BIOLOGY OF CORONAVIRUS INDUCED DEMYELINATION

间接证据表明病毒与多发性硬化症的病因有关 (Ms)和更直接的证据表明，病毒导致其他人类 脱髓鞘疾病。冠状病毒感染小鼠肝炎病毒 是研究病毒诱导慢性炎症的良好模型系统。 脱髓鞘。这个项目的长期目标是使用这些技术 以了解持续感染的机制 和病毒诱导的慢性中枢神经系统疾病在该动物模型中。 我们之前已经证明，脑内接种MHV-A59到4-6 周龄C57BL/6小鼠100%致慢性中枢神经系统疾病 几乎没有急性疾病或死亡率。原发性脱髓鞘可以是 最晚在感染后6个月检测到，并可通过以下方式增强 免疫抑制。而传染性病毒和病毒抗原则不能 在慢性病状态下检测到的病毒核酸确实持续存在。 在这个方案中，我们将研究病毒持续的机制和它的 与脱髓鞘的关系。使用原位杂交和 基因特异性克隆的MHV-A59cDNA作为检测病毒核酸的探针 用针对个体的抗血清进行免疫过氧化物酶染色 病毒蛋白我们将确定病毒基因组的部分或全部是 在转录和翻译水平上表达。 将进行环磷酰胺治疗，以确定病毒是否可以 从长期感染的动物中恢复过来 免疫抑制。将通过进行感染来研究病毒的趋向性 在体外培养富集少突胶质细胞。 作为一个更次要的目标，我们将调查可能的关系 在人类冠状病毒和女士之间我们将使用高度特异性和 冠状病毒核酸杂交敏感探针技术 多发性硬化症患者中枢神经系统的序列分析。因此，中枢神经系统的原位杂交 MS患者的尸检组织将使用克隆的DNA进行 与人类冠状病毒基因组同源的片段作为探针。

Indirect evidence implicates a virus in the etiology of Multiple Sclerosis (MS) and more direct evidence shows that viruses cause other human demyelinating diseases. Coronavirus mouse hepatitis virus (MHV) infection of mice is a good model system for the study of virus-induced chronic demyelination. The long term goal of this project is to use the techniques of molecular biology to understand the mechanisms of persistent infection and virus-induced chronic CNS disease in this animal model. We have previously shown that intracerebral inoculation of MHV-A59 into 4-6 week old C57BL/6 mice results in chronic CNS disease in 100% of the animals with very little acute disease or mortality. Primary demyelination can be detected as late as six months post infection and can be enhanced by immunosuppression. While infectious virus and viral antigens cannot be detected during the chronic disease state, viral nucleic acids do persist. In this proposal we will study the mechanism of viral persistence and its relationship to demyelination. Using in situ hybridization and gene-specific cloned MHV-A59 cDNAs as probes to detect viral nucleic acids and immunoperoxidase staining with antisera directed against individual viral proteins we will determine if part or all of the viral genome is expressed at the transcriptional and translational levels. Cyclophosphamide treatment will be carried out to determine if virus can be recovered from chronically infected animals that have been immunosuppressed. Viral tropism will be studied by carrying out infections in vitro in enriched oligodendrocyte cultures. As a more secondary goal we will investigate the possible relationship between human coronaviruses and MS. We will use the highly specific and sensitive technique of nucleic acid hybridization to probe for coronavirus sequences in the CNS of MS patients. Thus, in situ hybridization of CNS autopsy tissue from MS patients will be carried out using cloned DNA fragments homologous to human coronavirus genomes as probes.