Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario.

1 P Avenida da República Instituto Nacional de Investigação Agrária e Veterinária Quinta do Marquês Oeiras 2780 157 Portugal

Agence Nationale de Sécurité Sanitaire de l'Alimentation de l'Environnement et du Travail Laboratoire de Ploufragan Plouzané Niort Unité de Virologie Immunologie Parasitologie Avaires et Cunicoles 41 Rue de Beaucemaine BP 53 Ploufragan 22440 France

Animal Health Department Food Safety and Veterinary Institute Rruga Aleksandër Moisiu 10 Tirana 1001 Albania

Austrian Agency for Health and Food Safety Institute for Veterinary Disease Control Robert Koch Gasse 17 Mödling 2340 Austria

Avian Virology and Immunology Sciensano Rue Groeselenberg 99 Ukkel 1180 Ukkel Belgium

Biomedical Center Institute for Experimental Pathology University of Iceland Keldnavegi 3 112 Reykjavík Ssn 650269 4549 Keldur 851 Iceland

Croatian Veterinary Institute Poultry Centre Heinzelova 55 Zagreb 10000 Croatia

Department for Virus and Microbiological Special Diagnostics Statens Serum Institut 5 Artillerivej Copenhagen DK 2300 Denmark

Department of Agriculture Food and the Marine Central Veterinary Research Laboratory Backweston Campus Stacumny Lane Celbridge Co Kildare W23 X3PH Ireland

Department of Animal Health State Veterinary Institute Pod Dráhami 918 Zvolen 96086 Slovakia

Department of Microbiology National Veterinary Institute Travvägen 20 Uppsala 75189 Sweden

Department of Molecular Biology State Veterinary Institute Prague Sídlištní 136 24 Praha 6 Lysolaje 16503 Czech Republic

Department of Pathology and Wildlife Disease National Veterinary Institute Travvägen 20 Uppsala 75189 Sweden

Department of Poultry Diseases National Veterinary Research Institute Al Partyzantow 57 Puławy 24 100 Poland

Department of Veterinary and Animal Sciences University of Copenhagen Grønnegårdsvej 15 Frederiksberg 1870 Denmark

Department of Virology Wageningen Bioveterinary Research Houtribweg 39 Lelystad 8221 RA The Netherlands

Department of Viroscience Erasmus MC Dr Molewaterplein 40 Rotterdam 3015 GD The Netherlands

European Centre for Disease Prevention and Control Gustav 3 s boulevard 40 Solna 169 73 Sweden

European Food Safety Authority Via Carlo Magno 1A Parma 43126 Italy

European Reference Laboratory for Avian Influenza and Newcastle Disease Istituto Zooprofilattico Sperimentale delle Venezie viale dell'universita 10 Legnaro Padua 35020 Italy

Federal Department of Home Affairs FDHA Institute of Virology and Immunology IVI Sensemattstrasse 293 Mittelhäusern 3147 Switzerland

Finnish Food Authority Animal Health Diagnostic Unit Veterinary Virology Mustialankatu 3 Helsinki FI 00790 Finland

Icelandic Food and Veterinary Authority Austurvegur 64 Selfoss 800 Iceland

Immunology and Virology department Norwegian Veterinary Institute Arboretveien 57 Oslo Pb 64 N 1431 Ås Norway

Institute for Diagnosis and Animal Health Str Dr Staicovici 63 Bucharest 050557 Romania

Institute of Diagnostic Virology Friedrich Loeffler Institut Südufer 10 Greifswald Insel Riems 17493 Germany

Institute of Food Safety Animal Health and Environment Laboratory of Microbilogy and Pathology 3 Lejupes Street Riga 1076 Latvia

Kosovo Food and Veterinary Agency Sector of Serology and Molecular Diagnostics Kosovo Food and Veterinary Laboratory Str Lidhja e Pejes Prishtina 10000 Kosovo

Laboratory for Animal Health Virology Section Veterinary Services 79 Athalassa Avenue Aglantzia Nicosia 2109 Cyprus

Luxembourg Institute of Health Department of Infection and Immunity 29 Rue Henri Koch Esch sur Alzette 4354 Luxembourg

Luxembourgish Veterinary and Food Administration State Veterinary Laboratory 1 Rue Louis Rech Dudelange 3555 Luxembourg

Ministry of Agriculture Fisheries and Food Laboratorio Central de Veterinaria Ctra M 106 Km 1 4 Algete Madrid 28110 Spain

National Centre for Laboratory Research and Risk Assessment Kreutzwaldi 30 Tartu 51006 Estonia

National Food and Veterinary Risk Assessment Institute Kairiukscio str 10 Vilnius 08409 Lithuania

National Reference Laboratory for Avian Influenza and Newcastle Disease National Diagnostic and Research Veterinary Medical Institute 190 Lomsko Shose Blvd Sofia 1231 Bulgaria

Republican Center for Veterinary Diagnostics 3 street Murelor Chisinau 2051 Republic of Moldova

Thessaloniki Veterinary Centre Department of Avian Diseases 26th October Street 80 Thessaloniki 54627 Greece

University of Ljubljana Veterinary Faculty National Veterinary Institute Gerbičeva 60 Ljubljana 1000 Slovenia

Veterinary Diagnostic Directorate Laboratory of Virology National Food Chain Safety Office Tábornok utca 2 Budapest 1143 Hungary

Virological Molecular Diagnostic Laboratory Veterinary Sciences Division Department of Virology Agri Food and Bioscience Institute Stoney Road Belfast BT4 3SD Northern Ireland

WOAH FAO international reference laboratory for Avian Influenza and Newcastle Disease Virology Department Animal and Plant Health Agency Weybridge Woodham Lane New Haw Addlestone KT15 3NB United Kingdom

Zobrazit více v PubMed

Abed  Y., Goyette  N., and Boivin  G. (2005) ‘Generation and Characterization of Recombinant Influenza A (H1N1) Viruses Harboring Amantadine Resistance Mutations’, Antimicrobial Agents And Chemotherapy, 49: 556–9. PubMed PMC

Abolnik  C.  et al. (2022) ‘Wild Bird Surveillance in the Gauteng Province of South Africa during the High-Risk Period for Highly Pathogenic Avian Influenza Virus Introduction’, Viruses, 14: 2027. PubMed PMC

Adams  S. E.  et al. (2019) ‘Effect of Influenza H1N1 Neuraminidase V116A and I117V Mutations on NA Activity and Sensitivity to NA Inhibitors’, Antiviral Research, 169: 104539. PubMed

Adlhoch  C.  et al. (2022) ‘Avian Influenza Overview June–September 2022’, EFSA Journal, 20: 7597. PubMed PMC

Adlhoch  C.  et al. (2023a) ‘Avian Influenza Overview April–June 2023’, EFSA Journal. European Food Safety Authority, 21: e08191. 10.2903/J.EFSA.2023.8191 PubMed DOI PMC

Adlhoch  C.  et al. (2023b) ‘Avian Influenza Overview March–April 2023’, EFSA Journal. European Food Safety Authority, 21: e08039. 10.2903/j.efsa.2023.8039 PubMed DOI PMC

Agüero  M.  et al. (2023) ‘Highly Pathogenic Avian Influenza A(H5N1) Virus Infection in Farmed Minks, Spain, October 2022’, Eurosurveillance, 28: 2300001. PubMed PMC

Alkie  T. N.  et al. (2022) ‘A Threat from Both Sides: Multiple Introductions of Genetically Distinct H5 HPAI Viruses into Canada via Both East Asia-Australasia/Pacific and Atlantic Flyways’, Virus Evolution, 8: veac077. PubMed PMC

Ayllon  J.  et al. (2014) ‘A Single Amino Acid Substitution in the Novel H7N9 Influenza A Virus NS1 Protein Increases CPSF30 Binding and Virulence’, Journal of Virology, 88: 12146–51. PubMed PMC

Bielejec  F.  et al. (2016) ‘SpreaD3: Interactive Visualization of Spatiotemporal History and Trait Evolutionary Processes’, Molecular Biology and Evolution, 33: 2167–9. PubMed PMC

Bordes  L.  et al. (2023) ‘Highly Pathogenic Avian Influenza H5N1 Virus Infections in Wild Red Foxes (Vulpes Vulpes) Show Neurotropism and Adaptive Virus Mutations’, Microbiology Spectrum, 11: e02867–22. PubMed PMC

Bortz  E.  et al. (2011) ‘Host- and Strain-specific Regulation of Influenza Virus Polymerase Activity by Interacting Cellular Proteins’, MBio, 2: 10–128. PubMed PMC

Bourmakina  S. V., and García-Sastre  A. (2003) ‘Reverse Genetics Studies on the Filamentous Morphology of Influenza A Virus’, Journal of General Virology, 84: 517–27. PubMed

Bragstad  K.  et al. (2007) ‘First Introduction of Highly Pathogenic H5N1 Avian Influenza A Viruses in Wild and Domestic Birds in Denmark, Northern Europe’, Virology Journal, 4: 1–10. PubMed PMC

Briand  F. X.  et al. (2022) ‘Multiple Independent Introductions of Highly Pathogenic Avian Influenza H5 Viruses during the 2020-2021 Epizootic in France’, Transboundary and Emerging Diseases, 69: 4028–33. PubMed PMC

Bruno  A.  et al. (2023) ‘First Case of Human Infection with Highly Pathogenic H5 Avian Influenza a Virus in South America: A New Zoonotic Pandemic Threat for 2023?’, Journal of Travel Medicine, 30: taad032. PubMed PMC

Buranathai  C.  et al. (2007) ‘Surveillance Activities and Molecular Analysis of H5N1 Highly Pathogenic Avian Influenza Viruses from Thailand, 2004–2005’, Avian Diseases, 51: 194–200. PubMed

Bussey  K. A.  et al. (2010) ‘PB2 Residue 271 Plays A Key Role in Enhanced Polymerase Activity of Influenza A Viruses in Mammalian Host Cells’, Journal of Virology, 84: 4395–406. PubMed PMC

Caliendo  V.  et al. (2022) ‘Transatlantic Spread of Highly Pathogenic Avian Influenza H5N1 by Wild Birds from Europe to North America in 2021’, Scientific Reports, 12. PubMed PMC

Campbell  P. J.  et al. (2014) ‘The M Segment of the 2009 Pandemic Influenza Virus Confers Increased Neuraminidase Activity, Filamentous Morphology, and Efficient Contact Transmissibility to A/Puerto Rico/8/1934-Based Reassortant Viruses’, Journal of Virology, 88: 3802–14. PubMed PMC

Chen  H.  et al. (2006) ‘Properties and Dissemination of H5N1 Viruses Isolated during an Influenza Outbreak in Migratory Waterfowl in Western China’, Journal of Virology, 80: 5976–83. PubMed PMC

Chen  L. M.  et al. (2012) ‘In Vitro Evolution of H5N1 Avian Influenza Virus toward Human-type Receptor Specificity’, Virology, 422: 105–13. PubMed PMC

Chernomor  O.  et al. (2014) ‘Split Diversity in Constrained Conservation Prioritization Using Integer Linear Programming’, Methods in Ecology and Evolution, 6: 83–91. PubMed PMC

Chestakova  I. V.  et al. (2023) ‘High Number of HPAI H5 Virus Infections and Antibodies in Wild Carnivores in the Netherlands, 2020-2022’, Emerg Microbes Infect.  12:  2270068. PubMed PMC

Cheung  C. L.  et al. (2006) ‘Distribution of Amantadine-resistant H5N1 Avian Influenza Variants in Asia’, Journal of Infectious Diseases, 193: 1626–9. PubMed

Chizhmakov  I. V.  et al. (2003) ‘Differences in Conductance of M2 Proton Channels of Two Influenza Viruses at Low and High pH’, The Journal of Physiology, 546: 427–38. PubMed PMC

Domańska-Blicharz  K.  et al. (2023) ‘Outbreak of Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus in Cats, Poland, June to July 2023’, Eurosurveillance, 28: 2300366. PubMed PMC

Drummond  A. J., and Rambaut  A. (2007) ‘BEAST: Bayesian Evolutionary Analysis by Sampling Trees’, BMC Evolutionary Biology, 7: 214. PubMed PMC

Du  W.  et al. (2018) ‘Substrate Binding by the Second Sialic Acid-Binding Site of Influenza A Virus N1 Neuraminidase Contributes to Enzymatic Activity’, Journal of Virology, 92: 10–128. PubMed PMC

Du  W.  et al. (2021) ‘Second Sialic Acid-binding Site of Influenza A Virus Neuraminidase: Binding Receptors for Efficient Release’, The FEBS Journal, 288: 5598–612. PubMed PMC

Elgendy  E. M.  et al. (2017) ‘Identification of Polymerase Gene Mutations that Affect Viral Replication in H5N1 Influenza Viruses Isolated from Pigeons’, Journal of General Virology, 98: 6–17. PubMed

Elleman  C. J., and Barclay  W. S. (2004) ‘The M1 Matrix Protein Controls the Filamentous Phenotype of Influenza A Virus’, Virology, 321: 144–53. PubMed

Engelsma  M.  et al. (2022) ‘Multiple Introductions of Reassorted Highly Pathogenic Avian Influenza H5Nx Viruses Clade 2.3.4.4b Causing Outbreaks in Wild Birds and Poultry in the Netherlands, 2020-2021’, Microbiology Spectrum, 10: e02499–21. PubMed PMC

Floyd  T.  et al. (2021) ‘Encephalitis and Death in Wild Mammals at a Rehabilitation Center after Infection with Highly Pathogenic Avian Influenza A(H5N8) Virus’, Emerging Infectious Diseases, 27: 2856–63. PubMed PMC

Food Safety Authority, E., Adlhoch  C.  et al. (2023) ‘Avian Influenza Overview September – December 2022’, EFSA Journal, 21: e07786. PubMed PMC

Fornek  J. L.  et al. (2009) ‘A Single-amino-acid Substitution in A Polymerase Protein of an H5N1 Influenza Virus Is Associated with Systemic Infection and Impaired T-cell Activation in Mice’, Journal of Virology, 83: 11102–15. PubMed PMC

Fouchier  R. A. M.  et al. (2005) ‘Characterization of A Novel Influenza A Virus Hemagglutinin Subtype (H16) Obtained from Black-headed Gulls’, Journal of Virology, 79: 2814–22. PubMed PMC

Fusaro  A.  et al. (2019)Disentangling the Role of Africa in the Global Spread of H5 Highly Pathogenic Avian Influenza, Nature Communications, 10: 5310. PubMed PMC

Gabriel  G.  et al. (2005) ‘The Viral Polymerase Mediates Adaptation of an Avian Influenza Virus to a Mammalian Host’, The Proceedings of the National Academy of Sciences of the United States of America, 102: 18590–5. PubMed PMC

Gabriel  G., Herwig  A., and Klenk  H. D. (2008) ‘Interaction of Polymerase Subunit PB2 and NP with Importin Alpha1 Is A Determinant of Host Range of Influenza A Virus’, PLoS Pathogens, 4: e11. PubMed PMC

Gao  Y.  et al. (2009) ‘Identification of Amino Acids in HA and PB2 Critical for the Transmission of H5N1 Avian Influenza Viruses in a Mammalian Host’, PLoS Pathogens, 5: e1000709. PubMed PMC

Gao  W.  et al. (2019) ‘Prevailing I292V PB2 Mutation in Avian Influenza H9N2 Virus Increases Viral Polymerase Function and Attenuates IFN-β Induction in Human Cells’, Journal of General Virology, 100: 1273–81. PubMed PMC

Govorkova  E. A.  et al. (2022) ‘Global Update on the Susceptibilities of Human Influenza Viruses to Neuraminidase Inhibitors and the Cap-dependent Endonuclease Inhibitor Baloxavir, 2018-2020’, Antiviral Research, 200: 105281. PubMed PMC

Grant  M.  et al. (2022) ‘Highly Pathogenic Avian Influenza (HPAI H5Nx, Clade 2.3.4.4.b) In Poultry and Wild Birds in Sweden: Synopsis of the 2020–2021 Season’, Veterinary Sciences, 9: 344. PubMed PMC

Gubareva  L. V.  et al. (2017) ‘Drug Susceptibility Evaluation of an Influenza A(H7N9) Virus by Analyzing Recombinant Neuraminidase Proteins’, The Journal of Infectious Diseases, 216: S566–S574. PubMed PMC

Hagag  N. M.  et al. (2022) ‘Molecular Epidemiology and Evolutionary Analysis of Avian Influenza A(H5) Viruses Circulating in Egypt, 2019-2021’, Viruses, 14: 1758. PubMed PMC

Hatta  H.  et al. (2001) ‘Molecular Basis for High Virulence of Hong Kong H5N1 Influenza A Viruses’, Science, 293: 1840–2. PubMed

Hatta  M.  et al. (2007) ‘Growth of H5N1 Influenza A Viruses in the Upper Respiratory Tracts of Mice’, PLoS Pathogens, 3: 1374–9. PubMed PMC

He  G.  et al. (2008) ‘Amantadine-resistance among H5N1 Avian Influenza Viruses Isolated in Northern China’, Antiviral Research, 77: 72–6. PubMed

Herfst  S.  et al. (2012) ‘Airborne Transmission of Influenza A/H5N1 Virus between Ferrets’, Science, 336: 1534–41. PubMed PMC

Hoffmann  T. W.  et al. (2012) ‘Length Variations in the NA Stalk of an H7N1 Influenza Virus Have Opposite Effects on Viral Excretion in Chickens and Ducks’, Journal of Virology, 86: 584–8. PubMed PMC

Hu  M.  et al. (2016) ‘Amino Acid Substitutions V63I or A37S/I61T/V63I/V100A in the PA N-terminal Domain Increase the Virulence of H7N7 Influenza A Virus’, Scientific Reports, 6: 1–11. PubMed PMC

Hu  M.  et al. (2017a) ‘PA Nsubstitutions A37S, A37S/I61T and A37S/V63I Attenuate the Replication of H7N7 Influenza A Virus by Impairing the Polymerase and Endonuclease Activities’, Journal of General Virology, 98: 364–73. PubMed

Hu  M.SS et al. (2017b) ‘PB2 Substitutions V598T/I Increase the Virulence of H7N9 Influenza A Virus in Mammals’, Virology, 501: 92–101. PubMed

Human Infection Caused by Avian Influenza A (H5N1) - Chile [WWW Document], (n.d.) <https://www.who.int/emergencies/disease-outbreak-news/item/2023-DON461> Accessed 8 Aug 2023.

Hurt  A. C.  et al. (2011) ‘Increased Detection in Australia and Singapore of a Novel Influenza a(H1N1)2009 Variant with Reduced Oseltamivir and Zanamivir Sensitivity Due to a S247N Neuraminidase Mutation’, Eurosurveillance, 16: 19884. PubMed

Ilyushina  N. A., Govorkova  E. A., and Webster  R. G. (2005) ‘Detection of Amantadine-resistant Variants among Avian Influenza Viruses Isolated in North America and Asia’, Virology, 341: 102–6. PubMed

Imai  H.  et al. (2010) ‘The HA and NS Genes of Human H5N1 Influenza A Virus Contribute to High Virulence in Ferrets’, PLoS Pathogens, 6: e1001106. PubMed PMC

Investigation into the Risk to Human Health of Avian Influenza (influenza A H5N1) in England: Technical Briefing 4 - GOV.UK [WWW Document], (n.d.) <https://www.gov.uk/government/publications/avian-influenza-influenza-a-h5n1-technical-briefings/investigation-into-the-risk-to-human-health-of-avian-influenza-influenza-a-h5n1-in-england-technical-briefing-4> Accessed 21 Nov 2023.

Isoda  N.  et al. (2022) ‘Detection of New H5N1 High Pathogenicity Avian Influenza Viruses in Winter 2021-2022 in the Far East, Which are Genetically Close to Those in Europe’, Viruses, 14: 2168. PubMed PMC

Jackson  D.  et al. (2008) ‘A New Influenza Virus Virulence Determinant: The NS1 Protein Four C-terminal Residues Modulate Pathogenicity’, The Proceedings of the National Academy of Sciences of the United States of America, 105: 4381–6. PubMed PMC

Jiao  P.  et al. (2008) ‘A Single-amino-acid Substitution in the NS1 Protein Changes the Pathogenicity of H5N1 Avian Influenza Viruses in Mice’, Journal of Virology, 82: 1146–54. PubMed PMC

Katoh  K., and Standley  D. M. (2013) ‘MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability’, Molecular Biology and Evolution, 30: 772–80. PubMed PMC

Kim  J. H.  et al. (2010) ‘Role of Host-specific Amino Acids in the Pathogenicity of Avian H5N1 Influenza Viruses in Mice’, Journal of General Virology, 91: 1284–9. PubMed PMC

King  J.  et al. (2022) ‘Connect to Protect: Dynamics and Genetic Connections of Highly Pathogenic Avian Influenza Outbreaks in Poultry from 2016 to 2021 in Germany’, Viruses, 14: 1849. PubMed PMC

Kiso  M.  et al. (2020) ‘Baloxavir Marboxil Treatment of Nude Mice Infected with Influenza A Virus’, The Journal of Infectious Diseases, 221: 1699–702. PubMed PMC

Kode  S. S.  et al. (2019) ‘A Novel I117T Substitution in Neuraminidase of Highly Pathogenic Avian Influenza H5N1 Virus Conferring Reduced Susceptibility to Oseltamivir and Zanamivir’, Veterinary Microbiology, 235: 21–4. PubMed

Kuo  R.-L., and Krug  R. M. (2009) ‘Influenza a Virus Polymerase Is an Integral Component of the CPSF30-NS1A Protein Complex in Infected Cells’, Journal of Virology, 83: 1611–6. PubMed PMC

Labadie  K.  et al. (2007) ‘Host-range Determinants on the PB2 Protein of Influenza A Viruses Control the Interaction between the Viral Polymerase and Nucleoprotein in Human Cells’, Virology, 362: 271–82. PubMed

Lan  Y.  et al. (2010) ‘A Comprehensive Surveillance of Adamantane Resistance among Human Influenza A Virus Isolated from Mainland China between 1956 and 2009’, Antiviral Therapy, 15: 853–9. PubMed

Le  Q. M.  et al. (2009) ‘Selection of H5N1 Influenza Virus PB2 during Replication in Humans’, Journal of Virology, 83: 5278–81. PubMed PMC

Lee  D. H.  et al. (2017) ‘Evolution, Global Spread, and Pathogenicity of Highly Pathogenic Avian Influenza H5Nx Clade 2.3.4.4’, Journal of Veterinary Science, 18: 269–80. PubMed PMC

Leguia  M.  et al. (2023) ‘Highly Pathogenic Avian Influenza A (H5N1) in Marine Mammals and Seabirds in Peru’, Nat Commun.  14: 5489. PubMed PMC

Lemey  P.  et al. (2009) ‘Bayesian Phylogeography Finds Its Roots’, PLoS Computational Biology, 5: e1000520. PubMed PMC

Letsholo  S. L.  et al. (2022) ‘Emergence of High Pathogenicity Avian Influenza Virus H5N1 Clade 2.3.4.4b In Wild Birds and Poultry in Botswana’, Viruses, 14: 2601. PubMed PMC

Lewis  N. S.  et al. (2021) ‘Emergence and Spread of Novel H5N8, H5N5 and H5N1 Clade 2.3.4.4 Highly Pathogenic Avian Influenza in 2020’, Emerging Microbes & Infections, 10: 148–51. PubMed PMC

Leyson  C. M.  et al. (2021) ‘Multiple Gene Segments are Associated with Enhanced Virulence of Clade 2.3.4.4 H5N8 Highly Pathogenic Avian Influenza Virus in Mallards’, Journal of Virology, 95: 10–128. PubMed PMC

Li  K. S.  et al. (2004) ‘Genesis of a Highly Pathogenic and Potentially Pandemic H5N1 Influenza Virus in Eastern Asia’, Nature, 430: 209–13. PubMed

Li  Z.  et al. (2005) ‘Molecular Basis of Replication of Duck H5N1 Influenza Viruses in a Mammalian Mouse Model’, Journal of Virology, 79: 12058–64. PubMed PMC

Li  J.  et al. (2009) ‘Single Mutation at the Amino Acid Position 627 of PB2 that Leads to Increased Virulence of an H5N1 Avian Influenza Virus during Adaptation in Mice Can Be Compensated by Multiple Mutations at Other Sites of PB2’, Virus Research, 144: 123–9. PubMed

Li  J.  et al. (2018) ‘Three Amino Acid Substitutions in the NS1 Protein Change the Virus Replication of H5N1 Influenza Virus in Human Cells’, Virology, 519: 64–73. PubMed

Lindh  E.  et al. (2023) ‘Highly Pathogenic Avian Influenza A(H5N1) Virus Infection on Multiple Fur Farms in the South and Central Ostrobothnia Regions of Finland, July 2023’, Eurosurveillance, 28: 2300400. PubMed PMC

Lo  F. T.  et al. (2022) ‘Intercontinental Spread of Eurasian Highly Pathogenic Avian Influenza A(H5N1) to Senegal’, Emerging Infectious Diseases, 28: 234–7. PubMed PMC

The Global Consortium for H5N8 and Related Influenza, Lycett  S. J.  et al. (2016) ‘Role for Migratory Wild Birds in the Global Spread of Avian Influenza H5N8’, Science, 354: 213–7. PubMed PMC

Lycett  S. J.  et al. (2020) ‘Genesis and Spread of Multiple Reassortants during the 2016/2017 H5 Avian Influenza Epidemic in Eurasia’, The Proceedings of the National Academy of Sciences of the United States of America, 117: 20814–25. PubMed PMC

Madslien  K.  et al. (2021) ‘First Detection of Highly Pathogenic Avian Influenza Virus in Norway’, BMC Veterinary Research, 17: 218. PubMed PMC

Makalo  M. R. J.  et al. (2022) ‘Highly Pathogenic Avian Influenza (A/H5N1) Virus Outbreaks in Lesotho, May 2021’, Emerging Microbes & Infections, 11: 757–60. PubMed PMC

Manzoor  R.  et al. (2009) ‘PB2 Protein of a Highly Pathogenic Avian Influenza Virus Strain A/chicken/Yamaguchi/7/2004 (H5N1) Determines Its Replication Potential in Pigs’, Journal of Virology, 83: 1572–8. PubMed PMC

Mehle  A., and Doudna  J. A. (2008) ‘Inhibitory Activity Restricts the Function of an Avian-like Influenza Polymerase in Primate Cells’, Cell Host & Microbe, 4: 111–22. PubMed PMC

Meseko  C.  et al. (2023) ‘The Evolution of Highly Pathogenic Avian Influenza A (H5) in Poultry in Nigeria, 2021–2022’, Viruses, 15: 1387. PubMed PMC

Minin  V. N., and Suchard  M. A. (2008) ‘Counting Labeled Transitions in Continuous-time Markov Models of Evolution’, Journal of Mathematical Biology, 56: 391–412. PubMed

More  S.  et al. (2017) ‘Avian Influenza’, EFSA Journal. European Food Safety Authority, 15: e04991. PubMed PMC

Moreno  A.  et al. (2023) ‘Asymptomatic Infection with Clade 2.3.4.4b Highly Pathogenic Avian Influenza A(H5N1) in Carnivore Pets, Italy, April 2023’, Eurosurveillance, 28: 2300441. PubMed PMC

Munier  S.  et al. (2010) ‘A Genetically Engineered Waterfowl Influenza Virus with A Deletion in the Stalk of the Neuraminidase Has Increased Virulence for Chickens’, Journal of Virology, 84: 940–52. PubMed PMC

Nagy  A., Černíková  L., and Stará  M. (2022) ‘A New Clade 2.3.4.4b H5N1 Highly Pathogenic Avian Influenza Genotype Detected in Europe in 2021’, Archives of Virology, 167: 1455–9. PubMed

Nguyen  L.-T.  et al. (2015) ‘IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-likelihood Phylogenies’, Molecular Biology and Evolution, 32: 268–74. PubMed PMC

European Food Safety Authority, European Centre for Disease Prevention and Control, European Union Reference Laboratory for Avian Influenza, Adlhoch  C.  et al. (2023) ‘Avian Influenza Overview December 2022–March 2023’, EFSA Journal. European Food Safety Authority, 21: e07917. PubMed PMC

Oliver  I.  et al. (2022) ‘A Case of Avian Influenza A(H5N1) in England, January 2022’, Eurosurveillance, 27: 2200061. PubMed PMC

Olsen  B.  et al. (2006) ‘Global Patterns of Influenza a Virus in Wild Birds’, Science, 312: 384–8. PubMed

Ouoba  L. B.  et al. (2022) ‘Emergence of a Reassortant 2.3.4.4b Highly Pathogenic H5N1 Avian Influenza Virus Containing H9N2 PA Gene in Burkina Faso, West Africa, in 2021’, Viruses, 14: 1901. PubMed PMC

Pinto  R. M.s et al. (2022) ‘Zoonotic Avian Influenza Viruses Evade Human BTN3A3 Restriction’, Nature, 619: 338–47. PubMed

Pinto  R. M.  et al. (2023) ‘BTN3A3 Evasion Promotes the Zoonotic Potential of Influenza A Viruses’, Nature, 619: 338–47. PubMed

Pohlmann  A.  et al. (2022) ‘Has Epizootic Become Enzootic? Evidence for a Fundamental Change in the Infection Dynamics of Highly Pathogenic Avian Influenza in Europe, 2021’, MBio, 13: e00609–22. PubMed PMC

Puthavathana  P.  et al. (2005) ‘Molecular Characterization of the Complete Genome of Human Influenza H5N1 Virus Isolates from Thailand’, Journal of General Virology, 86: 423–33. PubMed

Rameix-Welti  M.-A.  et al. (2009) ‘Avian Influenza A Virus Polymerase Association with Nucleoprotein, but Not Polymerase Assembly, Is Impaired in Human Cells during the Course of Infection’, Journal of Virology, 83: 1320–31. PubMed PMC

Richard  M.  et al. (2017) ‘Mutations Driving Airborne Transmission of A/H5N1 Virus in Mammals Cause Substantial Attenuation in Chickens Only When Combined’, Scientific Reports, 71: 1–13. PubMed PMC

Rosone  F.  et al. (2023) ‘Seroconversion of a Swine Herd in a Free-Range Rural Multi-Species Farm against HPAI H5N1 2.3.4.4b Clade Virus’, Microorganisms, 11: 1162. PubMed PMC

Sagong  M.  et al. (2022) ‘Emergence of Clade 2.3.4.4b Novel Reassortant H5N1 High Pathogenicity Avian Influenza Virus in South Korea during Late 2021’, Transboundary and Emerging Diseases, 69: e3255–60. PubMed

Salaheldin  A. H.  et al. (2022) ‘Isolation of Genetically Diverse H5N8 Avian Influenza Viruses in Poultry in Egypt, 2019-2021’, Viruses, 14: 1431. PubMed PMC

Salomon  R.  et al. (2006) ‘The Polymerase Complex Genes Contribute to the High Virulence of the Human H5N1 Influenza Virus Isolate A/Vietnam/1203/04’, The Journal of Experimental Medicine, 203: 689–97. PubMed PMC

Samson  M.  et al. (2014) ‘Characterization of Drug-Resistant Influenza Virus A(H1N1) and A(H3N2) Variants Selected in Vitro with Laninamivir’, Antimicrobial Agents and Chemotherapy, 58: 5220–8. PubMed PMC

Sanogo  I. N.  et al. (2022) ‘Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus in Poultry, Benin, 2021’, Emerging Infectious Diseases, 28: 2534–37. PubMed PMC

Scheiffarth  G. (2003), Born to fly-Migratory Strategies and Stopover Ecology in the European Wadden Sea of a Long-distance Migrant, the Bar-tailed Godwit (Limosa Lapponica). Faculty of Mathematics and Science, Universität Oldenburg. <http://oops.uni-oldenburg.de/198/>

Scholtissek  C.  et al. (2002) ‘Cooperation between the Hemagglutinin of Avian Viruses and the Matrix Protein of Human Influenza A Viruses’, Journal of Virology, 76: 1781–6. PubMed PMC

Shapiro  B.  et al. (2011) ‘A Bayesian Phylogenetic Method to Estimate Unknown Sequence Ages’, Molecular Biology and Evolution, 28: 879–87. PubMed PMC

Shapiro  B., Rambaut  A., and Drummond  A. J. (2006) ‘Choosing Appropriate Substitution Models for the Phylogenetic Analysis of Protein-Coding Sequences’, Molecular Biology and Evolution, 23: 7–9. PubMed

Shinya  K.  et al. (2004) ‘PB2 Amino Acid at Position 627 Affects Replicative Efficiency, but Not Cell Tropism, of Hong Kong H5N1 Influenza A Viruses in Mice’, Virology, 320: 258–66. PubMed

Smith  G. J. D., and Donis  R. O. (2015) ‘Nomenclature Updates Resulting from the Evolution of Avian Influenza A(H5) Virus Clades 2.1.3.2a, 2.2.1, And 2.3.4 During 2013–2014’, Influenza and Other Respiratory Viruses, 9: 271–6. PubMed PMC

Song  W.  et al. (2014) ‘The K526R Substitution in Viral Protein PB2 Enhances the Effects of E627K on Influenza Virus Replication’, Nature Communications, 5: 1–12. PubMed PMC

Sonnberg  S., Webby  R. J., and Webster  R. G. (2013) ‘Natural History of Highly Pathogenic Avian Influenza H5N1’, Virus Research, 178: 63–77. PubMed PMC

Sorrell  E. M.  et al. (2010) ‘A 27-Amino-Acid Deletion in the Neuraminidase Stalk Supports Replication of an Avian H2N2 Influenza A Virus in the Respiratory Tract of Chickens’, Journal of Virology, 84: 11831–40. PubMed PMC

Soubies  S. M.  et al. (2010) ‘Species-specific Contribution of the Four C-terminal Amino Acids of Influenza A Virus NS1 Protein to Virulence’, Journal of Virology, 84: 6733–47. PubMed PMC

Spesock  A.  et al. (2011) ‘The Virulence of 1997 H5N1 Influenza Viruses in the Mouse Model Is Increased by Correcting a Defect in Their NS1 Proteins’, Journal of Virology, 85: 7048–58. PubMed PMC

Steel  J.  et al. (2009) ‘Transmission of Influenza Virus in a Mammalian Host Is Increased by PB2 Amino Acids 627K or 627E/701N’, PLoS Pathogens, 5: e1000252. PubMed PMC

Stevens  J.  et al. (2006) ‘Structure and Receptor Specificity of the Hemagglutinin from an H5N1 Influenza Virus’, Science, 312: 404–10. PubMed

Su  Y.  et al. (2008) ‘Analysis of a Point Mutation in H5N1 Avian Influenza Virus Hemagglutinin in Relation to Virus Entry into Live Mammalian Cells’, Archives of Virology, 153: 2253–61. PubMed

Subbarao  E. K., London  W., and Murphy  B. R. (1993) ‘A Single Amino Acid in the PB2 Gene of Influenza A Virus Is A Determinant of Host Range’, Journal of Virology, 67: 1761–4. PubMed PMC

Suchard  M. A.  et al. (2018) ‘Bayesian Phylogenetic and Phylodynamic Data Integration Using BEAST 1.10’, Virus Evolution, 4: vey016. PubMed PMC

Suchard  M. A., and Rambaut  A. (2009) ‘Many-core Algorithms for Statistical Phylogenetics’, Bioinformatics, 25: 1370–6. PubMed PMC

Suttie  A.  et al. (2019) ‘Inventory of Molecular Markers Affecting Biological Characteristics of Avian Influenza A Viruses’, Virus Genes, 55: 739–68. PubMed PMC

Swieton  E.  et al. (2020) ‘Sub-Saharan Africa and Eurasia Ancestry of Reassortant Highly Pathogenic Avian Influenza A(H5N8) Virus, Europe, December 2019 - Volume 26, Number 7—July 2020 - Emerging Infectious Diseases Journal - CDC’, Emerging Infectious Diseases, 26: 1557–61. PubMed PMC

Taft  A. S.  et al. (2015) ‘Identification of Mammalian-adapting Mutations in the Polymerase Complex of an Avian H5N1 Influenza Virus’, Nature Communications, 6: 7491. PubMed PMC

Tammiranta  N.  et al. (2023) ‘Highly Pathogenic Avian Influenza A (H5N1) Virus Infections in Wild Carnivores Connected to Mass Mortalities of Pheasants in Finland’, Infection Genetics & Evolution, 111: 105423. PubMed

Thi Hoang  D.  et al. (2017) ‘UFBoot2: Improving the Ultrafast Bootstrap Approximation’, Molecular Biology and Evolution, 35: 518–22. PubMed PMC

Threat Assessment Brief: First Identification of Human Cases of Avian Influenza A(H5N8) Infection [WWW Document], (n.d.), <https://www.ecdc.europa.eu/en/publications-data/threat-assessment-first-human-cases-avian-influenza-h5n8> Accessed Aug 8 2023.

U.S. Case of Human Avian Influenza A(H5) Virus Reported | CDC Online Newsroom | CDC [WWW Document], (n.d.), <https://www.cdc.gov/media/releases/2022/s0428-avian-flu.html> Accessed Aug 8 2023.

Verhagen  J. H., Fouchier  R. A. M., and Lewis  N. (2021) ‘Highly Pathogenic Avian Influenza Viruses at the Wild–Domestic Bird Interface in Europe: Future Directions for Research and Surveillance’, Viruses, 13: 212. PubMed PMC

Vreman  S.  et al. (2023) ‘Zoonotic Mutation of Highly Pathogenic Avian Influenza H5N1 Virus Identified in the Brain of Multiple Wild Carnivore Species’, Pathogens, 12: 168. PubMed PMC

Wang  W.  et al. (2010) ‘Glycosylation at 158N of the Hemagglutinin Protein and Receptor Binding Specificity Synergistically Affect the Antigenicity and Immunogenicity of a Live Attenuated H5N1 A/Vietnam/1203/2004 Vaccine Virus in Ferrets’, Journal of Virology, 84: 6570–7. PubMed PMC

Watanabe  Y.  et al. (2011) ‘Acquisition of Human-Type Receptor Binding Specificity by New H5N1 Influenza Virus Sublineages during Their Emergence in Birds in Egypt’, PLoS Pathogens, 7: e1002068. PubMed PMC

Webster  R. G.  et al. (1992) ‘Evolution and Ecology of Influenza A Viruses’, Microbiological Reviews, 56: 152–79. PubMed PMC

Webster  R. G., Kawaoka  Y., and Bean  W. J. (1989) ‘What Is the Potential of Avirulent Influenza Viruses to Complement a Cleavable Hemagglutinin and Generate Virulent Strains?’, Virology, 171: 484–92. PubMed

Wille  M.  et al. (2011) ‘Extensive Geographic Mosaicism in Avian Influenza Viruses from Gulls in the Northern Hemisphere’, PLoS One, 6: e20664. PubMed PMC

World Health Organization (2021) ‘Antigenic and Genetic Characteristics of Zoonotic Influenza A Viruses and Development of Candidate Vaccine Viruses for Pandemic Preparedness March 2021’.

World Health Organization (2023) ‘Avian Influenza Weekly Update Number 895 - 12 May 2023 - Human Infection with Avian Influenza A(H5) Viruses’.

Xiao  C.  et al. (2016) ‘B2–588 V Promotes the Mammalian Adaptation of H10N8, H7N9 and H9N2 Avian Influenza Viruses’, Scientific Reports, 6: 19474. PubMed PMC

Xie  R.  et al. (2023) ‘The Episodic Resurgence of Highly Pathogenic Avian Influenza H5 Virus’, Nature, 622: 810–7. PubMed

Xu  G.  et al. (2016) ‘Prevailing PA Mutation K356R in Avian Influenza H9N2 Virus Increases Mammalian Replication and Pathogenicity’, Journal of Virology, 90: 8105–14. PubMed PMC

Yamada  S.  et al. (2006) ‘Haemagglutinin Mutations Responsible for the Binding of H5N1 Influenza A Viruses to Human-type Receptors’, Nature, 444: 378–82. PubMed

Yamayoshi  S.  et al. (2014) ‘Virulence-Affecting Amino Acid Changes in the PA Protein of H7N9 Influenza A Viruses’, Journal of Virology, 88: 3127–34. PubMed PMC

Yamayoshi  S.  et al. (2018) ‘Enhanced Replication of Highly Pathogenic Influenza A(H7N9) Virus in Humans’, Emerging Infectious Diseases, 24: 746–50. PubMed PMC

Yang  Z. Y.  et al. (2007) ‘Immunization by Avian H5 Influenza Hemagglutinin Mutants with Altered Receptor Binding Specificity’, Science, 317: 825–8. PubMed PMC

Ye  H.  et al. (2022) ‘Divergent Reassortment and Transmission Dynamics of Highly Pathogenic Avian Influenza A(H5N8) Virus in Birds of China during 2021’, Frontiers in Microbiology, 13: 913551. PubMed PMC

Youk  S.  et al. (2023) ‘H5N1 Highly Pathogenic Avian Influenza Clade 2.3.4.4b In Wild and Domestic Birds: Introductions into the United States and Reassortments, December 2021-April 2022’, Virology, 587: 109860. PubMed

Zhang  J.  et al. (2018) ‘The D253N Mutation in the Polymerase Basic 2 Gene in Avian Influenza (H9N2) Virus Contributes to the Pathogenesis of the Virus in Mammalian Hosts’, Virologica Sinica, 33: 531–7. PubMed PMC

Zhang  G.  et al. (2023) ‘Bidirectional Movement of Emerging H5N8 Avian Influenza Viruses between Europe and Asia via Migratory Birds since Early 2020’, Molecular Biology and Evolution, 40: msad019. PubMed PMC

Zhou  H.  et al. (2009) ‘The Special Neuraminidase Stalk-motif Responsible for Increased Virulence and Pathogenesis of H5N1 Influenza A Virus’, PLoS One, 4: e6277. PubMed PMC

Zhu  W.  et al. (2015) ‘Residues 41V And/or 210D in the NP Protein Enhance Polymerase Activities and Potential Replication of Novel Influenza (H7N9) Viruses at Low Temperature’, Virology Journal, 12: 1–5. PubMed PMC

Enzootic Circulation, Massive Gull Mortality and Poultry Outbreaks during the 2022/2023 High-Pathogenicity Avian Influenza H5N1 Season in the Czech Republic