Cargando…
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...
Autores principales: | , , , , , , , , , , , , |
---|---|
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 |
_version_ | 1782434938498318336 |
---|---|
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. |
format | Online Article Text |
id | pubmed-4889061 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT wuxiaogang developmentofapcrbasedreversegeneticssystemforanattenuatedducktembusuvirusstrain AT shiying developmentofapcrbasedreversegeneticssystemforanattenuatedducktembusuvirusstrain AT yandawei developmentofapcrbasedreversegeneticssystemforanattenuatedducktembusuvirusstrain AT lixuesong developmentofapcrbasedreversegeneticssystemforanattenuatedducktembusuvirusstrain AT yanpixi developmentofapcrbasedreversegeneticssystemforanattenuatedducktembusuvirusstrain AT gaoxuyuan developmentofapcrbasedreversegeneticssystemforanattenuatedducktembusuvirusstrain AT zhangyuee developmentofapcrbasedreversegeneticssystemforanattenuatedducktembusuvirusstrain AT yulei developmentofapcrbasedreversegeneticssystemforanattenuatedducktembusuvirusstrain AT renchaochao developmentofapcrbasedreversegeneticssystemforanattenuatedducktembusuvirusstrain AT liguoxin developmentofapcrbasedreversegeneticssystemforanattenuatedducktembusuvirusstrain AT yanliping developmentofapcrbasedreversegeneticssystemforanattenuatedducktembusuvirusstrain AT tengqiaoyang developmentofapcrbasedreversegeneticssystemforanattenuatedducktembusuvirusstrain AT lizejun developmentofapcrbasedreversegeneticssystemforanattenuatedducktembusuvirusstrain |