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）：我的实验室的一个长期目标是确定神经侵袭性α-hepesviruss侵入并扩散在哺乳动物神经系统中的分子机制。在此提案中进行的工作集中在使用新成像技术（例如，两光子激光扫描显微镜和串行块面扫描电子显微镜）以揭示病毒粒子成分如何在神经元之间和之间移动。实验分配在两个目标之间，重点是使用成像技术来可视化感染如何从一个神经元传播到另一个神经元到另一个神经元（解离神经元；目标1）和体内（活动物和组织；目标2）。要测试的主要假设是，跨性神经元扩散是由从感染细胞释放的包裹的衣壳介导的，该细胞随后被突触未受感染的神经元所吸收。还考虑了两个对比的假设。在AIM 1中，病毒体结构将用荧光融合蛋白标记，神经元结构将用特定的染料和抗体标记，并使用两光子显微镜和常规共焦显微镜在活和固定分离的外周神经系统神经元中成像。为了有助于可视化发生跨神经元感染的部位，将使用选定的病毒突变体在扩散中有缺陷并用各种荧光记者标记。在AIM 2中，我将研究体内病毒侵袭和感染从周围神经系统（下颌神经节）传播到活动物，组织外植体和固定组织中的中枢神经系统。串行块面扫描电子显微镜将使50nm内注册的堆栈收集，以揭示PRV感染的下颌下腺神经节的3D图像。这些图像将补充并扩展使用两光子激光扫描显微镜进行的观察结果。从这些研究中获得的知识不仅可以更好地理解疱疹病毒发病机理，还提供了揭示神经元细胞生物学和神经回路方面的手段，这是病毒侵袭和扩散的关键。尽管现代药物和抗病毒药物，神经系统的病毒感染仍是毁灭性的，并且难以管理。通过揭示用于预防和治疗的新目标，了解与人类健康有关的宿主和病毒相互作用与人类健康有关。另一方面，神经入侵的相同特性为使用这种病毒和机制提供了使用病毒作为神经回路的示踪剂来理解神经系统组织的机会。公共卫生相关性：尽管多年来取得了很大进展，但即使在简单的轮廓中，我们仍然对疱疹病毒的跨神经元传播的分子机制也没有全面的了解。了解轴突和树突运输，组装，释放和吸收的机制将为操纵提供目标，从而可以大大扩展我们对病毒扩散的理解。从这些研究中获得的信息至少出于三个原因将很重要。首先，它将通过提供全新的基于成像的视角来提高我们对神经系统α-疱疹病毒感染和跨神经元传播的理解。其次，它将告知我们其他涉及轴突运输和功能的疾病，例如Charcot-Marie-type II型，遗传性痉挛性截瘫，淀粉样淀粉样性侧面硬化症以及其他周围神经病，例如后赫性神经神经痛。这些疾病可能会影响与病毒复制和在神经系统中传播的途径相似的途径。第三，它将为建造新的基因工程病毒菌株的神经追踪菌株提供关键信息。这些神经示踪剂将是阐明脑微路的强大工具，可以更好地理解神经系统功能。