NEUROTERATOGENIC MECHANISM OF LCM VIRUS INFECTION

这一研究生涯奖是一项计划，旨在促进丹尼尔·邦蒂乌斯博士发展成为一名独立的神经科学家。邦修斯博士是一名儿科神经科医生，对包括先天性病毒感染在内的环境因素对胎儿大脑发育的不利影响特别感兴趣。他的长期职业目标是成为一名内科科学家，能够在神经畸形学方面做出有意义的贡献。邦修斯博士将专注于分子神经生物学的研究技术，这些技术在神经畸形学领域具有关键的重要性。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膜脑膜炎病毒(LCMV)感染的神经畸形学的研究，获得新的研究技能。LCMV是一种流行的病毒，可严重损害发育中的人胎脑。将LCMV注射到新生大鼠的大脑中会导致小脑皮层和齿状回的选择性感染。这种感染引发了小脑的急性破坏和齿状颗粒细胞的延迟死亡。这项建议的目的是确定急性和延迟性病理改变的机制。第一个目标是确定病毒感染的动态以及颗粒细胞丢失的时间进程和程度。病毒定位将通过免疫细胞化学和原位杂交来确定，局部病毒载量将通过滴度和核糖核酸酶保护试验来确定。将使用体视学方法来确定神经元丢失的时间进程。第二个目的是验证病理变化是由免疫介导的假设，并确定涉及的免疫细胞类型和分子。将在免疫缺陷大鼠身上研究LCMV感染。特定免疫细胞类型的作用将通过免疫细胞化学和流式细胞仪分析来确定。将探讨细胞因子和一氧化氮过度产生在LCMV引起的急性和延迟性病理改变中的作用。第三个目的是探讨α-营养不良聚糖(a-DG)可能是新生大鼠脑内LCMV的细胞受体，并在体内感染的趋向性中发挥关键作用。免疫组织化学方法将显示发育脑中α-DG表达的地形图，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。A-DG在影响LCMV的趋向性和感染性方面的重要性将通过在脑片暴露于LCMV之前用抗体封闭a-DG来检验。

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.