The amino-terminal domain of ORF149 of koi herpesvirus is preferentially targeted by IgM from carp populations surviving infection

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 frgII(ORF149), ELISA using IgM-capture, Western blotting, and reduction of reactivity in CyHV-3-ELISA by pre-adsorption of sera with frgII(ORF149). The similarity of IgM-binding profiles between frgI(ORF149) (amino acid residues 42-629) and frgII(ORF149) (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.