Attenuation mechanisms of the P7-P8 live-attenuated cyvirus cyprinidallo2 vaccine potentially involve apoptosis non-inhibition feature: insights into virus pathogenesis

<i>Cyvirus cyprinidallo2</i> (CyHV-2) is an alloherpesvirus and the causative agent of herpesviral haematopoietic necrosis in goldfish. Whole-genome sequence comparison of the developed live-attenuated vaccine P7-P8 with virulent CyHV-2 strains revealed seven single-nucleotide polymorphisms, five deletions and one inversion in the ORFs, which may be involved in attenuation. A start codon loss in ORF 113, a putative apoptosis-inhibition gene, was observed in the mutations. <i>In vitro</i> assays indicated that apoptosis-related genes were upregulated in cells inoculated with the vaccine or virulent virus compared to uninfected cells. However, the vaccine group showed increased phosphatidylserine externalization and DNA damage, suggesting the apoptosis-inducing properties of P7-P8. In the <i>in vivo</i> experiment, histopathology demonstrated that vaccinated goldfish exhibited immune responses, such as leucocyte aggregation and melanomacrophage centre formation, without marked degeneration. Gene expression analysis showed upregulation of proinflammatory and granzyme B genes in vaccinated fish. In addition, the vaccine strain triggered apoptosis of the infected cells during the early stage of infection, potentially promoting virus clearance and preventing excessive virus replication. The results show that the P7-P8 potentially induces apoptosis and immune responses, contributing to low virus propagation without tissue damage. This study provides insights into CyHV-2 pathogenesis, suggesting that apoptosis-related genes can be the targets for vaccine development against alloherpesviruses in aquaculture species.