title: The amino-terminal domain of ORF149 of koi herpesvirus is preferentially targeted by IgM from carp populations surviving infection.
abstract: Recombinantly expressed fragments of the protein encoded by ORF149 (pORF149), a structural protein from the common- and koi-carp-infecting cyprinid herpesvirus-3 (CyHV-3) that was previously shown to be antigenic, were used to obtain evidence that its amino-terminal part contains immunodominant epitopes in fish populations that survived the infection. To obtain such evidence, nonspecific binding of carp serum tetrameric IgM had to be overcome by a novel ELISA protocol (rec2-ELISA). Rec2-ELISA involved pre-adsorption of carp sera with a heterologous recombinant fragment before incubation with pORF149 fragments and detection with anti-carp IgM monoclonal antibodies. Only in this way was it possible to distinguish between sera from uninfected and survivor carp populations. Although IgM from survivors recognised pORF149 fragments to a lesser degree than whole virus, specificity was confirmed by correlation of rec2- and CyHV-3-ELISAs, inhibition of rec2-ELISA by an excess of frgIIORF149, ELISA using IgM-capture, Western blotting, and reduction of reactivity in CyHV-3-ELISA by pre-adsorption of sera with frgIIORF149. The similarity of IgM-binding profiles between frgIORF149 (amino acid residues 42-629) and frgIIORF149 (42-159) and their reactivities with previously described anti-CyHV-3 monoclonal antibodies confirmed that most pORF149 epitopes were localised in its amino-terminal part.
authors.affiliation: Escuela Superior de Ingenieros de Montes, Universidad Politécnica de Madrid (UPM), Piscifactoría, Madrid, Spain.
authors.forename: F
authors.initials: F
authors.lastname: Torrent
authors.affiliation: Departamento de Biología Molecular, Universidad de León, Leon, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Villena
authors.affiliation: Graduate Institute of Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan.|GeneReach Biotechnology, Taichung, Taiwan.
authors.forename: P A
authors.initials: PA
authors.lastname: Lee
authors.affiliation: Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Südufer 10, 17493, Greifswald-Insel Riems, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Fuchs
authors.affiliation: Institute of Infectology, German Reference Laboratory for KHVD, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Südufer 10, 17493, Greifswald-Insel Riems, Germany.
authors.forename: S M
authors.initials: SM
authors.lastname: Bergmann
authors.affiliation: Departamento Biotecnología, Instituto Nacional Investigaciones y Tecnologías Agrarias y Alimentarias, INIA, Madrid, Spain. juliocoll@inia.es.
authors.forename: J M
authors.initials: JM
authors.lastname: Coll
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000914:Antibodies, Viral / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D002347:Carps / Q000276:immunology* / Q000821:virology*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D005393:Fish Diseases / Q000276:immunology*; D006564:Herpesviridae / Q000276:immunology*; D006566:Herpesviridae Infections / Q000276:immunology / Q000662:veterinary*; D016056:Immunodominant Epitopes / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D015678:Viral Structural Proteins / Q000276:immunology* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D016056:Immunodominant Epitopes; D007075:Immunoglobulin M; D015678:Viral Structural Proteins