Development of cell lines from brain, spleen and heart of ornamental blood parrot cichlid and their susceptibility to Tilapia tilapinevirus

Rastogi A.; Yadav M.K.; Joaquin M.P.C.; Verma D.K.; Swaminathan T.R.; Kushwaha B.; Paria A.; Pradhan P.K.; Sood N. · Aquaculture (2022) · DOI: 10.1016/j.aquaculture.2022.738711

Resumen

Blood parrot cichlid, a hybrid of Amphilophus citrinellus (♂) and Paraneetroplus synspilus (♀), is among the most popular ornamental fishes globally. In this study, three cell lines were established from brain, spleen, and heart tissues of the blood parrot cichlid using explant method and these were designated as parrot fish brain (PFB), parrot fish spleen (PFS) and parrot fish heart (PFH), respectively. All the cell lines have been subcultured for over 120 passages and these grow optimally at 28–32 °C in Leibovitz's L-15 medium containing 20% fetal bovine serum. PFB and PFH cells depict epithelial morphology whereas PFS cells consist of fibroblast-like cells. Although the chromosome number of blood parrot cichlid was 2n = 48, the cell lines exhibited heteroploidy and aneuploidy, suggesting transformation. The sequencing of amplified fragments of two mitochondrial genes, namely COI and 16S rRNA confirmed that the cell lines originated from blood parrot cichlid. The three cell lines were free of Mycoplasma contamination. Besides, their successful transfection with a GFP reporter gene suggested potential application in transient gene expression studies. All the three cell lines were susceptible to Tilapia tilapinevirus, also known as Tilapia lake virus (TiLV), which was indicated by appearance of cytopathic effects and RT-PCR. Of the three cell lines, PFS was found to be the most permissive with maximum TiLV copy number of 2.5 × 108 per 100 ng of total RNA, as determined by qRT-PCR. Importantly, the TiLV disease was successfully reproduced in naive tilapia after injection of cell culture supernatant from TiLV-infected PFS cell line, and this was evidenced by clinical signs, histopathological alterations, reisolation of virus and specific amplification in RT-PCR. The three newly established cell lines were submitted to National Repository on Fish Cell Lines at ICAR-NBFGR. This forms the first report of development of cell lines from blood parrot cichlid, and these would be useful for surveillance of diseases suspected to be of viral origin in this candidate species and also other ornamental cichlids.

Instituciones: Universidad Nacional de Tumbes