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. 2022 Apr 5;8:31. doi: 10.1038/s41421-022-00393-3

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a, b Scheme of the infection strategy with IHNV via SB injection. Fish were injected with 25 μL of virus (1 × 10⁵ TCID₅₀) and one group of surviving fish was sacrificed at 28 DPI (28DPI-S group) while another group was reinfected twice at 30 and 60 DPI with the same viral dose, and the resulting surviving fish were sacrificed at 75 days after the first infection (75DPI-S group). c Differential interference contrast (DIC) images of immunofluorescence staining on trout SB paraffin-sections from uninfected (control) fish, 28DPI-S and 75DPI-S fish, stained for IgT (green) and IgM (red) (Isotype-matched control antibody staining is shown in Supplementary Fig. S2c); nuclei (blue) were stained with DAPI (blue). SC SB cavity, SM SB mucosa, ML muscle layer. Scale bars, 20 μm. d The number of IgT⁺ and IgM⁺ B-cells in SB paraffin-sections of control, 28DPI-S and 75DPI-S fish counted from c (n = 9). e, f Concentration of IgT, IgM, and IgD protein in SB mucus (e) and serum (f) of control, 28DP-S, and 75DPI-S fish (n = 9–16). g, i Western blot analysis of IgT-, IgM-, and IgD-specific binding to IHNV in SB mucus (dilution 1/2) from 28DPI-S (g) and 75DPI-S fish (i). h, j IgT-, IgM- and IgD-specific binding to IHNV in dilutions of SB mucus from 28DPI-S (h) and 75DPI-S (j) fish evaluated by densitometric analysis of immunoblots and presented as relative values to those of control fish (n = 8–12). k, m Western blot analysis of IgT-, IgM-, and IgD-specific binding to IHNV in serum (dilution 1/10) from 28DPI-S (k) and 75DPI-S fish (m). l, n IgT-, IgM-, and IgD-specific binding to IHNV in dilutions of serum from 28DPI-S (l) and 75DPI-S (n) fish evaluated by densitometric analysis of immunoblots and presented as relative values to those of control fish (n = 8–12). Statistical differences were evaluated by one-way ANOVA with Bonferroni correction (d–f) and were performed by unpaired Student’s t-test (h, j, l, n). Data are representative of at least three independent experiments (mean ± SEM). *P < 0.05, **P < 0.01, and ***P < 0.001.