Advances and challenges of reverse genetics in mosquito-borne flaviviruses: focus on Japanese encephalitis virus

Mosquito-borne flaviviruses such as Japanese encephalitis virus (JEV), Zika virus, and dengue virus pose a significant threat to public health.Among these, JEV remains endemic across Asia, with the recent emergence of genotype V raising concerns about antigenic divergence and potential vaccine escape.Reverse genetics (RG), which enables precise engineering of viral genomes from cloned cDNA, has become an indispensable tool for studying viral pathogenesis, antigenicity, and immune evasion.This review focuses on the application of RG in flavivirus research, particularly in the context of JEV.We highlight how RG platforms have facilitated the development of infectious clones, the evaluation of genotype-specific vaccine responses, and the functional dissection of viral genes.While technical challenges such as genome instability remain, recent advances in cloning strategies are enhancing the stability and reproducibility of RG systems for flaviviruses.As these technologies continue to evolve, RG will play a central role in elucidating the pathogenic mechanisms of emerging viral genotypes and guiding the development of targeted vaccines and antiviral strategies.Ultimately, RG provides a critical framework that bridges basic virology and translational applications for the prevention and control of mosquitoborne viral diseases.