User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome
Bingyu Yan,
Xiaohui Zou,
Xinglong Liu,
Jiaming Zhao,
Wenfeng Zhang,
Xiaojuan Guo,
Min Wang,
Yingtao Lv,
Zhuozhuang Lu
Affiliations
Bingyu Yan
College of Marine Science and Biological Engineering, Qingdao University of Science and Technology, Qingdao 266042, China
Xiaohui Zou
State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China
Xinglong Liu
College of Marine Science and Biological Engineering, Qingdao University of Science and Technology, Qingdao 266042, China
Jiaming Zhao
State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China
Wenfeng Zhang
State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China
Xiaojuan Guo
State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China
Min Wang
State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China
Yingtao Lv
College of Marine Science and Biological Engineering, Qingdao University of Science and Technology, Qingdao 266042, China
Zhuozhuang Lu
State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China
A novel fowl adenovirus 4 (FAdV-4) has caused significant economic losses to the poultry industry in China since 2015. We established an easy-to-use reverse genetics system for modification of the whole right and partial left ends of the novel FAdV-4 genome, which worked through cell-free reactions of restriction digestion and Gibson assembly. Three recombinant viruses were constructed to test the assumption that species-specific viral genes of ORF4 and ORF19A might be responsible for the enhanced virulence: viral genes of ORF1, ORF1b and ORF2 were replaced with GFP to generate FAdV4-GFP, ORF4 was replaced with mCherry in FAdV4-GFP to generate FAdV4-GX4C, and ORF19A was deleted in FAdV4-GFP to generate FAdV4-CX19A. Deletion of ORF4 made FAdV4-GX4C form smaller plaques while ORF19A deletion made FAdV4-CX19A form larger ones on chicken LMH cells. Coding sequence (CDS) replacement with reporter mCherry demonstrated that ORF4 had a weak promoter. Survival analysis showed that FAdV4-CX19A-infected chicken embryos survived one more day than FAdV4-GFP- or FAdV4-GX4C-infected ones. The results illustrated that ORF4 and ORF19A were non-essential genes for FAdV-4 replication although deletion of either gene influenced virus growth. This work would help function study of genes on the right end of FAdV-4 genome and facilitate development of attenuated vaccines.
