Diversity of A(H5N1) clade avian influenza viruses with evidence of reassortment in Cambodia, 2014-2016

PLoS One. 2019 Dec 9;14(12):e0226108. doi: 10.1371/journal.pone.0226108. eCollection 2019.


In Cambodia, highly pathogenic avian influenza A(H5N1) subtype viruses circulate endemically causing poultry outbreaks and zoonotic human cases. To investigate the genomic diversity and development of endemicity of the predominantly circulating clade A(H5N1) viruses, we characterised 68 AIVs detected in poultry, the environment and from a single human A(H5N1) case from January 2014 to December 2016. Full genomes were generated for 42 A(H5N1) viruses. Phylogenetic analysis shows that five clade genotypes, designated KH1 to KH5, were circulating in Cambodia during this period. The genotypes arose through multiple reassortment events with the neuraminidase (NA) and internal genes belonging to H5N1 clade, clade or A(H9N2) lineages. Phylogenies suggest that the Cambodian AIVs were derived from viruses circulating between Cambodian and Vietnamese poultry. Molecular analyses show that these viruses contained the hemagglutinin (HA) gene substitutions D94N, S133A, S155N, T156A, T188I and K189R known to increase binding to the human-type α2,6-linked sialic acid receptors. Two A(H5N1) viruses displayed the M2 gene S31N or A30T substitutions indicative of adamantane resistance, however, susceptibility testing towards neuraminidase inhibitors (oseltamivir, zanamivir, lananmivir and peramivir) of a subset of thirty clade viruses showed susceptibility to all four drugs. This study shows that A(H5N1) viruses continue to reassort with other A(H5N1) and A(H9N2) viruses that are endemic in the region, highlighting the risk of introduction and emergence of novel A(H5N1) genotypes in Cambodia.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Bayes Theorem
  • Cambodia
  • Chickens
  • Genetic Variation*
  • Genotype
  • Hemagglutinins / classification
  • Hemagglutinins / genetics
  • Influenza A Virus, H5N1 Subtype / classification
  • Influenza A Virus, H5N1 Subtype / genetics*
  • Influenza A Virus, H5N1 Subtype / isolation & purification
  • Influenza in Birds / pathology
  • Influenza in Birds / virology
  • Phylogeny
  • Poultry Diseases / pathology
  • Poultry Diseases / virology
  • Reassortant Viruses / genetics*
  • Reassortant Viruses / isolation & purification
  • Selection, Genetic
  • Virulence / genetics


  • Hemagglutinins

Grant support

This publication is the result of work conducted under a cooperative agreement with the Office of the Assistant Secretary for Preparedness and Response in the U.S. Department of Health and Human Services (HHS), grant number IDSEP140020-01-00. Its contents and conclusions are solely the responsibility of the authors and do not represent the official views of HHS. The study was also funded, in part, by the US Agency for International Development (grant No. AID-442-G-14-00005) and partially funded through the UK Research and Innovation Global Challenges Research Fund to The Consortium of Animal Market Networks to Assess Risk of Emerging Infectious Diseases Through Enhanced Surveillance (CANARIES; grant No. GCRFNGR3\1497). Annika Suttie is funded by an Australian Government Research Training Program Scholarship and a Faculty of Science and Technology Research Scholarship from Federation University. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health. GlaxoSmithKline Biologicals SA provided support in the form of salary for an author [PB], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the ‘author contributions’ section. The authors are solely responsible for final content and interpretation.