马立克病病毒特超强变异株meq基因缺失株构建及作为疫苗候选株的鉴定  

Generation and Characterization of A Candidate MD Vaccine Strain with Meqdeletions Derived from A Hypervirulent Variant of the Marek's Disease Virus

作　　者：张卓 樊剑鸣[1] 滕蔓[2,3] 张多 刘金玲 郑鹿平 赵东 各思雨[1,2,3] 韩放 罗琴 柴书军 罗俊 ZHANG Zhuo;FAN Jianming;TENG Man;ZHANG Duo;LIU Jinling;ZHENG Luping;ZHAO Dong;GE Siyu;HAN Fang;LUO Qin;CHAI Shujun;LUO Jun(College of Public Health,Zhengzhou University,Zhengzhou 450001,China;Institute for Animal Health&UK-China Centre of Ercellence for Research on Avian Diseases,Henan Academy of Agricultural Sciences,Zhengzhou 450002,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;College of Veterinary Medicine,Henan University of Animal Husbandry andEconomy,Zhengzhou450046,China)

机构地区：[1]郑州大学公共卫生学院,郑州450001 [2]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,郑州450002 [3]龙湖现代免疫实验室,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,洛阳471003 [5]河南牧业经济学院动物医药学院,郑州450046

出　　处：《病毒学报》2024年第6期1341-1353,共13页Chinese Journal of Virology

基　　金：国家重点研发计划(项目号:2023YFE0106100),题目:中英禽病防控技术创新联合实验室;国家自然科学基金(项目号:U21A20260),题目:鸡马立克病病毒遗传进化与新型疫苗毒株设计的分子基础;河南省杰出青年科学基金(项目号:232300421009),题目:家禽免疫抑制病与肿瘤病流行病学研究;河南省农业科学院自主创新项目(项目号:2024ZC096)题目:鸡马立克病基因缺失疫苗株的表型鉴定和免疫评价。

摘　　要：马立克病(Marek’s disease,MD)是一种严重危害家禽健康养殖的免疫抑制病与肿瘤病,由致病性的血清1型MDV(Marek’s disease virus serotype 1,MDV-1)毒株感染引起。近年来,即使在已接种疫苗的鸡群中,MD病例的发生及危害在全球范围内仍呈不断上升趋势。此前研究发现,经典的MD疫苗已不能很好保护当前流行的MDV特超强毒株,尤其是新出现的特超强MDV(Hypervirulent MDV,HV-MDV)变异株,这意味着新一代高效的MD疫苗亟待研发。本研究中,以近期最新分离鉴定的HV-MDV变异株SDCW01为亲本毒株,首先将其在鸡胚成纤维细胞(Chicken embryo fibroblast,CEF)上连续传代以降低对宿主潜在的免疫抑制性,然后利用基于CRISPR/Cas9系统的基因编辑技术敲除致瘤基因meq,构建meq基因编辑缺失的疫苗候选毒株。经过PCR扩增鉴定、病毒克隆纯化、DNA测序、间接免疫荧光试验(IFA)、实时荧光定量PCR(RT-qPCR)及病毒体外增殖曲线测定等试验,证实获得一株meq基因完全缺失的MDV毒株,命名为SD01△meq。进一步传代培养及PCR鉴定结果显示,SD01△meq传代稳定性良好,meq基因的编辑缺失未发生回复突变,且不影响其它MDV基因的表达及体外复制能力。1日龄SPF鸡攻毒实验结果显示,与亲本毒株SDCW01相比,SD01△meq完全丧失了对宿主的免疫抑制和致病性,其诱导的MD发病率、致死率及肿瘤率均为0%。上述数据表明,本研究成功构建获得了一株meq基因编辑缺失疫苗候选毒株SD01△meq,为后续新型高效MD疫苗的研发奠定了重要基础。Marek's disease(MD)is caused by the pathogenic Marek's disease virus(MDV).MD is an important immunosuppressive and neoplastic disease that has led to serious losses to the worldwide poultry industry.In recent years,the frequency of MD outbreaks has increased significantly worldwide(even in MD-vaccinated chicken flocks).Previously,we found that widely used classical MD vaccines could no longer provide“ideal”protection against prevalent MDV strains,especially the newly emerged hypervirulent MDV(HV-MDV)variants.Hence,a new generation of highly efficacious MD vaccines must be developed urgently.We used an HV-MDV variant,SDCW01,isolated recently from tumor-bearing chickens that had been immunized with an MD vaccine,as the parental virus.It was first passaged on chicken embryonic fibroblasts for reducing its potential immunosuppression property to the host.Then,it was used for the generation of a meq gene-edited and deficient MD vaccine strain utilizing clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9-based gene editing.Subsequent experiments using polymerase chain reaction(PCR)amplification and identification,indirect immunofluorescence assay,real-time reverse transcription-quantitative PCR,and in vitro viral proliferation demonstrated that a meq gene that mutated the MDV strain completely named“SD01△meq”had been generated.Data on virus passage and from PCR analysis showed that SD01△meq was a stable virus without a reverse mutation,and that deletion of the meq gene did not affect MDV replication in vitro.Finally,challenge experiments using 1 day-old specific pathogenfree chickens showed that,compared with the parental virus SDCW01,SD01Δmeq had lost its immunosuppression,pathogenicity,and oncogenicity completely to hosts,with no deaths and absence of gross tumors.Our data indicated that a meq-deleted candidate MD vaccine strain had been generated.Our results provide an important foundation for the subsequent development of novel efficacious MD vaccines.

关 键 词：马立克病病毒 MEQ基因 CRISPR/Cas9 基因编辑 新型疫苗 

分 类 号：R373.9[医药卫生—病原生物学] S852.65[医药卫生—基础医学]