马立克病病毒meq基因缺失毒株HN302Δmeq的构建及致病性分析  

Generation and pathogenicity analysis of a meq-deleted mutant HN302ΔMeq of Marek's disease virus

作　　者：张志会 滕蔓[1,3] 刘金玲 郑鹿平 王伟东 张文凯 张多 李林燕 张卓 柴书军 樊剑鸣[2] 罗俊 ZHANG Zhihui;TENG Man;LIU Jinling;ZHENG Luping;WANG Weidong;ZHANG Wenkai;ZHANG Duo;LI Linyan;ZHANG Zhuo;CHAI Shujun;FAN Jianming;LUO Jun(Institute of Animal Health&UK-China Centre of Excellence for Research on Avian Diseases,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;College of Public Health,Zhengzhou University,Zhengzhou 450001,China;Longhu Laboratory,Zhengzhou 450046,China;Laboratory of Functional Microbiology and Animal Health,College of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471003,China)

机构地区：[1]河南省农业科学院动物疫病防控研究所中英禽病国际研究中心,河南郑州450002 [2]郑州大学公共卫生学院,河南郑州450001 [3]龙湖现代免疫实验室,河南郑州450046 [4]河南科技大学动物科技学院功能微生物与畜禽健康实验室,河南洛阳471003

出　　处：《畜牧与兽医》2024年第10期71-79,共9页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(U21A20260);国家重点研发计划(2023YFE0106100);河南省杰出青年科学基金(232300421009);河南省农业科学院自主创新项目(2024ZC096)。

摘　　要：马立克病(MD)是由马立克病病毒(MDV)感染引起的一种重要的家禽免疫抑制病与肿瘤病,严重危害世界养禽业。随着MDV持续进化及毒力增强,急需研发新型高效的MD疫苗以加强该病的有效防控。本研究利用CRISPR/Cas9基因编辑技术,以MDV变异株HN302为亲本毒株,成功构建了meq基因缺失的毒株HN302Δmeq,经过PCR扩增鉴定及测序分析,证实meq基因缺失且传代稳定。间接免疫荧光试验(IFA)和实时荧光定量PCR(RT-qPCR)分析均证实HN302Δmeq感染鸡胚成纤维细胞(CEF)中无meq基因表达,并且meq基因的缺失不影响其他病毒基因的表达。利用RT-qPCR分析HN302Δmeq在CEF中的体外增殖曲线,结果显示meq基因的缺失不影响MDV体外复制。1日龄SPF鸡攻毒试验结果显示,与亲本毒株HN302相比,HN302Δmeq对宿主无致病性,其诱导的MD发病率、死亡率和肿瘤发生率均为0,且未观察到明显的免疫器官萎缩。上述数据表明,HN302Δmeq的毒力明显丧失,具备继续作为疫苗候选株开展相关研究的可能性。本研究成功构建了MDV变异株HN302的meq基因缺失毒株,为后续MDV的致病机制及新型疫苗研究奠定了基础。Marek disease(MD),caused by Marek's disease virus(MDV),is one of the most serious avian immunosuppressive and neoplastic diseases,and it brings damage to the healthy development of global poultry industry.Due to the persistent evolution and increasing virulence of MDV,it is urgent to develop novel and high efficient MD vaccines against this disease.Herein,using the CRISPR/Cas9-based gene editing technique,we successfully constructed a meq-deleted MDV mutant named HN302Δmeq using the highly virulent MDV Chinese variant HN302 as a parental virus.The stable deletion of the meq gene from the viral genome of HN302Δmeq was confirmed by both of polymerase chain reaction(PCR)amplification and subsequent DNA sequencing.Indirect immunofluorescence assay(IFA)and reverse transcriptional realime quantitative PCR(RT-qPCR)analysis also confirmed that the Meq protein did not express in HN302Δmeq-infected CEFs,and the deletion of meq did not affect the expression of other MDV-1 genes.The in vitro proliferation curve of the virus determined by qPCR showed that deletion of meq did not affect virus replication.Animal experiments on 1-day-old SPF chickens showed that,compared with the parental HN302,the pathogenicity of HN302Δmeq had been significantly weakened,and all of the cumulative morbidity,mortality,and tumor incidence of MD in virus-challenged birds were 0,without any serious atrophy of immune organs.These data indicated that,compared wwith HN302,the virulence of HN302Δmeq was significantly omitted and it might possible to be used for future studies on MD vaccine candidate strains.In conclusion,we successfully constructed a meq-deleted mutant of the virulent MDV Chinese variant,of which the virulence was significantly eliminated;and this study might serve as an important basis for future research on MDV pathogenesis and development of novel MD vaccines.

关 键 词：鸡 马立克病病毒 MEQ基因 CRISPR/Cas9 基因编辑 致病性 

分 类 号：S852[农业科学—基础兽医学]