草鱼呼肠孤病毒反向遗传学系统构建及其毒力相关基因研究

Grass carp hemorrhagic disease caused by GCRV is a highly severe viral disease with high morbidity and mortality, and has resulted in great economic losses in grass carp culture in China. The genome of GCRV contains 11 double-stranded RNA segments. Typical characteristics of GCRV are high rate of variation, widely dispersed and different virulence strains, which have also become the major obstacles to further study in GCRV. Reverse genetics is a powerful tool to investigate gene functions in RNA viruses. However, to date, there is no report of a reverse genetics system for GCRV, and virulence-related genes of GCRV are not determined. Based on the previous research that different virulence GCRV strains have been obtained, this project is aimed to sequence the whole genomes and construct reverse genetic systems for virulent and avirulent GCRV strains. Using bioinformatics analysis method and sequence alignment between corresponding segments, a number of virulence-related candidate segments and key domains of GCRV will be selected. A series of recombinant virus will be generated by the reverse genetic system through the segment replacement, gene deletion and mutation. Finally, virulence-related genes of GCRV will be determined by comparing the virulence changes between recombinant viruses and wild-type viruses in vitro and in vivo. The results will provide a powerful technical platform for the study of functional genomics in GCRV, which will be helpful for better understanding of pathogenesis, designing novel attenuated live vaccines with high protective efficiency and pay the way for the prevention and control of grass carp hemorrhagic disease in China.

草鱼出血病是由草鱼呼肠孤病毒（GCRV）引起的严重病毒性疾病，对我国草鱼养殖业危害极大。GCRV是分节段的双链RNA病毒，具有易变异、分布广、致病性强弱不同的特点，这已成为对该病毒深入研究的主要障碍，而反向遗传学技术能很好解决这一难题，但目前未见成功建立GCRV反向遗传学系统的报道，GCRV毒力基因也不清楚。本项目拟在已获得致病性不同的GCRV强、弱毒株基础上，测定其全基因组序列；随后利用反向遗传学技术，分别构建强、弱毒株的反向遗传学系统。在此基础之上，采用生物信息学分析方法，将强、弱毒株基因组序列进行比对，推测出GCRV毒力相关的可能基因组节段和关键位点，通过基因置换、缺失和突变，构建系列病毒突变株；在鱼体和细胞水平上对比突变株、野生株的毒力变化，确定GCRV毒力基因和必须氨基酸位点。研究结果将为GCRV功能基因组研究、致病机理、新型弱毒活疫苗研制和病毒病有效防控提供技术支持和理论依据。

初步建立了草鱼呼肠病毒的“四质粒”反向遗传学系统，获得了GCRV的重组病毒；建立了GCRV的稀有鮈鲫感染模型，并通过该模型确定了22株不同GCRV的毒力强弱；系统分析比较了GCRV强弱毒株在细胞和鱼体中的病毒增殖动态，及毒力、致病性的差异，开展了病毒诱导引起的细胞凋亡研究；II型GCRV强弱毒株感染鱼体后的蛋白组学差异分析表明，强毒株感染导致宿主更多的免疫相关蛋白的下调，这可能是导致死亡率更高的重要原因；通过对已知的I型GCRV强弱毒株FAST蛋白的多序列比对分析，发现该蛋白的72位氨基酸（S to L）可能是潜在的毒力决定位点；从健康草鱼中分离鉴定了一株新的草鱼呼肠孤病毒（命名为HGCRV），序列分析表明该毒株与GCRV−873为代表的GCRV−I型毒株蛋白序列具有较高的同源性(69.57~96.71%)，而与GCRV−II型（GCRV−HZ08）和GCRV−III型（HGDRV）毒株的同源性相对较低，分别为22.65~45.85%和23.37~43.39%；表明该毒株属于I型GCRV毒株，但同现有毒株存在较大差异，同时该毒株对鱼不致病，可作为潜在的疫苗弱毒株。研究结果可为GCRV功能基因组研究、致病机理、新型弱毒活疫苗研制和病毒病有效防控提供技术支持和理论依据。