Host origin is a determinant of coevolution between gene segments of avian H9 influenza viruses.

Several emerging influenza viruses, including H7N9 and H5N6 viruses, trace their origins to reassortment with H9N2 viruses that contributed internal gene segments. However, the evolutionary constraints governing the reassortment of H9N2 viruses remain unknown. In seasonal human influenza A viruses, gene segments coevolve at both the nucleotide and amino acid levels. Here, we demonstrate that evolutionary relationships between gene segments, including polymerase subunits in human H3N2 viruses, differ from avian H9 viruses. Avian H9 viruses were characterized by little coevolution between gene segments or between polymerase subunits. Strikingly, protein trees built from avian H9 polymerase subunits diverge despite known functional constraints on polymerase evolution. The evolutionary divergence observed between gene segments of avian H9 viruses was consistent across isolates from different continents, suggesting that coevolution between H9 gene segments is not dependent on regionally defined avian lineages. Instead, coevolution between gene segments was only found in H9 viruses that crossed the species barrier into humans. Our study reveals the role of the host in the coevolution of influenza gene segments and suggests that high reassortment potential in avian species may be a consequence of evolutionary flexibility between gene segments.IMPORTANCEEmerging pandemic influenza viruses can contain a combination of viral gene segments from avian, swine, and/or human species through the process of reassortment. H9 viruses have been important gene segment donors to several avian viruses of concern, including H5N1 and H7N9. In this work, we found that H9 gene segments and proteins do not have constrained evolutionary relationships typical of human seasonal influenza viruses, suggesting a flexibility that could allow for greater reassortment potential. However, we also found that this observation was dependent upon the host species source, with greater evolutionary constraints in H9 viruses from human sources. Understanding such constraints that underlie viral reassortment is critical to predicting future viruses that may be feasible in nature and have pandemic potential.