【作者】 陈胜利；

【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】 新城疫是一种严重危害多种禽类健康的传染病，给世界养禽业造成巨大经济损失。该病的病原新城疫病毒（Newcastle disease virus， NDV）属于副黏病毒科（Paramyxovirudae）、禽腮腺炎病毒属（Avulavirus），是禽副黏病毒1型的唯一成员。病毒基因组为单链负股RNA，长度约为15.2kb。NDV对不同宿主的致病性存在很大差异。近年来，NDV感染和致病宿主范围正在不断扩大，给新城疫的防控带来新的挑战。干扰素系统是宿主抵抗病毒的第一道防线，近些年研究发现，副黏病毒的V蛋白能够有效拮抗宿主干扰素系统，对病毒的免疫逃避发挥功能，同时在病毒致病性和宿主限制方面发挥重要作用。与其他副黏病毒相似，NDV的P基因通过RNA编辑现象，编码产生的V蛋白在病毒拮抗宿主干扰素过程中发挥关键作用。已有研究均是对1株或2株NDV毒株V蛋白拮抗宿主干扰素作用进行研究报道，然而众多动物来源、不同毒力和基因型的NDV毒株V蛋白的分子进化特性及拮抗宿主干扰素功能差异目前尚不清楚。为了阐明不同动物来源NDV基因组演化特点、宿主特性改变的原因及V蛋白与病毒致病性和宿主特异性的关系，本研究在对实验室多种动物来源新城疫病毒分析的基础上，对3株NDV代表性毒株（2株朱鹮源和1株猪源）进行生物特性研究、全基因组克隆测序及遗传进化分析，同时对其他动物来源如鸡、鸽、鹅和野鸟源等22株不同NDV毒株V蛋白的分子特征、遗传变异规律及拮抗宿主干扰素功能差异进行了系统研究。具体研究内容和结果分为以下五个部分：1、2株朱鹮源新城疫病毒分离鉴定、全基因测序分析对2010年1月陕西省周至县楼观台珍稀野生动物救护饲养研究中心朱鹮暴发的疑似ND病料及实验室2006年2月收集的发病朱鹮病料，进行病毒分离鉴定、病毒致病指数测定，并运用RT-PCR技术对分离株进行全基因扩增并测序。利用DNAStar及MEGA4.0软件，对获得的全基因序列与NDV参考毒株的全基因组成、基因编码区及非编码区序列进行同源性分析、比较、并构建系统发育进化树。结果共分离鉴定出2株NDV，分别为Shaanxi06和Shaanxi10，病毒致病指数测定结果表明Shaanxi06为强毒，而Shaanxi10为中强毒。但F蛋白裂解位点区氨基酸序列均为¹¹²R-R-Q-K-R-F₁₁₇，与强毒株F0蛋白裂解序列相符。全基因组测序结果表明，Shaanxi06（GenBank登录号：KC853019）和Shaanxi10（GenBank登录号：KC853020）基因组全长15,192bp，分离株与NDV参考毒株基因组特征、蛋白编码区和非编码区序列并无太大差异，但2株朱鹮源NDV分离株具有一些新的特征。系统进化树表明，2株朱鹮源NDV分离株均为NDV class II群，Shaanxi06属于基因VIId亚型，而Shaanxi10属于一个新的基因亚型VIi，这2株朱鹮源NDV分离株与陕西省同一地区2006²010年其他禽类NDV流行株亲缘关系最近，而与我国传统的NDV疫苗毒La Sota、B1和clone30株及我国NDV标准强毒F48E9亲缘关系均较远。2、猪源新城疫病毒HX01株分离鉴定、全基因测序分析2009年12月从陕西省户县某养猪场疑似猪流感病死猪采集肺脏病料，进行病毒分离鉴定，致病性测定及运用RT-PCR技术对分离株进行全基因扩增并测序。利用DNAStar及MEGA4.0软件，对获得的全基因序列与NDV参考毒株的全基因组成、基因编码区及非编码区序列进行同源性分析、比较、并构建系统发育进化树。结果显示，分离出1株新城疫病毒，命名为HX01株。致病性测定结果显示分离毒株为NDV弱毒株。F蛋白裂解位点氨基酸组成为¹¹²G-R-Q-G-R-L¹¹⁷，符合弱毒特征。全基因组测序结果表明，HX01株（GenBank登录号：JF795531）基因组全长15,186bp，基因组特征与其他NDV参考毒株相似。HX01株与我国传统弱毒疫苗株La Sota及其他猪源NDV毒株同源性最高，与La Sota无论全基因水平还是各个基因水平，同源性均在99.5%以上。遗传进化表明，HX01株与La Sota、clone30等同属于基因II型。但是，HX01株HN基因具有独特性，在HN蛋白第2抗原表位出现S526N点突变和其他点突变N98K、I227T和S464P。此外，HN基因的终止序列出现A-T的点突变。这些突变在NDV进化中的作用有待进一步研究。3、新城疫病毒分离株V基因克隆测序、分子特征及遗传进化分析根据GenBank上已发表的NDV P基因序列，对20株不同宿主来源、毒力和基因型的NDV分离株和2株NDV参考株进行P基因克隆测序，根据测序结果对P基因RNA编辑位点进行定点突变，插入1个非模板G，获取不同NDV毒株V基因序列。利用DNAStar及MEGA4.0软件，对获得的V基因序列与NDV参考毒株的序列进行同源性分析、比较并构建系统发育进化树。结果显示，成功克隆22株不同宿主来源、毒力和基因型的NDV毒株V基因。20株NDV分离株V基因的核苷酸的同源性在80.4%¹00%，氨基酸的同源性在67.5%¹00%。20株NDV分离株与其他参考株V基因的同源性差异较大，在68.3%¹00.0%。不同基因型的毒株V基因的核苷酸和氨基酸的同源性较低，而相同基因型的NDV毒株V基因同源性高达99.1%以上。目前流行的基因VII型毒株与传统疫苗株、我国标准强毒株的V蛋白同源性相比，氨基酸序列发生了较大变异，并且基因VI型不同基因亚型间差异也较大。V蛋白结构分析表明，N端55¹05位和C端135¹72位氨基酸是V蛋白的高变区，而N端起始MATF/LTDAEI、132KKG134、177HRRE180、182SISW185、C端1个组氨酸和7个半胱氨酸残基形成的锌指结构基序在NDV毒株中高度保守。NDV V蛋白氨基酸变异呈现基因型一致性。遗传进化分析表明，根据V基因全长序列和V蛋白序列所作进化树与F基因（42⁴70nt）进化树高度一致，表明非结构基因V基因也可应用于NDV遗传进化分析和分子流行病学研究的候选靶基因。提示NDV的演化是所有基因（包括结构基因和非结构基因）同步演化的结果。4、新城疫病毒不同毒株对禽源DF-1细胞系IFN-β生成的研究选取了6株不同宿主来源、毒力和基因型的NDV毒株，构建V蛋白真核表达载体pCMV-3HA-V，转染DF-1细胞，通过双荧光素酶试验、RT-PCR和ELISA，检测NDV的V蛋白过表达对DF-1细胞IFN-β各个水平的影响。结果表明，不同NDV毒株编码的V蛋白均可不同程度的降低poly（I:C）诱导的IFN-β启动子活性、mRNA水平和蛋白水平。不同NDV毒株V蛋白的表达量相近，而它们的V蛋白抑制IFN-β能力存在很大差异。V蛋白抑制IFN-β能力从高到低依次pV-F48E9、pV-Sd-08、pV-Shaanxi06、pV-Gfw-10、pV-Shaanxi10和pV-HX01。F48E9强毒株V蛋白拮抗干扰素能力最强，而猪源HX01株最弱，朱鹮源毒株居中。NDV强毒株V蛋白抑制IFN-β能力强于中强毒和弱毒；NDV毒力越强，其V蛋白抑制IFN-β生成的能力越强，反之，则越弱。研究表明，NDV V蛋白与病毒致病性密切相关，与病毒的宿主来源并无关联。5、新城疫病毒V蛋白拮抗IFN-β生成功能域的初步研究为了探讨NDV V蛋白拮抗IFN-β功能区域及C端半胱氨酸富集区7个半胱氨酸残基在抑制IFN-β中发挥的作用。通过构建P蛋白，V蛋白N端、C端截短突变体，半胱氨酸突变体C1^C7真核表达载体，应用双荧光素酶试验、ELISA和real-time PCR，检测V蛋白突变体与野生型V蛋白过表达对DF-1细胞IFN-β各个水平的影响。参考PIV5V蛋白锌指结构，对NDV V蛋白锌指结构进行模拟。结果表明，各突变体均构建成功，并在DF-1细胞获得高效表达；NDV的V蛋白具有抑制poly（I:C）诱导的DF-1细胞IFN-β启动子活性、mRNA水平和蛋白水平的能力，而P蛋白虽具有与V蛋白共同的N端，但并不具有抑制IFN-β作用。NDV V蛋白抑制DF-1细胞IFN-β生成的主要功能域位于V蛋白的C端。V蛋白C端196位（C1）、221位（C6）和224位（C7）半胱氨酸残基对于V蛋白抑制DF-1细胞IFN-β是非必需的，而200位（C2）、212位（C3）、214位（C4）和217位（C5）是必需的关键残基。研究提示，NDV V蛋白C端的7个半胱氨酸残基在V蛋白抑制IFN-β生成中发挥不同的作用。对NDV V蛋白锌指结构进行模拟，结果表明NDV V蛋白C端结构域中的大锌指结构区域L2中的残基（C2、C3、C4和C5）对于V蛋白拮抗IFN-β必需的，而小锌指结构区域L1中的残基（C1、C6、C7）是非必需的。综上所述，本研究较为系统的阐明了不同宿主来源NDV基因组特征，病毒演化特点，遗传变异规律，为阐明病毒起源、流行特点及宿主特性改变的分子机制奠定了理论基础。同时对帮助NDV病毒逃避宿主干扰素的不同NDV毒株V蛋白的分子进化特性及其拮抗宿主干扰素功能差异进行系统的研究。本研究揭示了NDV V蛋白与病毒致病性的关系，为进一步探究NDV逃避干扰素机理， NDV V蛋白拮抗干扰素功能域及开发新城疫长效疫苗奠定了基础。更多还原

【Abstract】 Newcastle disease （ND） is one of the most serious infectious diseases of birds andeconomically important poultry diseases. Newcastle disease virus （NDV）, a sole member ofthe avian paramyxovirus serotype1（APMV-1）, belongs to the genus Avulavirus within theParamyxoviridae family and is the causative agent of ND. The enveloped virus possesses anegative-sense, single-stranded RNA genome approximately15.2kb in length. There isconsiderable virulence diversity among different hosts. In the past few years, the infection andhost range of NDV is expanding constantly, which bringing new challenges for the preventionand control of ND. Interferon （IFN） is the first line of defense against virus infection. It hasbecome clear that most paramyxoviruses encode V proteins function as an alpha interferonantagonist and play important role in viral pathogenesis. Similar to other paramyxovirus, Vprotein is generated by an RNA-editing event that occurs during the transcription of the NDVP gene and it plays a key role in antagonism alpha interferon. However, NDV strains contain agroup of complex viruses with different hosts, diverse virulence and many genotypes, themolecular evolution characteristics of their V proteins and effect on host interferon remainsunclear.To illustrate the genome evolution characteristics and the relationship between viral Vproteins, viral pathogenesis and host specificity among NDV isolates from different hosts.Based on analysis of many NDV isolates from different hosts in our lab, we selected threerepresentative NDV strains, which includes two from crested ibis and one from pig to studythe biology, genomic characterization and genetic variation of Newcastle disease virus strainsfrom different hosts, and further a total of twenty-two NDV strains were also compared toanalyze the molecular evolution characteristics of their V proteins and effect on hostinterferon, which including the other hosts derived virus such as chicken, goose, pigeon, andwild birds etc. This work contains five different parts and the details are presented infollowing paragraphs.1. Genomic characterization of two Newcastle disease virus strains isolated from Crested Ibis （Nipponia nippon） in ChinaTissue samples were obtained from ND-like disease of the crested ibis population in theShaanxi Rare and Wildlife Rescuing and Breeding Centre in February2006and January2010.Two Newcastle disease virus （NDV） isolates were collected from sick crested ibises and theirpathogenic and phylogenetic were investigated. Complete genome sequence of the twoisolates were obtained by RT-PCR and genomic analysis were conducted using DNAStar andMEGA4.0software. The pathogenic results indicated that the Shaanxi06isolate wasvelogenic, whereas the Shaanxi10isolate was mesogenic. They shared the same virulent motif¹¹²R-R-Q-K-R-F¹¹⁷ at the F protein cleavage site. Genomic characterization results showedthat both isolates consist of15,192nt, and there were tiny difference between the Shaanxi06（GenBank: KC853019）, Shaanxi10（GenBank: KC853020） isolates and other NDV referencestrains in genome characteristics, protein sequence coding and non-coding region of thegenome, but the two isolates owned new characteristics. The phylogenetic analysis revealedthat both isolates were clustered with class II NDVs, with one in genotype VIId and another ina novel genotype （provisionally designated as VIi）. The two isolates shared high homologywith the strains isolated from poultry flocks in the same region from2006to2010. However,both crested ibis isolates were genetically distinct from the traditional vaccine strains La Sota,B1and clone30strains, Chinese standard challenged strain F48E9and these isolatesclustered in different groups.2. Genomic characterization of a Newcastle disease virus strain HX01isolated from sickpigs in ChinaThe genomic characterization of one swine NDV isolate, HX01, is reported. HX01wasisolated from sick pigs suffering from an influenza-like disease in a commercial pig farm inShaanxi Province in December2009. The pathogenic tests and phylogenetic analysis wereinvestigated. Complete genome sequence of the swine isolate HX01was obtained by RT-PCRand genomic analysis was conducted using DNAStar and MEGA4.0software. Thepathogenic tests indicated that the isolate was a lentogenic NDV strain. HX01possessed a¹¹²G-R-Q-G-R-L¹¹⁷ lentogenic motif at the F protein cleavage site. The complete genomicsequence of HX01isolate consists of15,186nt （GenBank accession number: JF795531）,similar to other NDV reference strains in genome characteristics and is highly similar with theLa Sota vaccine strain （99.5%） and other swine-origin NDVs at either genome or single-genelevel. The phylogenetic analysis of F gene also showed that HX01belonged to genotype II,and is most closely related to the traditional vaccine strain La Sota, B1and clone30strains.New characteristics were found in the HN gene of HX01. A single amino acid change S526Nwas found within region2of the antigenic epitope in HN. In addition, a unique A-T mutation was observed in the HN gene end sequence in HX01. This finding may play an important rolein further research on NDV evolution.3. Cloning, molecular characteristics and genetic analysis of V proteins of Newcastledisease virus strainsBased on the published sequences of NDV P gene on GenBank, the P genes of twentyNDV strains of different hosts, genotypes and various virulence and two other NDV referencestrains were cloned and sequences, the corresponding V gene were obtained by inserting onenon-template G residue at the conserved editing sites of P gene of different NDV strains. Thesequence homology analysis, comparison with other reference strains and phylogeneticanalysis were conducted by using DNAStar and MEGA4.0software. The results showed thatV genes of twenty-two different genotypes and virulence NDV strains isolated from differenthosts were cloned and sequenced. The nucleotide similarity ranged from80.4%to100.0%,while deduced amino acid similarity ranged from67.5%to100%. The homology of the twentyNDV strains with other reference strains was from68.3%to100.0%. The results also indicatedthat the homology of V gene was high among same genotypes while low homology amongdifferent genotypes. There existed great variation in V protein among the current prevalentgenotype VII strains, traditional vaccine strain and Chinese standard challenged strain F48E9,also great differences among sub-genotypes within genotype VI strains. V protein structureanalysis showed that N-terminal residues55-105and C-terminal residues135-172wasthe most variable region, and the N-terminal MATF/LTDAEI, KKG （residues132-134）,HRRE （residues177-180）, SISW （residues182-185） and zinc finger structure region ofhistidine and seven cysteine residues was highly conservative in all NDV strain, somestrains with exception. The results also indicated that the genetic variation of V proteinsof different NDV strains were consistent with their genotypes. The phylogenetic analysisdemonstrated that the phylogenetic trees based on whole nucleotide sequence of V geneand V protein was highly consistent with that based on the47nt to420nt in the F gene’scoding region. The results indicated that the non-structural V gene can be applied ascandidate target gene to genetic analysis and molecular epidemiological studies in NDV,also suggested that genetic evolution of NDV is synchronous among all genes （includingstructural genes and nonstructural gene）.4. Differential interferon β production suppressing capacities of the V proteins fromdifferent Newcastle disease virus strains in DF-1cell linesTo study differential interferon β production suppressing capacities of the V proteinsfrom different Newcastle disease virus strains, seven NDV strains including different hosts,virulence and the genotypes were used to construct V protein eukaryotic expression vector pCMV-3HA-V. Reporter gene assay, RT-PCR and ELISA assays were applied to detectinterferon β in DF-1cells at different levels post transfection with various V constructs. Theresults showed that overexpression of V protein from different NDV strains were able tosuppress the IFN-β promoter activity, mRNA and protein levels in response to poly （I:C） inDF-1cells. The expression level of different V proteins was comparable to each other in DF-1cells, but their abilities to suppress IFN-β were different. The interferon β productionsuppressing capacities were from strong to weak in this order, pV-F48E9、pV-Sd-08、pV-Shaanxi06、pV-Gfw-10、pV-Shaanxi10and pV-HX01. The results indicated that the Vprotein of F48E9strain was most powerful in IFN-β antagonism, HX01is the most weakest,and the V proteins of other NDV strains were intermediate between them. It seems the Vproteins of velogenic strains were much stronger than that of mesogenic and lentogenicstrains in IFN-β suppressing capacities. The more virulent in virulence, more stronger ininhibiting IFN-β. It suggested that the V protein of NDV is closely related to the viralpathogenesis, but no correlation with the source of virus.5. Molecular determinants of V protein of Newcastle disease virus for interferon βproduction suppressing capabilityIn order to explore molecular determinants of V protein of Newcastle disease virus forinterferon β production suppressing capability and role of seven cysteine residues sited at theC-terminal cysteine-rich region of the NDV V protein. P protein, N-or C-terminal truncationsand seven cysteine mutants C1-C7were constructed and subcloned into plasmid pCMV-3HA.Western blotting, reporter gene assay, ELISA and real-time PCR were applied to detectinterferon β in DF-1cells at different levels post transfection with wide-type or mutant formsof NDV V proteins. Finally the zinc finger region of NDV V protein was modeled by usingPIV5V protein as a model, the results showed that the mutants were successful constructed,and high expression in DF-1cells. Overexpression of wide-type V protein of NDV were ableto suppress the IFN-β promoter activity, mRNA and protein levels in response to poly （I:C） inDF-1cells, but P protein, which shared the same N-terminal had no effect on suppressingIFN-β capability. The domain of NDV V protein was located at the C-terminal. Mutation ofcysteine196（C1）,221（C6）, or224（C7）, resulted in V proteins that retained the ability toinhibit IFN-β, indicating that these amino acids are not required for NDV V protein onsuppressing IFN-β capability. In contrast, mutation of cysteine200（C2）,213（C3）,214（C4）,or217（C5） resulted in V proteins defective in their ability to inhibit IFN-β, indicating thatthese residues are essential for NDV V protein on suppressing IFN-β capability. The resultsindicated that differential cysteine requirement observed with the NDV V protein. Schematicdiagram of the NDV V protein zinc finger structure showed that these residues of the smaller finger loops L1（C1, C6and C7） are not required for NDV V protein on suppressing IFN-βcapability while and these residues of the larger finger loops L2（C2, C3, C4and C5） wereessential.In summary, we firstly investigated and genomic characterization of the NDV strainsisolated from crested ibises and fully genomic analysis the swine NDV isolates with otherNDV strains. The genomic characterization of NDV strains of different hosts were useful toillustrate genome evolution, characteristics and genetic variation of NDV, also be helpful toclarify the virus origins, epidemic characteristics and molecular mechanism of the extensionof the host range. The study on molecular evolution characteristics and function on interferonβ production suppressing capacities of the V proteins from different Newcastle disease virusstrains can help us understand the role of NDV V protein in viral pathogenesis and theextension of the host range. The study reveal the relationship between the NDV V protein andviral pathogenesis, and it useful for further exploring the mechanisms of virus escapinginterferon, the function domain of NDV V protein on interferon antagonism, and long-termNewcastle disease vaccine develop.更多还原