本文關(guān)鍵詞：豬偽狂犬病病毒（HNX株）的生物學(xué)特性與比較基因組學(xué)研究　出處：《華中農(nóng)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： 偽狂犬病 豬偽狂犬病病毒HNX株 Bartha弱毒疫苗 致病力 全基因組測序 序列比對 進(jìn)化樹分析 重組

【摘要】：豬偽狂犬病(Pseudorabies,PR)由偽狂犬病病毒(Pseudorabies virus,PRV)引起,發(fā)病動物主要表現(xiàn)體溫升高、瘙癢、急性腦脊髓炎和繁殖障礙等主要臨床癥狀。該病危害各階段的豬,給世界多國養(yǎng)豬業(yè)造成了重大的經(jīng)濟(jì)損失。美國和部分歐盟國家通過實施根除計劃,凈化了家豬的PR。在我國,隨著PRV弱毒疫苗的使用,以及部分規(guī)模化種豬場實施PR的根除凈化計劃,PR得到有效的控制。但是自2011年10月以來,我國有超過20個省市區(qū)規(guī)�；i場暴發(fā)了PR,許多免疫過PRV弱毒疫苗的豬場也有發(fā)病報道,這給我國PR的防控帶來了巨大的挑戰(zhàn)。為了深入了解此次PR重新暴發(fā)的原因,開展了以下研究工作:1.PRV的病原流行病學(xué)調(diào)查2012年1-11月,從我國多省市386家規(guī)模化豬場送檢的1227份臨床組織樣品中檢測出13.73%(53/386)的豬場存在著PRV野毒感染,12.47%(153/1227)的樣品中存在著PRV野毒;其中4月,5月,11月送檢的樣品中PRV野毒的感染率都超過30.00%。選取8份PRV野毒感染的陽性樣品,對其gB、gC、gE、TK、RR1、RR2等毒力和免疫原性相關(guān)基因分別進(jìn)行測序、比對和進(jìn)化樹分析,結(jié)果顯示,野毒株與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變,各基因同源性都比Ea株這些基因同源性低,各基因都不在同一進(jìn)化分支。結(jié)果表明PRV野毒株與Ea株親緣關(guān)系比Bartha株近。2.PRV的分離鑒定PRV野毒感染樣品分別通過病毒的分離鑒定,測定其TCID50、一步生長曲線和病毒粒子的形態(tài)觀察等研究,分離鑒定出PRV HNX、HNB、HNQZ、HNQX、HNZK株等5株病毒,其第1-9代病毒的TCID50為10-5.0-10-6.5/0.1m L,HNX株與Fa株生長趨勢相似,病毒形態(tài)均為直徑150nm左右,橢球形,有囊膜和纖突的粒子。3.PRV對小鼠的致病力研究PRV HNX、HNB、Ea、Fa株對6周齡Balb/c小鼠的LD50分別為102.0TCID50、102.4TCID50,102.0TCID50、100.7TCID50;同劑量感染小鼠,HNX株和HNB株感染的小鼠在短時間內(nèi)出現(xiàn)嚴(yán)重瘙癢等臨床癥狀,能引起小鼠腦組織小膠質(zhì)細(xì)胞增多,神經(jīng)元變性壞死等嚴(yán)重的病理損傷。4.HNX株對PRV Bartha株母源抗體仔豬的致病力研究HNX株和Ea株分別頸部肌肉注射含有Bartha株母源抗體的仔豬,結(jié)果顯示,接種HNX株的仔豬,出現(xiàn)體溫升高,瘙癢等臨床癥狀,其鼻拭子和肛拭子中持續(xù)排毒21d,第28d大腦、肺、扁桃體、脾、肝和腎等組織中都檢測出低拷貝數(shù)的PRVDNA。而接種Ea株的仔豬,無明顯臨床癥狀,其鼻拭子和肛拭子中短暫排毒,第28d大腦、肺和扁桃體等組織中檢測出低拷貝數(shù)的PRV DNA。結(jié)果表明,HNX株對PRV Bartha株母源抗體的仔豬有很強(qiáng)的致病力,母源抗體不能保護(hù)仔豬抵御PRV HNX株感染,也不能阻止其排毒。5.HNX株對Bartha株疫苗免疫仔豬的致病力研究免疫Bartha株弱毒疫苗28d的仔豬和未免疫疫苗的PRV抗體陰性仔豬分別都滴鼻感染和頸部肌肉注射107TCID50的HNX株,結(jié)果顯示,所有仔豬感染HNX株后,都出現(xiàn)體溫升高、食欲不振、打噴嚏、呼吸困難、精神沉郁等臨床癥狀,其鼻拭子和肛拭子中持續(xù)排毒,第16d大腦、肺、扁桃體、脾和肝等組織中都有高拷貝數(shù)的PRV DNA,感染仔豬的大腦、扁桃體、肺、脾和腎等組織中都有嚴(yán)重的病變和病理損傷,免疫組化都出現(xiàn)陽性信號,其中扁桃體和肺組織中陽性信號最強(qiáng);未免疫疫苗的陰性仔豬出現(xiàn)神經(jīng)癥狀,50%的仔豬死亡。結(jié)果表明,PRV HNX株對抗體陰性仔豬和疫苗免疫的仔豬都有很強(qiáng)的致病力,Bartha株弱毒疫苗免疫,不能保護(hù)仔豬抵御HNX株的感染,不能阻止仔豬排毒,也不能阻止PRV HNX株在大腦、扁桃體、肺、脾和腎等組織中定植。6.HNX、HNB、Fa和Ea株全基因組測序研究利用第二代高通量測序技術(shù)和Sanger法分別對HNX、HNB、Fa株和Ea株進(jìn)行全基因組測序,其核苷酸序列數(shù)分別為142294bp、142255bp、141930bp和142334bp,GC含量分別為73.56%、73.61%、73.67%和73.60%,Gen Bank登錄號分別為KM189912、KM189914、KM189913和KU315430,都編碼70個基因,均包含獨特長序列區(qū)(UL),內(nèi)部重復(fù)序列(IRS),獨特短序列區(qū)(US)和末端重復(fù)序列(TRS)等4部分。7.PRV不同毒株比較基因組學(xué)研究對PRV HNX、HNB、Fa、Ea株與Bartha株等PRV毒株的全基因組和各基因序列比對分析,結(jié)果顯示,HNX株與HNB、Ea、Fa和Bartha株的同源性分別為98.1%、96.7%、96.4%和90.1%,Ea和Fa株與Bartha株的全基因組序列同源性分別為90.3%和90.7%,HNX、HNB、Ea、Fa株與Bartha株的所有基因核苷酸和氨基酸序列同源性分別為91.1%-99.9%和82.3%-99.7%;HNX、HNB、Fa、Ea株與國外分離株全基因組比對,其UL36基因、US1基因和非編碼區(qū)序列同源性較低,HNX和HNB株與Fa株各基因氨基酸比對,有26個基因出現(xiàn)了差異,其中UL49.5,UL36和US1基因氨基酸差異率最大;PRV全基因組進(jìn)化樹存在兩個獨立分支:中國分離株分支與國外分離株分支,中國分離株分支位于不同亞分支,中國新分離株與中國2012年前分離株的免疫原性基因分別位于兩個亞分支,而毒力相關(guān)基因位于同一個亞分支。結(jié)果表明,中國分離株之間全基因組同源性比Bartha、Becker、Kaplan株高,親緣關(guān)系較近,中國新分離株與2012年前中國分離株基因組免疫原性基因進(jìn)化出不同的亞分支,而毒力相關(guān)基因進(jìn)化關(guān)系未分化。8.PRV全基因組和部分基因序列重組分析研究對PRV HNX、HNB、Fa、Ea株和Bartha、Kaplan、Becker、TJ、ZJ01、He N1、JS-2012等株全基因組序列和部分基因進(jìn)行重組分析,結(jié)果顯示,PRV基因組在UL區(qū)、IR區(qū)、US區(qū)和TR區(qū)內(nèi)未出現(xiàn)基因片段重組,UL51、UL49.5、UL49、UL47、UL46、UL27、UL36、UL44、UL15、UL1、IE180、US1、US8等差異較大基因也未出現(xiàn)重組現(xiàn)象。結(jié)果表明,中國新分離PRV不同株之間以及與Bartha株之間沒有出現(xiàn)重組現(xiàn)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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S852.651
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本文編號：1440331