Fig 12

(A-C) Examination on the stimulatory role of zebrafish IFNφ1 and IFNφ2 in Env38 expression by in vivo administering recombinant IFNφ1 and IFNφ2 proteins. The stimulatory effect was examined by the increased percentage of Env38⁺ cells and the upregulated transcriptional expression of env38, isg15 and viperin genes in leukocytes from spleen, head kidney and peripheral blood of zebrafish i.p. injected with proportional dosage (2.5 μg, 5 μg and 10 μg) of the IFNφ1 (A) or IFNφ2 (B) through FCM analysis (A and B) and RT-qPCR (C), respectively. Zebrafish i.p. injected with mock PBS was used as control. (D and E) Examination on the stimulatory role of zebrafish IFNφ1 in Env38 expression by in vivo neutralization and rescue assays via administering mouse anti-IFNφ1 Ab (10 μg/fish) and recombinant IFNφ1 (10 μg/fish) back into the IFNφ1-neutralized zebrafish. The stimulatory effect was examined by the percentage of Env38⁺ cells (D) and the expression level of env38, isg15 and viperin genes (E) in leukocytes from spleen, head kidney and peripheral blood of zebrafish in each group under stimulation with SVCV (10⁵ TCID₅₀). Zebrafish in the control groups were treated with isotype mouse IgG and rescued with mock PBS. (F) Western blot analysis of the recombinant IFNφ1 and IFNφ2 proteins with anti-Flag Ab (1:5,000) from supernatant of HEK293T cells transfected with the pcDNA3.1-His-Flag-IFNφ1 and pcDNA3.1-His-Flag-IFNφ2 recombinant constructs or an empty control construct. In the FCM analysis, different treatments were presented at the top of each block diagram, and the data presented in each block diagram indicated the average percentage of Env38⁺ cells in each treatment group. RT-qPCRs were run in combination with the endogenous β-actin control. Error bars represented SEM. (*p < 0.05; **p < 0.01; ***p < 0.001; ns, no significant difference).