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

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.

Susceptibility of isogeneic ginbuna Carassius auratus langsdorfii Temminck et Schlegel to cyprinid herpesvirus‐2 (CyHV‐2) as a model species

“…As Shibata et al . () reported that total infectivity of the virus culture was lost after incubation at 35 °C for 1 day, it was thought that the virus in culture medium lost infectivity during incubation at 34 °C for 4 days in this study; however, the reappearance of CPE could be attributed to virus that was reactivated within the cells. The same phenomenon has been reported for KHV, which is closely related to CyHV‐2.…”

“…This observation is consistent with in vitro virus propagation in the goldfish fin RyuF‐2 cell line which occurred within 24 h after inoculation at 25 °C (Shibata et al . ). These infected fish were then kept at 34 °C, a temperature that is non‐permissive to virus replication, and virus propagation in fish tissues was halted.…”

“…CyHV-2 Sat-1 isolate ) was used in this study. The virus was propagated in RyuF-2 cells derived from the fin of Ryukin variety goldfish in medium 199 (M199: Sigma) supplemented with 5% foetal bovine serum (FBS: Gibco), penicillin, streptomycin (Sigma) and 0.2% kidney extract from healthy goldfish (M199-5 w/kid ex) at 25°C (Shibata et al 2015). The virus culture was divided into vials after passing through a 0.45-lm filter (Millipore) and was stored at À80°C.…”

“…Shibata et al . () reported that temperatures of 34 °C and over are non‐permissive of CyHV‐2. As high temperature treatment (HT) reduced CyHV‐2‐associated mortality of infected goldfish (Tanaka ), the treatment has also been applied to control the disease in some of the goldfish farms in Japan.…”

“…An alternative to reduce mortality due to CyHV-2 is by increasing, or decreasing the rearing water temperature to 32-35°C, or 13-15°C. Shibata et al (2015) reported that temperatures of 34°C and over are non-permissive of CyHV-2. As high temperature treatment (HT) reduced CyHV-2-associated mortality of infected goldfish (Tanaka 2005), the treatment has also been applied to control the disease in some of the goldfish farms in Japan.…”