RVFV infection in zebrafish larvae resulting in viral replication and innate immune response. (A) Schematic representation of the experimental timeline, conditions, and injection site. Drawings acquired through BioRender. (B) Viral RNA copies of RVFV_35/74 per zebrafish larva. Mean ± standard error of the mean (s.e.m.) of 5–8 pools of 10 larvae from 8 independent experiments. The dotted line shows limit of quantification (LOQ). (C) Titres of RVFV_35/74 infectious viral particles in zebrafish larvae. Mean ± s.e.m. of 6 pools of 10 larvae from 6 independent experiments. (D) Relative fold induction of infϕ1, mxa, isg-15, and rsad2 in RVFV_35/74 infected zebrafish larvae compared to PBS-injected zebrafish larvae. Results determined by RT-qPCR and normalized to 18 s, ef1α, and β-actin at 0 dpi. Mean ± s.e.m. of 8 pools of 10 larvae from 8 independent experiments. (E) Viral RNA copies of RVFV_35/74 per zebrafish larva after immersion in Danieau’s solution containing the vehicle, 1000 µM, or 10 mM 2′-FdC at 0 dpi. Mean ± s.e.m. of 5 pools of 10 larvae from 5 independent experiments. The dotted line shows LOQ. dpf = days post fertilization; dpi = days post infection; RVFV = Rift Valley fever virus; 2′-FdC = 2′-Fluoro-2′-deoxycytidine; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant.

Viral replication and dissemination of RVFV_eGFP in zebrafish larvae, with infection of the liver, sensory nervous system and vascular system. (A) Viral RNA copies of RVFV_eGFP per zebrafish larva. Mean ± s.e.m. of 6–8 pools of 10 larvae from 8 independent experiments. (B–D,F) Whole mount immunohistochemistry fluorescence images of RVFV_eGFP-infected zebrafish larvae at 10× magnification, using anti-GFP primary antibody and Hoechst 33342. (B) Wildtype zebrafish larva at 1 and 2 dpi showing progressing RVFV infection around the site of injection. (C) Tg (fabp10a:DsRed; nacre) zebrafish larvae demonstrating infection in the liver using anti-RFP primary antibody. (D) Wildtype zebrafish larva showing infection of the retina, optic tectum (yellow circle), the anterior and posterior macula (red circle), the ganglia (yellow arrows), and neuromasts (red arrows) of the lateral line. dpi = days post infection. (E) Viral RNA copies of RVFV_35/74 and RVFV_eGFP per zebrafish larva immersed in Danieau’s solution supplemented with ruxolitinib (25 µM) from 0 dpi. Inoculum: ~1 CCID₅₀ of RVFV_35/74 or ~15 CCID₅₀ of RVFV_eGFP. Mean ± s.e.m. of 8 pools of 10 larvae from 8 independent experiments. (F) Wildtype zebrafish larva at 6 dpi, showing extensive dissemination of RVFV_eGFP infection after immersion in Danieau’s solution supplemented with ruxolitinib (25 µM) from 0 dpi. Image has not been 3D-deconvoluted. dpi = days post infection; RVFV = Rift Valley fever virus; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant.

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.