Abstract
Megalocytiviruses are important emerging pathogens in both freshwater and marine finfish aquaculture. However, a limited number of piscine cell lines are persistently susceptible to these viruses, which greatly limits the study of megalocytiviruses. In this study, a new fibroblast-like cell line was established from an early primary culture from mandarin fish fry by a single cell cloning and was designated as MFF-8C1. The MFF-8C1 cells grow well in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum and had been subcultured more than 60 passages since the initial recovery culture in October 2009. Chromosomal analysis revealed that 91 % of the MFF-8C1 cells maintained a normal diploid chromosome number (2n = 48) in the 46th passage. Infection experiments showed that both freshwater-borne and marine-borne megalocytiviruses induce severe cytopathic effects in infected MFF-8C1 cells characterized by the rounding and enlargement of cells, which are highly consistent with the previous description of the infection in other susceptible cells with megalocytivirus. Megalocytivirus infections were further confirmed by a transmission electron microscopy. Furthermore, the MFF-8C1-cultured megalocytiviral suspension was highly virulent to infected mandarin fish. In summary, a new fibroblast cell line from mandarin fish fry that was highly permissive to megalocytiviruses was established. The MFF-8C1 cell line is a promising cellular substrate candidate for cell-cultured vaccine production of megalocytivirus.
References
Anderson IG, Prior HC, Rodwell BJ, Harris GO (1993) Iridovirus-like virions in imported dwarf gourami (Colisa lalia) with systemic amoebiasis. Aust Vet J 70:66–67
Chinchar VG, Essbayer S, He JG, Hyatt A, Miyazaki T, Seligy V, Williams T (2005) Family Iridoviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA (eds) Virus taxonomy: 8th report of the International Committee on the Taxonomy of Viruses. Elsevier Academic Press, San Diego, CA, pp 163–175
Chou HY, Hsu CC, Peng TY (1998) Isolation and characterization of a pathogenic iridovirus from cultured grouper (Epinephelus sp.) in Taiwan. Fish Pathol 33:201–206
Chua FHC, Ng ML, Ng KL, Loo JJ, Wee JY (1994) Investigation of outbreaks of a novel disease, ‘sleepy grouper disease’, affecting the brown-spotted grouper, Epinephelus tauvina Forskal. J Fish Dis 17:417–427
Dong C, Weng S, Shi X, Xu X, Shi N, He J (2008) Development of a mandarin fish Siniperca chuatsi fry cell line suitable for the study of infectious spleen and kidney necrosis virus (ISKNV). Virus Res 135:273–281
Dong C, Weng S, Luo Y, Huang M, Ai H, Yin Z, He J (2010) A new marine megalocytivirus from spotted knifejaw, Oplegnathus punctatus, and its pathogenicity to freshwater mandarinfish, Siniperca chuatsi. Virus Res 147:98–106
Dong C, Weng S, Li W, Li X, Yi Y, Liang Q, He J (2011) Characterization of a new cell line from caudal fin of koi, Cyprinus carpio koi, and first isolation of cyprinid herpesvirus 3 in China. Virus Res 161:140–149
Dong C, Xiong X, Luo Y, Weng S, Li Y, He J (2013) Efficacy of a formalin-killed cell vaccine against infectious spleen and kidney necrosis virus (ISKNV) and immunoproteomic analysis of its major immunogenic proteins. Vet Microbiol 162:419–428
He JG, Zeng K, Weng SP, Chan SM (2000) Systemic disease caused by an iridovirus-like agent in cultured mandarinfish, Siniperca chuatsi (Basillewsky), in China. J Fish Dis 23:219–222
Imajoh M, Sugiura H, Oshima S (2004) Morphological changes contribute to apoptotic cell death and are affected by caspase-3 and caspase-6 inhibitors during red sea bream iridovirus permissive replication. Virology 322:220–230
Imajoh M, Ikawa T, Oshima S (2007) Characterization of a new fibroblast cell line from a tail fin of red sea bream, Pagrus major, and phylogenetic relationships of a recent RSIV isolate in Japan. Virus Res 126:45–52
Inouye K, Yamano K, Maeno Y, Nakajima K, Matsuoka M, Wada Y, Sorimachi M (1992) Iridovirus infection of cultured red sea bream, Pagrus major. Fish Pathol 27:19–27
Iwamoto T, Nakai T, Mori K, Arimoto M, Furusawa I (2000) Cloning of the fish cell line SSN-1 for piscine nodaviruses. Dis Aquat Organ 43:81–89
Jung SJ, Oh MJ (2000) Iridovirus-like infection associated with high mortalities of striped beakperch, Oplegnathus fasciatus (Temminck et Schlegel), in southern coastal areas of the Korean peninsula. J Fish Dis 23:223–226
Kawakami H, Nakajima K (2002) Cultured finfish species affected by red sea bream iridoviral disease from 1996 to 2000. Fish Pathol 37:45–47
Kurita J, Nakajima K (2012) Review: Megalocytiviruses. Viruses 4:521–538
Ma H, Xie J, Weng S, Zhou T, He J (2012) Co-infection of megalocytivirus and viral nervous necrosis virus in a very severe mass mortality of juvenile orange-spotted groupers (Epinephelus coioides). Aquaculture 358–359:170–175
Marcos-López M, Feist SW, Hicks R, Noguera PA (2011) Systemic megalocytivirus infection in three-spined stickleback Gasterosteus aculeatus. Bull Eur Assoc Fish Pathol 31:227–234
Matsuoka S, Inouye K, Nakajima K (1996) Cultured fish species affected by red sea bream iridoviral disease from 1991 to 1995. Fish Pathol 31:233–234
Nakajima K, Sorimachi M (1994) Biological and physico-chemical properties of the iridovirus isolated from cultured red sea bream, Pagrus major. Fish Pathol 29:29–33
Pham PH, Lai YS, Lee FF, Bols NC, Chiou PP (2012) Differential viral propagation and induction of apoptosis by grouper iridovirus (GIV) in cell lines from three nonhost species. Virus Res 167:16–25
Reed LJ, Muench H (1938) A simple method of estimating fifty percent endpoints. Am J Hyg 27:493–497
Shuang F, Luo Y, Xiong XP, Weng S, Li Y, He J, Dong C (2013) Virions proteins of an RSIV-type megalocytivirus from spotted knifejaw Oplegnathus punctatus (SKIV-ZJ07). Virology 437:89–99
Subramaniam K, Shariff M, Omar AR, Hair-Bejo M (2012) Megalocytivirus infection in fish. Rev Aquac 4:221–233
Sudthongkong C, Miyata M, Miyazaki T (2002) Viral DNA sequences of genes encoding the ATPase and the major capsid protein of tropical iridovirus isolates which are pathogenic to fishes in Japan, South China Sea and Southeast Asian countries. Arch Virol 147:2089–2109
Zhang QY, Li ZQ, Gui JF (1999) Studies on morphogenesis and cellular interactions of Rana grylio virus in an infected fish cell line. Aquaculture 175:185–197
Acknowledgments
This research was supported by the National Basic Research Program of China under Grant No. 2012CB114406; Technology Planning Project of Guangdong Province under number 2011A020102002 and 2012A020800006, and the planning subject of ‘the twelfth five-year-plan’ in national science and technology for the rural development in China under number 2011BAD13B00.
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Dong, C., Shuang, F., Weng, S. et al. Cloning of a new fibroblast cell line from an early primary culture from mandarin fish (Siniperca chuatsi) fry for efficient proliferation of megalocytiviruses. Cytotechnology 66, 883–890 (2014). https://doi.org/10.1007/s10616-013-9642-7
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DOI: https://doi.org/10.1007/s10616-013-9642-7