Survival rates of zebrafish larvae challenged with NNV through different infection routes and NNV replication. (A) Schematic representation of the different infection routes used to determine the susceptibility of zebrafish larvae to NNV. (B) Kaplan–Meier survival curves of NNV-infected and uninfected larvae in 3- and 14-dpf larvae. Mortality was registered during the next 10 dpi. (C) Quantification of NNV capsid protein gene expression in 3-dpf larvae infected via the brain or intramuscularly at different sampling points through qPCR; data are presented as the mean ± SEM of biological replicates. Statistically significant differences are displayed as follows: ***, 0.0001 > p value > 0.001.

Image analysis of the swimming behavior of zebrafish larvae (3 and 14 dpf) infected with NNV via brain or IM. (A) Comparison of velocity, directionality, accumulated distance and Euclidean distance parameters between NNV-infected and the corresponding uninfected control larvae. Video tracking of zebrafish larvae was conducted at different times postinfection (3, 6, and 10 dpi). The fold change (FC) of infected larvae compared with their uninfected control (Control FC = 1, dotted lines) was calculated. The graphs represent the mean ± SEM of the biological replicates. Statistically significant differences are displayed as follows: ***, 0.0001 > p value > 0.001; **, 0.001 > p value > 0.01; *, 0.01 > p value > 0.05. (B) Example of maximal projection of the video recorded for 3-dpf larvae infected via brain and the corresponding controls at 6 dpi.

Whole-mount immunofluorescence of zebrafish larvae infected by intramuscular microinjection with NNV. Confocal images of the head from uninfected and NNV-infected larvae at 24 and 48 hpi. NNV particles are stained red, and cell nuclei are stained blue (DAPI). White arrows denote the position of NNV-infected cells.

Visualization and analysis of innate immune cell migration to the head of 3 pdf larvae infected through different routes. The transgenic zebrafish lines (A) Tg(lyz:DsRed2), (B) Tg(mpx:GFP) and (C) Tg(mpeg:mCherry) were used to analyse the migration of myeloid precursors with lysozyme activity, neutrophils and macrophages, respectively, to the cephalic region. Larvae were infected through the 4 infection routes analysed in this study, and the cells were counted at 1, 2 and 3 dpi. Fluorescent immune cells were counted using ImageJ, and the graphs represent the difference in fold change of the number of cells located in the head from infected larvae compared to their corresponding uninfected control larvae (control FC = 1 in the dotted line). Representative images of the three transgenic lines at 2 dpi were included. (D) Whole-mount immunofluorescence of Tg(mpx:GFP) transgenic larvae infected via the brain with NNV at 1 dpi; neutrophils are displayed in green, NNV are displayed in red, and nuclei are displayed in blue. No colocalization between NNV particles and neutrophils was observed. (E) Expression analysis of marker genes of the two major innate immune cells (mpx – neutrophils, marco – macrophages) in NNV-infected and uninfected larvae at different times postinfection. Each sample (5 biological replicates, pools of 4 larvae each) was normalized to the 18S gene. The normalized expression values were standardized against their respective controls (Control FC = 1, dotted lines). For (A–C, E), the graphs represent the mean ± SEM of the biological replicates. Statistically significant differences are displayed as follows: ***, 0.0001 > p value > 0.001; **, 0.001 > p value > 0.01; *, 0.01 > p value > 0.05.

Transcriptome analysis of 3-dpf zebrafish larvae infected with NNV via the brain. (A) RNA-Seq experimental design: Zebrafish larvae were microinjected via the brain, and three pools of infected and uninfected larvae were sampled at 1, 3, and 5 days postinfection for RNA isolation and Illumina sequencing. (B) Kaplan–Meier survival curves of NNV-infected and uninfected larvae conducted in parallel to RNA-Seq sampling. Statistically significant differences are displayed as follows: ****, p value < 0.0001. (C) Principal component analysis (PCA) of the samples.

Differentially expressed genes in zebrafish larvae infected with NNV. (A) Heatmaps representing the TPM expression values of the DEGs (FC > |2|; FDR < 0.05) modulated at 1, 3 and 5 dpi. Expression levels are represented as row-normalized values on a blue–red colour scale. (B) Stacked column chart reflecting the number and intensity (in FC value) of the DEGs identified at the 3 sampling points. (C) Venn diagram reflecting the common and exclusive DEGs at each sampling point.

GO terms and KEGG pathways enriched during NNV infection of zebrafish larvae. (A) GO biological process terms significantly enriched at 1, 3, and 5 dpi. (B) KEGG pathways enriched at 1, 3, and 5 dpi; the four common pathways significantly enriched over time are boxed.

Heatmap representing the DEGs linked to the type I IFN system at 1, 3, and 5 dpi. A heatmap was constructed with the TPM expression values of the DEGs. Expression levels are represented as row-normalized values on a white–purple colour scale.

Heatmaps representing the DEGs belonging to different immune categories at 1, 3 and 5 dpi: (A) cytokines; (B) pattern recognition receptors; (C) complement system; and (D) galectins. Heatmaps were constructed with the TPM expression values of the DEGs. Expression levels are represented as row-normalized values on a white–purple colour scale.