Recombinant virus recovery.

recVSV-ΔG-eGFP-G_RABV variants were recovered following a previously described protocol (84). Briefly, expression plasmids encoding VSV-N (0.5 μg plasmid DNA), VSV-P (0.4 μg plasmid DNA), and VSV-L (0.2 μg plasmid DNA) were cotransfected with plasmids containing the cDNA copies of parental recVSV-ΔG-eGFP-G_RABV or recVSV-ΔG-eGFP-G_RABV with mutant RABV G (1.0 μg plasmid DNA) into BSR-T7/5 cells (2.5 × 10⁵ cells/well) stably expressing T7 polymerase. Transfected cells were incubated at 37°C and monitored microscopically for the appearance of green fluorescence. Culture supernatants containing recovered progeny virions were transferred to Vero-E6 cells for generation of a P₀ stock. After amplification, cleared supernatants containing stocks of recovered viruses were stored in aliquots at −80°C, titers were determined by TCID₅₀ titration using eGFP fluorescence as the readout, and the integrity of the G protein-encoding open reading frame was verified through reverse transcription-PCR (RT-PCR) and Sanger sequencing.