Neuronal Spread of Herpesvirus Infection

疱疹病毒感染的神经元传播

• 批准号: 8055864
• 负责人: Lynn W. Enquist
• 金额: $ 33.87万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-15 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8055864
• 关键词: Affect; Amyloid; Anatomy; Antibodies; Antiviral Agents; Axonal Transport; Biological; Brain; Capsid; Cells; Cellular biology; Chimeric Proteins; Collection; Complement; Confocal Microscopy; Development; Disease; Dyes; Engineering; Face; Ganglia; Goals; Health; Hereditary Motor and Sensory-Neuropathy Type II; Hereditary Spastic Paraplegia; Herpesviridae; Herpesviridae Infections; Human; Image; Imaging technology; In Situ; In Vitro; Individual; Infection; Invaded; Knowledge; Label; Laboratories; Laser Scanning Microscopy; Lasers; Life; Mediating; Methods; Microscopy; Modern Medicine; Molecular; Movement; Mus; Nervous System Physiology; Nervous system structure; Neuraxis; Neurons; Pathogenesis; Pathway interactions; Peripheral Nervous System; Peripheral Nervous System Diseases; Postherpetic neuralgia; Prevention therapy; Primary Lateral Sclerosis; Property; Recombinants; Reporter; Scanning; Scanning Electron Microscopy; Site; Structure; Structure of superior cervical ganglion; Submandibular gland; Suid Herpesvirus 1; System; Testing; Three-Dimensional Image; Tracer; Viral; Virion; Virus; Virus Diseases; Work; animal tissue; base; in vivo; mutant; neural circuit; new technology; particle; relating to nervous system; research study; tissue fixing; tissue/cell culture; tool; two-photon; uptake

DESCRIPTION (provided by applicant): A long-term goal of my laboratory is to determine the molecular mechanisms by which neuroinvasive alpha-herpesviruses invade and spread in the mammalian nervous system. Work in this proposal centers on using new imaging technology (e.g., two-photon laser scanning microscopy and serial block face scanning electron microscopy) to reveal how virion components move inside and between neurons. Experiments are divided between two aims focusing on using imaging technology to visualize how infection spreads from one neuron to another in vitro (dissociated neurons; aim 1) and in vivo (living animals and tissues; aim 2). The primary hypothesis to be tested is that trans-neuronal spread is mediated by an enveloped capsid released from the infected cell that is subsequently taken up by the synaptically-opposed uninfected neuron. Two contrasting hypotheses are also considered. In aim 1, virion structures will be labeled with fluorescent fusion proteins, neuronal structures will be labeled with specific dyes and antibodies, and imaged using two-photon microscopy and conventional confocal microscopy in live and fixed dissociated peripheral nervous system neurons. To aid in visualizing sites where trans-neuronal infection occurs, selected viral mutants defective in spread and marked with various fluorescent reporters will be used. In aim 2, I will study in vivo viral invasion and spread of infection from the peripheral nervous system (the submandibular ganglia) to the central nervous system in living animals, tissue explants, and fixed tissues. Serial block face scanning electron microscopy will enable collection of stacks of in-register 50nm sections to reveal 3D images of PRV-infected submandibular gland ganglia. These images will complement and expand the observations made using two-photon laser scanning microscopy. The knowledge obtained from these studies enables not only a better understanding of herpesvirus pathogenesis, it also provides the means to reveal aspects of neuronal cell biology and neural circuitry that are key to viral invasion and spread. Despite modern medicine and antiviral drugs, viral infections of the nervous system are devastating and exceedingly difficult to manage. Understanding the host and viral interactions involved in neuroinvasion and resulting pathogenesis has relevance to human health by revealing new targets for prevention and therapy. On the other hand, the same properties of neuroinvasion provide opportunities to use such viruses and mechanisms to understand the organization of the nervous system using viruses as tracers of neural circuitry. PUBLIC HEALTH RELEVANCE: Despite considerable progress over the years, we still do not have a comprehensive understanding of the molecular mechanisms of trans-neuronal spread of herpesviruses, even in simple outline. Understanding the mechanisms of axonal and dendritic transport, assembly, release, and uptake will provide targets for manipulation that could substantially expand our understanding of viral spread. The information obtained from these studies will be important for at least three reasons. First, it will advance our understanding of alpha- herpesvirus infection of the nervous system and trans-neuronal spread of infection by providing an entirely new imaging-based perspective. Second, it will inform us about other diseases involving axonal transport and function such as Charcot-Marie-Tooth disease type II, hereditary spastic paraplegia, amyloid lateral sclerosis, and other peripheral neuropathies such as post-herpetic neuralgia. These diseases are likely to affect similar pathways as those engaged by viruses for their replication and spread in the nervous system. Third, it will provide critical information for the construction of new genetically engineering strains of virus for neural tracing. These neural tracers would be powerful tools to elucidate brain micro-circuitry, providing a better understanding of nervous system functions.

描述（由申请人提供）：我的实验室的长期目标是确定神经侵袭性α-疱疹病毒侵入哺乳动物神经系统并传播的分子机制。该提案的工作重点是使用新的成像技术（例如双光子激光扫描显微镜和串行块面扫描电子显微镜）来揭示病毒颗粒成分如何在神经元内部和神经元之间移动。实验分为两个目标，重点是使用成像技术在体外（分离的神经元；目标 1）和体内（活体动物和组织；目标 2）可视化感染如何从一个神经元传播到另一个神经元。要测试的主要假设是，跨神经元传播是由受感染细胞释放的有包膜衣壳介导的，该衣壳随后被突触相对的未受感染神经元吸收。还考虑了两种相反的假设。在目标 1 中，病毒粒子结构将用荧光融合蛋白标记，神经元结构将用特定染料和抗体标记，并使用双光子显微镜和传统共聚焦显微镜对活体和固定分离的周围神经系统神经元进行成像。为了帮助可视化发生跨神经元感染的位点，将使用选定的传播缺陷并标有各种荧光报告基因的病毒突变体。在目标 2 中，我将研究活体动物、组织外植体和固定组织中的体内病毒入侵和感染从周围神经系统（下颌下神经节）到中枢神经系统的传播。串行块面扫描电子显微镜将能够收集注册的 50 nm 切片堆栈，以显示 PRV 感染的下颌下腺神经节的 3D 图像。这些图像将补充和扩展使用双光子激光扫描显微镜进行的观察。从这些研究中获得的知识不仅可以更好地了解疱疹病毒的发病机制，还提供了揭示神经细胞生物学和神经回路的方法，这些对于病毒入侵和传播至关重要。尽管有现代医学和抗病毒药物，神经系统的病毒感染仍然具有破坏性，并且极难控制。了解神经侵袭中涉及的宿主和病毒相互作用以及由此产生的发病机制，通过揭示预防和治疗的新目标，与人类健康相关。另一方面，神经侵袭的相同特性提供了使用此类病毒和机制来了解神经系统组织的机会，使用病毒作为神经回路的示踪剂。公共卫生相关性：尽管多年来取得了相当大的进展，但我们仍然对疱疹病毒跨神经元传播的分子机制没有全面的了解，即使是简单的概述。了解轴突和树突运输、组装、释放和摄取的机制将为操作提供目标，从而大大扩展我们对病毒传播的理解。从这些研究中获得的信息非常重要，至少有三个原因。首先，它将通过提供全新的基于成像的视角，增进我们对神经系统的α-疱疹病毒感染和感染的跨神经元传播的理解。其次，它将告诉我们有关轴突运输和功能的其他疾病，例如 II 型腓骨肌萎缩症、遗传性痉挛性截瘫、淀粉样蛋白侧索硬化症和其他周围神经病，例如带状疱疹后神经痛。这些疾病可能会影响与病毒在神经系统中复制和传播相似的途径。第三，它将为构建用于神经追踪的新基因工程病毒株提供关键信息。这些神经示踪剂将成为阐明大脑微电路的强大工具，从而更好地理解神经系统功能。