{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T14:59:16Z","timestamp":1790693956621,"version":"4.1.0"},"reference-count":34,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2022,6,8]],"date-time":"2022-06-08T00:00:00Z","timestamp":1654646400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Highly pathogenic influenza A virus H5 subtype remains a risk for transmission in humans. The H5N8 subtype has caused multiple outbreaks in poultry in Europe over the past few winters. During one recent outbreak in poultry in Astrakhan, workers on the farm were also infected. So far, little is known about how this virus evolves and adapts to infect humans. Here, we performed a time-resolved phylogenetic analysis of 129 HA sequences representing all 1891 available H5N8 viruses collected from 2010 to 2020. We also conducted a whole-genome scan on the human virus at the protein level. We found that H5N8 viruses have spilled over in 34 European countries during the flu season of 2020\u20132021. These viruses underwent two significant evolutionary steps during 2015\u20132016 and after 2018. Furthermore, we characterized a number of critical mutations in all viral proteins except PB1-F2, which contribute to increased virulence and avian-to-human adaptation. Our findings suggested that the accumulated mutations under evolution led to quantitative and qualitative changes, likely allowing the virus to spread to humans. Given that the H5N8 virus is co-circulating with other H5 viruses in Europe, the risk of a pandemic should not be underestimated. Continental surveillance and pandemic preparedness are to be established.<\/jats:p>","DOI":"10.3390\/pathogens11060666","type":"journal-article","created":{"date-parts":[[2022,6,12]],"date-time":"2022-06-12T23:55:24Z","timestamp":1655078124000},"page":"666","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Evolutionary and Mutational Characterization of the First H5N8 Subtype Influenza A Virus in Humans"],"prefix":"10.3390","volume":"11","author":[{"given":"Lin","family":"Ding","sequence":"first","affiliation":[{"name":"Department of Cardiology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200240, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Basic Medical Sciences & Forensic Medicine, Hangzhou Medical College, Hangzhou 310059, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xue","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine III, University Hospital Heidelberg, 69120 Heidelberg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7330-2929","authenticated-orcid":false,"given":"Bingqian","family":"Qu","sequence":"additional","affiliation":[{"name":"Department of Veterinary Medicine, Paul Ehrlich Institute, 63225 Langen, Germany"},{"name":"European Virus Bioinformatics Center (EVBC), 07743 Jena, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,6,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Sutton, T.C. (2018). The Pandemic Threat of Emerging H5 and H7 Avian Influenza Viruses. Viruses, 10.","DOI":"10.3390\/v10090461"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"472","DOI":"10.1016\/S0140-6736(97)11212-0","article-title":"Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus","volume":"351","author":"Claas","year":"1998","journal-title":"Lancet"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"9363","DOI":"10.1073\/pnas.96.16.9363","article-title":"Molecular characterization of H9N2 influenza viruses: Were they the donors of the \u201cinternal\u201d genes of H5N1 viruses in Hong Kong?","volume":"96","author":"Guan","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1016\/j.vetmic.2012.12.025","article-title":"Characterization of three H5N5 and one H5N8 highly pathogenic avian influenza viruses in China","volume":"163","author":"Zhao","year":"2013","journal-title":"Vet. Microbiol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"299","DOI":"10.4142\/jvs.2016.17.3.299","article-title":"Molecular characterization of highly pathogenic avian influenza H5N8 viruses isolated from Baikal teals found dead during a 2014 outbreak in Korea","volume":"17","author":"Kim","year":"2016","journal-title":"J. Vet. Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"6521","DOI":"10.1128\/JVI.00728-15","article-title":"Intercontinental Spread of Asian-Origin H5N8 to North America through Beringia by Migratory Birds","volume":"89","author":"Lee","year":"2015","journal-title":"J. Virol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Global Consortium for H5N8 and Related Influenza Viruses (2016). Role for migratory wild birds in the global spread of avian influenza H5N8. Science, 354, 213\u2013217.","DOI":"10.1126\/science.aaf8852"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1368","DOI":"10.3201\/eid2308.170143","article-title":"Genesis of Influenza A(H5N8) Viruses","volume":"23","author":"Barman","year":"2017","journal-title":"Emerg. Infect. Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"20996","DOI":"10.2807\/1560-7917.ES2014.19.50.20996","article-title":"Comparing introduction to Europe of highly pathogenic avian influenza viruses A(H5N8) in 2014 and A(H5N1) in 2005","volume":"19","author":"Adlhoch","year":"2014","journal-title":"Eurosurveillance"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"20814","DOI":"10.1073\/pnas.2001813117","article-title":"Genesis and spread of multiple reassortants during the 2016\/2017 H5 avian influenza epidemic in Eurasia","volume":"117","author":"Lycett","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2100439","DOI":"10.2807\/1560-7917.ES.2021.26.24.2100439","article-title":"Isolation of clade 2.3.4.4b A(H5N8), a highly pathogenic avian influenza virus, from a worker during an outbreak on a poultry farm, Russia, December 2020","volume":"26","author":"Pyankova","year":"2021","journal-title":"Eurosurveillance"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2295","DOI":"10.3201\/eid2512.181472","article-title":"Highly Pathogenic Avian Influenza A(H5N8) Virus in Gray Seals, Baltic Sea","volume":"25","author":"Shin","year":"2019","journal-title":"Emerg. Infect. Dis."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Herve, S., Schmitz, A., Briand, F.X., Gorin, S., Queguiner, S., Niqueux, E., Paboeuf, F., Scoizec, A., Le Bouquin-Leneveu, S., and Eterradossi, N. (2021). Serological Evidence of Backyard Pig Exposure to Highly Pathogenic Avian Influenza H5N8 Virus during 2016\u20132017 Epizootic in France. Pathogens, 10.","DOI":"10.3390\/pathogens10050621"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"503","DOI":"10.1111\/zph.12841","article-title":"Exposure of wild boar to Influenza A viruses in Bavaria: Analysis of seroprevalences and antibody subtype specificity before and after the panzootic of highly pathogenic avian influenza viruses A (H5N8)","volume":"68","author":"Schulein","year":"2021","journal-title":"Zoonoses Public Health"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2856","DOI":"10.3201\/eid2711.211225","article-title":"Encephalitis and Death in Wild Mammals at a Rehabilitation Center after Infection with Highly Pathogenic Avian Influenza A(H5N8) Virus, United Kingdom","volume":"27","author":"Floyd","year":"2021","journal-title":"Emerg. Infect. Dis."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1016\/j.antiviral.2009.01.006","article-title":"Different neuraminidase inhibitor susceptibilities of human H1N1, H1N2, and H3N2 influenza A viruses isolated in Germany from 2001 to 2005\/2006","volume":"82","author":"Bauer","year":"2009","journal-title":"Antivir. Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"949","DOI":"10.1099\/vir.0.017459-0","article-title":"Emergence of H5N1 avian influenza viruses with reduced sensitivity to neuraminidase inhibitors and novel reassortants in Lao People\u2019s Democratic Republic","volume":"91","author":"Boltz","year":"2010","journal-title":"J. Gen. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1038\/nsmb.1566","article-title":"Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses","volume":"16","author":"Sui","year":"2009","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"8628","DOI":"10.1039\/c2ob25627d","article-title":"Synthesis and evaluation of novel 3-C-alkylated-Neu5Ac2en derivatives as probes of influenza virus sialidase 150-loop flexibility","volume":"10","author":"Rudrawar","year":"2012","journal-title":"Org. Biomol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/S0168-1702(99)00027-1","article-title":"Genetic analysis of mouse-adapted influenza A virus identifies roles for the NA, PB1, and PB2 genes in virulence","volume":"61","author":"Brown","year":"1999","journal-title":"Virus Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"224","DOI":"10.1006\/viro.1997.8580","article-title":"Avian influenza A viruses differ from human viruses by recognition of sialyloligosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site","volume":"233","author":"Matrosovich","year":"1997","journal-title":"Virology"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"3510","DOI":"10.1073\/pnas.1019744108","article-title":"Broadly neutralizing DNA vaccine with specific mutation alters the antigenicity and sugar-binding activities of influenza hemagglutinin","volume":"108","author":"Chen","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"12911","DOI":"10.1128\/JVI.01522-07","article-title":"Epitope mapping of the hemagglutinin molecule of a highly pathogenic H5N1 influenza virus by using monoclonal antibodies","volume":"81","author":"Kaverin","year":"2007","journal-title":"J. Virol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1174","DOI":"10.1080\/22221751.2022.2063076","article-title":"Novel H5N6 reassortants bearing the clade 2.3.4.4b HA gene of H5N8 virus have been detected in poultry and caused multiple human infections in China","volume":"11","author":"Gu","year":"2022","journal-title":"Emerg. Microbes Infect."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Imai, H., Shinya, K., Takano, R., Kiso, M., Muramoto, Y., Sakabe, S., Murakami, S., Ito, M., Yamada, S., and Le, M.T. (2010). The HA and NS genes of human H5N1 influenza A virus contribute to high virulence in ferrets. PLoS Pathog., 6.","DOI":"10.1371\/journal.ppat.1001106"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1760","DOI":"10.1080\/22221751.2021.1971568","article-title":"The C-terminus of non-structural protein 1 (NS1) in H5N8 clade 2.3.4.4 avian influenza virus affects virus fitness in human cells and virulence in mice","volume":"10","author":"Blaurock","year":"2021","journal-title":"Emerg. Microbes Infect."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"14170","DOI":"10.1038\/srep14170","article-title":"Dual E627K and D701N mutations in the PB2 protein of A(H7N9) influenza virus increased its virulence in mammalian models","volume":"5","author":"Zhu","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"180","DOI":"10.1086\/595736","article-title":"A novel neuraminidase deletion mutation conferring resistance to oseltamivir in clinical influenza A\/H3N2 virus","volume":"199","author":"Abed","year":"2009","journal-title":"J. Infect. Dis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2395","DOI":"10.1128\/AAC.01339-05","article-title":"Detection of influenza viruses resistant to neuraminidase inhibitors in global surveillance during the first 3 years of their use","volume":"50","author":"Monto","year":"2006","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Yeo, J.Y., and Gan, S.K. (2021). Peering into Avian Influenza A(H5N8) for a Framework towards Pandemic Preparedness. Viruses, 13.","DOI":"10.20944\/preprints202109.0506.v1"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"30494","DOI":"10.2807\/1560-7917.ES.2017.22.13.30494","article-title":"GISAID: Global initiative on sharing all influenza data\u2014From vision to reality","volume":"22","author":"Shu","year":"2017","journal-title":"Eurosurveillance"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2725","DOI":"10.1093\/molbev\/mst197","article-title":"MEGA6: Molecular Evolutionary Genetics Analysis version 6.0","volume":"30","author":"Tamura","year":"2013","journal-title":"Mol. Biol. Evol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Bouckaert, R., Vaughan, T.G., Barido-Sottani, J., Duchene, S., Fourment, M., Gavryushkina, A., Heled, J., Jones, G., Kuhnert, D., and De Maio, N. (2019). BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis. PLoS Comput. Biol., 15.","DOI":"10.1371\/journal.pcbi.1006650"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Qu, B., Li, X., Cardona, C.J., and Xing, Z. (2020). Reassortment and adaptive mutations of an emerging avian influenza virus H7N4 subtype in China. PLoS ONE, 15.","DOI":"10.1371\/journal.pone.0227597"}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/11\/6\/666\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:26:31Z","timestamp":1760138791000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/11\/6\/666"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,6,8]]},"references-count":34,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2022,6]]}},"alternative-id":["pathogens11060666"],"URL":"https:\/\/doi.org\/10.3390\/pathogens11060666","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,6,8]]}}}