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

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...

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Autores principales: Yan, Bingyu, Zou, Xiaohui, Liu, Xinglong, Zhao, Jiaming, Zhang, Wenfeng, Guo, Xiaojuan, Wang, Min, Lv, Yingtao, Lu, Zhuozhuang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150739/
https://www.ncbi.nlm.nih.gov/pubmed/32168853
http://dx.doi.org/10.3390/v12030301
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author Yan, Bingyu
Zou, Xiaohui
Liu, Xinglong
Zhao, Jiaming
Zhang, Wenfeng
Guo, Xiaojuan
Wang, Min
Lv, Yingtao
Lu, Zhuozhuang
author_facet Yan, Bingyu
Zou, Xiaohui
Liu, Xinglong
Zhao, Jiaming
Zhang, Wenfeng
Guo, Xiaojuan
Wang, Min
Lv, Yingtao
Lu, Zhuozhuang
author_sort Yan, Bingyu
collection PubMed
description 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.
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spelling pubmed-71507392020-04-20 User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome Yan, Bingyu Zou, Xiaohui Liu, Xinglong Zhao, Jiaming Zhang, Wenfeng Guo, Xiaojuan Wang, Min Lv, Yingtao Lu, Zhuozhuang Viruses Article 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. MDPI 2020-03-11 /pmc/articles/PMC7150739/ /pubmed/32168853 http://dx.doi.org/10.3390/v12030301 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Yan, Bingyu
Zou, Xiaohui
Liu, Xinglong
Zhao, Jiaming
Zhang, Wenfeng
Guo, Xiaojuan
Wang, Min
Lv, Yingtao
Lu, Zhuozhuang
User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome
title User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome
title_full User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome
title_fullStr User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome
title_full_unstemmed User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome
title_short User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome
title_sort user-friendly reverse genetics system for modification of the right end of fowl adenovirus 4 genome
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150739/
https://www.ncbi.nlm.nih.gov/pubmed/32168853
http://dx.doi.org/10.3390/v12030301
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