Growth of cyprinid herpesvirus 2 (CyHV-2) in cell culture and experimental infection of goldfish Carassius auratus

Herpesviral haematopoietic necrosis has caused great economic damage to goldfish Carassius auratus aquaculture in Japan. The existence of cyprinid herpesvirus 2 (CyHV-2), the causative agent, has also been reported from several other countries. To prevent spread to other areas, basic virological information such as viral kinetics in infected fish is essential. Experimental infection trials using reliably prepared CyHV-2 for defining viral kinetics are difficult to carry out because successful and sustainable propagation of this virus in cell culture has previously been limited. Here we describe a method for sustainable propagation of CyHV-2 in cell culture, and the results of fish infection experiments using the propagated virus. We found that goldfish fin (GFF) cells and standard Ryukin Takafumi (SRTF) cells established from goldfish fin can be used for continuous propagation of CyHV-2. Experimental infections using 2 varieties of goldfish, Ryukin and Edonishiki, were performed with the virus passaged 7 times in GFF cells. In transmission experiments with water temperature at 20 degrees C, cumulative mortality was 30% in Ryukin infected by immersion, and 90 and 100% in Edonishiki and Ryukin intraperitoneally injected with the virus, respectively. In an experiment carried out at 25 degrees C, 90% of Edonishiki challenged by immersion died. PCR detection of viral DNA from the organs of infected fish showed that systemic infection occurs and also that the kidney is a main viral multiplication site. Moreover, CyHV-2 was successfully re-isolated in GFF cells from the dead fish.