The Cell Biology of Theiler's Virus Persistence in CNS

描述(申请人提供)：泰勒氏病毒，一种微小核糖核酸病毒，导致遗传易感小鼠脊髓持续感染，并伴有慢性感染和原发脱髓鞘。因此，它为免疫介导的脱髓鞘提供了最好的动物模型之一。泰勒氏病毒首先感染神经元，但后来在少突胶质细胞和巨噬细胞/大胶质细胞中持续存在。这种从灰质到白质的转变与病毒特异性免疫反应的出现不谋而合，这表明免疫压力使病毒无法进入神经元，但无法将其从神经胶质细胞中清除。在这一应用中，我们建议测试一个模型，在该模型中，病毒依次从神经元到少突胶质细胞再到巨噬细胞，在面对免疫压力时，少突胶质细胞提供了必需的中间体。首先，我们将监测野生型C3H型和颤抖型和Rumpshaker小鼠中不同细胞类型的Theler‘s感染的时间进程，这两种小鼠具有不同的严重髓鞘缺陷并对持续感染具有抵抗力。我们将检查在耐药突变小鼠中，少突胶质细胞、巨噬细胞或两者是否从未感染，或仅短暂感染。骨髓移植实验将揭示来自颤栗和Rumpshaker小鼠的CNS或骨髓来源的细胞是否对持续感染具有抵抗力，而对少突胶质细胞和巨噬细胞的体外培养将揭示这些细胞对病毒感染的内在容许性是否会因突变而改变。其次，我们将确定髓鞘是否是病毒进入白质的门户。我们将在少突胶质细胞和神经元的混合培养中特异性地在神经元中表达病毒基因组，并跟踪病毒在有无髓鞘形成的情况下传播到少突胶质细胞。对女性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感染的频率。这将检验实现持久性的可能性，部分是通过确保可再生宿主种群存在的慢性炎症实现的。总而言之，这一模型可能证明了一种将感染性物质从神经元转移到少突胶质细胞，并从那里转移到免疫系统细胞的范例。

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.