新城疫病毒末端序列与毒力相关性研究

Newcastle disease（ND）which have been listed as an important animal disease by Office International des Epizooties（OIE）is one of the most serious infectious diseases in the worldwide poultry industry. It is becoming an urgent scientific issue to research on molecular pathogenic mechanisms of different Newcastle disease virus strains and to develope a new effective live vaccine deeply. Given the current domestic and international studies showed that lack of the research of the non-coding regulation region of NDV genome, especially the regulated function in terminal sequence, and particular relationship about the structure and function of terminal sequence, so this project will intend to use the reverse genetics to build the full-length cDNA infectious clones of different NDV strains to practice the substitution of the terminal sequence of the homologous/ heterlogous and the insertion of foreign genes, and to rescue the recombinant virus and to identify the related biological characterization, respectively. Through the research, we not only clarify the influence of terminal sequence on the virulence of NDV strains, but also clear the impact of homologous/heterogonous terminal sequence of NDV strains in replication, regulation of transcription, genetic stability of homologous/heterogonous virus strains, and the regulation of endogenous/exogenous protein expression，and also assess the potential of the different recombinant virus stains as a vaccine candidate virus. The study is important for the research of the molecular pathogenesis mechanisms of different NDV strains, and screening/reserving new live vaccine candidate virus and the scientific prevention and control of ND.

新城疫是危害世界养禽业最严重的动物传染病之一，OIE已将其列为必报名录中的重要动物疫病。深入研究不同禽源新城疫病毒株的毒力差异与致病分子机制以及新型活疫苗创制已成为亟待解决的重大科学问题。鉴于目前国内外对新城疫病毒基因组非编码区尤其是末端序列功能的研究甚少以及末端序列结构与功能的关系，本项目拟利用反向遗传操作技术构建不同禽源株、不同毒力新城疫毒株的全长cDNA感染性克隆，分别进行同源/异源末端序列的替换以及外源基因的插入，再进行重组病毒株的拯救和相关生物学特性鉴定。通过本项目的研究，不仅将阐明末端序列对新城疫病毒毒力的影响，同时将明确同源/异源新城疫病毒末端序列对同源/异源毒株的复制、调控转录、遗传稳定性的影响以及对内源/外源蛋白表达的调控作用，评估各株重组病毒作为疫苗候选种毒的潜力。对于研究新城疫病毒不同禽源株毒力差异及致病分子机制、筛选新型弱毒疫苗候选株以及防控新城疫具有重要科学意义。

本研究为阐明新城疫病毒末端序列与毒力的相关性，将鹅源NA-1株3’/5’末端非编码区单独和同时替换为La Sota株3’/5’末端，构建了3株全长cDNA感染性克隆，并成功拯救出有感染活性的病毒粒子（分别命名为rNA-L、rNA-T及rNA-LT株）。体外试验及生长动力学曲线表明，3株突变病毒中rNA-L的复制能力与亲本野毒株NA-1相似，而rNA-T和rNA-LT复制能力有轻微减弱；生物学特性分析结果显示3株获救突变病毒遗传稳定性良好。3’/5’末端序列同时替换和5’末端单独替换的毒株，较野毒株致病性稍有减弱但没有完全致弱为同La Sota一样的缓发型毒株，而3’末端单独替换毒株的致病性与NA-1相似。在此基础上，为进一步探究和开发更加有效的新型疫苗，通过将NA-1株的F蛋白裂解位点氨基酸序列替换为La Sota株F蛋白裂解位点相应序列成功拯救出突变致弱株rmNA-1。rmNA-1致病力显著降低，但依然保持亲本株相似的生长特性并具有良好遗传稳定性，表明rmNA-1具有作为基因Ⅶ型NDV弱毒疫苗侯选株及候选疫苗载体的潜力。以rmNA-1为载体，构建了表达GPV VP3蛋白重组新城疫病毒rmNA-VP3，VP3基因在NDV基因组中可稳定遗传，F裂解位点修饰碱基也保持稳定不变，以重组病毒rmNA-VP3免疫雏鹅可同时诱导坚实的GPV和NDV中和抗体，为今后外源基因的表达、活病毒载体疫苗、分子标记疫苗以及新型活疫苗的创制奠定了基础。