Fig 9

(A) The functional role of Env38 was verified by the impairment of the antiviral activity of Env38^-/- zebrafish after vaccination with formaldehyde-inactivated SVCV vaccine or infection with live SVCV. In both wildtype and knockout zebrafish, the immunized groups were i.p. inoculated with vaccine derived from 0.4% formaldehyde-inactivated SVCV (10⁶ TCID₅₀) and then challenged with SVCV (10⁵ TCID₅₀); whereas the unimmunized groups were challenged with SVCV (10⁵ TCID₅₀) without vaccination. Data points were from three independent experiments, and 30 zebrafish were used in each experiment (n = 30). Differences were analyzed using log-rank test. (B) Viral titer (TCID₅₀) was measured to evaluate the viral load in the wild type and knockout zebrafish with or without vaccinated immunoprotection. Viral samples were prepared from zebrafish at 4th day post SVCV (10⁵ TCID₅₀) infection. The TCID₅₀ was examined in EPC cells by using Reed-Muench method. Viral titers were log₁₀ transformed prior to statistical analysis. Data points were from three independent experiments, and three zebrafish were used for viral sample preparation in each experiment (n = 3). (C and D) The degree of CD4⁺ T and IgM⁺ B cell activation between WT and KO zebrafish under SVCV (10⁵ TCID₅₀) stimulation was represented by the percentage of CD4⁺CD154⁺ T and IgM⁺CD40⁺ B cells with mouse anti-CD154 (1:500), rabbit anti-CD4 (1:500) (C) and mouse anti-mIgM (1:2,000), rabbit anti-CD40 (1:500) (D) through determination with FCM analysis. The data presented in each block diagram indicated the average percentage of CD4⁺CD154⁺ T and IgM⁺CD40⁺ B cells in WT and KO zebrafish. (E) The degree of CD4⁺ T or IgM⁺B cell activation between WT and KO zebrafish under SVCV (10⁵ TCID₅₀) stimulation was represented by the transcriptional levels of cd154, lck, cd40 and IgM through RT-qPCR. The 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).