摘要
PRRS在世界范围内迅速蔓延而成为养猪业的重要威胁。2006年以来,“猪高热综合征”肆虐我国养猪业,高致病性猪蓝耳病是其主要致病病原之一。现有疫苗在免疫原性以及交叉保护性方面的作用均值得商榷,此外对于蓝耳病病毒的毒力因子的探寻仍无定论。本实验室拥有北美型经典弱毒PRRSV感染性克隆和中国第一株高致病性猪蓝耳病感染性克隆,构建了一系列结构蛋白嵌合感染性克隆,并获得嵌合病毒,为研究高致病性猪蓝耳病的毒力因子的定位和多价疫苗的研制奠定了基础。现将研究内容简述如下:
1.PRRSV嵌合感染性克隆的构建
高致病性蓝耳病近年来在我国广泛流行,目前尚无高效疫苗用于该病的防制。本研究以PRRSV弱毒感染性克隆-pAPRRS作为主要骨架,将高致病性蓝耳病江西分离株(JX143)的主要结构蛋白基因(ORF4-7)以及3′UTR区域替换入pAPPRS相应编码区域,构建了pSX12(ORF4-7-3′UTR)、p5NX12(ORF5-7-3′UTR)以及p56N12(ORF5-6)三个嵌合克隆。经DNA转染Marc-145细胞后拯救出嵌合病毒,同时对拯救病毒进行了病毒生物学特性分析,结果表明,嵌合病毒的生长曲线与亲本病毒差异相似。本研究成功构建了3个高致病性蓝耳病嵌合感染性克隆,并能拯救出活病毒,嵌合病毒在Marc-145细胞上能稳定传代。
2.嵌合病毒疫苗候选株的免疫原性及保护性实验
选取拯救病毒v56N12(ORF5-6)和vSX12(ORF4-7-3′UTR)免疫29日龄猪进行免疫原性试验,结果表明,以v56N12免疫后抗体水平相对较低,故免疫原性相对较差;而vSX12病毒免疫后14d血清ELISA抗体达到阳性值(IDEXX ELISA值s/p≥0.4),免疫后28 d中和抗体效价从原来的1:5以下达到1:15以上,说明vSX12具有良好的免疫原性并可进行免疫监测;免疫后28 d以强毒JX143株攻毒,结果vSX12免疫组未见发病,而未免疫攻毒对照组全部发病并有1头死亡,vSX12免疫猪攻毒后病毒血症持续10d后消失,而对照组攻毒后至少持续13 d,说明vSX12可对高致病性猪蓝耳病提供有效的免疫保护。本研究构建的这三个嵌合病毒为开发同时抗经典和变异株PRRSV感染的二价疫苗,以及探寻高致病性猪蓝耳病病毒的毒力因子奠定了基础。
Porcine Reproductive and Respiratory Syndrome(PRRS) is a severe infectious disease. PRRS is a economically significant infectious disease in the swine industry worldwide. from 2006,"Porcine High Fever Syndrome" has spreaded all over the Chinese swine industry,the highly pathogenic(HP) PRRSV is one of the major cause pathogen.As the virus genome recombinating and mutating,the biological type,antigen type and genotype of PRRSV variated quickly,current control for PRRS is insufficient.The available vaccines are lack of antigencity and cross protection.It is urgent that more efficient PRRS vaccine should be developed.At the same time,the virulence factor of PRRSV is not known clearly. Based on our established PRRSV infectious clone of North American strain and first highly pathogenic(HP) PRRSV infectious clone in China,chimeric structural protein infectious clones are constructed successfully,in order to provide the platform for searching virulent factor and multi- valence PRRS vaccine.The detailed experimentations are discussed as follows.
1.Construction of Chimeric Infectious Clones of PRRSV
In recent years,mass outbreaks of highly pathogenic(HP) porcine reproductive and respiratory syndrome virus(PRRSV) have spread all over the Chinese swine industry. There is no highly efficient PRRS vaccine to control it.Based on the first infectious cDNA clone of HP PRRSV strain pJX143 and that of an attenuated PRRSV,pAPRRS,we constructed several chimeric clones with various substitutions of structural protein genes (ORF4-7) and 3′UTR between attenuated pAPRRS and virulent pJX143.Upon transfection on Marc-145 cell lines,all chimeric constructs pSX12,p5NX12,and p56N12 were rescued. The rescued viruses maintain the similar virological properties,based on the results of the growth curve of the rescued viruses.In this study,three chimeric infectious clones are constructed successfully,the chimeric viruses can be passaged stably on Marc-145 cell.
2.The Immunogenicity and Protection Study of Chimeric Virus vaccine Candidates
To test if the chimeric viruses can be used as a vaccine candidate,the proper amount of vSX12 and v56N12 were used for vaccinated in pigs of 29 days old.As a result,the pigs vaccinated by vSX12 were all seroeonverted into positive 14-day-post vaccination,the neutralizing antibody of vSX12-vaccinated group increased from under 1:5 to 1:15 on 28th day,while v56N12 vaccinated pigs showed poor immuno responses.28 days post vaccination,the vSX12 group is challenged with the HP PRRSV JX143 strain,the vSX12-vaccinated group showed no signs of PRRS clinical symptom,and viremia period was shortened to 6 days.Our results demonstrated that 1) vSX12 chimeric virus is a good vaccine candidate;2) the virulence determinants of HP PRRSV probably located in coding regions other than ORF4-7 and 3′UTR,as our chimeric viruses were proved to be attenuated,this study could provide the significant platform for searching virulent determinants and developing multi- valence PRRS vaccine.
引文
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