title: Multiplex evaluation of influenza neutralizing antibodies with potential applicability to in-field serological studies.
abstract: The increased number of outbreaks of H5 and H7 LPAI and HPAI viruses in poultry has major public and animal health implications. The continuous rapid evolution of these subtypes and the emergence of new variants influence the ability to undertake effective surveillance. Retroviral pseudotypes bearing influenza haemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins represent a flexible platform for sensitive, readily standardized influenza serological assays. We describe a multiplex assay for the study of neutralizing antibodies that are directed against both influenza H5 and H7 HA. This assay permits the measurement of neutralizing antibody responses against two antigenically distinct HAs in the same serum/plasma sample thus increasing the amount and quality of serological data that can be acquired from valuable sera. Sera obtained from chickens vaccinated with a monovalent H5N2 vaccine, chickens vaccinated with a bivalent H7N1/H5N9 vaccine, or turkeys naturally infected with an H7N3 virus were evaluated in this assay and the results correlated strongly with data obtained by HI assay. We show that pseudotypes are highly stable under basic cold-chain storage conditions and following multiple rounds of freeze-thaw. We propose that this robust assay may have practical utility for in-field serosurveillance and vaccine studies in resource-limited regions worldwide.
authors.affiliation: Viral Pseudotype Unit (Medway), School of Pharmacy, University of Kent, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK.
authors.forename: Eleonora
authors.identifier: 0000-0002-8561-9966
authors.initials: E
authors.lastname: Molesti
authors.affiliation: Viral Pseudotype Unit (Fitzrovia), Faculty of Science and Technology, University of Westminster, London W1W 6UW, UK.
authors.forename: Edward
authors.identifier: 0000-0001-7041-5138
authors.initials: E
authors.lastname: Wright
authors.affiliation: FAO, OIE, and National Reference Laboratory for Newcastle Disease and Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell'Università 10, 35020 Legnaro, Italy.
authors.forename: Calogero
authors.initials: C
authors.lastname: Terregino
authors.affiliation: Viral Pseudotype Unit (Medway), School of Pharmacy, University of Kent, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK.
authors.forename: Rafat
authors.initials: R
authors.lastname: Rahman
authors.affiliation: FAO, OIE, and National Reference Laboratory for Newcastle Disease and Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell'Università 10, 35020 Legnaro, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Cattoli
authors.affiliation: Viral Pseudotype Unit (Medway), School of Pharmacy, University of Kent, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK.
authors.forename: Nigel J
authors.identifier: 0000-0002-7978-3815
authors.initials: NJ
authors.lastname: Temperton
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood / Q000276:immunology*; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology*; D002645:Chickens; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D006385:Hemagglutination Inhibition Tests; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000276:immunology; D006801:Humans; D009980:Influenza A virus / Q000145:classification / Q000235:genetics / Q000276:immunology*; D007252:Influenza Vaccines / Q000276:immunology; D005585:Influenza in Birds / Q000175:diagnosis / Q000276:immunology*; D007251:Influenza, Human / Q000175:diagnosis / Q000276:immunology*; D016086:Lentivirus / Q000235:genetics; D009439:Neuraminidase / Q000235:genetics / Q000276:immunology; D010802:Phylogeny; D012698:Serologic Tests / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D014764:Viral Proteins; C487630:NA protein, influenza A virus; D009439:Neuraminidase
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