DAO 115:223-232 (2015)  -  DOI: https://doi.org/10.3354/dao02885

In vitro characteristics of cyprinid herpesvirus 2: effect of kidney extract supplementation on growth

Tomoya Shibata1, Azusa Nanjo1, Masato Saito1, Keisuke Yoshii1, Takafumi Ito2, Teruyuki Nakanishi3, Takashi Sakamoto1, Motohiko Sano1,*

1Faculty of Marine Science, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan
2Aquatic Animal Health Division, National Research Institute of Aquaculture, Fisheries Research Agency, Tamaki, Mie 519-0423, Japan
3Department of Veterinary Medicine, Nihon University, Fujisawa, Kanagawa 252-0880, Japan
*Corresponding author:

ABSTRACT: Herpesviral hematopoietic necrosis caused by goldfish hematopoietic necrosis virus (now identified as cyprinid herpesvirus 2, CyHV-2) has contributed to economic losses in goldfish Carassius auratus culture and is becoming a major obstacle in Prussian carp C. gibelio aquaculture in China. Several reports have described difficulties in culturing the virus, with the total loss of infectivity within several passages in cell culture. We succeeded in propagating CyHV-2 with a high infectious titer in a RyuF-2 cell line newly derived from the fin of the Ryukin goldfish variety using culture medium supplemented with 0.2% healthy goldfish kidney extract. The addition of kidney extract to the medium enabled rapid virus growth, resulting in the completion of cytopathic effect (CPE) within 4 to 6 d at 25°C. The extract also enabled reproducible virus culture with a titer of 105-6 TCID50 ml-1. The virus cultured using this protocol showed pathogenicity in goldfish after intraperitoneal injection. The virus grew in RyuF-2 cells at 15, 20, 25, 30, and 32°C but not at 34°C or higher. Higher incubation temperatures allowed earlier development of CPE, but culture at 30 and 32°C yielded a lower virus titer than that obtained at other temperatures because of heat inactivation of the propagated virus during cultivation. Cell lines derived from goldfish and ginbuna C. langsdorfii showed high susceptibility to the virus; cell lines from carp were susceptible to the virus using a medium containing goldfish kidney extract, but EPC, FHM, and BF-2 cell lines did not produce any CPE, even in the presence of the extract.


KEY WORDS: Goldfish hematopoietic necrosis virus · GFHNV · Cyprinid herpesvirus 2 · Culture · Kidney extract · Growth temperature · Cell susceptibility · Carassius


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Cite this article as: Shibata T, Nanjo A, Saito M, Yoshii K and others (2015) In vitro characteristics of cyprinid herpesvirus 2: effect of kidney extract supplementation on growth. Dis Aquat Org 115:223-232. https://doi.org/10.3354/dao02885

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