改造水痘带状疱疹病毒构建新的神经环路示踪工具

Neural circuit is the foundation of brain function connectome and its damage leads to neuropathy. Neural circuit tracing is the key technology for mapping brain functional connectivity and elucidating the mechanisms for neuropathy. The development of novel neural circuit tracing system with high resolution, low pathogenicity and high safety will benefit the “Brain Project” of all nations. Because neurotropic viruses traverse neural pathways during naturally infections, it is extremely valuable to develop viral tools. Viral tools have following advantages: ability of viral replication to amplify the signals before transsynapse; ability to traverse multisynaptic pathway; viral spread can be highly specific to synaptically connected cells; high efficient markers (fluorescent proteins) can be expressed, so that the markers can be visualized without staining. Current using neural tracing viral tools have shortages. For example, pseudorabies virus has strong toxicity/ pathogenicity, causes server neural cell death and animal death within 1 week. Rabies virus has very small vial genome, and can’t be applied for expressing of interest genes. Varicella- zoster virus (VZV) is a neurotrophic virus with large DNA genome, and the vaccine strain is suitable for transforming into neural circuit tracers. We have inserted VZV genome into the bacterial artificial chromosome (BAC), constructed VZV-BAC. VZV-BAC is an effective platform for genetic modification of virus to further develop VZV based circuit tracer system. From VZV-BAC, we have obtained VZV-GFP, confirmed that VZV-GFP can label neural circuit efficiently, with low toxicity in neurons and in mouse brain. In this project, we will further modify VZV to obtain a viral tool system, including starting neuron specific monosynaptic tracer, neuron type specific tracer, and functional coupling multisynaptic tracer; characterize these VZV tools in cultured neurons, provide the information for further modification, such as spread direction, speed, efficiency of marking the neurons; test the labeling efficiency of mouse brain neural circuits;further optimize these viral tools. Our goal is to develop a novel circuit tracing tool system with specificity, high efficiency, low toxicity, and high safety; to visualize neural circuits with high resolution, help to understand the brain function and elucidate the mechanisms for brain disorders.

神经环路是脑功能的基础，其病变则发生脑疾病。神经环路的解析是理解脑功能联结图谱和脑疾病机制的关键。研发神经环路示踪工具，标记神经环路是解析神经环路的关键技术。基于病毒的示踪工具有信号强、跨突触等优点。但现有工具病毒毒性强或改造空间有限。水痘带状疱疹病毒（VZV）为大基因组DNA病毒，天然嗜神经元，其疫苗株有改造为神经环路示踪工具的优势。我们将VZV基因组插入细菌人工染色体（BAC），构建了有效改造VZV的平台VZV-BAC；经初步改造，获得VZV-GFP，证实VZV-GFP神经标记信号好、毒性低。在此基础上进一步改造，获得新工具病毒，包括起始细胞特异跨多/单级突触、功能耦联等工具；研究这些新工具病毒在体外神经元中的感染、传播特性和机理，为定向运输改造提供依据；检测在脑内不同环路中的标记效果。有望获得安全、高效、特异、低毒的新工具病毒，为神经环路的解析提供条件，促进脑功能和脑疾病机制研究。

神经环路是脑功能的基础，其病变则引发脑疾病。神经环路的解析是理解脑功能联结图谱和脑疾病机制的关键。改造天然的嗜神经病毒，发展优良的示踪病毒工具用于标记神经环路是解析神经环路的关键。水痘带状疱疹病毒（VZV）和Ⅰ型单纯疱疹病毒（HSV-1）具有天然嗜神经元、跨突触、改造空间大等作为神经环路示踪工具的优势，因此，本项目选择它们作为改造对象。.项目执行期间，主要进行了以下两方面内容的研究： .（1）建立了VZV的遗传改造平台VZV-BAC-GFP，改造了一系列工具病毒并测试其示踪效果,由于制备的VZV示踪工具病毒的滴度较低，其跨突触传播的效率相对较低；基于VZV遗传改造平台，鉴定了VZV的嗜神经因子ORF7作为皮膜蛋白参与神经细胞内病毒外膜的装配，其缺失严重的影响了病毒在神经细胞间的传播，VZV-7D不仅可以作为潜在的顺向跨单级示踪工具病毒，还可作为不具神经传播能力的新型神经减毒VZV疫苗而被进一步开发，研究结果在线发表于《Journal of Virology》。.（2）基于HSV-1-H129-BAC的遗传改造平台发展了高效标记的顺向跨多级突触示踪病毒工具H129-G4，这是目前报道的唯一可用于fMOST成像的顺向示踪病毒工具，对于解析全脑大范围神经环路连接具有重要价值；发展了首个顺向跨单级突触示踪病毒工具H129-dTK-tdT，标记直接输出环路，弥补了顺向跨单级示踪病毒工具的空白；相关研究成果发表于《Molecular Neurodegeneration》，并申请了3项国内专利，2项美国专利,其中一项于2019年12月3日获得授权。新工具立即引起多个神经科学实验室关注，上述工具目前已广泛用于神经科学研究，并合作发表多项成果。.我们将进一步优化本项目开发的顺向跨突触神经环路示踪病毒工具，为解析神经环路提供条件，促进脑功能和脑疾病的机制研究。