Archives of Virology

, Volume 163, Issue 5, pp 1195–1207 | Cite as

Spatial transmission of H5N6 highly pathogenic avian influenza viruses among wild birds in Ibaraki Prefecture, Japan, 2016–2017

  • Ryota Tsunekuni
  • Yuji Yaguchi
  • Yuki Kashima
  • Kaoru Yamashita
  • Nobuhiro Takemae
  • Junki Mine
  • Taichiro Tanikawa
  • Yuko Uchida
  • Takehiko Saito
Original Article


From 29 November 2016 to 24 January 2017, sixty-three cases of H5N6 highly pathogenic avian influenza virus (HPAIV) infections were detected in wild birds in Ibaraki Prefecture, Japan. Here, we analyzed the genetic, temporal, and geographic correlations of these 63 HPAIVs to elucidate their dissemination throughout the prefecture. Full-genome sequence analysis of the Ibaraki isolates showed that 7 segments (PB2, PB1, PA, HA, NP, NA, NS) were derived from G1.1.9 strains while the M segment was from G1.1 strains; both groups of strains circulated in south China. Pathological studies revealed severe systemic infection in dead swans (the majority of dead birds and the only species necropsied), thus indicating high susceptibility to H5N6 HPAIVs. Coalescent phylogenetic analysis using the 7 G1.1.9-derived segments enabled detailed analysis of the short-term evolution of these highly homologous HPAIVs. This analysis revealed that the H5N6 HPAIVs isolated from wild birds in Ibaraki Prefecture were divided into 7 groups. Spatial analysis demonstrated that most of the cases concentrated around Senba Lake originated from a single source, and progeny viruses were transmitted to other locations after the infection expanded in mute swans. In contrast, within just a 5-km radius of the area in which cases were concentrated, three different intrusions of H5N6 HPAIVs were evident. Multi-segment analysis of short-term evolution showed that not only was the invading virus spread throughout Ibaraki Prefecture but also that, despite the small size of this region, multiple invasions had occurred during winter 2016–2017.



In this research, we used the supercomputer of AFFRIT, MAFF, Japan.

Author contributions

RT designed the study, characterized viruses, and drafted the manuscript; YY, YK, and KY conducted pathology diagnosis and virus isolation; TS designed and coordinated the study and drafted the manuscript; NT, JM, TT, and YU characterized the viruses; all authors have read and approved the manuscript.


This study was part of a research project for improving food safety and animal health that was supported by the Ministry of Agriculture, Forestry, and Fisheries of Japan.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or live animals performed by any of the authors.

Supplementary material

705_2018_3752_MOESM1_ESM.pdf (105 kb)
Supplementary material 1 (PDF 105 kb) Online Resource 1: Accession numbers of sequences of Ibaraki isolates
705_2018_3752_MOESM2_ESM.pdf (396 kb)
Supplementary material 2 (PDF 396 kb) Online Resource 2: Pathologic findings in dead mute swans. Brain (a, b), lung (c, d,), and rachis (e, f) were collected from dead mute swans. Tissue sections were stained with hematoxylin and eosin (a, c, e). In immunohistochemical analysis for AIV M protein, the antigen stains red (b, d, f)
705_2018_3752_MOESM3_ESM.pdf (261 kb)
Supplementary material 3 (PDF 260 kb) Online Resource 3: Pathologic finding in a dead mute swan: hemorrhage in the conjunctiva (a). Tissue sections were stained with hematoxylin and eosin (b). In the immunohistochemical analysis for AIV M protein, the antigen stains red (c)
705_2018_3752_MOESM4_ESM.pdf (250 kb)
Supplementary material 4 (PDF 249 kb) Online Resource 4: Phylogenetic tree based on the HA gene. Branches in clade are filled in beige and that of the Ibaraki isolates is shown as a red line
705_2018_3752_MOESM5_ESM.pdf (314 kb)
Supplementary material 5 (PDF 313 kb) Online Resource 5: Maximum-likelihood phylogenetic tree based on HA gene
705_2018_3752_MOESM6_ESM.pdf (314 kb)
Supplementary material 6 (PDF 314 kb) Online Resource 6: Maximum-likelihood phylogenetic tree based on NA gene
705_2018_3752_MOESM7_ESM.pdf (313 kb)
Supplementary material 7 (PDF 312 kb) Online Resource 7: Maximum-likelihood phylogenetic tree based on PB2 gene
705_2018_3752_MOESM8_ESM.pdf (371 kb)
Supplementary material 8 (PDF 371 kb) Online Resource 8: Maximum-likelihood phylogenetic tree based on PB1 gene
705_2018_3752_MOESM9_ESM.pdf (367 kb)
Supplementary material 9 (PDF 366 kb) Online Resource 9: Maximum-likelihood phylogenetic tree based on PA gene
705_2018_3752_MOESM10_ESM.pdf (368 kb)
Supplementary material 10 (PDF 367 kb) Online Resource 10: Maximum-likelihood phylogenetic tree based on NP gene
705_2018_3752_MOESM11_ESM.pdf (310 kb)
Supplementary material 11 (PDF 310 kb) Online Resource 11: Maximum-likelihood phylogenetic tree based on M gene
705_2018_3752_MOESM12_ESM.pdf (366 kb)
Supplementary material 12 (PDF 366 kb) Online Resource 12: Maximum-likelihood phylogenetic tree based on NS gene
705_2018_3752_MOESM13_ESM.pdf (211 kb)
Supplementary material 13 (PDF 211 kb) Online Resource 13: Occurrence rates of branch posteriors of the maximum clade credibility (MCC) tree. The graph shows five ranges of posterior values: 0 to 0.2, >0.2 to 0.4, >0.4 to 0.6, >0.6 to 0.8, and >0.8 to 1. MCC trees based on the HA or NA segment were generated under the same conditions as that using 7 segments. The branch posteriors of the MCC tree generated from 7 segments differed significantly (P < 0.01, Mann–Whitney U test) from those of the MCC tree generated by using the HA or NA segment


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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • Ryota Tsunekuni
    • 1
  • Yuji Yaguchi
    • 2
  • Yuki Kashima
    • 2
  • Kaoru Yamashita
    • 2
  • Nobuhiro Takemae
    • 1
  • Junki Mine
    • 1
  • Taichiro Tanikawa
    • 1
  • Yuko Uchida
    • 1
  • Takehiko Saito
    • 1
  1. 1.Division of Transboundary Animal Disease, National Institute of Animal HealthNational Agriculture and Food Research OrganizationTsukubaJapan
  2. 2.Ibaraki Prefecture Kenpoku Livestock Hygiene Service CenterMitoJapan

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