Application of the CPER reverse genetics system for genetic engineering of rabies virus

Rabies virus is a mononegavirus, and plasmid-based reverse genetics systems have been widely used for its manipulation. Reporter rabies viruses generated by reverse genetics are useful for visualization of neuronal circuits. However, construction of full-length genome plasmids requires considerable time and cost, and introduction of artificial mutations is difficult. Recently, CPER has been established as a novel reverse genetics method, although its application to mononegaviruses remains limited. In this study, a CPER-based reverse genetics system for rabies virus was established. Infectious viruses rescued by CPER showed growth kinetics similar to those of parental strain. Mutant viruses containing point mutations, reporter viruses expressing fluorescent proteins, and chimeric viruses with exchanged G proteins were successfully generated. Full-genome sequencing revealed relatively frequent mutations in CPER-derived viruses. In addition, some strains could not be rescued, and PCR amplification introduced mutations into the viral genome.
(AI)