Influenza A Virus Data from Emperor Geese, Alaska

Environmental Fecal Sampling: Swab samples for viral screening were collected by applying a polyester-tipped applicator to the feces. All swabs were placed in cyrovials with chilled viral transport media supplemented with antibiotics. Samples were then placed directly in dry shippers charged with liquid nitrogen and transferred to -80˚ C freezers until testing.

Serology of Nesting Females: After hatch year female emperor geese were captured on nests using bow traps at locations adjacent to the Manokinak River, Alaska in May and June 2015-2017. Blood samples were collected from each bird via jugular venipuncture. Sampling procedures were reviewed by the Animal Care and Use Committee at the U.S. Geological Survey (USGS) Alaska Science Center (2008–17) and conducted under USGS federal bird banding permit #20022. Blood samples were kept at ambient temperature for < 12 hours and subsequently centrifuged for 15 minutes to separate serum. Sera were decanted into cryovials, placed in dry shippers charged with liquid nitrogen, and held at -80° C prior to analysis.

Environmental Fecal Sampling: All swab samples were initially screened at the University of Georgia using real-time reverse transcriptase polymerase chain reaction (rRT-PCR) targeting gene products for the influenza A virus matrix gene. All samples yielding Ct values < 45 for the IAV matrix gene were inoculated into specific pathogen-free embryonated chicken eggs for virus isolation. Amnio-allantoic fluids were collected after 120 hours and tested via hemagglutination assay. RNAs extracted from hemagglutination assay positive amnio-allantoic fluids were tested for the IAV matrix gene by rRT-PCR and resultant isolates were genomically sequenced at the Alaska Science Center using next-generation sequencing technology (Illumina platform, Illumina Inc., San Diego, CA).

Serology of Nesting Females (bELISA): Sera were screened for IAV antibodies using a combination of blocking enzyme-linked immunosorbent assay (bELISA), microneutralization (MN), and hemagglutinin inhibition (HI). First, sera were tested for antibodies to IAV nucleoproteins using a commercial bELISA (AI MultiS-Screen antibody test kit; Idexx Laboratories, Westbrook, ME) following manufacturer guidelines. Results of the bELISA assays indicate the relative concentration of antibodies in the sample. Negative control samples were also tested.

Serology of Nesting Females (Microneutralization): A subset of sera (n = 113) for which serum-sample-to-negative-control (S/N) absorbance values were < 0.5, interpreted as positive for IAV antibodies (i.e., evidence of previous IAV exposure), were subsequently tested for antibodies to 14 IAV subtypes using a microneutralization (MN) assay. Each positive serum sample was diluted two-fold in MEM on a 96 well v-bottom plate (final volume of 25 µl. well at dilutions 1:20 to 1:640). If cytopathic effect (CPE) was observed at the minimum 1:20 dilution, the sample was classified as negative. If no CPE was observed at the minimum 1:20 dilution, the positive titer was recorded as the highest dilution at which no CPE was observed. Values "<20" represent a negative result. Values greater than or equal to 20 represent a positive result. Alternatively stated a value of 20 or greater indicates that the goose had a positive titer of antibodies to the corresponding IAV subtype.

Serology of Nesting Females (Hemagglutination Inhibition): Using the same subset of sera from the MN assays, samples were screened for evidence of previous exposure to the clade 2.3.4.4 goose Guangdon Gs/GD lineage highly pathogenic (HP) IAV. Briefly, hemagglutination inhibition assays were performed using two antigens: (1) reverse genetics-A/blue-winged teal/Texas/AI12-4150/2012 (rgBWTE/TX; H5N2) that included North American H5 and N2 genes on a PR-8 backbone, and (2) inactivated A/gyrfalcon/Washington/41088-6/2014 (GYRFA/WA; H5N8). Antibody titers to North American (rgBWTE/TX) and clade 2.3.4.4 GsGD HP (GYRF/WA) H5 antigens were compared for each sera sample. A two dilution higher antibody titer bias towards GYRF/WA was used as the criteria to assign the detected antibody response as suggestive of exposure to clade 2.3.4.4 GsGD HP H5 IAV.