Molecular Characterization and Pathogenesis of H6N6 Low Pathogenic Avian Influenza Viruses Isolated from Mallard Ducks (Anas platyrhynchos) in South Korea

Abstract

The subtype H6N6 has been identified worldwide following the increasing frequency of avian influenza viruses (AIVs). These AIVs also have the ability to bind to human-like receptors, thereby increasing the risk of animal-human transmission. In September 2019, an H6N6 avian influenza virus—KNU2019-48 (A/Mallard (Anas platyrhynchos)/South Korea/KNU 2019-48/2019(H6N6))—was isolated from Anas platyrhynchos in South Korea. Phylogenetic analysis results revealed that the hemagglutinin (HA) gene of this strain belongs to the Korean lineage, whereas the neuraminidase (NA) and polymerase basic protein 1 (PB1) genes belong to the Chinese lineage. Outstanding internal proteins such as PB2, polymerase acidic protein, nucleoprotein, matrix protein, and non-structural protein belong to the Vietnamese lineage. Additionally, a monobasic amino acid (PRIETR↓GLF) at the HA cleavage site; non-deletion of the stalk region (residue 59–69) in the NA gene; and E627 in the PB2 gene indicate that the KNU2019-48 isolate is a typical low-pathogenic avian influenza (LPAI) virus. The nucleotide sequence similarity analysis of HA revealed that the highest homology (97.18%) of this isolate is to that of A/duck/Jiangxi/01.14 NCJD125-P/2015(H6N6), and the amino acid sequence of NA (97.38%) is closely related to that of A/duck/Fujian/10.11_FZHX1045-C/2016 (H6N6). An in vitro analysis of the KNU2019-48 virus shows a virus titer of not more than 2.8 Log10 TCID 50/mL until 72 h post-infection, whereas in the lungs, the virus is detected at 3 dpi (days post-infection). The isolated KNU2019-48 (H6N6) strain is the first reported AIV in Korea, and the H6 subtype virus has co-circulated in China, Vietnam, and Korea for half a decade. Overall, our study demonstrates that Korean H6N6 strain PB1-S375N, PA-A404S, and S409N mutations are infectious in humans and might contribute to the enhanced pathogenicity of this strain. Therefore, we emphasize the importance of continuous and intensive surveillance of the H6N6 virus not only in Korea but also worldwide.

Genetic characterization and evolution of H6N6 subtype avian influenza viruses

“…In September 2019, an H6N6-AIV (KNU2019-48) was isolated in South Korea, which was the first reported AIV in Korea. In fact, the H6-subtype viruses had co-circulated in China, Vietnam, and Korea for half a decade ( Durairaj et al, 2022 ). Therefore, the monitoring and prevention of the H6-subtype AIVs, especially H6N6 subtype, should not be taken lightly.…”

Evolution and Reassortment of H6 Subtype Avian Influenza Viruses

“…Meanwhile, a study found that PB2-E627K enhanced the replication of the H6N1 virus in mammals [ 41 ]. Other mutations: Kaliannan Durairal et al identified PB1 (S375N/T) and PA (A404S, S409N) human host markers in the genes of KNU2019-48 (H6N6) isolates [ 42 ], which may contribute to the adaptation of H6 subtype AIVs to mammals. Furthermore, although amino acid mutations that have been previously shown to be associated with receptor preferences for other AIV subtypes have been identified in H6 AIV, where a small number of results have been based only on currently published AIV human host markers and have not been validated in animal studies, so further investigation is needed to clarify the relationship between these mutations and H6 AIV human receptor preferences.…”

“…However, in recent years, M2-S31N and M2-V27I mutations have been found in some strains that are resistant to amantadine [ 25 , 45 ]. Mutant residues E119V, H275Y, R293K and N295S were found in the H6 AIV NA origin gene of Korean Anas platyrhynchos , indicating resistance to oseltamivir [ 42 ]. However, several H6 subtype AIV isolates have also been found to be susceptible to oseltamivir [ 46 ], so enhanced monitoring of the resistance of H6 subtype AIV against NA inhibitor drugs is needed to precisely select targeted antiviral drugs for efficient treatment.…”