野生水禽副粘病毒的分离鉴定及Ⅴ蛋白对抗宿主细胞先天免疫机制的研究

发布时间：2017-12-28 05:37

本文关键词：野生水禽副粘病毒的分离鉴定及Ⅴ蛋白对抗宿主细胞先天免疫机制的研究　出处：《东北林业大学》2016年博士论文　论文类型：学位论文

【摘要】：新城疫是由新城疫病毒(Newcastle disease virus,NDV)引起的一种烈性传染病,发病急,感染率和死亡率高(有时高达100%),它主要危害禽类。该病分布广泛,对养殖业危害极大。针对新城疫病毒的分子流行病学研究主要集中于家禽和水禽,而作为新城疫病毒的天然宿主,野生鸟类在病毒传播过程中所起到的作用并未引起足够的重视。野生鸟类作为许多病原携带者和自然宿主,可潜在传播人和动物的重要传染病。随着候鸟的迁徙,携带某些病毒的候鸟可能会将病毒传播到不同的地方而导致疾病的爆发和流行。因此,对野生鸟类进行常见病原的分子流行病学监测,具有重要的指示意义。为研究野生鸟类感染或携带新城疫病毒的情况,本研究对2011年10月至2013年5月期间收集自吉林省向海自然保护区的野生鸟类粪便拭子共计1002份,进行新城疫病毒的分子流行病学调查。一方面调查新城疫病毒的携带情况,进行病毒的分离与鉴定,并选取有代表性的毒株进行基因克隆和序列的分子遗传特征分析；另一方面,首次分离到6型禽副粘病毒(avian paramyxo virus virus type 6,APMV6),并对其进行全基因组测序。1、野生鸟类粪便拭子的PCR检测将粪便拭子按常规方法无菌处理后,接种SPF鸡胚,72h后收集尿囊液,提取尿囊液中的病毒RNA,用特异性的检测新城疫病毒和禽副粘病毒的PCR方法进行初步检测。实验结果表明野鸟存在NDV以及APMV6的感染。所有野生鸟类样品的NDV PCR阳性检出率为4.19%,APMV6 PCR阳性检出率为0.2%。野生鸟类新城疫病毒携带情况不具明显的季节性,不同年份野鸟的NDV检出率有一定差别,其中2011年NDV阳性检出率最高,为14.02%。2、野鸟源NDV的分离鉴定及F基因遗传演化分析将用NDV PCR检测引物初筛为阳性的尿囊液经适当处理后接种鸡胚成纤维细胞(CEF)培养增殖,进行蚀斑纯化。经特异性PCR、血凝试验及血凝抑制试验鉴定,确定共分离到42株野鸟源NDV。对42株病毒的毒力基因F基因进行部分克隆和序列测定,与GenBank上已发表的NDV参考株进行序列比对和分析。测序结果显示42株分离株均为弱毒株,其中3株属于class Ⅰ genotype2,另外39株属于class Ⅱ genotype Ⅰ。序列分析结果显示：3个class Ⅰ分离株F基因核苷酸序列相似性为97.5%-98.9%；核苷酸遗传进化分析表明,这3株野鸟源NDV分离株与南方家禽分离株亲缘关系较近。39株classⅡ分离株F基因核苷酸序列相似性为96.4%-100%,核苷酸遗传进化分析表明,毒株NEFU1301,NEFU1302, NEFU1102, NEFU1104, NEFU1113表现出高度的序列相似性,与2004-2006年间南方家禽分离株和远东及日本野鸟分离株亲缘关系较近。另外34株classⅡ分离株亲缘关系较近,处于另一个相对独立的集簇,但这39株分离株均与疫苗株亲缘关系较远。结果说明我国野生鸟类中存在NDV的感染,分离株均为弱毒株,提示强毒株在野鸟中并未广泛流行；弱毒株在远东地区、日本地区及中国东海岸线可能已经发生广泛交流；经免疫的家禽可能无法抵御来自野鸟所携带病毒的感染。本研究选择NDV代表株NEFU1106和NEFU1136进行全基因组序列,获得的GenBank登陆号分别为KF361507和KC894391。本研究既丰富了东北地区NDV的流行病学资料,又警示我们要对野生鸟类在NDV传播中所起的作用引起重视,为深入研究NDV的传播、遗传、进化等奠定基础。3、野鸟源APMV6的分离及其全基因组测序通过副粘病毒通用引物PCR检测及电镜观察,确定共分离到2株野鸟源APMV6,分别命名为JL190及JL127。设计特异性引物分16段扩增这两株病毒的基因组,通过软件拼接获得它们的全长基因组序列。其基因组全长为16236nt,获得的GenBank登录号分别为JX522537和KF267717；基因组结构为3'leader-NP-P-M-F-SH-HN-L-Trailer5',根据JL217的F基因及HN基因氨基酸序列构建系统发育树,结果表明,JL127株病毒与TW和FE株亲缘关系较近,与HK株亲缘关系较远。4、副粘病毒V蛋白对抗宿主细胞先天免疫机制的研究扩增新城疫病毒强毒株、弱毒株以及APMV6的V基因,将其克隆至真核表达载体pcDNA3.1中,构建真核表达质粒,分别与IFN-β-Luc、NF-κB-Luc、PRDⅢ/Ⅰ-Luc、AP-1-luc报告质粒共转染HEK-293T细胞,接种仙台病毒刺激细胞后,测定细胞内萤火虫荧光素酶及海肾荧光素酶活性。结果表明,新城疫病毒强弱毒株的V蛋白均能抑制宿主细胞干扰素p的产生,但强毒株V蛋白抑制PRDⅢ/Ⅰ启动子转录的作用要强于弱毒株V蛋白,构建的强弱毒株V蛋白嵌合体表明,强毒株V蛋白C末端在此过程中发挥重要作用；APMV6的V蛋白能通过降低NF-κB的活性显著抑制宿主细胞产生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.
【学位授予单位】：东北林业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65

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