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IRF7-deficient MDCK cell based on CRISPR/Cas9 technology for enhancing influenza virus replication and improving vaccine production
The influenza virus is a cause of seasonal epidemic disease and enormous economic injury. The best way to control influenza outbreaks is through vaccination. The Madin-Darby canine kidney cell line (MDCK) is currently approved to manufacture influenza vaccines. However, the viral load from cell-base...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
PeerJ Inc.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9508885/ https://www.ncbi.nlm.nih.gov/pubmed/36164603 http://dx.doi.org/10.7717/peerj.13989 |
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author | Mayuramart, Oraphan Poomipak, Witthaya Rattanaburi, Somruthai Khongnomnan, Kritsada Anuntakarun, Songtham Saengchoowong, Suthat Chavalit, Tanit Chantaravisoot, Naphat Payungporn, Sunchai |
author_facet | Mayuramart, Oraphan Poomipak, Witthaya Rattanaburi, Somruthai Khongnomnan, Kritsada Anuntakarun, Songtham Saengchoowong, Suthat Chavalit, Tanit Chantaravisoot, Naphat Payungporn, Sunchai |
author_sort | Mayuramart, Oraphan |
collection | PubMed |
description | The influenza virus is a cause of seasonal epidemic disease and enormous economic injury. The best way to control influenza outbreaks is through vaccination. The Madin-Darby canine kidney cell line (MDCK) is currently approved to manufacture influenza vaccines. However, the viral load from cell-based production is limited by host interferons (IFN). Interferon regulating factor 7 (IRF7) is a transcription factor for type-I IFN that plays an important role in regulating the anti-viral mechanism and eliminating viruses. We developed IRF7 knock-out MDCK cells (IRF7(−/ −) MDCK) using CRISPR/Cas9 technology. The RNA expression levels of IRF7 in the IRF7(−/ −) MDCK cells were reduced by 94.76% and 95.22% under the uninfected and infected conditions, respectively. Furthermore, the IRF7 protein level was also significantly lower in IRF7(−/ −) MDCK cells for both uninfected (54.85% reduction) and viral infected conditions (32.27% reduction) compared to WT MDCK. The differential expression analysis of IFN-related genes demonstrated that the IRF7(−/ −) MDCK cell had a lower interferon response than wildtype MDCK under the influenza-infected condition. Gene ontology revealed down-regulation of the defense response against virus and IFN-gamma production in IRF7(−/ −) MDCK. The evaluation of influenza viral titers by RT-qPCR and hemagglutination assay (HA) revealed IRF7(−/ −) MDCK cells had higher viral titers in cell supernatant, including A/pH1N1 (4 to 5-fold) and B/Yamagata (2-fold). Therefore, the IRF7(−/ −) MDCK cells could be applied to cell-based influenza vaccine production with higher capacity and efficiency. |
format | Online Article Text |
id | pubmed-9508885 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | PeerJ Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-95088852022-09-25 IRF7-deficient MDCK cell based on CRISPR/Cas9 technology for enhancing influenza virus replication and improving vaccine production Mayuramart, Oraphan Poomipak, Witthaya Rattanaburi, Somruthai Khongnomnan, Kritsada Anuntakarun, Songtham Saengchoowong, Suthat Chavalit, Tanit Chantaravisoot, Naphat Payungporn, Sunchai PeerJ Biochemistry The influenza virus is a cause of seasonal epidemic disease and enormous economic injury. The best way to control influenza outbreaks is through vaccination. The Madin-Darby canine kidney cell line (MDCK) is currently approved to manufacture influenza vaccines. However, the viral load from cell-based production is limited by host interferons (IFN). Interferon regulating factor 7 (IRF7) is a transcription factor for type-I IFN that plays an important role in regulating the anti-viral mechanism and eliminating viruses. We developed IRF7 knock-out MDCK cells (IRF7(−/ −) MDCK) using CRISPR/Cas9 technology. The RNA expression levels of IRF7 in the IRF7(−/ −) MDCK cells were reduced by 94.76% and 95.22% under the uninfected and infected conditions, respectively. Furthermore, the IRF7 protein level was also significantly lower in IRF7(−/ −) MDCK cells for both uninfected (54.85% reduction) and viral infected conditions (32.27% reduction) compared to WT MDCK. The differential expression analysis of IFN-related genes demonstrated that the IRF7(−/ −) MDCK cell had a lower interferon response than wildtype MDCK under the influenza-infected condition. Gene ontology revealed down-regulation of the defense response against virus and IFN-gamma production in IRF7(−/ −) MDCK. The evaluation of influenza viral titers by RT-qPCR and hemagglutination assay (HA) revealed IRF7(−/ −) MDCK cells had higher viral titers in cell supernatant, including A/pH1N1 (4 to 5-fold) and B/Yamagata (2-fold). Therefore, the IRF7(−/ −) MDCK cells could be applied to cell-based influenza vaccine production with higher capacity and efficiency. PeerJ Inc. 2022-09-21 /pmc/articles/PMC9508885/ /pubmed/36164603 http://dx.doi.org/10.7717/peerj.13989 Text en ©2022 Mayuramart et al. https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited. |
spellingShingle | Biochemistry Mayuramart, Oraphan Poomipak, Witthaya Rattanaburi, Somruthai Khongnomnan, Kritsada Anuntakarun, Songtham Saengchoowong, Suthat Chavalit, Tanit Chantaravisoot, Naphat Payungporn, Sunchai IRF7-deficient MDCK cell based on CRISPR/Cas9 technology for enhancing influenza virus replication and improving vaccine production |
title | IRF7-deficient MDCK cell based on CRISPR/Cas9 technology for enhancing influenza virus replication and improving vaccine production |
title_full | IRF7-deficient MDCK cell based on CRISPR/Cas9 technology for enhancing influenza virus replication and improving vaccine production |
title_fullStr | IRF7-deficient MDCK cell based on CRISPR/Cas9 technology for enhancing influenza virus replication and improving vaccine production |
title_full_unstemmed | IRF7-deficient MDCK cell based on CRISPR/Cas9 technology for enhancing influenza virus replication and improving vaccine production |
title_short | IRF7-deficient MDCK cell based on CRISPR/Cas9 technology for enhancing influenza virus replication and improving vaccine production |
title_sort | irf7-deficient mdck cell based on crispr/cas9 technology for enhancing influenza virus replication and improving vaccine production |
topic | Biochemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9508885/ https://www.ncbi.nlm.nih.gov/pubmed/36164603 http://dx.doi.org/10.7717/peerj.13989 |
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