口蹄疫兔化弱毒疫苗ZB株L蛋白N^2D、M^(143)L和E^(147)G氨基酸突变对病毒毒力影响的研究  

Foot-and-mouth disease virus of attenuated vaccine ZB strain L protein mutations N^2D, M^(143)L and E^(147)G do not impair viral virulence

作　　者：赵国洪 王继华[3] 李华春 何于雯 朱明旺[3] 信爱国 ZHAO Guo-hong;WANG Ji-hua;LI Hua-chun;HE Yu-wen;ZHU Ming-wang;XIN Ai-guo(National Professional Laboratory of Foot-and-mouth disease（Kunming）,Yunnan Animal Science and Veterinary Institute,Kunming 650224 China;Yunnan Tropical and Subtropical Animal Virus Disease Laboratory,Yunnan Animal Science and Veterinary Institute,Kunming 650224,China;Yunnan Provincial Research Center for Veterinary Biological Products,Baoshan 678000,China)

机构地区：[1]云南省畜牧兽医科学院国家口蹄疫专业实验室(昆明),云南昆明650224 [2]云南省畜牧兽医科学院云南省热带亚热带动物病毒病重点实验室,云南昆明650224 [3]云南省兽医生物制品研制中心,云南保山678000

出　　处：《中国预防兽医学报》2018年第9期822-827,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：云南省基础研究基金项目(2015FA036);国家自然科学基金项目(31760745)

摘　　要：口蹄疫病毒(FMDV)前导蛋白L^(pro)是病毒的毒力因子之一。前期研究发现,FMDV兔化弱毒疫苗ZB株传代减毒致弱后,前导蛋白L^(pro)内仅存在3个氨基酸点突变(N^2D、M^(143)L和E^(147)G)。为确定这3个突变对病毒生物学特性的影响,本研究利用弱毒疫苗ZB株感染性分子克隆为骨架,构建获得含有这3个氨基酸回复突变的重组病毒r ZBatt-L,并比较其与亲本病毒r ZBatt生物学特性。结果显示,r ZBatt-L和r ZBatt在BHK-21细胞中均可以产生细胞病变效应,形成大小相似的噬斑,而在牛原代肾细胞(BK)中均不产生CPE;二者在BHK-21细胞中的生长曲线与病毒RNA复制动态相似(p>0.05),对乳鼠的致病性也无显著差异;测定了r ZBatt-L和r ZBatt分别感染BK诱导I型干扰素(IFN-α和β)能力,结果显示r ZBatt-L和r ZBatt均可以在感染早期诱导IFN-α表达,而感染晚期抑制IFN-α表达。相对的是,r ZBatt-L和r ZBatt感染细胞后均可以诱导IFN-β含量增加,并且二者诱导IFN-β含量无差异(p>0.05)。结果表明,FMDV弱毒疫苗ZB株L^(pro)蛋白中的N^2D、M^(143)L和E^(147)G突变不改变病毒的生物学特性,对病毒毒力不产生明显的影响。本研究明确了L^(pro)蛋白突变不是ZB株致弱的关键氨基酸位点。Foot-and-mouth disease virus （FMDV） leader proteinase （L^pro） is one of the viral virulence factors. Previously, the only three amino acid mutations （N;D, M^143L and E^147G） had occurred in L^pro of FMDV attenuated vaccine ZB strain during the attenuation process. In this study, a chimeric virus （rZBatt-L） which encoded the L^pro of virulent ZB virus containing the NZD, M^143L and E^147G mutations was rescued. Comparison of rZBatt-L with its parental plasmid-derived rescued virus （rZBatt） demonstrated that these two viruses displayed similar plaque morphologies in BHK-2 1 cells, but no plaque formation was observed in primary fetal bovine kidney （BK） cells. Analysis of TCID50 growth curves and FMDV RNA levels revealed the similar replication kinetics between rZBatt-L and rZBatt 09〉0.05）. The pathogenicity in suckling mice of these two viruses showed no significant difference （p〉0.05）. Furthermore, the levels of type I interferon （IFN-a and IFN-β） in rZBatt-L or rZBatt virus-infected BK were detected. The production of IFN-α in BK were elevated in early-phase infection, but it was inhibited in the late infection phase. In contrast, the IFN-β levels were increased in BK infected with either rZBatt-L or rZBatt, without any significant difference （p〉0.05）. The rZBatt-L displayed similar characteristics as its parental virus rZBatt. These results indicated that the mutations in the L^pro protein of ZB attenuated vaccine virus were not to be major determinants of the attenuation phenotype.

关 键 词：口蹄疫病毒 兔化弱毒疫苗株 ZB株 前导蛋白 点突变 

分 类 号：S852.65[农业科学—基础兽医学]