{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T20:03:09Z","timestamp":1785787389860,"version":"3.56.0"},"reference-count":65,"publisher":"MDPI AG","issue":"20","license":[{"start":{"date-parts":[[2022,10,17]],"date-time":"2022-10-17T00:00:00Z","timestamp":1665964800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001349","name":"National University of Singapore","doi-asserted-by":"publisher","award":["MOH-OFYIRG18nov-0013"],"award-info":[{"award-number":["MOH-OFYIRG18nov-0013"]}],"id":[{"id":"10.13039\/501100001349","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001349","name":"National University of Singapore","doi-asserted-by":"publisher","award":["COVID19TUG21-0129"],"award-info":[{"award-number":["COVID19TUG21-0129"]}],"id":[{"id":"10.13039\/501100001349","id-type":"DOI","asserted-by":"publisher"}]},{"name":"NMRC Open Fund Young Individual Research Grant","award":["MOH-OFYIRG18nov-0013"],"award-info":[{"award-number":["MOH-OFYIRG18nov-0013"]}]},{"name":"NMRC Open Fund Young Individual Research Grant","award":["COVID19TUG21-0129"],"award-info":[{"award-number":["COVID19TUG21-0129"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Influenza remains one of the most prevalent viruses circulating amongst humans and has resulted in several pandemics. The prevention and control of H3N2 influenza is complicated by its propensity for evolution, which leads to vaccine mismatch and reduced vaccine efficacies. This study employed the strategy of serial passaging to compare the evolution of the human seasonal influenza strain A\/Singapore\/G2-31.1\/2014(H3N2) in MDCK-SIAT1 versus primary chick embryo fibroblast (CEF) cells. Genetic analysis of the HA, NS1, NA, and PB1 gene segments by Sanger sequencing revealed the presence of specific mutations and a repertoire of viral quasispecies following serial passaging. Most quasispecies were also found in PB1, which exhibited consistently high transversion-to-transition ratios in all five MDCK-SIAT1 passages. Most notably, passage 5 virus harbored the D457G substitution in the HA2 subunit, while passage 3 virus acquired K53Q and Q69H mutations in PB1-F2. An A971 variant leading to a non-synonymous R316Q substitution in PB1 was also identified in MDCK-SIAT1 passages 2 and 4. With an increasing number of passages, the proportion of D457G mutations progressively increased and was associated with larger virus plaque sizes. However, microneutralization assays revealed no significant differences in the neutralizing antibody profiles of human-influenza-immune serum samples against pre-passaged virus and passage 5 virus. In contrast, viable virus was only detected in passage 1 of CEF cells, which gave rise to multiple viral RNA quasispecies. Our findings highlight that serial passaging is able to drive differential adaptation of H3N2 influenza in different host species and may alter viral virulence. More studies are warranted to elucidate the complex relationships between H3N2 virus evolution, viral virulence changes, and low vaccine efficacy.<\/jats:p>","DOI":"10.3390\/ijms232012408","type":"journal-article","created":{"date-parts":[[2022,10,17]],"date-time":"2022-10-17T21:11:51Z","timestamp":1666041111000},"page":"12408","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Serial Passaging of Seasonal H3N2 Influenza A\/Singapore\/G2-31.1\/2014 Virus in MDCK-SIAT1 Cells and Primary Chick Embryo Cells Generates HA D457G Mutation and Other Variants in HA, NA, PB1, PB1-F2, and NS1"],"prefix":"10.3390","volume":"23","author":[{"given":"Daryl Zheng Hao","family":"Aw","sequence":"first","affiliation":[{"name":"NUHS Infectious Diseases Translational Research Program, Host and Pathogen Interactivity Laboratory, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keng Kai","family":"Heng","sequence":"additional","affiliation":[{"name":"NUS High School of Mathematics and Science, Singapore 129957, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jovian Yee Han","family":"Heok","sequence":"additional","affiliation":[{"name":"NUS High School of Mathematics and Science, Singapore 129957, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xin Yang","family":"Kong","sequence":"additional","affiliation":[{"name":"NUS High School of Mathematics and Science, Singapore 129957, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2258-0750","authenticated-orcid":false,"given":"Hui","family":"Chen","sequence":"additional","affiliation":[{"name":"Human Genetics, Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore 138672, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2046-4502","authenticated-orcid":false,"given":"Tong","family":"Zhang","sequence":"additional","affiliation":[{"name":"Human Genetics, Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore 138672, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weiwei","family":"Zhai","sequence":"additional","affiliation":[{"name":"Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2634-6213","authenticated-orcid":false,"given":"Vincent T. K.","family":"Chow","sequence":"additional","affiliation":[{"name":"NUHS Infectious Diseases Translational Research Program, Host and Pathogen Interactivity Laboratory, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,10,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1840","DOI":"10.1080\/21645515.2018.1462639","article-title":"H3N2 influenza viruses in humans: Viral mechanisms, evolution, and evaluation","volume":"14","author":"Allen","year":"2018","journal-title":"Hum. Vaccin. Immunother."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"669","DOI":"10.2105\/AJPH.2019.305557","article-title":"Fifty years of influenza A(H3N2) following the pandemic of 1968","volume":"110","author":"Jester","year":"2020","journal-title":"Am. J. Public Health"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1817","DOI":"10.1093\/cid\/ciz411","article-title":"Influenza vaccine effectiveness: Defining the H3N2 problem","volume":"69","author":"Belongia","year":"2019","journal-title":"Clin. Infect. Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1016\/j.it.2017.08.003","article-title":"From original antigenic sin to the universal influenza virus vaccine","volume":"39","author":"Henry","year":"2018","journal-title":"Trends Immunol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"335","DOI":"10.4049\/jimmunol.1801149","article-title":"Original antigenic sin: How first exposure shapes lifelong anti-influenza virus immune responses","volume":"202","author":"Zhang","year":"2019","journal-title":"J. Immunol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Bull, M.B., Cohen, C.A., Leung, N.H.L., and Valkenburg, S.A. (2021). Universally immune: How infection permissive next generation vaccines may affect population immunity and viral spread. Viruses, 13.","DOI":"10.3390\/v13091779"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"17221","DOI":"10.1073\/pnas.1920321117","article-title":"Original antigenic sin priming of influenza virus hemagglutinin stalk antibodies","volume":"117","author":"Arevalo","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e146138","DOI":"10.1172\/JCI146138","article-title":"Age-specific effects of vaccine egg adaptation and immune priming on A(H3N2) antibody responses following influenza vaccination","volume":"131","author":"Liu","year":"2021","journal-title":"J. Clin. Investig."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"6047","DOI":"10.1016\/j.vaccine.2020.06.021","article-title":"The impact of candidate influenza virus and egg-based manufacture on vaccine effectiveness: Literature review and expert consensus","volume":"38","author":"Rajaram","year":"2020","journal-title":"Vaccine"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3357","DOI":"10.1128\/jvi.71.4.3357-3362.1997","article-title":"Differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection","volume":"71","author":"Ito","year":"1997","journal-title":"J. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"823","DOI":"10.1002\/embj.201387442","article-title":"Role of receptor binding specificity in influenza A virus transmission and pathogenesis","volume":"33","author":"Fouchier","year":"2014","journal-title":"EMBO J."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"12578","DOI":"10.1073\/pnas.1712377114","article-title":"Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains","volume":"114","author":"Zost","year":"2017","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"8148","DOI":"10.1038\/ncomms9148","article-title":"Development of high-yield influenza A virus vaccine viruses","volume":"6","author":"Ping","year":"2015","journal-title":"Nat. Commun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1186\/s12929-020-0626-6","article-title":"Better influenza vaccines: An industry perspective","volume":"27","author":"Chen","year":"2020","journal-title":"J. Biomed. Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"10986","DOI":"10.1128\/JVI.01077-14","article-title":"Recent H3N2 influenza virus clinical isolates rapidly acquire hemagglutinin or neuraminidase mutations when propagated for antigenic analyses","volume":"88","author":"Chambers","year":"2014","journal-title":"J. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"836","DOI":"10.1016\/j.chom.2019.04.013","article-title":"Preventing an antigenically disruptive mutation in egg-based H3N2 seasonal influenza vaccines by mutational incompatibility","volume":"25","author":"Wu","year":"2019","journal-title":"Cell Host Microbe"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Choi, E.J., Lee, Y.J., Lee, J.M., Kim, Y.J., Choi, J.H., Ahn, B., Kim, K., and Han, M.G. (2020). The effect of mutations derived from mouse-adapted H3N2 seasonal influenza A virus to pathogenicity and host adaptation. PLoS ONE, 15.","DOI":"10.1371\/journal.pone.0227516"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1016\/j.micinf.2008.09.013","article-title":"Adaptation of human influenza H3N2 virus in a mouse pneumonitis model: Insights into viral virulence, tissue tropism and host pathogenesis","volume":"11","author":"Narasaraju","year":"2009","journal-title":"Microbes Infect."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"16616","DOI":"10.1038\/s41598-019-51877-4","article-title":"Synergystic PA and HA mutations confer mouse adaptation of a contemporary A\/H3N2 influenza virus","volume":"9","author":"Baz","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Powell, H., and Pekosz, A. (2020). Neuraminidase antigenic drift of H3N2 clade 3c.2a viruses alters virus replication, enzymatic activity and inhibitory antibody binding. PLoS Pathog., 16.","DOI":"10.1101\/2020.02.20.957399"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3133","DOI":"10.1093\/molbev\/msw190","article-title":"Dynamic convergent evolution drives passage adaptation across 48 years\u2019 history of H3N2 influenza evolution","volume":"33","author":"Chen","year":"2016","journal-title":"Mol. Biol. Evol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1198","DOI":"10.1093\/cid\/ciy1065","article-title":"Passage adaptation correlates with the reduced efficacy of the influenza vaccine","volume":"69","author":"Chen","year":"2019","journal-title":"Clin. Infect. Dis."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Chen, H., Wang, J., Liu, Y., Quek, I., Shih, C.C., Wu, D., Fu, Z., Lee, R.T.C., Xu, M., and Chow, V.T. (2022). MADE: A computational tool for predicting vaccine effectiveness for the influenza A(H3N2) virus adapted to embryonated eggs. Vaccines, 10.","DOI":"10.3390\/vaccines10060907"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Hamamoto, I., Takaku, H., Tashiro, M., and Yamamoto, N. (2013). High yield production of influenza virus in Madin Darby canine kidney (MDCK) cells with stable knockdown of IRF7. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0059892"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"997","DOI":"10.4014\/jmb.1712.12007","article-title":"Generation of a high-growth influenza vaccine strain in MDCK cells for vaccine preparedness","volume":"28","author":"Kim","year":"2018","journal-title":"J. Microbiol. Biotechnol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"56","DOI":"10.7774\/cevr.2020.9.1.56","article-title":"Comparison of antigenic mutation during egg and cell passage cultivation of H3N2 influenza virus","volume":"9","author":"Park","year":"2020","journal-title":"Clin. Exp. Vaccine Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"222","DOI":"10.1016\/j.jiac.2018.08.012","article-title":"High genetic stability in MDCK-SIAT1 passaged human influenza viruses","volume":"25","author":"Matsumoto","year":"2019","journal-title":"J. Infect. Chemother."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1038\/s41541-021-00415-3","article-title":"Enhanced isolation of influenza viruses in qualified cells improves the probability of well-matched vaccines","volume":"6","author":"Peck","year":"2021","journal-title":"NPJ Vaccines"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2189","DOI":"10.1128\/JCM.00398-08","article-title":"MDCK-SIAT1 cells show improved isolation rates for recent human influenza viruses compared to conventional MDCK cells","volume":"46","author":"Oh","year":"2008","journal-title":"J. Clin. Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"941","DOI":"10.1007\/s10529-016-2069-4","article-title":"Comparison between MDCK and MDCK-SIAT1 cell lines as preferred host for cell culture-based influenza vaccine production","volume":"38","author":"Abdoli","year":"2016","journal-title":"Biotechnol. Lett."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"419","DOI":"10.1007\/s10616-012-9495-5","article-title":"Impact of chicken-origin cells on adaptation of a low pathogenic influenza virus","volume":"65","author":"Shahsavandi","year":"2013","journal-title":"Cytotechnology"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"40698","DOI":"10.1038\/srep40698","article-title":"Proteome response of chicken embryo fibroblast cells to recombinant H5N1 avian influenza viruses with different neuraminidase stalk lengths","volume":"7","author":"Li","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1016\/j.ijid.2019.10.024","article-title":"Molecular insights into evolution, mutations and receptor-binding specificity of influenza A and B viruses from outpatients and hospitalized patients in Singapore","volume":"90","author":"Ivan","year":"2020","journal-title":"Int. J. Infect. Dis."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Wu, N.C., Zost, S.J., Thompson, A.J., Oyen, D., Nycholat, C.M., McBride, R., Paulson, J.C., Hensley, S.E., and Wilson, I.A. (2017). A structural explanation for the low effectiveness of the seasonal influenza H3N2 vaccine. PLoS Pathog., 13.","DOI":"10.1371\/journal.ppat.1006682"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"287","DOI":"10.1038\/s41586-019-1530-7","article-title":"Structures of influenza A virus RNA polymerase offer insight into viral genome replication","volume":"573","author":"Fan","year":"2019","journal-title":"Nature"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"684","DOI":"10.1093\/infdis\/jiu186","article-title":"Association between antibody titers and protection against influenza virus infection within households","volume":"210","author":"Tsang","year":"2014","journal-title":"J. Infect. Dis."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"3778","DOI":"10.1016\/j.vaccine.2008.04.048","article-title":"High titer growth of human and avian influenza viruses in an immortalized chick embryo cell line without the need for exogenous proteases","volume":"26","author":"Smith","year":"2008","journal-title":"Vaccine"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"8661","DOI":"10.1016\/j.vaccine.2011.08.122","article-title":"Immortalized chick embryo cell line adapted to serum-free growth conditions and capable of replicating human and reassortant H5N1 influenza strains for vaccine production","volume":"29","author":"Coussens","year":"2011","journal-title":"Vaccine"},{"key":"ref_39","first-page":"5","article-title":"Efficient replication but low titer growth of influenza virus in immortalised chick embryo fibroblasts cell line","volume":"73","author":"Muvunyi","year":"2016","journal-title":"Rwanda Med. J."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"6583","DOI":"10.1016\/j.vaccine.2014.06.045","article-title":"Performance characteristics of qualified cell lines for isolation and propagation of influenza viruses for vaccine manufacturing","volume":"32","author":"Donis","year":"2014","journal-title":"Vaccine"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1080\/21645515.2016.1231261","article-title":"Reassortment of high-yield influenza viruses in vero cells and safety assessment as candidate vaccine strains","volume":"13","author":"Zhou","year":"2017","journal-title":"Hum. Vaccin. Immunother."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"693204","DOI":"10.3389\/fmicb.2021.693204","article-title":"Molecular evolution of the influenza A virus non-structural protein 1 in interspecies transmission and adaptation","volume":"12","author":"Evseev","year":"2021","journal-title":"Front. Microbiol."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Lee, R.T.C., Chang, H.H., Russell, C.A., Lipstitch, M., and Maurer-Stroh, S. (2019). Influenza A hemagglutinin passage bias sites and host specificity mutations. Cells, 8.","DOI":"10.3390\/cells8090958"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"2831","DOI":"10.1002\/j.1460-2075.1986.tb04576.x","article-title":"Mutations blocking the transport of the influenza virus hemagglutinin between the rough endoplasmic reticulum and the Golgi apparatus","volume":"5","author":"Schuy","year":"1986","journal-title":"EMBO J."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"4504","DOI":"10.1128\/JVI.00057-15","article-title":"Influenza hemagglutinin (HA) stem region mutations that stabilise or destabilise the structure of multiple HA subtypes","volume":"89","author":"Galloway","year":"2015","journal-title":"J. Virol."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Suntronwong, N., Klinfueng, S., Vichiwattana, P., Korkong, S., Thongmee, T., Vongpunsawad, S., and Poovorawan, Y. (2017). Genetic and antigenic divergence in the influenza A(H3N2) virus circulating between 2016 and 2017 in Thailand. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0189511"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"458","DOI":"10.1007\/s11262-019-01675-x","article-title":"Genetic characterization of hemagglutinin (HA) gene of influenza A viruses circulating in Southwest India during 2017 season","volume":"55","author":"Jagadesh","year":"2019","journal-title":"Virus Genes"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1016\/j.ygeno.2012.06.003","article-title":"Highly conserved influenza A virus epitope sequences as candidates of H3N2 flu vaccine targets","volume":"100","author":"Wu","year":"2012","journal-title":"Genomics"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"280","DOI":"10.1016\/j.jviromet.2013.08.022","article-title":"The highly conserved HA2 protein of the influenza A virus induces a cross protective immune response","volume":"194","author":"Lee","year":"2013","journal-title":"J. Virol. Methods"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1038\/mi.2014.59","article-title":"Targeting the HA2 subunit of influenza A virus hemagglutinin via CD40L provides universal protection against diverse subtypes","volume":"8","author":"Fan","year":"2014","journal-title":"Mucosal Immunol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"8359","DOI":"10.1128\/JVI.01122-12","article-title":"Influenza virus protein PB1-F2 inhibits the induction of type I interferon by binding to MAVS and decreasing mitochondrial membrane potential","volume":"86","author":"Varga","year":"2012","journal-title":"J. Virol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1140","DOI":"10.1111\/j.1462-5822.2008.01116.x","article-title":"The proapoptotic influenza A virus protein PB1-F2 regulates viral polymerase activity by interaction with the PB1 protein","volume":"10","author":"Mazur","year":"2008","journal-title":"Cell. Microbiol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"692","DOI":"10.3389\/fmicb.2017.00692","article-title":"Amino acid residues 68\u201371 contribute to influenza A virus PB1-F2 protein stability and functions","volume":"8","author":"Cheng","year":"2017","journal-title":"Front. Microbiol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"503","DOI":"10.1128\/JVI.01373-13","article-title":"A novel cytotoxic sequence contributes to influenza A viral protein PB1-F2 pathogenicity and predisposition to secondary bacterial infection","volume":"88","author":"Alymova","year":"2014","journal-title":"J. Virol."},{"key":"ref_55","doi-asserted-by":"crossref","unstructured":"Varga, Z.T., Ramos, I., Hai, R., Schmolke, M., Garc\u00eda-Sastre, A., Fernandez-Sesma, A., and Palese, P. (2011). The influenza virus protein PB1-F2 inhibits the induction of type I interferon at the level of the MAVS adaptor protein. PLoS Pathog., 7.","DOI":"10.1371\/journal.ppat.1002067"},{"key":"ref_56","doi-asserted-by":"crossref","unstructured":"Zeng, Y., Xu, S., Wei, Y., Zhang, X., Wang, Q., Jia, Y., Wang, W., Han, L., Chen, Z., and Wang, Z. (2021). The PB1 protein of influenza A virus inhibits the innate immune response by targeting MAVS for NBR1-mediated selective autophagic degradation. PLoS Pathog., 17.","DOI":"10.1371\/journal.ppat.1009300"},{"key":"ref_57","doi-asserted-by":"crossref","unstructured":"Lyons, D.M., and Lauring, A.S. (2018). Mutation and epistasis in influenza virus evolution. Viruses, 10.","DOI":"10.3390\/v10080407"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"773","DOI":"10.1099\/jgv.0.001263","article-title":"Epistatic interactions can moderate the antigenic effect of substitutions in haemagglutinin of influenza H3N2 virus","volume":"100","author":"Koel","year":"2019","journal-title":"J. Gen. Virol."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"e621","DOI":"10.1016\/j.ijid.2012.04.010","article-title":"Comparison of neutralizing antibody and cell-mediated immune responses to pandemic H1N1 2009 influenza virus before and after H1N1 2009 influenza vaccination of elderly subjects and healthcare workers","volume":"16","author":"Hsu","year":"2012","journal-title":"Int. J. Infect. Dis."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"E8276","DOI":"10.1073\/pnas.1806133115","article-title":"Deep mutational scanning of hemagglutinin helps predict evolutionary fates of human H3N2 influenza variants","volume":"115","author":"Lee","year":"2018","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_61","first-page":"A-4I","article-title":"Growth and maintenance of chick embryo fibroblasts (CEF)","volume":"4","author":"Hernandez","year":"2010","journal-title":"Curr. Protoc. Microbiol."},{"key":"ref_62","doi-asserted-by":"crossref","unstructured":"Takahashi, T., Song, J., Suzuki, T., and Kawaoka, Y. (2013). Mutations in NA that induced low pH-stability and enhanced the replication of pandemic (H1N1) 2009 influenza A virus at an early stage of the pandemic. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0064439"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.jcv.2015.04.019","article-title":"A simplified Sanger sequencing method for full genome sequencing of multiple subtypes of human influenza A viruses","volume":"68","author":"Deng","year":"2015","journal-title":"J. Clin. Virol."},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"e19712","DOI":"10.2196\/19712","article-title":"Effectiveness of a mobile-based influenza-like illness surveillance system (FluMob) among health care workers: Longitudinal study","volume":"8","author":"Lwin","year":"2020","journal-title":"JMIR Mhealth Uhealth"},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"5.6.1","DOI":"10.1002\/0471250953.bi0506s47","article-title":"Comparative protein structure modeling using MODELLER","volume":"47","author":"Webb","year":"2014","journal-title":"Curr. Protoc. Bioinform."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/20\/12408\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:55:47Z","timestamp":1760144147000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/20\/12408"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,10,17]]},"references-count":65,"journal-issue":{"issue":"20","published-online":{"date-parts":[[2022,10]]}},"alternative-id":["ijms232012408"],"URL":"https:\/\/doi.org\/10.3390\/ijms232012408","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,10,17]]}}}