The VHSV infection experiment in viperin^-/- zebrafish. rVHSV immersion experiment for larval zebrafish. (A) WT control larvae, (A’)viperin^-/- control larvae. (B) WT, (C)viperin^-/- fish were injured by caudal fin amputation method and then immersed in rVHSV. Migration of virus was analyzed after 24, 48, and 72 h postinfection (hpi). According to the experiment, rVHSV rapidly migrated into the internal tissue region of viperin^-/- fish compared to WT fish. A clinical score represents the quantitative analysis of pathogenic symptoms. (D) Mortality recording for injury immersion experiment, according to the analysis, the viperin^-/- fish had significantly higher mortality. (E) Morphology under VHSV infections. Adult WT and viperin^-/- fish showed no differences in their morphology without VHSV infections. When infected with VHSV, both fish showed abnormalities such as edema, hemorrhage, and spinal defects causing head protrusions above the body axis (14 dpi). (F) A mortality experiment was conducted for the viperin^-/- fish (n = 15/group). Two doses of VHSV were intraperitoneally injected into the fish (high dose 5 × 10⁶ TCID₅₀/fish and the low dose 1 × 10⁶ TCID₅₀/fish). Fish mortalities were recorded from 4 dpi onward. Mortality data were analyzed by the Kaplan–Meier (KM) method, and statistical significance was plotted using the Mantel–Cox test (p < 0.05). (G) VHSV copy number between the VHSV-injected viperin^-/- and WT fish over time is shown, indicating a higher VHSV copy number in the viperin^-/- fish. NP gene (H) expression in the viperin^-/- fish shows significantly elevated levels compared to that in WT fish. VHSV, Viral hemorrhagic septicemia virus; rVHSV, Recombinant viral hemorrhagic septicemia virus; WT, wild type: dpi, days post infection; NP, VHSV Nucleoprotein.

Metabolic alterations in the viperin^-/- fish. (A) ROS quantification in vitro. viperin-overexpressing cells were analyzed for ROS production, and results are indicating a higher ROS production with the presence of Viperin. (B) The expression of metabolic related genes under VHSV infection in adult zebrafish. According to the analysis, the expression of hif1a, soda, and ldha is downregulated in viperin^-/- fish. Expression of genes responsible for lipid synthesis, acaca and fasna, has upregulated. (C) Lipid analysis in vitro, the viperin-overexpressing cells had significantly lower levels of lipids under virus infection. (D) Cholesterol analysis in vivo, viperin^-/- fish had a significantly higher amount of cholesterol compared to that in WT fish. ROS, Reactive oxygen species; hif1a, hypoxia-inducible factor-1; soda, superoxide dismutase; ldha, Lactate dehydrogenase; acc, Acetyl-CoA carboxylase; fasn, fatty acid synthase; ifn, interferon.

Neutrophil and macrophage recruitment in viperin^-/- fish. (A) Zebrafish lines viperin^-/-Tg(mpx:mcherry) and viperin^-/-Tg(mpeg:egfp) were generated to track neutrophils and macrophages, respectively. The caudal fin was amputated of these lines and immersed in poly I:C. (B) Analysis of neutrophil recruitment, (C) the recruitment of the macrophages to the site of infection was observed in a time-dependent manner (I-Wt PBS, II-Wt+Poly I:C, III-viperin^-/- + PBS, and IV-viperin^-/- + Poly I:C). Dynamics of the neutrophils (D), macrophages (E) at the wound site showed time-dependent differences between the WT and viperin^-/- fish. Expression analysis of neutrophil marker genes mpx(F) and nox(G) in the adult zebrafish with VHSV infection showed significant upregulation of the marker genes in viperin^-/- fish compared to those in WT fish. Gene expression analysis for the marker gene csf1a(H) for macrophages, indicating significant downregulation in viperin^-/- fish. Poly I:C, Polyinosinic:polycytidylic acid; hpi, Hours post infection; mpx, myeloid-specific peroxidase; nox, NADPH oxidase; csf1a, colony stimulating factor 1.