In 2021/2022, the re-emergence of highly pathogenic avian influenza (HPAI) occurred in Europe. The outbreak was seeded from two sources: resident and reintroduced viruses, which is unprecedented in the recorded history of avian influenza. The dominant subtype was H5N1, which replaced the H5N8 subtype that had predominated in previous seasons. In this study, we present a whole genome sequence and a phylogenetic analysis of 57 H5N1 HPAI and two low pathogenic avian influenza (LPAI) H5N1 strains collected in the Czech Republic during 2021/2022. Phylogenetic analysis revealed close relationships between H5N1 genomes from poultry and wild birds and secondary transmission in commercial geese. The genotyping showed considerable genetic heterogeneity among Czech H5N1 viruses, with six different HPAI genotypes, three of which were apparently unique. In addition, second-order reassortment relationships were observed with the direct involvement of co-circulating H5N1 LPAI strains. The genetic distance between Czech H5N1 HPAI and the closest LPAI segments available in the database illustrates the profound gaps in our knowledge of circulating LPAI strains. The changing dynamics of HPAI in the wild may increase the likelihood of future HPAI outbreaks and present new challenges in poultry management, biosecurity, and surveillance.

State Veterinary Institute Prague Sídlištní 136 24 165 03 Prague Czech Republic

See more in PubMed

EFSA (European Food Safety Authority) ECDC (European Centre for Disease Prevention and Control) EURL (European Reference Laboratory for Avian Influenza) Adlhoch C., Fusaro A., Gonzales J.L., Kuiken T., Marangon S., Niqueux É., Staubach C., et al. Scientific report: Avian influenza overview March–June 2022. EFSA J. 2022;20:7415. PubMed

EFSA (European Food Safety Authority) ECDC (European Centre for Disease Prevention and Control) EURL (European Reference Laboratory for Avian Influenza) Brown I., Mulatti P., Smietanka K., Staubach C., Willeberg P., Adlhoch C., Candiani D., et al. Scientific report on the avian influenza overview October 2016–August 2017. EFSA J. 2017;15:5018. PubMed PMC

EFSA (European Food Safety Authority) ECDC (European Centre for Disease Prevention and Control) EURL (European Reference Laboratory for Avian Influenza) Adlhoch C., Fusaro A., Gonzales J.L., Kuiken T., Marangon S., Niqueux É., Staubach C., et al. Scientific report: Avian influenza overview December 2021–March 2022. EFSA J. 2022;20:7289. PubMed PMC

Poultrymed. [(accessed on 21 October 2022)]. Available online: https://web.archive.org/web/20221021094717/https://www.poultrymed.com/Poultrymed/Templates/showpage.asp?DBID=1&LNGID=1&TMID=178&FID=2948&PID=0&IID=79782.

Lewis N.S., Banyard A.C., Whittard E., Karibayev T., Al Kafagi T., Chvala I., Byrne A., Meruyert Akberovna S., King J., Harder T., et al. Emergence and spread of novel H5N8, H5N5 and H5N1 clade 2.3.4.4 highly pathogenic avian influenza in 2020. Emerg. Microbes Infect. 2021;10:148–151. doi: 10.1080/22221751.2021.1872355. PubMed DOI PMC

EFSA (European Food Safety Authority) ECDC (European Centre for Disease Prevention and Control) EURL (European Reference Laboratory for Avian Influenza) Adlhoch C., Fusaro A., Gonzales J.L., Kuiken T., Marangon S., Niqueux É., Staubach C., et al. Scientific report: Avian influenza overview August–December 2020. EFSA J. 2020;18:6379.

EFSA (European Food Safety Authority) ECDC (European Centre for Disease Prevention and Control) EURL (European Reference Laboratory for Avian Influenza) Adlhoch C., Fusaro A., Gonzales J.L., Kuiken T., Marangon S., Niqueux É., Staubach C., et al. Scientific report: Avian influenza overview December 2020–February 2021. EFSA J. 2021;19:6497.

EFSA (European Food Safety Authority) ECDC (European Centre for Disease Prevention and Control) EURL (European Reference Laboratory for Avian Influenza) Adlhoch C., Fusaro A., Gonzales J.L., Kuiken T., Marangon S., Niqueux É., Staubach C., et al. Scientific report: Avian influenza overview February–May 2021. EFSA J. 2021;19:6951. PubMed

EFSA (European Food Safety Authority) ECDC (European Centre for Disease Prevention and Control) EURL (European Reference Laboratory for Avian Influenza) Adlhoch C., Fusaro A., Gonzales J.L., Kuiken T., Marangon S., Niqueux É., Staubach C., et al. Scientific report: Avian influenza overview May–September 2021. EFSA J. 2022;20:7122. PubMed PMC

Pohlmann A., King J., Fusaro A., Zecchin B., Banyard A.C., Brown I.H., Byrne A.M.P., Beerens N., Liang Y., Heutink R., et al. Has Epizootic Become Enzootic? Evidence for a Fundamental Change in the Infection Dynamics of Highly Pathogenic Avian Influenza in Europe, 2021. mBio. 2022;30:e0060922. doi: 10.1128/mbio.00609-22. PubMed DOI PMC

Nagy A., Černíková L., Stará M. A new clade 2.3.4.4b H5N1 highly pathogenic avian influenza genotype detected in Europe in 2021. Arch. Virol. 2022;167:1455–1459. doi: 10.1007/s00705-022-05442-6. PubMed DOI

EFSA (European Food Safety Authority. ECDC (European Centre for Disease Prevention and Control) EURL (European Reference Laboratory for Avian Influenza) Adlhoch C., Fusaro A., Gonzales J.L., Kuiken T., Marangon S., Niqueux É., Staubach C., et al. Scientific report: Avian influenza overview September–December 2021. EFSA J. 2021;19:7108.

Nagy A., Černíková L., Stará M., Hofmannová L., Sedlák K. Genotype Uniformity, Wild Bird-to-Poultry Transmissions, and Farm-to-Farm Carryover during the Spread of the Highly Pathogenic Avian Influenza H5N8 in the Czech Republic in 2021. Viruses. 2022;14:1411. doi: 10.3390/v14071411. PubMed DOI PMC

Nagy A., Dán Á., Černíková L., Vitásková E., Křivda V., Horníčková J., Masopust R., Sedlák K. Microevolution and independent incursions as main forces shaping H5 Hemagglutinin diversity during a H5N8/H5N5 highly pathogenic avian influenza outbreak in Czech Republic in 2017. Arch. Virol. 2018;163:2219–2224. doi: 10.1007/s00705-018-3833-7. PubMed DOI

Nagy A., Černíková L., Kunteová K., Dirbáková Z., Thomas S.S., Slomka M.J., Dán Á., Varga T., Máté M., Jiřincová H., et al. A universal RT-qPCR assay for “One Health” detection of influenza A viruses. PLoS ONE. 2021;16:e0244669. doi: 10.1371/journal.pone.0244669. PubMed DOI PMC

Slomka M.J., Pavlidis T., Banks J., Shell W., McNally A., Essen S., Brown I.H. Validated H5 Eurasian real-time reverse transcriptase-polymerase chain reaction and its application in H5N1 outbreaks in 2005–2006. Avian Dis. 2007;51:373–377. doi: 10.1637/7664-060906R1.1. PubMed DOI

Slomka M.J., Coward V.J., Banks J., Löndt B.Z., Brown I.H., Voermans J., Koch G., Handberg K.J., Jørgensen P.H., Cherbonnel-Pansart M., et al. Identification of sensitive and specific avian influenza polymerase chain reaction methods through blind ring trials organized in the European Union. Avian Dis. 2007;51:227–234. doi: 10.1637/7674-063006R1.1. PubMed DOI

Payungporn S., Chutinimitkul S., Chaisingh A., Damrongwantanapokin S., Buranathai C., Amonsin A., Theamboonlers A., Poovorawan Y. Single step multiplex real-time RT-PCR for H5N1 influenza A virus detection. J. Virol. Methods. 2006;131:143–147. doi: 10.1016/j.jviromet.2005.08.004. PubMed DOI

Arctic Network. [(accessed on 21 October 2022)]. Available online: https://web.archive.org/web/20221021094307/https://artic.network/

Samtools. [(accessed on 21 October 2022)]. Available online: https://web.archive.org/web/20221021094039/http://www.htslib.org/

Influenza Research Database. [(accessed on 21 October 2022)]. Available online: https://web.archive.org/save/https://www.fludb.org/brc/analysis_landing.spg?decorator=influenza.

Katoh K., Rozewicki J., Kazunori K.D. MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief. Bioinform. 2019;20:1160–1166. doi: 10.1093/bib/bbx108. PubMed DOI PMC

Larsson A. AliView: A fast and lightweight alignment viewer and editor for large datasets. Bioinformatics. 2014;30:3276–3278. doi: 10.1093/bioinformatics/btu531. PubMed DOI PMC

Hall T.A. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 1999;41:95–98.

Trifinopoulos J., Nguyen L.T., Von Haeseler A., Minh B.Q. W-IQ-TREE: A fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res. 2016;44:W232–W235. doi: 10.1093/nar/gkw256. PubMed DOI PMC

EMMBOS: Union Manual. [(accessed on 21 October 2022)]. Available online: https://web.archive.org/web/20221021093806/https://www.bioinformatics.nl/cgi-bin/emboss/help/union.

To T.-H., Jung M., Lycett S., Gascuel O. Fast dating using least-squares criteria and algorithms. Syst. Biol. 2016;65:82–97. doi: 10.1093/sysbio/syv068. PubMed DOI PMC

Li J., Dohna H., Cardona C.J., Miller J., Carpenter T.E. Emergence and Genetic Variation of Neuraminidase Stalk Deletions in Avian Influenza Viruses. PLoS ONE. 2011;6:e14722. doi: 10.1371/journal.pone.0014722. PubMed DOI PMC

Yang Z.Y., Wei C.J., Kong W.P., Wu L., Xu L., Smith D.F., Nabel G.J. Immunization by avian H5 influenza hemagglutinin mutants with altered receptor binding specificity. Science. 2007;317:825–828. doi: 10.1126/science.1135165. PubMed DOI PMC

Chen L.M., Blixt O., Stevens J., Lipatov A.S., Davis C.T., Collins B.E., Cox N.J., Paulson J.C., Donis R.O. In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity. Virology. 2012;422:105–113. doi: 10.1016/j.virol.2011.10.006. PubMed DOI PMC

Watanabe Y., Ibrahim M.S., Ellakany H.F., Kawashita N., Mizuike R., Hiramatsu H., Sriwilaijaroen N., Takagi T., Suzuki Y., Ikuta K. Acquisition of Human-Type Receptor Binding Specificity by New H5N1 Influenza Virus Sublineages during Their Emergence in Birds in Egypt. PLoS Pathog. 2011;7:e1002068. doi: 10.1371/journal.ppat.1002068. PubMed DOI PMC

Li J., Ishaq M., Prudence M., Xi X., Hu T., Liu Q., Guo D. 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 Res. 2009;144:123–129. doi: 10.1016/j.virusres.2009.04.008. PubMed DOI

Fan S., Deng G., Song J., Tian G., Suo Y., Jiang Y., Guan Y., Bu Z., Kawaoka Y., Chen H. Two amino acid residues in the matrix protein M1 contribute to the virulence difference of H5N1 avian influenza viruses in mice. Virology. 2009;384:28–32. doi: 10.1016/j.virol.2008.11.044. PubMed DOI

Gabriel G., Herwig A., Klenk H.D. Interaction of polymerase subunit PB2 and NP with importin alpha1 is a determinant of host range of influenza A virus. PLoS Pathog. 2008;4:e11. doi: 10.1371/journal.ppat.0040011. PubMed DOI PMC

Bataille A., Van der Meer F., Stegeman A., Koch G. Evolutionary Analysis of Inter-Farm Transmission Dynamics in a Highly Pathogenic Avian Influenza Epidemic. PLoS Pathog. 2011;7:e1002094. doi: 10.1371/journal.ppat.1002094. PubMed DOI PMC

Bouwstra R.J., Koch G., Heutink R., Harders F., Van der Spek A., Elbers A.R., Bossers A. Phylogenetic analysis of highly pathogenic avian influenza A(H5N8) virus outbreak strains provides evidence for four separate introductions and one between-poultry farm transmission in the Netherlands, November 2014. Eurosurveillance. 2015;20:21174. doi: 10.2807/1560-7917.ES2015.20.26.21174. PubMed DOI

Venkatesh D., Brouwer A., Goujgoulova G., Ellis R., Seekings J., Brown I.H., Lewis N.S. Regional Transmission and Reassortment of 2.3.4.4b Highly Pathogenic Avian Influenza (HPAI) Viruses in Bulgarian Poultry 2017/18. Viruses. 2020;12:605. doi: 10.3390/v12060605. PubMed DOI PMC

Webster R.G., Guan Y., Peiris M., Walker D., Krauss S., Zhou N.N., Govorkova E.A., Ellis T.M., Dyrting K.C., Sit T., et al. Characterization of H5N1 influenza viruses that continue to circulate in geese in southeastern China. J. Virol. 2002;76:118–126. doi: 10.1128/JVI.76.1.118-126.2002. PubMed DOI PMC

Xiang B., Liang J., You R., Han L., Mei K., Chen L., Chen R., Zhang Y., Dai X., Gao P., et al. Pathogenicity and transmissibility of a highly pathogenic avian influenza virus H5N6 isolated from a domestic goose in Southern China. Vet. Microbiol. 2017;212:16–21. doi: 10.1016/j.vetmic.2017.10.022. PubMed DOI

Śmietanka K., Świętoń E., Kozak E., Wyrostek K., Tarasiuk K., Tomczyk G., Konopka B., Welz M., Domańska-Blicharz K., Niemczuk K. Highly Pathogenic Avian Influenza H5N8 in Poland in 2019–2020. J. Vet. Res. 2020;64:469–476. doi: 10.2478/jvetres-2020-0078. PubMed DOI PMC

Park M.J., Cha R.M., Kye S.J., Lee Y.N., Kim N.Y., Baek Y.G., Heo G.B., Sagong M., Lee K.N., Lee Y.J., et al. Pathogenicity of H5N8 High Pathogenicity Avian Influenza Virus in Chickens and Ducks from South Korea in 2020–2021. Viruses. 2021;13:1903. doi: 10.3390/v13101903. PubMed DOI PMC

Scoizec A., Niqueux E., Thomas R., Daniel P., Schmitz A., Le Bouquin S. Airborne Detection of H5N8 Highly Pathogenic Avian Influenza Virus Genome in Poultry Farms, France. Front. Vet. Sci. 2018;5:15. doi: 10.3389/fvets.2018.00015. PubMed DOI PMC

Filaire F., Lebre L., Foret-Lucas C., Vergne T., Daniel P., Lelièvre A., De Barros A., Jbenyeni A., Bolon P., Paul M., et al. Highly Pathogenic Avian Influenza A(H5N8) Clade 2.3.4.4b Virus in Dust Samples from Poultry Farms, France, 2021. Emerg. Infect. Dis. 2022;28:1446–1450. doi: 10.3201/eid2807.212247. PubMed DOI PMC

Munster V.J., Baas C., Lexmond P., Waldenström J., Wallensten A., Fransson T., Rimmelzwaan G.F., Beyer W.E., Schutten M., Olsen B., et al. Spatial, temporal, and species variation in prevalence of influenza A viruses in wild migratory birds. PLoS Pathog. 2007;3:e61. doi: 10.1371/journal.ppat.0030061. PubMed DOI PMC

Wallensten A., Munster V.J., Latorre-Margalef N., Brytting M., Elmberg J., Fouchier R.A., Fransson T., Haemig P.D., Karlsson M., Lundkvist A., et al. Surveillance of influenza A virus in migratory waterfowl in northern Europe. Emerg. Infect. Dis. 2007;13:404–411. doi: 10.3201/eid1303.061130. PubMed DOI PMC

Dugan V.G., Chen R., Spiro D.J., Sengamalay N., Zaborsky J., Ghedin E., Nolting J., Swayne D.E., Runstadler J.A., Happ G.M., et al. The evolutionary genetics and emergence of avian influenza viruses in wild birds. PLoS Pathog. 2008;4:e1000076. doi: 10.1371/journal.ppat.1000076. PubMed DOI PMC

Nagy A., Cerníková L., Jiřincová H., Havlíčková M., Horníčková J. Local-scale diversity and between-year “frozen evolution” of avian influenza A viruses in nature. PLoS ONE. 2014;9:e103053. doi: 10.1371/journal.pone.0103053. PubMed DOI PMC

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