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Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)

BACKGROUND: Cytomegalovirus (CMV) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. Developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recip...

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Autores principales: Balakrishnan, Krishnan Nair, Abdullah, Ashwaq Ahmed, Bala, Jamilu Abubakar, Jesse, Faez Firdaus Abdullah, Abdullah, Che Azurahanim Che, Noordin, Mustapha Mohamed, Mohd-Azmi, Mohd Lila
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590257/
https://www.ncbi.nlm.nih.gov/pubmed/33109247
http://dx.doi.org/10.1186/s12985-020-01436-5
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author Balakrishnan, Krishnan Nair
Abdullah, Ashwaq Ahmed
Bala, Jamilu Abubakar
Jesse, Faez Firdaus Abdullah
Abdullah, Che Azurahanim Che
Noordin, Mustapha Mohamed
Mohd-Azmi, Mohd Lila
author_facet Balakrishnan, Krishnan Nair
Abdullah, Ashwaq Ahmed
Bala, Jamilu Abubakar
Jesse, Faez Firdaus Abdullah
Abdullah, Che Azurahanim Che
Noordin, Mustapha Mohamed
Mohd-Azmi, Mohd Lila
author_sort Balakrishnan, Krishnan Nair
collection PubMed
description BACKGROUND: Cytomegalovirus (CMV) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. Developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recipients. In the present study, with the aim of exploring new strategies to counteract CMV replication, several anti-CMV siRNAs targeting IE2 and DNA polymerase gene regions were characterized and used as in combinations for antiviral therapy. METHODS: The rat embryo fibroblast (REF) cells were transfected with multi siRNA before infecting with CMV strain ALL-03. Viral growth inhibition was measured by tissue culture infectious dose (TCID50), cytopathic effect (CPE) and droplet digital PCR (ddPCR) while IE2 and DNA polymerase gene knockdown was determined by real-time PCR. Ganciclovir was deployed as a control to benchmark the efficacy of antiviral activities of respective individual siRNAs. RESULTS: There was no significant cytotoxicity encountered for all the combinations of siRNAs on REF cells analyzed by MTT colorimetric assay (P > 0.05). Cytopathic effects (CPE) in cells infected by RCMV ALL-03 had developed significantly less and at much slower rate compared to control group. The expression of targeted genes was downregulated successfully resulted in significant reduction (P < 0.05) of viral mRNA and DNA copies (dpb + dpc: 79%, 68%; dpb + ie2b: 68%, 60%; dpb + dpc + ie2b: 48%, 42%). Flow cytometry analysis showed a greater percentage of viable and early apoptosis of combined siRNAs-treated cells compared to control group. Notably, the siRNAs targeting gene regions were sequenced and mutations were not encountered, thereby avoiding the formation of mutant with potential resistant viruses. CONCLUSIONS: In conclusion. The study demonstrated a tremendous promise of innovative approach with the deployment of combined siRNAs targeting at several genes simultaneously with the aim to control CMV replication in host cells.
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spelling pubmed-75902572020-10-27 Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03) Balakrishnan, Krishnan Nair Abdullah, Ashwaq Ahmed Bala, Jamilu Abubakar Jesse, Faez Firdaus Abdullah Abdullah, Che Azurahanim Che Noordin, Mustapha Mohamed Mohd-Azmi, Mohd Lila Virol J Research BACKGROUND: Cytomegalovirus (CMV) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. Developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recipients. In the present study, with the aim of exploring new strategies to counteract CMV replication, several anti-CMV siRNAs targeting IE2 and DNA polymerase gene regions were characterized and used as in combinations for antiviral therapy. METHODS: The rat embryo fibroblast (REF) cells were transfected with multi siRNA before infecting with CMV strain ALL-03. Viral growth inhibition was measured by tissue culture infectious dose (TCID50), cytopathic effect (CPE) and droplet digital PCR (ddPCR) while IE2 and DNA polymerase gene knockdown was determined by real-time PCR. Ganciclovir was deployed as a control to benchmark the efficacy of antiviral activities of respective individual siRNAs. RESULTS: There was no significant cytotoxicity encountered for all the combinations of siRNAs on REF cells analyzed by MTT colorimetric assay (P > 0.05). Cytopathic effects (CPE) in cells infected by RCMV ALL-03 had developed significantly less and at much slower rate compared to control group. The expression of targeted genes was downregulated successfully resulted in significant reduction (P < 0.05) of viral mRNA and DNA copies (dpb + dpc: 79%, 68%; dpb + ie2b: 68%, 60%; dpb + dpc + ie2b: 48%, 42%). Flow cytometry analysis showed a greater percentage of viable and early apoptosis of combined siRNAs-treated cells compared to control group. Notably, the siRNAs targeting gene regions were sequenced and mutations were not encountered, thereby avoiding the formation of mutant with potential resistant viruses. CONCLUSIONS: In conclusion. The study demonstrated a tremendous promise of innovative approach with the deployment of combined siRNAs targeting at several genes simultaneously with the aim to control CMV replication in host cells. BioMed Central 2020-10-27 /pmc/articles/PMC7590257/ /pubmed/33109247 http://dx.doi.org/10.1186/s12985-020-01436-5 Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Balakrishnan, Krishnan Nair
Abdullah, Ashwaq Ahmed
Bala, Jamilu Abubakar
Jesse, Faez Firdaus Abdullah
Abdullah, Che Azurahanim Che
Noordin, Mustapha Mohamed
Mohd-Azmi, Mohd Lila
Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)
title Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)
title_full Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)
title_fullStr Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)
title_full_unstemmed Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)
title_short Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)
title_sort multiple gene targeting sirnas for down regulation of immediate early-2 (ie2) and dna polymerase genes mediated inhibition of novel rat cytomegalovirus (strain all-03)
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590257/
https://www.ncbi.nlm.nih.gov/pubmed/33109247
http://dx.doi.org/10.1186/s12985-020-01436-5
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