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

描述（由申请方提供）：本项目的广泛和长期目标是阐明与流行性BSE相关的传染性vCJD因子的性质：传染性如何传播，如何准确和快速滴定，以及如何确定这种vCJD因子。这里正在开发的方法和模式对公共卫生和预防至关重要。它们还为解析这些传染性病原体的基本分子组分和基本结构奠定了基础。TSE病原体有许多不同的菌株，特别是vCJD是有问题的，因为它对许多物种（包括人类）的毒力已被证明。它还能够通过无症状献血者的输血在人与人之间传播，这是令人担忧的另一个原因。此外，vCJD因子在许多不同物种中传代后保留其特征和身份，因此vCJD的动物模型对于解决该传染因子在人群中的传播和诊断是高度相关的。 该R21旨在建立新的实验动物和组织培养模型，以更彻底地定义vCJD感染因子，特别是其在不同组织中传播的能力，其中晚期病理标记物如异常宿主朊蛋白（PrP）无法检测到。我们还试图找到更快的方法来检测这种代理使用创新的共培养策略。 根据耶鲁大学的初步数据，我们已经开发出迄今为止最快的vCJD动物模型，这种小鼠模型可以为vCJD传播的特定特征提供关键信息。此外，有限的初步研究强烈表明，vCJD因子可以在简化的鼠源组织培养物中感染和复制。这种培养模型为解决感染因子提供了新的机会，而使用复杂的变性脑组织是不可能的。本申请旨在1）验证动物和培养vCJD模型的病原体特异性，2）进一步开发用于vCJD病原体的菌株特异性诊断的培养物中的创新性重复感染试验，以及3）使用直接培养和重复感染方法快速揭示可验证具有感染性但缺乏病理性PrP的样品（如血液）中的感染性。4)我们还将检查vCJD感染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，这些颗粒在许多TSE感染的大脑中有记录，但在对照组大脑中没有。我们已经在培养物中发现了类似的病毒样颗粒，具有高滴度的羊瘙痒病感染性，因此我们怀疑这些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.