{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,10]],"date-time":"2026-04-10T23:17:27Z","timestamp":1775863047895,"version":"3.50.1"},"reference-count":46,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T00:00:00Z","timestamp":1767139200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2023YFE0106100"],"award-info":[{"award-number":["2023YFE0106100"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Scientific and technological research project of higher education institutions in Hebei Province China","award":["ZC2025027"],"award-info":[{"award-number":["ZC2025027"]}]},{"name":"Natural science foundation of Cangzhou City, Hebei Province, China","award":["20221001004D"],"award-info":[{"award-number":["20221001004D"]}]},{"name":"Shandong Provincial Poultry Industry and Technology System","award":["SDAIT-11-16"],"award-info":[{"award-number":["SDAIT-11-16"]}]},{"name":"Taishan Industrial Experts Program, Binzbou Young Scientist Program","award":["QMX2024002"],"award-info":[{"award-number":["QMX2024002"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background: Large-scale production of poultry viral vaccines increasingly requires robust suspension cell platforms. However, most avian cell lines, including DF-1, are strictly anchorage-dependent, limiting scalability. Aquaporin-1 (AQP1) regulates cell\u2013cell adhesion and membrane dynamics, making it a potential target for engineering suspension growth. This study aimed to generate a stable DF-1 suspension cell line via AQP1 disruption and evaluate its potential for enhanced infectious bursal disease virus (IBDV) production. Methodology: DF-1 cells were engineered using a CRISPR\/Cas9 ribonucleoprotein system to create a truncated AQP1 gene. DF-1\/AQP1\u2212 cells were assessed for morphology, tumorigenicity in nude mice, and genetic stability across 20 passages. Suspension growth, cell density, and viability were measured. Cells were infected with IBDV strain BJQ902, and viral titers were compared with wild-type DF-1 and monolayer DF-1\/AQP1\u2212 cells. Results: DF-1\/AQP1\u2212 cells maintained normal morphology, were non-tumorigenic, and retained stable AQP1 mutations. They grew as true suspension cultures without adaptation, reaching 4.0 \u00d7 106 cells\/mL with &gt;95% viability. Suspension DF-1\/AQP1\u2212 cells cells produced significantly higher viral titers (9.0 log TCID50\/mL; 8.63 log EID50\/mL) than both monolayer DF-1\/AQP1\u2212 and wild-type DF-1 cells. Virus production time was shortened in suspension cultures. Conclusions: Targeted AQP1 disruption converts DF-1 cells into a stable, non-tumorigenic suspension cell line with markedly enhanced IBDV production, providing a scalable platform for next-generation avian vaccine manufacturing.<\/jats:p>","DOI":"10.3390\/vaccines14010052","type":"journal-article","created":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T16:34:23Z","timestamp":1767198863000},"page":"52","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Engineering AQP1-Deficient DF-1 Suspension Cells for High-Yield IBDV Production and Vaccine Scale-Up"],"prefix":"10.3390","volume":"14","author":[{"given":"Bingmei","family":"Dong","sequence":"first","affiliation":[{"name":"College of Life Science, Cangzhou Normal University, Guofeng South Avenue 16#, Cangzhou 061001, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ruonan","family":"Wang","sequence":"additional","affiliation":[{"name":"Shandong Binzhou Institute of Animal Husbandry and Veterinary Sciences, Binzhou 256600, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yu","family":"Guan","sequence":"additional","affiliation":[{"name":"Shandong Binzhou Institute of Animal Husbandry and Veterinary Sciences, Binzhou 256600, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiubao","family":"Zhao","sequence":"additional","affiliation":[{"name":"Shandong Binzhou Institute of Animal Husbandry and Veterinary Sciences, Binzhou 256600, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ronghua","family":"Li","sequence":"additional","affiliation":[{"name":"College of Life Science, Cangzhou Normal University, Guofeng South Avenue 16#, Cangzhou 061001, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qingqing","family":"Xu","sequence":"additional","affiliation":[{"name":"Shandong Binzhou Institute of Animal Husbandry and Veterinary Sciences, Binzhou 256600, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hui","family":"Li","sequence":"additional","affiliation":[{"name":"Shandong Binzhou Institute of Animal Husbandry and Veterinary Sciences, Binzhou 256600, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qingfang","family":"Gao","sequence":"additional","affiliation":[{"name":"Shandong Lvdu Biological Technology Co., Ltd., Binzhou 256600, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shengjie","family":"Yao","sequence":"additional","affiliation":[{"name":"College of Life Science, Cangzhou Normal University, Guofeng South Avenue 16#, Cangzhou 061001, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shuyu","family":"Song","sequence":"additional","affiliation":[{"name":"College of Life Science, Cangzhou Normal University, Guofeng South Avenue 16#, Cangzhou 061001, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9008-2075","authenticated-orcid":false,"given":"Ashenafi Kiros","family":"Wubshet","sequence":"additional","affiliation":[{"name":"Shandong Binzhou Institute of Animal Husbandry and Veterinary Sciences, Binzhou 256600, China"},{"name":"College of Veterinary and Animal Sciences, Mekelle University, Mekelle 2084, Ethiopia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6422-5901","authenticated-orcid":false,"given":"Na","family":"Tang","sequence":"additional","affiliation":[{"name":"Shandong Binzhou Institute of Animal Husbandry and Veterinary Sciences, Binzhou 256600, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Piku\u0142a, A., Lisowska, A., and Doma\u0144ska-Blicharz, K. (2023). Epidemiology of Infectious Bursal Disease Virus in Poland during 2016\u20132022. Viruses, 15.","DOI":"10.3390\/v15020289"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Pikula, A., Lisowska, A., Jasik, A., and Perez, L.J. (2021). The Novel Genetic Background of Infectious Bursal Disease Virus Strains Emerging from the Action of Positive Selection. Viruses, 13.","DOI":"10.3390\/v13030396"},{"key":"ref_3","first-page":"9","article-title":"Infectious bursal disease virus in poultry: Current status and future prospects","volume":"7","author":"Alkie","year":"2016","journal-title":"Vet. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e70","DOI":"10.4142\/jvs.23297","article-title":"Immuno-protective effect of commercial IBD vaccines against emerging novel variant infectious bursal disease virus in specific-pathogen-free chickens","volume":"25","author":"Aliyu","year":"2024","journal-title":"J. Vet. Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2373521","DOI":"10.1080\/21645515.2024.2373521","article-title":"Innovations in cell culture-based influenza vaccine manufacturing\u2014from static cultures to high cell density cultivations","volume":"20","author":"Zinnecker","year":"2024","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Park, S., Kim, J.Y., Ryu, K.H., Kim, A.Y., Kim, J., Ko, Y.J., and Lee, E.G. (2021). Production of a Foot-and-Mouth Disease Vaccine Antigen Using Suspension-Adapted BHK-21 Cells in a Bioreactor. Vaccines, 9.","DOI":"10.3390\/vaccines9050505"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"5392033","DOI":"10.1155\/2022\/5392033","article-title":"Comparative Safety, Immunogenicity, and Efficacy of CEF Cell-Based and DF-1 Cell Line Adapted Infectious Bursal Disease Vaccines in Specific-Pathogen-Free Chickens","volume":"2022","author":"Workineh","year":"2022","journal-title":"J. Immunol. Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"241","DOI":"10.37201\/req\/117.2021","article-title":"Effectiveness of cell culture-based influenza vaccines compared with egg-based vaccines: What does the literature say?","volume":"35","author":"Salamanca","year":"2022","journal-title":"Rev. Esp. Quimioter."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"607","DOI":"10.1586\/erv.09.19","article-title":"Vero cell platform in vaccine production: Moving towards cell culture-based viral vaccines","volume":"8","author":"Barrett","year":"2009","journal-title":"Expert Rev. Vaccines"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"7193","DOI":"10.1016\/j.vaccine.2010.08.059","article-title":"Production and antigenic properties of influenza virus from suspension MDCK-siat7e cells in a bench-scale bioreactor","volume":"28","author":"Chu","year":"2010","journal-title":"Vaccine"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"14802","DOI":"10.1073\/pnas.0905912106","article-title":"Conversion of MDCK cell line to suspension culture by transfecting with human siat7e gene and its application for influenza virus production","volume":"106","author":"Chu","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","first-page":"39\u201351; discussion 73\u201374","article-title":"Growth and immunogenicity of influenza viruses cultivated in Vero or MDCK cells and in embryonated chicken eggs","volume":"98","author":"Govorkova","year":"1999","journal-title":"Dev. Biol. Stand."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2716","DOI":"10.1016\/S0264-410X(00)00508-9","article-title":"The human cell line PER.C6 provides a new manufacturing system for the production of influenza vaccines","volume":"19","author":"Pau","year":"2001","journal-title":"Vaccine"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3444","DOI":"10.1016\/S0264-410X(01)00053-6","article-title":"Comparison of large-scale mammalian cell culture systems with egg culture for the production of influenza virus A vaccine strains","volume":"19","author":"Tree","year":"2001","journal-title":"Vaccine"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1159\/000510585","article-title":"Study on the Dynamic Proliferation of JEV in BHK-21 Cells","volume":"64","author":"Zhang","year":"2021","journal-title":"Intervirology"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1083\/jcb.133.2.391","article-title":"Adhesion-dependent cell cycle progression linked to the expression of cyclin D1, activation of cyclin E-cdk2, and phosphorylation of the retinoblastoma protein","volume":"133","author":"Zhu","year":"1996","journal-title":"J. Cell Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"6074","DOI":"10.1016\/j.vaccine.2006.05.023","article-title":"Wave microcarrier cultivation of MDCK cells for influenza virus production in serum containing and serum-free media","volume":"24","author":"Genzel","year":"2006","journal-title":"Vaccine"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"5736","DOI":"10.1016\/j.vaccine.2008.08.015","article-title":"Microcarrier-based MDCK cell culture system for the production of influenza H5N1 vaccines","volume":"26","author":"Hu","year":"2008","journal-title":"Vaccine"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3062","DOI":"10.1002\/bit.28210","article-title":"Challenges and opportunities in downstream separation processes for mesenchymal stromal cells cultured in microcarrier-based stirred suspension bioreactors","volume":"119","author":"Mawji","year":"2022","journal-title":"Biotechnol. Bioeng."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1007\/978-1-0716-2376-3_2","article-title":"Culturing Suspension Cancer Cell Lines","volume":"2508","author":"Longjohn","year":"2022","journal-title":"Methods Mol. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1049\/enb2.12015","article-title":"Recent advances of integrated microfluidic suspension cell culture system","volume":"5","author":"Kerk","year":"2021","journal-title":"Eng. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"106154","DOI":"10.1016\/j.pep.2022.106154","article-title":"Adaptation process of engineered cell line FCHO\/IL-24 stably secreted rhIL-24 in serum-free suspension culture","volume":"199","author":"Hua","year":"2022","journal-title":"Protein Expr. Purif."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"468","DOI":"10.1080\/14760584.2023.2214219","article-title":"Suspended cell lines for inactivated virus vaccine production","volume":"22","author":"Zhang","year":"2023","journal-title":"Expert Rev. Vaccines"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1034","DOI":"10.1016\/j.bbrc.2005.01.066","article-title":"Transient strong reduction of PTEN expression by specific RNAi induces loss of adhesion of the cells","volume":"328","author":"Obata","year":"2005","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Calamita, G. (2023). Advances in Aquaporins. Cells, 12.","DOI":"10.3390\/cells12020303"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1007\/978-981-19-7415-1_3","article-title":"Expression Regulation and Trafficking of Aquaporins","volume":"1398","author":"Zhao","year":"2023","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"4443","DOI":"10.1002\/jcp.29320","article-title":"Aquaporin-2 and Na(+)\/H(+) exchanger isoform 1 modulate the efficiency of renal cell migration","volume":"235","author":"Pizzoni","year":"2020","journal-title":"J. Cell. Physiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"6980","DOI":"10.1096\/fj.201802068RR","article-title":"Aquaporins differentially regulate cell-cell adhesion in MDCK cells","volume":"33","author":"Login","year":"2019","journal-title":"FASEB J."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1492","DOI":"10.1016\/j.bbagen.2013.09.033","article-title":"Human aquaporins: Regulators of transcellular water flow","volume":"1840","author":"Day","year":"2014","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"7955","DOI":"10.3390\/cimb46080470","article-title":"Aquaporin Modulation by Cations, a Review","volume":"46","author":"Mom","year":"2024","journal-title":"Curr. Issues Mol. Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e12978","DOI":"10.1111\/ahe.12978","article-title":"Distributions and expressions of Aquaporin-5 and 7 in the testes of developing male chicks","volume":"53","author":"Kara","year":"2024","journal-title":"Anat. Histol. Embryol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"104560","DOI":"10.1016\/j.psj.2024.104560","article-title":"Water homeostasis gene expression in the kidney of broilers divergently selected for water conversion ratio","volume":"104","author":"Lassiter","year":"2025","journal-title":"Poult. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2208683","DOI":"10.1080\/22221751.2023.2208683","article-title":"Pteropine orthoreoviruses use cell surface heparan sulphate as an attachment receptor","volume":"12","author":"Tan","year":"2023","journal-title":"Emerg. Microbes Infect."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2026","DOI":"10.1080\/15384101.2019.1637202","article-title":"Inhibitory effect of AQP1 silencing on adhesion and angiogenesis in ectopic endometrial cells of mice with endometriosis through activating the Wnt signaling pathway","volume":"18","author":"Shu","year":"2019","journal-title":"Cell Cycle"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"L489","DOI":"10.1152\/ajplung.00410.2015","article-title":"Influence of AQP1 on cell adhesion, migration, and tumor sphere formation in malignant pleural mesothelioma is substratum- and histological-type dependent","volume":"310","author":"Jagirdar","year":"2016","journal-title":"Am. J. Physiol. Lung Cell. Mol. Physiol."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Zhang, W., Wang, X., Gao, Y., and Qi, X. (2022). The Over-40-Years-Epidemic of Infectious Bursal Disease Virus in China. Viruses, 14.","DOI":"10.3390\/v14102253"},{"key":"ref_37","first-page":"410","article-title":"Pros and Cons on Use of Live Viral Vaccines in Commercial Chicken Flocks","volume":"67","author":"Ganapathy","year":"2024","journal-title":"Avian Dis."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/j.vetmic.2016.11.022","article-title":"Advances in vaccine research against economically important viral diseases of food animals: Infectious bursal disease virus","volume":"206","author":"Jackwood","year":"2017","journal-title":"Vet. Microbiol."},{"key":"ref_39","first-page":"456","article-title":"Veterinary Autogenous Vaccines for Poultry in Europe-Many Ways to Crack an Egg","volume":"67","author":"Sulejmanovic","year":"2024","journal-title":"Avian Dis."},{"key":"ref_40","first-page":"261","article-title":"Immunogenicity and Efficacy Evaluation of Vero Cell-Adapted Infectious Bursal Disease Virus LC-75 Vaccine Strain","volume":"12","author":"Kebede","year":"2021","journal-title":"Vet. Med."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1943","DOI":"10.1007\/s00705-021-05099-7","article-title":"Protective effects of the NLRP3 inflammasome against infectious bursal disease virus replication in DF-1 cells","volume":"166","author":"He","year":"2021","journal-title":"Arch. Virol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"108620","DOI":"10.1016\/j.vetmic.2020.108620","article-title":"Naturally occurring cell-adapted classic strain of infectious bursal disease virus","volume":"243","author":"Wang","year":"2020","journal-title":"Vet. Microbiol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1016\/j.jbiosc.2024.02.005","article-title":"Establishment of a novel cell line, CHO-MK, derived from Chinese hamster ovary tissues for biologics manufacturing","volume":"137","author":"Masuda","year":"2024","journal-title":"J. Biosci. Bioeng."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Rodrigues, A.F., Fernandes, P., Laske, T., Castro, R., Alves, P.M., Genzel, Y., and Coroadinha, A.S. (2020). Cell Bank Origin of MDCK Parental Cells Shapes Adaptation to Serum-Free Suspension Culture and Canine Adenoviral Vector Production. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21176111"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Chia, M.Y., Lin, C.Y., Chen, P.L., Lai, C.C., Weng, T.C., Sung, W.C., Hu, A.Y.-C., and Lee, M.-S. (2022). Characterization and Immunogenicity of Influenza H7N9 Vaccine Antigens Produced Using a Serum-Free Suspension MDCK Cell-Based Platform. Viruses, 14.","DOI":"10.3390\/v14091937"},{"key":"ref_46","first-page":"240","article-title":"Enhancing recombinant antibody yield in Chinese hamster ovary cells","volume":"36","author":"Yang","year":"2024","journal-title":"Tzu Chi Med. J."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/1\/52\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,5]],"date-time":"2026-01-05T13:54:49Z","timestamp":1767621289000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/1\/52"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,31]]},"references-count":46,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["vaccines14010052"],"URL":"https:\/\/doi.org\/10.3390\/vaccines14010052","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,31]]}}}