Figure 3

The effect of stat1a and stat1b knockout on RVFV viral replication, dissemination, and innate immune response. (A) Schematic representation of the creation of stat1a^−/− and stat1b^−/− larvae. Created in BioRender. (B) The relative mRNA expression fold change in stat1a and stat1b in stat1a^−/− zebrafish larvae. (C) The relative mRNA expression fold change in stat1a and stat1b in stat1b^−/− zebrafish larvae. (D) Survival curves of stat1a^+/+ (n = 69), stat1a^+/− (n = 129), and stat1a^−/− (n = 74) larvae of the F2 generation. (E) Survival curves of stat1b^+/+ (n = 71), stat1b^+/− (n = 92), and stat1b^−/− (n = 55) larvae of the F2 generation. (F) Viral RNA copies of RVFV_35/74 per stat1a^−/− zebrafish larva. Inoculum: ~1 CCID₅₀ of RVFV_35/74. Mean ± s.e.m. of 5–6 pools of 10 larvae from 6 independent experiments. (H) Viral RNA copies of RVFV_35/74 per stat1b^−/− zebrafish larva. Inoculum: ~1 CCID₅₀ of RVFV_35/74. Mean ± s.e.m. of 6 pools of 10 larvae from 6 independent experiments. (G,I) Whole mount immunohistochemistry fluorescence images of RVFV_eGFP infected stat1a^−/− (G) and stat1b^−/− (I) zebrafish larvae at 10× magnification, using anti-GFP primary antibody (green) and Hoechst 33342 (blue). Inoculum: ~15 CCID₅₀ of RVFV_eGFP. Stat1a^−/− zebrafish larva at 4 dpi, showing extensive dissemination of RVFV_eGFP infection. Stat1b^−/− zebrafish larva at 3 dpi, showing limited dissemination of RVFV_eGFP infection. (J) Fold induction of gene expression relative to wildtype larvae of hematopoietic markers in stat1a^−/− and stat1b^−/− larvae, respectively, at 6 dpf. Results determined by RT-qPCR and normalized to 18s, ef1α, and β-actin mRNA. dpi = days post infection; RVFV = Rift Valley fever virus; WT = wildtype; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant.