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Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes
The chikungunya virus (CHIKV) is an emerging pathogen with widespread distribution in regions of Africa, India, and Asia that threatens to spread into temperate climates with the introduction of its major vector, Aedes albopictus. CHIKV causes a disease frequently misdiagnosed as dengue fever, with...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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MDPI
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4926183/ https://www.ncbi.nlm.nih.gov/pubmed/27294950 http://dx.doi.org/10.3390/v8060163 |
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author | Mishra, Priya Furey, Colleen Balaraman, Velmurugan Fraser, Malcolm J. |
author_facet | Mishra, Priya Furey, Colleen Balaraman, Velmurugan Fraser, Malcolm J. |
author_sort | Mishra, Priya |
collection | PubMed |
description | The chikungunya virus (CHIKV) is an emerging pathogen with widespread distribution in regions of Africa, India, and Asia that threatens to spread into temperate climates with the introduction of its major vector, Aedes albopictus. CHIKV causes a disease frequently misdiagnosed as dengue fever, with potentially life-threatening symptoms that can result in a longer-term debilitating arthritis. The increasing risk of spread from endemic regions via human travel and commerce and the current absence of a vaccine put a significant proportion of the world population at risk for this disease. In this study we designed and tested hammerhead ribozymes (hRzs) targeting CHIKV structural protein genes of the RNA genome as potential antivirals both at the cellular and in vivo level. We employed the CHIKV strain 181/25, which exhibits similar infectivity rates in both Vero cell cultures and mosquitoes. Virus suppression assay performed on transformed Vero cell clones of all seven hRzs demonstrated that all are effective at inhibiting CHIKV in Vero cells, with hRz #9 and #14 being the most effective. piggyBac transformation vectors were constructed using the Ae. aegypti t-RNA(val) Pol III promoted hRz #9 and #14 effector genes to establish a total of nine unique transgenic Higgs White Eye (HWE) Ae. aegypti lines. Following confirmation of transgene expression by real-time polymerase chain reaction (RT-PCR), comparative TCID(50)-IFA analysis, in situ Immuno-fluorescent Assays (IFA) and analysis of salivary CHIKV titers demonstrated effective suppression of virus replication at 7 dpi in heterozygous females of each of these transgenic lines compared with control HWE mosquitoes. This report provides a proof that appropriately engineered hRzs are powerful antiviral effector genes suitable for population replacement strategies |
format | Online Article Text |
id | pubmed-4926183 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-49261832016-07-06 Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes Mishra, Priya Furey, Colleen Balaraman, Velmurugan Fraser, Malcolm J. Viruses Article The chikungunya virus (CHIKV) is an emerging pathogen with widespread distribution in regions of Africa, India, and Asia that threatens to spread into temperate climates with the introduction of its major vector, Aedes albopictus. CHIKV causes a disease frequently misdiagnosed as dengue fever, with potentially life-threatening symptoms that can result in a longer-term debilitating arthritis. The increasing risk of spread from endemic regions via human travel and commerce and the current absence of a vaccine put a significant proportion of the world population at risk for this disease. In this study we designed and tested hammerhead ribozymes (hRzs) targeting CHIKV structural protein genes of the RNA genome as potential antivirals both at the cellular and in vivo level. We employed the CHIKV strain 181/25, which exhibits similar infectivity rates in both Vero cell cultures and mosquitoes. Virus suppression assay performed on transformed Vero cell clones of all seven hRzs demonstrated that all are effective at inhibiting CHIKV in Vero cells, with hRz #9 and #14 being the most effective. piggyBac transformation vectors were constructed using the Ae. aegypti t-RNA(val) Pol III promoted hRz #9 and #14 effector genes to establish a total of nine unique transgenic Higgs White Eye (HWE) Ae. aegypti lines. Following confirmation of transgene expression by real-time polymerase chain reaction (RT-PCR), comparative TCID(50)-IFA analysis, in situ Immuno-fluorescent Assays (IFA) and analysis of salivary CHIKV titers demonstrated effective suppression of virus replication at 7 dpi in heterozygous females of each of these transgenic lines compared with control HWE mosquitoes. This report provides a proof that appropriately engineered hRzs are powerful antiviral effector genes suitable for population replacement strategies MDPI 2016-06-09 /pmc/articles/PMC4926183/ /pubmed/27294950 http://dx.doi.org/10.3390/v8060163 Text en © 2016 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 Mishra, Priya Furey, Colleen Balaraman, Velmurugan Fraser, Malcolm J. Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes |
title | Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes |
title_full | Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes |
title_fullStr | Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes |
title_full_unstemmed | Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes |
title_short | Antiviral Hammerhead Ribozymes Are Effective for Developing Transgenic Suppression of Chikungunya Virus in Aedes aegypti Mosquitoes |
title_sort | antiviral hammerhead ribozymes are effective for developing transgenic suppression of chikungunya virus in aedes aegypti mosquitoes |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4926183/ https://www.ncbi.nlm.nih.gov/pubmed/27294950 http://dx.doi.org/10.3390/v8060163 |
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