Continuous cell lines from kidney and spleen of Japanese eel, Anguilla japonica, with capacity for antiviral immune response.

In this study, two stable cell lines were established from the kidney (AJK) and spleen (AJSP) tissues of Japanese eel (Anguilla japonica) using the tissue explant method, with the aim of supporting immunological research in this species. Following isolation, AJK and AJSP cells were cultured in DMEM/F-12 medium supplemented with 20 % fetal bovine serum (FBS) at 28 °C, with passaging every 3-4 days. After the fifth passage, the cells were maintained in DMEM/F-12 containing 15 % FBS for passage culture. Ribosomal RNA gene sequencing confirmed the origin of both cell lines as A. japonica. Karyotype analysis revealed that the AJK and AJSP cell lines each possessed 38 chromosomes, including 10 metacentric, 10 submetacentric, and 18 acrocentric chromosomes. To assess the suitability of these cell lines for gene function studies, we introduced a GFP-LC3 expression plasmid into AJK and AJSP cells using various transfection reagents. Flow cytometry analysis showed maximum transfection efficiencies of 21.43 ± 0.33 % in AJK and 9.30 ± 0.21 % in AJSP cells. Furthermore, co-transfection of the interferon regulatory factor (IRF) AjIRF1 with interferon (IFN) promoter constructs (AjIFNa or AjIFNc1) led to increased promoter activity in both cell lines, demonstrating their potential for functional gene analysis. In addition, both cell lines supported infection and efficient replication of Anguilla herpesvirus 1 (AngHV-1), and exhibited immune responses following viral challenge, indicating their applicability in studies of viral infection and host antiviral immune responses. Collectively, the AJK and AJSP cell lines established in this study provide a valuable cell model for further investigation into the immune responses against pathogenic infections in eels.