【作者】 宋庆庆；

【导师】 刘秀梵；

【作者基本信息】 扬州大学 ， 预防兽医学， 2013， 博士

【摘要】 新城疫是一种重要的家禽传染病,该病病原体为新城疫病毒(Newcastle disease virus, NDV),又称禽副粘病毒1型(Avian paramyxovirus-1, APMV-1),属于副粘病毒科Avulavirus病毒属中的一员。尽管NDV只有一个血清型,但却存在多个基因型。按不同分离株基因组之间的分歧度大小,NDV被分为11个基因型(Ⅰ-Ⅺ)；根据最新提议的分类标准,该病毒进一步被分成16个基因型(Ⅰ-ⅩⅥ)和更多的基因亚型。作为副粘病毒科一员的NDV其主要进化动力是米源于RNA依赖性的RNA聚合酶缺乏校对功能而造成的RNA复制错配率高,但也有学者表示不同分离株之间也会发生重组现象,使该病毒能以一种更迅速的方式进化。在这两种进化动力中,RNA依赖性的RNA聚合酶的高变异率是毋庸置疑的,但是对于负链RNA病毒来讲同源重组似乎并不多见。本研究通过对NDV基因组一系列的数据集进行生物信息学分析来估测影响NDV变异的主要进化动力。另外,根据不同分离株的毒力差异,NDV又可以分为低毒力、中等毒力和强毒力三种致病型。本研究在全基因组比对分析时发现一株基因Ⅲ型强毒分离株JS/7/05/Ch的基因组与Ⅰ系苗Mukteswar株相似度高达99%以上,但其毒力却明显高于后者,为了探究影响这两株Ⅲ型NDV毒力差异的关键性基因,本文详细比对了两株病毒基因组之间的差异,并对主要差异基因的生物学意义进行了研究。1非自然重组对新城疫病毒遗传进化分析的影响通过对本实验室于2005年到2009年间分离的100多株NDV的F和HN基因进行遗传进化分析,我们并未发现同源重组事件的存在,但近几年越来越多的关于NDV的同源重组的案例被报道,其中有些基因组甚至发生多重重组现像。鉴于同源重组在NDV遗传进化过程中所起的作用存在不同的观点,本研究首先从GenBank数据库中下载到80条NDV的全基因组序列(2011年4月18日之前释放的序列)并使用重组分析软件(Recombination Detection Program, RDP)进行分析。结果显示有8条全基因组序列具有重组现象,在这8株预测的同源重组子代病毒中有5株病毒发生了多重重组现象。本研究对其中的两条含有重组片段的全基因组序列(China/Guangxi09/2003和NDV/03/018)进行遗传进化分析,结果同样显示GenBank数据库中的全基因组序列均含有两种不同基因型的DNA片段。为确保重组事件的真实性,我们对这两株NDV进行空斑纯化后重新测序并再次进行重组分析。结果显示,新获得的全基因组并不存在同源重组片段,我们分析认为原始数据中的重组现象很可能是测序者对混合感染的分离样品或者实验室污染的样品测序时造成的人为重组事件。为证实人为重组存在的可能性,我们将两株不同基因型的NDV进行人为混合,然后使用一对引物对该混合样品扩增M基因并测序,结果表明这对引物不仅可以扩增出两种不同基因型的M基因片段,而且还可以在同一段PCR产物中同时扩增出两种基因型的DNA片段。这充分证实对NDV基因组进行PCR时Taq酶存在模板转换的现象,导致不同基因型的序列被扩增到一起而产生非自然的人为重组现象。以上结果充分表明GenBank数据中NDV全基因组序列中的同源重组并非都是自然重组事件,而这些非自然的人为重组现象会对NDV遗传进化动力学分析造成一定的误导作用。2鸽副粘病毒1型进化动力学特点分析鸽源NDV,又称鸽副粘病毒1型(pigeon paramyxo virus-1, PPM V-1),是APMV-1的变异分支,20世纪80年代中期该变异株病毒通过鸽群开始向世界各国蔓延。虽然PPMV-1的F蛋白裂解位点是典型的强毒类型,但多数PPMV-1对鸡的致病力却是中等毒力或低毒力。基于这个特点我们推断PPMV-1应该具有其独特的进化动力学基础。遗传进化分析显示几乎所有分离于鸽群的NDV均聚拢在同一个分支—-Genotype Ⅵ b,说明PPMV-1很可能已经在鸽群中获得适应性变异导致该分支对鸽子具有明显的宿主特异性；与之相对的是基因Ⅶd型,该分支的NDV却具有最广的宿主谱。731株已知宿主来源的NDV的F基因编码区序列的遗传关系进一步的证实了PPMV-1对鸽子的特嗜性和基因Ⅶd亚型具有最广的宿主范围。通过125株不含重组片段的全基因组序列比对我们发现PPMV-1在整个基因组水平上与经典的APMV-1相比有19个氨基酸位点的差异。贝叶斯方法估算结果显示M基因的年平均进化率都高于其他5个基因,并且.通过最近共同祖先分析推断PPMV-1最早可能出现于20世纪60年代,要早于有记载的第一株PPMV-1的分离时间。适应性分析表明虽然PPMV-1的6个基因均处在负向选择的模式下进化,但各基因均存在不同数口的正向选择位点。PPMV-1的HN基因中的正向选择位点是6个基因中最少的,这预示着该基因受到的宿主免疫压最小。历史种群大小重建显示PPMV-1在其进化过程中有两个较大的拐点,20世纪80年代PPMV-1经历爆炸式增长过程,而到了最近几年PPMV-1的种群大小又开始逐渐下降。总之,本研究结果显示PPMV-1已经与其主要宿主鸽子形成了相互适应的关系,并推断其具有相对独特的进化特点。3基因Ⅲ型强毒株JS/7/05/Ch与同型Ⅰ系苗Mukteswar株全基因组比对分析基因Ⅲ型NDV临床分离株JS/7/05/Ch为典型的强毒,但其基因组却与Ⅰ系苗Mukteswar株具有99%以上的相似性,据此我们分析该强毒应该由Ⅰ系苗Mukteswar株进化而来。本研究分别对JS/7/05/Ch与Mukteswar两株NDV分别进行空斑纯化,并对得到的四株空斑纯化株JS-7122、JS-1217、Muk-4和Muk-5进行IVPI值的测定。结果显示JS-7122和JS-1217的IVPI值分别是2.2和2.15,而Muk-4和Muk-5的IVPI值分别为0.03和0.09。为了探索影响Mukteswar株NDV毒力增强的关键性基因,我们对JS-7122和Muk-4进行全基因组测序并对所测序列进行比对分析,结果显示两株病毒的差异位点主要集中在NP、 HN和L基因上,P、M和F基因则完全一致。NP基因有3个核甘酸的差异,其中第1433nt变异引起氨基酸位点的变化；HN基因有6个核苷酸位点的差异并均引起氨基酸的变化；L基因有5个核苷酸位点的差异,但均未引起氨基酸的突变。通过三维结构模拟可以发现HN蛋白的4个差异位点(第19位和29位无法进行模拟)均在HN蛋白的表面,并且494和495位的氨基酸止处于HN蛋白的抗原表位site12内,我们推测该基因很可能是导致这两株Ⅲ型NDV毒力差异主要因素。4Mukteswar株毒力增强分子机制研究参照JS-7122和Muk-4株NDV的全基因组设计8对引物,经RT-PCR方法从两株Ⅲ型NDV感染的尿囊液中扩增得到目的片段。首先将扩增的NP、PM依次克隆到pCR2.1载体中构建上游中间质粒pNPM;然后将MF、FH和HL片段依次克隆到pCR2.1载体中构建中游中间质粒pMFHL;最后将L1、L2和L3片段依次克隆到pCR2.1载体中构建下游中间质粒pL123。上、下游中间质粒经Spe Ⅰ和FspAⅠ酶切后,各自回收目的片段后进行连接,获得中间质粒pNPML;将该质粒使用Age Ⅰ和FspAⅠ进行酶切后,回收目的片段与同样酶切的pMFHL进行连接,获得两株Ⅲ型NDV的全长克隆质粒,然后通过特异性酶切反应将NDV的基因组全长质粒转移到TVT7R(0.0)载体中,成功构建两株Ⅲ型NDV的全长转录质粒。在上述基础上,利用Age Ⅰ和FspAⅠ两个酶切位点将两株Ⅲ型NDV的HN基因进行互换,共得到四个感染性全长质粒,分别是含有母本病毒基因组的TVT/JS-7122、TVT/Muk-4和HN基因互换的感染性质粒JS/MHN、Mu/JHN。将这四个感染性全长质粒与三个辅助质粒(pCI-L、pCI-NP和pCI-P)共转染能稳定表达T7RNA聚合酶的BSR-T7/5细胞,成功拯救出了四株NDV。MDT和IVPI测定结果显示拯救的病毒与原始母本病毒相似,但表达JS-7122株HN蛋白病毒的IVPI值要明显高于表达Muk-4株HN蛋白的毒株,该实验结果表明HN基因是Mukteswar株毒力增强的主要分子基础。更多还原

【Abstract】 Newcastle disease (ND) is one of the most devastating diseases in poultry. The causative agent, Newcastle disease virus (NDV), is a member of the Avulavirus genus in the Paramyxoviridae family. Although NDV has only one serotype, substantial antigenic and genetic diversity have been previously recognized. At least11genotypes (genotypes I to XI) have been described for NDVs. According to the latest proposed classification, NDV have been divided into16genotypes and more sub-genotypes. The main dynamics of evolution in nonsegmented RNA viruses is due to the inherent error rate of RNA replication associated with the RNA-dependent RNA polymerase. and some researchers consider recombination may play an important role in the evolution of NDV. While polymerase error is believed to be the main driving force for NDV evolution, it has been established that recombination in nonsegmented negative-sense RNA viruses, including NDV. is rare. In this study. A comprehensive dataset of NDV genome sequences was evaluated using bioinformatics to characterize the evolutionary forces affecting NDV genomes. Furthermore, NDV strains can be categorized according to their virulence into low (lentogenic), intermediate (mesogenic) or highly (velogenic) virulent strains. Base on the alignment of genomes, we found that two genotype III viruses, JS/7/05/Ch and Mukteswar possessing significant difference in virulence, shared more than99%identities in nucleotide sequence. The IVPI score of JS/7/05/Ch was remarkably higher than that of Mukteswar, even they shared the consistent cleavage site of virulent NDV strains. In order to elucidate the molecular mechanism responsible for the increased virulence of JS/7/05/Ch. the genomic difference between these two strains were thoroughly analyzed and identified. 1. Non-natural Recombination may influenced the evolutionary analysis of NDVDuring2005to2009. we characterized more than100NDV strains in our laboratory, but no recombinant strains were detected on the basis of the analysis of F and HN gene sequences. However, in recent years, more and more recombination events have been reported for NDVs. with the recombination occurring throughout the whole genome, even some recombinants of NDVs involved multiple genotypes have been reported. This controversy has prompted us to investigate whether the recombination events in NDVs are as common as has been reported. Eighty complete genomic sequences of NDV retrieved from GenBank (released before18Apr2011) were analyzed using RDP program. Our analysis indicated at least8strains were predicted to be recombinants in the80NDVs, including5strains generated from multi-recombination. Phylogenetic tree also confirmed the existence of recombination events in two available NDVs NDV/03/018and China/Guangxi09/2003. Using strains China/Guangxi09/2003and NDV/03/018as examples, we attempted to validate whether the unexpected high recombination rate was due to inaccuracy of some of the NDV sequences deposited in GenBank. The two viruses were plaque purified three times on primary chicken embryo fibroblasts and resequenced in this study. Surprisingly, the putative recombinations detected with the old versions of sequences in GenBank (DQ485230and GQ338310) did not exist with new seqences from purified sequences, indicating the artificial natue of the recombinations caused by polymerase template switching in these samples or laboratory-generated recombinants. To provide evidence for a potential non-natural recombinations resulting in a mosaic sequence, we performed reverse transcription-PCR (RT-PCR) amplification of the M gene from a mixed sample of different La Sota and ZJ1viruses, which represent genotypes Ⅱ and Ⅶ. respectively. PCR products were cloned into pGEM-T vector (Promega). and multiple clones were sequenced. Clones carrying either genotype Ⅱ or Ⅶ sequences were identified. Notably, the two genotype sequences could also be identified in the same clone. These results suggest that non-natural recombination events can be induced with samples containing mixed virus genotypes or strains, probably through polymerase template switching during the PCR procedure. Collectively, some recombinant genome sequences in the GenBank are not nature-recombinations, which will influence the evolution analysis of NDV.2. The evolutionary dynamics of pigeon paramyxovirus-1Pigeon NDV. also named Pigeon paramyxovirus-1(PPMV-1). is an antigenic variant of avian paramyxovirus type1(APMV-1) of chickens that is responsible for an autonomous Newcastle disease (ND)-like entity in pigeons. During the1980s, this virus variants spread worldwide among racing and show pigeons. The F proteins of PPMV-1strains bear a poly-basic cleavage site motif characteristic of virulent NDV strains. However, most of the PPMV-1strains show low virulence based on the pathogenicity assays in chickens. Considering the unique feature of the PPMV-1. a comprehensive dataset of avian paramyxovirus-1(APMV-1) were evaluated using bioinformatics to characterize the evolutionary forces affecting PPMV-1genomes. Phylogenetic analysis of142full-length Newcastle disease virus genomes indicated that Pigeon paramyxo virus-1(PPMV-1) isolates all clustered to the same clade (sub-genotype VIb), suggesting that PPMV-1have a high host specificity to the pigeon (Columba livia). Phylogenetic analysis of731sequences of the complete coding region of F genes reinforced the suggestion that PPMV-1is host-specific to pigeons, while genotype Ⅶd owns the widest range of host. According to the alignment of genome sequences without recombination,19specific amino acid residues were identified to differentiate PPMV-1from "classical" APMV-1. Matrix protein gene displayed higher yearly rates of change than the other five genes and the Time to Most Recent Common Ancestry (TMRCA) analysis showed that PPMV-1may appear early in1960s. The population history of PPMV-1displayed two major inflexion points, indicating its population size fluctuations. In1980s, the population size of PPMV-1underwent an explosive growth, whereas it showed a sudden decline in recent years. Additionally, compared to the other five genes, less positive selection sites were present in the HN gene, suggesting that HN gene suffered less immune pressure from the host. Result in this study showed that pigeon paramyxovirus-1is host-specific to pigeons and assumes a unique evolutionary history.3. Comparative analysis of the entire genomes of JS/7/05/Ch and MukteswarDuring the genome alignment, we found that two genotype III viruses, JS/7/05/Ch and Mukteswar showed significant difference in virulence while they shared more than99%identities in their genome sequences. The IVPI scores of the two cloned strains of JS/7/05/Ch JS-7122and JS-1217were2.2and2.15. while those of the two cloned strains of Mukteswar Muk-4and Muk-5were0.03and0.09, respectively. To elucidate the crtical genetic regions responsible for the incresed virulence of JS/7/05/Ch, the cloned strains JS-7122and Muk-4were selected for their entire genome sequence analysis. The alignment result showed that the main differences between these two NDV genomes were located in NP. HN and L genes while their P, M and F gene sequences were completely the same. There are3nucleotide mutations in NP gene with one site (1433nt) inducing the amino acid (AA) change and5mutations in L gene without involvement of AA substitutions. Notably, all the6mutations in HN gene caused the A A substitutions. The three-dimensional structures of HN protein of the two strains showed that there were4mutations on the surface of the protein. Furthermore, the AA substitutions at positions494and495are included in antigenic site12, which is also an important receptor-binding site of HN protein. Therefore, we surmised that HN gene may play a key role in the virulence increase of Mukteswar strain.4. The molecular mechanism in the virulence increase of Muktswar strainEight fragments from genomes of JS-7122and Muk-4were amplified and cloned into pCR2.1vector with the designed primers. The two fragments, NP and PM, were subcloned into pCR2.1vector sequentially to construct the plasmid pNPM. As the clone strategy mentioned above, the plasmid pMFHL composed fragments of MF, FH and HL while the plasmid pL123composed fragments L1, L2and L3. The plasmids pNPM and pL123were digested with Spe I and FspA I, and the targeted fragments from pNPM were purified and ligated into the pL123to construct the plasmid pNPML. The resultant plasmid was digested with Age I and FspA I, and then the fragment of interest was cloned into plasmid pMFHL, digested with the same enzymes, to construct the plasmid pALL which contained the full-length cDNA of NDV. Finally, the full-length cDNA was transferred to TVT7R(0.0) vector to construct full-length NDV infectious clone using specific enzymes. With the above clone steps, two infectious clones TVT/Muk-4, TVT/JS-7122were obtained. According to the genomic difference between the NDVs JS-7122and Muk-4, The HN genes of them were exchanged via molecular manipulations, resulting in two chimeric infectious clones Mu/JHN and JS/MHN, respectively. The four infectious clones (TVT/Muk-4, TVT/JS-7122, Mu/JHN and JS/MHN) with three helper plasmids, pCI-NP, pCI-P and pCI-L, were cotranfected into BSR-T7/5cell expressing T7RNA polymerase. After inoculation of transfected cell culture into embroynated chicken eggs from one specific pathogen free (SPF) flock, four NDV strains (rJS-7122, rMuk-4, rJS/MHN and rMu/JHN) were rescued successfully. The pathogenicity of the newly generated NDVs were characterized and the results showed that the virulence of rJS-7122and rMu/JHN were much higher than that of rMuk-4and rJS/MHN, suggesting that HN gene is the key factor for virulence increase of Mukteswar strain.更多还原