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 phosphoprotein（P）and matrix protein（M）regions on RABV transcription and replication levels，in order to study the pathogenic 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 mutant 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 extremely 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 intracellular 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.