PDF(Pubmed)
Abstract：
Avian infectious bronchitis is an acute respiratory disease of poultry of particular concern for global food security. Investigation of infectious bronchitis virus (IBV), the causative agent of avian infectious bronchitis, via reverse genetics enables deeper understanding of virus biology and a rapid response to emerging variants. Classic methods of reverse genetics for IBV can be time consuming, rely on recombination for the introduction of mutations, and, depending on the system, can be subject to genome instability and unreliable success rates. In this study, we have applied data-optimized Golden Gate Assembly design to create a rapidly executable, flexible, and faithful reverse genetics system for IBV. The IBV genome was divided into 12 fragments at high-fidelity fusion site breakpoints. All fragments were synthetically produced and propagated in E. coli plasmids, amenable to standard molecular biology techniques for DNA manipulation. The assembly can be carried out in a single reaction, with the products used directly in subsequent viral rescue steps. We demonstrate the use of this system for generation of point mutants and gene replacements. This Golden Gate Assembly-based reverse genetics system will enable rapid response to emerging variants of IBV, particularly important to vaccine development for controlling spread within poultry populations.
摘要：
禽传染性支气管炎是家禽的急性呼吸道疾病，对全球粮食安全特别关注。传染性支气管炎病毒（IBV）的调查，禽类传染性支气管炎的病原体,通过反向遗传学可以更深入地了解病毒生物学和对新出现的变异的快速反应.IBV反向遗传学的经典方法可能很耗时，依靠重组来引入突变，and,取决于系统，可能会受到基因组不稳定和不可靠的成功率的影响。在这项研究中,我们应用了数据优化的金门装配设计来创建一个快速可执行的，灵活,和忠实的IBV反向遗传学系统。在高保真融合位点断点处将IBV基因组分成12个片段。所有片段都是在大肠杆菌质粒中合成和繁殖的，适用于DNA操作的标准分子生物学技术。组装可以在单个反应中进行，产品直接用于随后的病毒救援步骤。我们演示了该系统用于生成点突变体和基因替换的用途。这种基于金门组装的反向遗传学系统将能够对IBV的新兴变体做出快速反应，对于控制家禽种群内传播的疫苗开发尤为重要。
