NEUROTERATOGENIC MECHANISM OF LCM VIRUS INFECTION

这个研究职业奖是一个计划，以促进丹尼尔邦修斯博士发展成为一个独立的神经科学家。 Bonthius博士是一名儿科神经学家，对环境因素（包括先天性病毒感染）对胎儿大脑发育的不良影响特别感兴趣。 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医生科学家。 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，技术研讨会的组合，并通过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，获得他的新的研究技能。 LCMV是一种流行性病毒，可严重损害发育中的人胎脑。 将LCMV注射到新生大鼠脑中导致小脑皮质和齿状回的选择性感染。 这种感染引发小脑的急性破坏和齿状颗粒细胞的延迟死亡。 本提案的目的是确定潜在的机制的急性和延迟的病理变化。 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幅度。 将通过免疫细胞化学和原位杂交确定病毒定位，并通过滴度和RNA酶保护试验确定区域病毒载量。体视学方法将用于定义神经元损失的时间过程。 第二个目的是检验病理变化是免疫介导的假设，并确定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的过度产生在LCMV引起的急性和迟发性病理变化中的作用将被探讨。 第三个目的是探索α-肌营养不良蛋白聚糖（α-DG）是LCMV在新生大鼠脑中的细胞受体并且在体内感染的向性中起关键作用的可能性。 发育中的脑中的a-DG表达的地形图将通过免疫组织化学显示，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α-DG。 将通过在将脑切片暴露于LCMV之前用抗体阻断a-DG来检查a-DG在影响LCMV的向性和感染性中的重要性。

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.