SRP172887 PRJNA508785 Plasticity of amino acid 226 in the receptor-binding site of an H9 subtype influenza A virus. Influenza A viruses (IAVs) remain a significant public health threat causing more than 300,000 hospitalizations in the United states during 2015-2016 season alone. While only a few IAVs of avian origin have been associated with human infections, the ability of these viruses to cause zoonotic infections further increases the public health risk of influenza. Of these, H9N2 viruses in Asia are of particular importance as they have contributed internal gene segments to other emerging zoonotic IAVs. Notably, recent H9N2 viruses have acquired molecular markers that allow for a transition from “avian-like” to “human-like” sialic acid (SA) receptor recognition via a single amino acid change at position 226 (H3 numbering), from glutamine (Q226) to leucine (L226), within the HA's receptor-binding site (RBS). We sought to determine the plasticity of the amino acid 226 and the biological effects of alternative amino acids at this position on variant viruses. We created a library of viruses with the potential of having any of the 20 amino acids at position 226 on a prototypic H9 HA subtype IAV. We isolated H9 variant viruses that carried natural and alternative amino acids at position 226, some of which have yet to be identified in any reported influenza subtype. Interestingly, some identified amino acids conferred a broader receptor-binding profile to resulting viruses. Furthermore, in vivo fitness studies revealed some variant amino acids to confer an in vivo replication advantage. This study shows the plasticity of position 226 on the HA of H9 influenza viruses and the resulting effect of single amino acid changes on the phenotype of variants in vivo and in vitro.