Construction and modification of a low-copy plasmid-based infectious clone for GI-19 genotype IBV via Red/ET...

Avian infectious bronchitis virus (IBV), a Gamma coronavirus (γ-CoV), is one of the most economically important pathogens affecting the global poultry industry. In this study, we established a novel reverse genetics platform of infectious clone of GI-19 genotype IBV. This platform integrates low-copy plasmid vectors with Red/ET recombineering technology, combined with positive and counter-selection strategies, providing a streamlined and efficient workflow for genome cDNA assembly, precise genetic modification, and rapid screening of recombinant viruses. Specifically, we developed a one-step method for the assembly of multiple segments through RecE/RecT-mediated recombination using a p15A-CmR low-copy plasmid vector. Applying this system, an infectious clone of the GI-19 genotype IBV, designated p15A-CmR-D90 was constructed and rescued successfully. This strategy significantly improves cloning stability and operational flexibility, enabling seamless assembly of full-length IBV genome cDNA. Furthermore, we established a precise and high-efficiency strategy for targeted genetic modification of the IBV infectious clone, based on a two-step Redα/Redβ-mediated recombination with both positive and counter selection. This approach enables accurate mutation, insertion, deletion, or replacement of genetic elements within the IBV genome, supporting fine-tuned genetic manipulation. The robustness of the platform was demonstrated through the successful generation and rescue of a recombinant virus, rD90-∆5a/EGFP, in which the non-essential 5a gene of the D90 strain was replaced by enhanced green fluorescent protein (EGFP). Collectively, the method and strategy established in this study provide a versatile and scalable technical foundation for IBV and CoVs reverse genetics and recombinant vaccine development.