{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T19:11:00Z","timestamp":1791486660491,"version":"4.3.4"},"reference-count":57,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2025,8,6]],"date-time":"2025-08-06T00:00:00Z","timestamp":1754438400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2025,8,6]],"date-time":"2025-08-06T00:00:00Z","timestamp":1754438400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"name":"Fraunhofer-Institut f\u00fcr Zelltherapie und Immunologie IZI"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title>\n          <jats:p>West Nile virus (WNV), Usutu virus (USUV) and tick-borne encephalitis virus (TBEV) are worldwide endemic zoonotic orthoflaviviruses, often co-circulating in the same areas. Serological studies in animals, mostly birds and horses, are important means to monitor the spread of these viruses and the infection risks for humans. However, cross-reactive antibodies to these structurally similar flaviviruses frequently impact serological differentiation in enzyme-linked immunosorbent assays (ELISAs), hence time-consuming virus neutralization tests (VNTs) have to be employed in laboratories with high biosafety level. This study presents ELISAs using recombinant flavivirus E proteins with point mutations in the conserved fusion loop domain (Equad proteins) for differentiating IgY or IgG antibodies against WNV, USUV or TBEV in ducks, geese, chickens and horses. Panels of 169 duck and goose sera, 101 chicken sera and 136 horse sera were tested in Equad ELISAs, which resulted in high sensitivity and specificity, further improved by a pre-absorption step for the differentiation of WNV and USUV antibodies. Equad ELISAs for poultry and horse sera enable the reliable differentiation of WNV, USUV and TBEV specific antibodies without the need for VNTs, which has important implications for conducting seroprevalence studies as well as for veterinary routine diagnosis.<\/jats:p>","DOI":"10.1038\/s41598-025-14448-4","type":"journal-article","created":{"date-parts":[[2025,8,6]],"date-time":"2025-08-06T11:34:34Z","timestamp":1754480074000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Serological differentiation of West Nile, Usutu, and tick-borne encephalitis virus antibodies in birds and horses using mutant E protein ELISAs"],"prefix":"10.1038","volume":"15","author":[{"given":"Anne","family":"Schwarzer","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ute","family":"Ziegler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jasmin","family":"Fertey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Markus","family":"Kreuz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas W.","family":"Vahlenkamp","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martin H.","family":"Groschup","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sebastian","family":"Ulbert","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,8,6]]},"reference":[{"key":"14448_CR1","doi-asserted-by":"publisher","unstructured":"Colpitts, T. M., Conway, M. J., Montgomery, R. R. & Fikrig, E. West nile virus: biology, transmission, and human infection. Clin. Microbiol. Rev. 25, 635\u2013648. https:\/\/doi.org\/10.1128\/cmr.00045 (2012).","DOI":"10.1128\/cmr.00045"},{"key":"14448_CR2","doi-asserted-by":"publisher","first-page":"24","DOI":"10.1016\/j.prevetmed.2008.06.023","volume":"88","author":"K Brugger","year":"2009","unstructured":"Brugger, K. & Rubel, F. Simulation of climate-change scenarios to explain Usutu-virus dynamics in Austria. Prev. Vet. Med. 88, 24\u201331. https:\/\/doi.org\/10.1016\/j.prevetmed.2008.06.023 (2009).","journal-title":"Prev. Vet. Med."},{"key":"14448_CR3","doi-asserted-by":"publisher","unstructured":"Semenza, J. C. & Suk, J. E. Vector-borne diseases and climate change: a European perspective. FEMS Microbiol. Lett. 365 https:\/\/doi.org\/10.1093\/femsle\/fnx244 (2018).","DOI":"10.1093\/femsle\/fnx244"},{"key":"14448_CR4","doi-asserted-by":"publisher","unstructured":"Ziegler, U. et al. Spread of West nile virus and Usutu virus in the German bird population, 2019\u20132020. Microorganisms 10 https:\/\/doi.org\/10.3390\/microorganisms10040807 (2022).","DOI":"10.3390\/microorganisms10040807"},{"key":"14448_CR5","doi-asserted-by":"publisher","unstructured":"Bergmann, F. et al. Longitudinal study of the occurrence of Usutu virus and West nile virus infections in birds in a zoological garden in Northern Germany. Pathogens 12 https:\/\/doi.org\/10.3390\/pathogens12060753 (2023).","DOI":"10.3390\/pathogens12060753"},{"key":"14448_CR6","doi-asserted-by":"publisher","unstructured":"Schopf, F. et al. Circulation of West Nile virus and Usutu virus in birds in Germany, 2021 and 2022. Infectious diseases (London, England), 1\u201322; (2024). https:\/\/doi.org\/10.1080\/23744235.2024.2419859","DOI":"10.1080\/23744235.2024.2419859"},{"key":"14448_CR7","doi-asserted-by":"publisher","first-page":"39","DOI":"10.1016\/j.antiviral.2018.12.005","volume":"162","author":"U Ziegler","year":"2019","unstructured":"Ziegler, U. et al. West nile virus epizootic in germany, 2018. Antiviral Res. 162, 39\u201343. https:\/\/doi.org\/10.1016\/j.antiviral.2018.12.005 (2019).","journal-title":"Antiviral Res."},{"key":"14448_CR8","doi-asserted-by":"publisher","first-page":"20","DOI":"10.53854\/liim-3101-4","volume":"31","author":"C D\u2019Amore","year":"2022","unstructured":"D\u2019Amore, C. et al. West nile virus diffusion in temperate regions and climate change. A systematic review. Le Infezioni Med. 31, 20\u201330. https:\/\/doi.org\/10.53854\/liim-3101-4 (2022).","journal-title":"Le Infezioni Med."},{"key":"14448_CR9","doi-asserted-by":"publisher","first-page":"e32604","DOI":"10.1371\/journal.pone.0032604","volume":"7","author":"N Becker","year":"2012","unstructured":"Becker, N. et al. Epizootic emergence of Usutu virus in wild and captive birds in Germany. PloS One. 7, e32604. https:\/\/doi.org\/10.1371\/journal.pone.0032604 (2012).","journal-title":"PloS One"},{"key":"14448_CR10","doi-asserted-by":"publisher","unstructured":"Michel, F. et al. Evidence for West nile virus and Usutu virus infections in wild and resident birds in germany, 2017 and 2018. Viruses 11 https:\/\/doi.org\/10.3390\/v11070674 (2019).","DOI":"10.3390\/v11070674"},{"key":"14448_CR11","doi-asserted-by":"publisher","unstructured":"Cl\u00e9, M. et al. Usutu virus: A new threat? Epidemiol. Infect. 147 (e232). https:\/\/doi.org\/10.1017\/S0950268819001213 (2019).","DOI":"10.1017\/S0950268819001213"},{"key":"14448_CR12","doi-asserted-by":"publisher","first-page":"23","DOI":"10.1016\/j.antiviral.2019.01.014","volume":"164","author":"D Ruzek","year":"2019","unstructured":"Ruzek, D. et al. Tick-borne encephalitis in Europe and russia: review of pathogenesis, clinical features, therapy, and vaccines. Antiviral Res. 164, 23\u201351. https:\/\/doi.org\/10.1016\/j.antiviral.2019.01.014 (2019).","journal-title":"Antiviral Res."},{"key":"14448_CR13","doi-asserted-by":"publisher","unstructured":"Dobler, G., Euringer, K., Kaier, K. & Borde, J. P. Serological protection rates against TBEV infection in blood donors from a highly endemic region in Southern Germany. Vaccines 11 https:\/\/doi.org\/10.3390\/vaccines11030522 (2023).","DOI":"10.3390\/vaccines11030522"},{"key":"14448_CR14","doi-asserted-by":"publisher","unstructured":"Brugman, V. A. et al. The role of culex pipiens L. (Diptera: Culicidae) in virus transmission in Europe. Int. J. Environ. Res. Public Health. 15 https:\/\/doi.org\/10.3390\/ijerph15020389 (2018).","DOI":"10.3390\/ijerph15020389"},{"key":"14448_CR15","doi-asserted-by":"publisher","first-page":"370","DOI":"10.1056\/NEJMra030476","volume":"351","author":"T Solomon","year":"2004","unstructured":"Solomon, T. Flavivirus encephalitis. N. Engl. J. Med. 351, 370\u2013378. https:\/\/doi.org\/10.1056\/NEJMra030476 (2004).","journal-title":"N. Engl. J. Med."},{"key":"14448_CR16","doi-asserted-by":"publisher","first-page":"637","DOI":"10.1007\/s00705-004-0463-z","volume":"150","author":"KM van der Meulen","year":"2005","unstructured":"van der Meulen, K. M., Pensaert, M. B. & Nauwynck, H. J. West nile virus in the vertebrate world. Arch. Virol. 150, 637\u2013657. https:\/\/doi.org\/10.1007\/s00705-004-0463-z (2005).","journal-title":"Arch. Virol."},{"key":"14448_CR17","doi-asserted-by":"publisher","first-page":"1167","DOI":"10.3201\/eid1108.050289a","volume":"11","author":"EB Hayes","year":"2005","unstructured":"Hayes, E. B. et al. Epidemiology and transmission dynamics of West nile virus disease. Emerg. Infect. Dis. 11, 1167\u20131173. https:\/\/doi.org\/10.3201\/eid1108.050289a (2005).","journal-title":"Emerg. Infect. Dis."},{"key":"14448_CR18","doi-asserted-by":"publisher","first-page":"571","DOI":"10.3109\/23744235.2016.1164890","volume":"48","author":"S David","year":"2016","unstructured":"David, S. & Abraham, A. M. Epidemiological and clinical aspects on West nile virus, a globally emerging pathogen. Infect. Dis. (London England). 48, 571\u2013586. https:\/\/doi.org\/10.3109\/23744235.2016.1164890 (2016).","journal-title":"Infect. Dis. (London England)"},{"key":"14448_CR19","doi-asserted-by":"publisher","unstructured":"Zannoli, S. & Sambri, V. West nile virus and Usutu virus Co-Circulation in europe: epidemiology and implications. Microorganisms 7 https:\/\/doi.org\/10.3390\/microorganisms7070184 (2019).","DOI":"10.3390\/microorganisms7070184"},{"key":"14448_CR20","doi-asserted-by":"publisher","first-page":"772","DOI":"10.1089\/vbz.2012.1236","volume":"13","author":"L Barbic","year":"2013","unstructured":"Barbic, L. et al. Demonstration of Usutu virus antibodies in horses, Croatia. Vector Borne Zoonotic Dis. (Larchmont N Y). 13, 772\u2013774. https:\/\/doi.org\/10.1089\/vbz.2012.1236 (2013).","journal-title":"Vector Borne Zoonotic Dis. (Larchmont N Y)"},{"key":"14448_CR21","doi-asserted-by":"publisher","unstructured":"Michelitsch, A., Wernike, K., Klaus, C., Dobler, G. & Beer, M. Exploring the reservoir hosts of Tick-Borne encephalitis virus. Viruses 11 https:\/\/doi.org\/10.3390\/v11070669 (2019).","DOI":"10.3390\/v11070669"},{"key":"14448_CR22","doi-asserted-by":"publisher","first-page":"157","DOI":"10.1016\/j.jviromet.2008.10.016","volume":"155","author":"S Zainah","year":"2009","unstructured":"Zainah, S. et al. Performance of a commercial rapid dengue NS1 antigen immunochromatography test with reference to dengue NS1 antigen-capture ELISA. J. Virol. Methods. 155, 157\u2013160. https:\/\/doi.org\/10.1016\/j.jviromet.2008.10.016 (2009).","journal-title":"J. Virol. Methods"},{"key":"14448_CR23","doi-asserted-by":"publisher","first-page":"788","DOI":"10.3390\/v16050788","volume":"16","author":"P Girl","year":"2024","unstructured":"Girl, P. et al. Comparison of five serological methods for the detection of West nile virus antibodies. Viruses 16, 788. https:\/\/doi.org\/10.3390\/v16050788 (2024).","journal-title":"Viruses"},{"key":"14448_CR24","doi-asserted-by":"publisher","unstructured":"Beck, C. et al. A High-Performance Multiplex Immunoassay for Serodiagnosis of Flavivirus-Associated Neurological Diseases in Horses. BioMed research international 678084; (2015). https:\/\/doi.org\/10.1155\/2015\/678084 (2015).","DOI":"10.1155\/2015\/678084"},{"key":"14448_CR25","doi-asserted-by":"publisher","first-page":"6049","DOI":"10.3390\/ijerph10116049","volume":"10","author":"C Beck","year":"2013","unstructured":"Beck, C. et al. Flaviviruses in europe: complex circulation patterns and their consequences for the diagnosis and control of West nile disease. Int. J. Environ. Res. Public Health. 10, 6049\u20136083. https:\/\/doi.org\/10.3390\/ijerph10116049 (2013).","journal-title":"Int. J. Environ. Res. Public Health"},{"issue":"Pt 1","key":"14448_CR26","doi-asserted-by":"publisher","first-page":"37","DOI":"10.1099\/0022-1317-70-1-37","volume":"70","author":"CH Calisher","year":"1989","unstructured":"Calisher, C. H. et al. Antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera. J. Gen. Virol. 70 (Pt 1), 37\u201343. https:\/\/doi.org\/10.1099\/0022-1317-70-1-37 (1989).","journal-title":"J. Gen. Virol."},{"key":"14448_CR27","doi-asserted-by":"publisher","first-page":"e0005936","DOI":"10.1371\/journal.pntd.0005936","volume":"11","author":"C Beck","year":"2017","unstructured":"Beck, C. et al. Improved reliability of serological tools for the diagnosis of West nile fever in horses within Europe. PLoS Negl. Trop. Dis. 11, e0005936. https:\/\/doi.org\/10.1371\/journal.pntd.0005936 (2017).","journal-title":"PLoS Negl. Trop. Dis."},{"key":"14448_CR28","doi-asserted-by":"publisher","first-page":"11828","DOI":"10.1128\/JVI.00643-07","volume":"81","author":"T Oliphant","year":"2007","unstructured":"Oliphant, T. et al. Induction of epitope-specific neutralizing antibodies against West nile virus. J. Virol. 81, 11828\u201311839. https:\/\/doi.org\/10.1128\/JVI.00643-07 (2007).","journal-title":"J. Virol."},{"key":"14448_CR29","doi-asserted-by":"publisher","first-page":"212","DOI":"10.1111\/j.1600-065X.2008.00676.x","volume":"225","author":"MS Diamond","year":"2008","unstructured":"Diamond, M. S., Pierson, T. C. & Fremont, D. H. The structural immunology of antibody protection against West nile virus. Immunol. Rev. 225, 212\u2013225. https:\/\/doi.org\/10.1111\/j.1600-065X.2008.00676.x (2008).","journal-title":"Immunol. Rev."},{"key":"14448_CR30","doi-asserted-by":"publisher","first-page":"975398","DOI":"10.3389\/fcimb.2022.975398","volume":"12","author":"KR Chan","year":"2022","unstructured":"Chan, K. R. et al. Serological cross-reactivity among common flaviviruses. Front. Cell. Infect. Microbiol. 12, 975398. https:\/\/doi.org\/10.3389\/fcimb.2022.975398 (2022).","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"14448_CR31","doi-asserted-by":"publisher","first-page":"3167","DOI":"10.1128\/JCM.01143-07","volume":"45","author":"JA Roberson","year":"2007","unstructured":"Roberson, J. A., Crill, W. D. & Chang, G. J. J. Differentiation of West nile and st. Louis encephalitis virus infections by use of noninfectious virus-like particles with reduced cross-reactivity. J. Clin. Microbiol. 45, 3167\u20133174. https:\/\/doi.org\/10.1128\/JCM.01143-07 (2007).","journal-title":"J. Clin. Microbiol."},{"key":"14448_CR32","doi-asserted-by":"publisher","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). https:\/\/doi.org\/10.1186\/1471-2334-14-246 (2014).","DOI":"10.1186\/1471-2334-14-246"},{"key":"14448_CR33","doi-asserted-by":"publisher","first-page":"e0004218","DOI":"10.1371\/journal.pntd.0004218","volume":"9","author":"A Rockstroh","year":"2015","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, e0004218. https:\/\/doi.org\/10.1371\/journal.pntd.0004218 (2015).","journal-title":"PLoS Negl. Trop. Dis."},{"key":"14448_CR34","doi-asserted-by":"publisher","first-page":"1111","DOI":"10.3201\/eid2501.180605","volume":"25","author":"A Rockstroh","year":"2019","unstructured":"Rockstroh, A. et al. Dengue virus IgM serotyping by ELISA with Recombinant mutant envelope proteins. Emerg. Infect. Dis. 25, 1111\u20131115. https:\/\/doi.org\/10.3201\/eid2501.180605 (2019).","journal-title":"Emerg. Infect. Dis."},{"key":"14448_CR35","doi-asserted-by":"publisher","first-page":"2779","DOI":"10.1111\/tbed.14429","volume":"69","author":"BS Berneck","year":"2022","unstructured":"Berneck, B. S. et al. Serological differentiation of West nile virus- and Usutu virus-induced antibodies by envelope proteins with modified cross-reactive epitopes. Transbound. Emerg. Dis. 69, 2779\u20132787. https:\/\/doi.org\/10.1111\/tbed.14429 (2022).","journal-title":"Transbound. Emerg. Dis."},{"key":"14448_CR36","doi-asserted-by":"publisher","first-page":"e99","DOI":"10.1038\/emi.2017.87","volume":"6","author":"A Rockstroh","year":"2017","unstructured":"Rockstroh, A. et al. Specific detection of dengue and Zika virus antibodies using envelope proteins with mutations in the conserved fusion loop. Emerg. Microbes Infections. 6, e99. https:\/\/doi.org\/10.1038\/emi.2017.87 (2017).","journal-title":"Emerg. Microbes Infections"},{"key":"14448_CR37","doi-asserted-by":"publisher","first-page":"2466","DOI":"10.3201\/eid2709.204706","volume":"27","author":"EF Oliveira-Filho","year":"2021","unstructured":"Oliveira-Filho, E. F. et al. Ecologic determinants of West nile virus Seroprevalence among equids, Brazil. Emerg. Infect. Dis. 27, 2466\u20132470. https:\/\/doi.org\/10.3201\/eid2709.204706 (2021).","journal-title":"Emerg. Infect. Dis."},{"key":"14448_CR38","doi-asserted-by":"publisher","first-page":"639","DOI":"10.1089\/vbz.2009.0236","volume":"10","author":"D Seidowski","year":"2010","unstructured":"Seidowski, D. et al. West nile virus monitoring of migratory and resident birds in Germany. Vector Borne Zoonotic Dis. (Larchmont N Y). 10, 639\u2013647. https:\/\/doi.org\/10.1089\/vbz.2009.0236 (2010).","journal-title":"Vector Borne Zoonotic Dis. (Larchmont N Y)"},{"key":"14448_CR39","doi-asserted-by":"publisher","unstructured":"Holicki, C. M. et al. Pathogenicity of West nile virus lineage 1 to German poultry. Vaccines 8 https:\/\/doi.org\/10.3390\/vaccines8030507 (2020).","DOI":"10.3390\/vaccines8030507"},{"key":"14448_CR40","doi-asserted-by":"publisher","unstructured":"Reemtsma, H. et al. Pathogenesis of West nile virus lineage 2 in domestic geese after experimental infection. Viruses 14 https:\/\/doi.org\/10.3390\/v14061319 (2022).","DOI":"10.3390\/v14061319"},{"key":"14448_CR41","doi-asserted-by":"publisher","first-page":"959","DOI":"10.3390\/pathogens12070959","volume":"12","author":"H Reemtsma","year":"2023","unstructured":"Reemtsma, H. et al. A prior Usutu virus infection can protect geese from severe West nile disease. Pathogens 12, 959. https:\/\/doi.org\/10.3390\/pathogens12070959 (2023).","journal-title":"Pathogens"},{"key":"14448_CR42","doi-asserted-by":"publisher","first-page":"499","DOI":"10.1111\/tbed.13704","volume":"68","author":"F Michel","year":"2021","unstructured":"Michel, F. et al. Role of ducks in the transmission cycle of tick-borne encephalitis virus? Transbound. Emerg. Dis. 68, 499\u2013508. https:\/\/doi.org\/10.1111\/tbed.13704 (2021).","journal-title":"Transbound. Emerg. Dis."},{"key":"14448_CR43","doi-asserted-by":"publisher","first-page":"372","DOI":"10.1089\/vbz.2011.0709","volume":"12","author":"JY Yeh","year":"2012","unstructured":"Yeh, J. Y. et al. A diagnostic algorithm to serologically differentiate West nile virus from Japanese encephalitis virus infections and its validation in field surveillance of poultry and horses. Vector-Borne Zoonotic Dis. 12, 372\u2013379. https:\/\/doi.org\/10.1089\/vbz.2011.0709 (2012).","journal-title":"Vector-Borne Zoonotic Dis."},{"key":"14448_CR44","doi-asserted-by":"publisher","unstructured":"Netto, E. M. et al. High Zika virus Seroprevalence in salvador, Northeastern Brazil limits the potential for further outbreaks. mBio 8 https:\/\/doi.org\/10.1128\/mbio.01390-17 (2017).","DOI":"10.1128\/mbio.01390-17"},{"key":"14448_CR45","doi-asserted-by":"publisher","unstructured":"Kvapil, P. et al. A Sentinel serological study in selected zoo animals to assess early detection of West nile and Usutu virus circulation in Slovenia. Viruses 13 https:\/\/doi.org\/10.3390\/v13040626 (2021).","DOI":"10.3390\/v13040626"},{"key":"14448_CR46","doi-asserted-by":"publisher","first-page":"40","DOI":"10.1016\/j.vetmic.2017.02.007","volume":"203","author":"JPS Neo","year":"2017","unstructured":"Neo, J. P. S. & Tan, B. H. The use of animals as a surveillance tool for monitoring environmental health hazards, human health hazards and bioterrorism. Vet. Microbiol. 203, 40\u201348. https:\/\/doi.org\/10.1016\/j.vetmic.2017.02.007 (2017).","journal-title":"Vet. Microbiol."},{"key":"14448_CR47","doi-asserted-by":"publisher","first-page":"127","DOI":"10.1111\/j.1749-6632.2001.tb02691.x","volume":"951","author":"H Bin","year":"2001","unstructured":"Bin, H. et al. West nile fever in Israel 1999\u20132000: from geese to humans. Ann. N. Y. Acad. Sci. 951, 127\u2013142. https:\/\/doi.org\/10.1111\/j.1749-6632.2001.tb02691.x (2001).","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"14448_CR48","doi-asserted-by":"publisher","first-page":"824","DOI":"10.3201\/eid3104.241293","volume":"31","author":"C Fontoura-Gon\u00e7alves","year":"2025","unstructured":"Fontoura-Gon\u00e7alves, C. et al. Dynamics of bagaza, West nile, and Usutu viruses in Red-Legged partridges, portugal, 2018\u20132022. Emerg. Infect. Dis. 31, 824\u2013828. https:\/\/doi.org\/10.3201\/eid3104.241293 (2025).","journal-title":"Emerg. Infect. Dis."},{"key":"14448_CR49","doi-asserted-by":"publisher","unstructured":"Klaus, C., Ziegler, U., Kalthoff, D., Hoffmann, B. & Beer, M. Tick-borne encephalitis virus (TBEV) - findings on cross reactivity and longevity of TBEV antibodies in animal Sera. BMC Vet. Res. 10 https:\/\/doi.org\/10.1186\/1746-6148-10-78 (2014).","DOI":"10.1186\/1746-6148-10-78"},{"key":"14448_CR50","doi-asserted-by":"publisher","first-page":"1801","DOI":"10.1111\/tbed.12569","volume":"64","author":"NB Cleton","year":"2017","unstructured":"Cleton, N. B. et al. A serological protein microarray for detection of multiple Cross-Reactive flavivirus infections in horses for veterinary and public health surveillance. Transbound. Emerg. Dis. 64, 1801\u20131812. https:\/\/doi.org\/10.1111\/tbed.12569 (2017).","journal-title":"Transbound. Emerg. Dis."},{"key":"14448_CR51","doi-asserted-by":"publisher","first-page":"825","DOI":"10.1089\/vbz.2017.2172","volume":"17","author":"TBH Piyasena","year":"2017","unstructured":"Piyasena, T. B. H. et al. Differential diagnosis of flavivirus infections in horses using viral envelope protein domain III antigens in Enzyme-Linked immunosorbent assay. Vector-Borne Zoonotic Dis. 17, 825\u2013835. https:\/\/doi.org\/10.1089\/vbz.2017.2172 (2017).","journal-title":"Vector-Borne Zoonotic Dis."},{"key":"14448_CR52","doi-asserted-by":"publisher","first-page":"168","DOI":"10.1016\/j.vetmic.2013.08.013","volume":"167","author":"J Angenvoort","year":"2013","unstructured":"Angenvoort, J., Brault, A. C., Bowen, R. A. & Groschup, M. H. West nile viral infection of equids. Vet. Microbiol. 167, 168\u2013180. https:\/\/doi.org\/10.1016\/j.vetmic.2013.08.013 (2013).","journal-title":"Vet. Microbiol."},{"key":"14448_CR53","doi-asserted-by":"publisher","unstructured":"Gothe, L. M. R. et al. Horses as sentinels for the circulation of flaviviruses in Eastern-Central Germany. Viruses 15 https:\/\/doi.org\/10.3390\/v15051108 (2023).","DOI":"10.3390\/v15051108"},{"key":"14448_CR54","doi-asserted-by":"publisher","first-page":"665","DOI":"10.1128\/CDLI.12.5.665-667.2005","volume":"12","author":"SEJ Gibbs","year":"2005","unstructured":"Gibbs, S. E. J. et al. Persistence of antibodies to West nile virus in naturally infected rock pigeons (Columba livia). Clin. Diagn. Lab. Immunol. 12, 665\u2013667. https:\/\/doi.org\/10.1128\/CDLI.12.5.665-667.2005 (2005).","journal-title":"Clin. Diagn. Lab. Immunol."},{"key":"14448_CR55","doi-asserted-by":"publisher","unstructured":"Bergmann, F. et al. Vaccination of zoo birds against West nile Virus-A field study. Vaccines 11 https:\/\/doi.org\/10.3390\/vaccines11030652 (2023).","DOI":"10.3390\/vaccines11030652"},{"key":"14448_CR56","doi-asserted-by":"publisher","first-page":"1174","DOI":"10.1111\/2041-210X.12583","volume":"7","author":"CA Fassbinder-Orth","year":"2016","unstructured":"Fassbinder-Orth, C. A. et al. Immunoglobulin detection in wild birds: effectiveness of three secondary anti-avian IgY antibodies in direct ELISAs in 41 avian species. Methods Ecol. Evol. 7, 1174\u20131181. https:\/\/doi.org\/10.1111\/2041-210X.12583 (2016).","journal-title":"Methods Ecol. Evol."},{"key":"14448_CR57","doi-asserted-by":"publisher","unstructured":"Cadar, D. & Simonin, Y. Human Usutu Virus Infections in Europe: A New Risk on Horizon? Viruses 15; (2022). https:\/\/doi.org\/10.3390\/v15010077","DOI":"10.3390\/v15010077"}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-025-14448-4.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-025-14448-4","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-025-14448-4.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,8,6]],"date-time":"2025-08-06T11:34:35Z","timestamp":1754480075000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-025-14448-4"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,6]]},"references-count":57,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["14448"],"URL":"https:\/\/doi.org\/10.1038\/s41598-025-14448-4","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,6]]},"assertion":[{"value":"4 April 2025","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"31 July 2025","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"6 August 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":"S.U. is on a patent application that includes the proteins described in this study. This does not alter his adherence to policies on sharing data and materials. The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. A.S., U.Z., J.F., M.K., T.W.V. and M.H.G. declare no potential conflict of interest.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}},{"value":"Duck, goose and chicken sera were used according to the ethical approvals by the administration of the federal state of Mecklenburg-Western Pomerania, Germany (LALLF reference numbers 7221.3-2-042\/17, 7221.3-2-003\/23, 7221.3-1-075\/16, 7221.3-1.1-018\/18 and 7221.3-1-031\/20) and in accordance with the relevant guidelines. All methods are reported in accordance with ARRIVE guidelines. Blood samples from horses were collected during routine diagnostic testing by veterinarians for which, according to German law, ethical approval was not required.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval"}}],"article-number":"28752"}}