Identification of key residues involved in the neuraminidase antigenic variation of H9N2 influenza virus

Abstract

Influenza A H9N2 virus causes economic loss to the poultry industry and has likely contributed to the genesis of H5N1 and H7N9 viruses. The neuraminidase (NA) of H9N2 virus, like haemagglutinin, is under antibody selective pressure and may undergo antigenic change; however, its antigenic structure remains to be elucidated. In this study, we used monoclonal antibodies (mAbs) to probe the H9N2 viral NA residues that are key for antibody binding/inhibition. These mAbs fell into three groups based on their binding/inhibition of the NA of H9N2 viruses isolated during 1999-2019: group I only bounded the NA of the early 2000 H9N2 viruses but possessed no neutralizing ability, group II bounded and inhibited the NA of H9N2 viruses isolated before 2012, and group III reacted with most or all tested H9N2 viruses. We showed that NA residue 356 is key for the recognition by group I mAbs, residues 344, 368, 369, and 400 are key for the binding/inhibition of NA by group II antibodies, whereas residues 248, 253, and the 125/296 combination are key for neutralizing antibodies in group III. Our findings highlighted NA antigenic change of the circulating H9N2 viruses, and provided data for a more complete picture of the antigenic structure of H9N2 viral NA.

Keywords: H9N2; Influenza virus; antigenic change; key residues; monoclonal antibodies; neuraminidase.

Figure 1.

Phylogenetic analysis of NA genes of 19 H9N2 field strains isolated from 1999 to 2019. Green dots represent vaccine strains. Red dot represents XXM H9N2 virus and black dots represent the other 18 field strains used in this study. The phylogenetic tree was constructed with MEGA X in neighbour-joining method and 1000 boot-strap replicates.

Figure 2.

The impact of N356D mutation on reactivity of group I mAbs. (A) Amino acid sequence alignment of NA sequences (342–379, N2 numbering) of 19 field strains was analysed with MEGA X. The D356 in NA of A/Chicken/Jiangsu/C1/2002 (H9N2) virus was marked in dark blue. (B) Plasmids of WT NA gene (pCAGGS-NA (N356)) and mutant NA gene (pCAGGS-NA (D356)) were transfected into COS-1 cells and examined with 4 mAbs in IFA.

Figure 3.

NI ability of group II mAbs to escape mutants and WT XXM H9N2 virus. NA inhibitory effect of mAbs A2A3, A4C6, A5D12 and B4D6 on escape mutants was measured in ELLA. A to D represent results of A2A3, A4C6, A5D12 and B4D6.

Figure 4.

NI ability of group III mAbs A3C9 and A6A7 on escape mutants and WT XXM H9N2 virus. NA inhibitory effect of mAbs A3C9 and A6A7 on escape mutants was measured in ELLA. A represents result of A3C9 and B represents result of A6A7.

Figure 5.

Locations of the 9 identified key residues on NA. The NA head structure of N2 subtype (PDB:1NN2) was analysed with Swiss PDB Deep-viewer. The 9 residues were labelled with various colours in an NA monomer. The structure is shown in the top view (A) and bottom view (B).