The amino-terminal domain of ORF149 of koi herpesvirus is preferentially targeted by IgM from carp populations surviving infection
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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.
KeywordsRecombinant Fragment ELISA Protocol Carp Population Carp Serum Infectious Haematopoietic Necrosis Virus
Thanks go to Paula Perez Gonzalez, who helped with the experimental work. Thanks are due to Dra. D. Chee (Animal & Plant Health Laboratories, Aquatic Animal health Section Laboratories Group, Singapore) for providing sera from carp surviving CyHV-3, to Dra. K. Thompson (AquaTic Diagnostics, Ltd) for providing some of the anti-CyHV-3 monoclonal antibodies, and Dra. P. Fernandez (MAGRAMA, Algete, Madrid, Spain) for her help with CyHV-3-U. We also thank J.L. Gonzalez and his team (CBC, SL) for obtaining common carp sera samples from all over Spain and from imported koi, and P. Encinas, B. Chinchilla and Dr. E. Gomez-Casado, who helped with Spanish koi sera at UPM (Madrid). This work was supported by RTA2013-00008-00-00 of INIA, and AGL2014-51773-C3 and AGL2014-53190 REDC of the Ministerio de Economía y Competitividad of Spain.
Compliance with ethical standards
This study was funded by RTA2013-00008-00-00 of the Instituto Nacional de Investigaciones y Tecnología Agraria y Alimentaria, INIA, and AGL2014-51773-C3 and AGL2014-53190 REDC of the Ministerio de Economía y Competitividad of Spain. The authors declare that they have no conflict of interest. All applicable international, national and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants by any of the authors.
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