Identification of amino acids in H9N2 influenza virus neuraminidase that are critical for the binding of two mouse monoclonal antibodies

• Generation and characterization of two mAbs binding different epitopes in the NA of a Eurasian H9N2 virus.
• Identify three critical amino acid positions in antigenic sites/epitopes in NA of H9N2 virus through characterizing the mAb escape mutants.
• Natural mutations at these three positions have occurred in the field.

Neuraminidase (NA) is one of the major glycoproteins on the surface of influenza virus. It cleaves the linkage between haemagglutinin and cell surface receptors, and thus helps the release and spread of influenza virus. Despite the importance of H9N2 virus in influenza pandemic preparedness, the antigenic characteristics of its surface glycoproteins, especially NA, remains to be investigated. In the present study, we characterized two monoclonal antibodies (mAbs), 1D1 and 1G8, which are against the NA of an H9N2 virus A/Chicken/Jiangsu/X1/2004 (X1). We examined the inhibitory effect of these mAbs in two NA inhibition assays: enzyme-linked lectin assay (ELLA) and 2′-(4-methylumbelliferyl)-a-d-N-acetylneuraminic acid (Mu-NANA) assay. In ELLA, which uses a large molecule fetuin (molecular weight: 50 kd) as substrate, both antibodies effectively inhibit the NA activity of X1 virus. However, in Mu-NANA assay, which uses the small molecule Mu-NANA (molecular weight: 489 d) as substrate, antibody 1G8 inhibits the NA activity, while antibody 1D1 does not. Three amino acid mutations, at positions 198, 199 and 338, respectively, were detected in the NA of escape mutants of X1 virus selected with the two antibodies. Natural mutations at these three positions have occurred, indicative of immune pressure on H9N2 virus in the field. Our findings lay a basis for detailed investigation on the antigenic structure of H9N2 virus NA, which may be helpful for developing NA-based antibody reagents as well as vaccines.