Establishment and identification of spleen cell line from tiger puffer fish (Takifugu rubripes) and immune transcriptome response to IHNV

The tiger puffer fish (Takifugu rubripes) is highly valued both economically and for its culinary appeal. However, it currently faces a significant risk of disease. This study aimed to establish a spleen cell line to aid in disease prevention and control for this species. We established a new stable cell line (TRSC) from the spleen tissue of the tiger puffer fish. The TRSC cells exhibited stable growth over more than 90 generations in L-15 medium supplemented with 4 ng/mL recombinant human basic fibroblast growth factor (bFGF) and 2 ng/mL recombinant human leukemia inhibitory factor (LIF) at 24 degrees C. Chromosomal analysis revealed a diploid number of 2n = 44. The TRSC cells were susceptibly infected by both viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV), exhibiting a clear cytopathic effect (CPE). After 48 h of infection, alterations in cell morphology and disintegration were observed. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis revealed differential replication of the two viruses in TRSC cells. Transcriptome sequencing identified 1308 differentially expressed genes (DEGs) following IHNV infection, with 668 genes upregulated and 640 downregulated. These DEGs were significantly enriched in MAPK signaling pathway, p53 signaling pathway, and DNA replication and so on. The establishment of the TRSC cell line provides valuable insights into viral infection mechanisms, aids in the identification of key immune genes, and offers a more effective approach to disease control in aquaculture, supporting the sustainable development of the industry.