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 (SO₃), 3_6_2_0, 4_3_1_2 (SO₃), 7_5_2_0 (SO₃), 3_6_2_1 (SO₃), 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.

Keywords: A(H3N2); antibody recognition; glycoforms; glycosylation possessing; receptor binding.

FIGURE 1

Immunofluorescence assay for detecting H3N2 clinical specimens. The nucleoproteins (NPs) of H3N2 viruses were determined by the D3 Ultra DFA Respiratory Virus Screen and ID Kit. H3N2 strains (LZP, GMU03) and B10- or HK4801-infected cells were fixed in 4% paraformaldehyde at 37°C for 30 min. FITC-labeled mouse anti-NP antibody was used to detect nucleoprotein. The cells were observed using a fluorescence microscope.

FIGURE 2

Hemagglutination titers of H3N2 recombinant viruses. To figure out whether HA protein of LZP led to unable to bind the RBCs, the seven segments of LZP (NA, PB1, PB2, PA, NP, M, NS) were used as a backbone to rescue recombinant viruses (LZP-B10, LZP-HK4801, LZP-GMU03) bearing the HA genes of B10, HK4801, and GMU03, respectively. The recombinant viruses and LZP were determined by HA assay. A: PBS; B: LZP; C: LZP-GMU03; D: LZP-B10; E: LZP-HK4801.

FIGURE 3

Prevalence of T and K residues at HA1 position 160 in the world since 1968. The sequences of 62330 clinical strains worldwide from 1968 to 2019 were collected from GISAID. The T and K residues at HA1 position 160 were analyzed. The y-axis on the left: the number of HA1-160T and HA1-160K; The y-axis on the right: the percentage of HA1-160T and HA1-160K.

FIGURE 4

Hemagglutination titers of H3N2 mutant strains. To distinguish whether the possessing of glycosylation motif site at position 158 failed to bind RBCs, recombinant virus containing HA1-160K, HA1-158K, and LZP were determined by HA assay. A: PBS; B: LZP; C: T160K; D: N158K.

FIGURE 5

Worldwide prevalence of the glycosylation motif site at position 158 from 1968 to 2019. The sequences of 62,330 clinical strains worldwide from 1968 to 2019 were collected from GISAID. The substitutes at position 158–160 were analyzed.

FIGURE 6

Prevalence of the N-X-T/K motif in Guangdong, China from 2012 to 2019. The sequences of 106 clinical strains in Guangdong, China from 2012 to 2019 were collected from GISAID. The substitutes N-X-T/K at position 158–160 were analyzed. The y-axis on the left: the number of the substitutes N-X-T and N-X-K at HA1 residues 158–160; The y-axis on the right: the percentage of the substitutes N-X-T and N-X-K at HA1 residues 158–160.

FIGURE 7

Overlaid extracted compound chromatograms (ECCs) of identified HA1 glycopeptides containing the glycosylation site at position 158. HA1 proteins extracted from influenza were digested with chymotrypsin at 25°C for 16 h, followed by profiling the glycopeptides by C18 chip-Q-TOF-MS. The glycoforms at position 158 were then identified based on high-resolution MS data. (A): 4_3_1_0, (B): 3_6_2_0, (C): 5_6_0_1, (D): 4_3_1_2 (SO3), (E): 3_3_0_1, (F): 7_5_2_0 (SO3), (G): 4_4_3_0, (H): 3_6_2_1 (SO3), (I): 6_7_0_0 (SO3), (J): 3_7_0_2.