Characterization of a primary cell culture from lymphoid organ of Litopenaeus vannamei and use for studies on WSSV replication

•Gelatin coating is important to promote cell migration and survival in vitro.•Cholesterol (600 μg/ml) enhances cell survival and performance significantly.•l-Glutathione (1000 μg/ml) enhances cell survival and performance significantly.•99% identity by sequencing blast proved that cultured cells were from L. vannamei.•Cultured cells were susceptible to WSSV and viral antigens were found from 9 hpi.

Shrimp aquaculture is a booming agro-industry worldwide. Due to intensification of shrimp farming, pathogens emerge. Control of these pathogens especially viral pathogens is essential for a further expansion of this industry. Until now, the lack of shrimp cell lines has limited research on shrimp viral pathogens. In this context, a primary culture from the lymphoid organ of Litopenaeus vannamei was developed and standardized as a platform for further research on white spot syndrome virus (WSSV). Explants from the lymphoid organ of L. vannamei were cultured in 2 × L-15 (Leibovitz-15) medium supplemented with 20% fetal bovine serum (FBS), 10% Chen's salt, penicillin (100 IU/ml) & streptomycin (100 μg/ml), gentamicin (50 μg/ml) and fungizone (0.25 μg/ml) with a pH of 7.5. Gelatin (0.1%)-coated culture plates promoted the migration of cells from the explants and cell survival. 600 μg/ml cholesterol and 1000 μg/ml l-glutathione (GSH) both enhanced cell survival and performance in vitro. Susceptibility of lymphoid organ cells for infection with white spot syndrome virus (WSSV) was determined by indirect immunofluorescence (IIF) staining by monoclonal antibodies against VP28 (W29) and goat anti-mouse IgG-FITC. FITC positive signals in the nuclei starting from 9 h post inoculation (hpi) demonstrated the susceptibility of the cells for WSSV infection in these cultures. This cell culture system will be used in the future as a tool for studying host–virus interactions in WSSV pathogenesis.