Subtype Diversity of Influenza A Virus in North American Waterfowl: a Multidecade Study

Abstract

The discovery in 1976 of waterfowl as the primary reservoir of influenza A viruses (IAVs) has since spurred decades of waterfowl surveillance efforts by researchers dedicated to understanding the ecology of IAV and its subsequent threat to human and animal health. Here, we employed a multidecade, continental-scale approach of surveillance data to understand trends of seasonal IAV subtype diversity. Between 1976 and 2015, IAVs were detected in 8,427 (10.8%) of 77,969 samples from migratory waterfowl throughout the Central and Mississippi Migratory Flyways in the United States and Canada. A total of 96 hemagglutinin (HA)/neuraminidase (NA) subtype combinations were isolated, which included most HA (H1 to H14) and all 9 NA subtypes. We observed an annual trend of high influenza prevalence, involving a few dominant subtypes, on northern breeding grounds during summer with progressively lowered influenza prevalence, comprised of a highly diverse profile of subtypes, as waterfowl migrate toward southern wintering grounds. Isolates recovered during winter had the highest proportion of mixed and rare HA/NA combinations, indicating increased opportunity for reassortment of IAVs. In addition, 70% of H5 and 49% of H7 IAV isolates were recovered from samples collected during fall and spring, respectively; these are subtypes that can have significant implications for public health and agriculture sectors. Annual cyclical dominance of subtypes on northern breeding grounds is revealed through the longitudinal nature of this study. Our novel findings exhibit the unrealized potential for discovery using existing IAV surveillance data.IMPORTANCE Wild aquatic birds are the primary natural reservoir of influenza A viruses (IAVs) and are therefore responsible for the dispersal and maintenance of IAVs representing a broad range of antigenic and genetic diversity. The aims of IAV surveillance in waterfowl not only relate to understanding the risk of spillover risk to humans, but also to improving our understanding of basic questions related to IAV evolution and ecology. By evaluating several decades of surveillance data from wild aquatic birds sampled along North American migratory flyways, we discovered an annual trend of increasing subtype diversity during southbound migration, peaking on southern wintering grounds. Winter sampling revealed the highest proportion of mixed and rare infections that suggest higher opportunity for spillover. These findings allow improvements to surveillance efforts to robustly capture IAV diversity that will be used for vaccine development and cultivate a more thorough understanding of IAV evolution and persistence mechanisms.

Keywords: ecology; hemagglutinin; influenza A virus; neuraminidase; season; subtype diversity; waterfowl; wild birds.

FIG 1

Spatial and temporal distribution of influenza A virus (IAV) sampling sites of waterfowl in North America between 1976 and 2015. Latitude of map on the left (A) approximately aligns with the latitude shown on the y axis on the right (B) for a clear context. The red color gradation of circles gives IAV prevalence, with the darkest shade indicating ≥30% prevalence. The size of circles represents the relative number of samples collected at a specific study site or latitude. The average latitude of sampling sites was 51.2°N during summer (June to August), 41.0°N during fall (September to November), 35.5°N during winter (December to February), and 28.3°N during spring (March to May). Migratory activity in fall and spring relates to the wider range of sampling latitudes: summer range, 21.2°; fall, 26.2°; winter, 15.7°; and spring, 27.1°.

FIG 2

Stacked bar graphs showing total number of IAV samples collected from waterfowl per year between 1976 and 2015 in North America. (A) The number of IAV isolates recovered each year is indicated in red, and IAV-negative samples are indicated in blue. IAV isolates recovered by decade are as follows: 1976 to 1985, 2,749 (24%) IAV isolates from 11,361 waterfowl; 1986 to 1995, 304 (11%) IAV isolates from 2,721 waterfowl; 1996 to 2005, 303 (8%) IAV isolates from 3,624 waterfowl; and 2006 to 2015, 5,071 (8%) IAV isolates recovered from 60,263 waterfowl. (B) The number of samples collected each year by state or province (see key for color coding). Locations are given in descending order from north to south by latitude of the sampling site. Sampling expanded to locations throughout the extent of the Mississippi Migratory Flyway in 2006.

FIG 3

Distribution of 96 IAV virus subtype combinations isolated from migratory waterfowl in North America between 1976 and 2015. All subtype combinations shown in salmon color are classified as rare (recovered ≤10 times in the data set). The remaining subtype combinations in blue darken relative to the frequency of recovery.

FIG 4

Influenza A virus hemagglutinin (HA) subtype diversity from migratory waterfowl sampled during (A) summer (June to August), (B) fall (September to November), (C) winter (December to February), and (D) spring (March to May) between 1976 and 2015. Each pie segment represents the proportion of the respective HA subtype (color-coded) isolated in the respective location and season. Size of circles are relative to the number of samples collected at a location. An annual trend is evident of progressively increased HA subtype diversity during southbound migration, peaking during winter.

FIG 5

Temporal distribution of IAV subtypes of HA and NA recovered from migratory waterfowl in North America between 1976 and 2015. The 7,797 single-subtype combination isolates are color-coded by season of recovery (3-month intervals; summer: June to August).

FIG 6

Stacked bars representing IAV isolates per year from waterfowl sampled in North America between 1976 and 2015, stratified by season sampled (3-month intervals; summer: June to August). The black line indicates subtype diversity, calculated by the number of distinct subtype combinations detected each year. Expansion of IAV surveillance efforts to locations associated with fall migration, winter, and spring migration led to higher diversity of IAV subtypes recovered than in years with predominant summer sampling.

FIG 7

Cyclical trends in the annual proportion of respective IAV HA subtypes isolated during summer (June to August) from migratory waterfowl in North America. (A) H3 and H4 demonstrated an apparent inverse cyclical trend and (B) H1 demonstrated an apparent cyclical trend. No other HA subtypes appeared to cycle during summer, and data for other seasons were limited. Restriction by season assumes that waterfowl were sampled in regionally similar areas each year.