Influenza A virus recovery, diversity, and intercontinental exchange: A multi-year assessment of wild bird sampling at Izembek National Wildlife Refuge, Alaska

<div><p>Western Alaska is a potential point-of-entry for foreign-origin influenza A viruses (IAVs) into North America via migratory birds. We sampled waterfowl and gulls for IAVs at Izembek National Wildlife Refuge (NWR) in western Alaska, USA, during late summer and autumn months of 2011–2015, to evaluate the abundance and diversity of viruses at this site. We collected 4842 samples across five years from 25 species of wild birds resulting in the recovery, isolation, and sequencing of 172 IAVs. With the intent of optimizing sampling efficiencies, we used information derived from this multi-year effort to: 1) evaluate from which species we consistently recover viruses, 2) describe viral subtypes of isolates by host species and year, 3) characterize viral gene segment sequence diversity with respect to host species, and assess potential differences in the viral lineages among the host groups, and 4) examine how evidence of intercontinental exchange of IAVs relates to host species. We consistently recovered viruses from dabbling ducks (<i>Anas spp</i>.), emperor geese (<i>Chen canagica</i>) and glaucous-winged gulls (<i>Larus glaucescens</i>). There was little evidence for differences in viral subtypes and diversity from different waterfowl hosts, however subtypes and viral diversity varied between waterfowl host groups and glaucous-winged gulls. Furthermore, higher proportions of viral sequences from northern pintails (<i>Anas acuta</i>), emperor geese and glaucous-winged gulls were grouped in phylogenetic clades that included IAV sequences originating from wild birds sampled in Asia as compared to non-pintail dabbling ducks, a difference that may be related to intercontinental migratory tendencies of host species. Our summary of research and surveillance efforts at Izembek NWR will assist in future prioritization of which hosts to sample and swab types to collect in Alaska and elsewhere in order to maximize isolate recovery, subtype and sequence diversity for resultant viruses, and detection of evidence for intercontinental viral exchange.</p></div

Summary of surveillance sampling of wild birds for influenza A viruses at Izembek National Wildlife Refuge, Alaska, USA 2011–2015.

<p>Summary of surveillance sampling of wild birds for influenza A viruses at Izembek National Wildlife Refuge, Alaska, USA 2011–2015.</p

Summary of subtype combinations detected from IAV isolates originating from samples collected at Izembek National Wildlife Refuge in Alaska, USA 2011–2015.

<p>Summary of subtype combinations detected from IAV isolates originating from samples collected at Izembek National Wildlife Refuge in Alaska, USA 2011–2015.</p

Competition between influenza A virus subtypes through heterosubtypic immunity modulates re-infection and antibody dynamics in the mallard duck

<div><p>Our overall hypothesis is that host population immunity directed at multiple antigens will influence the prevalence, diversity and evolution of influenza A virus (IAV) in avian populations where the vast subtype diversity is maintained. To investigate how initial infection influences the outcome of later infections with homologous or heterologous IAV subtypes and how viruses interact through host immune responses, we carried out experimental infections in mallard ducks (<i>Anas platyrhynchos</i>). Mallards were pre-challenged with an H3N8 low-pathogenic IAV and were divided into six groups. At five weeks post H3N8 inoculation, each group was challenged with a different IAV subtype (H4N5, H10N7, H6N2, H12N5) or the same H3N8. Two additional pre-challenged groups were inoculated with the homologous H3N8 virus at weeks 11 and 15 after pre-challenge to evaluate the duration of protection. The results showed that mallards were still resistant to re-infection after 15 weeks. There was a significant reduction in shedding for all pre-challenged groups compared to controls and the outcome of the heterologous challenges varied according to hemagglutinin (HA) phylogenetic relatedness between the viruses used. There was a boost in the H3 antibody titer after re-infection with H4N5, which is consistent with original antigenic sin or antigenic seniority and suggest a putative strategy of virus evasion. These results imply competition between related subtypes that could regulate IAV subtype population dynamics in nature. Collectively, we provide new insights into within-host IAV complex interactions as drivers of IAV antigenic diversity that could allow the circulation of multiple subtypes in wild ducks.</p></div

Phylogeny of PB2 nucleotide sequences.

<p>The Maximum Likelihood phylogeny of PB2 nucleotide sequences originating from wild bird samples collected in Asia and North America (2011–2015) including those collected from Izembek National Wildlife Refuge (NWR) and sequenced as a part of this study. This phylogeny illustrates the delineation/naming of clades for the purpose of assessing diversity of viruses across host species and the identification of lineages for which there is evidence of intercontinental viral exchange. Clades are collapsed for visualization purposes; the height of collapsed clades (triangles) 1.1.1.1 and 1.1.2 are not proportional to the rest of the phylogeny and instead reduced for presentation purposes. Pie charts at right of clades are proportional in size to the total number of sequences within each clade and color-coded based on the geographical origins of sequences: Izembek NWR = light blue, elsewhere in North America = dark blue, Asia = orange. Clades 2.2.1 and 2.2.2.2.2.1, marked with ‘***,’ are examples of clades providing evidence of intercontinental dispersal of viruses (i.e. designated ‘Asian/mixed origin” via our methods). Clade 2.2.2.2.1, marked with ‘¥,’ comprised of 62 sequence from Asia and North America associated with recent outbreak of highly pathogenic H5 intercontinental group clade A viruses. Sequences identified as “undesignated” did not meet minimum criteria for clade designations (e.g. ≥ 4 sequences). Sequences originating from Izembek NWR in each clade are represented with silhouettes to the right of pie charts corresponding to the species/group from which isolates were derived as used in our data summary and analyses (northern pintail, other dabbling duck, emperor goose, glaucous winged gulls, and sea duck; as described in our methods, several analyses omitted data from viruses isolated from sea ducks). Numbers overlaid on silhouettes provide the number of viral sequences from Izembek NWR for each host species group per clade. For the complete PB2 phylogeny with individual strain names see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195327#pone.0195327.s002" target="_blank">S1 Fig</a>.</p

Distribution of influenza A virus (IAV) nucleoprotein antigen in the gastrointestinal tract and bursa of Fabricius of mallards exposed to a H14N5 IAV through intranasal inoculation or contact exposure.

<p>Viral antigen was not identified in larynx, trachea, lung, brain, kidney, adrenal glands, pectoral skeletal muscle, heart, or liver. ¹– = no staining; + = staining of a few cells; ++ = staining of moderate numbers of cells; +++ = staining of numerous cells.</p

Pre-challenge reduces cloacal (CL) viral shedding after heterologous challenge.

<p>Birds were pre-challenged (dashed lines) or not (solid lines) with H3N8 and challenged with different LPIAV viruses/strains. CL shedding showing the variation in intensity of shedding: 40 –mean Ct-values and SE for the different heterologous challenge groups: H4N5, H10N7, H6N2. Continuous lines and circles indicate the control groups and discontinuous lines indicate the H3N8 pre-challenged groups.</p

Photomicrographs of the ileum of a mallard intranasally inoculated with H14N5 influenza A virus (A/blue-winged teal/Texas/AI13-1028(H14N5)) at 2 days post-inoculation.

<p>A. No microscopic lesions are present in the mucosal villi (hematoxylin and eosin stain, bar = 100 µm). B. Serial section of the ileum showing immunolabeling for avian influenza virus nucleoprotein (brown) in epithelial cells lining the tips of the villi and rarely within cells of underlying the lamina propria (Immunoperoxidase labeling, hematoxylin counterstain, bar = 100 µm).</p

Genomic Characterization of H14 Subtype Influenza A Viruses in New World Waterfowl and Experimental Infectivity in Mallards (<i>Anas platyrhynchos</i>)

<div><p>Recent repeated isolation of H14 hemagglutinin subtype influenza A viruses (IAVs) in the New World waterfowl provides evidence to suggest that host and/or geographic ranges for viruses of this subtype may be expanding. In this study, we used genomic analyses to gain inference on the origin and evolution of H14 viruses in New World waterfowl and conducted an experimental challenge study in mallards (<i>Anas platyrhynchos</i>) to evaluate pathogenicity, viral replication, and transmissibility of a representative viral strain in a natural host species. Genomic characterization of H14 subtype IAVs isolated from New World waterfowl, including three isolates sequenced specifically for this study, revealed high nucleotide identity among individual gene segments (e.g. ≥95% shared identity among H14 HA gene segments). In contrast, lower shared identity was observed among internal gene segments. Furthermore, multiple neuraminidase subtypes were observed for H14 IAVs isolated in the New World. Gene segments of H14 viruses isolated after 2010 shared ancestral genetic lineages with IAVs isolated from wild birds throughout North America. Thus, genomic characterization provided evidence for viral evolution in New World waterfowl through genetic drift and genetic shift since purported introduction from Eurasia. In the challenge study, no clinical disease or lesions were observed among mallards experimentally inoculated with A/blue-winged teal/Texas/AI13-1028/2013(H14N5) or exposed via contact with infected birds. Titers of viral shedding for mallards challenged with the H14N5 IAV were highest at two days post-inoculation (DPI); however shedding was detected up to nine DPI using cloacal swabs. The distribution of viral antigen among mallards infected with H14N5 IAV was largely restricted to enterocytes lining the villi in the lower intestinal tract and in the epithelium of the bursa of Fabricius. Characterization of the infectivity of A/blue-winged teal/Texas/AI13-1028/2013(H14N5) in mallards provides support for similarities in viral replication and shedding as compared to previously described waterfowl-adapted, low pathogenic IAV strains in ducks.</p></div

Hemagglutinin and Neuraminidase subtypes identified by year.

<p>Totals and proportions of influenza A virus subtypes (Hemagglutinin [HA, A]; Neuraminidase [NA, B]) identified per year from virus isolation positive samples collected at Izembek National Wildlife Refuge, Alaska, USA. The number of sequences identified for each of the surface glycoproteins are 38, 26, 28, 44, and 43 for the years 2011–2015, respectively. The total numbers of HA and NA sequences (179 each) exceed the total number of isolates genomically sequenced (172) because two HA and two NA sequences were identified from seven mixed infection samples.</p