{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,16]],"date-time":"2026-06-16T16:53:08Z","timestamp":1781628788295,"version":"3.54.5"},"reference-count":73,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2025,12,29]],"date-time":"2025-12-29T00:00:00Z","timestamp":1766966400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2025,12,30]],"date-time":"2025-12-30T00:00:00Z","timestamp":1767052800000},"content-version":"vor","delay-in-days":1,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100023685","name":"Fraunhofer-Institut f\u00fcr Translationale Medizin und Pharmakologie ITMP","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100023685","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    The aim of this study was to develop a rapid and easy-to-adapt protocol for recombinant production of Orthoflavivirus proteins. We introduced the new-generation pOpiE2 vector for virus-free transient transfection of\n                    <jats:italic>Trichopluisa ni<\/jats:italic>\n                    High Five insect cells to produce the envelope protein ectodomain of Yellow Fever Virus Asibi and 17D strains. We achieved high yields in the range of 6\u20137\u00a0mg\/L within five working days. In-depth biophysical analysis demonstrated highest protein purity, homogeneity, stability, and affinities to specific antibodies in the low nanomolar range. A combination of peptide mass fingerprint and intact mass spectrometry characterized the copurified precursor chaperone peptide pr and discovered a new posttranslational processing site for the recombinantly produced protein.\n                  <\/jats:p>","DOI":"10.1038\/s41598-025-33406-8","type":"journal-article","created":{"date-parts":[[2025,12,29]],"date-time":"2025-12-29T15:34:57Z","timestamp":1767022497000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Rapid virus-free production of recombinant yellow fever virus envelope protein and its in-depth biophysical analysis"],"prefix":"10.1038","volume":"15","author":[{"given":"Luca","family":"Schelle","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wolfgang","family":"Kuttenlochner","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Victoria","family":"Sanchez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Barbara","family":"Steigenberger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katja","family":"Finkl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marius","family":"Schmid","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Celine","family":"Douat","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Einar","family":"Halldorsson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maren","family":"Schubert","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sabine","family":"Suppmann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,12,29]]},"reference":[{"key":"33406_CR1","unstructured":"Countries with risk of yellow fever transmission. and countries requiring proof of vaccination against yellow fever; WHO report; International travel and Health, Annex 1\u201318, November (2022). revised on 3 January 2023."},{"key":"33406_CR2","doi-asserted-by":"crossref","unstructured":"Angerami, R. N., Chaves, T. D. S. & Rodr\u00edguez-Morales, A. J. Yellow fever outbreaks in South america: current epidemiology, legacies of the recent past and perspectives for the near future. New Microbes New. Infections, 65. (2025).","DOI":"10.1016\/j.nmni.2025.101580"},{"key":"33406_CR3","doi-asserted-by":"crossref","unstructured":"Coulter, F. J. & Messer, W. B. Breakthroughs and insights: A comprehensive review of yellow fever vaccine breakthrough infection across 8 decades. Vaccine 43, (2025).","DOI":"10.1016\/j.vaccine.2024.126423"},{"issue":"1","key":"33406_CR4","doi-asserted-by":"publisher","first-page":"36","DOI":"10.1038\/s44298-025-00114-z","volume":"3","author":"FS Madere","year":"2025","unstructured":"Madere, F. S. et al. Flavivirus infections and diagnostic challenges for dengue, West nile and Zika viruses. Npj Viruses. 3 (1), 36 (2025).","journal-title":"Npj Viruses"},{"key":"33406_CR5","doi-asserted-by":"crossref","unstructured":"Hale, G. L. Flaviviruses and the traveler: around the world and to your Stage. A review of West Nile, yellow Fever, Dengue, and Zika viruses for the practicing pathologist. Mod. Pathol. 36(6). (2023).","DOI":"10.1016\/j.modpat.2023.100188"},{"issue":"10","key":"33406_CR6","doi-asserted-by":"publisher","first-page":"2427","DOI":"10.3390\/microorganisms11102427","volume":"11","author":"E Diani","year":"2023","unstructured":"Diani, E. et al. Vector-Transmitted flaviviruses: an antiviral molecules overview. Microorganisms 11 (10), 2427 (2023).","journal-title":"Microorganisms"},{"issue":"6","key":"33406_CR7","doi-asserted-by":"publisher","first-page":"796","DOI":"10.1038\/s41564-020-0714-0","volume":"5","author":"TC Pierson","year":"2020","unstructured":"Pierson, T. C. & Diamond, M. S. The continued threat of emerging flaviviruses. Nat. Microbiol. 5 (6), 796\u2013812 (2020).","journal-title":"Nat. Microbiol."},{"issue":"8","key":"33406_CR8","doi-asserted-by":"publisher","first-page":"4448","DOI":"10.1021\/acs.chemrev.7b00719","volume":"118","author":"NJ Barrows","year":"2018","unstructured":"Barrows, N. J. et al. Biochemistry and molecular biology of flaviviruses. Chem. Rev. 118 (8), 4448\u20134482 (2018).","journal-title":"Chem. Rev."},{"issue":"2","key":"33406_CR9","doi-asserted-by":"publisher","first-page":"168","DOI":"10.1016\/j.coviro.2012.02.011","volume":"2","author":"TC Pierson","year":"2012","unstructured":"Pierson, T. C. & Diamond, M. S. Degrees of maturity: the complex structure and biology of flaviviruses. Curr. Opin. Virol. 2 (2), 168\u2013175 (2012).","journal-title":"Curr. Opin. Virol."},{"key":"33406_CR10","doi-asserted-by":"publisher","first-page":"23","DOI":"10.1002\/0470058005.ch3","volume":"277","author":"E Harris","year":"2006","unstructured":"Harris, E., Holden, K. L., Edgil, D., Polacek, C. & Clyde, K. Molecular biology of flaviviruses. Novartis Found. Symp. 277, 23\u201339 (2006). discussion 40, 71\u2009\u2013\u200923.","journal-title":"Novartis Found. Symp."},{"key":"33406_CR11","doi-asserted-by":"crossref","unstructured":"Dey, D., Poudyal, S., Rehman, A. & Hasan, S. S. Structural and biochemical insights into flavivirus proteins. Virus Res. 296, 198343 (2021).","DOI":"10.1016\/j.virusres.2021.198343"},{"issue":"7","key":"33406_CR12","doi-asserted-by":"publisher","first-page":"615","DOI":"10.3390\/life11070615","volume":"11","author":"R Zhao","year":"2021","unstructured":"Zhao, R. et al. Flavivirus: from structure to therapeutics development. Life 11 (7), 615 (2021).","journal-title":"Life"},{"key":"33406_CR13","doi-asserted-by":"crossref","unstructured":"Mancini, N. Monoclonal antibodies and flaviviruses: a possible option? mBio 13(3), e0082422 (2022).","DOI":"10.1128\/mbio.00824-22"},{"key":"33406_CR14","doi-asserted-by":"crossref","unstructured":"Cervantes Rincon, T. et al. Human antibodies in Mexico and Brazil neutralizing tick-borne flaviviruses. Cell Rep. 43(6), 114298 (2024).","DOI":"10.1016\/j.celrep.2024.114298"},{"key":"33406_CR15","doi-asserted-by":"crossref","unstructured":"Santos-Peral, A. et al. Prior flavivirus immunity skews the yellow fever vaccine response to cross-reactive antibodies with potential to enhance dengue virus infection. Nat. Commun. 15(1), 1696 (2024).","DOI":"10.1038\/s41467-024-45806-x"},{"issue":"7","key":"33406_CR16","doi-asserted-by":"publisher","first-page":"678","DOI":"10.1016\/j.tim.2023.11.016","volume":"32","author":"M Ceconi","year":"2024","unstructured":"Ceconi, M., Arien, K. K. & Delputte, P. Diagnosing arthropod-borne flaviviruses: non-structural protein 1 (NS1) as a biomarker. Trends Microbiol. 32 (7), 678\u2013696 (2024).","journal-title":"Trends Microbiol."},{"key":"33406_CR17","doi-asserted-by":"crossref","unstructured":"Mora-Cardenas, E. et al. Comparative specificity and sensitivity of NS1-based serological assays for the detection of flavivirus immune response. PLoS Negl. Trop. Dis. 14(1), e0008039 (2020).","DOI":"10.1371\/journal.pntd.0008039"},{"issue":"6","key":"33406_CR18","doi-asserted-by":"publisher","first-page":"1598","DOI":"10.1016\/j.celrep.2019.01.036","volume":"26","author":"H Puerta-Guardo","year":"2019","unstructured":"Puerta-Guardo, H. et al. Flavivirus NS1 triggers Tissue-Specific vascular endothelial dysfunction reflecting disease tropism. Cell. Rep. 26 (6), 1598\u20131613 (2019).","journal-title":"Cell. Rep."},{"issue":"4","key":"33406_CR19","doi-asserted-by":"publisher","first-page":"2337","DOI":"10.1128\/JVI.06072-11","volume":"86","author":"VC Luca","year":"2012","unstructured":"Luca, V. C., AbiMansour, J., Nelson, C. A. & Fremont, D. H. Crystal structure of the Japanese encephalitis virus envelope protein. J. Virol. 86 (4), 2337\u20132346 (2012).","journal-title":"J. Virol."},{"issue":"2","key":"33406_CR20","doi-asserted-by":"publisher","first-page":"818","DOI":"10.1128\/JVI.01950-12","volume":"87","author":"VC Luca","year":"2013","unstructured":"Luca, V. C., Nelson, C. A. & Fremont, D. H. Structure of the St. Louis encephalitis virus postfusion envelope trimer. J. Virol. 87 (2), 818\u2013828 (2013).","journal-title":"J. Virol."},{"issue":"2","key":"33406_CR21","doi-asserted-by":"publisher","first-page":"438","DOI":"10.1016\/j.celrep.2018.12.065","volume":"26","author":"X Lu","year":"2019","unstructured":"Lu, X. et al. Double lock of a human neutralizing and protective monoclonal antibody targeting the yellow fever virus envelope. Cell. Rep. 26 (2), 438\u2013446 (2019). e435.","journal-title":"Cell. Rep."},{"issue":"1","key":"33406_CR22","doi-asserted-by":"publisher","first-page":"129","DOI":"10.1016\/j.pep.2010.06.015","volume":"74","author":"LCM Tan","year":"2010","unstructured":"Tan, L. C. M. et al. Rapid purification of Recombinant dengue and West nile virus envelope domain III proteins by metal affinity membrane chromatography. Protein Exp. Purif. 74 (1), 129\u2013137 (2010).","journal-title":"Protein Exp. Purif."},{"key":"33406_CR23","doi-asserted-by":"publisher","first-page":"57","DOI":"10.1016\/j.pep.2017.03.004","volume":"133","author":"M Combe","year":"2017","unstructured":"Combe, M. et al. Expression, refolding and bio-structural analysis of a tetravalent Recombinant dengue envelope domain III protein for serological diagnosis. Protein Exp. Purif. 133, 57\u201365 (2017).","journal-title":"Protein Exp. Purif."},{"issue":"33","key":"33406_CR24","doi-asserted-by":"publisher","first-page":"4287","DOI":"10.1016\/j.vaccine.2017.04.052","volume":"35","author":"M Yang","year":"2017","unstructured":"Yang, M., Dent, M., Lai, H. F., Sun, H. Y. & Chen, Q. Immunization of Zika virus envelope protein domain III induces specific and neutralizing immune responses against Zika virus. Vaccine 35 (33), 4287\u20134294 (2017).","journal-title":"Vaccine"},{"key":"33406_CR25","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1016\/j.nbt.2018.05.002","volume":"46","author":"NK Tripathi","year":"2018","unstructured":"Tripathi, N. K., Karothia, D., Shrivastava, A., Banger, S. & Kumar, J. S. Enhanced production and immunological characterization of Recombinant West nile virus envelope domain III protein. New Biotechnol. 46, 7\u201313 (2018).","journal-title":"New Biotechnol."},{"issue":"5","key":"33406_CR26","doi-asserted-by":"publisher","first-page":"696","DOI":"10.1016\/j.chom.2016.04.013","volume":"19","author":"LP Dai","year":"2016","unstructured":"Dai, L. P. et al. Structures of the Zika virus envelope protein and its complex with a flavivirus broadly protective antibody. Cell. Host Microbe. 19 (5), 696\u2013704 (2016).","journal-title":"Cell. Host Microbe"},{"issue":"4","key":"33406_CR27","doi-asserted-by":"publisher","first-page":"3187","DOI":"10.1007\/s42770-024-01509-5","volume":"55","author":"FD Morgado","year":"2024","unstructured":"Morgado, F. D., Cahu, R., de Jesus, D. C., Chaves, L. C. D. & Ribeiro, B. M. Insect cell production of chimeric virus-like particles based on human immunodeficiency virus GAG proteins and yellow fever virus envelope protein. Brazilian J. Microbiol. 55 (4), 3187\u20133197 (2024).","journal-title":"Brazilian J. Microbiol."},{"key":"33406_CR28","doi-asserted-by":"crossref","unstructured":"Kanai, R. et al. Crystal structure of West Nile virus envelope glycoprotein reveals viral surface epitopes. J. Virol. 80(22), 11000\u201311008 (2006).","DOI":"10.1128\/JVI.01735-06"},{"key":"33406_CR29","doi-asserted-by":"crossref","unstructured":"Zhang, F. Q., Ma, W. Y., Zhang, L., Aasa-Chapman, M. & Zhang, H. Y. Expression of particulate-form of Japanese encephalitis virus envelope protein in a stably transfected drosophila cell line. Virol. J. 4, 17 (2007).","DOI":"10.1186\/1743-422X-4-17"},{"issue":"12","key":"33406_CR30","doi-asserted-by":"publisher","first-page":"6986","DOI":"10.1073\/pnas.0832193100","volume":"100","author":"Y Modis","year":"2003","unstructured":"Modis, Y., Ogata, S., Clements, D. & Harrison, S. C. A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc. Natl. Acad. Sci. U.S.A. 100 (12), 6986\u20136991 (2003).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"issue":"46","key":"33406_CR31","doi-asserted-by":"publisher","first-page":"6308","DOI":"10.1016\/j.vaccine.2017.09.063","volume":"35","author":"J Sun","year":"2017","unstructured":"Sun, J., Li, M., Wang, Y. N., Hao, P. & Jin, X. Elaboration of tetravalent antibody responses against dengue viruses using a subunit vaccine comprised of a single consensus dengue envelope sequence. Vaccine 35 (46), 6308\u20136320 (2017).","journal-title":"Vaccine"},{"issue":"10","key":"33406_CR32","doi-asserted-by":"publisher","first-page":"7333","DOI":"10.1007\/s11033-020-05772-1","volume":"47","author":"L Lopes-Luz","year":"2020","unstructured":"Lopes-Luz, L. et al. Dengue and Zika virus multi-epitope antigen expression in insect cells. Mol. Biol. Rep. 47 (10), 7333\u20137340 (2020).","journal-title":"Mol. Biol. Rep."},{"key":"33406_CR33","doi-asserted-by":"publisher","first-page":"1006","DOI":"10.1099\/vir.0.000052","volume":"96","author":"RK Du","year":"2015","unstructured":"Du, R. K. et al. Glycoprotein E of the Japanese encephalitis virus forms virus-like particles and induces syncytia when expressed by a baculovirus. J. Gen. Virol. 96, 1006\u20131014 (2015).","journal-title":"J. Gen. Virol."},{"issue":"1404","key":"33406_CR34","first-page":"365","volume":"2","author":"H. Yamaji","year":"2016","unstructured":"Yamaji, H. & Konishi, E. Production of Japanese Encephalitis Virus-Like Particles Using Insect Cell Expression Systems. Vaccine Design: Methods and Protocols. Vaccines Veterinary Dis. 2 (1404), 365\u2013375 (2016).","journal-title":"Vaccines Veterinary Dis."},{"issue":"6","key":"33406_CR35","doi-asserted-by":"publisher","first-page":"657","DOI":"10.1016\/j.jbiosc.2012.06.012","volume":"114","author":"H Yamaji","year":"2012","unstructured":"Yamaji, H. et al. Production of Japanese encephalitis virus-like particles using the baculovirus-insect cell system. J. Biosci. Bioeng. 114 (6), 657\u2013662 (2012).","journal-title":"J. Biosci. Bioeng."},{"key":"33406_CR36","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1016\/j.virusres.2015.02.008","volume":"201","author":"GY Niu","year":"2015","unstructured":"Niu, G. Y., Pang, Z., Guan, C., Qi, J. & Li, D. X. Dengue virus envelope domain III protein based on a tetravalent antigen secreted from insect cells: potential use for serological diagnosis. Virus Res. 201, 73\u201378 (2015).","journal-title":"Virus Res."},{"key":"33406_CR37","doi-asserted-by":"crossref","unstructured":"Abbo, S. R. et al. Virus-like particle vaccine with authentic quaternary epitopes protects against Zika virus-induced viremia and testicular damage. J. Virol. 99(4), e0232224 (2025).","DOI":"10.1128\/jvi.02322-24"},{"key":"33406_CR38","doi-asserted-by":"crossref","unstructured":"Tomatis, C. et al. Baculovirus surface display of a chimeric E-NS1 protein of YFV protects against YFV infection. Vaccine 42(25), 126045 (2024).","DOI":"10.1016\/j.vaccine.2024.06.013"},{"key":"33406_CR39","doi-asserted-by":"crossref","unstructured":"Malogolovkin, A. et al. Enhanced Zika virus-like particle development using baculovirus spp. Constructs. J. Med. Virol. 95(1),  e28252 (2023).","DOI":"10.1002\/jmv.28252"},{"key":"33406_CR40","doi-asserted-by":"crossref","unstructured":"Marfia, J. I. et al. Development and validation of dengue virus envelope protein domain III IgG antibody enzyme-linked immunosorbent assay. J. Immunol. Methods 542, 113887 (2025).","DOI":"10.1016\/j.jim.2025.113887"},{"key":"33406_CR41","doi-asserted-by":"crossref","unstructured":"Shin, M. et al. Zika virus baculovirus-expressed envelope protein elicited humoral and cellular immunity in immunocompetent mice. Sci. Rep. 12(1), 660 (2022).","DOI":"10.1038\/s41598-021-04713-7"},{"key":"33406_CR42","doi-asserted-by":"crossref","unstructured":"Rouvinski, A. et al. Covalently linked dengue virus envelope glycoprotein dimers reduce exposure of the immunodominant fusion loop epitope. Nat. Commun. 8, 15411 (2017).","DOI":"10.1038\/ncomms15411"},{"key":"33406_CR43","doi-asserted-by":"crossref","unstructured":"Rockstroh, A. et al. Recombinant Envelope-Proteins with mutations in the conserved fusion loop allow specific serological diagnosis of Dengue-Infections. Plos Negl. Trop. Dis. 9(11), e0004218 (2015).","DOI":"10.1371\/journal.pntd.0004218"},{"key":"33406_CR44","doi-asserted-by":"crossref","unstructured":"Zheng, A., Umashankar, M. & Kielian, M. In vitro and in vivo studies identify important features of dengue virus pr-E protein interactions. Plos Pathog. 6(10), e1001157 (2010).","DOI":"10.1371\/journal.ppat.1001157"},{"issue":"25","key":"33406_CR45","doi-asserted-by":"publisher","first-page":"6052","DOI":"10.1016\/j.cell.2021.11.010","volume":"184","author":"A Sharma","year":"2021","unstructured":"Sharma, A. et al. The epitope arrangement on flavivirus particles contributes to Mab C10\u2019s extraordinary neutralization breadth across Zika and dengue viruses. Cell 184 (25), 6052\u20136066 (2021).","journal-title":"Cell"},{"issue":"2","key":"33406_CR46","doi-asserted-by":"publisher","first-page":"71","DOI":"10.1016\/j.jsb.2018.03.004","volume":"203","author":"P Stolt-Bergner","year":"2018","unstructured":"Stolt-Bergner, P. et al. Baculovirus-driven protein expression in insect cells: A benchmarking study. J. Struct. Biol. 203 (2), 71\u201380 (2018).","journal-title":"J. Struct. Biol."},{"issue":"4","key":"33406_CR47","doi-asserted-by":"publisher","first-page":"102572","DOI":"10.1016\/j.xpro.2023.102572","volume":"4","author":"A Schutz","year":"2023","unstructured":"Schutz, A. et al. A concise guide to choosing suitable gene expression systems for Recombinant protein production. STAR. Protoc. 4 (4), 102572 (2023).","journal-title":"STAR. Protoc."},{"issue":"1","key":"33406_CR48","doi-asserted-by":"publisher","first-page":"47","DOI":"10.1038\/nmeth.1800","volume":"9","author":"A del Valle Rodr\u00edguez","year":"2012","unstructured":"del Valle Rodr\u00edguez, A., Didiano, D. & Desplan, C. Power tools for gene expression and clonal analysis in drosophila. Nat. Methods. 9 (1), 47\u201355 (2012).","journal-title":"Nat. Methods"},{"key":"33406_CR49","doi-asserted-by":"publisher","first-page":"61","DOI":"10.1016\/j.jbiotec.2013.11.018","volume":"171","author":"X Shen","year":"2014","unstructured":"Shen, X., Hacker, D. L., Baldi, L. & Wurm, F. M. Virus-free transient protein production in Sf9 cells. J. Biotechnol. 171, 61\u201370 (2014).","journal-title":"J. Biotechnol."},{"key":"33406_CR50","doi-asserted-by":"publisher","first-page":"67","DOI":"10.1016\/j.jbiotec.2015.10.007","volume":"216","author":"X Shen","year":"2015","unstructured":"Shen, X. et al. A simple plasmid-based transient gene expression method using high five cells. J. Biotechnol. 216, 67\u201375 (2015).","journal-title":"J. Biotechnol."},{"issue":"10","key":"33406_CR51","doi-asserted-by":"publisher","first-page":"1560","DOI":"10.1128\/CVI.00087-10","volume":"17","author":"M Kuwahara","year":"2010","unstructured":"Kuwahara, M. & Konishi, E. Evaluation of extracellular subviral particles of dengue virus type 2 and Japanese encephalitis virus produced by spodoptera Frugiperda cells for use as vaccine and diagnostic antigens. Clinical Vaccine Immunology: CVI. 17 (10), 1560\u20131566 (2010).","journal-title":"Clinical Vaccine Immunology: CVI"},{"issue":"3","key":"33406_CR52","doi-asserted-by":"publisher","first-page":"1071","DOI":"10.1007\/s00253-012-4371-y","volume":"97","author":"H Yamaji","year":"2013","unstructured":"Yamaji, H. et al. Efficient production of Japanese encephalitis virus-like particles by Recombinant lepidopteran insect cells. Appl. Microbiol. Biotechnol. 97 (3), 1071\u20131079 (2013).","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"33406_CR53","doi-asserted-by":"crossref","unstructured":"Bleckmann, M. et al. Identifying parameters to improve the reproducibility of transient gene expression in high five cells. Plos One 14(6), e0217878 (2019).","DOI":"10.1371\/journal.pone.0217878"},{"issue":"1","key":"33406_CR54","doi-asserted-by":"publisher","first-page":"21393","DOI":"10.1038\/s41598-020-78425-9","volume":"10","author":"J Korn","year":"2020","unstructured":"Korn, J. et al. Baculovirus-free insect cell expression system for high yield antibody and antigen production. Sci. Rep. 10 (1), 21393 (2020).","journal-title":"Sci. Rep."},{"issue":"Edition","key":"33406_CR55","first-page":"2305","volume":"3","author":"M Schubert","year":"2021","unstructured":"Schubert, M. et al. Reproducible and easy production of mammalian proteins by transient gene expression in high five insect cells. Struct. Proteom. 3 (Edition), 2305\u20132129 (2021).","journal-title":"Struct. Proteom."},{"issue":"1","key":"33406_CR56","doi-asserted-by":"publisher","first-page":"81","DOI":"10.1007\/s00253-017-8623-8","volume":"102","author":"C Oliveira","year":"2018","unstructured":"Oliveira, C. & Domingues, L. Guidelines to reach high-quality purified Recombinant proteins. Appl. Microbiol. Biotechnol. 102 (1), 81\u201392 (2018).","journal-title":"Appl. Microbiol. Biotechnol."},{"issue":"1","key":"33406_CR57","doi-asserted-by":"publisher","first-page":"2795","DOI":"10.1038\/s41467-021-23167-z","volume":"12","author":"A de Marco","year":"2021","unstructured":"de Marco, A. et al. Quality control of protein reagents for the improvement of research data reproducibility. Nat. Commun. 12 (1), 2795 (2021).","journal-title":"Nat. Commun."},{"key":"33406_CR58","doi-asserted-by":"crossref","unstructured":"Vaney, M. C. et al. Evolution and activation mechanism of the flavivirus class II membrane-fusion machinery. Nat. Commun. 13(1), 3718 (2022).","DOI":"10.1038\/s41467-022-31111-y"},{"key":"33406_CR59","doi-asserted-by":"crossref","unstructured":"Crampon, E. et al. New insight into flavivirus maturation from structure\/function studies of the yellow fever virus envelope protein complex. mBio 14(5), e0070623 (2023).","DOI":"10.1128\/mbio.00706-23"},{"issue":"24","key":"33406_CR60","doi-asserted-by":"publisher","first-page":"13975","DOI":"10.1128\/Jvi.78.24.13975-13986.2004","volume":"78","author":"WD Crill","year":"2004","unstructured":"Crill, W. D. & Chang, G. J. J. Localization and characterization of flavivirus envelope glycoprotein cross-reactive epitopes. J. Virol. 78 (24), 13975\u201313986. https:\/\/doi.org\/10.1128\/Jvi.78.24.13975-13986.2004 (2004).","journal-title":"J. Virol."},{"key":"33406_CR61","doi-asserted-by":"crossref","unstructured":"Musso, D. & Despres, P. Serological diagnosis of Flavivirus-Associated human infections. Diagnostics (Basel) 10(5), 302 (2020).","DOI":"10.3390\/diagnostics10050302"},{"issue":"5","key":"33406_CR62","doi-asserted-by":"publisher","first-page":"460","DOI":"10.1056\/NEJMoa044029","volume":"353","author":"MP Busch","year":"2005","unstructured":"Busch, M. P. et al. Screening the blood supply for West nile virus RNA by nucleic acid amplification testing. N. Engl. J. Med. 353 (5), 460\u2013467 (2005).","journal-title":"N. Engl. J. Med."},{"issue":"9","key":"33406_CR63","doi-asserted-by":"publisher","first-page":"1505","DOI":"10.1002\/jmv.24735","volume":"89","author":"D Musso","year":"2017","unstructured":"Musso, D. et al. Molecular detection of Zika virus in blood and RNA load determination during the French Polynesian outbreak. J. Med. Virol. 89 (9), 1505\u20131510 (2017).","journal-title":"J. Med. Virol."},{"issue":"2","key":"33406_CR64","doi-asserted-by":"publisher","first-page":"177","DOI":"10.1128\/Cvi.00078-07","volume":"15","author":"M Niedrig","year":"2008","unstructured":"Niedrig, M., K\u00fcrsteiner, O., Herzog, C. & Sonnenberg, K. Evaluation of an indirect Immunofluorescence assay for detection of Immunoglobulin M (IgM) and IgG antibodies against yellow fever virus. Clin. Vaccine Immunol. 15 (2), 177\u2013181. https:\/\/doi.org\/10.1128\/Cvi.00078-07 (2008).","journal-title":"Clin. Vaccine Immunol."},{"key":"33406_CR65","doi-asserted-by":"crossref","unstructured":"Rathore, A. P. S. & St John, A. L. Cross-Reactive immunity among flaviviruses. Front. Immunol. 11, 334 (2020).","DOI":"10.3389\/fimmu.2020.00334"},{"key":"33406_CR66","doi-asserted-by":"crossref","unstructured":"Hou, B. H., Chen, H., Gao, N. & An, J. Cross-reactive immunity among five medically important mosquito-borne flaviviruses related to human diseases. Viruses 14(6), 1213 (2022).","DOI":"10.3390\/v14061213"},{"key":"33406_CR67","doi-asserted-by":"publisher","first-page":"132","DOI":"10.1016\/j.coviro.2017.06.009","volume":"24","author":"FA Rey","year":"2017","unstructured":"Rey, F. A., Stiasny, K. & Heinz, F. X. Flavivirus structural heterogeneity: implications for cell entry. Curr. Opin. Virol. 24, 132\u2013139 (2017).","journal-title":"Curr. Opin. Virol."},{"issue":"22","key":"33406_CR68","doi-asserted-by":"publisher","first-page":"12067","DOI":"10.1128\/JVI.05559-11","volume":"85","author":"S Mukherjee","year":"2011","unstructured":"Mukherjee, S., Lin, T. Y., Dowd, K. A., Manhart, C. J. & Pierson, T. C. The infectivity of prm-containing partially mature West nile virus does not require the activity of cellular furin-like proteases. J. Virol. 85 (22), 12067\u201312072 (2011).","journal-title":"J. Virol."},{"key":"33406_CR69","doi-asserted-by":"crossref","unstructured":"Chabierski, S. et al. Distinguishing West nile virus infection using a recombinant envelope protein with mutations in the conserved fusion-loop. Bmc Infect. Dis. 14, 246 (2014).","DOI":"10.1186\/1471-2334-14-246"},{"key":"33406_CR70","doi-asserted-by":"crossref","unstructured":"Lotke, R., Petersen, M. & Sauter, D. Restriction of viral glycoprotein maturation by cellular protease inhibitors. Viruses 16(3), 332 (2024).","DOI":"10.3390\/v16030332"},{"key":"33406_CR71","doi-asserted-by":"crossref","unstructured":"Braun, E. & Sauter, D. Furin-mediated protein processing in infectious diseases and cancer. Clin. Transl. Immunol. 8, e1073 (2019).","DOI":"10.1002\/cti2.1073"},{"key":"33406_CR72","doi-asserted-by":"crossref","unstructured":"Jaron, M. et al.  Viruses 14(10), 2087 (2022).","DOI":"10.3390\/v14102087"},{"issue":"1","key":"33406_CR73","doi-asserted-by":"publisher","first-page":"14874","DOI":"10.1038\/s41598-024-65316-6","volume":"14","author":"V Lampinen","year":"2024","unstructured":"Lampinen, V. et al. Production of norovirus-, rotavirus-, and enterovirus-like particles in insect cells is simplified by plasmid-based expression. Sci. Rep. 14 (1), 14874 (2024).","journal-title":"Sci. Rep."}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-025-33406-8","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-025-33406-8.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-025-33406-8.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,16]],"date-time":"2026-06-16T15:56:10Z","timestamp":1781625370000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-025-33406-8"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,29]]},"references-count":73,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["33406"],"URL":"https:\/\/doi.org\/10.1038\/s41598-025-33406-8","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,29]]},"assertion":[{"value":"11 August 2025","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"18 December 2025","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"29 December 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":"The authors declare no competing interests.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"45666"}}