Flexibility In Vitro of Amino Acid 226 in the Receptor-Binding Site of an H9 Subtype Influenza A Virus and Its Effect In Vivo on Virus Replication, Tropism, and Transmission

Abstract

Influenza A viruses (IAVs) remain a significant public health threat, causing more than 300,000 hospitalizations in the United States during the 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 terminal sialic acid (SA) receptor recognition via a single amino acid change at position 226 (H3 numbering), from glutamine (Q226) to leucine (L226), within the hemagglutinin (HA) receptor-binding site (RBS). We sought to determine the plasticity of amino acid 226 and the biological effects of alternative amino acids 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 viruses that carried naturally occurring amino acids, variants found in other subtypes, and variants not found in any subtype at position 226. Fitness studies in quails revealed that some natural amino acids conferred anin vivoreplication advantage. This study shows the flexibility of position 226 of the HA of H9 influenza viruses and the resulting effect of single amino acid changes on the phenotype of variantsin vivoandin vitro.IMPORTANCEA single amino acid change at position 226 in the hemagglutinin (HA) from glutamine (Q) to leucine (L) has been shown to play a key role in receptor specificity switching in various influenza virus HA subtypes, including H9. We tested the flexibility of amino acid usage and determined the effects of such changes. The results reveal that amino acids other than L226 and Q226 are well tolerated and that some amino acids allow for the recognition of both avian and human influenza virus receptors in the absence of other changes. Our results can inform better avian influenza virus surveillance efforts as well as contribute to rational vaccine design and improve structural molecular dynamics algorithms.

H9 Influenza Viruses: An Emerging Challenge

“…The acquisition of HA L216 in H9N2 viruses is a sign of land-based poultry adaptation (Choi et al 2004;Li et al 2005;Xu et al 2007a), consistent with the observation that both SA α2,3Gal and SA α2,6Gal receptors are present in the respiratory tract of some of these species (Gambaryan et al 2002;Wan and Perez 2006;Guo et al 2007;Kimble et al 2010;Yu et al 2011b). Other amino acids at position 216 are tolerated and have been found in nature, but do not seem favored in poultry (Obadan et al 2019). Coincidentally, most Asian-origin poultry-adapted H9N2 strains can replicate in ferrets, although airborne transmission is typically inefficient (Wan et al 2008;Sorrell et al 2009;Kimble et al 2011Kimble et al , 2014Qiao et al 2012;Scotch et al 2014; SJCEIRS H9 Working Group 2013).…”

“…Noteworthy, the HA protein of most poultry-adapted H9N2 strains contain leucine at position 216 (L216, L226 in H3 HA numbering) within the receptor-binding site (RBS). L216 provides preferential binding to sialic acid (SA) linked to galactose by α2,6 linkage (SA α2,6Gal), which is typical of human-origin influenza viruses (Matrosovich et al 2001;Wan and Perez 2007;Obadan et al 2019). The second-most common amino acid signature at this position is glutamine Q216 (Q226 in H3 numbering) (Obadan et al 2019).…”

“…L216 provides preferential binding to sialic acid (SA) linked to galactose by α2,6 linkage (SA α2,6Gal), which is typical of human-origin influenza viruses (Matrosovich et al 2001;Wan and Perez 2007;Obadan et al 2019). The second-most common amino acid signature at this position is glutamine Q216 (Q226 in H3 numbering) (Obadan et al 2019). H9N2 strains with Q216 preferentially bind the α2,3 linkage SA receptors (SA α2,3Gal), more typical of avian-origin influenza viruses, but some show dual receptor specificity (Wan and Perez 2007;Obadan et al 2019).…”

“…The second-most common amino acid signature at this position is glutamine Q216 (Q226 in H3 numbering) (Obadan et al 2019). H9N2 strains with Q216 preferentially bind the α2,3 linkage SA receptors (SA α2,3Gal), more typical of avian-origin influenza viruses, but some show dual receptor specificity (Wan and Perez 2007;Obadan et al 2019).…”

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

“…Here we perform a comprehensive, quantitative analysis often testing changes at residues in multiple different virus backbones and in multiple combinations to investigate strain-specific and compensatory effects. For example, residue 226 has been shown several times in an older G1-Eastern lineage virus to increase binding to human-like receptors [17, 32], with a similar effect when we investigate a contemporary virus of the same lineage (i.e. HK/33982), however in a G1-Western backbone (i.e.…”