Characterization of PR8 and Color-flu systemically infected AB zebrafish. (A) Decreased survival of AB zebrafish systemically infected with three different lots of PR8 IAV at 2 dpf compared to vehicle (HBSS) controls (p < 0.0001 for each lot comparison). Survival rates of PR8-infected embryos were not significantly different between lots (p = 0.0603). (B) Decreased survival of AB zebrafish systemically infected with two different lots of PR8 IAV at 3 dpf compared to vehicle controls (p < 0.0001 for each lot comparison). Survival rates of PR8-infected embryos were not significantly different between lots (p = 0.1442). (C) Decreased survival of AB zebrafish systemically infected with eCFP-PR8 or Venus-PR8 (3.2 × 102 TCID₅₀/mL) compared to vehicle controls (p < 0.0001 for each comparison). Survival rates of eCPF-PR8- or Venus-PR8-infected zebrafish were not significantly different strains (p = 0.5238). (D) Increased TCID₅₀ viral titer in Color-flu systemically infected AB zebrafish at 24 and 48 hpi compared to 0 hpi. eCFP-PR8-, mCherry-PR8-, and Venus-PR8-infected zebrafish had increased viral titers at 24 and 48 hpi compared to 0 hpi (adjusted p-values = 0.0001 and <0.0001, respectively, for eCFP-PR8; 0.0019 and 0.0001, respectively, for eGFP-PR8; 0.0047 and <0.0001, respectively, for mCherry-PR8; and 0.0003 and <0.0001, respectively, for Venus-PR8). Survival studies were conducted with four independent experiments (n = 4) and 50 larvae per sample group. TCID₅₀ assays were carried out with three independent experiments (n = 3) and 25 larvae per group for each time point. Not significant (ns), p > 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Characterization of PR8 and Color-flu systemic infection across zebrafish lines. (A) Decreased survival was greater in casper than AB or EK lines with 2 dpf systemic PR8 infection. PR8-infected zebrafish had a decreased survival rate compared to vehicle (HBSS) controls (p < 0.0001 for all lines). Casper zebrafish had a lower survival rate than EK (p = 0.0024) and AB (p = 0.0055) according to the log-rank Mantel–Cox test. (B) Decreased survival rates were observed in AB, EK, and casper with 3 dpf PR8 infection compared to controls (p < 0.0001 for all lines). No significant (ns) difference in survival rate was detected between PR8-infected AB, EK, and casper larvae. (C) Increased TCID₅₀ viral titer in 2 dpf PR8-infected AB, EK, and casper zebrafish at 24, 48, and 72 hpi compared to 0 hpi (adj. p-value < 0.001 for all comparisons except for AB 24 hpi (adj. p-value = 0.0006) and AB 96 hpi (adj. p-value = 0.0003)). (D) Increased TCID₅₀ viral titer in 3 dpf PR8-infected AB, EK, and casper zebrafish at 72 hpi compared to 0 hpi (adj. p-value = 0.0323, 0.0211, and 0.0452, respectively) and casper zebrafish at 48 hpi compared to 0 hpi (adj. p-value = 0.0063). (E) Decreased survival with 3 dpf mVenus-PR8-infected AB, EK, and casper zebrafish compared to HBSS controls (p < 0.0001 for all lines). As observed with PR8 infection, casper zebrafish had the lowest survival rate (67.7%), which was lower than that of AB (94.5%, p < 0.0001) and EK (85.5%, p < 0.0002). (F) mVenus-PR8-infected AB, EK, and casper zebrafish had an increased viral titer at 24 and 48 hpi compared to 0 hpi (adjusted p-values = 0.0012 and 0.0022, respectively, for AB; 0.0025 and 0.0027, respectively, for EK; and 0.0045 and 0.0066, respectively, for Venus-PR8). mVenus-PR8-infected AB and EK zebrafish had an increased viral titer at 72 hpi compared to 0 hpi (adjusted p-value = 0.0236, and 0.0281, respectively). Survival studies were conducted with four independent (n = 4) experiments and 50 larvae per sample group. TCID₅₀ assays were carried out with three independent (n = 3) experiments and 25 larvae per group for each time point. Not significant (ns), p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Confocal imaging of Color-flu-infected zebrafish. (A) Representative images of larvae at 24 h post systemic injection of HBSS, eCFP-PR8, eGFP-PR8, mCherry-PR8, and mVenus-PR8 at 3 dpf, at 4× resolution. Scale bar: 300 mm. BF: brightfield. (B) Representative images of Tg(mpeg1:eGFP;lyz:dsRed) larvae at 24 h post systemic injection of HBSS, eCFP-PR8 at 3 dpf at 10× resolution, showing macrophages (green), neutrophils (red), and eCFP-PR8 (blue). Infected skeletal muscle was detected (white arrowheads). Scale bar: 700 μm.

Ramipril and MDIVI-1 increase survival in systemically infected PR8 and Color-flu-infected 3 dpf zebrafish. (A) Increased survival in PR8-infected AB zebrafish treated with 0.2 nM ramipril compared to DMSO controls (**** p < 0.0001). (B) Increased survival in PR8-infected AB zebrafish treated with 7 nM MDIVI-1 compared to DMSO controls (p < 0.0001). (C) Increased survival in mVenus-PR8-infected AB zebrafish treated with 0.2 nM ramipril or 7 nM MDIVI-1 compared to DMSO controls (p < 0.0001). Survival studies were conducted with n = 4 and 50 larvae per sample group.

Ramipril and MDIVI-1 treatments lower viral burden in IAV-infected zebrafish. (A) Viral titers for both ramipril (0.2 nM)- and MDIVI-1 (7 nM)-treated 2 dpf zebrafish infected with PR8 were significantly higher at 24 and 48 hpi when compared to 0 hpi, but not different at 72 hpi. For the DMSO-treated zebrafish, viral titers were significantly increased at 24, 48, and 72 hpi (p < 0.0001 for each comparison). For the ramipril-treated zebrafish, viral titers were significantly increased at 24 (p < 0.0001) and 48 hpi (p = 0.0022). For the MDIVI-1-treated zebrafish, viral titers were also significantly increased at 24 and 48 hpi (p < 0.0001 for each comparison). (B) Viral titers for ramipril- and MDIVI-1-treated 3 dpf zebrafish infected with PR8 were significantly lower by 48 hpi (p < 0.0001 for each comparison). (C) Viral titers for ramipril- and MDIVI-1-treated 3 dpf zebrafish infected with mVenus-PR8 were significantly lower at 48 hpi (p < 0.0001 for each comparison). TCID₅₀ assays were conducted using n = 3 and 25 larvae per group for each time point. Not significant (ns), p > 0.05; ** p < 0.01; **** p < 0.0001.

Ramipril and MDIVI-1 treatment alters the respiratory burst response in IAV systemically infected zebrafish at 48 hpi. (A) Respiratory burst response in larvae treated with DMSO, ramipril (0.2 nM), and MDIVI-1 (7 nM) at 48 hpi following systemic injection at 3 dpf with PR8 or HBSS. PR8 infection decreased the response over HBSS in DMSO-treated controls (adjusted p = 0.0008). Both ramipril and MDIVI-1 treatment remedied the reduction in respiratory burst response, as the PR8-infected larvae had the same response as HBSS-injected controls (comparisons were not significant (ns)). The protein kinase C inhibitor bisindolylmaleimide I (BisI) was used as a positive control as it suppresses the respiratory burst response (adj. p < 0.0001 for all comparisons). (B) Respiratory burst response in larvae treated with DMSO, ramipril (0.2 nM), and MDIVI-1 (7 nM) at 48 hpi following systemic injection at 3 dpf with mVenus-PR8 or HBSS. Similar to the PR8-infected larvae, mVenus-PR8 infection decreased the response over HBSS in DMSO-treated controls (adj. p = 0.0040). Ramipril treatment resulted in a higher respiratory burst response with mVenus-PR8 infection than HBSS controls (adj. p = 0.0049). MDIVI-1 treatment remedied the reduction in respiratory burst response as the mVenus-PR8-infected larvae had the same response as HBSS injected controls. The BisI controls were different to the DMSO (adj. p < 0.0001 for both), ramipril (adj. p = 0.0003 for HBSS, and adj. p < 0.0001 for mVenus-PR8), and MDIVI-1 (adj. p = 0.0006 for HBSS, ns for mVenus-PR8) groups. Not significant (ns), p > 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Quantification of the number of neutrophils (A), macrophages (B), and the relative abundance of virus infected cells (C) via fluorescent confocal imaging of Tg(mpeg1:eGFP;lyz:dsRed) larvae at 48 h post injection by eCFP-PR8 or HBSS following treatment with DMSO, ramipril, and MDIVI-1 per optical cross section. For each larva, 10 five-micron optical cross sections were analyzed, which together totaled 50 microns (n = 4 representative larvae). (A) The number of neutrophils increased with eCFP-PR8 infection following DMSO treatment over HBSS controls (adjusted p-value = 0.0145), and with ramipril treatment (adj. p-value = 0.0010), but not with MDIVI-1 treatment. (B) The number of macrophages decreased in eCFP-PR8-infected larvae treated with DMSO over HBSS controls (adj. p-value = 0.0020), but was not different with ramipril or MDIVI-1 treatment. MDIVI-1 treatment increased the number of macrophages compared to the DMSO controls (adj. p-value < 0.0001), and ramipril-treated larvae (adj. p-value = 0.0126). (C) The extent of viral infection was higher in eCFP-PR8-infected larvae treated with DMSO, ramipril, and MDIVI-1 (adj. p-value < 0.0001 for all comparisons). The level of virus infection was lower with ramipril (adj. p-value < 0.0001) and MDIVI-1 (adj. p-value = 0.0008) treatment compared to the DMSO-treated controls. Not significant (ns), p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.