Genetic Determinants of Receptor-Binding Preference and Zoonotic Potential of H9N2 Avian Influenza Viruses

Abstract

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, showing a preference for human-like receptors. Here we take a mutagenesis approach, making viruses with single or multiple substitutions in H9 haemagglutinin and test 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 changes at residues 135, 137, 147, 157, 158, 184, 188, and 192 can also modulate virus receptor avidity and that substitutions that increased or decreased the net positive charge around the haemagglutinin receptor-binding site show increases and decreases in avidity, respectively. The motifs we identify as increasing preference for the human-receptor will help guide future H9N2 surveillance efforts and facilitate our understanding of the emergence of influenza viruses with increased zoonotic potential. IMPORTANCE As of 2020, over 60 infections of humans by H9N2 influenza viruses have been recorded in countries where the virus is endemic. Avian-like cellular receptors are the primary target for these viruses. However, given that human infections have been detected on an almost monthly basis since 2015, there may be a capacity for H9N2 viruses to evolve and gain the ability to target human-like cellular receptors. Here we identify molecular signatures that can cause viruses to bind human-like receptors, and we identify the molecular basis for the distinctive preference for sulphated receptors displayed by the majority of recent H9N2 viruses. This work will help guide future surveillance by providing markers that signify the emergence of viruses with enhanced zoonotic potential as well as improving understanding of the basis of influenza virus receptor-binding.

Coinfection of Chickens with H9N2 and H7N9 Avian Influenza Viruses Leads to Emergence of Reassortant H9N9 Virus with Increased Fitness for Poultry and a Zoonotic Potential

“…Compared to the H7N9 parental virus, the binding preference of the genotype 122 H9N9 virus was comparable for the 6SLN but weaker for 3SLN sialoglycans, but the reassortant H9N9 again showed its increased binding toward the 3SLN(6-su) receptor analogue. Viruses with preferable binding toward 3SLN(6-su) may have an increased propensity for circulation in terrestrial poultry ( 77 , 78 ), and this observation was reflected in the successful generation and transmission of these H9N9 genotypes in chickens in our in vivo experiment. Previous analysis of N9 NA showed that hemadsorption sites could be responsible for increasing the overall avidity of the virus toward the sialoglycans ( 79 , 80 ).…”

“…The results suggested that the reassortant H9N9 virus has a relatively more acid stable HA, similar avidity for human-like (6SLN) and less avidity for avian-like (3SLN) compared to Anhui/13 H7N9 virus. Further, the H9N9 virus has strongest binding toward 3SLN(6-su) receptors, thereby maintaining adaptation of such viruses for poultry ( 78 ) while also enabling additional zoonotic potential ( 83 , 84 ).…”

Identification of Hemagglutinin Mutations Caused by Neuraminidase Antibody Pressure

“…However, mutations in the HA head and RBS variations would help virus evade these antibodies ( 21 ). Single or combined mutations at positions 166, 168, 198, 201, and 234 were involved in switching the antigenic phenotype and receptor specificity of seasonal H3N2 virus and H9N2 IAV ( 34 – 36 ). The HA mutations identified in this study also decreased the HI titers of serum samples from challenged mice, H9-specific MAbs, and chicken serum, which indicated that HA mutations caused by NA antibody pressure can also help virus evade neutralizing antibodies against HA.…”

Airborne Transmission of Avian Origin H9N2 Influenza A Viruses in Mammals

“…The presence of Q227 in combination with either aspartic acid (D) or glutamic acid (E) at position 190 favors binding α2,6SAs receptors. Other changes such as A160D/N, Q156R, T205A, V245I, V216L, D208E, T212I, R1721 and S175N also enhance the human-like receptor binding in vitro [76,77]. The terminal sialic acid linkage is not the only factor that affects the binding of IAV HA to the host cell; other features such as the sialic acid structure (e.g., N-acetylneuraminic acid versus N-glycolylneuraminic acid) or length may also play a role (reviewed in [55]).…”