Protection of cultured Cyprinus carpio against a lethal viral disease by an attenuated virus vaccine

Massive mortality of koi and common carp--Cyprinus carpio species--has been observed since 1998 in many countries worldwide, resulting in severe economic losses. The cause of the disease is an as yet unclassified large DNA virus, designated carp nephritis gill necrosis virus (CNGV) or koi herpes virus (KHV). Previously, we demonstrated that the wild type CNGV lost its pathogenecity following serial transfer in cell culture, and that clones isolated from the attenuated population can be used as a prophylactic vaccine. Here, we describe the basic conditions required for proper fish immunization so that a protection protocol may be devised. We demonstrated that carps are very sensitive to the pathogenic and the attenuated viruses, and short immersion of fish in water containing the viruses is sufficient for infection. The infection of fish with the pathogenic and the attenuated viruses is temperature-restricted; fish held at the non-permissive temperature, immediately following infection, were not affected by the pathogenic virus, and were not rendered resistant to the disease. Thus, propagation of the virus in the fingerlings is a pre-requisite for immunization. In order to increase the number of random mutations in the genome of the attenuated virus, and thus, reduce the possibility of the attenuated virus reverting to pathogenic, we irradiated it and selected additional clones appropriate for vaccination. The results of our study suggest that a safe and efficient prophylactic vaccine can be developed by selecting an appropriate attenuated virus.