Abstract

The host-specific infection of Avian Astrovirus (AAstVs) has posed significant challenges to the poultry industry, resulting in substantial economic losses. However, few reports exist on the functional consequences of genome diversity, cross-species infectivity and mechanisms governing virus replication of AAstVs, making it difficult to develop measures to control astrovirus transmission. Reverse genetics technique can be used to study the function of viruses at the molecular level, as well as investigating pathogenic mechanisms and guide vaccine development and disease treatment. Herein, the reverse genetics technique of goose astrovirus GAstV/JS2019 strain was developed based on use of a reconstructed vector including CMV promotor, hammerhead ribozyme (HamRz), hepatitis delta virus ribozyme (HdvRz), and SV40 tail, then the cloned viral genome fragments were connected using Red/ET recombineering. The recombinant rGAstV-JS2019 was readily rescued by transfected the infectious clone plasmid into LMH cells. Importantly, the rescued rGAstV/JS2019 exhibited similar growth kinetics comparable to those of the parental GAstV/JS2019 isolate in cultured cells. Our research results provide an alternative and more effective reverse genetic tool for a detailed understanding of viral replication, pathogenic mechanisms, and molecular mechanisms of evolution.

中文翻译：

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通过 Red/ET 组装快速拯救鹅星状病毒基因组

摘要

禽星状病毒（AAstVs）的宿主特异性感染给家禽业带来了重大挑战，造成了巨大的经济损失。然而，关于基因组多样性、跨物种感染性和控制 AAstV 病毒复制机制的功能性后果的报道很少，这使得制定控制星状病毒传播的措施变得困难。反向遗传学技术可用于在分子水平上研究病毒的功能，以及研究致病机制并指导疫苗开发和疾病治疗。本文利用包含CMV启动子、锤头核酶(HamRz)、丁型肝炎病毒核酶(HdvRz)和SV40尾巴的重建载体，开发了鹅星状病毒GAstV/JS2019株的反向遗传学技术，然后克隆了病毒基因组片段。使用 Red/ET 重组工程进行连接。通过将感染性克隆质粒转染至LMH细胞中，重组rGAstV-JS2019很容易被拯救。重要的是，拯救的 rGAstV/JS2019 在培养细胞中表现出与亲本 GAstV/JS2019 分离株相似的生长动力学。我们的研究结果为详细了解病毒复制、致病机制和进化分子机制提供了一种替代的、更有效的反向遗传工具。