Establishment of a Hybrid Snakehead Spleen Cell Line for Viral Studies

A spleen-derived cell line was established from the spleen of hybrid snakehead (♀Channa argus × ♂Channa maculata) (abbreviated as CAMsp), a species of considerable economic importance in China's freshwater aquaculture, which is severely impacted by the hybrid snakehead rhabdovirus (HSHRV), largemouth bass ranavirus (LMBV), and infectious spleen and kidney necrosis virus (ISKNV). The establishment of a stable in vitro culture system is imperative for the effective isolation, identification, and study of fish viruses. CAMsp, generated through trypsin digestion, had successfully undergone over 80 passages since its initial culture. This cell line exhibited rapid proliferation in Leibovitz's-15 medium (L-15) supplemented with 10% fetal bovine serum at 28°C, achieving monolayer formation within 24 h at a passage ratio of 1:3. Chromosomal analysis of CAMsp at the 60th passage identified a chromosome count of 42; the chromosome number in hybrid snakehead somatic cells is 44, 45, or 46, suggesting chromosomal alterations. Inoculation of CAMsp monolayers with HSHRV, LMBV, and ISKNV resulted in characteristic cytopathic effects (CPE), including cell rounding, aggregation, and eventual detachment. Transmission electron microscopy (TEM) confirmed viral replication and revealed extensive cytopathological changes within the infected cells, demonstrating the susceptibility of the CAMsp cell line to all three viruses. Viral titers, determined by TCID₅₀ assay at 7 days post-infection (dpi), reached 10^9.25 ± 10^0.36 TCID₅₀/mL for LMBV, 10⁶·¹⁵ ± 10^0.25 TCID₅₀/mL for ISKNV, and 10⁸·³³ ± 10^0.12 TCID₅₀/mL for HSHRV, indicating efficient viral propagation in this cell line. The CAMsp cell line serves as a valuable model for studying certain fish viruses, virus-host interactions, and disease prevention strategies.