改造乙型脑炎病毒作为神经环路示踪工具的探索性研究

Elucidation of the structures of neurocircuits is the prerequisite to understand the mechanisms of brain functions and brain diseases. Thanks to the unique advantages, neurotropic virus based tracers are the major tools in structural studies of neurocircuits. However, the shortcomings of the available systems, such as high toxicity, low safety, complicated preparation procedure, and low infect efficiency, hindered broader usages. In this study, we plan to take advantage of my research experience in virology and the recent advances in the relevant fields to develop new tracer overcoming the drawbacks described above. .Japanese encephalitis virus (JEV) belongs to the genus Flavivirus of the family Flaviviridae. The genome is a single-stranded, positive-sense RNA of about 12 kb in length, encoding a large polyprotein of about 3,400 amino acids. After translation, the polyprotein is processed by host and viral proteases into three structural proteins and seven nonstructural proteins. The structural and nonstructural proteins are for virion assembly and viral replication, respectively. The reverse genetic systems of JEV, such as full length infectious clone, replicon and single round infection of virus-like particle, are braodly available. The glycoprotein (G) of Rabies virus (RV) and Vesicular stomatitis virus (VSV) could package each other and other viruses, such as Lentivirus, Adeno-associated virus (AAV), etc. The viruses packaged with RV-G and VSV-G can efficiently infect neurons and spread retrogradely and anterogradely along synaptic connections, respectively. .In this study，we will use the methods of reverse genetics, fluorescence microscope and immunohistochemistry to develop vaccine strain of JEV into tools for neurocircuit studies, such as helper viral vector for rapid exogenous gene expression, and trans-mono-synaptic and trans-multi-synaptic tracers. These tools will be useful in the analysis of neural circuits of primate and non-primate species, and the development of viral vaccine and drug screening.

神经环路的结构和功能是神经科学的基本问题。近年来，嗜神经病毒以其独特的优点成为神经环路结构研究的重要工具，但存在毒性大、安全性低、制备方法复杂、感染效率低等问题，在一定程度上限制了相关研究。申请人拟利用多年的病毒学研究经历，针对上述问题，以具有嗜神经、毒性低、安全性高、制备过程简捷等特点的乙型脑炎病毒疫苗株为对象，采用病毒反向遗传、荧光显微成像、免疫组化等技术，分别建立可实现外源蛋白表达、跨单级和多级突触的新型示踪工具系统：1）构建具有单次感染性的病毒样颗粒，建立快速表达外源蛋白的辅助工具病毒；2）构建缺失结构蛋白基因的复制子，结合狂犬病毒和水泡性口炎病毒膜蛋白介导病毒定向跨突触的特性，建立特异逆行、顺行跨单级突触的回路示踪体系；3）构建带有荧光蛋白基因的重组乙型脑炎病毒，建立跨多级突触的回路示踪工具。该系统既可用于非灵长类和灵长类动物的神经环路标记，也可用于病毒疫苗和药物筛选等方面的研究

神经环路的结构和功能是神经科学的基本问题。近年来，嗜神经病毒以其独特的优点成为神经环路结构研究的重要工具。乙型脑炎病毒能感染啮齿类和灵长类神经细胞细胞，因此具备开发为标记啮齿类，尤其是啮齿类神经环路的工具的潜力，因此，制备可视化的重组乙型脑炎病毒是本项目的核心。在重组病毒的制备过程中，发现其不能稳定携带绿色荧光蛋白基因，这也是目前没有可视化乙型脑炎病毒的原因。因此，采取了一系列筛选方法，鉴定出一个适应性突变，其能使重组病毒稳定携带绿色荧光蛋白基因。通过一系列体外和体内的分析实验，表明其能点亮被标记的神经细胞。目前获得的数据对于下一步分析其标记鼠脑和猴脑神经和神经网络的特性具有十分重要的意义。