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Article

User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome

1
College of Marine Science and Biological Engineering, Qingdao University of Science and Technology, Qingdao 266042, China
2
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
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Department of Laboratory Medicine, School of Public Health and Management, Weifang Medical University, Weifang 261053, China
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Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan 430071, China
5
Chinese Center for Disease Control and Prevention-Wuhan Institute of Virology, Chinese Academy of Sciences Joint Research Center for Emerging Infectious Diseases and Biosafety, Wuhan 430071, China
*
Authors to whom correspondence should be addressed.
Viruses 2020, 12(3), 301; https://doi.org/10.3390/v12030301
Received: 7 February 2020 / Revised: 29 February 2020 / Accepted: 9 March 2020 / Published: 11 March 2020
(This article belongs to the Section Animal Viruses)
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. View Full-Text
Keywords: fowl adenovirus; reverse genetics system; Gibson assembly; restriction enzyme; essential gene fowl adenovirus; reverse genetics system; Gibson assembly; restriction enzyme; essential gene
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MDPI and ACS Style

Yan, B.; Zou, X.; Liu, X.; Zhao, J.; Zhang, W.; Guo, X.; Wang, M.; Lv, Y.; Lu, Z. User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome. Viruses 2020, 12, 301. https://doi.org/10.3390/v12030301

AMA Style

Yan B, Zou X, Liu X, Zhao J, Zhang W, Guo X, Wang M, Lv Y, Lu Z. User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome. Viruses. 2020; 12(3):301. https://doi.org/10.3390/v12030301

Chicago/Turabian Style

Yan, Bingyu, Xiaohui Zou, Xinglong Liu, Jiaming Zhao, Wenfeng Zhang, Xiaojuan Guo, Min Wang, Yingtao Lv, and Zhuozhuang Lu. 2020. "User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome" Viruses 12, no. 3: 301. https://doi.org/10.3390/v12030301

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