New Animal and Culture Models to rapidly evaluate infectivity of the vCJD Agent

描述(由申请人提供)：该项目的广泛和长期目标是澄清与流行性疯牛病有关的传染性vCJD病原体的性质：传染性如何传播，如何准确和快速地进行滴定，以及如何确定这种vCJD病原体。这里正在开发的方法和模式对公共卫生和预防至关重要。它们还为解决这些感染性病原体的基本分子成分和基本结构奠定了基础。有许多不同的TSE毒剂菌株，特别是vCJD是有问题的，因为它已被证明对包括人类在内的许多物种具有毒力。它还能够通过无症状捐赠者的输血在人与人之间传播，这是另一个令人担忧的原因。此外，vCJD病原体在许多不同物种中传代后仍保持其特征和特性，因此vCJD动物模型对于解决这种感染性病原体在人类群体中的传播和诊断具有高度的相关性。 R21试图建立新的实验动物和组织培养模型，以更彻底地确定vCJD感染源，特别是它在不同组织中传播的能力，在这些组织中，它不能被异常宿主蛋白(PrP)等晚期病理标志物检测到。我们还试图通过创新的共培养策略找到更快的方法来检测这种病原体。 基于耶鲁大学的初步数据，我们建立了迄今为止最快的vCJD动物模型，该小鼠模型可以为vCJD的特定病原体传播特征提供关键信息。此外，有限的初步研究强烈表明，vCJD试剂可以在小鼠来源的简化组织培养中感染和复制。这种培养模式为解决感染性病原体打开了新的机会，而使用复杂的变性脑组织是不可能的。这项应用旨在1)验证动物和培养vCJD模型的试剂特异性，2)进一步开发创新的培养重叠感染试验，用于vCJD病原体的菌株特异性诊断，以及3)使用直接培养和重叠感染方法来快速揭示血液等经证实具有传染性但缺乏病理PrP的样本中的传染性。4)我们还将检查感染vCJD的培养物的超微结构，以确定它们是否含有25 nm的病毒样颗粒，这些颗粒已在许多TSE感染的大脑中发现，但未在对照脑组织中发现。我们在具有高滴度瘙痒病传染性的培养物中发现了类似的病毒样颗粒，因此我们怀疑这种颗粒也可以帮助诊断、预防和基本了解这些不可避免的致命感染。

DESCRIPTION (provided by applicant): The broad and long-term objective of this project is to clarify the nature of infectious vCJD agent that is linked to epidemic BSE: how infectivity spreads, how it can be accurately and rapidly titered, and how this vCJD agent can be identified with certainty. The approaches and models under development here are critical for public health and prevention. They also lay the groundwork for resolving the essential molecular components and fundamental structure of these infectious agents. There are many distinct strains of TSE agents, and vCJD in particular is problematic because of its proven virulence for many species, including humans. It has also been able to transmit from person to person by transfusion from asymptomatic donors, a further reason for concern. The vCJD agent, moreover, retains its characteristics and identity after passage in many different species, and therefore animal models of vCJD are highly relevant for addressing the spread and diagnosis of this infectious agent in the human population. This R21 seeks to establish new experimental animal and tissue culture models to define the vCJD infectious agent more thoroughly, particularly its ability to spread in different tissues where it is not detectable by late pathological markers such as abnormal host prion protein (PrP). We also seek to find faster ways to detect this agent using innovative co-culture strategies. Based on preliminary data at Yale, we have developed the quickest animal model of vCJD to date, and this mouse model can yield critical information for vCJD agent-specific features of spread. Additionally, limited pilot studies here strongly suggest the vCJD agent can infect and replicate in simplified tissue cultures of murine origin. This culture model opens new opportunities for the resolution of the infectious agent that are not possible using complex degenerating brain tissue. This application aims 1) to verify the agent-specificity of both the animal and culture vCJD models, 2) to further develop innovative superinfection tests in culture for strain-specific diagnosis of the vCJD agent, and 3) to use direct culture and superinfection approaches to rapidly reveal infectivity in samples such as blood that are verifiably infectious, but that lack pathological PrP. 4) We will also examine vCJD-infected cultures ultrastructurally to find if they contain the 25nm viruslike particles that have been documented in many TSE-infected, but not control brains. We have discovered comparable viruslike particles in cultures with high titers of scrapie infectivity, and thus we suspect such particles can also aid in the diagnosis, prevention and fundamental understanding of these inevitably lethal infections.