草鱼呼肠孤病毒（GD108株）fiber蛋白介导的病毒吸附机制研究

Grass carp reovirus (GCRV) infection can lead to high mortality to grass carp and cause huge losses to the aquaculture. There is little understanding of the specific recognition and binding mechanisms between GCRV and host. In our previous work, grass carp reovirus Guangdong strain (GCRV-GD108) was isolated, and its fiber protein was proved could function as the cell attachment protein in GCRV-GD108. However, it is not clear that the fiber protein binds to what kind of proteins on grass carp cells to initial the infection. On this basis, the project intends to study the mechanism of GCRV-GD108 fiber protein recognizing and binding host. The proteins in grass carp cells interacting with GCRV-GD108 fiber will be isolated and identified by yeast two-hybrid cDNA library and Co-immunoprecipitation; and the functions as receptors of the isolated proteins will be verified using RNAi and eukaryotic expression techniques; interaction of the GCRV-GD108 fiber protein with the receptor proteins will be identified by surface plasmon resonance and pull down; in vitro and in vivo binding of GCRV-GD108 to the proteins will be confirmed by fluorescence-linked immunosorbent assay and immunocytochemistry. The study will reveal the attachment mechanisms during GCRV-GD108 earlier infection, and will lay the foundation for deeply understanding the infection mechanism of GCRV, and also will open up the prospect of new strategies for preventing and controlling of grass carp hemorrhage.

草鱼呼肠孤病毒（GCRV）可感染草鱼导致高死亡率，给草鱼养殖业造成巨大损失，但目前对其入侵机制了解甚少。申请人前期分离鉴定了草鱼呼肠孤病毒广东株（GCRV-GD108），发现其在GCRV流行株中具代表性，并已证实GCRV-GD108 fiber蛋白是病毒的细胞吸附蛋白。但病毒fiber蛋白与草鱼细胞中何种蛋白结合开启病毒感染尚未清楚。本研究拟开展GCRV-GD108 fiber 蛋白介导的病毒吸附机制研究，通过酵母双杂交及免疫共沉淀技术筛选草鱼细胞中与病毒fiber互作蛋白；利用RNAi及真核表达实验验证其受体功能；进一步通过表面等离子共振、pull down、荧光免疫吸附及免疫细胞化学等技术明确fiber蛋白与筛选蛋白的互相作用，以期阐明GCRV-GD108在感染初期吸附宿主细胞的分子机制。本研究将为阐明GCRV感染机制提供科学依据，并为草鱼出血病的有效防控提供新思路。

草鱼呼肠孤病毒（GCRV）可感染草鱼导致高死亡率，给草鱼养殖业造成巨大损失。草鱼呼肠孤病毒广东株（GCRV-GD108）在GCRV流行株中具代表性，但其入侵感染机制尚未清楚，亟待开展GCRV入侵宿主机制以及防治策略的研究。本项目开展了草鱼吻端成纤维细胞（PSF）中GCRV-GD108 fiber互作蛋白的分离鉴定；对病毒候选受体JAM-A及NgR1进行表达分析， gcjam-a1在未受精卵中表达，并且其表达水平在草鱼及细胞中受病毒影响最大，推测其可能为GCRV-GD108的主要受体。NgR1与草鱼神经系统的发育有关，在草鱼幼鱼脑组织中的表达量变化随病毒感染显著，在PSF细胞中NgR1 的表达变化与病毒的感染直接相关；对草鱼呼肠孤病毒（GCRV-GD108）感染PSF细胞前后的转录组进行测序，相对于未感染的PSF细胞，GCRV-GD108感染PSF细胞后得到显著差异表达基因共31个，涉及到病原感染、蛋白转运等，这是首次通过转录组分析揭示草鱼感染PSF细胞的分子机制；对GCRV-GD108基因组编码蛋白分析，选取6个病毒节段进行原核重组表达，通过Elisa实验分析其免疫原性，免疫保护实验检测蛋白的免疫保护率，筛选到重组S7编码蛋白在实验室保护实验中对草鱼的免疫保护率达到100%，可作为抗草鱼出血病的优质高效基因工程亚单位疫苗的；同时制备GCRV fiber蛋白的多抗及单抗，建立一种ELISA检测GCRV的方法，能特异检测患病草鱼中的草鱼呼肠孤病毒。. GCRV感染及入侵机制的研究一直是鱼类病毒学的研究热点，本项目研究不仅加深对草鱼呼肠孤病毒吸附宿主机制的认识，还可为其他水生呼肠孤病毒病毒的入侵机制研究提供科学依据，推动相关领域的科研进展。筛选到的草鱼呼肠孤病毒基因工程亚单位疫苗，进一步将在草鱼主养区推广应用将提高养殖区草鱼成活率，提高生产效益，增加渔民收入。