Phenotypic and Genetic Characterization of Avian Influenza H5N2 Viruses with Intra- and Inter-Duck Variations in Taiwan

Human infections with avian influenza viruses (AIVs) have frequently raised global concerns of emerging, interspecies-transmissible viruses with pandemic potential. Waterfowl, the predominant reservoir of influenza viruses in nature, harbor precursors of different genetic lineages that have contributed to novel pandemic influenza viruses in the past.Two duck influenza H5N2 viruses, DV518 and DV413, isolated through virological surveillance at a live-poultry market in Taiwan, showed phylogenetic relatedness but exhibited different replication capabilities in mammalian Madin-Darby Canine Kidney (MDCK) cells. This study characterizes the replication properties of the two duck H5N2 viruses and the determinants involved.The DV518 virus replicated more efficiently than DV413 in both MDCK and chicken DF1 cells. Interestingly, the infection of MDCK cells by DV518 formed heterogeneous plaques with great differences in size [large (L) and small (S)], and the two viral strains (p518-L and p518-S) obtained from plaque purification exhibited distinguishable replication kinetics in MDCK cells. Nonetheless, both plaque-purified DV518 strains still maintained their growth advantages over the plaque-purified p413 strain. Moreover, three amino acid substitutions in PA (P224S), PB2 (E72D), and M1 (A128T) were identified in intra-duck variations (p518-L vs p518-S), whereas other changes in HA (N170D), NA (I56T), and NP (Y289H) were present in inter-duck variations (DV518 vs DV413). Both p518-L and p518-S strains had the N170D substitution in HA, which might be related to their greater binding to MDCK cells. Additionally, polymerase activity assays on 293T cells demonstrated the role of vRNP in modulating the replication capability of the duck p518-L viruses in mammalian cells.These results demonstrate that intra-host phenotypic variation occurs even within an individual duck. In view of recent human infections by low pathogenic AIVs, this study suggests possible determinants involved in the stepwise selection of virus variants from the duck influenza virus population which may facilitate inter-species transmission