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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...

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Autores principales: Mayuramart, Oraphan, Poomipak, Witthaya, Rattanaburi, Somruthai, Khongnomnan, Kritsada, Anuntakarun, Songtham, Saengchoowong, Suthat, Chavalit, Tanit, Chantaravisoot, Naphat, Payungporn, Sunchai
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2022
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.
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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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