{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,23]],"date-time":"2026-08-23T18:10:31Z","timestamp":1787508631568,"version":"build-2736575974"},"reference-count":73,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2025,5,5]],"date-time":"2025-05-05T00:00:00Z","timestamp":1746403200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2025,5,5]],"date-time":"2025-05-05T00:00:00Z","timestamp":1746403200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100003624","name":"Ministry of Agriculture, Food and Rural Affairs","doi-asserted-by":"crossref","award":["RS-2024-00399869"],"award-info":[{"award-number":["RS-2024-00399869"]}],"id":[{"id":"10.13039\/501100003624","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003624","name":"Ministry of Agriculture, Food and Rural Affairs","doi-asserted-by":"crossref","award":["RS-2024-00399869"],"award-info":[{"award-number":["RS-2024-00399869"]}],"id":[{"id":"10.13039\/501100003624","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003624","name":"Ministry of Agriculture, Food and Rural Affairs","doi-asserted-by":"crossref","award":["RS-2024-00399869"],"award-info":[{"award-number":["RS-2024-00399869"]}],"id":[{"id":"10.13039\/501100003624","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003624","name":"Ministry of Agriculture, Food and Rural Affairs","doi-asserted-by":"crossref","award":["RS-2024-00399869"],"award-info":[{"award-number":["RS-2024-00399869"]}],"id":[{"id":"10.13039\/501100003624","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003624","name":"Ministry of Agriculture, Food and Rural Affairs","doi-asserted-by":"crossref","award":["RS-2024-00399869"],"award-info":[{"award-number":["RS-2024-00399869"]}],"id":[{"id":"10.13039\/501100003624","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"crossref","award":["NRF-2022M3A9I2017587"],"award-info":[{"award-number":["NRF-2022M3A9I2017587"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"crossref","award":["NRF-2022M3A9I2017587"],"award-info":[{"award-number":["NRF-2022M3A9I2017587"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"crossref","award":["NRF-2022M3A9I2017587"],"award-info":[{"award-number":["NRF-2022M3A9I2017587"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"crossref","award":["NRF-2022M3A9I2017587"],"award-info":[{"award-number":["NRF-2022M3A9I2017587"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"crossref","award":["NRF-2022M3A9I2017587"],"award-info":[{"award-number":["NRF-2022M3A9I2017587"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"crossref","award":["NRF-2022M3A9I2017587"],"award-info":[{"award-number":["NRF-2022M3A9I2017587"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Animal and Plant Quarantine Agency in the Republic of Korea","award":["Z-1543418-2021-22-0102"],"award-info":[{"award-number":["Z-1543418-2021-22-0102"]}]},{"name":"Animal and Plant Quarantine Agency in the Republic of Korea","award":["Z-1543418-2021-22-0102"],"award-info":[{"award-number":["Z-1543418-2021-22-0102"]}]},{"name":"Animal and Plant Quarantine Agency in the Republic of Korea","award":["Z-1543418-2021-22-0102"],"award-info":[{"award-number":["Z-1543418-2021-22-0102"]}]},{"name":"Animal and Plant Quarantine Agency in the Republic of Korea","award":["Z-1543418-2021-22-0102"],"award-info":[{"award-number":["Z-1543418-2021-22-0102"]}]},{"name":"Animal and Plant Quarantine Agency in the Republic of Korea","award":["Z-1543418-2021-22-0102"],"award-info":[{"award-number":["Z-1543418-2021-22-0102"]}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Virol J"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The global spread, frequent antigenic changes, and pandemic potential of clade 2.3.4.4b highly pathogenic avian influenza H5N1 underscore the urgent need for robust cross-protective vaccines. Here, we developed a clade 2.3.4.4b H5N1 whole inactivated virus (WIV) vaccine strain with improved structural stability, productivity, and safety. By analyzing the evolutionary trends of clade 2.3.4.4b H5N1 viruses, we identified a key mutation (R90K) that increases heat stability while preserving antigenicity. Additionally, the PB2 gene of PR8 was replaced with a prototypical avian PB2 gene to increase replication efficiency in embryonated chicken eggs and reduce replication efficiency in mammalian cells, thereby improving productivity and biosafety. We found that our optimized clade 2.3.4.4b H5N1 vaccine strain (22W_KY), inactivated with binary ethylenimine (BEI), had superior antigen internalization into respiratory epithelial cells compared to those inactivated with formaldehyde or beta-propiolactone. Following intranasal administration to mice, the BEI-inactivated 22W_KY also elicited significantly stronger systemic IgG, mucosal IgA, and T-cell responses, especially in the lungs. Protective efficacy studies revealed that the BEI-inactivated 22W_KY vaccine provided complete protection against heterologous viral challenges and significant protection against heterosubtypic viral challenges, with no weight loss and complete suppression of the viral load in the respiratory tract in 2 of 3 mice. These results indicate that the BEI-inactivated 22W_KY vaccine could serve as a promising candidate for a safe, stable, cost-efficient, and broadly protective intranasal influenza vaccine against zoonotic and pandemic threats.<\/jats:p>","DOI":"10.1186\/s12985-025-02760-4","type":"journal-article","created":{"date-parts":[[2025,5,4]],"date-time":"2025-05-04T23:01:06Z","timestamp":1746399666000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Intranasally administered whole virion inactivated vaccine against clade 2.3.4.4b H5N1 influenza virus with optimized antigen and increased cross-protection"],"prefix":"10.1186","volume":"22","author":[{"given":"Jin-Ha","family":"Song","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seung-Eun","family":"Son","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ho-Won","family":"Kim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seung-Ji","family":"Kim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Se-Hee","family":"An","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chung-Young","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hyuk-Joon","family":"Kwon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kang-Seuk","family":"Choi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,5,5]]},"reference":[{"issue":"S1","key":"2760_CR1","doi-asserted-by":"publisher","first-page":"269","DOI":"10.4142\/jvs.2017.18.S1.269","volume":"18","author":"D-H Lee","year":"2017","unstructured":"Lee D-H, Bertran K, Kwon J-H, Swayne DE. Evolution, global spread, and pathogenicity of highly pathogenic avian influenza H5Nx clade 2.3.4.4. J Vet Sci. 2017;18(S1):269\u201380.","journal-title":"J Vet Sci"},{"issue":"7984","key":"2760_CR2","doi-asserted-by":"publisher","first-page":"810","DOI":"10.1038\/s41586-023-06631-2","volume":"622","author":"R Xie","year":"2023","unstructured":"Xie R, Edwards KM, Wille M, Wei X, Wong S-S, Zanin M, et al. The episodic resurgence of highly pathogenic avian influenza H5 virus. Nature. 2023;622(7984):810\u20137.","journal-title":"Nature"},{"issue":"12","key":"2760_CR3","doi-asserted-by":"publisher","first-page":"1389","DOI":"10.3390\/v12121389","volume":"12","author":"S Jeong","year":"2020","unstructured":"Jeong S, Lee D-H, Kwon J-H, Kim Y-J, Lee S-H, Cho AY, et al. Highly pathogenic avian influenza clade 2.3.4.4b subtype H5N8 virus isolated from Mandarin Duck in South Korea, 2020. Viruses. 2020;12(12):1389.","journal-title":"Viruses"},{"issue":"1","key":"2760_CR4","doi-asserted-by":"publisher","first-page":"58","DOI":"10.1038\/s41421-023-00571-x","volume":"9","author":"P Huang","year":"2023","unstructured":"Huang P, Sun L, Li J, Wu Q, Rezaei N, Jiang S, et al. Potential cross-species transmission of highly pathogenic avian influenza H5 subtype (HPAI H5) viruses to humans calls for the development of H5-specific and universal influenza vaccines. Cell Discovery. 2023;9(1):58.","journal-title":"Cell Discovery"},{"key":"2760_CR5","doi-asserted-by":"crossref","unstructured":"Webby RJ, Uyeki TM. An update on highly pathogenic avian influenza a (H5N1) virus, clade 2.3. 4.4 B. J Infect Dis. 2024:jiae379.","DOI":"10.1093\/infdis\/jiae379"},{"issue":"1","key":"2760_CR6","doi-asserted-by":"publisher","first-page":"3082","DOI":"10.1038\/s41467-023-38415-7","volume":"14","author":"A Kandeil","year":"2023","unstructured":"Kandeil A, Patton C, Jones JC, Jeevan T, Harrington WN, Trifkovic S, et al. Rapid evolution of A (H5N1) influenza viruses after intercontinental spread to North America. Nat Commun. 2023;14(1):3082.","journal-title":"Nat Commun"},{"issue":"3","key":"2760_CR7","doi-asserted-by":"publisher","first-page":"2300001","DOI":"10.2807\/1560-7917.ES.2023.28.3.2300001","volume":"28","author":"M Ag\u00fcero","year":"2023","unstructured":"Ag\u00fcero M, Monne I, S\u00e1nchez A, Zecchin B, Fusaro A, Ruano MJ, et al. Highly pathogenic avian influenza A(H5N1) virus infection in farmed Minks, Spain, October 2022. Eurosurveillance. 2023;28(3):2300001.","journal-title":"Eurosurveillance"},{"issue":"12","key":"2760_CR8","doi-asserted-by":"publisher","first-page":"2451","DOI":"10.3201\/eid2912.230464","volume":"29","author":"EJ Elsmo","year":"2023","unstructured":"Elsmo EJ, W\u00fcnschmann A, Beckmen KB, Broughton-Neiswanger LE, Buckles EL, Ellis J, et al. Highly pathogenic avian influenza A (H5N1) virus clade 2.3. 4.4 B infections in wild terrestrial mammals, united States, 2022. Emerg Infect Dis. 2023;29(12):2451.","journal-title":"Emerg Infect Dis"},{"issue":"8","key":"2760_CR9","doi-asserted-by":"publisher","first-page":"1660","DOI":"10.3201\/eid3008.240481","volume":"30","author":"R Stimmelmayr","year":"2024","unstructured":"Stimmelmayr R, Rotstein D, Torchetti MK, Gerlach R. Highly pathogenic avian influenza virus A (H5N1) clade 2.3. 4.4 B infection in Free-Ranging Polar Bear, Alaska, USA. Emerg Infect Dis. 2024;30(8):1660.","journal-title":"Emerg Infect Dis"},{"issue":"1","key":"2760_CR10","doi-asserted-by":"publisher","first-page":"5489","DOI":"10.1038\/s41467-023-41182-0","volume":"14","author":"M Leguia","year":"2023","unstructured":"Leguia M, Garcia-Glaessner A, Mu\u00f1oz-Saavedra B, Juarez D, Barrera P, Calvo-Mac C, et al. Highly pathogenic avian influenza A (H5N1) in marine mammals and seabirds in Peru. Nat Commun. 2023;14(1):5489.","journal-title":"Nat Commun"},{"issue":"4","key":"2760_CR11","doi-asserted-by":"publisher","first-page":"786","DOI":"10.3201\/eid2904.221538","volume":"29","author":"W Puryear","year":"2023","unstructured":"Puryear W, Sawatzki K, Hill N, Foss A, Stone JJ, Doughty L, et al. Highly pathogenic avian influenza A (H5N1) virus outbreak in new England seals, united States. Emerg Infect Dis. 2023;29(4):786.","journal-title":"Emerg Infect Dis"},{"issue":"1","key":"2760_CR12","doi-asserted-by":"publisher","first-page":"9516","DOI":"10.1038\/s41467-024-53766-5","volume":"15","author":"MM Uhart","year":"2024","unstructured":"Uhart MM, Vanstreels RE, Nelson MI, Olivera V, Campagna J, Zavattieri V, et al. Epidemiological data of an influenza A\/H5N1 outbreak in elephant seals in Argentina indicates mammal-to-mammal transmission. Nat Commun. 2024;15(1):9516.","journal-title":"Nat Commun"},{"key":"2760_CR13","doi-asserted-by":"crossref","unstructured":"Pulit-Penaloza JA, Belser JA, Brock N, Kieran TJ, Sun X, Pappas C et al. Transmission of a human isolate of clade 2.3. 4.4 B A (H5N1) virus in ferrets. Nature. 2024:1\u20133.","DOI":"10.1038\/s41586-024-08246-7"},{"key":"2760_CR14","doi-asserted-by":"crossref","unstructured":"Peacock T, Moncla L, Dudas G, VanInsberghe D, Sukhova K, Lloyd-Smith JO et al. The global H5N1 influenza panzootic in mammals. Nature. 2024:1\u20132.","DOI":"10.1038\/s41586-024-08054-z"},{"issue":"6","key":"2760_CR15","doi-asserted-by":"publisher","first-page":"e0128982","DOI":"10.1371\/journal.pone.0128982","volume":"10","author":"A Johnson","year":"2015","unstructured":"Johnson A, Chen L-M, Winne E, Santana W, Metcalfe MG, Mateu-Petit G, et al. Identification of influenza A\/PR\/8\/34 donor viruses imparting high hemagglutinin yields to candidate vaccine viruses in eggs. PLoS ONE. 2015;10(6):e0128982.","journal-title":"PLoS ONE"},{"issue":"1","key":"2760_CR16","doi-asserted-by":"publisher","first-page":"10205","DOI":"10.1038\/s41598-017-09560-z","volume":"7","author":"C-Y Lee","year":"2017","unstructured":"Lee C-Y, An S-H, Kim I, Go D-M, Kim D-Y, Choi J-G, et al. Prerequisites for the acquisition of mammalian pathogenicity by influenza A virus with a prototypic avian PB2 gene. Sci Rep. 2017;7(1):10205.","journal-title":"Sci Rep"},{"issue":"S1","key":"2760_CR17","doi-asserted-by":"publisher","first-page":"299","DOI":"10.4142\/jvs.2017.18.S1.299","volume":"18","author":"J-W Jang","year":"2017","unstructured":"Jang J-W, Lee C-Y, Kim I-h, Choi J-G, Lee Y-J, Yuk S-S, et al. Optimized clade 2.3. 2.1 C H5N1 recombinant-vaccine strains against highly pathogenic avian influenza. J Vet Sci. 2017;18(S1):299\u2013306.","journal-title":"J Vet Sci"},{"issue":"2","key":"2760_CR18","doi-asserted-by":"publisher","first-page":"162","DOI":"10.3390\/vaccines10020162","volume":"10","author":"S-H An","year":"2022","unstructured":"An S-H, Son S-E, Song J-H, Hong S-M, Lee C-Y, Lee N-H, et al. Selection of an optimal Recombinant Egyptian H9N2 avian influenza vaccine strain for poultry with high antigenicity and safety. Vaccines. 2022;10(2):162.","journal-title":"Vaccines"},{"issue":"10","key":"2760_CR19","doi-asserted-by":"publisher","first-page":"8840","DOI":"10.3390\/ijms24108840","volume":"24","author":"S-H An","year":"2023","unstructured":"An S-H, Hong S-M, Song J-H, Son S-E, Lee C-Y, Choi K-S, et al. Engineering an optimal Y280-Lineage H9N2 vaccine strain by tuning PB2 activity. Int J Mol Sci. 2023;24(10):8840.","journal-title":"Int J Mol Sci"},{"issue":"5","key":"2760_CR20","doi-asserted-by":"publisher","first-page":"315","DOI":"10.1038\/nri3665","volume":"14","author":"A Iwasaki","year":"2014","unstructured":"Iwasaki A, Pillai PS. Innate immunity to influenza virus infection. Nat Rev Immunol. 2014;14(5):315\u201328.","journal-title":"Nat Rev Immunol"},{"issue":"6","key":"2760_CR21","doi-asserted-by":"publisher","first-page":"936","DOI":"10.1002\/eji.202249823","volume":"52","author":"M Cokari\u0107 Brdov\u010dak","year":"2022","unstructured":"Cokari\u0107 Brdov\u010dak M, Materljan J, \u0160usti\u0107 M, Ravli\u0107 S, Ru\u017ei\u0107 T, Lisni\u0107 B, et al. ChAdOx1-S adenoviral vector vaccine applied intranasally elicits superior mucosal immunity compared to the intramuscular route of vaccination. Eur J Immunol. 2022;52(6):936\u201345.","journal-title":"Eur J Immunol"},{"issue":"2","key":"2760_CR22","doi-asserted-by":"publisher","first-page":"e52","DOI":"10.1016\/S1473-3099(21)00472-2","volume":"22","author":"D Mostaghimi","year":"2022","unstructured":"Mostaghimi D, Valdez CN, Larson HT, Kalinich CC, Iwasaki A. Prevention of host-to-host transmission by SARS-CoV-2 vaccines. Lancet Infect Dis. 2022;22(2):e52\u20138.","journal-title":"Lancet Infect Dis"},{"issue":"4","key":"2760_CR23","doi-asserted-by":"publisher","first-page":"236","DOI":"10.1038\/s41577-021-00583-2","volume":"22","author":"EC Lavelle","year":"2022","unstructured":"Lavelle EC, Ward RW. Mucosal vaccines\u2014fortifying the frontiers. Nat Rev Immunol. 2022;22(4):236\u201350.","journal-title":"Nat Rev Immunol"},{"key":"2760_CR24","doi-asserted-by":"publisher","first-page":"121180","DOI":"10.1016\/j.ijpharm.2021.121180","volume":"609","author":"H Xu","year":"2021","unstructured":"Xu H, Cai L, Hufnagel S, Cui Z. Intranasal vaccine: factors to consider in research and development. Int J Pharm. 2021;609:121180.","journal-title":"Int J Pharm"},{"issue":"8","key":"2760_CR25","doi-asserted-by":"publisher","first-page":"592","DOI":"10.1038\/nri3251","volume":"12","author":"N Lycke","year":"2012","unstructured":"Lycke N. Recent progress in mucosal vaccine development: potential and limitations. Nat Rev Immunol. 2012;12(8):592\u2013605.","journal-title":"Nat Rev Immunol"},{"issue":"5","key":"2760_CR26","doi-asserted-by":"publisher","first-page":"595","DOI":"10.1586\/erv.12.31","volume":"11","author":"MA Rose","year":"2012","unstructured":"Rose MA, Zielen S, Baumann U. Mucosal immunity and nasal influenza vaccination. Expert Rev Vaccines. 2012;11(5):595\u2013607.","journal-title":"Expert Rev Vaccines"},{"issue":"1","key":"2760_CR27","doi-asserted-by":"publisher","first-page":"8053","DOI":"10.1038\/s41467-023-43842-7","volume":"14","author":"RS Thwaites","year":"2023","unstructured":"Thwaites RS, Uruchurtu AS, Negri VA, Cole ME, Singh N, Poshai N, et al. Early mucosal events promote distinct mucosal and systemic antibody responses to live attenuated influenza vaccine. Nat Commun. 2023;14(1):8053.","journal-title":"Nat Commun"},{"issue":"8","key":"2760_CR28","doi-asserted-by":"publisher","first-page":"1316","DOI":"10.1038\/s41564-019-0443-4","volume":"4","author":"SC David","year":"2019","unstructured":"David SC, Norton T, Tyllis T, Wilson JJ, Singleton EV, Laan Z, et al. Direct interaction of whole-inactivated influenza A and Pneumococcal vaccines enhances influenza-specific immunity. Nat Microbiol. 2019;4(8):1316\u201327.","journal-title":"Nat Microbiol"},{"issue":"11","key":"2760_CR29","doi-asserted-by":"publisher","first-page":"5904","DOI":"10.1128\/JVI.00296-15","volume":"89","author":"T Qin","year":"2015","unstructured":"Qin T, Yin Y, Yu Q, Huang L, Wang X, Lin J, et al. CpG oligodeoxynucleotides facilitate delivery of whole inactivated H9N2 influenza virus via transepithelial dendrites of dendritic cells in nasal mucosa. J Virol. 2015;89(11):5904\u201318.","journal-title":"J Virol"},{"issue":"7","key":"2760_CR30","doi-asserted-by":"publisher","first-page":"2351","DOI":"10.1016\/j.biomaterials.2011.11.068","volume":"33","author":"Y Wu","year":"2012","unstructured":"Wu Y, Wei W, Zhou M, Wang Y, Wu J, Ma G, et al. Thermal-sensitive hydrogel as adjuvant-free vaccine delivery system for H5N1 intranasal immunization. Biomaterials. 2012;33(7):2351\u201360.","journal-title":"Biomaterials"},{"key":"2760_CR31","doi-asserted-by":"publisher","first-page":"1132129","DOI":"10.3389\/fimmu.2023.1132129","volume":"14","author":"J Liu","year":"2023","unstructured":"Liu J, Yan X-D, Li X-Q, Du Y-H, Zhu L-L, Ye T-T, et al. Chrysanthemum Sporopollenin: A novel vaccine delivery system for nasal mucosal immunity. Front Immunol. 2023;14:1132129.","journal-title":"Front Immunol"},{"issue":"13","key":"2760_CR32","doi-asserted-by":"publisher","first-page":"30494","DOI":"10.2807\/1560-7917.ES.2017.22.13.30494","volume":"22","author":"Y Shu","year":"2017","unstructured":"Shu Y, McCauley J. GISAID: global initiative on sharing all influenza data\u2013from vision to reality. Eurosurveillance. 2017;22(13):30494.","journal-title":"Eurosurveillance"},{"key":"2760_CR33","doi-asserted-by":"crossref","unstructured":"Kwon H-J, Cho S-H, Ahn Y-J, Kim J-H, Yoo H-S, Kim S-J. Characterization of a chicken embryo-adapted H9N2 subtype avian influenza virus. Open Veterinary Sci J. 2009;3(1).","DOI":"10.2174\/1874318800903010009"},{"issue":"1","key":"2760_CR34","doi-asserted-by":"publisher","first-page":"5359","DOI":"10.1038\/s41598-020-62036-5","volume":"10","author":"C-Y Lee","year":"2020","unstructured":"Lee C-Y, An S-H, Choi J-G, Lee Y-J, Kim J-H, Kwon H-J. Rank orders of mammalian pathogenicity-related PB2 mutations of avian influenza A viruses. Sci Rep. 2020;10(1):5359.","journal-title":"Sci Rep"},{"key":"2760_CR35","doi-asserted-by":"crossref","unstructured":"Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG. A DNA transfection system for generation of influenza A virus from eight plasmids. Proceedings of the National Academy of Sciences. 2000;97(11):6108-13.","DOI":"10.1073\/pnas.100133697"},{"issue":"7","key":"2760_CR36","doi-asserted-by":"publisher","first-page":"714","DOI":"10.1021\/es60130a004","volume":"11","author":"MA Hamilton","year":"1977","unstructured":"Hamilton MA, Russo RC, Thurston RV. Trimmed Spearman-Karber method for estimating median lethal concentrations in toxicity bioassays. Environ Sci Technol. 1977;11(7):714\u20139.","journal-title":"Environ Sci Technol"},{"issue":"6","key":"2760_CR37","doi-asserted-by":"publisher","first-page":"e02011","DOI":"10.1128\/JVI.02011-18","volume":"93","author":"AO Obadan","year":"2019","unstructured":"Obadan AO, Santos J, Ferreri L, Thompson AJ, Carnaccini S, Geiger G, et al. Flexibility\u2009<\u2009i\u2009>\u2009In vitro\u2009of amino acid 226 in the Receptor-Binding site of an H9 subtype influenza A virus and its effect\u2009<\u2009i\u2009>\u2009in vivo\u2009on virus replication, tropism, and transmission. J Virol. 2019;93(6):e02011\u201318.","journal-title":"J Virol"},{"key":"2760_CR38","doi-asserted-by":"crossref","unstructured":"Qin C, Li W, Li Q, Yin W, Zhang X, Zhang Z et al. Real-time dissection of dynamic uncoating of individual influenza viruses. Proceedings of the National Academy of Sciences. 2019;116(7):2577-82.","DOI":"10.1073\/pnas.1812632116"},{"issue":"22","key":"2760_CR39","doi-asserted-by":"publisher","first-page":"13029","DOI":"10.1128\/JVI.01430-14","volume":"88","author":"S Stauffer","year":"2014","unstructured":"Stauffer S, Feng Y, Nebioglu F, Heilig R, Picotti P, Helenius A. Stepwise priming by acidic pH and a high K\u2009+\u2009concentration is required for efficient uncoating of influenza A virus cores after penetration. J Virol. 2014;88(22):13029\u201346.","journal-title":"J Virol"},{"issue":"2","key":"2760_CR40","doi-asserted-by":"publisher","first-page":"01707","DOI":"10.1128\/jvi","volume":"94","author":"G Wang","year":"2020","unstructured":"Wang G, Jiang L, Wang J, Zhang J, Kong F, Li Q, et al. The G protein-coupled receptor FFAR2 promotes internalization during influenza A virus entry. J Virol. 2020;94(2):01707\u201319. https:\/\/doi.org\/10.1128\/jvi.","journal-title":"J Virol"},{"key":"2760_CR41","first-page":"100916","volume":"25","author":"MH Shihan","year":"2021","unstructured":"Shihan MH, Novo SG, Le Marchand SJ, Wang Y, Duncan MK. A simple method for quantitating confocal fluorescent images. Biochem Biophys Rep. 2021;25:100916.","journal-title":"Biochem Biophys Rep"},{"key":"2760_CR42","unstructured":"Organization WH. WHO manual on animal influenza diagnosis and surveillance. World Health Organization; 2002."},{"key":"2760_CR43","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1016\/j.jviromet.2014.09.003","volume":"210","author":"L Couzens","year":"2014","unstructured":"Couzens L, Gao J, Westgeest K, Sandbulte M, Lugovtsev V, Fouchier R, et al. An optimized enzyme-linked lectin assay to measure influenza A virus neuraminidase Inhibition antibody titers in human Sera. J Virol Methods. 2014;210:7\u201314.","journal-title":"J Virol Methods"},{"issue":"4","key":"2760_CR44","doi-asserted-by":"publisher","first-page":"781","DOI":"10.3390\/vaccines8040781","volume":"8","author":"S-H An","year":"2020","unstructured":"An S-H, Hong S-M, Son S-E, Song J-H, Lee C-Y, Choi J-G, et al. Improvement of PR8-Derived Recombinant clade 2.3.4.4c H5N6 vaccine strains by optimization of internal genes and H103Y mutation of hemagglutinin. Vaccines. 2020;8(4):781.","journal-title":"Vaccines"},{"issue":"24","key":"2760_CR45","doi-asserted-by":"publisher","first-page":"3589","DOI":"10.1016\/j.vaccine.2023.05.011","volume":"41","author":"E Kehagia","year":"2023","unstructured":"Kehagia E, Papakyriakopoulou P, Valsami G. Advances in intranasal vaccine delivery: A promising non-invasive route of immunization. Vaccine. 2023;41(24):3589\u2013603.","journal-title":"Vaccine"},{"key":"2760_CR46","doi-asserted-by":"crossref","unstructured":"Caserta LC, Frye EA, Butt SL, Laverack M, Nooruzzaman M, Covaleda LM et al. Spillover of highly pathogenic avian influenza H5N1 virus to dairy cattle. Nature. 2024:1\u20138.","DOI":"10.1038\/s41586-024-07849-4"},{"issue":"2","key":"2760_CR47","doi-asserted-by":"publisher","first-page":"299","DOI":"10.3201\/eid3002.231274","volume":"30","author":"Y-R Seo","year":"2024","unstructured":"Seo Y-R, Cho AY, Si Y-J, Lee S-I, Kim D-J, Jeong H, et al. Evolution and spread of highly pathogenic avian influenza A (H5N1) clade 2.3. 4.4 B virus in wild birds, South Korea, 2022\u20132023. Emerg Infect Dis. 2024;30(2):299.","journal-title":"Emerg Infect Dis"},{"issue":"4","key":"2760_CR48","doi-asserted-by":"publisher","first-page":"1153","DOI":"10.1016\/j.bbamem.2013.10.004","volume":"1838","author":"CM Mair","year":"2014","unstructured":"Mair CM, Ludwig K, Herrmann A, Sieben C. Receptor binding and pH stability\u2014How influenza A virus hemagglutinin affects host-specific virus infection. Biochim Et Biophys Acta (BBA)-Biomembranes. 2014;1838(4):1153\u201368.","journal-title":"Biochim Et Biophys Acta (BBA)-Biomembranes"},{"issue":"3","key":"2760_CR49","doi-asserted-by":"publisher","first-page":"1569","DOI":"10.1128\/JVI.02332-15","volume":"90","author":"J Baumann","year":"2016","unstructured":"Baumann J, Kouassi NM, Foni E, Klenk H-D, Matrosovich M. H1N1 swine influenza viruses differ from avian precursors by a higher pH optimum of membrane fusion. J Virol. 2016;90(3):1569\u201377.","journal-title":"J Virol"},{"issue":"20","key":"2760_CR50","doi-asserted-by":"publisher","first-page":"10602","DOI":"10.1128\/JVI.00939-15","volume":"89","author":"W Wang","year":"2015","unstructured":"Wang W, DeFeo CJ, Alvarado-Facundo E, Vassell R, Weiss CD. Intermonomer interactions in hemagglutinin subunits HA1 and HA2 affecting hemagglutinin stability and influenza virus infectivity. J Virol. 2015;89(20):10602\u201311.","journal-title":"J Virol"},{"issue":"4","key":"2760_CR51","doi-asserted-by":"publisher","first-page":"995","DOI":"10.1096\/fj.06-7052hyp","volume":"21","author":"PS Rachakonda","year":"2007","unstructured":"Rachakonda PS, Veit M, Korte T, Ludwig K, B\u00f6ttcher C, Huang Q, et al. The relevance of salt bridges for the stability of the influenza virus hemagglutinin. FASEB J. 2007;21(4):995\u20131002.","journal-title":"FASEB J"},{"issue":"6139","key":"2760_CR52","doi-asserted-by":"publisher","first-page":"1463","DOI":"10.1126\/science.1236787","volume":"340","author":"W Zhang","year":"2013","unstructured":"Zhang W, Shi Y, Lu X, Shu Y, Qi J, Gao GF. An airborne transmissible avian influenza H5 hemagglutinin seen at the atomic level. Science. 2013;340(6139):1463\u20137.","journal-title":"Science"},{"issue":"2","key":"2760_CR53","doi-asserted-by":"publisher","first-page":"218","DOI":"10.1002\/jps.10296","volume":"92","author":"DT Brandau","year":"2003","unstructured":"Brandau DT, Jones LS, Wiethoff CM, Rexroad J, Middaugh CR. Thermal stability of vaccines. J Pharm Sci. 2003;92(2):218\u201331.","journal-title":"J Pharm Sci"},{"issue":"2","key":"2760_CR54","doi-asserted-by":"publisher","first-page":"e01379","DOI":"10.1128\/jvi.01379-22","volume":"97","author":"Z Wan","year":"2023","unstructured":"Wan Z, Zhao Z, Sang J, Jiang W, Chen J, Tang T, et al. Amino acid variation at hemagglutinin position 193 impacts the properties of H9N2 avian influenza virus. J Virol. 2023;97(2):e01379\u201322.","journal-title":"J Virol"},{"issue":"1","key":"2760_CR55","doi-asserted-by":"publisher","first-page":"152","DOI":"10.1038\/s41541-024-00947-4","volume":"9","author":"Y Zhang","year":"2024","unstructured":"Zhang Y, Cui P, Shi J, Zeng X, Jiang Y, Chen Y, et al. A broad-spectrum vaccine candidate against H5 viruses bearing different sub-clade 2.3. 4.4 HA genes. Npj Vaccines. 2024;9(1):152.","journal-title":"Npj Vaccines"},{"issue":"17","key":"2760_CR56","doi-asserted-by":"publisher","first-page":"9904","DOI":"10.1128\/JVI.01023-13","volume":"87","author":"Y Li","year":"2013","unstructured":"Li Y, Bostick DL, Sullivan CB, Myers JL, Griesemer SB, StGeorge K, et al. Single hemagglutinin mutations that alter both antigenicity and receptor binding avidity influence influenza virus antigenic clustering. J Virol. 2013;87(17):9904\u201310.","journal-title":"J Virol"},{"key":"2760_CR57","doi-asserted-by":"crossref","unstructured":"Chou Y-y, Vafabakhsh R, Do\u011fanay S, Gao Q, Ha T, Palese P. One influenza virus particle packages eight unique viral RNAs as shown by FISH analysis. Proceedings of the National Academy of Sciences. 2012;109(23):9101-6.","DOI":"10.1073\/pnas.1206069109"},{"issue":"4","key":"2760_CR58","doi-asserted-by":"publisher","first-page":"e1009171","DOI":"10.1371\/journal.ppat.1009171","volume":"17","author":"J Gao","year":"2021","unstructured":"Gao J, Wan H, Li X, Rakic Martinez M, Klenow L, Gao Y, et al. Balancing the influenza neuraminidase and hemagglutinin responses by exchanging the vaccine virus backbone. PLoS Pathog. 2021;17(4):e1009171.","journal-title":"PLoS Pathog"},{"issue":"5","key":"2760_CR59","doi-asserted-by":"publisher","first-page":"824","DOI":"10.1128\/am.18.5.824-833.1969","volume":"18","author":"JC Hierholzer","year":"1969","unstructured":"Hierholzer JC, Suggs MT, Hall EC. Standardized viral hemagglutination and hemagglutination-inhibition tests. II. Description and statistical evaluation. Appl Microbiol. 1969;18(5):824\u201333.","journal-title":"Appl Microbiol"},{"issue":"1\u20132","key":"2760_CR60","doi-asserted-by":"publisher","first-page":"91","DOI":"10.1016\/j.virusres.2004.02.019","volume":"103","author":"I Stephenson","year":"2004","unstructured":"Stephenson I, Wood J, Nicholson K, Charlett A, Zambon M. Detection of anti-H5 responses in human Sera by HI using horse erythrocytes following MF59-adjuvanted influenza A\/Duck\/Singapore\/97 vaccine. Virus Res. 2004;103(1\u20132):91\u20135.","journal-title":"Virus Res"},{"issue":"778","key":"2760_CR61","doi-asserted-by":"publisher","first-page":"eads1273","DOI":"10.1126\/scitranslmed.ads1273","volume":"16","author":"M Hatta","year":"2024","unstructured":"Hatta M, Hatta Y, Choi A, Hossain J, Feng C, Keller MW, et al. An influenza mRNA vaccine protects ferrets from lethal infection with highly pathogenic avian influenza A (H5N1) virus. Sci Transl Med. 2024;16(778):eads1273.","journal-title":"Sci Transl Med"},{"key":"2760_CR62","doi-asserted-by":"publisher","first-page":"909297","DOI":"10.3389\/fimmu.2022.909297","volume":"13","author":"M-C Bernard","year":"2022","unstructured":"Bernard M-C, Waldock J, Commandeur S, Strau\u00df L, Trombetta CM, Marchi S, et al. Validation of a harmonized enzyme-Linked-Lectin-Assay (Ella-Ni) based neuraminidase Inhibition assay standard operating procedure (Sop) for quantification of N1 influenza antibodies and the use of a calibrator to improve the reproducibility of the Ella-Ni with reverse genetics viral and Recombinant neuraminidase antigens: A flucop collaborative study. Front Immunol. 2022;13:909297.","journal-title":"Front Immunol"},{"issue":"2","key":"2760_CR63","doi-asserted-by":"publisher","first-page":"1022","DOI":"10.4049\/jimmunol.0902147","volume":"186","author":"K El Bakkouri","year":"2011","unstructured":"El Bakkouri K, Descamps F, De Filette M, Smet A, Festjens E, Birkett A, et al. Universal vaccine based on ectodomain of matrix protein 2 of influenza A: Fc receptors and alveolar macrophages mediate protection. J Immunol. 2011;186(2):1022\u201331.","journal-title":"J Immunol"},{"issue":"11","key":"2760_CR64","doi-asserted-by":"publisher","first-page":"e01646","DOI":"10.1128\/jvi.01646-22","volume":"97","author":"WF Rijnink","year":"2023","unstructured":"Rijnink WF, Stadlbauer D, Puente-Massaguer E, Okba NM, Kirkpatrick Roubidoux E, Strohmeier S, et al. Characterization of non-neutralizing human monoclonal antibodies that target the M1 and NP of influenza A viruses. J Virol. 2023;97(11):e01646\u201322.","journal-title":"J Virol"},{"key":"2760_CR65","doi-asserted-by":"publisher","first-page":"277","DOI":"10.1016\/j.ebiom.2016.04.029","volume":"8","author":"HA Vanderven","year":"2016","unstructured":"Vanderven HA, Ana-Sosa-Batiz F, Jegaskanda S, Rockman S, Laurie K, Barr I, et al. What Lies beneath: antibody dependent natural killer cell activation by antibodies to internal influenza virus proteins. EBioMedicine. 2016;8:277\u201390.","journal-title":"EBioMedicine"},{"key":"2760_CR66","doi-asserted-by":"publisher","first-page":"1457","DOI":"10.3389\/fimmu.2019.01457","volume":"10","author":"TA Von Holle","year":"2019","unstructured":"Von Holle TA, Moody MA. Influenza and antibody-dependent cellular cytotoxicity. Front Immunol. 2019;10:1457.","journal-title":"Front Immunol"},{"issue":"10","key":"2760_CR67","doi-asserted-by":"publisher","first-page":"1305","DOI":"10.1038\/nm.3350","volume":"19","author":"S Sridhar","year":"2013","unstructured":"Sridhar S, Begom S, Bermingham A, Hoschler K, Adamson W, Carman W, et al. Cellular immune correlates of protection against symptomatic pandemic influenza. Nat Med. 2013;19(10):1305\u201312.","journal-title":"Nat Med"},{"issue":"5","key":"2760_CR68","doi-asserted-by":"publisher","first-page":"426","DOI":"10.3390\/vaccines9050426","volume":"9","author":"ED Yu","year":"2021","unstructured":"Yu ED, Grifoni A, Sutherland A, Voic H, Wang E, Frazier A, et al. Balanced cellular and humoral immune responses targeting multiple antigens in adults receiving a quadrivalent inactivated influenza vaccine. Vaccines. 2021;9(5):426.","journal-title":"Vaccines"},{"issue":"5","key":"2760_CR69","doi-asserted-by":"publisher","first-page":"489","DOI":"10.1016\/j.micinf.2011.01.007","volume":"13","author":"SA Valkenburg","year":"2011","unstructured":"Valkenburg SA, Rutigliano JA, Ellebedy AH, Doherty PC, Thomas PG, Kedzierska K. Immunity to seasonal and pandemic influenza A viruses. Microbes Infect. 2011;13(5):489\u2013501.","journal-title":"Microbes Infect"},{"issue":"6","key":"2760_CR70","doi-asserted-by":"publisher","first-page":"e2074","DOI":"10.1002\/rmv.2074","volume":"29","author":"A Sabbaghi","year":"2019","unstructured":"Sabbaghi A, Miri SM, Keshavarz M, Zargar M, Ghaemi A. Inactivation methods for whole influenza vaccine production. Rev Med Virol. 2019;29(6):e2074.","journal-title":"Rev Med Virol"},{"key":"2760_CR71","doi-asserted-by":"publisher","first-page":"23","DOI":"10.1016\/j.coviro.2021.03.001","volume":"48","author":"MS Borau","year":"2021","unstructured":"Borau MS, Stertz S. Entry of influenza A virus into host cells\u2014Recent progress and remaining challenges. Curr Opin Virol. 2021;48:23\u20139.","journal-title":"Curr Opin Virol"},{"issue":"6","key":"2760_CR72","doi-asserted-by":"publisher","first-page":"695","DOI":"10.1586\/erv.12.38","volume":"11","author":"I Delrue","year":"2012","unstructured":"Delrue I, Verzele D, Madder A, Nauwynck HJ. Inactivated virus vaccines from chemistry to prophylaxis: merits, risks and challenges. Expert Rev Vaccines. 2012;11(6):695\u2013719.","journal-title":"Expert Rev Vaccines"},{"key":"2760_CR73","doi-asserted-by":"crossref","unstructured":"Broo K, Wei J, Marshall D, Brown F, Smith TJ, Johnson JE et al. Viral capsid mobility: a dynamic conduit for inactivation. Proceedings of the National Academy of Sciences. 2001;98(5):2274-7.","DOI":"10.1073\/pnas.051598298"}],"container-title":["Virology Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12985-025-02760-4.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1186\/s12985-025-02760-4\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12985-025-02760-4.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,5,14]],"date-time":"2025-05-14T22:02:15Z","timestamp":1747260135000},"score":1,"resource":{"primary":{"URL":"https:\/\/virologyj.biomedcentral.com\/articles\/10.1186\/s12985-025-02760-4"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,5,5]]},"references-count":73,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["2760"],"URL":"https:\/\/doi.org\/10.1186\/s12985-025-02760-4","relation":{},"ISSN":["1743-422X"],"issn-type":[{"value":"1743-422X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,5,5]]},"assertion":[{"value":"31 January 2025","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"23 April 2025","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"5 May 2025","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"All animal experiments were performed in strict accordance with the guidelines set by the Institutional Animal Care and Use Committee (IACUC). The SPF chicken experiments used for the collection of immune sera were approved by the IACUC of Seoul National University (SNU-231102-2) and conducted in an Animal Biosafety Level 1 (ABSL-1) facility at the College of Veterinary Medicine, Seoul National University (Seoul, Republic of Korea). Mouse studies were approved by the IACUC of Seoul National University (SNU-240305-2) and conducted in an Animal Biosafety Level 2 (ABSL-2) facility at the Animal Center for Pharmaceutical Research, Seoul National University (Seoul, Republic of Korea). All SPF chickens and mice were housed in negative-pressure isolators with HEPA filter setting.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"131"}}