Rapid reconstruction of infectious bronchitis virus expressing fluorescent protein from its nsp2 gene based on transformation-associated recombination platform

作者：Yingfei Li, Linqing Duan, Lihua Tang, Min Huang, Ye Zhao, Guozhong Zhang, Jing Zhao · 发表于：Journal of Virology · 年份：2025 · DOI：10.1128/jvi.00535-25 · 被引用次数：9 · 研究领域：Animal Virus Infections Studies、Virus-based gene therapy research、SARS-CoV-2 and COVID-19 Research

ABSTRACT 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 ...