Characterization and Evolutionary Analysis of a Novel H3N2 Influenza A Virus Glycosylation Motif in Southern China

Abstract

An influenza A (H3N2) virus epidemic occurred in China in 2017 and the causative strain failed to bind red blood cells (RBCs), which may affect receptor binding and antibody recognition. The objective of this study was to analyze the genetic characteristics and glycosylation changes of this novel H3N2 strain. We directly sequenced the hemagglutinin (HA) genes of H3N2 clinical specimens collected from patients with acute respiratory tract infection during 2017 in Guangdong, China. We aligned these sequences with those of A/Hong Kong/1/1968 (H3N2) and A/Brisbane/10/2007 (H3N2). Glycosylation changes were analyzed by C18 Chip-Q-TOF-MS. A/China/LZP/2017 (H3N2) was negative by HA assay, but was positive by quantitative real-time Polymerase Chain Reaction (qPCR) and direct immunofluorescence assay (DFA). We found that the HA1 residue 160T of A/China/LZP/2017 (H3N2) could block virus binding to receptors on RBCs. Furthermore, the ASN (N)-X-Thr (T) motif at HA1 residues 158-160, encoding a glycosylation site as shown by C18 Chip-Q-TOF-MS, predominated worldwide and played a critical role in RBC receptor binding. Ten glycoforms at HA1 residue 158 were identified [4_3_1_0, 5_6_0_1, 3_3_0_1, 4_4_3_0, 6_7_0_0 (SO 3), 3_6_2_0, 4_3_1_2 (SO 3), 7_5_2_0 (SO 3), 3_6_2_1 (SO 3), and 3_7_0_2]. Glycosylation changes at HA1 residues 158-160 of a circulating influenza A (H3N2) virus in Guangdong, China, in 2017 blocked binding to RBC receptors. Changes to these HA1 residues may have reduced protective antibody responses as well. Understanding these critical epitopes is important for selecting vaccine strains.

Antigenic and virological properties of an H3N2 variant that continues to dominate the 2021–22 Northern Hemisphere influenza season

“…Recombinant HA proteins engineered to possess I160 (2a2 and 2a1 –glycan) also migrated with a lower molecular weight compared to HAs with T160 (2a1 and 2a2 +glycan) ( Figure S1 B), consistent with a loss of a glycosylation site with the T160I substitution. Influenza virus entry into human cells is mediated by HA binding to α2,6-linked sialoglycans ( Thompson and Paulson, 2021 ), and previous studies have demonstrated that the 158–160 HA glycosylation site impacts HA sialic acid binding specificity ( Zeng et al., 2020 ). To determine if the 2a2 H3N2 HA has altered receptor specificity, we measured HA binding to a panel of α2,3- and α2,6-linked sialoside glycans ( Figures 1 C and Table S1 ).…”

mRNA vaccines encoding computationally optimized hemagglutinin elicit protective antibodies against future antigenically drifted H1N1 and H3N2 influenza viruses isolated between 2018-2020

“…Viruses from clade 3c.3a typically lack this glycosylation at site 160, exposing antigenic epitopes which may be covered up in 3c.2a viruses that have this glycosylation ( 5 , 66 ). The glycosylation at site 160 impacts the HA sialic acid binding specificity, which plays a role in the HA protein binding to host cell receptors ( 65 , 67 ). NG2 and Kan/17 HA proteins also differ by 11 amino acids and 4 of those residues reside in antigenic sites of the H3 HA protein.…”

Insect Cell-Based Quadrivalent Seasonal Influenza Virus-like Particles Vaccine Elicits Potent Immune Responses in Mice

“…1 VLPs were harvested at 72 h post infection of the High Five cells. 2 Hemagglutination assay is not suitable for quantifying HA protein of recent influenza H3N2 viruses [21][22][23]. 3 HA concentration was not determined because samples were aggregated.…”

“… 1 Estimated HA yield (mg/L) was based on the HA content (µg/mL) and volume (1.2 mL) after purification using 40 mL working volume and calculated by converting working volume from 40 mL to one liter. 2 Hemagglutination assay is not suitable for quantifying HA protein of recent influenza H3N2 viruses [ 21 , 22 , 23 ]. …”