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野生鸟类新城疫病毒的分离鉴定及其F与HN基因的分子特征研究
Isolation and Identification of Newcastle Disease Virus from Wild Birds and Study on Molecular Character of the Isolates
【作者】 张鹏；
【导师】 杨增岐；
【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2009， 硕士
【摘要】 新城疫（Newcastle disease,ND）又名亚洲鸡瘟,是由新城疫病毒（Newcastle diseasvirus,NDV）引起的一种多病型的高度接触性传染病,该病在世界范围内广泛流行。OIE规定,引起新城疫的新城疫病毒的特征为（1）新城疫病毒的颅内接种指数（intracerebralpathogenicity indices,ICPI）大于0.7;或（2）新城疫病毒的融合蛋白C端的裂解位点（即113-117位）包含3个或以上的碱性氨基酸（R或K）。野生鸟类是新城疫病毒的重要宿主,研究野生鸟类中新城疫病毒流行情况,对于了解野生鸟类在新城疫传播过程中的作用以及防控新城疫的爆发都有着重要的意义。1.本研究从位于秦岭北麓的陕西省楼观台自然保护区及其周边地区采集野生鸟类（包括5个目6个科8个种以及3个未定种）咽拭子和泄殖腔拭子样品各49份,通过鸡胚分离法,血凝试验,血凝抑制试验和RT-PCR等方法进行新城疫病毒的分离鉴定,结果从采集的野生鸟类样品中共分离到新城疫病毒5株,分离率为10.2%,5株病毒分别来源孔雀,天鹅,珠颈斑鸠,灰椋鸟和一个未定种。本研究表明在该生态区野生鸟类中新城疫病毒的感染率较高。2.通过OIE规定的方法,对5个野生鸟类新城疫分离株的MDT,ICPI和IPVI进行测定,结果表明NDV/heiniao/CH（SHX）/3/2008为速发型强毒,其毒力高于F48E9;NDV/SpottedDove/CH（SHX）/4/2008为中发型的毒株;而NDV/Peafowl/CH（SHX）/1/2008,NDV/WhooperSwan/CH（SHX）/2/2008,NDV/White-cheekedStarling/CH（SHX）/1/2008的毒力不能确定。然而这些毒株的ICPI均接近和大于0.7,对家禽养殖业都有潜在的威胁。3.对5株野生鸟类NDV分离株F基因进行克隆测序,与已发表的国内外不同时期的NDV毒株HN基因进行核苷酸和推导的氨基酸遗传变异分析及分子特性研究,结果显示,所有分离株与F48E9的亲缘关系最近,其F基因的同源性在98.7%～99.4%之间,其推导氨基酸的同源性在98.3%～99.4%之间,而与其他参考株,疫苗毒和目前流行的基因Ⅶ型毒株的关系较远;其F蛋白裂解位点的氨基酸序列均为112R-R-Q-R-R-F117,符合强毒株的特征。遗传进化分析显示,这5株新城疫病毒分离株均为基因Ⅸ型。4.对5株野生鸟类NDV分离株的HN基因进行克隆测序,与已发表的国内外不同时期的NDV毒株HN基因进行核苷酸和推导的氨基酸遗传变异分析及分子特性研究。结果显示,所有分离株与国内参考强毒株F48E9和国外参考强毒株Herts-33在HN基因C末端终止密码子的位置相同,符合强毒株的特征。从核酸和氨基酸同源性来看,分离株与F48E9的核苷酸同源性和氨基酸的同源性99.6%～99.8%,99.1%～99.7%之间,与LaSota,B1和Clone30的同源性在90.2%～91.8%,87.9%～88.5%之间,ZJ1的同源性在84.8%～85.0%,89.3%～89.7%之间;从系谱进化分析来看,这5个毒株和F48E9关系较近,同属一支,与LaSota,ZJ1,Texas-33等疫苗株和参考毒株关系较远。
【Abstract】 Newcastle disease（ND）,otherwise,Asian Fowl pest,was a highly contagious disese of poultry caused by Newcastle disease virus（NDV） and spreaded widely in the World.By the means of OIE,the characterization of NDV which could lead to birds occurred Newcastle disease was:（1） having intracerebral pathogenicity indices（ICPI） of 0.7 or more in day-old chickens（Gallus gallus） and/or:（2）having multiple basic amino acids（at least three arginine（R）or lysine（K） residues） at the C-terminus of the fusion protein cleavage site,starting at position 113,along with a phenylalanine at position 117.Wild birds may be natural reservoirs of NDV.Thus,understanding the epidemic pattern in wild birds has the significant meaning in preventing and controlling the outbreak of ND.Also it can help us illustrate the role of wide bird plays in NDV transmiting.1 By using the methods of chicken embryo inoculation,haemagglutination,haemagglutination inhibition（H1）and RT-PCR,5 stains of NDV were isolated and idenificated from 49 wild birds’ tracheals and cloacal swabs.The result showed that the overall separation rate of NDV was 10.2%,and the NDV strains were isolated from Peafowl,WhooperSwan, heiniao,SpottedDove and White-cheekedStarling.So,this study indicates that the wild birds being the carrier of NDV are very common in this area.2 The virulence of 5 isolated NDV stains was evaluated by the classical pathogenicity tests, including mean death time（MDT）,intracerebral pathogenicity index（ICPI） and intravenous pathogenicity index（IVPI）.MDT,ICPI and IVPI of the isolates were 37.2h～120h, 0.425～1.638 and 2.04～2.76,respectively.These results indicated that the NDV isolated from heiniao was virulent,the NDV isolated from SpottedDove was mesogenic viruses, but the virulence of NDV isolated from Peafowl,WhooperSwan and White-cheekedStarling were indefinite.3 The N-terminal of F gene of all isolates were amplified by PCR,sequenced and analyzed for their genetic relationship by performing the alignment and constructing phylogenetic tree based on the sequences.The results indicated that The nucleotide and amino acid homology of F gene of NDV isolates and F48E9 was 98.7%-99.4%and 98.3%-99.4% respectively.The deduced amino acid sequences of F protein at the cleavage sites of all the isolates were 112R-R-Q-R-R-F117,with the motif characteristics of the virulent NDV strains.The phylogenetic tree based on the nucleotide sequences of F gene revealed that the 5 isolates of NDV and F48E9 belong to subgenotypeⅨ.4 The ORF of HN gene of all isolates were amplified by PCR,sequenced and analyzed for their genetic relationship by performing the alignment and constructing phylogenetic tree based on the sequences.The nucleotide and amino acid homology of NDV isolates and F48E9 was 99.6%-99.8%and 99.1%-99.7%respectively,but 90.2%-91.8%and 87.9%-88.5%compared with LaSota,Clone 30 and B1.Compared with ZJI strains the homology was 84.8%-85.0%and 89.3%-89.7%.All the isolated C-terminus of HN gene have the same termination codons,which is coincided with the reference virulent strains F48E9 and Hert-33,belong to subgenotype C.The phylogenetic tree based on the nucleotide sequences of HN gene revealed that the 5 isolates of NDV and F48E9 were the same subgenotype viruses.