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Egypt is a hotspot for H5- and H9-subtype avian influenza A virus (AIV) infections and co-infections in poultry by both subtypes have been frequently reported. However, natural genetic reassortment of these subtypes has not been reported yet. Here, we evaluated the genetic compatibility and replication efficiency of reassortants between recent isolates of an Egyptian H5N1 and a H9N2 AIV (H5N1 EGY and H9N2 EGY ). All internal viral proteins-encoding segments of the contemporaneous G1-like H9N2 EGY , expressed individually and in combination in the genetic background of H5N1 EGY , were genetically compatible with the other H5N1 EGY segments. At 37 °C the replication efficiencies of H5N1 EGY reassortants expressing the H9N2 EGY polymerase subunits PB2 and PA (H5N1 PB2-H9N2EGY, H5N1 PA-H9N2EGY ) were higher than the wild-type H5N1 EGY in Madin-Darby canine kidney (MDCK-II) cells. This could not be correlated to viral polymerase activity as this was found to be improved for H5N1 PB2-H9N2EGY, but reduced for H5N1 PA-H9N2EGY . At 33 °C and 39 °C, H5N1 PB2-H9N2EGY and H5N1 PA-H9N2EGY replicated to higher levels than the wild-type H5N1 EGY in human Calu-3 and A549 cell lines. Nevertheless, in BALB/c mice both reassortants caused reduced mortality compared to the wild-type H5N1 EGY . Genetic analysis of the polymerase-encoding segments revealed that the PA H9N2EGY and PB2 H9N2EGY encode for a distinct uncharacterized mammalian-like variation (367K) and a well-known mammalian signature (591K), respectively. Introducing the single substitution 367K into the PA of H5N1 EGY enabled the mutant virus H5N1 PA-R367K to replicate more efficiently at 37 °C in primary human bronchial epithelial (NHBE) cells and also in A549 and Calu-3 cells at 33 °C and 39 °C. Furthermore, H5N1 PA-R367K caused higher mortality in BALB/c mice. These findings demonstrate that H5N1 (Clade 2.2.1.2) reassortants carrying internal proteins-encoding segments of G1-like H9N2 viruses can emerge and may gain improved replication fitness. Thereby such H5N1/H9N2 reassortants could augment the zoonotic potential of H5N1 viruses, especially by acquiring unique mammalian-like aa signatures.

viruses

PA from a Recent H9N2 (G1-Like) Avian Influenza A Virus (AIV) Strain Carrying Lysine 367 Confers Altered Replication Efficiency and Pathogenicity to Contemporaneous H5N1 in Mammalian Systems