Transition in genetic constellations of H3N8 and H4N6 low-pathogenic avian influenza viruses isolated from an overwintering site in Japan throughout different winter seasons

  • Ahmed Magdy Khalil
  • Natsuko Nishi
  • Isshu Kojima
  • Wataru Fukunaga
  • Masakazu Kuwahara
  • Tatsunori Masatani
  • Tsutomu Matsui
  • Makoto OzawaEmail author
Original Article


The Izumi plain in Kagoshima Prefecture, Japan, is an overwintering site for migratory ducks and endangered cranes. We have surveyed avian influenza viruses (AIVs) in this area since 2012 and isolated low-pathogenic AIVs (LPAIVs) of various subtypes every winter season. H3N8 LPAIVs were isolated during the 2012/13 and 2016/17 seasons, and H4N6 LPAIVs were isolated during the 2012/13 and 2013/14 seasons. In the 2017/18 season, one H3N8 and two H4N6 LPAIV strains were isolated from environmental water samples. Genetic and phylogenetic analysis for each gene segment from these H3N8 and H4N6 LPAIVs suggested that our isolates were genetic reassortants generated by intermixing between AIVs circulating not only in Eurasia but also in Africa and/or North America. Comparison of the genetic constellations of our three isolates with their counterparts isolated during previous seasons from the Izumi plain revealed a drastic transition in the genetic constellations of both subtypes. These findings emphasize the importance of continuous surveillance of AIVs on the Izumi plain.



We thank the City of Izumi for supporting the sample collection. This work was supported by grants from the Project of the NARO Bio-oriented Technology Research Advancement Institution (“R&D Matching Funds on the Field for Knowledge Integration and Innovation”) and by the contracted research activity for crane conservation with the City of Izumi, Japan. This research was commissioned by the Kagoshima Crane Conservation Committee.

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interest.

Research involving human participants and/or animals

This research did not involve human or animal participants.


  1. 1.
    Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y (1992) Evolution and ecology of influenza A viruses. Microbiol Rev 56(1):152–179PubMedPubMedCentralGoogle Scholar
  2. 2.
    Neumann G, Kawaoka Y (2015) Transmission of influenza A viruses. Virology 479–480:234–246CrossRefGoogle Scholar
  3. 3.
    Fouchier RA, Munster V, Wallensten A, Bestebroer TM, Herfst S, Smith D, Rimmelzwaan GF, Olsen B, Osterhaus AD (2005) Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol 79(5):2814–2822CrossRefGoogle Scholar
  4. 4.
    Yasuhiro Y (2017) Hooded Crane Nabe-zuru (Jpn) Grus monacha. Bird Research News, Japan Bird Research Association Vol.4, No.1. Accessed 22 June 2019
  5. 5.
    Mutsuyuki U (2010) White naped-crane Manazuru (Jpn) Grus Vibio. Bird Research News, Japan Bird Research Association Vol.7 No.1. Accessed 22 June 2019
  6. 6.
    BirdLife International (2016) Grus monacha. The IUCN Red List of Threatened Species 2016: e.T22692151A93337861.
  7. 7.
    BirdLife International (2018) Antigone vipio. The IUCN Red List of Threatened Species 2018: e.T22692073A131927305.
  8. 8.
    Olsen B, Munster VJ, Wallensten A, Waldenstrom J, Osterhaus AD, Fouchier RA (2006) Global patterns of influenza A virus in wild birds. Science 312(5772):384–388CrossRefGoogle Scholar
  9. 9.
    Okuya K, Kawabata T, Nagano K, Tsukiyama-Kohara K, Kusumoto I, Takase K, Ozawa M (2015) Isolation and characterization of influenza A viruses from environmental water at an overwintering site of migratory birds in Japan. Arch Virol 160(12):3037–3052CrossRefGoogle Scholar
  10. 10.
    Nakagawa H, Okuya K, Kawabata T, Matsuu A, Takase K, Kuwahara M, Toda S, Ozawa M (2018) Genetic characterization of low pathogenic avian influenza viruses isolated on the Izumi plain in Japan: possible association of dynamic movements of wild birds with AIV evolution. Arch Virol 163(4):911–923CrossRefGoogle Scholar
  11. 11.
    Sakoda Y, Ito H, Uchida Y, Okamatsu M, Yamamoto N, Soda K, Nomura N, Kuribayashi S, Shichinohe S, Sunden Y, Umemura T, Usui T, Ozaki H, Yamaguchi T, Murase T, Ito T, Saito T, Takada A, Kida H (2012) Reintroduction of H5N1 highly pathogenic avian influenza virus by migratory water birds, causing poultry outbreaks in the 2010–2011 winter season in Japan. J Gen Virol 93(Pt 3):541–550CrossRefGoogle Scholar
  12. 12.
    Ozawa M, Matsuu A, Tokorozaki K, Horie M, Masatani T, Nakagawa H, Okuya K, Kawabata T, Toda S (2015) Genetic diversity of highly pathogenic H5N8 avian influenza viruses at a single overwintering site of migratory birds in Japan, 2014/15. Euro Surveill 20(20):21132CrossRefGoogle Scholar
  13. 13.
    Okamatsu M, Ozawa M, Soda K, Takakuwa H, Haga A, Hiono T, Matsuu A, Uchida Y, Iwata R, Matsuno K, Kuwahara M, Yabuta T, Usui T, Ito H, Onuma M, Sakoda Y, Saito T, Otsuki K, Ito T, Kida T (2017) Characterization of highly pathogenic avian influenza virus A(H5N6), Japan, November 2016. Emerg Infect Dis. 23(4):691–695CrossRefGoogle Scholar
  14. 14.
    Ozawa M, Matsuu A, Khalil AM, Nishi N, Tokorozaki K, Masatani T, Horie M, Okuya K, Ueno K, Kuwahara M, Toda S (2019) Phylogenetic variations of highly pathogenic H5N6 avian influenza viruses isolated from wild birds in the Izumi plain, Japan, during the 2016–17 winter season. Transbound Emerg Dis 66(2):797–806CrossRefGoogle Scholar
  15. 15.
    Okuya K, Kanazawa N, Kanda T, Kuwahara M, Matsuu A, Horie M, Masatani T, Toda S, Ozawa M (2017) Genetic characterization of an avian H4N6 influenza virus isolated from the Izumi plain, Japan. Microbiol Immunol 61(11):513–518CrossRefGoogle Scholar
  16. 16.
    Tsukamoto K, Ashizawa H, Nakanishi K, Kaji N, Suzuki K, Okamatsu M, Yamaguchi S, Mase M (2008) Subtyping of avian influenza viruses H1 to H15 on the basis of hemagglutinin genes by PCR assay and molecular determination of pathogenic potential. J Clin Microbiol 46(9):3048–3055CrossRefGoogle Scholar
  17. 17.
    Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR (2001) Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 146(12):2275–2289CrossRefGoogle Scholar
  18. 18.
    Review of latest available evidence on potential transmission of avian influenza (H5N1) through water and sewage and ways to reduce the risks to human health (2007) Water, Sanitation and Health Public Health and Environment Geneva 2006. Accessed 30 Oct 2019
  19. 19.
    Maeda Y, Tohya Y, Nakagami Y, Yamashita M, Sugimura T (2001) An occurrence of salmonella infection in cranes at the Izumi Plains, Japan. J Vet Med Sci 63(8):943–944CrossRefGoogle Scholar
  20. 20.
    Daniel R, Hosseini PR, Mazet JA, Daszak P, Goldstein T (2015) Evolutionary dynamics and global diversity of influenza A virus. J Virol 89(21):10993–11001. CrossRefGoogle Scholar
  21. 21.
    Winker K, McCracken KG, Gibson DD, Pruett CL, Meier R, Huettmann F, Wege M, Kulikova IV, Zhuravlev YN, Perdue ML, Spackman E, Suarez D, Swayne DE (2007) Movements of birds and avian influenza from Asia into Alaska. Emerg Infect Dis. 13(4):547–552CrossRefGoogle Scholar
  22. 22.
    Ramey AM, Reeves AB, Sonsthagen SA, TeSlaa JL, Nashold S, Donnelly T, Casler B, Hall JS (2015) Dispersal of H9N2 influenza A viruses between East Asia and North America by wild birds. Virology 482(2015):79–83CrossRefGoogle Scholar
  23. 23.
    Schmolke M, Manicassamy B, Pena L, Sutton T, Hai R, Varga ZT, Hale BG, Steel J, Perez DR, Garcia-Sastre A (2011) Differential contribution of PB1-F2 to the virulence of highly pathogenic H5N1 influenza A virus in mammalian and avian species. PLoS Pathog 7(8):e1002186CrossRefGoogle Scholar
  24. 24.
    Chakrabarti AK, Pasricha G (2013) An insight into the PB1F2 protein and its multifunctional role in enhancing the pathogenicity of the influenza A viruses. Virology 440(2):97–104CrossRefGoogle Scholar
  25. 25.
    Conenello GM, Zamarin D, Perrone LA, Tumpey T, Palese P (2007) A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence. PLoS Pathog 3(10):1414–1421CrossRefGoogle Scholar
  26. 26.
    García-Sastre A, Egorov A, Matassov D, Brandt S, Levy DE, Durbin JE, Palese P, Muster T (1998) Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 252(2):324–330CrossRefGoogle Scholar
  27. 27.
    Obenauer JC, Denson PK, Mehta X, Su S, Mukatira DB, Finkelstein X, Xu J, Wang J, Ma Y, Fan KM, Rakestraw Webster RG, Hoffmann E, Krauss S, Zheng Z, Zhang J, Naeve CW (2006) Large-scale sequence analysis of avian influenza isolates. Science 311(5767):1576–1580CrossRefGoogle Scholar
  28. 28.
    Sheng M, Sala C (2001) PDZ domains and the organization of supramolecular complexes. Annu Rev Neurosci 24:1–29CrossRefGoogle Scholar
  29. 29.
    Jackson D, Hossain MJ, Hickman D, Perez DR, Lamb RA (2008) A new influenza virus virulence determinant: the NS1 protein four C-terminal residues modulate pathogenicity. Proc Natl Acad Sci USA 105(11):4381–4386CrossRefGoogle Scholar
  30. 30.
    Imai M, Watanabe T, Hatta M, Das SC, Ozawa M, Shinya K, Zhong G, Hanson A, Katsura H, Watanabe S, Li C, Kawakami E, Yamada S, Kiso M, Suzuki Y, Maher EA, Neumann G, Kawaoka Y (2012) Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Nature 486(7403):420–428CrossRefGoogle Scholar
  31. 31.
    Nidom CA, Takano R, Yamada S, Sakai-Tagawa Y, Daulay S, Aswadi D, Suzuki T, Suzuki Y, Shinya K, Iwatsuki-Horimoto K, Muramoto Y, Kawaoka Y (2010) Influenza A (H5N1) viruses from pigs, Indonesia. Emerg Infect Dis 16(10):1515–1523CrossRefGoogle Scholar
  32. 32.
    Yang H, Chen LM, Carney PJ, Donis RO, Stevens J (2010) Structures of receptor complexes of a North American H7N2 Influenza Hemagglutinin with a loop deletion in the receptor binding site. PLoS Pathog 6(9):e1001081CrossRefGoogle Scholar
  33. 33.
    Bussey KA, Bousse TL, Desmet EA, Kim B, Takimoto T (2010) PB2 residue 271 plays a key role in enhanced polymerase activity of Influenza A Viruses in mammalian host cells. J Virol 84:4395–4406CrossRefGoogle Scholar
  34. 34.
    Hatta M, Gao P, Halfmann P, Kawaoka Y (2001) Molecular basis for high virulence of Hong Kong H5N1 Influenza A viruses. Science 293:1840–1842CrossRefGoogle Scholar
  35. 35.
    Min JY, Santos C, Fitch A, Twaddle A, Toyoda Y, DePasse JV, Ghedin E, Subbarao K (2013) Mammalian adaptation in the PB2 gene of Avian H5N1 Influenza Virus. J Virol 87(19):10884–10888CrossRefGoogle Scholar
  36. 36.
    Steel J, Lowen AC, Mubareka S, Palese P (2009) Transmission of Influenza Virus in a mammalian host is increased by PB2 amino acids 627 K or 627E/701N. PLoS Pathog 5(1):e1000252CrossRefGoogle Scholar
  37. 37.
    Yamada S, Hatta M, Staker BL, Watanabe S, Imai M, Shinya K, Sakai-Tagawa Y, Ito M, Ozawa M, Watanabe T, Sakabe S, Li C, Kim JH, Myler PJ, Phan I, Raymond A, Smith E, Stacy R, Nidom CA, Lank SM, Wiseman RW, Bimber BN, O’Connor DH, Neumann G, Stewart LJ, Kawaoka Y (2010) Biological and structural characterization of a host-adapting amino acid in Influenza Virus. PLoS Pathog 6(8):e1001034CrossRefGoogle Scholar
  38. 38.
    Hay AJ, Zambon MC, Wolstenholme AJ, Skehel JJ, Smith MH (1986) Molecular basis of resistance of influenza A viruses to amantadine. J Antimicrob Chemother 18(Suppl B):19–29CrossRefGoogle Scholar
  39. 39.
    Pinto LH, Holsinger LJ, Lamb RA (1992) Influenza virus M2 protein has ion channel activity. Cell 69:517–528CrossRefGoogle Scholar
  40. 40.
    Abed Y, Baz M, Boivin G (2006) Impact of neuraminidase mutations conferring influenza resistance to neuraminidase inhibitors in the N1 and N2 genetic backgrounds. Antiviral Therapy 11(8):971–976PubMedGoogle Scholar
  41. 41.
    Aoki FY, Boivin G, Roberts N (2007) Influenza virus susceptibility and resistance to oseltamivir. Antivir Therapy 12(4):603–616Google Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Laboratory of Animal Hygiene, Joint Faculty of Veterinary MedicineKagoshima UniversityKagoshimaJapan
  2. 2.United Graduate School of Veterinary ScienceYamaguchi UniversityYamaguchiJapan
  3. 3.Department of Zoonotic Diseases, Faculty of Veterinary MedicineZagazig UniversityZagazigEgypt
  4. 4.Joint Graduate School of Veterinary MedicineKagoshima UniversityKagoshimaJapan
  5. 5.Transboundary Animal Diseases Research Center, Joint Faculty of Veterinary MedicineKagoshima UniversityKagoshimaJapan
  6. 6.Matsuoka Research Institute for ScienceKoganeiJapan
  7. 7.Kagoshima Crane Conservation CommitteeKagoshimaJapan

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