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（由申请人提供）：小鼠中枢神经系统（CNS）的小鼠肝炎病毒（MHV）感染被广泛用于对人类的脑炎和多发性硬化症进行建模。 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在大脑中，没有引起抗原特异性CD8+ T细胞反应。基于初步数据，我假设这两个特征源于JHM专业化对神经元扩散的专业化，从而通过将病毒扩散限制为CNS中的抗原呈现细胞和/或排水淋巴结的抗原呈现细胞来降低CD8+ T细胞的反应。我建议更好地定义这种专业化的机制，以改善对潜在神经毒动病毒的识别，研究JHM分离株对神经元电路跟踪的潜力，并提高我们对有效呈现CNS衍生抗原的要求的理解。 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确定CEACAM1A非依赖性在神经元中是否仅需要RIS或神经元特异性传播机制。 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室内。接下来，我将研究JHM是否在颅内（I.C.）接种后是否无法诱导大脑中的CD8+ T细胞反应，因为它无法扩散到颅内或颅外肛门肛门呈现细胞。首先，我将使用各种RNA，蛋白质和功能分析来确认THA JHM感染激活骨髓来源的树突状细胞（DC）进行抗原表现。接下来，我将通过淋巴细胞神经扰动性病毒（LCMV）GP33抗原特异性T细胞的淋巴结炎炎感染C57BL/6小鼠。腹膜内（i.p.）用LCMV GP33表演表达JHM（JHM-GP33）并评估大脑中的抗GP33 CD8+ T细胞反应，以确定颅外病毒复制是否可以挽救颅内CD8+ T细胞反应。最后，我将接种分娩的小鼠I.N.使用野生型JHM，注入DCS 感染JHM-GP33 I.C.并评估大脑中的GP33特异性T细胞反应，以确定受感染DC的颅内抗原表现是否挽救了颅内CD8+反应。这些实验将大大增强我们对神经动力毒气的理解，并开放对神经元病毒间扩散和中枢神经系统抗原表现的新途径。 公共卫生相关性：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的脑炎和多发性硬化症。这两个疾病过程都受到大脑中神经元扩散的效率和抗病毒CD8+ T细胞反应的影响。拟议的项目将通过定义MHV极其神经性肺部分离物在CNS中的适应性的适应方式来增强我们对这些模型系统的理解，从而影响CNS中的神经元扩散和CD8+ T细胞反应的启动。