本文關(guān)鍵詞：野生水禽副粘病毒的分離鑒定及Ⅴ蛋白對(duì)抗宿主細(xì)胞先天免疫機(jī)制的研究　出處：《東北林業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 新城疫病毒(NDV) 禽副粘病毒6型(APMV6) 分子流行病學(xué) V蛋白 先天免疫

【摘要】：新城疫是由新城疫病毒(Newcastle disease virus,NDV)引起的一種烈性傳染病,發(fā)病急,感染率和死亡率高(有時(shí)高達(dá)100%),它主要危害禽類。該病分布廣泛,對(duì)養(yǎng)殖業(yè)危害極大。針對(duì)新城疫病毒的分子流行病學(xué)研究主要集中于家禽和水禽,而作為新城疫病毒的天然宿主,野生鳥類在病毒傳播過程中所起到的作用并未引起足夠的重視。野生鳥類作為許多病原攜帶者和自然宿主,可潛在傳播人和動(dòng)物的重要傳染病。隨著候鳥的遷徙,攜帶某些病毒的候鳥可能會(huì)將病毒傳播到不同的地方而導(dǎo)致疾病的爆發(fā)和流行。因此,對(duì)野生鳥類進(jìn)行常見病原的分子流行病學(xué)監(jiān)測(cè),具有重要的指示意義。為研究野生鳥類感染或攜帶新城疫病毒的情況,本研究對(duì)2011年10月至2013年5月期間收集自吉林省向海自然保護(hù)區(qū)的野生鳥類糞便拭子共計(jì)1002份,進(jìn)行新城疫病毒的分子流行病學(xué)調(diào)查。一方面調(diào)查新城疫病毒的攜帶情況,進(jìn)行病毒的分離與鑒定,并選取有代表性的毒株進(jìn)行基因克隆和序列的分子遺傳特征分析；另一方面,首次分離到6型禽副粘病毒(avian paramyxo virus virus type 6,APMV6),并對(duì)其進(jìn)行全基因組測(cè)序。1、野生鳥類糞便拭子的PCR檢測(cè)將糞便拭子按常規(guī)方法無菌處理后,接種SPF雞胚,72h后收集尿囊液,提取尿囊液中的病毒RNA,用特異性的檢測(cè)新城疫病毒和禽副粘病毒的PCR方法進(jìn)行初步檢測(cè)。實(shí)驗(yàn)結(jié)果表明野鳥存在NDV以及APMV6的感染。所有野生鳥類樣品的NDV PCR陽(yáng)性檢出率為4.19%,APMV6 PCR陽(yáng)性檢出率為0.2%。野生鳥類新城疫病毒攜帶情況不具明顯的季節(jié)性,不同年份野鳥的NDV檢出率有一定差別,其中2011年NDV陽(yáng)性檢出率最高,為14.02%。2、野鳥源NDV的分離鑒定及F基因遺傳演化分析將用NDV PCR檢測(cè)引物初篩為陽(yáng)性的尿囊液經(jīng)適當(dāng)處理后接種雞胚成纖維細(xì)胞(CEF)培養(yǎng)增殖,進(jìn)行蝕斑純化。經(jīng)特異性PCR、血凝試驗(yàn)及血凝抑制試驗(yàn)鑒定,確定共分離到42株野鳥源NDV。對(duì)42株病毒的毒力基因F基因進(jìn)行部分克隆和序列測(cè)定,與GenBank上已發(fā)表的NDV參考株進(jìn)行序列比對(duì)和分析。測(cè)序結(jié)果顯示42株分離株均為弱毒株,其中3株屬于class Ⅰ genotype2,另外39株屬于class Ⅱ genotype Ⅰ。序列分析結(jié)果顯示：3個(gè)class Ⅰ分離株F基因核苷酸序列相似性為97.5%-98.9%；核苷酸遺傳進(jìn)化分析表明,這3株野鳥源NDV分離株與南方家禽分離株親緣關(guān)系較近。39株classⅡ分離株F基因核苷酸序列相似性為96.4%-100%,核苷酸遺傳進(jìn)化分析表明,毒株NEFU1301,NEFU1302, NEFU1102, NEFU1104, NEFU1113表現(xiàn)出高度的序列相似性,與2004-2006年間南方家禽分離株和遠(yuǎn)東及日本野鳥分離株親緣關(guān)系較近。另外34株classⅡ分離株親緣關(guān)系較近,處于另一個(gè)相對(duì)獨(dú)立的集簇,但這39株分離株均與疫苗株親緣關(guān)系較遠(yuǎn)。結(jié)果說明我國(guó)野生鳥類中存在NDV的感染,分離株均為弱毒株,提示強(qiáng)毒株在野鳥中并未廣泛流行；弱毒株在遠(yuǎn)東地區(qū)、日本地區(qū)及中國(guó)東海岸線可能已經(jīng)發(fā)生廣泛交流；經(jīng)免疫的家禽可能無法抵御來自野鳥所攜帶病毒的感染。本研究選擇NDV代表株NEFU1106和NEFU1136進(jìn)行全基因組序列,獲得的GenBank登陸號(hào)分別為KF361507和KC894391。本研究既豐富了東北地區(qū)NDV的流行病學(xué)資料,又警示我們要對(duì)野生鳥類在NDV傳播中所起的作用引起重視,為深入研究NDV的傳播、遺傳、進(jìn)化等奠定基礎(chǔ)。3、野鳥源APMV6的分離及其全基因組測(cè)序通過副粘病毒通用引物PCR檢測(cè)及電鏡觀察,確定共分離到2株野鳥源APMV6,分別命名為JL190及JL127。設(shè)計(jì)特異性引物分16段擴(kuò)增這兩株病毒的基因組,通過軟件拼接獲得它們的全長(zhǎng)基因組序列。其基因組全長(zhǎng)為16236nt,獲得的GenBank登錄號(hào)分別為JX522537和KF267717；基因組結(jié)構(gòu)為3'leader-NP-P-M-F-SH-HN-L-Trailer5',根據(jù)JL217的F基因及HN基因氨基酸序列構(gòu)建系統(tǒng)發(fā)育樹,結(jié)果表明,JL127株病毒與TW和FE株親緣關(guān)系較近,與HK株親緣關(guān)系較遠(yuǎn)。4、副粘病毒V蛋白對(duì)抗宿主細(xì)胞先天免疫機(jī)制的研究擴(kuò)增新城疫病毒強(qiáng)毒株、弱毒株以及APMV6的V基因,將其克隆至真核表達(dá)載體pcDNA3.1中,構(gòu)建真核表達(dá)質(zhì)粒,分別與IFN-β-Luc、NF-κB-Luc、PRDⅢ/Ⅰ-Luc、AP-1-luc報(bào)告質(zhì)粒共轉(zhuǎn)染HEK-293T細(xì)胞,接種仙臺(tái)病毒刺激細(xì)胞后,測(cè)定細(xì)胞內(nèi)螢火蟲熒光素酶及海腎熒光素酶活性。結(jié)果表明,新城疫病毒強(qiáng)弱毒株的V蛋白均能抑制宿主細(xì)胞干擾素p的產(chǎn)生,但強(qiáng)毒株V蛋白抑制PRDⅢ/Ⅰ啟動(dòng)子轉(zhuǎn)錄的作用要強(qiáng)于弱毒株V蛋白,構(gòu)建的強(qiáng)弱毒株V蛋白嵌合體表明,強(qiáng)毒株V蛋白C末端在此過程中發(fā)揮重要作用；APMV6的V蛋白能通過降低NF-κB的活性顯著抑制宿主細(xì)胞產(chǎn)生IFN-β。
[Abstract]:Newcastle disease is a severe infectious disease caused by Newcastle disease virus (NDV). It is characterized by acute infection, high infection rate and high mortality rate (sometimes up to 100%), which mainly endangering poultry. The disease is widely distributed and is very harmful to the breeding industry. The molecular epidemiology of Newcastle disease virus is mainly concentrated in poultry and waterfowl. However, as a natural host of Newcastle disease virus, the role of wild birds in the process of virus transmission has not attracted enough attention. As a host of pathogenic carriers and natural hosts, wild birds can potentially spread important infectious diseases of humans and animals. As migratory birds migrate, migratory birds with certain viruses may spread the virus to different places and lead to the outbreak and epidemic of the disease. Therefore, the molecular epidemiological monitoring of the common pathogens of wild birds is of great significance. In order to study the infection of wild birds or the occurrence of Newcastle disease virus, we collected 1002 fecal swabs from wild birds in Xianghai Nature Reserve from October 2011 to May 2013, and carried out a molecular epidemiological investigation of Newcastle disease virus. With a survey of Newcastle disease virus, virus isolation and identification, selection and analysis of molecular genetic characteristics of representative strains were cloned and sequence; on the other hand, the first 6 isolates of avian paramyxovirus type (avian paramyxo virus virus type 6, APMV6), and whole genome sequencing the. 1, the PCR detection of fecal swabs in wild birds was conducted. After fecal swabs were sterilized by routine methods, SPF chicken embryos were inoculated, and then the allantoic fluid was collected after 72h. The virus RNA in allantoic fluid was extracted, and the PCR method for detection of Newcastle disease virus and avian paramyxovirus was preliminarily detected. The experimental results show that the presence of NDV and APMV6 infection in wild birds. The positive rate of NDV PCR in all wild bird samples was 4.19%, and the positive rate of APMV6 PCR was 0.2%. Newcastle disease virus in wild birds carried out without obvious seasonal, wild birds in different years the detection rate of NDV had some differences, which in 2011 the highest positive rate of NDV was 14.02%. The analysis will use the NDV PCR primer screening for positive allantoic fluid by appropriate treatment after inoculation of chick embryo fibroblast evolution of 2 wild birds, isolation and identification of NDV and F genes (CEF) proliferation, by plaque purification. The specificity of PCR, hemagglutination test and hemagglutination inhibition test to determine the identification, 42 strains were isolated from wild birds in NDV. The F gene of the virulence gene of 42 strains of virus was partially cloned and sequenced, and the sequence was compared and analyzed with the published NDV reference strain on GenBank. The results of sequencing showed that 42 isolates were all weak strains, 3 of which belonged to class I genotype2, and the other 39 belonged to class II genotype I. The results of sequence analysis showed that 3 class isolates of F gene nucleotide sequence similarity of nucleotide 97.5%-98.9%; phylogenetic analysis indicated that these 3 strains of NDV isolates from wild birds and poultry isolates of Southern close relationship. 39 strains of class II isolates F gene nucleotide sequence similarity to 96.4%-100% nucleotide phylogenetic analysis indicated that strains NEFU1301, NEFU1302, NEFU1102, NEFU1104, NEFU1113 showed high sequence similarity with 2004-2006 from southern poultry isolates and isolates of wild birds in the Far East and Japan closer relationship. The other 34 class II isolates were in a relatively close relationship and were in another relatively independent cluster, but the 39 isolates were closely related to the vaccine strain. The result shows that there is NDV infection in wild birds in China, isolates were weakly virulent strains, suggesting that in the wild is not widely popular; attenuated in the Far East, Japan and Chinese East Coast may have occurred through extensive exchanges; immunization of poultry may not be able to resist the virus infection from wild birds. In this study, the genome sequences of NDV, NEFU1106 and NEFU1136 were selected, and the number of GenBank landings was KF361507 and KC894391, respectively. This study not only enriched the epidemiological data of NDV in Northeast China, but also warned us to pay attention to the role of wild birds in NDV transmission, and lay the foundation for further research on the transmission, inheritance and evolution of NDV. The separation of 3, APMV6 isolated and whole genome sequencing by paramyxovirus universal primer PCR detection and electron microscope, identified a total of 2 strains isolated from wild birds in APMV6, named JL190 and JL127. Specific primers were designed to amplify the genome of the two viruses by 16 segments, and their full-length genome sequences were obtained by software splicing. The genome length is 16236nt, the GenBank accession number were JX522537 and KF267717; genome structure of 3'leader-NP-P-M-F-SH-HN-L-Trailer5', according to the F gene and the amino acid sequence of JL217 HN gene phylogenetic tree, the results show that the more recent JL127 virus and TW FE strain and phylogenetic relationship, far genetic relationship with HK strain. Study 4, paramyxovirus V proteins against host cell innate immune mechanisms of the amplification of virulent Newcastle disease virus, attenuated APMV6 and V gene, cloned into eukaryotic expression vector pcDNA3.1, to construct eukaryotic expression plasmid, -Luc, NF- and IFN- respectively, Beta Kappa B-Luc, PRD III, AP-1-luc I / -Luc reporter plasmids were transfected into HEK-293T cells, the cells stimulated by Sendai virus inoculation, determination of firefly luciferase and Renilla luciferase activity in cells. The results show that the strength of strain of Newcastle disease virus V protein could inhibit host cell interferon P, but virulent strain V protein inhibited stronger PRD III / I promoter transcription in attenuated V protein, the strength of strain V protein chimeras showed that virulent strain V protein play an important role in the process of C terminal APMV6; V protein can produce IFN- by reducing the NF- Beta Kappa B activity was significantly inhibited in host cells.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.65

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