Reverse Genetics of Orthoflaviviruses: Strategies for Constructing Functional or Infectious cDNA

Reverse genetics (RG) systems have transformed RNA virus research by enabling the recovery of recombinant viruses entirely from cloned cDNA. These platforms allow precise, hypothesis-driven manipulation of viral genomes and support systematic phenotypic analyses in vitro and in vivo. Orthoflaviviruses, a genus that includes numerous zoonotic arboviruses associated with neurological and visceral disease, present distinct challenges for RG development. RNA-launched systems, which rely on in vitro transcription of infectious RNA from a full-length functional cDNA clone, have long been the standard approach. However, the technical demands of RNA synthesis and handling have increased interest in DNA-launched systems that initiate virus recovery directly from a full-length infectious cDNA clone. Despite these advances, constructing genetically stable full-length orthoflavivirus cDNA clones remains difficult because certain genomic regions are unstable or toxic during bacterial propagation. To address these obstacles, conventional bacteria-based cloning has been supplemented with bacteria-free strategies such as long-distance reverse transcription-PCR, circular polymerase extension reaction, Gibson isothermal assembly, and infectious subgenomic amplicons. This commentary summarizes the key features, strengths, and limitations of current RNA- and DNA-launched RG systems for orthoflaviviruses, with emphasis on the technical challenges unique to this group. Together, these perspectives provide a framework for selecting and implementing RG approaches tailored to orthoflaviviruses.