Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aquaculture Derivation of two tilapia (Oreochromis niloticus) cell lines for efficient propagation of Tilapia Lake Virus (TiLV) Raja Swaminathan Thangaraj a, ⁎ , Charan Ravi a , Raj Kumar a , Arathi Dharmaratnam a , Basheer Valaparambil Saidmuhammed a , Pravata Kumar Pradhan b , Neeraj Sood b a Peninsular and Marine Fish Genetic Resources Centre, ICAR-NBFGR, CMFRI Campus, Kochi 682 018, Kerala, India b ICAR National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow 226 002, Uttar Pradesh, India ARTICLE INFO Keywords: Cell lines Isolation Oreochromis niloticus Tilapia Lake Virus ABSTRACT Tilapia Lake Virus (TiLV) has been associated with disease outbreaks in cultured tilapia worldwide. In this study, we developed and characterized two cell lines from the brain (OnlB) and liver (OnlL) of the Nile tilapia, Oreochromis niloticus for the efficient propagation of TiLV. Both the cells grew well in Leibovitz's – 15 (L-15) medium supplemented with 20% fetal bovine serum (FBS) and have been sub-cultured more than 45 times. Chromosome analysis of the cells revealed that both lines had normal diploid number (2n = 44). TiLV was isolated from diseased tilapia and continuously propagated for 20 passages in these cell lines. The maximum TiLV titer was 10 7.3 ± 0.05 and 10 7.0 ± 0.96 TCID 50 /ml, in OnlL and OnlB respectively. The TiLV isolate consistently produced the same CPE in all passages. In vivo challenge experiments using the TiLV infected cell culture su- pernatant reproduced symptoms of the disease in healthy tilapia, with mortality commencing 10 days post- infection and we were able to isolate TiLV from the challenged fish The above results suggest both cell lines are highly permissive for propagating TiLV and could be important tools for studying the molecular pathogenesis of TiLV infection. 1. Introduction Tilapia are the second most farmed finfish species after carps and likely to be the most important cultured fish in the 21st Century (Fitzsimmons, 2000). The global production of tilapia in 2015 is esti- mated at 6.4 million metric tons (MMT), (FAO, 2017a) and the Nile tilapia, Oreochromis niloticus is the 6th most cultured species in the world (Reantaso, 2017). Tilapia are considered to be relatively resistant to a number of diseases encountered in other farmed fishes (Del-Pozo et al., 2017), but the emergence of Tilapia Lake Virus (TiLV) disease, the first major disease epidemic reported in tilapia aquaculture, has put the global tilapia industry at risk (Jansen and Mohan, 2017; FAO, 2017b). Though the disease has been confirmed from seven countries, namely Israel (Eyngor et al., 2014; Bacharach et al., 2016), Ecuador (Ferguson et al., 2014; Del-Pozo et al., 2017), Colombia (Tsofack et al., 2017), Egypt (Fathi et al., 2017; Nicholson et al., 2017), Thailand (Surachetpong et al., 2017), Malaysia (Amal et al., 2018), India (Behera et al., 2018) and Ugandan and Tanzanian parts of Lake Victoria (Mugimba et al., 2018), it is likely to be present in many more countries (Dong et al., 2017a). The disease is usually associated with high mor- talities (Ferguson et al., 2014; Eyngor et al., 2014; Dong et al., 2017a; Behera et al., 2018) and is characterized by skin erosions, ocular ab- normalities and distended abdomen. Internally, the lesions are mainly localized in liver and brain (Eyngor et al., 2014). In a recent study, Liamnimitr et al. (2018) confirmed that the mucus could be used for nonlethal sampling for the detection of TiLV by RT-qPCR and cell cul- ture. Tattiyapong et al. (2018) developed a rapid and trustworthy RT PCR assay for the detection of TiLV in clinical cases as well in asymp- tomatic tilapia. Tilapia Tilapinevirus (TiLV) initially called Tilapia Lake Virus classified in the Orthomyxovirus group is now classed into a new unassigned group of its own termed Tilapinevirus (Adams et al., 2017). Cell lines are essential for isolating viruses and studying virus-host interactions (Crane and Hyatt, 2011). Fish viruses are generally host specific, which makes a cell line derived from a particular fish species more appropriate for studying the viruses reported from that species (Pandey, 2013). Therefore, the establishment of susceptible, homo- logous and tissues-specific cell lines is considered necessary for iso- lating viral pathogens. In vitro culture of TiLV has been carried out using the E-11 cell line derived from snakehead (Iwamoto et al., 2000) as well as OmB and TmB cell lines derived from O. mossambicus (Gardell et al., 2014; Lewis and Marks, 1985). However, no cell lines from Nile tilapia are available for the continuous propagation of TiLV. Keeping https://doi.org/10.1016/j.aquaculture.2018.04.012 Received 29 December 2017; Received in revised form 31 March 2018; Accepted 8 April 2018 ⁎ Corresponding author at: Peninsular and Marine Fish Genetic Resources Centre, ICAR-NBFGR, CMFRI Campus, P.O. Number 1603, Kochi 682 018, Kerala, India. E-mail address: Thangaraj.swaminathan@icar.gov.in (R.S. Thangaraj). Aquaculture 492 (2018) 206–214 Available online 10 April 2018 0044-8486/ © 2018 Elsevier B.V. All rights reserved. T