Variability in H9N2 haemagglutinin receptor-binding preference and the pH of fusion

Abstract

H9N2 avian influenza viruses are primarily a disease of poultry; however, they occasionally infect humans and are considered a potential pandemic threat. Little work has been performed to assess the intrinsic biochemical properties related to zoonotic potential of H9N2 viruses. The objective of this study, therefore, was to investigate H9N2 haemagglutinins (HAs) using two well-known correlates for human adaption: receptor-binding avidity and pH of fusion. Receptor binding was characterized using bio-layer interferometry to measure virus binding to human and avian-like receptor analogues and the pH of fusion was assayed by syncytium formation in virus-infected cells at different pHs. We characterized contemporary H9N2 viruses of the zoonotic G1 lineage, as well as representative viruses of the zoonotic BJ94 lineage. We found that most contemporary H9N2 viruses show a preference for sulphated avian-like receptor analogues. However, the ‘Eastern' G1 H9N2 viruses displayed a consistent preference in binding to a human-like receptor analogue. We demonstrate that the presence of leucine at position 226 of the HA receptor-binding site correlated poorly with the ability to bind a human-like sialic acid receptor. H9N2 HAs also display variability in their pH of fusion, ranging between pH 5.4 and 5.85 which is similar to that of the first wave of human H1N1pdm09 viruses but lower than the pH of fusion seen in zoonotic H5N1 and H7N9 viruses. Our results suggest possible molecular mechanisms that may underlie the relatively high prevalence of human zoonotic infection by particular H9N2 virus lineages.

Genetic determinants of receptor-binding preference and zoonotic potential of H9N2 avian influenza viruses

“…Additionally, Q226 favoured 3SLN binding as can be seen by the mutant UDL1/08 L226Q and the difference in avidity between Em/R66 E190A/Q226L and E190A alone (Figure 2C, F – blue and brown lines). In the background of Em/R66, Q226L showed a large increase in 6SLN binding (Figure 2E – mint line), however a matching reduction in 6SLN binding by UDL1/08 L226Q was not seen (Figure 2B), indicating this is probably dependent on the context of the other amino acids in the H9 HA RBS as we had previously predicted [21].…”

“…The virus A/chicken/Pakistan/UDL-01/2008 (UDL1/08) displays high binding avidity to 3SLN(6su), but not 3SLN (avian-like), with residual binding to the human-like receptor 6SLN (Figure 1A), similar to the majority of contemporary H9N2 viruses [20–22]. The virus A/chicken/Emirates/R66/2002 (Em/R66) binds to both 3SLN(6su) and 3SLN but has no detectable binding to 6SLN (Figure 1B), similar to conventional avian-adapted H5N1 and H7 viruses [21, 23]. Finally, A/Hong Kong/33982/2009 (HK/33982) binds to all three receptor analogues, but with an appreciable preference for 6SLN, similar to early human pandemic H3N2 viruses and zoonotic H7N9 viruses (Figure 1C) [21, 24].…”

“…The virus A/chicken/Emirates/R66/2002 (Em/R66) binds to both 3SLN(6su) and 3SLN but has no detectable binding to 6SLN (Figure 1B), similar to conventional avian-adapted H5N1 and H7 viruses [21, 23]. Finally, A/Hong Kong/33982/2009 (HK/33982) binds to all three receptor analogues, but with an appreciable preference for 6SLN, similar to early human pandemic H3N2 viruses and zoonotic H7N9 viruses (Figure 1C) [21, 24]. To test the molecular basis of these different receptor preferences, libraries of individual or multiple reciprocal mutants were generated between these viruses with a particular focus on positions 190, 226 and 227, as well as several other nearby RBS residues.…”

“…In a previous study, we tested the receptor-binding preference of several H9N2 viruses and described notable receptor preference variability amongst circulating H9N2 viruses which we hypothesised was due to amino acid variability at residues 190, 226 and 227 [21]. In this study we take three H9N2 viruses that are representative of different receptor-binding profiles, including a virus isolated from a human with a natural preference for α2,6-linked SA, and generate recombinant virus libraries with HA amino acid substitutions that represent reciprocal changes between the progenitor H9N2 viruses.…”

The molecular basis of antigenic variation among A(H9N2) avian influenza viruses

“…In this study substitutions with the largest impact of antigenicity were associated with two distinct mechanisms of immune evasion. Of the eight most significant mutants, four (T127N, L150S, T188N and D189N) possessed additional glycosylation sites and four (T145I, L216Q, I217Q, and I249V) possessed substitutions either known to influence receptorbinding avidity in H9N2 viruses or determined to do so in this study through analysis of reciprocal mutants, or both 9,[31][32][33]35 . These observations demonstrate the potential of alternative mechanisms, other than reduction of antibody recognition by altering epitope biophysics, in facilitating immune escape and determining antigenic diversity of H9N2 viruses.…”