Molecular Detection and Characterization of Highly Pathogenic H5N1 Avian Influenza Viruses in Wild Birds Inhabiting Western Alaska Provides Evidence for Three Independent Viral Introductions

Combined oral-pharyngeal and cloacal swab samples from hunter-harvested waterfowl (Anseriformes spp.) and environmental fecal swab samples from monospecific flocks of either emperor geese (Chen canagica) or glaucous-winged gulls (Larus glaucescens) were collected within and around Izembek National Wildlife Refuge during September–October 2022. Samples were deposited into viral transport media, placed in dry shippers charged with liquid nitrogen within 24 hours, shipped, and stored frozen at -80°C before laboratory analysis. In 2022, sampling was primarily carried out by Evan Buck.

Viral RNA was extracted from the samples using a commercially available magnetic bead kit (MagMax, Thermofisher) on an automated Kingfisher Duo using the manufacturer's protocol. With every extraction, positive and negative controls were used. Extracted RNA was tested for the Matrix gene. The primers used to amplify the Matrix gene were as follows: "M+25" 5'-AGATGAGTCTTCTAACCGAGGTCG-3', "M-124" 5'-TGCAAAAACATCTTCAAGTCTCTG-3'. Amplified target was detected with a FAM intercolated probe with the sequence: 5'-TCAGGCCCCCTCAAAGCCGA-3'. Master mix for the qPCR included reagents in the AgPath-ID One-step RT-PCR kit (Applied Biosystems) and the aforementioned primers and probe in the following volumes per reaction: reaction buffer, 12.5uL; M+25 primer (20uM), 0.25uL; M-124 primer (20uM), 0.25uL; probe (6uM), 0.25uL; 25x RT-PCR enzyme, 1uL; detection enhancer, 1.67uL; nanopure water, 1.08uL, sample RNA, 8uL. Thermal cycling conditions on a Bio-Rad CFX96 real time quantitative PCR machine were: 10 minutes 45°C, 10 minutes 94°C, followed by 45 cycles of 1 second denaturation at 94°C and 30 seconds annealing at 60°C, with fluorescence data collected at the annealing step. Cycle threshold (Ct) values were automatically generated by the thermal cycler.

RNA extracts with positive qPCR results for the matrix gene were amplified for the influenza A genome with reverse transcription PCR for sequencing. The consensus 5' and 3' influenza A primers are: "MBTuni-12" 5'-ACGCGTGATCAGCRAAAGCAGG-3' and "MBTuni-13" 5'-ACGCGTGATCAGTAGAAACAAGG-3'. Master mix for the PCR was made using reagents from the SuperScript III One-Step RT-PCR System with Platinum Taq high Fidelity (Invitrogen) and the previously listed primers in the following volumes per reaction: 2x reaction mix, 12.5uL; Superscript III RT Platinum Taq Mix, 0.5uL; MBTuni-12 (10uM), 0.5uL; MBTuni-13 (10uM), 0.5uL; nanopure water, magnesium sulfate (5mM), 1uL; RNAse inhibitor (13.3U/uL), 0.5uL; sample RNA, 2uL. Thermal cycling conditions on a Bio-Rad T100 were: 60 minutes 42°C, 2 minutes 94°C, 5 cycles of 15 seconds 94°C, 30 seconds 45°C, and 3 minutes 68°C, 30 cycles of 15 seconds 94°C, 30 seconds 57°C, 3 minutes 68°C, and a final extension for 3 minutes at 68°C. PCR product was prepared for sequencing using the Nextera XT Library Preparation Kit (Illumina) following manufacturer's instructions, and sequenced on a MiSeq (Illumina) and subsquently a NextSeq 1000 (Illumina). Sequencing platforms generated fastq files for analysis.