【作者】 陆小龙；

【导师】 刘秀梵； 刘晓文；

【作者基本信息】 扬州大学 ， 预防兽医学， 2022， 博士

【摘要】 新城疫(Newcastle disease,ND)是由新城疫病毒(Newcastle disease virus,NDV)强毒株引起的危害家禽和野鸟的重要传染病,对全球养禽业造成重大经济损失。迄今为止,疫苗接种已被证明是控制新城疫流行的最有效方式。当前包括我国在内的多个国家均在家禽中广泛接种新城疫疫苗,其中I系苗Mukteswar曾作为紧急免疫疫苗被广泛应用。NDV作为RNA病毒,其聚合酶缺乏校正活性,同时在高水平免疫选择压力下,基因突变引起毒力增强的事件时有报道。本实验室前期在发病鸡中分离到一株NDV强毒株JS/7/05/Ch,全基因组测序结果表明与基因Ⅲ型中等毒力疫苗株Mukteswar高度同源,仅在NP和HN上分别有1和6个氨基酸位点差异;与Mukteswar相比,JS/7/05/Ch通过静脉接种途径感染后,对鸡致病性更强,但是通过滴鼻点眼途径(自然感染途径)感染后没有显著差异;确定了 HN蛋白氨基酸变异是导致NDV毒力差异的关键因素,然而其内在机制尚不清楚。为此,本研究系统性地分析了 HN蛋白突变对基因Ⅲ型NDV生物学特性的影响;鉴定了与不同来源HN蛋白差异互作的宿主蛋白,并进一步阐明了病毒感染与宿主蛋白间的相互影响;分析了基因Ⅲ型NDV经静脉接种途径介导的体内感染特性差异,旨在阐明不同毒力基因Ⅲ型NDV的感染机制。1.不同毒力基因Ⅲ型NDV的生物学特性研究Mukteswar和JS/7/05/Ch的HN蛋白存在6处氨基酸差异,结合NCBI上已公布的基因Ⅲ型NDV HN基因序列,我们发现Mukteswar来源的N19和A29与JS/7/05/Ch来源的T145、1266和D494为基因Ⅲ型NDVHN蛋白的优势氨基酸,而E495和K495各占一半比例,而这些氨基酸的变异改变了HN蛋白的三维结构。蛋白修饰结果显示,HN蛋白突变影响着NDV诱导的赖氨酸相关蛋白修饰水平,这提示着HN蛋白翻译后的功能可能存在差异。进一步研究发现HN蛋白突变影响着NDV生物学特性,主要表现为强毒株JS/7/05/Ch具备更强的受体结合活性、膜融合活性、溶血活性、F蛋白裂解活性以及噬斑形成能力,但其神经氨酸酶活性较弱;强毒株JS/7/05/Ch在HD11和PBMC细胞上均具备显著的复制优势,而在CEF细胞中仅在感染前期具有复制优势,感染后期的复制能力减弱,这可能与其诱导的高水平的细胞死亡有关。综上所述,本章节揭示了 HN蛋白突变对基因Ⅲ型NDV生物学特性的调控机制,有助于从病原学角度解释基因Ⅲ型NDV感染机制的差异。2.Vimentin与不同毒力基因Ⅲ型NDV HN的互作机制研究经质谱分析鉴定到一株骨架蛋白——波形蛋白(Vimentin)与不同毒力基因Ⅲ型NDV HN蛋白存在差异互作,表现为Mukteswar HN蛋白可与Vimentin互作,而JS/7/05/Ch HN蛋白不能,说明氨基酸的变异削弱了 JS/7/05/Ch HN蛋白与Vimentin的互作。在CEF细胞上,强毒株JS/7/05/Ch感染能显著下调Vimentin的表达水平,中等毒力株Mukteswar与替换JS/7/05/Ch HN基因的重组毒株JS/MukHN对Vimentin表达的影响不明显,而Vimentin对3株NDV的复制和吸附均起增强作用;强毒株JS/7/05/Ch感染CEF细胞后更早地引起Vimentin的重排,但在感染后期3株NDV均能显著诱导Vimentin的重排且在核周围发生聚集,无显著差异;另外CEF细胞Vimentin的重排区域未完全与病毒的复制重合,说明不是基因Ⅲ型NDV的病毒复制中心。在HD11细胞上,JS/7/05/Ch能够更显著地下调Vimentin的表达,而Mukteswar和JS/MukHN对Vimentin表达的影响较弱;HD11细胞中的Vimentin促进了 JS/7/05/Ch的吸附和复制,但对Mukteswar和JS/MukHN毒株无明显影响;JS/7/05/Ch感染HD11细胞后能快速诱导Vimentin发生重排并形成明显的“笼状”结构,且重排的Vimentin成为病毒复制中心,而Mukteswar和JS/MukHN的感染未能诱导Vimentin重排。此外在CEF和HD11细胞中,Vimentin的重排和病毒的复制均呈相互依赖的关系,即Vimentin重排的破坏会削弱病毒的复制,而病毒的失活则会阻碍Vimentin的重排。我们进一步对Vimentin生物学功能进行检测,发现强毒株JS/7/05/Ch诱导更强的细胞损伤、凋亡和自噬的水平,同时细胞凋亡和自噬对强毒株感染的影响也更显著;而Vimentin的敲低促进了 NDV诱导的细胞凋亡和自噬。值得注意的是,HN蛋白突变显著削弱了 JS/7/05/Ch对NF-κ1B的活化,这对病毒复制及其诱导的细胞活性、凋亡和自噬均起重要作用。综上所述,本章节鉴定了与不同毒力基因Ⅲ型NDV HN蛋白差异互作的宿主蛋白Vimentin,分析了 Vimentin与NDV感染间的差异互作,为进一步了解不同毒力基因Ⅲ型NDV的致病机制提供了重要线索。3.不同毒力基因Ⅲ型新城疫病毒的体内感染特性研究为了阐明HN蛋白突变影响基因Ⅲ型NDV对鸡致病性的内在机制,我们以携带差异HN蛋白的基因Ⅲ型NDV通过静脉注射途径感染SPF鸡,分析病毒体内感染特性的差异。与Mukteswar和JS/MukHN相比,强毒株JS/7/05/Ch感染鸡后导致更严重的临床症状和死亡率,排毒时间更早且比例更高,病毒经外周血高效复制后扩散至全身多个脏器从而造成严重的感染;JS/7/05/Ch具有更强的组织嗜性,主要引起肺脏和脾脏的炎性细胞浸润和胸腺的T淋巴细胞耗竭,且对体内脏器中的巨噬细胞亲嗜性更强,表现为病毒与肺脏、脾脏和胸腺的巨噬细胞存在明显的共定位。为了验证HN蛋白突变对NDV体内感染后Vimentin的影响,我们对不同组织中Vimentin及其相关功能蛋白进行了检测,结果发现JS/7/05/Ch在法氏囊、脾脏、胸腺和腺胃中显著下调Vimentin的表达水平,同时增强了法氏囊、胸腺和腺胃中自噬蛋白活化以及脾脏、胸腺和腺胃中凋亡蛋白活化,这与体外研究结果一致。此外,我们还通过转录组测序发现不同毒力基因Ⅲ型NDV感染后对宿主基因存在差异调控,其中差异表达的基因主要富集在细胞骨架和血液相关功能上,进一步佐证了基因Ⅲ型NDV在静脉接种后造成毒力差异以及与细胞骨架蛋白存在差异的互作。综上所述,本章节全面探讨了基因Ⅲ型NDV对鸡的致病性差异,揭示了 HN蛋白突变从多方面影响NDV的体内感染,进一步阐明了基因Ⅲ型NDV的毒力和致病性差异机制。更多还原

【Abstract】 Newcastle disease(ND),caused by virulent strains of Newcastle disease virus(NDV),is an important infectious disease of poultry and wild birds.It brings heavy economic losses to the global poultry industry.Vaccination has been proven to be the most effective way to control the epidemic of ND till now,and ND vaccine is widely administered in poultry in several countries around the world including China.Mukteswar,the classical I ND vaccine,was once widely used as an emergency immunization vaccine.As an RNA virus,genetic mutations increasing virulence have been reported from time to time due to the lack of proofreading activity and the high immune pressure.A virulent NDV strain JS/7/05/Ch was previously isolated from infected chickens in our laboratory.Whole genome sequencing showed that JS/7/05/Ch was highly homologous to the genotype Ⅲ mesogenic vaccine strain Mukteswar only with one amino acid(aa)difference in NP and six aa differences in HN.Compared with Mukteswar,JS/7/05/Ch showed more pathogenic following intravenous inoculation,but showed no significant difference after infection through the natural route(oculonasal inoculation).The aa differences in the HN protein was identified as the critical factor leading to enhanced virulence of Mukteswar.However,its internal mechanism is still unclear.Therefore,this study systematically analyzed the effect of the mutant HN protein on the biological activity of genotype Ⅲ NDV.Meanwhile,this study screened and identified the host proteins that differentially interacted with different HN proteins,which further elucidated the interaction between virus infection and host proteins.Moreover,this study evaluated the difference of infection characteristics of genotype Ⅲ ND Vs with different virulence through intravenous inoculation.The aim of this study was to elucidate the infection mechanism of genotype Ⅲ NDVs with different virulence.1.Biological characteristics of genotype Ⅲ NDVs with different virulenceMukteswar-and JS/7/05/Ch-type HN protein showed 6 aa differences.Combined with HN gene sequences of genotype Ⅲ NDVs published on NCBI,we found that N19 and A29 from Mukteswar-type HN and T145,1266 and D494 from JS/7/05/Ch-type HN were the dominant aa.Besides,both of E495 from Mukteswar-type HN and K495 from JS/7/05/Chtype HN accounted for half of the total,respectively.These aa variations affects the threedimensional structure of HN protein.The protein modification results showed that the mutant HN could affect NDV-induced protein modification,predicting that the posttranslational function of HN protein may be different.Subsequently,the biological activity assays showed that the virulent strain JS/7/05/Ch mediated the stronger receptor-binding activity,membrane fusion activity,hemolysis activity,and F protein lysis activity,but its neuraminidase activity was weaker.Moreover,the virulent strain JS/7/05/Ch showed an obvious replication advantage both in HD11 and PBMC cells,whereas it only replicated efficiently at the early stage of infection and the replication level was significantly reduced at the late stage of infection in the CEF cells,which could be associated with the high-level cell death.Additionally,the virulent strain JS/7/05/Ch induced plaque formation with larger diameter,indicating the stronger infectivity of JS/7/05/Ch.In summary,this chapter revealed the effects of the mutant HN on biological functions of genotype Ⅲ NDV5 which could help explain the differences in the infection mechanism of genotype Ⅲ NDV from the virus perspective.2.The interaction mechanism between vimentin and HN proteins of genotype Ⅲ NDVs with different virulenceVimentin,a skeleton protein that interacted differently with genotype Ⅲ NDV HN proteins,was identified through mass spectrometry analysis.Mukteswar-type HN protein could interact with vimentin,but JS/7/05/Ch-type HN protein could not,indicating that the aa variation significantly weakened the interaction between JS/7/05/Ch-type HN and vimentin.In CEF cells,the virulent strain JS/7/05/Ch obviously downregulated the level of vimentin,while two mesogenic strains Mukteswar and JS/MukHN(HN-replacement recombinant strain)showed no significant effect on the levels of vimentin.Simultaneously,vimentin in CEF cells positively regulated the viral adsorption and replication of three NDV strains.JS/7/05/Ch infection induced vimentin rearrangement earlier in CEF cells,but all three NDV strains could induce vimentin rearrangement around the nucleus without significant difference.In addition,the rearrangement region of vimentin in CEF cells did not completely coincide with the viral replication,indicating that the rearranged vimentin in CEF cells was not the viral replication center of genotype Ⅲ NDV.In HD 11 cells,JS/7/05/Ch downregulated the levels of vimentin more significantly compared with that in CEF cells,while Mukteswar and JS/MukHN only took a weak effect on the expression of vimentin.Vimentin in HD11 cells significantly affected the adsorption and replication of JS/7/05/Ch but exhibited no obvious effect on Mukteswar and JS/MukHN infection.JS/7/05/Ch infection rapidly downregulated the levels of vimentin and formed obvious "cage" structure in HD 11 cells,and the rearranged vimentin became the center of viral replication.By contrast,Mukteswar and JS/MukHN could not induce vimentin rearrangement.Besides,vimentin rearrangement and virus replication were mutually dependent both in CEF and HD 11 cells.That is,disruption of the vimentin rearrangement would impair virus replication,whereas inactivation of the virus would hinder vimentin rearrangement.Furthermore,we deterrmined vimentin-related biological functions.Briefly,the virulent strain JS/7/05/Ch induced the stronger cell damage,apoptosis and autophagy,and the.effects of apoptosis and autophagy on JS/7/05/Ch infection was more significant.Simultaneously,vimentin knock-down enhanced NDV-induced apoptosis and autophagy.Notably,JS/7/05/Ch enhanced the cell death,apoptosis and autophagy by weakening the activation of NF-κB.To conclude,this chapter identified a host protein vimentin that differentially interacted with genotype Ⅲ NDV HN proteins and analyzed the differential regulation between vimentin and NDV infection.This study provides important clues for further understanding the pathogenic mechanism of genotype Ⅲ NDVs with different virulence.3.In vivo infection characteristics of genotype Ⅲ NDVs with different virulenceTo elucidate the underlying mechanism of the mutant HN protein affecting the pathogenicity of genotype III NDV in chickens,SPF chickens were infected with genotype III NDVs carrying differential HN proteins by intravenous route.Thereby,we analyzed the differences in infection characteristics of genotype III NDV in vivo.Compared with Mukteswar and JS/MukHN,the virulent strain JS/7/05/Ch caused more severe clinical symptoms and mortality,induced earlier and higher rates of virus shedding.Notably,JS/7/05/Ch efficiently replicated in the peripheral blood and then spread to multiple organs throughout the body,subsequently causing serious infection.Moreover,JS/7/05/Ch exhibited stronger tissue tropism,which mainly caused inflammatory cell infiltration in the lung and spleen and T lymphocyte depletion in the thymus.Simultaneously,JS/7/05/Ch showed a stronger affinity for macrophages in vivo organs,which was characterized by obvious colocalization of the virus with macrophages in the lung,spleen,and thymus.To verify the effect of the mutant HN protein on the regulation of vimentin in vivo by NDV,we detected the vimentin level and its related functional proteins in different tissues of infected chickens.The results showed that JS/7/05/Ch significantly downregulated the vimentin level in the bursa,spleen,thymus and glandular stomach,and enhanced the activation of autophagic proteins in the bursa,thymus and glandular stomach,as well as apoptotic proteins in the spleen,thymus and glandular stomach.These results were consistent with in vitro results.Furthermore,transcriptome sequencing revealed significant differences in the regulation of host genes by genotype III NDVs with distinct virulence.The differentially expressed genes were mainly enriched in blood and cytoskeleton functions,which further supported the differences in virulence of NDV after intravenous inoculation and the differential interaction with cytoskeletal proteins.In conclusion,this chapter comprehensively investigated the differences in pathogenicity of genotype III NDV in chickens and revealed that the mutant HN protein affected in vivo infection of NDV in multiple ways.This study further elucidates the mechanism of virulence and pathogenicity differences of genotype III NDV.更多还原