猪伪狂犬病病毒（HNX株）的生物学特性与比较基因组学研究

发布时间：2018-01-18 09:22

  本文关键词：猪伪狂犬病病毒（HNX株）的生物学特性与比较基因组学研究　出处：《华中农业大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 伪狂犬病 猪伪狂犬病病毒HNX株 Bartha弱毒疫苗 致病力 全基因组测序 序列比对 进化树分析 重组

【摘要】：猪伪狂犬病(Pseudorabies,PR)由伪狂犬病病毒(Pseudorabies virus,PRV)引起,发病动物主要表现体温升高、瘙痒、急性脑脊髓炎和繁殖障碍等主要临床症状。该病危害各阶段的猪,给世界多国养猪业造成了重大的经济损失。美国和部分欧盟国家通过实施根除计划,净化了家猪的PR。在我国,随着PRV弱毒疫苗的使用,以及部分规模化种猪场实施PR的根除净化计划,PR得到有效的控制。但是自2011年10月以来,我国有超过20个省市区规模化猪场暴发了PR,许多免疫过PRV弱毒疫苗的猪场也有发病报道,这给我国PR的防控带来了巨大的挑战。为了深入了解此次PR重新暴发的原因,开展了以下研究工作:1.PRV的病原流行病学调查2012年1-11月,从我国多省市386家规模化猪场送检的1227份临床组织样品中检测出13.73%(53/386)的猪场存在着PRV野毒感染,12.47%(153/1227)的样品中存在着PRV野毒;其中4月,5月,11月送检的样品中PRV野毒的感染率都超过30.00%。选取8份PRV野毒感染的阳性样品,对其gB、gC、gE、TK、RR1、RR2等毒力和免疫原性相关基因分别进行测序、比对和进化树分析,结果显示,野毒株与Bartha株相比,g B、g C和RR1基因都存在氨基酸的缺失或插入突变,各基因同源性都比Ea株这些基因同源性低,各基因都不在同一进化分支。结果表明PRV野毒株与Ea株亲缘关系比Bartha株近。2.PRV的分离鉴定PRV野毒感染样品分别通过病毒的分离鉴定,测定其TCID50、一步生长曲线和病毒粒子的形态观察等研究,分离鉴定出PRV HNX、HNB、HNQZ、HNQX、HNZK株等5株病毒,其第1-9代病毒的TCID50为10-5.0-10-6.5/0.1m L,HNX株与Fa株生长趋势相似,病毒形态均为直径150nm左右,椭球形,有囊膜和纤突的粒子。3.PRV对小鼠的致病力研究PRV HNX、HNB、Ea、Fa株对6周龄Balb/c小鼠的LD50分别为102.0TCID50、102.4TCID50,102.0TCID50、100.7TCID50;同剂量感染小鼠,HNX株和HNB株感染的小鼠在短时间内出现严重瘙痒等临床症状,能引起小鼠脑组织小胶质细胞增多,神经元变性坏死等严重的病理损伤。4.HNX株对PRV Bartha株母源抗体仔猪的致病力研究HNX株和Ea株分别颈部肌肉注射含有Bartha株母源抗体的仔猪,结果显示,接种HNX株的仔猪,出现体温升高,瘙痒等临床症状,其鼻拭子和肛拭子中持续排毒21d,第28d大脑、肺、扁桃体、脾、肝和肾等组织中都检测出低拷贝数的PRVDNA。而接种Ea株的仔猪,无明显临床症状,其鼻拭子和肛拭子中短暂排毒,第28d大脑、肺和扁桃体等组织中检测出低拷贝数的PRV DNA。结果表明,HNX株对PRV Bartha株母源抗体的仔猪有很强的致病力,母源抗体不能保护仔猪抵御PRV HNX株感染,也不能阻止其排毒。5.HNX株对Bartha株疫苗免疫仔猪的致病力研究免疫Bartha株弱毒疫苗28d的仔猪和未免疫疫苗的PRV抗体阴性仔猪分别都滴鼻感染和颈部肌肉注射107TCID50的HNX株,结果显示,所有仔猪感染HNX株后,都出现体温升高、食欲不振、打喷嚏、呼吸困难、精神沉郁等临床症状,其鼻拭子和肛拭子中持续排毒,第16d大脑、肺、扁桃体、脾和肝等组织中都有高拷贝数的PRV DNA,感染仔猪的大脑、扁桃体、肺、脾和肾等组织中都有严重的病变和病理损伤,免疫组化都出现阳性信号,其中扁桃体和肺组织中阳性信号最强;未免疫疫苗的阴性仔猪出现神经症状,50%的仔猪死亡。结果表明,PRV HNX株对抗体阴性仔猪和疫苗免疫的仔猪都有很强的致病力,Bartha株弱毒疫苗免疫,不能保护仔猪抵御HNX株的感染,不能阻止仔猪排毒,也不能阻止PRV HNX株在大脑、扁桃体、肺、脾和肾等组织中定植。6.HNX、HNB、Fa和Ea株全基因组测序研究利用第二代高通量测序技术和Sanger法分别对HNX、HNB、Fa株和Ea株进行全基因组测序,其核苷酸序列数分别为142294bp、142255bp、141930bp和142334bp,GC含量分别为73.56%、73.61%、73.67%和73.60%,Gen Bank登录号分别为KM189912、KM189914、KM189913和KU315430,都编码70个基因,均包含独特长序列区(UL),内部重复序列(IRS),独特短序列区(US)和末端重复序列(TRS)等4部分。7.PRV不同毒株比较基因组学研究对PRV HNX、HNB、Fa、Ea株与Bartha株等PRV毒株的全基因组和各基因序列比对分析,结果显示,HNX株与HNB、Ea、Fa和Bartha株的同源性分别为98.1%、96.7%、96.4%和90.1%,Ea和Fa株与Bartha株的全基因组序列同源性分别为90.3%和90.7%,HNX、HNB、Ea、Fa株与Bartha株的所有基因核苷酸和氨基酸序列同源性分别为91.1%-99.9%和82.3%-99.7%;HNX、HNB、Fa、Ea株与国外分离株全基因组比对,其UL36基因、US1基因和非编码区序列同源性较低,HNX和HNB株与Fa株各基因氨基酸比对,有26个基因出现了差异,其中UL49.5,UL36和US1基因氨基酸差异率最大;PRV全基因组进化树存在两个独立分支:中国分离株分支与国外分离株分支,中国分离株分支位于不同亚分支,中国新分离株与中国2012年前分离株的免疫原性基因分别位于两个亚分支,而毒力相关基因位于同一个亚分支。结果表明,中国分离株之间全基因组同源性比Bartha、Becker、Kaplan株高,亲缘关系较近,中国新分离株与2012年前中国分离株基因组免疫原性基因进化出不同的亚分支,而毒力相关基因进化关系未分化。8.PRV全基因组和部分基因序列重组分析研究对PRV HNX、HNB、Fa、Ea株和Bartha、Kaplan、Becker、TJ、ZJ01、He N1、JS-2012等株全基因组序列和部分基因进行重组分析,结果显示,PRV基因组在UL区、IR区、US区和TR区内未出现基因片段重组,UL51、UL49.5、UL49、UL47、UL46、UL27、UL36、UL44、UL15、UL1、IE180、US1、US8等差异较大基因也未出现重组现象。结果表明,中国新分离PRV不同株之间以及与Bartha株之间没有出现重组现象。
[Abstract]:Pseudorabies (Pseudorabies, PR) by pseudorabies virus (Pseudorabies virus, PRV), the incidence of animal mainly fever, itching, acute encephalomyelitis and reproductive disorders and other major clinical symptoms. All stages of the pig harm of the disease, resulting in significant economic losses to the pig industry. Many countries in the world and part of United States EU countries through the implementation of the eradication program, purification of swine PR. in our country, with the use of PRV attenuated virus vaccine, and the eradication of purification part of scale pig breeding farm implement PR, PR has been effectively controlled. But since October 2011, China has more than 20 provinces and cities in large-scale pig farm outbreak of PR, many immune PRV attenuated vaccine of swine have reported the incidence, which brings a great challenge to the prevention and control of PR in China. In order to cause the outbreak of PR re understanding, carry out the following research work: the pathogeny of 1.PRV flow Epidemiological investigation in 2012 1-11 months, detected 13.73% from 1227 provinces and cities of China's 386 large-scale pig farm clinical tissue samples (53/386) from the existence of PRV virus infection, 12.47% (153/1227) are PRV wild virus samples; in April, May, November, submission of samples PRV wild virus infection rate of more than 30.00%. selected 8 positive samples were infected with PRV virus, the gB, gC, gE, TK, RR1, RR2, virulence and immunogenicity related genes were sequenced, alignment and phylogenetic tree analysis. The results showed that the wild strain and the Bartha strain by G B. G C and RR1 gene are amino acid deletions or insertions, all these genes than Ea strains of low homology of each gene homologous genes are not in the same evolutionary branch. The results showed that PRV wild strain and Ea strain relationship of infected samples by virus than Bartha strain isolation and identification of PRV near.2.PRV wild virus Isolation and identification, to determine the TCID50, morphological observation and so on one step growth curve and virus particles, the isolation and identification of PRV HNX, HNB, HNQZ, HNQX, HNZK and other 5 strains of virus strains, the 1-9 TCID50 10-5.0-10-6.5/0.1m L virus, HNX strain and Fa strain growth trend is similar to that of virus morphology were diameter about 150nm, ellipsoid, envelope and spike particle.3.PRV pathogenicity in mice of PRV HNX, HNB, Ea, Fa strains of 6 week old Balb/c mice LD50 102.0TCID50102.4TCID50102.0TCID50100.7TCID50 respectively; the same dose of infected mice, HNX and HNB strains of mice infected with severe itching and other symptoms in a short time inside, can cause brain microglia increased, neuronal degeneration and necrosis and other serious pathological damage of.4.HNX strain of PRV Bartha strain of maternal antibody in piglets the pathogenicity of HNX strain and Ea strain respectively, neck muscle injection containing Bartha strain The maternal antibody in piglets, results showed that the piglets inoculated with HNX strain, appeared elevated body temperature, itching and other symptoms, the continuous Detox 21d nasal swabs and anal swabs, 28d brain, lung, tonsil, spleen, liver and kidney were detected in low copy number of PRVDNA. and a Ea strain of piglets, no obvious clinical symptoms, the nasal swabs and anal swabs in brief detoxification, 28d brain, detect the low copy number of PRV DNA. showed that the lung and tonsil tissue in piglets of HNX strain to PRV strain Bartha maternal antibody has strong pathogenicity, maternal antibody can not protect piglets against PRV HNX strain infection, also cannot prevent the detoxification of.5.HNX piglets pathogenic study on immune Bartha Bartha strain vaccine in piglets strains of attenuated vaccine 28d and vaccine PRV antibody negative piglets were infected intranasally and cervical muscle injection of 107TCID50 HNX strain, the results show In all the piglets infected with HNX strain, there are fever, loss of appetite, sneezing, dyspnea, depression and other symptoms, continue its detoxification nasal swabs and anal swabs, 16d brain, lung, tonsil, spleen and liver tissues with high copy number of PRV DNA, infection pig brain, tonsil, lung lesions, and severe pathological damage of the spleen and kidney, immunohistochemistry were positive, the positive signal of tonsil and lung tissue in which the strongest immune vaccine; no negative piglets had neurological symptoms, the death of 50% piglets. The results showed that the piglets of PRV HNX strain the antibody negative piglets and vaccines are highly pathogenic Bartha strain attenuated vaccine, can not protect against HNX infection of piglets, piglets can prevent detoxification, also cannot stop PRV HNX strain in the brain, lung, tonsil, spleen and kidney in the colonization of.6.HNX, HNB, Fa and Ea strains of whole genome sequencing research using second generation high-throughput sequencing technology and Sanger method respectively to HNX, HNB, Fa and Ea strains of whole genome sequencing, the sequence number is 142294bp, 142255bp, 141930bp and 142334bp, GC contents were 73.56%, 73.61%, 73.67% and 73.60%, Gen Bank the accession number was KM189912, KM189914, KM189913 and KU315430, all 70 genes encoding, contains a unique long sequence region (UL), internal repeats (IRS), a unique short sequence region (US) and terminal repeats (TRS) of PRV HNX, HNB, Fa etc. The 4 part.7.PRV comparison of different strains genomics, Ea strain and Bartha strain and PRV strain genome and gene sequence analysis showed that HNX strain and HNB, Ea, Fa and Bartha strains homology were 98.1%, 96.7%, 96.4% and 90.1%, Ea and Fa strains with homologous genomic sequence of Bartha strain was 90.3% and 90.7%, HNX, HNB, Ea, Fa and Bartha strains all nucleotide and amino acid sequence homology were 91.1%-99.9% and 82.3%-99.7%; HNX, HNB, Fa, Ea strains with foreign isolates than the whole genome, the UL36 gene, US1 gene sequence and non encoding region with low homology, HNX and HNB strains with the Fa strain of amino acid sequences of each gene, 26 genes had differences, including UL49.5, UL36 and US1 gene amino acid difference rate; PRV whole genome phylogenetic tree has two independent branches: Chinese isolates and isolates of foreign branch branch branch, Chinese isolates in different sub branches, Chinese new isolates of immune with the Chinese before 2012 isolates of genes were located in two sub branches, and Virulence Related Genes in the same sub branch. The results showed that the Chinese genome homology between isolates than Bartha, Becker, Kaplan of plant height, close relationship, in In 2012 China new isolates and isolates of immunogenic genes evolved in different sub branches, and virulence related gene evolution of undifferentiated.8.PRV genome and partial sequence analysis of recombinant HNX research on PRV, HNB, Fa, Ea and Bartha strains, Kaplan, Becker, TJ, ZJ01, He and N1. JS-2012 strain genome sequence and partial gene recombination analysis, results showed that the PRV genome in the UL region, IR region, US region and TR region does not appear within the gene fragment, UL51, UL49.5, UL49, UL47, UL46, UL27, UL36, UL44, UL15, UL1, IE180, US1, does not appear too US8 and other large differences in gene recombination. The results show that the new China separation among different PRV strains and Bartha strains did not appear among the restructuring phenomenon.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.651
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本文编号：1440331