Establishment and characterization of a novel cell line from spleen of humpback grouper (Cromileptes altivelis) and its application in pathogens susceptibility

The humpback grouper (Cromileptes altivelis) is a marine fish of significant commercial value and has been identified as a potential candidate for marine aquaculture. In this research, a cell line named as CAS cells was developed from the spleen tissue of the humpback grouper. This cell line has been successfully passaged for over 100 passages, with most cells exhibiting a fibroblast-like morphology. After being cryopreserved in liquid nitrogen for three months, the survival rate of the cells was over 80 %. The sequencing of the COI and 18S rRNA genes has verified that the cell line is sourced from the humpback grouper. The CAS cells exhibited vigorous proliferation in L-15 medium supplemented with 20 % FBS at 26 °C. Chromosomal analysis indicated that the CAS cells possess a diploid chromosome number of 2n = 48. Furthermore, the expression of the GFP reporter gene in CAS cells was successfully demonstrated. Toxicity assays indicated that Vibrio harveyi exhibited greater toxicity to CAS cells compared to Photobacterium damselae. P. damselae exhibited marked host cell surface adherence and induced apoptotic morphology. Moreover, experiments on viral susceptibility showed that CAS cells were sensitive to ISKNV-HN and NNV, as evidenced by cytopathic effects and viral protein production within the cells, along with an increase in the expression of immune-related genes. Consequently, our research confirms that this cell line is appropriate for in vitro genetic modification and represents a significant resource for virus isolation and the investigation of pathogenesis.