Amino Acids in Hemagglutinin Antigenic Site B Determine Antigenic and Receptor Binding Differences between A(H3N2)v and Ancestral Seasonal H3N2 Influenza Viruses

Abstract

Influenza A H3N2 variant [A(H3N2)v] viruses, which have caused human infections in the UnitedStates in recent years, originated from human seasonal H3N2 viruses that were introduced into North American swine in the mid-1990s, but they are antigenically distinct from both the ancestral and current circulating H3N2 strains. A reference A(H3N2)v virus, A/Minnesota/11/2010 (MN/10), and a seasonal H3N2 strain, A/Beijing/32/1992 (BJ/92), were chosen to determine the molecular basis for the antigenic difference between A(H3N2)v and the ancestral viruses. Viruses containing wild-type and mutant MN/10 or BJ/92 hemagglutinins (HAs) were constructed and probed for reactivity with ferret antisera against MN/10 and BJ/92 in hemagglutination inhibition assays. Among the amino acids that differ between the MN/10 and BJ/92 HAs, those in antigenic site A had little impact on the antigenic phenotype. Within antigenic site B, mutations at residues 156, 158, 189, and 193 of MN/10 HA to those in BJ/92 switched the MN/10 antigenic phenotype to that of BJ/ 92. Mutations at residues 156, 157, 158, 189, and 193 of BJ/92 HA to amino acids present in MN/10 were necessary for BJ/92 to become antigenically similar to MN/ 10. The HA amino acid substitutions responsible for switching the antigenic phenotype also impacted HA binding to sialyl receptors that are usually present in the human respiratory tract. Our study demonstrates that antigenic site B residues play a critical role in determining both the unique antigenic phenotype and receptor specificity of A(H3N2)v viruses, a finding that may facilitate future surveillance and risk assessment of novel influenza viruses. IMPORTANCE Influenza A H3N2 variant [A(H3N2)v] viruses have caused hundreds of human infections in multiple states in the UnitedStates since 2009. Most cases have been children who had contact with swine in agricultural fairs. These viruses originated from human seasonal H3N2 viruses that were introduced into the U.S. swine population in the mid-1990s, but they are different from both these ancestral viruses and current circulating human seasonal H3N2 strains in terms of their antigenic characteristics as measured by hemagglutination inhibition (HI) assay. In this study, we identified amino acids in antigenic site B of the surface glycoprotein hemagglutinin (HA) that explain the antigenic difference between A(H3N2)v and the ancestral H3N2 strains. These amino acid mutations also alter binding to minor human-type glycans, suggesting that host adaptation may contribute to the selection of antigenically distinct H3N2 variants which pose a threat to public health.

Pathogenesis and Transmission of Genetically Diverse Swine-Origin H3N2 Variant Influenza A Viruses from Multiple Lineages Isolated in the United States, 2011–2016

“…Like their human influenza virus precursors, H3N2v viruses possess key residues (190D, 226V/I, and 228S) at the receptor binding site (RBS) responsible for binding to ␣2,6-linked sialic acids, the preferred receptor for human influenza viruses, suggesting that H3N2v viruses have an inherent property to target the human respiratory epithelium. However, compared to human seasonal viruses, most H3N2v viruses have HA substitutions at positions (e.g., positions 156, 157, 158, 189, and 193) adjacent to the RBS, which have recently been shown to affect binding to selected human glycan analogs (42). Whether these substitutions affect H3N2v virus binding or replication in human respiratory tissues in vivo needs further investigation.…”

The use of plant lectins to regulate H1N1 influenza A virus receptor binding activity

“…The affinity of each virus for biotinylated 3′- and 6′-sialylglycopolymers was measured in a direct binding assay as described previously [ 12 , 17 ]. Briefly, plates were pre-coated with each virus at 4°C for 16 h, followed by washing with 0.05% Tween 20 in PBS (PBS-T).…”

Comparison of the Efficacy of N9 Neuraminidase-Specific Monoclonal Antibodies against Influenza A(H7N9) Virus Infection

“…Selected hybridomas were cultured in a CELLine device (BD Biosciences, San Jose, CA), and MAbs were purified using protein G columns (GE Healthcare, Uppsala, Sweden). The reactivity of MAbs with N9 was confirmed by ELISA, using NA that was transiently expressed on 293T cells by transfecting cells with pCAGGS plasmid containing the NA gene of AH/13 or G70C (23,34,50) or purified H6N9 GD/16 virus. The residues on N9 that are critical for MAb binding were identified by selecting antibody escape mutants of H6N9 AH/13 virus and sequencing the NA gene of the resultant viruses (as described in the following paragraph) or by performing ELISAs with plasmid-transfected 293T cells (23) or purified parent H6N9 AH/13 virus and escape mutants.…”