Li Yingfei, Duan Linqing, Tang Lihua, Huang Min, Zhao Ye, Zhang Guozhong, Zhao Jing
National Key Laboratory of Veterinary Public Health Security, College of Veterinary Medicine, China Agricultural University, Beijing, China.
Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China.
J Virol. 2025 Jul 22;99(7):e0053525. doi: 10.1128/jvi.00535-25. Epub 2025 Jun 5.
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 for seamless assembly, we obtained a stable full-length genome clone with an efficiency exceeding 95%. Optimizing cultivation temperature and selecting appropriate 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.
Traditional reverse genetics systems for infectious bronchitis virus (IBV) are often hindered by assembly difficulties 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.
用于大基因组RNA病毒的传统反向遗传学策略受到多个技术瓶颈的阻碍，包括对特定限制酶切位点的依赖、繁琐的多步克隆以及细菌系统中过大DNA的遗传不稳定性。在此，我们通过对转化相关重组（TAR）技术进行系统优化，建立了一种用于传染性支气管炎病毒（IBV）的通用反向遗传学平台。通过将IBV基因组划分为七个重叠片段并采用无缝组装，我们获得了一个稳定的全长基因组克隆，效率超过95%。优化培养温度并选择合适的菌株是关键改进措施，可最大程度减少细菌复制过程中的诱变，确保构建体的保真度。拯救出的QX型IBV毒株在鸡胚中的复制和致病性与临床分离株相当，同时保留了工程标记且无额外突变。成功拯救出Mass型IBV毒株进一步证实了该平台的可行性，表明其具有广泛的适用性。值得注意的是，我们首次拯救出了一种表达mNeonGreen荧光蛋白的报告病毒，该蛋白通过猪捷申病毒1 2A蛋白酶切割位点连接，位于IBV nsp2基因上游紧邻处。这种设计使得报告基因能够从病毒多聚蛋白中自主分离，而无需删除任何病毒基因。重组病毒至少连续传代10次仍稳定保持这种插入，将nsp2位点标记为IBV中容纳外源基因的灵活位点。本研究不仅为γ冠状病毒建立了基于TAR的通用反向遗传学平台，还为基于可视化的高通量抗病毒药物筛选提供了有力工具。
传染性支气管炎病毒（IBV）的传统反向遗传学系统常常受到组装困难以及细菌繁殖过程中病毒基因组不稳定性的阻碍。在此，我们开发了一种基于转化相关重组的平台，用于IBV基因组的无缝组装，并在12天内快速拯救病毒。此外，我们在病毒基因组的5'UTR和nsp2之间鉴定出一个新的外源基因插入位点，能够在不删除任何病毒基因的情况下稳定表达荧光蛋白，确保病毒复制不受影响。该系统为追踪IBV感染、研究病毒嗜性和筛选抗病毒药物提供了有力工具，从而推动冠状病毒研究和家禽疾病防控。
