Establishment of a novel and highly permissive cell line for the efficient replication of cyprinid herpesvirus 2 (CyHV-2).
Haematopoietic necrosis of gibel carp (Carassius auratus gibelio) is caused by cyprinid herpesvirus 2 (CyHV-2) and has caused huge economic losses in aquaculture worldwide. Currently the isolation and propagation of CyHV-2 in vitro is very difficult due to the lack of permissive cell lines. Studies on the pathogenesis of CyHV-2 have been hampered because the virus has not been extensively characterized. In this study, a novel cell line from the brain of gibel carp, denoted GiCB, has been established and characterized. Sustainable propagation of CyHV-2 in the GiCB cell line has been confirmed by virus infection and titration, PCR, transmission electron microscopy, immunofluorescence assay and fluorescence in situ hybridization. The GiCB cells showed typical cytopathic effect by day 6 post-infection with CyHV-2 including cell shrinkage, rounding, and cell fusion with cytoplasmic vacuolization. The virus titer reached 10(7.5 ± 0.37)TCID₅₀/ml and has been successfully passaged over 50 times in the GiCB cell line. Electron microscopy analysis revealed the complete replication of CyHV-2 in GiCB cells. CyHV-2-infected GiCB cells reacted strongly with polyclonal antibodies against CyHV-2 and CyHV-2 RNA in cells hybridized specifically with the virus RNA probes. Additionally, an experimental infection demonstrated that CyHV-2 produced in GiCB cells caused 100% mortality in gibel carp. All the results provide solid evidence that the GiCB cell line is highly permissive for the isolation and propagation of CyHV-2. This is a significant advancement that will promote additional research on CyHV-2 infection in fish in the future.
Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, PR China.
Jie
J
Ma
Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, PR China.
Nan
N
Jiang
Research Division, Clear Springs Foods, Inc., PO Box 712, Buhl, ID 83316, USA.
Scott E
SE
LaPatra
Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis 97331, USA.
Ling
L
Jin
Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, PR China.
Jin
J
Xu
Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, PR China.
Yuding
Y
Fan
Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, PR China.
Yong
Y
Zhou
Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, Hubei 430223, PR China. Electronic address: zlb@yfi.ac.cn.
Lingbing
L
Zeng
D000818:Animals; D017756:Aquaculture; D001921:Brain / Q000166:cytology* / Q000821:virology; D002460:Cell Line / Q000166:cytology / Q000821:virology*; D004265:DNA Helicases / Q000235:genetics; D004279:DNA, Viral / Q000302:isolation & purification; D005393:Fish Diseases / Q000821:virology; D015967:Gene Expression Regulation, Viral; D006054:Goldfish / Q000821:virology*; D006564:Herpesviridae / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D006566:Herpesviridae Infections / Q000662:veterinary / Q000821:virology; D017404:In Situ Hybridization, Fluorescence; D007668:Kidney / Q000821:virology; D011817:Rabbits; D013154:Spleen / Q000821:virology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D004279:DNA, Viral; D014764:Viral Proteins; D004265:DNA Helicases
Brain; Cell line; Cyprinid herpesvirus 2 (CyHV-2); Gibel carp (Carassius auratus gibelio); Replication