3个非复制型痘苗病毒基因修饰株细胞生物学特性及小鼠体内毒力比较  

Biological characteristics in vitro and mouse toxicities differences among three attenuated poxvirus vaccine candidates

作　　者：豆亚美 任皎[1] 赵莉[1] 袁航 孟昕[1] 田厚文[1] 王文玲[1] 谭文杰[1] Dou Yamei;Ren Jiao;Zhao Li;Yuan Hang;Meng Xin;Tian Houwen;Wang Wenling;Tan Wenjie(NHC Key Laboratory of Medical Virology and Viral Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Beijing Institute of Biological Products CO.,LTD,Beijing 101100,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所、国家卫生健康委员会医学病毒与病毒病重点实验室,北京102206 [2]北京生物制品研究所有限责任公司,北京101100

出　　处：《中华实验和临床病毒学杂志》2023年第2期146-151,共6页Chinese Journal of Experimental and Clinical Virology

基　　金：国家重点研发计划(2021YFC2300101)。

摘　　要：目的通过对三个非复制型痘苗病毒修饰株的细胞生物学特性及小鼠体内毒力对比研究,为天花/猴痘疫苗替代产品的研制提供参考依据。方法在BHK-21/CEF中对复制型痘苗病毒天坛株(Vaccinia virus Tiantan strain,VTT)和复制缺陷型痘苗天坛株(non-replicating Tiantan Vaccinia virus strain,NTV)及其修饰株NTV-C7L、NTV-△F1L-C7L、NTV-K1L进行扩增纯化及Western blot鉴定后,采用免疫噬斑实验对各病毒株在细胞中的毒力和扩散能力进行评价;通过复制动力学曲线比较各毒株之间的复制差异,采用小鼠滴鼻实验进行体重变化观察,比较病毒的毒力水平。结果Western blot结果证明扩增纯化的痘苗病毒各毒株均正确。免疫噬斑及复制动力学曲线表明三株NTV基因修饰株在CEF中的复制能力与NTV相近;在Vero细胞间的扩散能力和复制能力均有所提高,但复制倍数都小于100倍;在MRC-5中复制水平与NTV相比得到明显增强,其中NTV-C7L的复制倍数达20000倍以上;小鼠体内毒力结果显示三个NTV基因修饰株与NTV相比体重变化无统计学意义。结论三个NTV基因修饰株在人源性细胞MRC-5中恢复了复制能力,但在小鼠中的毒力与NTV相近,具备了作为天花/猴痘疫苗换代产品候选株的初步条件。Objective Through the study of the cell biological characteristics and virulence in mice in vivo of three non-replicating vaccinia virus modified strains to provide reference for the development of smallpox/monkeypox vaccine replacement products.Methods Replicating vaccinia virus Tiantan strain VTT and non-replicating Tiantan Vaccinia Strain NTV were studied in BHK-21/CEF and its modified strains NTV-C7L,NTV-△F1L-C7L,NTV-K1L were amplified,purified,and identified by Western blotting.The virulence and diffusivity of each strain in cells were evaluated by immune-plaque assay.The replication dynamics curves were used to compare the replication differences between the strains,and the weight loss was observed by intranasal route in the mouse model to compare the virulence levels of the viruses.Results In this study,Western blotting result proved that the amplified and purified vaccinia virus strains were correct.Immunophagocytosis and replication kinetics showed that the replication capacity of the three NTV modified strains in CEF was similar to that of NTV.The diffusion ability and replication ability between Vero cells were improved,but the replication multiple was less than 100 times.The replication level of MRC-5 was significantly enhanced compared with that of NTV,and the replication ratio of NTV-C7L was more than 20000 times.The virulence in mice showed that the body weight of the three NTV modified strains had no statistical significance compared with that of NTV.Conclusions The three NTV modified strains recovered their replication ability in human MRC-5 cells,but their virulence in mice was similar to that of NTV,which provided the preliminary conditions for being candidate strains of smallpox/monkeypox vaccine replacement products.

关 键 词：痘苗病毒 复制缺陷 天花/猴痘 毒力 疫苗 

分 类 号：R392[医药卫生—免疫学]