【作者】 吴双；

【导师】 刘秀梵；

【作者基本信息】 扬州大学 ， 预防兽医学， 2010， 博士

【摘要】 新城疫(Newcastle disease, ND)为能感染多种禽类的一种急性、高度传染性疫病,给世界养禽业造成巨大的经济损失。新城疫病毒(Newcastle disease virus, NDV)属于副粘病毒科,禽腮腺炎病毒属,其基因组由单股负链RNA构成,基因组结构为3′-NP-P-M-F-HN-L-5′,依次编码6种结构蛋白:核衣壳蛋白(NP)、磷蛋白(P)、基质蛋白(M)、融合蛋白(F)、血凝素-神经氨酸酶蛋白(HN)和大分子蛋白(L),其中病毒囊膜表面的两种糖蛋白F和HN是构成NDV致病性的主要分子基础。本研究对2005-2008年间分离自华东地区的79株NDV的F基因和HN基因序列进行了测序和遗传进化分析,发现分离株在基因型和毒力上具有明显的多样性,但基因Ⅶd亚型仍然是我国流行的优势基因型;在NDV的遗传进化分析基础上,选取了三株不同遗传背景的NDV进行体内外的致病性试验,结果表明:基因Ⅶ型NDV对鸽、鸡和鹅均呈现出高致病性,鸽源Ⅵb亚型NDV具有明显宿主感染特异性;为了进一步探讨NDV在细胞分子水平上的致病机制以及三株NDV之间的致病性差异,本研究还运用比较蛋白质组方法对上述三株NDV感染细胞后的差异表达蛋白进行了初步分析。1. 2005-2008年我国华东地区新城疫病毒分离株的遗传进化分析对2005-2008年间从我国华东地区发病的鸡、鹅、鸽等禽类中所分离出的79株NDV进行了生物学特性和基因型鉴定。F和HN基因的遗传进化分析结果一致表明:79个分离株中,有3株弱毒为class I谱系;76株为class II谱系,其中基因Ⅰ型NDV弱毒1株,基因Ⅱ型NDV弱毒12株,基因Ⅲ型NDV强毒2株,基因Ⅵ型中等毒力NDV 4株,其它57株NDV强毒分离株均属于基因Ⅶd亚型。Ⅶd亚型毒株占到了所有分离强毒株的96%以上,表明Ⅶd亚型NDV仍然是我国当前流行的优势基因型。序列比对结果显示,基因Ⅰ型、Ⅱ型和Ⅲ型分离株与疫苗株V4、La Sota和Mukteswar的同源性分别为99.6%、96.2~99%和99.9%,由此,这些毒株可能来源于临床上广泛使用的疫苗株;4株基因Ⅵb亚型NDV均为鸽源分离株,暗示该亚型NDV具有强的宿主感染特异性。在遗传进化树上,57个Ⅶd亚型NDV可进一步分为Ⅶd1和Ⅶd2两个分支,近两年分离的大多数毒株属于Ⅶd2分支,与Ⅶd1分支中NDV相比,Ⅶd2中的NDV在F蛋白分别出现了4处突变:N145K、Q279H、K387R和F512I;在HN蛋白也出现3处突变,分别为:T102I、A118E和T443M。我国新城疫病毒分离株在毒力与基因型所呈现出的多样性,为研究不同遗传背景毒株之间的致病性差异提供了理想的生物材料。2.不同遗传背景NDV体内外致病性在NDV的遗传进化分析基础上选取鹅源毒株JS-5-05-Go(基因Ⅶd亚型)、鸽源毒株WX-10-07-Pi(基因Ⅵb亚型)和鸡源毒株JS-7-05-Ch(基因Ⅲ型)三个不同遗传背景的NDV进行体内外的致病性试验。三株不同遗传背景NDV均以0.01MOI接种量感染鸡胚成纤维细胞(CEF)、鸭胚成纤维细胞(DEF)和鹅胚成纤维细胞(GEF),结果显示JS-5-05-Go感染三种细胞的上清HA峰值均比其它两株病毒高,WX-10-07-Pi感染后三种细胞上清HA峰值相同,JS-7-05-Ch感染后三种细胞上清HA峰值差异显著;三株NDV感染GEF细胞崩解过程均比DEF缓慢。动物试验中,我们以上述三株NDV和标准强毒株F48E8经自然感染途径分别感染鸽、SPF鸡和鹅三种试验动物,结果显示,仅JS-5-05-Go和F48E8引起感染鸽的死亡,且只有JS-5-05-Go和WX-10-07-Pi毒株导致鸽泄殖腔出现间歇排毒。WX-10-07-Pi攻毒组鸽泄殖腔排毒时间最长、且病毒检出率较其它组要高,因此与其它基因型NDV相比,基因Ⅵb亚型NDV更易在鸽群中感染传播。SPF鸡感染试验结果表明,JS-7-05-Ch虽为强毒株,但是对鸡的致病性较弱,但其余3株NDV对鸡的致病力与其毒力呈正相关。四株NDV对鹅的致病力差异显著,致病力从强到弱依次为F48E8、JS-5-05-Go、JS-7-05-Ch和WX-10-07-Pi,WX-10-07-Pi几乎不引发鹅的临床症状;F48E8和JS-5-05-Go试验组在9种脏器组织中病毒检测率、喉头和泄殖腔排毒差异不显著。3.不同遗传背景新城疫病毒感染CEF的比较蛋白质组研究为了在细胞蛋白水平研究三株不同遗传背景NDV和宿主细胞之间的相互作用关系,本研究以感染了NDV的CEF为研究对象,采用双向凝胶电泳(2-DE)结合基质辅助激光解析电离飞行时间质谱鉴定技术(MALDI-TOF-MS)的比较蛋白质组研究方法,分析三株NDV感染CEF后24h和48h两个时间点细胞蛋白表达动态。双向凝胶图谱的差异表达分析显示,CEF感染三株NDV后,共有43个蛋白点出现了显著的差异表达,包括持续上调、持续下调、先期上调后期下调以及先期下调后期上调四种表达方式,部分蛋白点在不同的感染组中表达水平有所差异。采用MALDI-TOF-MS质谱法对43个差异表达蛋白点进行质谱鉴定,结果成功鉴定了41个蛋白点,对应39种蛋白质。根据蛋白的功能不同,这些差异表达蛋白涉及病毒结构蛋白(2%)、细胞骨架(43%)、应激反应(10%)、蛋白质翻译和合成(7%)、RNA加工和合成(12%)、能量代谢(7%)、泛素-蛋白酶体途径(2%)、信号传导(12%)以及一些功能未知蛋白(7%)。应用Western-blot分析方法验证了β-actin和α-tubulin的蛋白表达水平,其结果与2-DE的分析数据完全相符。根据差异表达蛋白的功能,推测在新城疫病毒感染细胞时,病毒可通过热休克蛋白27(HSP27)、硫氧还蛋白域5(TXNDC5)和核磷蛋白(NPM1)等蛋白的上调表达来抑制或延缓细胞的早期凋亡;同时,翻译延长因子Tu(EF-Tu)和40S核糖体蛋白SA(PRSA)上调表达有利于病毒自身蛋白质的合成;另外,细胞对于NDV的感染也具有一定的防御机制,热休克蛋白90kDa(GRP94)上调表达导致细胞凋亡增加。比较蛋白质给学试验结果显示,不同遗传背景NDV感染细胞中部分蛋白的表达水平存在明显差异。细胞微丝蛋白β-肌动蛋白(ACTB)和细胞质类型肌动蛋白8(ACTG8)、信号传导蛋白核仁素(C23)、含有TPR结构域的小的富含谷氨酰胺的蛋白(SGT)和14-3-3E蛋白(14-3-3E)以及参与蛋白翻译和合成的TXNDC5等与WX-10-07-Pi本身感染特性有关;细胞骨架蛋白中α-微管蛋白(TUBA)、角蛋白19(KRT19)、网素3(PLS3)和肌动蛋白加帽蛋白(CAPZA2),RNA加工蛋白中的不均一核糖核蛋白K(hnRNPK)以及功能未知的类人含46卷曲螺旋结构域(CCDC46)等与JS-7-05-Ch自身感染特性有关;而细胞骨架蛋白中波形纤维蛋白(VIM)和Β1-链微管蛋白(TUBA1C)、热应激蛋白Grp94、蛋白翻译蛋白EF-Tu以及早期内体抗原1(EEA1)等蛋白表达水平的变化与JS-5-05-Go本身感染特性有关。全文小结:(1) 2005-2008年间共分离鉴定NDV 79株,分离株在基因型和毒力上具有明显的多样性。基因Ⅶd亚型仍然是我国流行的优势基因型,该亚型目前已出现了Ⅶd1和基因Ⅶd2两个分支。(2)基因Ⅰ型、Ⅱ型、Ⅲ型NDV田间分离株与当前使用的疫苗毒株关系密切,推测这些田间分离株是疫苗株的变异株。与其它基因型毒株相比,鸽源Ⅵb亚型NDV具有明显的宿主感染特异性。(3)本研究首次应用比较蛋白质组方法研究不同遗传背景NDV感染CEF细胞后的差异表达的蛋白,质谱鉴定41个差异表达的蛋白,对应39种差异表达蛋白。(4) NDV感染细胞后改变了细胞骨架、影响泛素-蛋白酶体途径的稳定、破坏了宿主翻译机制、抑制细胞RNA的加工和能量代谢。(5)不同遗传背景NDV感染细胞后均出现与其本身特性相关的蛋白质。(6) NDV除通过膜融合进入细胞外,还可能通过胞吞途径进入细胞。与其它两株病毒相比,胞吞作用在JS-5-05-Go进入细胞的过程中,可以发挥更长时间的效应。更多还原

【Abstract】 Newcastle disease (ND) is an acute and highly contagious avian disease with worldwide distribution that can cause substantial economic losses. The causative agent, Newcastle disease virus (NDV), is a member of the genus Avulavirus in the family Paramyxoviridae. The genome of NDV is a non-segmented, single-stranded, negative-sense RNA. The NDV genome contains six genes (in the following order: 3′-NP-P-M-F-HN-L-5′) which code for nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN) and an RNA dependent RNA polymerase (L), respectively. F and HN are the two important membrane glycoproteins which directly determine the virus virulence and pathogenicity. To elucidate the molecular epidemiology of recent NDV isolates, seventy-nine Newcastle disease viruses (NDV) isolated from clinical specimens of different poultry species including chickens, pigeons, geese and ostriches in Eastern China during 2005–2008 were characterized biologically and phylogenetically. The results showed that these viruses represented diverse phylogenetic and pathogenic phenotypes, while, the subgenotypeⅦd NDV are still the dominant strains circulating in China. Based on the phylogenic analysis of NDV isolates, three NDVs with different genetic background were chosen to conduct the pathogenicity tests both in vivo and in vitro. The results displayed that subgenotypeⅦd NDV possessed high pathogenicity to chickens, geese and pigeons, while the subgenotypeⅥb NDV only exhibited host-specificity to pigeons. To further evaluate the difference in the pathogenicity induced by these three NDVs, the differential expression of proteins in the virus-infected cells was also analyzed via comparative proteome.1. Characterization of the NDVs isolated during 2005-2008Seventy-nine Newcastle disease viruses (NDVs) isolated from clinical specimens of different poultry species including chickens, pigeons, geese and ostriches in Eastern China during 2005–2008 were characterized biologically and phylogenetically. Both the F and HN gene sequences analysis showed that these isolates could be divided into two separate clusters: classes I (3) and II (76). Three class I viruses and one genotypeⅠand 12 genotypeⅡviruses of class II were avirulent, four genotypeⅥb viruses isolated from pigeons were moderately virulent, and two genotypeⅢviruses and 57 genotypeⅦd viruses were highly virulent. The sequence identity of genotype,ⅡandⅢwith field isolates vaccine strains Queensland V4, La Sota and Mukteswar were 99.6%, 96.2-99%, and 99.9% respectively, implying that they were derivatives of the corresponding vaccine strains widely used in Chinese poultry flocks. The four genotypeⅥb NDVs were all pigeon-origin, indicating the host-specific preference of viruses in this genotype. On the phylogenic tree, the 57 subgenotypeⅦd isolates were further divided into two subgroups,Ⅶd1 andⅦd2, in which most of the strains isolated in recent two years belonged toⅦd2. Compared ofⅦd1 viruses, the viruses inⅦd2 presented the four amino acid sequence mutations of N145K, Q279H, K387R and G520V in F protein and three mutations of T102I, A118E and T443M in HN, respectively. These NDV isolates exhibited the diversity both in phylogenesis and pathogenicity, and provided suitable biomaterials to fully investigate the pathogenesis of NDVs with different genetic background.2. Pathogenicity of NDVs with different genetic backgroundBased on the genetic and biological characteristics, three NDV isolates with different host origins and genotypes, including a goose isolate JS-5-05-Go (subgenotypeⅦd), a pigeon isolate WX-10-07-Pi (subgenotypeⅥb ) and a chicken isolate JS-7-05-Ch (genotypeⅢ), were seleted to study their pathogenicity. On the cellular level, chicken embryo fibroblast (CEF), duck embryo fibroblast (DEF) and goose embryo fibroblast (GEF) were infected with these viruses respectively at an MOI of 0.01. The results showed that the peak HA titers in culture supernatants induced by JS-5-05-Go were higher than that induced by the other two viruses. HA titers in culture supernatants of three avian embryo fibroblasts induced by WX-10-07-Pi were similar, whereas, those induced by JS-7-05-Ch were different. Additionally, GEF exhibited a slower disintegration process than DEF or CEF afterinoculation with the three viruses. To evaluate the pathogenicity of those NDVs with different genetic background in vivo, pigeons, specific-pathogen-free (SPF) chickens and geese were infected with the three viruses via natural route, with a virulent NDV strain F48E8 as a control virus. The results showed that only JS-5-05-Go and F48E8 could cause the death in the experimental pigeons. Pigeons inoculated with WX-10-07-Pi and JS-5-05-Go shed the virus intermittently, while pigeons infected with WX-10-07-Pi experienced much longer time in virus shedding and showed higher rate of virus isolation when compared with pigeons infected by other two strains. Therefore, genotypeⅥb NDV strain exhibited host-specifity to pigeons and could spread more easily in pigeons than other genotypes. The results in SPF chickens by natural route of infection showed that virulent strain JS-7-05-Ch was not highly pathogenic to chickens, whereas, the pathogenicity of the other three strains was closely related with their virulence. According to the extent of pathological changes, four viruses presented obvious variation in pathogenicity for geese and F48E8 demonstrated the highest pathogenicity, followed by JS-5-05-Go, JS-7-05-Ch and WX-10-07-Pi. Notably, no clinical sign was detected in geese infected with WX-10-07-Pi. Although F48E8 and JS-5-05-Go showed variation in pathogenicity for geese, no apparent differences in virus shedding and virus distribution in nine tissues were detected.3. Comparative proteome analysis of CEFs infected with NDVs of different genetic backgroundTo elucidate the interactions between host cells and NDVs with different genetic background on the cellular protein level, the proteomics technology of two-dimensional gel electrophoresis (2-DE) together with matrix associated laser dissociation / ionization time of flight mass spectrometry (MALDI-TOF-MS) was used to analyze proteomes of CEFs infected with viruses of different genetic background. Protein samples of CEFs were harvested at 24h and 48h postinoculation and prepared for 2-DE analysis. The results showed that 43 protein spots in total expressed differentially during virus infection, including persistent up-regulated protein spots, persistent down-regulated protein spots, up-regulated at antephase and down-regulated at anaphase protein spots and down-regulated at antephase and up-regulated at anaphase protein spots. Some proteins have differentially expressed levels in three NDV-infected groups. With MALDI-TOF-MS, 41 protein spots representing 39 proteins were successfully identified. Cellular functional protein analysis showed that these differentially expressed proteins related to viral protein (2%), cytoskeleton (41%), stress response (10%), protein translation and elongation (7%), RNA processing and biosynthesis (12%), energy metabolism-associated proteins (7%), signal transduction (12%), ubiquitin-proteasome pathway (2%)and other proteins (7%). The result of Western-blot analysis further demonstrated thatβ-actin andα-tubulin showed difference in expression during virus infection, confirming the results of 2-DE. Based on the protein functions, we concluded that in NDV infected CEFs, the up-regulated expression proteins, heat shock 27kDa protein 1 (HSP27), thioredoxin domain containing 5 (TXNDC5) and nucleophosmin 1 (NPM1) may inhibit or delay early apoptosis, and that the up-regulated expression of Elongation factor Tu, mitochondrial precursor (EF-Tu) and 40S ribosomal protein SA, (34/67 kDa laminin receptor) (PRSA) may be conducive to the synthesis of viral proteins. Furthermore, certain kinds of host defense mechanism might be developed to antagonize virus infection, such as the increased level of apoptosis induced by up-regulated heat shock protein 90kDa beta, member 1, (GRP94).Changes in the expression level of cellular microfilament-associated proteins beta-actin (ACTB) and Actin, cytoplasmic type 8 (ACTG8), signal transduction protein nucleolin (C23), small glutamine-rich tetratricopeptide (SGT) and 14-3-3 protein epsilon (14-3-3E) and translation and elongation protein TXNDC5 were related to the infection of WX-10-07-Pi. Alterations of the expression of cytoskeleton protein TUBA, keratin 19 (KRT19), plastin 3 (T isoform) (PLS3) and capping protein (actin filament) muscle Z-line, alpha 2 (CAPZA2), RNA processing protein heterogeneous nuclear ribonucleoprotein K (hnRNPK) and functionally unidentified protein similar to coiled-coil domain containing 46 (CCDC46) were associated with the infection of JS-7-05-Ch. Cytoskeleton protein vimentin (VIM) and Tubulin alpha-1 chain (TUBA1C), heat shock protein Grp94, protein translation and elongation EF-Tu and early endosome antigen 1 (EEA1) were considered the symbol proteins in the cells infected by JS-5-05-Go. Taken together, the data showed that cells could give arise to different responses to the infection of NDVs with different genetic background.Conclusions:(1) Seventy nine Newcastle disease viruses isolated during 2005-2008 were characterized, indicating diversify in phylogenesis and virulence. In China, genotypeⅦNDV was still the predominant genotype which could be divided into two subgroupsⅦd1 andⅦd2.(2) GenotypeⅠ,ⅡandⅢfiled isolates in this study shared high homology with vaccine stains V4, LaSota and Mukteswar, respectively, indicating that they were derivatives of the vaccines viruses. Compared with other genotype strains, subgenotypeⅥb NDV exhibited host-specificity to pigeons.(3) In this study, we first used the comparative proteome approach to investigate the differentially expressed proteins of chicken embryo fibroblasts infected by NDVs with different genetic backgrounds, and successfully identified 41 differentially expressed spots representing 39 proteins.(4) NDV infection induced a series of cellular responses, including alteration of cytoskeleton networks, disorders in the ubiquitin-proteasome pathway, disruption of the host translation mechanism, and inhibition of RNA processing and energy metabolism.(5) Symbolic proteins had been found which was related to the infected by the viruses with different genetic background.(6) The entry into cells of NDV is believed to occur by direct fusion at the plasma membrane. In addition, NDV may enter host cells by an endocytic pathway.更多还原