The cell biology of Theiler's virus persistence in CNS

描述（由申请人提供）：Theiler病毒是一种小病毒，可引起遗传易感小鼠脊髓的持续性感染，并伴有慢性感染和原发性脱髓鞘。因此，它为免疫介导的脱髓鞘提供了最好的动物模型之一。塞勒氏病毒首先感染神经元，但后来持续存在于少突胶质细胞和巨噬细胞/大胶质细胞中。这种从灰质到白质的转变与病毒特异性免疫反应的出现相吻合，这表明免疫压力使病毒远离神经元，但无法将其从神经胶质细胞中清除。在这个应用中，我们建议测试一个模型，在这个模型中，病毒依次从神经元到少突胶质细胞再到巨噬细胞，而少突胶质细胞在面对免疫压力时提供了一种强制性的中间物。首先，我们将监测野生型C3H和shiverer和rumpshaker小鼠的各种细胞类型的Theiler感染的时间过程，这两种小鼠具有不同的严重髓磷脂缺陷，并且对持续感染具有抗性。我们将检查是否少突胶质细胞，巨噬细胞或两者从未感染，或只是短暂感染，在耐药突变小鼠。骨髓移植实验将揭示来自颤颤鼠和颤鼠的中枢神经系统或骨髓来源的细胞是否赋予对持续感染的抵抗力，而体外培养的少突胶质细胞和巨噬细胞将揭示这些细胞对病毒感染的固有容许性是否被突变改变。其次，我们将确定髓磷脂是否是病毒进入白质的入口。我们将在少突胶质细胞和神经元的混合培养中特异性表达病毒基因组，并在髓鞘形成成功与否的情况下追踪病毒在少突胶质细胞中的传播。用雌性rumpshaker杂合子进行的实验将使我们有可能在体内使用自然嵌合体，因为rumpshaker突变是x连锁的。最后，我们将使用体内标记方法来监测血源性巨噬细胞向炎性中枢神经系统病变的迁移，并确定这些新到达的细胞被感染的频率。这将测试持久性实现的可能性，部分是通过慢性炎症来确保可再生宿主种群的存在。总之，这个模型可能证明感染性物质从神经元到少突胶质细胞的功能转移的一个范例，从那里再到免疫系统的细胞。

DESCRIPTION (provided by applicant): Theiler's virus, a picornavirus, causes a persistent infection of the spinal cord of genetically susceptible mice that is accompanied by chronic infection and primary demyelination. As such, it provides one of the best animal models for immune-mediated demyelination. Theiler's virus first infects neurons, but later persists in oligodendrocytes and macrophage/macroglial cells. This shift from gray to white matter coincides with the appearance of virus-specific immune responses, suggesting that immune pressure keeps the virus out of neurons but is unable to clear it from glial cells. In this application, we propose to test a model in which the virus traffics sequentially from neurons to oligodendrocytes to macrophages, with oligodendrocytes providing an obligatory intermediate in the face of immune pressure. First, we will monitor the time course of Theiler's infection of various cell types in wild-type C3H and in shiverer and rumpshaker mice, which have different severe myelin defects and are resistant to persistent infection. We will examine whether oligodendrocytes, macrophages or both are never infected, or infected only transiently, in the resistant mutant mice. Bone marrow transfer experiments will reveal whether CNS- or bone marrow-derived cells from shiverer and rumpshaker mice confer resistance to persistent infection and in vitro culture of oligodendrocytes and macrophages will reveal whether the intrinsic permissiveness of these cells to viral infection is altered by the mutations. Second, we will determine whether myelin is the portal of entry of the virus into the white matter. We will express viral genomes specifically in neurons in mixed cultures of oligodendrocytes and neurons, and track viral spread into oligodendrocytes in the presence and absence of successful myelination. Experiments performed with female rumpshaker heterozygotes will afford us the possibility to work with natural chimeras in vivo, because the rumpshaker mutation is X-linked. Finally, we will use in vivo labeling methods to monitor the migration of blood-borne macrophages into inflammatory CNS lesions, and determine the frequency with which these newly arrived cells become infected. This will test the possibility that persistence is achieved, in part, by the chronic inflammation that ensures the existence of a renewable host population. Together, this model may prove a paradigm for the functional transfer of infectious material from neurons to oligodendrocytes, and from thence to cells of the immune system.