Development, applications, and future prospects of RNA virus reverse genetics technology

Abstract

Viral reverse genetics enables the rescue of infectious virions from cloned cDNA and serves as a core technique for mapping viral genotype–phenotype relationships, dissecting RNA viral life cycles, and developing antiviral countermeasures. This review systematically summarizes the evolution, technical framework and optimization strategies of RNA virus reverse genetics, with three prototype viruses covering all major RNA genome types: SARS-CoV-2 (+ssRNA), non-segmented negative-sense Newcastle disease virus (NDV), and segmented negative-sense influenza A virus. Core modules including infectious clone construction, diverse promoter systems, hammerhead/HDV ribozymes, and solutions for large unstable genomes such as BAC and ISA are elaborated. We summarize its irreplaceable applications in vaccine development, pathogenesis research, virus-host interaction analysis and high-throughput antiviral screening. Current bottlenecks include low rescue efficiency, cDNA genetic instability and biosafety hazards. We also introduce non-infectious surrogate platforms and establish a three-tier antiviral screening pipeline. Future advances will integrate CRISPR/Cas editing, standardized modular tools, biosafety engineering, AI and big data. Distinct from previous reviews focusing on single viral genera, this work conducts cross-type horizontal comparisons and summarizes universal technical obstacles and tailored optimizations, offering comprehensive references for basic virology, accelerated vaccine innovation and precise antiviral design.