Reverse genetics strategies for coronaviruses: platform construction and applications in vaccine development

The continuous emergence of novel coronaviruses, characterized by high mutation rates and frequent recombination events, poses severe threats to global "One Health." Notably, the recent outbreak of the recombinant feline coronavirus (FCoV-23) and the persistence of SARS-CoV-2 variants underscore the urgent need to understand viral pathogenesis and cross-species transmission mechanisms. Reverse genetics technology serves as a critical platform for bridging genomic sequencing to functional virology, enabling targeted mutagenesis and the generation of recombinant viruses. However, the construction of reverse genetics systems for coronaviruses is often hampered by their exceptionally large genomes and the instability of viral cDNA sequences in bacterial hosts due to cytotoxicity. This review moves beyond a simple enumeration of methods to systematically compare current reverse genetics strategies-including in vitro ligation, bacterial artificial chromosome (BAC) systems, and transformation-associated recombination (TAR)-across different viral genera. Furthermore, we critically evaluate the application of these platforms in deciphering pathogenic mechanisms and developing next-generation vaccines, with a specific focus on overcoming technical bottlenecks and designing broad-spectrum countermeasures against emerging cross-species threats.