Evidence of a fixed internal gene constellation in influenza A viruses isolated from wild birds in Argentina (2006-2016)

Abstract

Wild aquatic birds are the major reservoir of influenza A virus. Cloacal swabs and feces samples (n = 6595) were collected from 62 bird species in Argentina from 2006 to 2016 and screened for influenza A virus. Full genome sequencing of 15 influenza isolates from 6 waterfowl species revealed subtypes combinations that were previously described in South America (H1N1, H4N2, H4N6 (n = 3), H5N3, H6N2 (n = 4), and H10N7 (n = 2)), and new ones not previously identified in the region (H4N8, H7N7 and H7N9). Notably, the internal gene segments of all 15 Argentine isolates belonged to the South American lineage, showing a divergent evolution of these viruses in the Southern Hemisphere. Time-scaled phylogenies indicated that South American gene segments diverged between ~ 30 and ~ 140 years ago from the most closely related influenza lineages, which include the avian North American (PB1, HA, NA, MP, and NS-B) and Eurasian lineage (PB2), and the equine H3N8 lineage (PA, NP, and NS-A). Phylogenetic analyses of the hemagglutinin and neuraminidase gene segments of the H4, H6, and N8 subtypes revealed recent introductions and reassortment between viruses from the Northern and Southern Hemispheres in the Americas. Remarkably and despite evidence of recent hemagglutinin and neuraminidase subtype introductions, the phylogenetic composition of internal gene constellation of these influenza A viruses has remained unchanged. Considering the extended time and the number of sampled species of the current study, and the paucity of previously available data, our results contribute to a better understanding of the ecology and evolution of influenza virus in South America.

Fig. 1. Avian species sampled for IAV detection from 2006 to 2016 in Argentina.

Species belong to orders Anseriformes (upper 14 species), Charadriiformes (middle 10 species) and Pscittaciformes (bottom species). Right panel: only species with more than 5 samples collected are shown. Left panel: number of isolates per species obtained from RT-qPCR positive samples

Fig. 2. Avian-origin influenza virus subtypes obtained from avian species in Argentina (2006–2016).

The gradient in color intensity indicates the number of isolates obtained for each subtype combination. Novel IAV subtypes, reported for the first time in South America, are shown in red

Fig. 3. Number of IAV isolates in South America. The color intensity gradient indicates the number of IAVs obtained from wild birds in each country.

Argentina (n = 23), Bolivia (n = 1), Brazil (n = 10), Chile (n = 20), Colombia (n = 2), and Peru (n = 30). Bird migration routes are indicated in green and yellow. Red dots represent all sites sampled for IAV in Argentina

Fig. 4. Time of divergence of the South American (SAm) lineage.

Each dot represents the time of the most recent common ancestor, or node, between the SAm lineage and the most closely related lineage of each of 10,000 trees inferred for each segment. The identity of the most closely related lineage is shown by the colors. Mean tMRCA and 95% HPDs are represented by whiskers. Eurasian (EAs), North American (NAm)

Fig. 5. Genomic constellations of avian-origin IAVs from South America.

IAVs by host-order and country color-coded as South American lineage (red), North America lineage (blue), Eurasian lineage (green), other global avian lineages (gray), and no sequence data available (white)

Fig. 6. Phylogenetic distance (number of base substitutions per site) between a reference sequence from an IAV from Argentina and other global LPAIV sequences used for phylogenetic analysis.

Each dot represents a single sequence (virus) and it is colored by its source (geographic or host). Argentinean IAVs are shown with red asterisks. The continuous distribution of viruses within their main lineages is shown by the violin shapes for lineages with more than 2 observations: avian South American (SAm), North America (NAm), Eurasian (EAs). Argentinean isolates used as references and shown at zero distance: 432/H1N1 (H1 and N1), 1737/H5N3 (H5 and N3), 188/H7N7 (H7), 1174A/H6N2 (N2), 1227/H4N6 (N6), 1588/H7N9 (N9). Detailed phylogenies are provided in the supplementary material