【作者】 段旭基；

【导师】 杨增岐；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2013， 博士

【摘要】 新城疫（Newcastle disease ND）因其全球广泛分布和对养禽业危害巨大著称为禽类主要的传染病，其病原新城疫病毒（Newcastle disease virus，NDV）被列入禽副黏病毒1型，属禽腮腺炎病毒属（Avulavirus）、副黏病毒科（Paramyxovirudae），单股负链有囊膜RNA病毒，长约15.2kb。NDV F蛋白裂解位点序列和HN蛋白是其主要的毒力决定因素。基于致病指数，NDV毒株被分为强毒型（velogenic）、中毒型（mesogenic）、无毒（或弱毒）型（apathogenic, or lentogenic）。“早期”基因Ⅸ型NDV基因组长15192nt，包括中国官方标准强毒株F48E9，代表了NDV独特地域分布型。基因Ⅸ型NDV自1946年在国内流行以来，直到上世纪80年代免疫计划实施，在过去的30年里基因Ⅸ型引起家禽ND零星散发，然而，近年来，基因Ⅸ型NDV从水禽中分离。尽管不同地域分布的不同基因型NDV在全球范围内演义持续性同步进化，但基因Ⅸ型NDV流行病学和进化依然是个谜团，这归咎于已报道不同宿主数量不多的分离株。研究野鸟源和家禽源基因Ⅸ型NDV流行病学和遗传进化，对于阐述基因Ⅸ型NDV总体进化规律、野鸟与家禽在NDV进化中扮演的角色以及防控ND暴发具有重要意义，研究内容和结果为以下四部分：1.秦岭北麓野鸟源基因Ⅸ型NDV病原学监测和分离株毒力分析为评估秦岭北麓野鸟源基因Ⅸ型NDV流行及毒力情况，对2008年8月到2011年7月间采集于健康野鸟的374份咽喉、泄殖腔拭子进行NDV分离与鉴定，对分离株进行生物学特性和遗传进化分析。获得5株NDV（包括2株候鸟源），分别命名为Peafowl/China/08、WhooperSwan/China/08、Blackbird/China/08、SpottedDove/China/08、White-cheekedStarling/China/08。F基因（535bp）序列分析显示，5株NDV均具有强毒F蛋白裂解位点序列112R-R-Q-R-R-F117，致病指数MDT、ICPI和IVPI差异较大，分别为37.2h～64.8h，0.425～1.638，2.04～2.76，珠颈斑鸠（鸠鸽属）分离株SpottedDove/China/08显示F蛋白强毒裂解位点序列¹¹²R-R-Q-R-R-F¹¹⁷与弱毒ICPI的不统一。F基因（535bp）同源性分析显示5株NDV同源性高达99.8%～100%。F基因（42～420nt，374bp）进化树分析显示5株野鸟NDV分离毒均属于我国特有的基因IX型NDV，与国内早期基因Ⅸ型NDV流行株F48E9亲缘关系很近（核苷酸同源性为99.4%～99.6%），而与传统疫苗株La Sota和V4及当前国内家禽中流行的基因Ⅶ型NDV毒株亲缘关系较远。研究表明相当数量种属的野鸟对基因Ⅸ型NDV有自然抵抗力，野鸟被证实为基因Ⅸ型NDV强毒株携带者，可能充当NDV“飞行器”角色，加速基因Ⅸ型NDV跨区传播，并一度在秦岭北麓多种野鸟中流行。2.陕西省部分商业鸡群基因Ⅸ型NDV病原学监测和分离株毒力分析为了评价陕西省商业鸡群基因Ⅸ型新城疫病毒（NDV）流行及毒力情况，对2009年6月到2012年7月间采集于陕西省境内34个商业鸡群的160份组织和咽喉-泄殖腔拭子进行NDV分离与鉴定，从长安和榆林两蛋鸡场分离获得2株NDV，分别命名为Layer/China/Changan/10和Layer/China/Yulin/10，并对分离株进行生物学特性和遗传进化分析。2株NDV致病指数MDT、ICPI各不相同，分别为56.3h和7.2h，1.603和1.838。F基因（535bp）推导氨基酸同源性分析显示2株家禽NDV均具有强毒融合（F）蛋白裂解位点112R-R-Q-R-R-F117，且两株同源性高达99.8%，与前期5株野鸟基因Ⅸ型NDV同源性高达99.6%～99.8%。F基因高变区（42～420nt，374bp）分析显示，2株家禽NDV属于我国特有的基因Ⅸ型NDV，与国内早期基因Ⅸ型NDV流行株F48E9亲缘关系很近（核苷酸同源性为99.3%～99.4%），而与传统疫苗株La Sota和V4及当前家禽中流行的基因Ⅶ型NDV毒株亲缘关系较远。本研究从野鸟源采样区周边商业鸡群分离到基因Ⅸ型NDV毒株，表明基Ⅸ型NDV强毒株仍可引起鸡群ND暴发，证实基因Ⅸ型NDV在秦岭北麓陕西省家禽中流行。3.基因Ⅸ型NDV分离株主要毒力基因遗传进化分析对7株基因Ⅸ型NDV分离株F和HN基因ORF进行克隆测序，以GenBank不同时期登录家禽和水禽基因Ⅸ型毒株为参考，进行同源性、氨基酸置换类型、遗传变异、进化距离和分子特性研究。H和HN基因同源性分析显示7株分离毒F基因同源高达99.6%～99.9%，与参考株同源性为99.6～99.9%；7株分离毒HN基因同源高达99.3%～100%，与参考株HN基因同源性为99.3～99.9%；包括分离株在内，大部分基因Ⅸ型毒株享有同源性99.0%以上，表明基因Ⅸ型NDV存在种间传播风险。23株基因Ⅸ型F基因间估算平均进化距离为0.012，7株基因Ⅸ型NDV毒株估算进化距离均接近（低于）估算平均进化距离0.012。14株基因Ⅸ型毒株HN基因估算平均进化距离0.007，除Layer/China/Changan/10株，其他6株分离株HN基因估算进化距离均接近于与基因Ⅸ型毒株间HN基因估算平均进化距离0.007。遗传进化研究表明7株分离毒F、HN基因存在少量无意义的点突变和氨基酸置换，其主要毒力基因演义无明显差异的同步进化。而基因Ⅸ型内较大进化距离表明水禽在基因Ⅸ型NDV进化中扮演着积极角色。4.不同禽类基因Ⅸ型NDV分离株基因组遗传进化研究为了深度评估野鸟源和家禽源基因Ⅸ型NDV分离株遗传进化关系，以GenBank已登录基因Ⅸ型NDV毒株和和本实验室SpottedDove/China/08株全基因序列为参考，运用RT-PCR技术对野鸟源Blackbird/China/08株和家禽源Layer/China/Yulin/10株进行全基因扩增测序。运用DNAstar5.0及MEGA5.2软件，对Blackbird/China/08株和Layer/China/Yulin/10株进行分析，结果显示两株基因Ⅸ型NDV病毒基因组全长均为15192bp，基因组结构均包含3′-Leader-NP-P-M-F-HN-L-Trailer-5′；F蛋白的裂解位点及HN蛋白长度均符合NDV强毒特征；全基因序列进化树显示两株NDV依旧属于基因Ⅸ型NDV。基因Ⅸ型NDV毒株遗传进化距离研究表明，NP、P、M、F、HN、L和V基因进化水平并不是完全与基因组进化水平保持一致，而是相对接近（略高于或低于）与基因组进化水平，HN基因和V基因的进化水平要略高于NP、P、M、F和L基因。基因组进化水平显示基因Ⅸ型NDV是极度保守的，但不否认一些水禽基因Ⅸ型NDV毒株在基因Ⅸ型进化中先行地位；V基因间进化距离作为NDV毒力增强标准有待证实。基因Ⅸ型分离株Blackbird/China/08、Layer/China/Yulin/10和SpottedDove/China/08株基因组进化水平低于（接近）基因Ⅸ型毒株估总体平均进化水平，基因Ⅸ型毒株中家禽源毒株受免疫选择压力影响进化水平要高于野鸟源基因IX型毒株；野鸟被推测是基因Ⅸ型NDV强毒株贮存宿主，加速了基因Ⅸ型NDV扩散和传播。综上所述，本研究与2008年8月至2012年7月间在秦岭北麓及陕西省境内分离到7株不同禽源、毒力差异的基因Ⅸ型NDV毒株，包括一株分离于珠颈斑鸠（鸠鸽属）的弱毒株SpottedDove/China/08。野鸟被推测为基因Ⅸ型NDV强毒株贮存宿主，在加速基因Ⅸ型NDV扩散传播中起了“飞行器”作用；在过去30年里，野鸟演义了基因Ⅸ型NDV进化史和流行病史；基因Ⅸ型NDV进化水平相对较低，各基因进化水平并不是完全与基因组进化水平保持一致，而是相对接近（略高于或低于）与基因组进化水平，HN基因和V基因的进化水平要略高于NP、P、M、F和L基因，家禽源毒株因疫苗免疫压力影响，其进化水平高于野鸟源基因Ⅸ型毒株；本研究基因Ⅸ型分离株进化水平低（或接近）于基因Ⅸ型NDV总体进化水平；证实基因Ⅸ型NDV一度在秦岭北麓和关中地区家禽和野鸟间流行；一些水禽源基因Ⅸ型NDV毒株在基因Ⅸ型进化中处于先行；V基因间进化距离作为NDV毒力增强标准有待证实。更多还原

【Abstract】 Newcastle disease is known as the primary infectious diseases in poultry due to its worldwidedistribution and devastating effects, the causative agent Newcastle disease virus （NDV）,designated as avian paramyxovirus serotype1（APMV-1）, belongs to the Avulavirus genusand the Paramyxoviridae family. The enveloped NDV contains a negative-sensesingle-stranded RNA genome of¹5.2kb. F protein cleavage site motif and HN were themost important determinant for NDV virulence. NDV isolates are categorized as being highlypathogenic （velogenic）, intermediate （mesogenic）, or apathogenic （lentogenic） strainsbased on their pathogenicity in chickens.The ‘early’ genotype Ⅸ NDV with15,192nt in the genome, containing the Chinesestandard F48E9strain, represents a unique geographic expansion of the NDV. This genotypevirus has been endemic in China since1946and a strict vaccination policy was adopted in the1980s, in the past30years, it have been implicated in sporadic ND cases in poultry flocks,however, recent reports suggest that genotype Ⅸ strains have been accidentally isolated fromwaterfowl. Although different of NDV genotypes have simultaneously evolved at variousgeographical locations throughout the world, the epidemiology and evolution of genotype Ⅸviruses remain an enigma due to several isolates from different hosts that have been reported.Thus, evaluate the epidemic and evolution status of genotype Ⅸ NDV from the wild andpoultry will be of great significance to prevent and controll the outbreak of ND, which olsoillustrate the general evolution status in genotype Ⅸ NDV and the exactly function of wildbird in the evolution and transmittion of virulent genotype Ⅸ NDV. The main result as follow:1. Etiology surveillance and virulence analysis of genotype Ⅸ NDV strains isolated fromwild birds in northern Qinling Mountains.This study aimed to evaluate the epidemiological status of genotype Ⅸ Newcastledisease virus （NDV） from healthy wild birds and virulence of isolates in the northernQinling Mountains and Guanzhong Areas from August,2008to July,2011.374larynx andcloaca swabs from healthy wild birds were used to isolate and identify genotype Ⅸ NDV,which were subsequently characterized pathotypically and genotypically.5NDV isolates were isolated and named the Peafowl/China/08, WhooperSwan/China/08, Blackbird/China/08,SpottedDove/China/08and White-cheekedStarling/China/08, respectively. Sequences analysisof partial F gene（535bp）revealed that all the5isolates shared common F0protein cleavagesite motif112R-R-Q-R-R-F117equivalent to virulent NDV. The5isolates including2frommigrants exhibited different pathogenicity indices, with MDT, ICPI and IVPI ranging37.2h～64.8h,0.425～1.638,2.04～2.76, respectively. One novel isolate from theSpotted-necked Dove（family Columbidae）exhibited discordance between its lentogenic ICPIand its virulent proteolytic cleavage site motif¹¹²R-R-Q-R-R-F¹¹⁷. Homology analysis basedon partial F gene （535bp） revealed that the5isolates shared homology as high as99.8%～100%with each other. Phylogenetic analysis based on variant region of F gene（42～420nt,374bp） revealed all the5isolates clustered to genotype Ⅸ NDV, together with the Chineseearly prevalent strain F48E9（homology99.4%～99.6%）, distinguished the traditional La-Sota and V4vaccine strains, or currently prevalent genotype Ⅶ NDV in poultry. This studyrevealed that asymptomatic wild bird species had natural resistance to genotype Ⅸ NDV, andacted as natural carriers of virulent genotype Ⅸ NDV, which may charge NDV "aircraft" inaccelerated spread of these viruses. The result indicated that these genotype Ⅸ NDV isolatesfrom wild bird had been circulating in Qinling Mountains and Guanzhong area for someperiod of time, also posed threat for poultry and enviroment.2. Etiology surveillance and virulence of the genotype Ⅸ NDV strains isolated fromcommercial poultry flocks in Shaanxi Province.This study was conducted to evaluate the status of genotype Ⅸ Newcastle disease virus（NDV） from commerical poultry flock in Shaanxi.160tissue samples, larynx and cloacaswabs were involved in34flocks in the duration of Jun,2009and July,2012.2isolates wereabtained from layer flocks ND outbreaks in Chang’an and Yuling, namelyLayer/China/Yulin/10and Layer/China/Changan/10, which were subsequently characterizedpathotypically and genotypically. The2isolates exhibited different pathogenicity indices, withMDT, ICPI ranging37.2h～56.5h,1.603～1.838, respectively. Sequences analysis of partialF gene （535bp） revealed that the2poultry isolates shared common F protein cleavage sitemotif112R-R-Q-R-R-F117equivalent to virulent NDV, which shared as high as99.8%homology with each other,99.6%～99.8%with the previous5wild bird isolates.Phylogenetic analysis based on variant region of F gene（42～420nt,374bp） revealed all the2isolates clustered to genotype Ⅸ NDV, together with the Chinese early prevalent strainF48E9（homology99.3%～99.4%）, distinguished the traditional LaSota and V4vaccinestrains, or currently prevalent genotype Ⅶ NDV in poultry. This study confirmed that virulentgenotype Ⅸ viruses were responsible for ND outbreak in commercial poultry flocks around the location of wild bird samples collection, also suggested that genotype Ⅸ viruses had everbeen epidemic in poultry in the northern Qinling Mountains.3. Genetic evolution analysis of the virulence gene of genotype Ⅸ NDV isoates.The ORF of F and HN gene of the7isolates were amplified by RT-PCR, sequenced andanalyzed for theri homology, deduced amino acid residue substitution，genetic relationship byperforming the alignment and constructing phylogenetic tree based on the sequences. Thehomology analysis within genotype Ⅸ NDV revealed that they share homology of99.6%～99.9%with each other, and94.4%～99.9%with other genotype Ⅸ NDV reference strains inF gene, while they shared homology of99.3%～100%with each other,99.3%～99.9%withother genotype Ⅸ NDV reference strains in HN gene. Most of the previous genotype Ⅸstrainshared homology more than99.0%, which indicated interspecies risk. The F gene distanceestimation analysis involving23sequences in genotype Ⅸ NDV, including the7isolates and16reference strains, showed the overall average value for the estimated evolutionary distancewas0.012, and each estimated average evolution distance of the7isolates was＜theoverall average value （0.012）. The HN gene distance estimation analysis involving14sequences in genotype Ⅸ NDV, including the7isolates and7reference strains, showed theoverall average value for the estimated evolutionary distance was0.007. The each estimatedaverage evolution distance of the7isolates was approximate the overall average value （0.012） in HN sequences except the Layer/China/Changan/10. Even though the7isolatesunderwent several amino acid mutations in the fusion protein, evidence of continuousevolutionary divergence did exist in the genotype Ⅸ NDV, which was always regarded as aconservative genotype, and in which the larger evolution divergence indicated the waterfowlproduced positive effect on evolution within this genotype.4. Genomic analysis of genotype Ⅸ NDV isolates from different fowl.To futher evaluate the genetic evolution realationship of genotype Ⅸ NDV isolates fromwild and poultry, Two complete genome sequence of the wild bird isolate Blackbird/China/08and the poultry isolate Layer/China/Yulin/10were obtained by RT-PCR and genomic analysiswas conducted using DNAStar5.0and MEGA5.2software, which was referenced againstprevious genome sequence of SpottedDove/China/08and8genotype Ⅸ NDV sequencesavailable in GenBank. Genomic sequence showed that both isolates consist of15,192nt, andgeneomic arrangement is3’Leader-N-P-M-F-HN-L-Trailer-5’. Both the characteristics of Fprotein cleavage site motif and length of HN protein of the two isolates were consistent withvirulent NDV. Phylogenetic analysis of complete genome indicated the both isolates stillclustered to the genotype Ⅸ clusters. The results of genetic evolutionary distance showed thatthere were slightly difference in evolutionary distance among each gene and whole genome evolution, HN gene and V gene is slightly higher than that of other genes. The results suggestthat the genomic evolution is highly conservative among genotype Ⅸ NDV viruses, andwaterfowl may play an important role in NDV evolution, the hypothesis that V gene as a markof NDV virulence needs further validation. Each genome evolution levels of these isolatesBlackbird/China/08,Layer/China/Yulin/10and SpottedDove/China/08were below （nearly） theestimated average evolution level with genotype Ⅸ NDV. The genome evolution levels ofpoultry were higher then the wild,due to the immune pressure selection against by poultryflock. Wild birds were confirmed nature reservoir of virulent genotype Ⅸ NDV strains, whichaccelerated the proliferation and spread up.In conclusion, this work was conducted for genotype Ⅸ NDV isoltates from differentfowl in the duration of Aug,2008and July,2012,7isolates from different fowls exhibiteddifferent virulence in despite of pocessin the common F protein cleavage site motif112R-R-Q-R-R-F117. Remarkable, a novel lentogenic NDV isolate from the Spotted-neckedDove （family Columbidae） exhibited discordance between its lentogenic ICPI and itsvirulent proteolytic cleavage site motif112R-R-Q-R-R-F117. The results suggest that thegenomic evolution is highly conservative among genotype Ⅸ NDV viruses, and waterfowlmay play an important role in NDV evolution, the hypothesis that V gene as a mark of NDVvirulence needs further validation. Each genome evolution levels of these isolatesBlackbird/China/08, Layer/China/Yulin/10and SpottedDove/China/08were below （nearly）the astimate average evolution level with genotype Ⅸ NDV. The genome evolution levels ofpoultry were higher then the wild, due to the immune pressure selection against by poultryflock. It was supposed that wild birds were nature reservoir of virulent genotype Ⅸ NDVstrains, and accelerated the spread of genotype Ⅸ NDV viruses. The hpothesis that V gene asa mark of NDV virulence need further validation.更多还原