Journal of Virological Methods 137 (2006) 309–316 Establishment of embryonic cell line from sea bass (Lates calc for virus isolation V. Parameswaran a , Ravi Shukla b , Ramesh Bhonde b , A.S. Sahul Hameed a,∗ a Aquaculture Biotechnology Division, Department of Zoology, C. Abdul Hakeem College, Melvisharam 632509, Vellore District, Ta b Tissue Engineering & Banking Laboratory, National Centre for Cell Science, Pune 411007, India Received 8 April 2006; received in revised form 3 July 2006; accepted 4 July 2006 Available online 21 August 2006 Abstract A continuous cell line was established from blastula stage embryos of sea bass (Lates calcarifer). The sea bass embryon in Leibovitz’s L-15 supplemented with 15% fetal bovine serum. The embryonic cell line was sub-cultured more than 70 pa of 1.5 years and is designated as Sahul Indian sea bass embryonic (SISE) cell line. The cells were able to grow at temper 32 ◦ C with an optimum temperature of 28 ◦ C. The growth rate of sea bass embryonic cells increased as the FBS proportion increa 20% at 28 ◦ C with optimum growth at the concentration of 15 or 20%. Polymerase chain reaction products were obtained fr and blastula of sea bass with primer sets of microsatellite markers of sea bass. Four fish viruses were tested on this cell line to determin susceptibility to these viruses and this cell line was found to be susceptible to IPNV VR-299 and nodavirus, and the infect cytopathic effect (CPE) and RT-PCR. Further, this cell line was characterized by immunocytochemistry using confocal-las (CFLSM), transfection with pEGFP-N1, proliferate marker (BrdU). © 2006 Elsevier B.V. All rights reserved. Keywords: Lates calcarifer; Embryonic cells; Characterization; Fish viruses; Susceptibility 1. Introduction Tissue culture and the development of cell lines from fish are of priority interest for pathogen detection, toxicological studies, carcinogenesis, cellular physiology, and genetic regulation and expression. Since 1962, some 157 fish cell lines have been estab- lished which represent 34 families of fish ( Fryer and Lannon, 1994). A comprehensive list of most fish cell lines developed before 1980 has been published ( Wolf and Ahne, 1982). Most of fish cell lines were derived from freshwater or anadromous fish species. The limited number of reports on viruses from marine fish compared with those from freshwater fish are due to the shortage of fish cell lines derived from marine fish. The study of marine fish cell lines has developed rapidly in recent years and at least 17 cell lines from tissues of commercially important marine fish have been described since 1980 (Fernandez et al., 1993). A large number of cell lines have been established in fresh water ∗ Corresponding author. Tel.: +91 4172 269487 (O)/233730 (R); fax: +91 4172 266487. E-mail address: cah sahul@hotmail.com (A.S.S. Hameed). fish (Fryer and Lannon, 1994; Hong et al., 2004), but rel only a few cell lines were developed in marine fish ( Toch al., 1989; Fernandez-Puentes et al., 1993a,b; Bejar et al. Tong et al., 1997; Chi et al., 1999,2005; Chang et al., 2001; Chen et al., 2003a,b, 2004, 2005; Kang et al., 2003). It is tial to develop species-specific cell lines from marine fish use for viral diagnosis. Availability of cell lines from diffe organs or tissues of fish would be very helpful for studyi entry and propagation in the host. This information is im for developing vaccines and prophylactic measures. In th text,an attempt was made to establish an embryonic cell from the blastula stage embryo of sea bass and also to st susceptibility of this cell line to different fish viruses. 2. Materials and methods 2.1.Primary culture and routine maintenance Blastula-stage sea bass embryos were harvested appro imately 6 h after fertilization and prepared for cell culture. A group ofabout100–200 embryos were disinfected with 0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.07.006