Development of a live attenuated vaccine candidate against herpesviral hematopoietic necrosis of goldfish

Abstract

Herpesviral hematopoietic necrosis causes huge economic loss in goldfish Carassius auratus and gibel carp C. auratus gibelio farming. To develop a live attenuated vaccine as a preventive measure against the disease, the causative virus cyprinid herpesvirus 2 (CyHV-2) was attenuated by serial subculture in two cell lines. The CyHV-2 SaT-1 isolate routinely propagated in the goldfish cell line RyuF-2 was serially propagated in the CFS cell line derived from ginbuna C. auratus langsdorfii, a non-natural host. Virus propagated in CFS showed no lethality in goldfish by immersion administration and provided high protective efficacy in subsequent virulent virus challenge, but virus propagated once in RyuF-2 caused skin hemorrhage in some fish after administration. The virus was further attenuated by subculturing several times in the KF-1 cell line from carp Cyprinus carpio, also a non-natural host. Viruses subcultured 7 or 10 times in CFS were propagated 5 or 8 times in KF-1 to obtain further attenuated viruses abbreviated as P7-P5, P7-P8, P10-P5 and P10-P8 (Passage number in CFS – KF-1). P7-P5, P7-P8 and P10-P5 showed high protective efficacy of >80% relative percentage survival in the vaccine trial, but P10-P8 was not highly effective. P7-P8 was selected as a vaccine candidate for further virulence reversion tests. In the in vitro reversion test, the candidate subcultured 5 times in RyuF-2 caused no pathogenicity in goldfish, revealing no signs of virulence reversion. Furthermore, in the in vivo test for virulence reversion, the candidate was propagated through goldfish 6 times by intraperitoneal injection with kidney homogenate of the inoculated fish. During in vivo passages in goldfish, no abnormality and mortality of the inoculated fish were observed, and virus DNA copy number in the kidney and caudal fin decreased from 104 to 108 copies/mg at the first passage to ~103 copies/mg at the second passage. These results demonstrated that P7-P8 can be a promising live attenuated vaccine candidate against the disease.