Peacock TP*, Sealy JE*, Harvey WT*, Benton DJ, Reeve R & Iqbal M (2021) Journal of Virology 95:e01651-20 doi: 10.1128/JVI.01651-20  PDF *Contributed equally

Receptor recognition and binding is the first step of viral infection and a key determinant of host specificity. The inability of avian influenza viruses to effectively bind human-like sialylated receptors is a major impediment to their efficient transmission in humans and pandemic capacity. Influenza H9N2 viruses are endemic in poultry across Asia and parts of Africa, where they occasionally infect humans and are therefore considered viruses with zoonotic potential. We previously described H9N2 viruses, including several isolated from human zoonotic cases, which showed a preference for human-like receptors. Here, we take a mutagenesis approach, making viruses with single or multiple substitutions in H9 hemagglutinin and testing binding to avian and human receptor analogues using biolayer interferometry. We determine the genetic basis of preferences for alternative avian receptors and for human-like receptors, describing amino acid motifs at positions 190, 226, and 227 that play a major role in determining receptor specificity, and several other residues such as 159, 188, 193, 196, 198, and 225 that play a smaller role. Furthermore, we show that changes at residues 135, 137, 147, 157, 158, 184, 188, and 192 can also modulate virus receptor avidity, and substitutions that increased or decreased the net positive charge around the hemagglutinin receptor-binding site show increases and decreases in avidity, respectively. The motifs we identify as increasing preference for the human-like receptor will help guide future H9N2 surveillance efforts and facilitate our understanding of the emergence of influenza viruses with increased zoonotic potential.