ArticlePoultry science2026
Construction and modification of a low-copy plasmid-based infectious clone for GI-19 genotype IBV via Red/ET recombineering: A simplified and efficient reverse genetics system for coronavirus.
Article in Poultry science, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
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.
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