title: Identification of amino acids in H9N2 influenza virus neuraminidase that are critical for the binding of two mouse monoclonal antibodies.
abstract: 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: 50kd) 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.
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Zhimin
authors.initials: Z
authors.lastname: Wan
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Ye
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Sang
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Hongxia
authors.initials: H
authors.lastname: Shao
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Qian
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Wenjie
authors.initials: W
authors.lastname: Jin
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China. Electronic address: aijian@yzu.edu.cn.
authors.forename: Aijian
authors.initials: A
authors.lastname: Qin
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China. Electronic address: hqwan129@yahoo.com.
authors.forename: Hongquan
authors.initials: H
authors.lastname: Wan
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000914:Antibodies, Viral / Q000378:metabolism*; D000939:Epitopes / Q000378:metabolism; D053127:Influenza A Virus, H9N2 Subtype / Q000235:genetics*; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D009439:Neuraminidase / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000939:Epitopes; D009439:Neuraminidase
keywords: Amino acid; Escape mutant; H9N2 virus; Neuraminidase