Rapid Rescue of Goose Astrovirus Genome via Red/ET Assembly

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.