NEUROTERATOGENIC MECHANISM OF LCM VIRUS INFECTION

LCM病毒感染的神经致畸机制

• 批准号: 6358299
• 负责人: Daniel J. Bonthius
• 金额: $ 1.31万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6358299
• 关键词: blocking antibody; cerebellar cortex; cytokine; dentate gyrus; disease /disorder model; granule cell; immunocytochemistry; in situ hybridization; laboratory rat; lymphocytic choriomeningitis; lymphocytic choriomeningitis virus; microorganism immunology; newborn animals; nitric oxide; pathologic process; teratogens; virus infection mechanism; virus receptors

This research career award is a plan to foster the development of Dr. Daniel Bonthius into an independent neuroscientist. Dr. Bonthius is a pediatric neurologist with a special interest in the adverse effects of environmental agents, including congenital viral infections, on fetal brain development. His long term career goal is to become a physician scientist capable of making meaningful contributions in neuroteratology. The research techniques on which Dr. Bonthius will focus are those of molecular neurobiology, which are of key importance in the field of neuroteratology. Dr. Bonthius will acquire his new research skills through a combination of didactic courses, skills workshops, technical seminars, and through his research into the neuroteratology of lymphocytic choriomeningitis virus (LCMV) infection. LCMV is a prevalant virus which can serverely damage the developing human fetal brain. Injection of LCMV into the neonatal rat brain results in a selective infection of the cerebellar cortex and dentate gyrus. This infection triggers an acute distruction of the cerebellum and a delayed mortality of dentate granule cells. The objective of this proposal is to identify the mechanisms underlying the acute and the delayed pathologic changes. The first aim is to determine the dynamics of the viral infection and the time course and magnitude of the granule cell loss. Viral localization will be determined by immunocytochemistry and insitu hybridization and regional viral load will be determined by titer and Rnase protection assay. Stereological methods will be used to define the time course of the neuronal loss. The second aim is to test the hypothesis that the pathologic changes are immune mediated and to identify the immune cell types and molecules involved. LCMV infection will be studied in immune deficient rats. The role of specific immune cell types will be determine by immunocytochemistry and FACS analysis. The role of cytokines and of nitric oxide over- production in both the acute and delayed pathologic changes induced by LCMV will be explored. The third aim is to explore the possibility that alpha-dystroglycan (a-DG) is the cellular receptor for LCMV in the neonatal rat brain and plays a critical role in the tropism of the infection in vivo. The topography of a-DG expression in the developing brain will be demonstrated by immunohistochemistry and compared with the spatial distribution of LCMV infection. Double labeling experiments will determine whether LCMV infected brain cells universally express a-DG. The importance of a-DG in influencing the tropism and infectivity of LCMV will be examined by blocking a-DG with an antibody prior to exposure of brain slices to LCMV.

这项研究事业奖旨在促进丹尼尔·邦修斯博士成为一名独立的神经科学家。Bonthius博士是一名儿科神经学家，对环境因素的不利影响特别感兴趣，包括先天性病毒感染，对胎儿大脑发育的影响。他的长期职业目标是成为一名能够在神经畸形学方面做出有意义贡献的内科科学家。Bonthius博士将专注于分子神经生物学的研究技术，这在神经畸形学领域至关重要。Bonthius博士将通过教学课程、技能研讨会、技术研讨会以及他对淋巴细胞性脉络丛脑膜炎病毒（LCMV）感染的神经畸形学的研究来获得他新的研究技能。LCMV是一种流行的病毒，可以严重损害发育中的人类胎儿大脑。在新生大鼠脑内注射LCMV可导致小脑皮层和齿状回的选择性感染。这种感染引起小脑的急性破坏和齿状颗粒细胞的延迟死亡。本建议的目的是确定潜在的机制，急性和延迟的病理改变。第一个目的是确定病毒感染的动力学和颗粒细胞损失的时间过程和大小。病毒定位将通过免疫细胞化学和原位杂交确定，区域病毒载量将通过滴度和Rnase保护试验确定。体视学方法将用于确定神经元丢失的时间过程。第二个目的是验证病理变化是免疫介导的假设，并确定所涉及的免疫细胞类型和分子。将在免疫缺陷大鼠中研究LCMV感染。特异性免疫细胞类型的作用将通过免疫细胞化学和FACS分析来确定。细胞因子和一氧化氮过量产生在LCMV引起的急性和延迟病理改变中的作用将被探讨。第三个目的是探索α -肌营养不良聚糖（a- dg）是否可能是新生大鼠大脑中LCMV的细胞受体，并在体内感染的趋向性中发挥关键作用。a-DG在发育脑中的表达将通过免疫组织化学来证实，并与LCMV感染的空间分布进行比较。双标记实验将确定LCMV感染的脑细胞是否普遍表达a-DG。通过在脑切片暴露于LCMV之前用抗体阻断a-DG，将检验a-DG在影响LCMV的趋向性和感染性中的重要性。