Mechanisms of Neuronal Spread of Neurotropic Mouse Hepatitis Virus

嗜神经性小鼠肝炎病毒的神经传播机制

• 批准号: 8382887
• 负责人: JUDITH M PHILLIPS
• 金额: $ 12.84万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8382887
• 关键词: Affect; Antigen Presentation; Antigen-Presenting Cells; Antigens; Antiviral Agents; Axon; Biological Assay; Biological Models; Body part; Bone Marrow; Brain; C57BL/6 Mouse; CD8B1 gene; Cell Shape; Cells; Central Nervous System Diseases; Cervical lymph node group; Cytotoxic T-Lymphocytes; Data; Defective Viruses; Dendritic Cells; Disease; Effectiveness; Encephalitis; Environment; Epitopes; Failure; Genes; Human; Immune; Immune response; In Vitro; Infection; Learning; Lymphocyte; MHVR; Mediating; Membrane; Membrane Fusion; Microglia; Modeling; Multiple Sclerosis; Murine hepatitis virus; Mus; Mutant Strains Mice; Mutation; Neuraxis; Neuroglia; Neurons; Neuropathogenesis; Pattern; Peptides; Peripheral; Physiologic pulse; Prevention; Process; Production; Proteins; RNA; Site; Study models; Surface; Synapses; T cell response; T-Lymphocyte; Transgenic Organisms; Viral; Viral Antigens; Viral Encephalitis; Viral hepatitis; Virus; Virus Diseases; base; cytokine; cytotoxic; improved; intraperitoneal; lymph nodes; mouse model; mutant; neuronal cell body; neurotropic; neurovirulence; pathogen; receptor; research study; response; stem; tissue culture; trait

DESCRIPTION (provided by applicant): Mouse hepatitis virus (MHV) infection of the mouse central nervous system (CNS) is widely used to model both encephalitis and multiple sclerosis in humans. Unlike the weakly neurovirulent A59 strain of MHV, which spreads from the brain to other parts of the body, the CNS-adapted JHM isolates, which are extremely neurovirulent but replicate poorly outside the CNS, share two strain-specific and apparently independent neurovirulence traits: the ability to spread among neurons lacking the canonical MHV receptor Ceacam1a and the ability to replicate in the brain without eliciting an antigen-specific CD8+ T-cell response. Based on preliminary data, I hypothesize that both of these traits stem from the specialization of JHM for interneuronal spread, which reduces the CD8+ T-cell response by limiting viral spread to antigen-presenting cells in the CNS and/or draining lymph nodes. I propose to better define the mechanisms of this specialization in order to improve identification of potentially neurovirulent viruses, investigate the potential of JHM isolates for neuronal circui tracing, and improve our understanding of the requirements for effective presentation of CNS-derived antigen. I will first examine the neurovirulence in C57BL/6 and ceacam1a-/- mice, the abilities to spread in C57BL/6 and ceacam1a-/- neuron cultures, and the patterns of spread following intranasal (i.n.) inoculation in C57BL/6 mice of mutant JHM viruses defective for receptor-independent spread (RIS) in non- murine cells in order to determine whether Ceacam1a-independent spread in neurons requires merely RIS or a neuron-specific mechanism of spread. I will then examine the spread across synapses, release of virus from neuronal cell bodies and axons in compartmented cultures, and membrane fusion of infected neurons of JHM, A59, rA59/SJHM (a chimeric A59 virus with the JHM spike gene), and, if relevant, the RIS mutants in cultured primary C57BL/6 neurons to determine whether JHM spreads interneuronally. I will next investigate whether JHM fails to induce a CD8+ T-cell response in the brain after intracranial (i.c.) inoculation because it fails to spread to intra- or extracranial anigen-presenting cells. First, I will use a variety of RNA, protein, and functional assays to confirm tha JHM infection activates bone marrow-derived dendritic cells (DCs) for antigen presentation. Next, I will infect C57BL/6 mice adoptively transferred with lymphochoriomeningitis virus (LCMV) gp33 antigen-specific T cells simultaneously i.c. and intraperitoneally (i.p.) with LCMV gp33 epitope-expressing JHM (JHM-gp33) and evaluate the anti-gp33 CD8+ T cell response in the brain to determine whether extracranial viral replication rescues the intracranial CD8+ T cell response. Finally, I will inoculate adoptively transferred mice i.n. with wild-type JHM, inject DCs infected with JHM-gp33 i.c., and evaluate the gp33-specific T cell response in the brain to determine whether intracranial antigen presentation by infected DCs rescues the intracranial CD8+ response. These experiments will greatly enhance our understanding of neurovirulence and open new avenues of exploration of interneuronal viral spread and CNS antigen presentation. PUBLIC HEALTH RELEVANCE: Mouse hepatitis virus (MHV) infection of the mouse central nervous system (CNS) is widely used to model encephalitis and multiple sclerosis in humans. Both of these disease processes are affected by the efficiency of interneuronal spread and the antiviral CD8+ T cell response in the brain. The proposed project will enhance our understanding of these model systems by defining the ways in which the adaptations of an extremely neurovirulent isolate of MHV to spread in the CNS influence both interneuronal spread and priming of the CD8+ T cell response.

描述（由申请方提供）：小鼠中枢神经系统（CNS）的小鼠肝炎病毒（MHV）感染广泛用于模拟人类脑炎和多发性硬化。与弱神经毒力的MHV A59株不同，它从大脑传播到身体的其他部位，CNS适应的JHM分离株具有极强的神经毒力，但在CNS外复制很差，共有两个菌株特异性和明显独立的神经毒力特征：在缺乏典型MHV受体Ceacam 1a的神经元中传播的能力和在脑中复制而不引发抗原的能力-特异性CD 8 + T细胞应答。基于初步的数据，我假设这两个性状干从专业化的JHM神经元间的传播，减少了CD 8 + T细胞反应，限制病毒传播到抗原呈递细胞在中枢神经系统和/或引流淋巴结。我建议更好地定义这种专业化的机制，以提高潜在的神经毒力病毒的识别，调查潜在的JHM分离株神经元circui跟踪，并提高我们的理解的要求，有效地介绍中枢神经系统衍生抗原。我将首先检查C57 BL/6和ceacam 1a-/-小鼠的神经毒力，在C57 BL/6和ceacam 1a-/-神经元培养物中传播的能力，以及鼻内（i.n.）在C57 BL/6小鼠中接种在非鼠细胞中的受体非依赖性扩散（RIS）缺陷的突变JHM病毒，以确定在神经元中的Ceacam 1a非依赖性扩散是否仅需要RIS或神经元特异性扩散机制。然后，我将检查跨突触的传播，从神经元细胞体和轴突在隔室培养中释放病毒，以及JHM，A59，rA 59/SJHM（与JHM刺突基因嵌合的A59病毒）感染的神经元的膜融合，以及如果相关的话，培养的原代C57 BL/6神经元中的RIS突变体，以确定JHM是否在神经元间传播。接下来，我将研究JHM在颅内（i.c.）因为它不能扩散到颅内或颅外的血管递呈细胞。首先，我将使用各种RNA，蛋白质和功能检测来证实JHM感染激活骨髓来源的树突状细胞（DC）进行抗原呈递。接下来，我将用淋巴脉络丛脑膜炎病毒（LCMV）gp 33抗原特异性T细胞同时i.c.感染过继转移的C57 BL/6小鼠。和腹膜内（i. p.）用表达LCMV gp 33表位的JHM（JHM-gp 33）感染，并评价脑中的抗gp 33 CD 8 + T细胞应答，以确定颅外病毒复制是否挽救颅内CD 8 + T细胞应答。最后，我将通过i.n.用野生型JHM， 用JHM-gp 33 i.c.感染，并评估脑中的gp 33特异性T细胞应答以确定感染的DC的颅内抗原呈递是否挽救颅内CD 8+应答。这些实验将大大提高我们的神经毒力的理解，并开辟新的途径探索神经元间的病毒传播和中枢神经系统抗原呈递。 公共卫生关系：小鼠中枢神经系统（CNS）的小鼠肝炎病毒（MHV）感染被广泛用于模拟人类脑炎和多发性硬化。这两种疾病过程都受到脑中神经元间扩散和抗病毒CD 8 + T细胞应答效率的影响。拟议的项目将提高我们对这些模型系统的理解，通过定义的方式，在其中的适应极其neurovirulent分离的MHV在中枢神经系统中传播的影响神经元间的传播和启动的CD 8 + T细胞反应。