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

【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 4^th 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.