鸭源新城疫病毒部分中国分离株生物学特性研究

发布时间：2018-09-01 15:12

【摘要】：新城疫是由新城疫病毒引起的严重威胁养禽业的一种病毒性传染病。新城疫病毒属于副粘病毒科禽腮腺炎病毒属。水禽特别是鸭和鹅通常被认为是新城疫病毒潜在的宿主,即使感染对鸡具有高死亡率特征的强毒株,也很少甚至不表现任何临床症状。但是,在过去十几年中许多国家和地区都有水禽感染新城疫病毒的报道。本次研究中的19株新城疫病毒均是从鸭的样品中分离,其中18株病毒是从临床健康鸭的泄殖腔拭子中分离,1株病毒是从发病鸭的肾脏中分离。所有的样品稀释后接种9~10日龄SPF鸡胚尿囊腔,然后通过血凝试验和血凝抑制试验对鸡胚尿囊液进行血清学鉴定。强毒株用蚀斑纯化法在原代鸡胚成纤维细胞中纯化,弱毒株用有限稀释法在鸡胚中纯化。将纯化后的病毒扩繁后,提取RNA并用一步法RT-PCR分段扩增病毒全基因组,将PCR产物亚克隆到p MD18-T载体并进行序列测定、拼接、比对和分析。19株NDV毒株基因组序列分析表明,这些病毒的基因组分为3种长度,15186 nt,15192 nt和15198 nt,符合新城疫病毒复制所需的“六碱基原则”,19株病毒基因组的基因顺序均为3?-NP-P-M-F-HN-L-5?;所有序列的差异性在0%到28.3%之间;F蛋白裂解位点是预测NDV毒力的重要依据,19株病毒中,Md/CH/LGD/1/2005的裂解位点为112RRQKRF117,具有典型的强毒株裂解位点特征,与class II系基因VIId型毒株相似;Du/CH/LAH/224/2011和Du/CH/LAH/209/2011的裂解位点分别为112GKQGRL117和112GRQGRL117,具有典型的弱毒株分裂位点特征,分别与class II系基因I型和II型疫苗毒株相似;16株病毒的裂解位点均为112ERQERL117,具有典型的弱毒株裂解位点特征,在野鸟和家鸭分离株中均有报道。根据F基因高变区和全基因组序列的系统进化分析表明,至少有4种基因型的新城疫病毒在国内家鸭的体内存在:Md/CH/LGD/1/2005属于class II系基因VIId型,是上个世纪末开始大多数新城疫爆发的主要基因型;Du/CH/LAH/224/2011属于class II系基因Ib,与CBU2374毒株类似;Du/CH/LAH/209/2011属于class II系基因II型;其余16株病毒属于class I系基因1b型,是中国水禽的流行基因型。根据19株鸭源新城疫病毒的分子特征和遗传进化分析,选出7株鸭源新城疫病毒进行致病性试验;并根据F基因高变区构建的系统进化树,选出8株不同宿主的毒株作为参考,同样进行致病性试验。Md/CH/LGD/1/2005的ICPI值为1.88,MDT值为50 h,鉴定为强毒株。其他毒株的ICPI值在0到0.34之间,MDT值在125h到168h,鉴定为弱毒株。与此同时,我们还选择病毒的宿主(鸭)作为实验动物,来进行致病性试验。根据19株鸭源新城疫病毒的遗传进化分析和致病性试验的特点,选出6株鸭源新城疫病毒,并从实验室分离到的毒株中选出不同宿主不同基因型的17株病毒,制备单因子血清,然后进行交叉血凝抑制试验。结果表明新城疫病毒株的抗原相关性可能与基因型有关,而与宿主无关。以上结果表明,鸭源新城疫病毒与鸽源新城疫病毒不同,鸭源新城疫病毒在遗传进化和抗原性方面没有形成独立的进化分支,据此推测鸭源新城疫病毒可能来源于其他禽类。新城疫病毒存在于鸭体内,可能是为了适应新的宿主,新城疫病毒发生了一定程度的变异,因此鸭和鸭胚能更真实地反应鸭源新城疫病毒的致病性。鸭源新城疫病毒在新城疫病毒进化中起到重要的作用。
[Abstract]:Newcastle disease (NDV) is a viral infectious disease caused by Newcastle disease virus (NDV) which is a serious threat to poultry industry. Newcastle disease virus (NDV) belongs to the paramyxovirus family avian mumps virus. Waterfowls, especially ducks and geese, are often considered as potential hosts of NDV, and rarely, if not manifested, even if infected with a virulent strain with high mortality to chickens. In this study, 19 strains of Newcastle disease virus were isolated from duck samples, 18 of which were isolated from cloacal swabs of clinically healthy ducks, and one strain was isolated from kidneys of infected ducks. The samples were diluted and inoculated into the allantoic cavity of 9-10 day-old SPF chicken embryos. The chicken embryo allantoic fluid was serologically identified by hemagglutination test and hemagglutination inhibition test. The virulent strain was purified in the primary chicken embryo fibroblasts by plaque purification method, and the attenuated strain was purified in the chicken embryos by limited dilution method. Genome sequence analysis of 19 NDV strains showed that the genomes of these viruses were divided into three lengths, 15186 nt, 15192 nt and 15198 nt, which accorded with the "six base principle" for the replication of Newcastle disease virus. The genome sequence of the strains was 3? -NP-P-M-F-HN-L-5?; all the sequences were between 0% and 28.3%. The F protein cleavage site was an important basis for predicting NDV virulence. Of the 19 strains, the cleavage site of Md/CH/LGD/1/2005 was 112RRQKRF117, which had typical characteristics of the cleavage site of the virulent strain and was similar to the type VIId strain of the class II gene. Similarly, the splitting sites of Du/CH/LAH/224/2011 and Du/CH/LAH/209/2011 were 112 GKQGRL117 and 112 GRQGRL117, respectively, with typical splitting site characteristics of attenuated strains, similar to class II genotype I and type II vaccine strains; the splitting sites of 16 strains were 112 ERQERL117, with typical splitting site characteristics of attenuated strains in wild birds and families. According to phylogenetic analysis of F gene hypervariable region and whole genome sequence, at least four genotypes of NDV were found in domestic ducks: Md/CH/LGD/1/2005 belonged to class II gene VIId, which was the main genotype of most NDV outbreaks since the end of last century; Du/CH/LAH/224/2. 011 belongs to class II gene Ib, similar to CB2374 strain; Du/CH/LAH/209/2011 belongs to class II gene type II; the remaining 16 strains belong to class I gene 1b, which is the epidemic genotype of Chinese waterfowls. According to the molecular characteristics and genetic evolution analysis of 19 strains of Duck-Derived Newcastle disease virus, 7 strains of Duck-Derived Newcastle disease virus were selected for pathogenicity test. The ICPI value of Md/CH/LGD/1/2005 was 1.88, the MDT value was 50 h, and the other strains were identified as virulent strains. The ICPI value of other strains ranged from 0 to 0.34, and the MDT value ranged from 125 h to 168 h. At the same time, we also selected weak strains. According to the characteristics of genetic evolution and pathogenicity test of 19 strains of Newcastle disease virus from ducks, six strains of Newcastle disease virus from ducks were selected, and 17 strains of virus with different genotypes were selected from the strains isolated in the laboratory. The single factor serum was prepared and then crossed. The results showed that the antigenic correlation of Newcastle disease virus strain may be related to genotype, but not to host. The above results showed that, unlike pigeon-borne Newcastle disease virus, duck-borne Newcastle disease virus did not form an independent evolutionary branch in genetic evolution and antigenicity. Newcastle disease virus exists in ducks. It may be that Newcastle disease virus has mutated to a certain extent in order to adapt to the new host. Therefore, duck and duck embryos can more truly reflect the pathogenicity of Newcastle disease virus from ducks. Newcastle disease virus from ducks plays an important role in the evolution of Newcastle disease virus.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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