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

  • 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。

     

    Abstract: 【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 4th day 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】The combined substitution of P gene and M gene of strong strain GX01 can improve the transmission ability of the virus and that the two were synergistic,and it has no effect on the virulence of the weak strain rRC-HL.

     

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