牛轮状病毒NSP4基因毒力位点分析及其毒力致弱株的获得

牛轮状病毒(Bovine Rotavirus, BRV)是犊牛急性腹泻的主要病原之一，给养牛业造成很大的经济损失。BRV通过消化道感染，主要防控手段为减毒疫苗经口服或直肠接种激发的肠道局部粘膜免疫反应，但国内尚无有效安全的BRV口服弱毒疫苗。NSP4基因是RV引起腹泻的重要毒力因子，在病毒致病过程中起关键作用。经对人轮状病毒NSP4基因功能研究发现部分毒力相关位点，但在完整病毒上相应位点是否有效，尚待进一步研究。本研究拟对BRV NSP4基因部分位点进行定点突变，利用反向遗传拯救NSP4基因突变的BRV，经RNAi和蚀斑克隆技术筛选并富集阳性重组病毒；通过动物感染实验，检测突变病毒的毒力改变和致腹泻效果变化，最终获得毒力致弱病毒株，为牛轮状病毒引起的犊牛腹泻的防治奠定坚实的基础。

Bovine rotavirus (Bovine Rotavirus, BRV) is one of the main pathogens of calves with acute diarrhea, can cause great economic losses to the cattle industry. Because BRV infectes animals through the digestive tract, so we can use attenuated vaccine by the oral or rectal inoculation to stimulate intestinal mucosal immune response, but now we have no effective and safe BRV oral attenuated vaccine in china. The rotavirus non-structural protein-4 (NSP4) is the first recognized rotavirus-encoded enterotoxin and has been implicated in a number of biological functions in rotavirus infection and pathogenesis. We have found some virulence-associated sites by human rotavirus NSP4 gene function studies, but the effectiveness of corresponding sites in the intact virus is not clear. In this study,we will change BRV NSP4 gene part sites by directed mutagenesis, rescue the recombinant BRV with reverse genetics via mutation of virulent site of NSP4 gene, then screen and enrich positive recombinant virus by RNAi and plaque cloning technology. Finally,we will get a virulence attenuated virus strain after detecting the rescued virus's virulence and diarrhea effect changes in animal infection experiments with a view to lay the foundation for prevention and treatment of BRV.