狂犬病病毒P和M基因重组突变体的生物学特性分析  

Biological characterization of recombinant mutants of P and M genes of rabies virus

作　　者：张银 金硕 栗朵朵 Abraha Bahlbi Kiflu 韩菲 武梦思 凌小清 梁晶晶[1,2] 李晓宁 罗廷荣[1,2] ZHANG Yin;JIN Shuo;LI Duo-duo;Abraha Bahlbi Kiflu;HAN Fei;WU Meng-si;LING Xiao-qing;LIANG Jing-jing;LI Xiao-ning;LUO Ting-rong(College of Animal Science and Veterinary Medicine,Guangxi University,Nanning,Guangxi 530004,China;State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,Nanning,Guangxi 530004,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]亚热带农业生物资源保护利用国家重点实验室,广西南宁530004

出　　处：《南方农业学报》2022年第9期2634-2642,共9页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31902311);广西自然科学基金项目(2020GXNSFAA297212)。

摘　　要：【目的】探索狂犬病病毒(RABV)强毒株P基因替换及P-M基因联合替换至弱毒株获得的重组突变体在宿主细胞内的传播能力、复制与转录能力及致病性,为掌握RABV强毒株P蛋白和M蛋白的生物学特性打下基础。【方法】以RABV广西街毒株GX01为研究对象,利用反向遗传技术将其P基因分别替换到弱毒株rRC-HL和r RC-HL(GX01M)株的对应位置,通过间接免疫荧光试验(IFA)及基因测序鉴定拯救的病毒,并测定重组突变体的多步生长曲线、荧光灶面积及N基因表达水平等,同时通过4周龄昆明小鼠攻毒试验验证P基因及P-M基因联合替换后对RABV致病性的影响。【结果】利用反向遗传技术能成功拯救重组突变体rRC-HL(GX01P)和rRC-HL(GX01P-M),通过IFA测定病毒荧光灶面积及多步生长曲线发现,r RC-HL(GX01P-M)株在BSR/T7-9细胞上的生长能力及传播能力均较rRC-HL(GX01P)株和rRC-HL(GX01M)株强。在复制与转录水平上,rRC-HL(GX01P)株的N基因相对表达量高于r RC-HL(GX01M)株,但低于rRC-HL(GX01P-M)株。在昆明小鼠攻毒试验中,重组突变体rRC-HL(GX01P)和rRC-HL(GX01P-M)攻毒后第4 d昆明小鼠开始出现精神不振、行动迟缓等症状,其致病性无明显差异;昆明小鼠体质量均呈一过性减轻,之后恢复正常并呈上升趋势;攻毒15 d内均未见昆明小鼠死亡,即重组突变体rRC-HL(GX01P)和rRCHL(GX01P-M)的致病性较弱。【结论】强毒株GX01的P基因与M基因联合替换可提高病毒传播能力,且二者具有协同性,但对弱毒株rRC-HL的毒力无影响。【Objective】To explore the transmission ability,replication and transcriptional capacity and pathogenicity of recombinant mutants obtained by P-gene substitution and combined P-M gene substitution of rabies virus(RABV)strong strain to weak strain in host cells,and to lay the foundation for mastering the biological properties of P and M proteins of RABV strong strain.【Method】The RABV Guangxi street strain GX01 was used as the target,and its P gene was replaced into the corresponding positions of the weak strains rRC-HL and rRC-HL(GX01M)respectively by reverse genetic technique. The rescued virus was identified by indirect immunofluorescence assay(IFA)and gene sequencing,and the multi-step growth curve,fluorescence foci area and N gene expression level of the recombinant mutant were measured,while the effect of combined replacement of P gene and P-M gene on the pathogenicity of RABV was verified by 4-week-old Kunming mice attack test.【Result】The recombinant mutants rRC-HL(GX01P)and rRC-HL(GX01P-M)could be successfully rescued using reverse genetic techniques. The viral fluorescence foci area and multi-step growth curve determined by IFA revealed that the rRC-HL(GX01P-M)strain was more capable of growth and propagation on BSR/T7-9cells than the rRC-HL(GX01P)strain and the rRC-HL(GX01M)strain. At the replication and transcription levels,the relative expression of N genes was higher in the r RC-HL(GX01P)strain than the rRC-HL(GX01M)strain but lower than the rRC-HL(GX01P-M)strain. In the attack test of Kunming mice,the recombinant mutant r RC-HL(GX01P)and rRCHL(GX01P-M)started to show signs of mental depression and slow movement at the 4thday after the attack,and there was no significant difference in their pathogenicity;the body weight of Kunming mice both showed a transient reduction,and then returned to normal and showed an increasing trend;no death was observed in Kunming mice within 15 d of the attack,i.e.,the recombinant The mutants rRC-HL(GX01P)and r RC-HL(GX01P-M)were less pathogenic.【Conclusion】

关 键 词：狂犬病病毒(RABV) P蛋白 M蛋白 联合替换 生物学特性 

分 类 号：S852.659.5[农业科学—基础兽医学]