The engineered ORF56-57 deletion mutant leads to attenuation of an Asian koi herpesvirus strain

Koi herpesvirus (KHV) poses a significant threat to the koi and common carp aquaculture industry due to its high mortality rate among infected fish. In this study, we successfully isolated an Asian KHV strain designated as SS09 and investigated the potential attenuation of the SS09 through the deletion of open reading frames 56 and 57 (ORF56-57). By utilizing homologous recombination technology, we generated a mutant strain with a deletion of ORF56-57, known as SS09 Delta 56-57. The mutant strain exhibited slightly reduced viral replication efficiency in common carp brain (CCB) cells. Furthermore, challenge trials conducted on healthy koi populations revealed that fish infected with the SS09 Delta 56-57 did not show mortality, whereas the mortality rate of fish infected with the wild strain was nearly 100%. This suggests that the deletion of ORF56-57 can lead to the attenuation of the Asian KHV strain. To further validate the feasibility of SS09 Delta 56-57 as novel vaccine candidates or therapeutic in-terventions against KHV, we conducted immersion immunization of koi fish with three different doses of SS09 Delta 56-57. Serum neutralizing antibody titers were determined, and challenge tests were performed three weeks after immunization. The results showed that SS09 Delta 56-57 at different doses exhibited a relative percentage survival (RPS) ranging from 70.0% to 96.6%. The neutralization titer of anti-serum in fish following 3 weeks of inoculation also displayed a dose-dependent response. In summary, owing to its outstanding immunogenicity, safety, and efficacy, SS09 Delta 56-57 can be regarded as a promising candidate vaccine strain for KHV disease.