Novel Approach for Insertion of Heterologous Sequences into Full-Length ZIKV Genome Results in Superior Level of Gene Expression and Insert Stability
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture Systems
2.2. Construction of Infectious ZIKV Clones
2.3. Virus
2.4. DNA Transfections
2.5. Plaque-Forming and Focus-Forming Assays
2.6. Infections
2.7. Viral Replication Kinetics
2.8. eGFP and Luciferase Reporter Assays
2.9. Reporter Virus Stability Assessment
3. Results
3.1. Mapping the Regulatory Elements That Constitute Replication Promoter of the C Gene of ZIKV
3.2. Development of ZIKV Clones Expressing nLuc Gene in the Duplicated Capsid Gene Region (dCGR) and Duplicated E/NS1 Junction Region
3.3. Characterization of the nLuc-Carrying Viruses In Vero Cells
3.4. Assessment of nLuc Insert Stability in Viral Genomes during Repeated Passages in Cell Culture
3.5. Construction of eGFP-Expressing ZIKV and Characterization of Reporter Viruses in Cell Culture
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Volkova, E.; Tsetsarkin, K.A.; Sippert, E.; Assis, F.; Liu, G.; Rios, M.; Pletnev, A.G. Novel Approach for Insertion of Heterologous Sequences into Full-Length ZIKV Genome Results in Superior Level of Gene Expression and Insert Stability. Viruses 2020, 12, 61. https://doi.org/10.3390/v12010061
Volkova E, Tsetsarkin KA, Sippert E, Assis F, Liu G, Rios M, Pletnev AG. Novel Approach for Insertion of Heterologous Sequences into Full-Length ZIKV Genome Results in Superior Level of Gene Expression and Insert Stability. Viruses. 2020; 12(1):61. https://doi.org/10.3390/v12010061
Chicago/Turabian StyleVolkova, Evgeniya, Konstantin A. Tsetsarkin, Emilia Sippert, Felipe Assis, Guangping Liu, Maria Rios, and Alexander G. Pletnev. 2020. "Novel Approach for Insertion of Heterologous Sequences into Full-Length ZIKV Genome Results in Superior Level of Gene Expression and Insert Stability" Viruses 12, no. 1: 61. https://doi.org/10.3390/v12010061
APA StyleVolkova, E., Tsetsarkin, K. A., Sippert, E., Assis, F., Liu, G., Rios, M., & Pletnev, A. G. (2020). Novel Approach for Insertion of Heterologous Sequences into Full-Length ZIKV Genome Results in Superior Level of Gene Expression and Insert Stability. Viruses, 12(1), 61. https://doi.org/10.3390/v12010061