Supplementary Material for: Emergence of a Novel Reassortant H5N3 Avian Influenza Virus in Korean Mallard Ducks in 2018

<b><i>Introduction:</i></b> The avian influenza (AI) virus causes a highly contagious disease which is common in wild and domestic birds and sporadic in humans. Mutations and genetic reassortments among the 8 negative-sense RNA segments of the viral genome alter its pathogenic potential, demanding well-targeted, active surveillance for infection control. <b><i>Methods:</i></b> Wild duck fecal samples were collected during the 2018 bird health annual surveillance in South Korea for tracking variations of the AI virus. One low-pathogenic avian influenza H5N3 reassortment virus (A/mallard duck/South Korea/KNU18-91/2018 [H5N3]) was isolated and genomically characterized by phylogenetic and molecular analyses in this study. <b><i>Results:</i></b> It was devoid of polybasic amino acids at the hemagglutinin (HA) cleavage site and exhibited a stalk region without deletion in the neuraminidase (<i>NA</i>) gene and NA inhibitor resistance-linked E/D627K/N and D701N marker mutations in the <i>PB2</i> gene, suggesting its low-pathogenic AI. It showed a potential of a reassortment where only <i>HA</i> originated from the H5N3 poultry virus of China and other genes were derived from Mongolia. In phylogenetic analysis, HA was different from that of the isolate of H5N3 in Korea, 2015. In addition, this novel virus showed adaptation in Madin-Darby canine kidney cells, with 8.05 ± 0.14 log10 50% tissue culture infectious dose (TCID50) /mL at 36 h postinfection. However, it could not replicate in mice well, showing positive growth at 3 days postinfection (dpi) (2.1 ± 0.13 log10 TCID50/mL) but not at 6 dpi. <b><i>Conclusions:</i></b> The HA antigenic relationship of A/mallard duck/South Korea/KNU18-91/2018 (H5N3) showed differences toward one of the old low-pathogenic H5N3 viruses in Korea. These results indicated that a novel reassortment low-pathogenic avian influenza H5N3 subtype virus emerged in South Korea in 2018 via novel multiple reassortments with Eurasian viruses, rather than one of old Korean H5N3 strains.