Fig. 1. Parameters of hydrogen nano-bubbles in nano-bubble hydrogen water (nano-HW). A Sizes of hydrogen bubbles in nano-HW were measured with a Nano Sight LM10V-HS system and analyzed using Zetasizer software. B Concentration of hydrogen nano-bubbles in nano-HW samples at different time post-treatment. The experiment was repeated for two times.

Fig. 2. Antiviral activity of nano-HW against SVCV in zebrafish. A Survival of zebrafish in the presence and absence of HW treatment. Size- and age-matched zebrafish (0.6 ?± ?0.1 ?g) were injected with 10 ??L of SVCV suspension (?2 ?× ?108 ?PFU/mL) per fish, and then treated with nano-HW. B Viral gene expression in nano-HW-treated and untreated zebrafish. The relative mRNA expression of SVCV G protein was examined by qRT-PCR in mixed tissues from 3 fish on 1, 3, 5 and 7 dpi. C The virus titer in nano-HW-treated and untreated zebrafish. The viral titer was measured by standard plaque assay with EPC cells. Viral particles in mixed tissues were titered on days 1, 3, 5, and 7 post-infection. Each value was represented as mean ?± ?SEM of three fishes. ?P ?< ?0.05, ??P ?< ?0.01, ???P ?< ?0.001, ????P ?< ?0.0001. ns means no significant difference.

Fig. 3. Histopathology of tissues from nano-HW-treated and untreated zebrafish. Tissue section of intestines (A and B) and brain (C?D) on day 5 post-infection were subjected to H&E stain. Images represent the 20 ?× ?(Scale bar ?= ?100 ??m in A and C) or 40 ?× ?(Scale bar ?= ?50 ??m in B and D) of H&E staining.

Fig. 4. Effects of nano-HW on SVCV-Induced ROS production in zebrafish. ATotal reactive oxygen species were measured in mixed tissues (kidney, intestine, brain, liver and spleen) at 1, 3, 5, and 7 dpi with 2, 7-dichlorofuorescin diacetate (DCFH-DA) probes. B Superoxide anion was monitored in mixed tissues (kidney, intestine, brain, liver and spleen) using dihydroethidium (DHE) at 1, 3, 5, and 7 dpi. Each value was represented as mean ?± ?SEM of three fish. ?P ?< ?0.05, ??P ?< ?0.01, ???P ?< ?0.001, ????P ?< ?0.0001. ns means no significant difference.

Fig. 5. Effect of nano-HW on the inflammatory response in SVCV- infected zebrafish. The IL-1? (A), IL-8 (B), and TNF-? (C) mRNA levels were examined in mixed tissues (kidney, intestine, brain, liver and spleen) of SVCV- infected zebrafish on 1, 3, 5 and 7 dpi. Each value represented the mean ?± ?SEM of three fish. The experiment was repeated for three times. ?P ?< ?0.05, ??P ?< ?0.01, ???P ?< ?0.001, ????P ?< ?0.0001. ns means no significant difference.

Fig. 6. Effect of nano-HW on the inflammatory response in SVCV- infected ZF4 cells. ZF4 cells were cultured in DMEM/F-12 medium or nano-HW-prepared DMEM-F12 medium to reach to a logarithmic growth. ZF4 cells were infected with SVCV (1 MOI), and the mRNA expression levels of IL-1? (A), IL-8 (B), and TNF-? (C) were quantified by qRT-PCR at 12 hpi. The SVCV titer (D) of infected ZF4 cells was determined at 6, 12, 24, and 36 hpi. All data are representatives of three independent experiments. ?P ?< ?0.05, ??P ?< ?0.01, ???P ?< ?0.001, ????P ?< ?0.0001. ns means no significant difference.