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Novel Approach for Insertion of Heterologous Sequences into Full-Length ZIKV Genome Results in Superior Level of Gene Expression and Insert Stability

Zika virus (ZIKV) emerged in the Americas in 2015, presenting unique challenges to public health. Unlike other arboviruses of the Flaviviridae family, it is transmissible by sexual contact, which facilitates the spread of the virus into new geographic areas. Additionally, ZIKV can be transmitted fro...

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Autores principales: Volkova, Evgeniya, Tsetsarkin, Konstantin A., Sippert, Emilia, Assis, Felipe, Liu, Guangping, Rios, Maria, Pletnev, Alexander G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019263/
https://www.ncbi.nlm.nih.gov/pubmed/31947825
http://dx.doi.org/10.3390/v12010061
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author Volkova, Evgeniya
Tsetsarkin, Konstantin A.
Sippert, Emilia
Assis, Felipe
Liu, Guangping
Rios, Maria
Pletnev, Alexander G.
author_facet Volkova, Evgeniya
Tsetsarkin, Konstantin A.
Sippert, Emilia
Assis, Felipe
Liu, Guangping
Rios, Maria
Pletnev, Alexander G.
author_sort Volkova, Evgeniya
collection PubMed
description Zika virus (ZIKV) emerged in the Americas in 2015, presenting unique challenges to public health. Unlike other arboviruses of the Flaviviridae family, it is transmissible by sexual contact, which facilitates the spread of the virus into new geographic areas. Additionally, ZIKV can be transmitted from mother to fetus, causing microcephaly and other severe developmental abnormalities. Reliable and easy-to-work-with clones of ZIKV expressing heterologous genes will significantly facilitate studies aimed at understanding the virus pathogenesis and tissue tropism. Here, we developed and characterized two novel approaches for expression of heterologous genes of interest in the context of full-length ZIKV genome and compared them to two previously published strategies for ZIKV-mediated gene expression. We demonstrated that among the four tested viruses expressing nLuc gene, the virus constructed using a newly developed approach of partial capsid gene duplication (PCGD) attained the highest titer in Vero cells and resulted in the highest level of nLuc expression. Suitability of the PCGD approach for expression of different genes of interest was validated by replacing nLuc sequence with that of eGFP gene. The generated constructs were further characterized in cell culture. Potential applications of ZIKV clones stably expressing heterologous genes include development of detection assays, antivirals, therapeutics, live imaging systems, and vaccines.
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spelling pubmed-70192632020-03-04 Novel Approach for Insertion of Heterologous Sequences into Full-Length ZIKV Genome Results in Superior Level of Gene Expression and Insert Stability Volkova, Evgeniya Tsetsarkin, Konstantin A. Sippert, Emilia Assis, Felipe Liu, Guangping Rios, Maria Pletnev, Alexander G. Viruses Article Zika virus (ZIKV) emerged in the Americas in 2015, presenting unique challenges to public health. Unlike other arboviruses of the Flaviviridae family, it is transmissible by sexual contact, which facilitates the spread of the virus into new geographic areas. Additionally, ZIKV can be transmitted from mother to fetus, causing microcephaly and other severe developmental abnormalities. Reliable and easy-to-work-with clones of ZIKV expressing heterologous genes will significantly facilitate studies aimed at understanding the virus pathogenesis and tissue tropism. Here, we developed and characterized two novel approaches for expression of heterologous genes of interest in the context of full-length ZIKV genome and compared them to two previously published strategies for ZIKV-mediated gene expression. We demonstrated that among the four tested viruses expressing nLuc gene, the virus constructed using a newly developed approach of partial capsid gene duplication (PCGD) attained the highest titer in Vero cells and resulted in the highest level of nLuc expression. Suitability of the PCGD approach for expression of different genes of interest was validated by replacing nLuc sequence with that of eGFP gene. The generated constructs were further characterized in cell culture. Potential applications of ZIKV clones stably expressing heterologous genes include development of detection assays, antivirals, therapeutics, live imaging systems, and vaccines. MDPI 2020-01-03 /pmc/articles/PMC7019263/ /pubmed/31947825 http://dx.doi.org/10.3390/v12010061 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
Volkova, Evgeniya
Tsetsarkin, Konstantin A.
Sippert, Emilia
Assis, Felipe
Liu, Guangping
Rios, Maria
Pletnev, Alexander G.
Novel Approach for Insertion of Heterologous Sequences into Full-Length ZIKV Genome Results in Superior Level of Gene Expression and Insert Stability
title Novel Approach for Insertion of Heterologous Sequences into Full-Length ZIKV Genome Results in Superior Level of Gene Expression and Insert Stability
title_full Novel Approach for Insertion of Heterologous Sequences into Full-Length ZIKV Genome Results in Superior Level of Gene Expression and Insert Stability
title_fullStr Novel Approach for Insertion of Heterologous Sequences into Full-Length ZIKV Genome Results in Superior Level of Gene Expression and Insert Stability
title_full_unstemmed Novel Approach for Insertion of Heterologous Sequences into Full-Length ZIKV Genome Results in Superior Level of Gene Expression and Insert Stability
title_short Novel Approach for Insertion of Heterologous Sequences into Full-Length ZIKV Genome Results in Superior Level of Gene Expression and Insert Stability
title_sort novel approach for insertion of heterologous sequences into full-length zikv genome results in superior level of gene expression and insert stability
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019263/
https://www.ncbi.nlm.nih.gov/pubmed/31947825
http://dx.doi.org/10.3390/v12010061
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