: Traditional reverse genetics strategies for large-genome RNA viruses are hindered by multiple technical bottlenecks, including dependence on specific restriction enzyme sites, cumbersome multi-step cloning, and genetic instability of oversized DNA in bacterial systems. Herein, we established a universal reverse genetics platform for infectious bronchitis virus (IBV) through systematic optimization of the transformation-associated recombination (TAR) technology. By dividing the IBV genome into seven overlapping fragments and employing Saccharomyces cerevisiae for seamless assembly, we obtained a stable full-length genome clone with an efficiency exceeding 95%. Optimizing cultivation temperature and selecting appropriate Escherichia coli strains were key improvements that minimized mutagenesis during bacterial replication, ensuring fidelity of the constructs. The rescued QX-type IBV strain replicated and induced pathogenicity in chicken embryos comparably to clinical isolates, while retaining engineered markers without additional mutations. The platform's feasibility was further confirmed by successfully rescuing the Mass-type IBV strain, demonstrating its broad applicability. Notably, we pioneeringly rescued a reporter virus expressing the mNeonGreen fluorescent protein, linked via the porcine teschovirus 1 2A proteolytic cleavage site, immediately upstream of the IBV nsp2 gene. This design enabled autonomous separation of the reporter from viral polyproteins without the deletion of any viral gene. The recombinant virus stably maintained this insertion for at least 10 passages, marking the nsp2 site as a flexible locus for foreign gene accommodation in IBV. This study not only establishes a universal TAR-based reverse genetics platform for gamma-coronaviruses but also provides a powerful tool for visualization-based high-throughput antiviral drug screening.

Objective: Traditional reverse genetics systems for infectious bronchitis virus (IBV) are often hindered by assembly difficulties in vitro and viral genome instability during bacterial propagation. Here, we developed a transformation-associated recombination-based platform for seamless IBV genome assembly and rapid virus rescue within 12 days. Additionally, we identified a novel foreign gene insertion site between the 5' UTR and nsp2 in the viral genome, enabling stable fluorescent protein expression without deleting any viral genes, ensuring that virus replication is not affected. This system provides a powerful tool for tracking IBV infection, studying viral tropism, and screening antivirals, thereby advancing coronavirus research and poultry disease control.