title: Characterization of cross-clade monoclonal antibodies against H5N1 highly pathogenic avian influenza virus and their application to the antigenic analysis of diverse H5 subtype viruses.
abstract: H5N1 highly pathogenic avian influenza viruses (HPAIVs) are a threat to both animal and public health and require specific and rapid detection for prompt disease control. We produced three neutralizing anti-hemagglutinin (HA) monoclonal antibodies (mAbs) using two clades (2.2 and 2.5) of the H5N1 HPAIV isolated in Japan. Blocking immunofluorescence tests showed that each mAb recognized different epitopes; 3B5.1 and 3B5.2 mAbs against the clade 2.5 virus showed cross-clade reactivity to all 26 strains from clades 1, 2.2, 2.3.2.1, 2.3.2.1a, b, c and 2.3.4, suggesting that the epitope(s) recognized are conserved. Conversely, the 1G5 mAb against the clade 2.2 virus showed reactivity to only clades 1, 2.3.4 and 2.5 strains. An analysis of escape mutants, and some clades of the H5N1 viruses recognized by 3B5.1 and 3B5.2 mAbs, suggested that the mAbs bind to an epitope, including amino acid residues at position 162 in the HA1 protein (R162 and K162). Unexpectedly, however, when five Eurasian-origin H5 low-pathogenic AIV (LPAIV) strains with R162 were examined (EA-nonGsGD clade) as well as two American-origin strains (Am-nonGsGD clade), the mAb recognized only EA-nonGsGD clade strains. The R162 and K162 residues in the HA1 protein were highly conserved among 36 of the 43 H5N1 clades reported, including clades 2.3.2.1a and 2.3.2.1c that are currently circulating in Asia, Africa and Europe. The amino acid residues (158-PTIKRSYNNTNQE-170) in the HA1 protein are probably an epitope responsible for the cross-clade reactivity of the mAbs, considering the epitopes reported elsewhere. The 3B5.1 and 3B5.2 mAbs may be useful for the specific detection of H5N1 HPAIVs circulating in the field.
authors.affiliation: Diagnostic Center for Animal Health and Food Safety, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Inada, Obihiro, Hokkaido, 080-8555, Japan.|Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, 73170, Thailand.
authors.forename: Dulyatad
authors.initials: D
authors.lastname: Gronsang
authors.affiliation: National Institute of Veterinary Research, Dong Da, Hanoi, 10000, Vietnam.
authors.forename: Anh N
authors.initials: AN
authors.lastname: Bui
authors.affiliation: National Institute of Veterinary Research, Dong Da, Hanoi, 10000, Vietnam.
authors.forename: Dai Q
authors.initials: DQ
authors.lastname: Trinh
authors.affiliation: National Institute of Veterinary Research, Dong Da, Hanoi, 10000, Vietnam.
authors.forename: Vuong N
authors.initials: VN
authors.lastname: Bui
authors.affiliation: National Institute of Veterinary Research, Dong Da, Hanoi, 10000, Vietnam.
authors.forename: Khong V
authors.initials: KV
authors.lastname: Nguyen
authors.affiliation: National Institute of Veterinary Research, Dong Da, Hanoi, 10000, Vietnam.|Sub-Department of Animal Health, Ha Dong, Hanoi, 10000, Vietnam.
authors.forename: Minh X
authors.initials: MX
authors.lastname: Can
authors.affiliation: Research and Education Center for Prevention of Global Infectious Diseases of Animals, Tokyo University of Agriculture and Technology, Tokyo, 183-8509, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Omatsu
authors.affiliation: Research and Education Center for Prevention of Global Infectious Diseases of Animals, Tokyo University of Agriculture and Technology, Tokyo, 183-8509, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Mizutani
authors.affiliation: Research and Education Center for Prevention of Global Infectious Diseases of Animals, Tokyo University of Agriculture and Technology, Tokyo, 183-8509, Japan.|Bioresources and Environmental Sciences, Ishikawa Prefectural University, Nonoichi, Ishikawa, 921-8836, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Nagai
authors.affiliation: Research and Education Center for Prevention of Global Infectious Diseases of Animals, Tokyo University of Agriculture and Technology, Tokyo, 183-8509, Japan.
authors.forename: Yukie
authors.initials: Y
authors.lastname: Katayama
authors.affiliation: Diagnostic Center for Animal Health and Food Safety, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Inada, Obihiro, Hokkaido, 080-8555, Japan.|Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, 73170, Thailand.
authors.forename: Rapeewan
authors.initials: R
authors.lastname: Thampaisarn
authors.affiliation: Diagnostic Center for Animal Health and Food Safety, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Inada, Obihiro, Hokkaido, 080-8555, Japan.
authors.forename: Haruko
authors.initials: H
authors.lastname: Ogawa
authors.affiliation: Diagnostic Center for Animal Health and Food Safety, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Inada, Obihiro, Hokkaido, 080-8555, Japan. imaiku@obihiro.ac.jp.
authors.forename: Kunitoshi
authors.identifier: http://orcid.org/0000-0003-2158-0545
authors.initials: K
authors.lastname: Imai
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology*; D000914:Antibodies, Viral / Q000097:blood*; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology*; D005260:Female; D006385:Hemagglutination Inhibition Tests; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000276:immunology*; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D000914:Antibodies, Viral; D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus