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Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2

Zika virus (ZIKV) has had detrimental effects on global public health in recent years. This is because the management of the disease has been limited, in part because its pathogenic mechanisms are not yet completely understood. Infectious clones are an important tool that utilize reverse genetics; t...

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Autores principales: Qin, Zhiran, Chen, Yangyang, Yu, Jianhai, He, Xiaoen, Liu, Xuling, Zhu, Li, Wu, Qinghua, Wan, Chengsong, Zhang, Bao, Zhao, Wei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8567171/
https://www.ncbi.nlm.nih.gov/pubmed/34745057
http://dx.doi.org/10.3389/fmicb.2021.752578
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author Qin, Zhiran
Chen, Yangyang
Yu, Jianhai
He, Xiaoen
Liu, Xuling
Zhu, Li
Wu, Qinghua
Wan, Chengsong
Zhang, Bao
Zhao, Wei
author_facet Qin, Zhiran
Chen, Yangyang
Yu, Jianhai
He, Xiaoen
Liu, Xuling
Zhu, Li
Wu, Qinghua
Wan, Chengsong
Zhang, Bao
Zhao, Wei
author_sort Qin, Zhiran
collection PubMed
description Zika virus (ZIKV) has had detrimental effects on global public health in recent years. This is because the management of the disease has been limited, in part because its pathogenic mechanisms are not yet completely understood. Infectious clones are an important tool that utilize reverse genetics; these can be used to modify the ZIKV genomic RNA at the DNA level. A homologous recombination clone was used to construct pWSK29, a low copy plasmid that contained sequences for a T7 promoter, the whole genome of ZIKV ZKC2 strain, and a hepatitis delta virus ribozyme. High fidelity PCR was then used to amplify the T7 transcription template. The transcript was then transfected into susceptible cells via lipofection to recover the ZIKV ZKC2 strain. Finally, the virulence of rZKC2 was evaluated both in vitro and in vivo. The rZKC2 was successfully obtained and it showed the same virulence as its parent, the ZIKV ZKC2 strain (pZKC2), both in vitro and in vivo. The 3730 (NS2A-D62G) mutation site was identified as being important, since it had significant impacts on rZKC2 recovery. The 4015 (NS2A, A157V) mutation may reduce virus production by increasing the interferon type I response. In this study, one of the earliest strains of ZIKV that was imported into China was used for infectious clone construction and one possible site for antiviral medication development was discovered. The use of homologous recombination clones, of PCR products as templates for T7 transcription, and of lipofection for large RNA transfection could increase the efficiency of infectious clone construction. Our infectious clone provides an effective tool which can be used to explore the life cycle and medical treatment of ZIKV.
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spelling pubmed-85671712021-11-05 Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2 Qin, Zhiran Chen, Yangyang Yu, Jianhai He, Xiaoen Liu, Xuling Zhu, Li Wu, Qinghua Wan, Chengsong Zhang, Bao Zhao, Wei Front Microbiol Microbiology Zika virus (ZIKV) has had detrimental effects on global public health in recent years. This is because the management of the disease has been limited, in part because its pathogenic mechanisms are not yet completely understood. Infectious clones are an important tool that utilize reverse genetics; these can be used to modify the ZIKV genomic RNA at the DNA level. A homologous recombination clone was used to construct pWSK29, a low copy plasmid that contained sequences for a T7 promoter, the whole genome of ZIKV ZKC2 strain, and a hepatitis delta virus ribozyme. High fidelity PCR was then used to amplify the T7 transcription template. The transcript was then transfected into susceptible cells via lipofection to recover the ZIKV ZKC2 strain. Finally, the virulence of rZKC2 was evaluated both in vitro and in vivo. The rZKC2 was successfully obtained and it showed the same virulence as its parent, the ZIKV ZKC2 strain (pZKC2), both in vitro and in vivo. The 3730 (NS2A-D62G) mutation site was identified as being important, since it had significant impacts on rZKC2 recovery. The 4015 (NS2A, A157V) mutation may reduce virus production by increasing the interferon type I response. In this study, one of the earliest strains of ZIKV that was imported into China was used for infectious clone construction and one possible site for antiviral medication development was discovered. The use of homologous recombination clones, of PCR products as templates for T7 transcription, and of lipofection for large RNA transfection could increase the efficiency of infectious clone construction. Our infectious clone provides an effective tool which can be used to explore the life cycle and medical treatment of ZIKV. Frontiers Media S.A. 2021-10-21 /pmc/articles/PMC8567171/ /pubmed/34745057 http://dx.doi.org/10.3389/fmicb.2021.752578 Text en Copyright © 2021 Qin, Chen, Yu, He, Liu, Zhu, Wu, Wan, Zhang and Zhao. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Qin, Zhiran
Chen, Yangyang
Yu, Jianhai
He, Xiaoen
Liu, Xuling
Zhu, Li
Wu, Qinghua
Wan, Chengsong
Zhang, Bao
Zhao, Wei
Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2
title Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2
title_full Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2
title_fullStr Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2
title_full_unstemmed Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2
title_short Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2
title_sort rapid construction of an infectious clone of the zika virus, strain zkc2
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8567171/
https://www.ncbi.nlm.nih.gov/pubmed/34745057
http://dx.doi.org/10.3389/fmicb.2021.752578
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