Construction of rabies virus mutant strain rRC-HL(GX074P1M1)and its biological characteristics
[Objective]The objective of this study was to construct the rabies virus(RABV)mutant strain rRC-HL(GX074P1M1)and investigate its biological characteristics,analyzed the impact of combined mutations in the phospho-protein(P)and matrix protein(M)regions on RABV transcription and replication levels,in order to study the patho-genic mechanisms of RABV and provide theoretical basis for targeted prevention and treatment.[Method]Using reverse genetics technology,the P protein P1 region(amino acids at positions 48-78)and M protein M1 region(amino acids at positions 1-22)of the RABV street strain GX074 were co-embedded into the corresponding positions of the attenuated strain RC-HL.The virus titers and the relative expression levels of the nucleoprotein(N)gene,P gene,and M gene,as well as their proteins were determined for the mutant strain and control strains[RC-HL,GX074,rRC-HL(GX074PM),and CVS-11]after infection of BSR/T7-9 cells using indirect immunofluorescence assay(IFA),real-time fluorescence quantitative PCR and Western blotting.[Result]The mutant strain rRC-HL(GX074P1M1)was successfully obtained through virus rescue.Multi-step virus growth curve assays showed that the virus titer of the mutant strain rRC-HL(GX074P1M1)was higher than that of the parental strains RC-HL and GX074 within 24-96 h post infection of BSR/T7-9 cells.Real-time fluorescence quantitative PCR revealed that the relative expression levels of the N gene,P gene and M gene of the mutant strain rRC-HL(GX074P1M1)were significantly(P＜0.05)or extremely significantly(P＜0.01,the same below)higher than those of the parental strains RC-HL and GX074 at 24 and 48 h post infection.Western blotting results indicated that at 24 h post infection,the relative expression levels of N protein,P protein and M proteins in the mu-tant strain rRC-HL(GX074P1M1)were slightly higher than those in the parental strain RC-HL.At 48 h post infection,the relative expression levels of N protein,P protein and M proteins in the mutant strain rRC-HL(GX074P1M1)were ex-tremely significantly higher than those in the parental strains RC-HL and GX074.[Conclusion]The mutant strain rRC-HL(GX074P1M1),obtained by co-embedding the P1 and M1 regions of the RABV street strain GX074 into the attenuated strain RC-HL,exhibits higher replication and transcription levels than the parental strains,indicating a stronger intracellu-lar proliferation capability.This suggests that the P protein P1 region and M protein M1 region of the street strain GX074 play synergistic effects in promoting viral transcription and replication.
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