Objective To compare the efficiency of cytomegalovirus （CMV）and T7 promoters in rescuing rabies virus (RABV), improve the success rate of virus rescue and provide evidence and technical support for future research of RABV pathogenesis and vaccine development.

Methods The transcription efficiency of the two promoters in BHK-21 and BSR-T7/5 cells were evaluated, respectively. Then, four minigenome (MG) systems with different promoter combinations were generated by cross-combining MG and helper plasmids of RABV ERA-M strain, each of which was constructed to be promoted by either the CMV or T7 promoter. After transfection, the intensity and distribution of enhanced green fluorescent protein (eGFP) fluorescence were used as criteria for the preliminary screening of efficient promoter combination. Subsequently, full-length infectious clones promoted by CMV or T7 promoters were constructed, and the efficiency of RABV rescue for each promoter combination was assessed based on virus RNA level, N protein expression level, and virus titer.

Results In vitro experiments showed that the transcription of the target gene promoted by T7 promoter had significantly higher efficiency (P<0.05), and strong eGFP signals were only observed in the MG system containing the helper plasmids with T7 promoter. Additionally, in the RABV rescue experiments, the helper plasmids construct under the T7 promoter showed elevated levels of viral RNA and viral titers compared with those under the CMV promoter (P<0.05), as well as obviously higher N protein expression levels. Finally, the virus titer in the dual T7 promoter combination group was higher than that in the other groups on day 9 after transfection (P<0.001).

Conclusion In the BSR-T7/5 cell line, the T7 promoter demonstrated a strong advantage in transcriptional efficiency, and the dual T7 promoter combination represents a superior strategy for efficient RABV rescue. These findings provide a solid foundation for further research into RABV pathogenesis and the development of novel vaccines.