Long-term surveillance of H7 influenza viruses in American wild aquatic birds: are the H7N3 influenza viruses in wild birds the precursors of highly pathogenic strains in domestic poultry?

Abstract

The emergence of influenza A virus (IAV) in domestic avian species and associated transmissions to mammals is unpredictable. In the Americas, the H7 IAVs are of particular concern, and there have been four separate outbreaks of highly pathogenic (HP) H7N3 in domestic poultry in North and South America between 2002 and 2012, with occasional spillover into humans. Here, we use long-term IAV surveillance in North American shorebirds at Delaware Bay, USA, from 1985 to 2012 and in ducks in Alberta, Canada, from 1976 to 2012 to determine which hemagglutinin (HA)-neuraminidase (NA) combinations predominated in Anseriformes (ducks) and Charadriiformes (shorebirds) and whether there is concordance between peaks of H7 prevalence and transmission in wild aquatic birds and the emergence of H7 IAVs in poultry and humans. Whole-genome sequencing supported phylogenetic and genomic constellation analyses to determine whether HP IAVs emerge in the context of specific internal gene segment sequences. Phylogenetic analysis of whole-genome sequences of the H7N3 influenza viruses from wild birds and HP H7N3 outbreaks in the Americas indicate that each HP outbreak was an independent emergence event and that the low pathogenic (LP) avian influenza precursors were most likely from dabbling ducks. The different polybasic cleavage sites in the four HP outbreaks support independent origins. At the 95% nucleotide percent identity-level phylogenetic analysis showed that the wild duck HA, PB1, and M sequences clustered with the poultry and human outbreak sequences. The genomic constellation analysis strongly suggests that gene segments/virus flow from wild birds to domestic poultry.

Figure 1

H7N3 HPAI domestic poultry outbreaks in the Americas relative to H7N3 wild bird isolates in North America between 1976 and 2012. H7N3 isolates obtained for ducks (red) and shorebirds (blue) is given as a percentage of the total number of annual influenza virus isolates sampled from each wild bird taxonomic Order. The four HPAI domestic poultry outbreaks that occurred in the Americas during this time period are marked with arrows.

Figure 2

Outbreaks of low pathogenic H7 influenza virus subtype in domestic poultry in the Americas between 1976 and 2013.

Figure 3

Avian influenza virus H7 subtype evolution in the Americas and the emergence of HP viruses in a whole-genome context. For all sampled wild bird sequences, all available highly pathogenic H7N3 strains for which the majority of internal genes were also sequenced, and a number of reference genomes, an HA nucleotide phylogeny was inferred using the GTR-IG substitution method, a lognormal relaxed clock, and a skygrid coalescent model in BEAST v1.8 with a chain length of 100 million. Runs were evaluated in Tracer to ensure reasonable effective sample size scores, and a maximum clade credibility tree was constructed using Tree Annotator. Estimates for the most recent common ancestor with the outgroup sequences (1877) and between the North and South American viruses (1955) are provided, along with the 95% HPD ranges (also indicated as horizontal blue bars). A genome constellation analysis was performed by generating gene clusters for each segment using a 90% nucleotide identity cutoff. Cluster assignments are represented by one colored box for each segment, creating a genome constellation for each virus. Coloring between columns is independent, and the total number of colors in a column reflects the number of clusters generated for that gene segment. Black indicates that the gene sequences were unavailable. Strain names are colored by taxonomic Order. Human and/or poultry outbreak strains are boxed in red, and HP strains are marked with red circles based on their HA cleavage site sequences.

Figure 4

Genome constellation analyses at a 95% nucleotide identity cutoff further demonstrate the uniqueness of each HP H7N3 outbreak in the Americas. A genome constellation analysis using a 95% nucleotide identity cutoff was performed on a subset of strains, including the human and poultry outbreak strains and some of the wild bird strains (all from the Order Anseriformes) having the closest cophenetic distances in the Bayesian HA phylogeny to the outbreak strains. Cluster assignments are represented by one colored box for each segment, creating a genome constellation for each virus. Coloring between columns is independent, and the total number of colors in a column reflects the number of clusters generated for that gene segment. Black indicates that the gene sequences were unavailable. Strain names are colored by taxonomic Order, and HP strains are marked with red circles based on their HA1/HA2 cleavage site sequences, which are provided on the left. Critical basic residues are colored in red.