Cyprinid Herpesvirus 3 ORF57 is an essential virulence factor

The genus Cyprinivirus comprises phylogenetically related viruses causing important economic losses in aquaculture. Its archetype species, the cyprinid herpesvirus 3 (CyHV 3) is the causative agent of mass mortalities in koi and common carp worldwide. Recently, we reported the development of a recombinant attenuated vaccine against CyHV 3 for mass vaccination of carp. This attenuated vaccine encodes a deletion encompassing ORF56 and ORF57. In the present study, we investigated the relative contribution of ORF56 and ORF57 in the safety observed for the ORF56-57 double deleted genotype. Single deleted recombinants were produced based on deletion-insertion of a galK cassette, inserting stop codons in frame with the sequence upstream of the deletion and disrupting the frame of the remaining sequence downstream of the deletion. Inoculation of these recombinants to carp demonstrated that the absence of ORF56 protein expression did not affect virulence; while ORF57 deletion led to an attenuation comparable to the one observed for the ORF56-57 deletion. To further demonstrate the role of ORF57 as a key virulence factor, a recombinant unable to express ORF57 protein was produced by insertion of multiple stop codons in the ORF. In vivo testing of this recombinant revealed a safety-efficacy profile comparable to the one observed for the vaccine candidate deleted for ORF56 and ORF57. Altogether, the present study demonstrates the role of ORF57 as a key virulence factor of CyHV 3 and identifies ORF57 orthologues encoded by other pathogenic cypriniviruses as gene candidates for production of attenuated recombinant vaccines.