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Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain

The infectious disease caused by the duck Tembusu virus (DTMUV) has resulted in massive economic losses to the Chinese duck industry in China since 2010. Research on the molecular basis of DTMUV pathogenicity has been hampered by the lack of a reliable reverse genetics system for this virus. Here we...

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Autores principales: Wu, Xiaogang, Shi, Ying, Yan, Dawei, Li, Xuesong, Yan, Pixi, Gao, Xuyuan, Zhang, Yuee, Yu, Lei, Ren, Chaochao, Li, Guoxin, Yan, Liping, Teng, Qiaoyang, Li, Zejun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889061/
https://www.ncbi.nlm.nih.gov/pubmed/27248497
http://dx.doi.org/10.1371/journal.pone.0156579
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author Wu, Xiaogang
Shi, Ying
Yan, Dawei
Li, Xuesong
Yan, Pixi
Gao, Xuyuan
Zhang, Yuee
Yu, Lei
Ren, Chaochao
Li, Guoxin
Yan, Liping
Teng, Qiaoyang
Li, Zejun
author_facet Wu, Xiaogang
Shi, Ying
Yan, Dawei
Li, Xuesong
Yan, Pixi
Gao, Xuyuan
Zhang, Yuee
Yu, Lei
Ren, Chaochao
Li, Guoxin
Yan, Liping
Teng, Qiaoyang
Li, Zejun
author_sort Wu, Xiaogang
collection PubMed
description The infectious disease caused by the duck Tembusu virus (DTMUV) has resulted in massive economic losses to the Chinese duck industry in China since 2010. Research on the molecular basis of DTMUV pathogenicity has been hampered by the lack of a reliable reverse genetics system for this virus. Here we developed a PCR-based reverse genetics system with high fidelity for the attenuated DTMUV strain FX2010-180P. The rescued virus was characterized by using both indirect immunofluorescence assays (IFA) and whole genome sequencing. The rescued virus (rFX2010-180P) grew to similar titers as compared with the wild-type virus in DF-1 cells, and had similar replication and immunogenicity properties in ducks. To determine whether exogenous proteins could be expressed from DTMUV, both an internal ribosomal entry site (IRES) and the enhanced green fluorescent protein (eGFP) gene were introduced between the NS5 gene and the 3' non-coding sequence of FX2010-180P. A recombinant DTMUV expressing eGFP was rescued, but eGFP expression was unstable after 4 passages in DF-1 cells due to a deletion of 1,294 nucleotides. The establishment of a reliable reverse genetics system for FX2010-180P provides a foundation for future studies of DTMUV.
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spelling pubmed-48890612016-06-10 Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain Wu, Xiaogang Shi, Ying Yan, Dawei Li, Xuesong Yan, Pixi Gao, Xuyuan Zhang, Yuee Yu, Lei Ren, Chaochao Li, Guoxin Yan, Liping Teng, Qiaoyang Li, Zejun PLoS One Research Article The infectious disease caused by the duck Tembusu virus (DTMUV) has resulted in massive economic losses to the Chinese duck industry in China since 2010. Research on the molecular basis of DTMUV pathogenicity has been hampered by the lack of a reliable reverse genetics system for this virus. Here we developed a PCR-based reverse genetics system with high fidelity for the attenuated DTMUV strain FX2010-180P. The rescued virus was characterized by using both indirect immunofluorescence assays (IFA) and whole genome sequencing. The rescued virus (rFX2010-180P) grew to similar titers as compared with the wild-type virus in DF-1 cells, and had similar replication and immunogenicity properties in ducks. To determine whether exogenous proteins could be expressed from DTMUV, both an internal ribosomal entry site (IRES) and the enhanced green fluorescent protein (eGFP) gene were introduced between the NS5 gene and the 3' non-coding sequence of FX2010-180P. A recombinant DTMUV expressing eGFP was rescued, but eGFP expression was unstable after 4 passages in DF-1 cells due to a deletion of 1,294 nucleotides. The establishment of a reliable reverse genetics system for FX2010-180P provides a foundation for future studies of DTMUV. Public Library of Science 2016-06-01 /pmc/articles/PMC4889061/ /pubmed/27248497 http://dx.doi.org/10.1371/journal.pone.0156579 Text en © 2016 Wu et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Wu, Xiaogang
Shi, Ying
Yan, Dawei
Li, Xuesong
Yan, Pixi
Gao, Xuyuan
Zhang, Yuee
Yu, Lei
Ren, Chaochao
Li, Guoxin
Yan, Liping
Teng, Qiaoyang
Li, Zejun
Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain
title Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain
title_full Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain
title_fullStr Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain
title_full_unstemmed Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain
title_short Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain
title_sort development of a pcr-based reverse genetics system for an attenuated duck tembusu virus strain
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889061/
https://www.ncbi.nlm.nih.gov/pubmed/27248497
http://dx.doi.org/10.1371/journal.pone.0156579
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