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: 8660280
• 负责人: JUDITH M PHILLIPS
• 金额: $ 13.38万
• 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/8660280
• 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.

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）感染被广泛用于模拟人类脑炎和多发性硬化症。与从大脑传播到身体其他部位的弱神经毒力 MHV A59 毒株不同，中枢神经系统适应的 JHM 分离株具有极高的神经毒力，但在中枢神经系统外的复制能力很差，它们具有两种毒株特异性且明显独立的神经毒力特征：在缺乏典型 MHV 受体 Ceacam1a 的神经元中传播的能力以及复制的能力 不引起抗原特异性 CD8+ T 细胞反应。根据初步数据，我假设这两个特征都源于 JHM 对神经元间传播的专门化，它通过限制病毒传播到中枢神经系统和/或引流淋巴结中的抗原呈递细胞来减少 CD8+ T 细胞反应。我建议更好地定义这一专业化的机制，以提高对潜在神经毒力病毒的识别，研究 JHM 分离株用于神经元循环追踪的潜力，并提高我们对有效呈递中枢神经系统衍生抗原的要求的理解。我将首先检查 C57BL/6 和 ceacam1a-/- 小鼠的神经毒力、在 C57BL/6 和 ceacam1a-/- 神经元培养物中的传播能力，以及在非小鼠细胞中存在受体非依赖性传播 (RIS) 缺陷的突变 JHM 病毒鼻内 (i.n.) 接种后的传播模式，以便 确定神经元中不依赖 Ceacam1a 的传播是否只需要 RIS 或神经元特异性传播机制。然后，我将检查突触间的传播、隔室培养物中神经元细胞体和轴突中病毒的释放，以及 JHM、A59、rA59/SJHM（具有 JHM 刺突基因的嵌合 A59 病毒）感染神经元的膜融合，以及培养的原代 C57BL/6 神经元中的 RIS 突变体（如果相关），以确定 JHM 是否传播 神经元间。接下来我将研究 JHM 在颅内 (i.c.) 接种后是否无法在大脑中诱导 CD8+ T 细胞反应，因为它无法扩散到颅内或颅外抗原呈递细胞。首先，我将使用各种 RNA、蛋白质和功能测定来确认 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，我将同时用淋巴绒毛膜脑膜炎病毒 (LCMV) gp33 抗原特异性 T 细胞经皮下感染过继转移的 C57BL/6 小鼠。并腹膜内（i.p.）使用表达 LCMV gp33 表位的 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，我会将过继转移的小鼠接种到体内。使用野生型 JHM，注射 DC 注射 JHM-gp33 感染，并评估大脑中 gp33 特异性 T 细胞反应，以确定受感染 DC 的颅内抗原呈递是否可以挽救颅内 CD8+ 反应。这些实验将极大地增强我们对神经毒力的理解，并为探索神经元间病毒传播和中枢神经系统抗原呈递开辟新途径。