Fig 5

(A) RT-qPCR analysis for the expression of env38 gene in spleen, head kidney, intestine, liver, skin, gill, brain and heart tissues of zebrafish stimulated with SVCV (10⁵ TCID₅₀) or mock PBS (control) for 7 days. The expression of env38 mRNA in spleen in PBS treatment was arbitrarily set at 1. Negative control group was treated with mock PBS. Each sample was obtained from at least 10 fish. (B) Cellular distribution of Env38 on leukocytes from spleen, head kidney and peripheral blood of zebrafish stimulated with SVCV (10⁵ TCID₅₀). The Env38⁺ APCs were sorted from the leukocytes by FCM and qualitatively examined by RT-PCR with a panel of cellular markers of various immune cells, including CSF-1R and FcεRIγ (markers of monocytes/macrophages), CD83 and CD80 (markers of dendritic cells), membrane IgM (markers of B lymphocytes), MHC-II (markers of antigen presenting cells) as well as TCR-α and TCR-β (markers of T lymphocytes). Total leukocyte sample was used as a control. (C) Flow cytometry analysis of the percentages of Env38⁺ cells and MHC-II⁺Env38⁺ cells in lymphatic and myeloid leukocytes from spleen, head kidney and peripheral blood of zebrafish stimulated with SVCV (10⁵ TCID₅₀) for 7 days. The numbers above the outlined areas indicated the percentage of cells in each group. In control groups, zebrafish were i.p. with the same amount of mock PBS. In the RT-qPCR assay, PCRs were run in combination with the endogenous β-actin control. Error bars represent SEM. (*p < 0.05; **p < 0.01; ***p < 0.001; ns, no significant difference).