随后在人类中出现变异型克雅氏病就是明证。朊病毒疾病最常见的传播途径是口服接种朊病毒感染物质，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了朊病毒传染性。确定朊病毒感染个体的大脑和不同组织以及血液中存在多少传染性是很重要的。这对于了解传染性朊病毒的生物学，确保供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的感染因子，检测其存在的唯一可靠方法是用感染了朊病毒的样本接种实验动物，观察受体是否发生朊病毒疾病。不可避免地，这导致大量实验动物，特别是小鼠和绵羊，被用来评估哺乳动物朊病毒的传染性，并因此死于晚期朊病毒疾病。这项提议的目的是尽可能减少和取代用老鼠和羊来测量朊病毒对无脊椎动物宿主——黑腹果蝇的感染性。反过来，这将有助于改进朊病毒传染性实验，通过使用不太有知觉的动物，这些动物不知道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痒病。我们已经培育出了绵羊PrP转基因果蝇，并证明这些果蝇对绵羊朊病毒的毒性作用很敏感。成年苍蝇在幼虫时期被喂食具有朊病毒传染性的食物，它们的移动能力下降，并且在它们的大脑中出现了错误折叠形式的PrP。在本方案的实验中，我们打算验证我们的新朊病毒蝇模型的敏感性，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来测量朊病毒的感染性。我们使用PrP转基因果蝇将产生人性化，高质量，可复制的基于朊病毒的科学，同时协助3Rs战略。使用PrP转基因果蝇将产生高质量、有统计学意义的数据。可以很容易地生产和饲养大量苍蝇，并且可以使用经济上可行的模型同时测试相关数量的重复。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，这样就不需要为其他实验动物注射朊病毒材料来获得用于本研究的样本。我们有许多这些样本的初步测试结果，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.