【作者】 徐怀英；

【导师】 刘金华； 秦卓明；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2014， 博士

【摘要】 新城疫(Newcastle disease, ND)是危害中国养禽业最重要的疫病之一。由ND引起的死亡、呼吸道症状和产蛋下降等十分严重,即使一些经过多次免疫、抗体水平较高的鸡群,仍时有发病。因此,了解NDV的流行动态,掌握我国流行的NDV的基因特性及生物学特性,探讨病毒变异对抗原性的影响,及时制定科学合理的防疫措施,对防控ND在家禽中的大流行具有重要意义。2006～2013年间,在山东省家禽NDV流行病学监测过程中从不同宿主家禽中分离并鉴定55株NDV,利用蚀斑纯化技术在鸡胚成纤维细胞上完成了49株NDV的纯化,进行了1日龄雏鸡脑内接种致病指数(ICPI)、最小致死量致死鸡胚平均死亡时间(MDT)和6周龄鸡静脉接种致病指数(IVPI)生物学毒力测定,证实有44株强毒,5株中等毒力,6株弱毒。通过对不同宿主红细胞(RBC)凝集试验表明,9株鸭源NDV均能凝集4种RBC,而鸡源NDV仅6株基因Ⅱ型NDV能凝集四种RBC,而其它鸡源和鸽源NDV强毒均不能凝集马RBC。通过对NDV流行毒F、HN等基因测序、同源性比较和基因分型,结果表明：55株NDV中,有43株基因Ⅶ型,5株Ⅵ型,6株基因Ⅱ型,1株基因Ⅸ型,基因Ⅶ型仍然为主要的流行毒株,且主要为d亚型。同源性比较证实,Ⅶ型NDV流行株与疫苗株LaSota等的同源性愈来愈低,不足80%；即便同属于基因Ⅶd亚型依然存在由点突变形成的累积性变化,且这种规律伴随着年代的增加愈来愈明显。通过对NP、P、M、F、HN和L6个编码基因和各段非编码区核苷酸绘制系统进化树,不同基因的分类结果基本相同,说明NDV在基因的遗传演化规律方面是一致的。选择16个NDV流行株和3个经典株(LaSota、Clone30和F48E9),分别制备单因子血清,利用血凝抑制(HI)进行交叉反应,比较了流行株与疫苗毒LaSota株和中国经典强毒F48E9间的抗原性交叉反应,结果显示我国鸡群中流行的基因Ⅶ型流行毒与LaSota之间的相关指数为：0.31≤R≤0.66,证实流行病毒株已在抗原性上逐渐偏离疫苗LaSota株。免疫攻毒保护试验证明,基因Ⅶ型毒株免疫组攻毒后的临床症状、病毒分离率及排毒量均显著低于传统疫苗组(LaSota)。综上所述,NDV的分子变异是编码基因与非编码序列同步性变异的结果。基因Ⅶd亚型NDV是山东省家禽中的优势流行株,其F和HN基因氨基酸的突变具有累积性；传统疫苗LaSota与流行毒抗原性已出现较大差异,基因Ⅶd亚型疫苗在免疫保护、抑制病毒复制等方面具有更好的效果。更多还原

【Abstract】 Newcastle (Newcastle disease, ND) is one of China’s most prominent disease hazards in the poultry industry. The death, respiratory symptoms and decreased egg production caused by ND are very common, even in chickens after repeated immunization. Therefore, to understand the epidemic dynamic of NDV, genetic characteristics and biological characteristics of grasp of China’s popular NDV, to explore the effect of virus antigenicity, formulate scientific and reasonable prevention measures, has important significance for the prevention and control of ND outbreak.Between2006to2013,55NDV isolates have been isolated and identified from different hosts in Shandong Province during the epidemiological surveillance of poultry. Then, fourty-nine NDV isolates which had successfully purified by plaque-purification in chicken embryo fibroblasts, were conducted and determined biological virulence include1-day-old chicks intracerebral pathogenicity index (ICPI), the minimum lethal dose lethal embryo mean death time (MDT) and6-week-old chickens intravenous pathogenicity index (IVPI). The results showed that55NDV, there are44of virulent, five moderate virulence, six of attenuated.. Through the different host red blood cell (RBC) agglutination tests showed that9strains isolated from duck and only6Chicken NDV genotype II NDV can agglutination four kinds of RBC, while other sources NDV from chicken and pigeon don’t have the character of agglutination horse RBC.Sequencing, genotyping and gene homology analysis were conducted on the F and HN genes of55isolates. The results showed that55strains belonged to four genotypes (Ⅱ, IX,Ⅵ, and Ⅶ),6isolates belonged to genotype Ⅱ,5strains (isolated from pigeon) to genotype VI,1isolate to IX, and the others to subtype Ⅶd. Amino acid sequence comparison confirmed that the strains between the same genotype with high homology, while homology rate of less than80%between genotype II LaSota strain and advantages. Meanwhile, even in the same genotype, there were still changes of point mutation accumulation, and this rule accompanied by an increase of more and more obvious. Genetic evolution analysis based on6coding genes and non-coding genes indicated that variation of NDV occurred throughout the genome.16epidemic NDV isolates and three classic strain (LaSota, Clone30and F48E9) were respectively prepared for definite factor positive sera by SPF chicken, and carried out the hemagglutination inhibition (HI) cross-reaction tests. The results showed that the relevant index of chickens popular genotype Ⅶ strains and LaSota is:0.31≤R≤0.66, confirmed pandemic strain were gradually deviated from antigenically LaSota vaccine strain. Protective efficacy tests proved that genotype VII strains immunohistochemistry symptoms, virus isolation rate and detoxification after the attack were significantly lower than the traditional vaccine (LaSota).In summary, the synchronous variation of the non-coding sequences and encoding genes results in the NDV molecular variation. Subtype Ⅶd NDVs are advantages epidemic stains in Shandong Province, with cumulative mutation of amino acids in functional F and HN genes; tradition LaSota vaccine and popular virus have appeared quite different antigenic, and genetic Ⅶd subtype vaccine have a better protection effect in inhibition of virus replication.更多还原