Influenza A Viruses from Wild Birds in Guatemala Belong to the North American Lineage

The role wild bird species play in the transmission and ecology of avian influenza virus (AIV) is well established; however, there are significant gaps in our understanding of the worldwide distribution of these viruses, specifically about the prevalence and/or significance of AIV in Central and South America. As part of an assessment of the ecology of AIV in Guatemala, we conducted active surveillance in wild birds on the Pacific and Atlantic coasts. Cloacal and tracheal swab samples taken from resident and migratory wild birds were collected from February 2007 to January 2010.1913 samples were collected and virus was detected by real time RT-PCR (rRT-PCR) in 28 swab samples from ducks (Anas discors). Virus isolation was attempted for these positive samples, and 15 isolates were obtained from the migratory duck species Blue-winged teal. The subtypes identified included H7N9, H11N2, H3N8, H5N3, H8N4, and H5N4. Phylogenetic analysis of the viral sequences revealed that AIV isolates are highly similar to viruses from the North American lineage suggesting that bird migration dictates the ecology of these viruses in the Guatemalan bird population

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

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

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

Oropharyngeal and cloacal viral shedding in six mallards intranasally inoculated with H14N5 influenza A virus (A/blue-winged teal/Texas/AI13-1028(H14N5)).

<p>Two mallards were euthanized and necropsied at 2 days post-inoculation (DPI), thus shedding data from 3 DPI on is based on four mallards. Gray bars represent the percentage of mallards that were virus isolation positive on each sampling day (left Y-axis). Black points on 2, 4, and 6 DPI represent the mean viral titers with 95% confidence intervals (right Y-axis). Virus isolation positive swab samples that had a concentration of virus below the detectable limit of the titration assay were listed as 10¹ log₁₀ EID₅₀/mL.</p

Maximum likelihood phylogenetic tree showing inferred relationship among nucleotide sequences for the hemagglutinin gene of influenza A viruses of the H14 subtype.

<p>Bootstrap support values ≥70 are shown. Isolates from Guatemala and Texas genetically characterized as part of the current study are indicated with an arrow. The isolate used to experimentally inoculate mallards is identified with a dagger (†).</p

Sites for avian influenza surveillance in wild birds, Guatemala, 2007–2010.

<p>A = Active surveillance (mist nets, live captured birds), K = Hunter-killed.</p

Phylogenetic trees for H5 and H7 HA genes.

<p>Trees were generated in PAUP 4.0b10 using the Neighbor-Joining method with 1000 bootstrap replicates (bootstrap values above 70% are shown). Scale bar on the bottom-left indicates number of nucleotide substitutions per site.</p

Positive species for influenza type A by rRT-PCR and viral isolates obtained in this study.

*<p>Percentage of positive samples obtained by real-time RT-PCR (rRT-PCR) and Virus Isolation (VI) based on the total number of sampled birds.</p><p>N/D: Non-Determined.</p

Phylogenetic trees for N2 and N8 NA genes.

<p>Trees were generated in PAUP 4.0b10 using Neighbor-Joining method with 1000 bootstrap replicates (bootstrap values above 70% are shown). Scale bar on the bottom-left indicates number of nucleotide substitutions per site.</p