Temporal Viral Viability Data from Avian Influenza A Viruses Maintained in Alaska Wetlands Under Experimental and Environmental Conditions

Sample collection from wild birds: Swab samples for viral screening were collected by applying a polyester-tipped applicator to the cloaca and oropharynx of hunter harvested wild birds. All swabs were placed in 4 ml of distilled water. 20 sub-samples were made from each sample. Each sub-sample was created by adding 0.2 ml aliquot of the initial sample to 1.8 ml of either distilled water or 0.22-micron filtered water from four unique wetlands within Izembek National Wildlife Refuge, Alaska, USA. Four sub-samples were made for each of the 5 water treatment types. All of the sub-samples in distilled water and one of four sub-samples of each filtered surface water type including negative controls were shipped as quickly as possible to the University of Georgia for initial influenza A virus (IAV) screening and storage. The other sub-samples were submerged in steel drums in approximately one meter of water within each of the four wetlands. Sample tubes were sorted and submerged in the corresponding wetland from which the filtered water in each tube was collected.

Sample IAV screening: 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 (Spackman et al. 2002). All samples were also inoculated into specific pathogen-free embryonated chicken eggs for virus isolation (Fouchier el al. 2000). 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).

Wetland water temperature: During September 2019, a perforated steel drum containing a Hobo® TidbiT® temperature logger (Onset Computer Corp; Bourne, Massachusetts) was anchored within each of the four field sites at a depth of approximately 0.5–1.0 m as measured from substrate to the top of the water column. The temperature loggers were programed to record temperature in degrees Celsius every hour on the hour for the duration of the study. Once retrieved (September 2020), the data was uploaded to a mobile device and downloaded as a comma-separated values (CSV) file.

Wetland water measurements and collections: We used a YSI (Yellow Springs, OH) EXO water quality meter (calibrated daily) to determine pH, dissolved oxygen, specific conductance, temperature, barometric pressure, and turbidity of water at each field site. All other water metrics were measured by collecting water samples directly from each wetland in air-tight acid-washed plastic bottles. In addition to the untreated samples collected, surface water was collected from each field site and pre-filtered through 100 micron and 1 micron pour-through filters (Duda Energy LLC; Decatur, Alabama) and finally filtered through a 0.22 µm vacuum filter (Corning®; Corning, New York). All water samples were sent immediately on ice packs to the U.S. Geological Redox Chemistry Laboratory in Boulder, Colorado for testing.

Water sample testing and characterization: Standard techniques were used at the U.S. Geological Redox Chemistry Laboratory in Boulder, Colorado to characterize chemical concentrations of surface waters (alkalinity, aluminum, ammonium, antimony, arsenic, barium, beryllium, boron, bromine, calcium, chlorine, cobalt , dissolved organic carbon, fluorine, iron, lead, lithium, manganese, magnesium, molybdenum, nickel, nitrite, phosphorous, potassium, rubidium, silicon dioxide, sodium, strontium, sulfate, total dissolved nitrogen, tungsten, uranium, vanadium)(U.S. Environmental Protection Agency 1993, U.S. Environmental Protection Agency 1994, Brinton et al. 1996, Barringer et al. 1996).

Genomic Sequencing: Total RNA was also extracted from isolates using MagMAX™-96 AI/ND Viral RNA Isolation Kits. Complementary DNA was amplified from virus RNA extracts, visualized, and purified using methods reported in Reeves et al. (2018) at the USGS Alaska Science Center. Using a Nextera XT DNA library preparation kit (Illumina, Inc., San Diego, CA), indexed libraries were pooled and sequenced on an Illumina MiSeq using either 500 or 600-cycle reagent kits with paired-end reads. Sequence reads were assembled using a customized workflow on Geneious R11 (Biomatters Ltd. Auckland, New Zealand) using reference data for IAVs obtained from GenBank (Clark et al. 2016).