Infection model of largemouth bass with LMBV.

(A) Fish were administered an intraperitoneal injection of 100 μL (1D×D106 TCID50) of the virus solution and then sacrificed at intervals of 1, 4, 7, 14, 21, and 28 days post-infection (DPI) for tissue sampling. The timeline uses a red line to indicate fish that died or diseased symptoms of viral infection, while a green line represents fish that remained healthy, showing normal behavior and no signs of viral infection. (B) Clinical observations following infection with LMBV. The black and red arrows indicate skin ulceration and subdermal hemorrhage of infected fish, respectively. (C, D) Spleen size (C) and spleen/body weight ratio (D) following LMBV infection (n = 9). (E) Cumulative survival of LMBV-infected and control fish. (F) LMBV-MCP gene copies (Log10) were measured via qPCR in head kidney (HK), spleen (SP), and gut (n = 6). (G) Immunofluorescence staining of LMBV in paraffin sections of the HK, SP, and gut from fish infected for 4 days and control fish (n = 6). LMBV was stained using an anti-LMBV-MCP monoclonal antibody (red). DAPI stains the cell nuclei blue. Scale bars, 50 μm. (H) The virus particles in the HK, SP, and gut were analyzed by transmission electron microscopy (TEM). Scale bars, 500Cnm. (I) Cytopathic effect (CPE) of LMBV on EPC cells following exposure to supernatant of HK, SP, and gut homogenates from 4 days infected fish. Scale bars, 100 μm. (J) Histological examination of HK, SP, and gut from LMBV-infected and control fish at 4 DPI (n = 6). The red arrow indicates erythrocyte infiltration. The black asterisk indicates cellular necrosis in HK and SP. The black arrow indicates epithelial cell shedding in gut. Scale bars, 50 μm. Data are shown as meanC±CSEM. An unpaired Student’s t-test was used. ***p < 0.001.

The dynamic immune response of largemouth bass upon LMBV infection.

(A–C) Heatmaps display q PCR results for expression of selected immune genes in the HK (A), SP (B), and gut (C) of LMBV-infected and control fish (n = 9). Color values: log2 (mean fold change). (D) Venn diagrams visualize the overlapping genes that are either upregulated or downregulated in the HK and gut of largemouth bass at 4 or 28 DPI when compared to control fish (n = 6). (E) Heat map analysis of all DEGs associated with immune in the HK and gut of largemouth bass compared to control fish at 4 or 28 DPI. (F) GO enrichment analysis of the DEGs in the HK and gut of largemouth bass at 4 or 28 DPI versus control fish. (G) KEGG enrichment analysis of the DEGs in the HK and gut of largemouth bass compared to control fish at 4 or 28 DPI. (H, I) Representative adaptive immune, innate, and antiviral response genes modulated by LMBV infection in the HK (H) and gut (I) of largemouth bass at 4 or 28 DPI.

IgM+ B cells and LMBV-specific sIgM responses of largemouth bass upon LMBV infection.

(A) Diagram illustrating the infection method using LMBV through intraperitoneal injection. Fish received a 100 μL injection of virus (1C×C106 TCID50). The surviving fish from one group were sacrificed at 28DPI, those from another group were re-injected with the same dose of virus at 30 DPI and then sacrificed 42 DPI. These groups are referred to as the 28DPI-S group and the 42DPI-S group, respectively. (B, D) Immunofluorescence staining of IgM (green) in paraffin sections of the largemouth bass HK (B) and gut (D) from control, 28DPI-S, and 42DPI-S fish. DAPI stains the cell nuclei blue. Scale bars, 20Cμm. (C, E) The number of IgM+ B cells the HK (C) and gut (E) from control, 28DPI-S, and 42DPI-S fish were quantified, as counted from B (n = 12). (F, H) Representative flow cytometry dot-plots demonstrate the proliferation of IgM+ B cells in the leukocytes of the HK (F) and gut (H) of control, 28DPI-S, and 42DPI-S fish. Each dot plot shows the percentage of lymphocytes that are proliferative B cells (EdU). (G, I) The percentage of EdU+ cells among the total IgM+ B cell populations in the HK (G) and gut (I) of control, 28DPI-S, and 42DPI-S fish (n = 12). (J, K) IgM protein concentration in serum (J) and gut mucus (K) of fish in the control, 28DP-S, and 42DPI-S group (nlJ= 12). (L, M) LMBV-specific IgM titers of serum (L) and gut mucus (M) from 28DPI-S and 42DPI-S fish (n = 12). Data are shown as meanC±CSEM. An unpaired Student’s t-test was used. *p C< C0.05, **p C< C0.01, and ***p < 0.001.

Resistance to reinfection in largemouth bass that survived LMBV infection.

(A) Strategy to obtain the control and survivor fish. Control fish (Con group) were obtained previously injection with EPC cells culture supernatant. At 42 days, fish were challenged with 100DμL of LMBV (1D×D106 TCID50), and 4 days post-challenge fish (control challenge or CC group) were sacrificed for sampling. 42 DPI survivor fish (42DPI-S group) were injected twice with LMBV (100 μL, 1C×C106 TCID50) at 0 and 28 days, respectively. At 42 days, fish were challenged with LMBV (100 μL, 1C×C106 TCID50), and 4 days post-challenge fish (42DPI-S challenge or SC group) were sacrificed for sampling. (B) Cumulative survival rates of the Con, CC, 42DPI-S, and 42DPI-SC. (C, D) LMBV-MCP gene copies (Log10) were quantified in HK (C), gut (D) of Con, CC, 42DPI-S, and 42DPI-SC using qPCR. (E, G) Immunofluorescence staining of LMBV in HK (E) and gut (G) paraffin-sections from Con, CC, 42DPI-S, and 42DPI-SC fish (n = 6). LMBV (red) was stained with an anti-LMBV-MCP mAb; nuclei (blue) were stained with DAPI. Scale bars, 20Cμm. (F, H) Quantification of LMBV in HK (F) and gut (H) paraffin-sections from Con, CC, 42DPI-S, and 42DPI-SC fish counted from E (nlJ=6). (I, K) Histological examination of HK (I) and gut (K) from Con, CC, 42DPI-S, and 42DPI-SC fish. The red arrow indicates erythrocyte infiltration. The black arrow indicates epithelial cell shedding in gut. The black asterisk indicates cellular necrosis in HK. (J, L) Pathology score of HK (J) and gut (L) from Con, CC, 42DPI-S, and 42DPI-SC fish (n = 6). Data are shown as meanC±CSEM. An unpaired Student’s t-test was used. *p C< C0.05, **p C< C0.01, and ***p < 0.001.

Depletion of IgM+ B cells in fish significantly enhances tissue viral load and increases mortality following LMBV infection.

(A) Schematic of the experimental strategy used to deplete bass IgM+ B cells. In brief, immune fish (those that survived two infections with 100CμL of LMBV at (1C×C106 TCID50 via intraperitoneal injection) were injected with anti-bass IgM mAbs (IgM-depleted immune fish) or either isotype control antibodies (non-depleted immune fish). One day after antibody injection, both non-depleted and IgM-depleted fish were infected with 100CμL of LMBV at (1C×C106 TCID50 via intraperitoneal injection via intraperitoneal injection. Fish from different groups were then analyzed at 2, 4, and 30 DPI for IgM titers, viral load, pathological changes, and mortality, respectively. (B, C) LMBV-specific IgM titers of serum (B) and gut mucus(C) from non-depleted and IgM+ B cell-depleted 42DPI-S fish at 2 DPI (n = 9). (D) H&E staining of largemouth bass HK, SP, and gut from non-depleted and IgM+ B cell-depleted 42DPI-S fish. Red arrow indicates erythrocyte infiltration. Black asterisk indicates cellular necrosis in HK, SP, and gut. Scale bars, 50Cμm. (E) Histological score of HK, SP, and gut in non-depleted and IgM+ B cell-depleted 42DPI-S fish at 4 DPI. (F) LMBV was detected using anti-LMBV mAb (red) in HK, SP, and gut of 42DPI-S fish, both non-depleted (left) and IgM+ B cell-depleted (right). Nuclei were stained with DAPI (blue). Scale bars, 20Cμm. (G) Quantification of LMBV-infected cells was performed in the HK, SP, and gut of both non-depleted and IgM+ B cell-depleted 42DPI-S fish, as counted from F. (H–J) LMBV gene copies (Log10) were quantified by qPCR in HK (H), SP (I), and gut (J) (n = 9). (K) Cumulative survival of non-depleted and IgM+ B cell-depleted 42DPI-S fish infected with LMBV. Data are shown as meanC±CSEM. An unpaired Student’s t-test and log-rank (Mantel-Cox) test in K were used. *p C< C0.05, **p C< C0.01, and ***p < 0.001.

Viral neutralization exerted by LMBV-specific sIgM of serum and gut mucus.

(A) Schematic of the experimental strategy. Magnetic protein G beads were incubated with anti-bass IgM mAbs to produce protein G beads coated with these antibodies. IgM in 42DPI-S serum and gut mucus was removed by incubating these samples with protein G beads coated with anti-bass IgM mAbs. Thereafter, serum and gut mucus obtained from control, 42DPI-S, or 42DPI-S-IgMDEP fish, and each of these was incubated with LMBV and then added to EPC cells. EPC cells were treated with different serum, gut mucus, or medium alone, resulting in three distinct treatment groups for the EPC cells. (including: Control (control serum/gut mucus-LMBV-EPC cells), 42DPI-S (42DPI-S serum/gut mucus-LMBV-EPC cells), and 42DPI-S-IgMDEP (42DPI-S-IgMDEP serum/gut mucus-LMBV-EPC cells)) were analyzed at 2 and 4 days after addition for EPC cell viability and LMBV loads. (B, C) Cell viability in the different groups was measured using the colorimetric alamarBlue assay and expressed as relative values compared to those of EPC cells that were treated with serum (B) and gut mucus (C) (n = 12). (D) The cellular cytopathic effect in the different groups was assessed by the crystal violet stain. (E, F) LMBV-MCP gene copies (Log10) were quantified by qPCR in EPC cells from the serum treatment group(E) and the gut mucus group (F) (n = 12). (G, H) The LMBV-MCP protein expression in EPC cells from the treatment with serum group (G) and gut mucus group (H) was evaluated by western blot. (I, J) Relative expression of LMBV-MCP protein in EPC cells from the serum treatment group (I) and the gut mucus group (J) was assessed using densitometric analysis of immunoblots (n = 12). (K, M) The LMBV-MCP protein in EPC cells from the serum treatment group (K) and the gut mucus group (M) was identified via immunofluorescence using the anti-LMBV-MCP mAb. Scale bars, 100Cμm. (L, N) The number of virally infected cells, as determined from K and M (nlJ= 12). Data are shown as meanC±CSEM. One-way analysis of ANOVA was used. *p C< C0.05, **p C< C0.01, and ***p < 0.001.

Time-of-addition experiments.

(A) EPC cells or LMBV was treated with serum or gut mucus from control, 42DPI-S, or 42DPI-S-IgM DEP fish at different times before and after infection. Samples were harvested at 24 hpi. G1-G8 respectively represent serum/gut mucus treatments during the different time periods. (B, C) LMBV-MCP gene copies (Log10) was quantified by qPCR in EPC cells from the treatment with serum (B) or gut mucus (C) (n = 6). (D, F) The LMBV-MCP protein in EPC cells from the treatment with serum (D) or gut mucus (F) was detected by western blot using anti-LMBV-MCP monoclonal antibody and anti-β-actin antibody (n = 6). (E, G) Expression of LMBV-MCP/β-actin in EPC cells from the treatment with serum (B) or gut mucus at different stage of LMBV infection group. Values were normalized against those for group 1 (G1, no treatment). Data are shown as mean ± SEM. One-way analysis of ANOVA was used. **p < 0.01 and ***p < 0.001.

Schematic of the proposed function and mechanism of teleost sIgM in response to LMBV infection.

In a healthy state, the gut contains few IgM+ B cells and lower levels of sIgM protein. Following the primary LMBV infection, the gut exhibits severe pathological changes, including erythrocyte infiltration, epithelial cell shedding, and a strong innate and adaptive immune response. Antigens (Ag) from LMBV are captured by antigen-presenting cells (APCs) and subsequently presented to naive CD4+ T cells. These antigen-specific CD4+ T cells then activate IgM+ B cells, prompting their proliferation. Subsequently, activated B cells may differentiate into plasma cells or memory IgM+ B cells. Upon secondary LMBV infection, plasma cells produce substantial quantities of LMBV-specific IgM. Critically, these virus-specific sIgM from both mucosal and systemic sources has the ability to neutralize the virus by directly binding viral particles and blocking host cell entry, thereby effectively reducing the proliferation of viruses within tissues. Consequently, the IgM-mediated neutralization confers protection against LMBV-induced tissue damage and significantly reduced mortality during secondary infection.

Phylogeny of the Ig heavy chain isotypes in jawed vertebrates, related to Introduction.

IgM stands out as the isotype conserved among jawed vertebrates.

Isotype control staining for anti-LMBV-MCP in largemouth bass head kidney (HK), spleen (SP), and gut paraffin sections, related to Figure 1.

Immunofluorescence staining of LMBV using isotype control antibodies for anti-LMBV-MCP in the HK, SP, and gut paraffin-sections, stained for LMBV (red); nuclei (blue) were stained with DAPI. Scale bars, 50 μm.

Validation of anti-bass IgM mAb by mass spectrometry, related to Figure 3.

(A) Affinity-purified IgM (∼1 μg) from largemouth bass serum was separated by 4-15% SDS-PAGE under reducing conditions and visualized with Coomassie blue staining. The band corresponding to the IgM heavy chain was excised for in-gel tryptic digestion followed by mass analysis. (B) Quantitative mass spectrometry showed that IgM heavy-chain peptides accounted for 97.3% of the total signal. (C) Database searching of mass spectra against the NCBI non-redundant database revealed 54 peptide matches to the IgM heavy chain of largemouth bass (QLF98876.1), comprising 72.2% of the IgM heavy chain sequence. The numbered amino acid sequence with highlighted peptide matches is shown in highlight green. (D) Mass spectrum of IgM peptides.

Validation and characterization of anti-bass IgM mAb, related to Figure 3.

(A, B) Serum samples were analyzed by western blot with anti-bass IgM (A) or an isotype-matched control antibody (B) under non-reducing conditions (NRD) or reducing conditions (RD). (C–F) Tissue lysates from various sources (i.e., spleen, skin, gill, and gut) were analyzed by western blot with a monoclonal antibody targeting largemouth bass IgM or an isotype-matched control antibody under NRD. (G) Flow cytometry of head kidney stained with anti-bass IgM mAb. (H, I) Gene-expression profiles of sorted IgM+ B cells and IgM− B cells among head kidney leukocytes.

Isotype control staining for anti-IgM in largemouth bass head kidney (HK), spleen (SP), and gut paraffin sections, related to Figure 3.

Immunofluorescence staining of IgM using isotype control antibodies for anti-bass IgM in the HK, SP, and gut paraffin-sections, stained for IgM (green); nuclei (blue) were stained with DAPI (blue). Scale bars, 20 μm.

Flow cytometry analysis of IgM+ B cells response in largemouth bass upon LMBV infection, related to Figure 3.

Head kidney (HK) and gut leukocytes of largemouth bass that injected with LMBV were isolated at 28 and 42 DPI. (A, B) Frequencies of IgM+ B cells in gated lymphocyte population of largemouth bass HK (A) and gut (B) after infection with LMBV. (C, D) Percentage of IgM+ cells from the HK (C) and gut (D) leukocytes populations in control, 28DPI-S, and 42DPI-S fish (n = 12). Statistical differences were evaluated by unpaired Student’s t-test. Data in C and D are representative of at least three independent experiments (mean ± SEM). **p < 0.01 and ***p C< 0.001.

Analysis of IgM+ B cells and IgM concentration in largemouth bass after IgM+ B cells depletion treatment, related to Figure 5.

(A–C) Flow cytometry of blood (A), spleen (B), and gut (C) leukocytes from control (left) and IgM+ B cell-depleted (right) fish after 3 days depletion treatment. Numbers adjacent to outlined areas indicate the percentage of IgM+ B cells in the lymphocyte population. (D–F) The percentage of IgM+ B cells within the lymphocyte population of blood (D), spleen (E), and gut (F) in control or IgMCdepleted fish (n = 6). (G, H) The depletion effects of IgM from the serum (G) or gut mucus (H) of control or IgMCdepleted fish was detected by western blot using anti-bass mAb (n = 6).C: Control group; D: IgMCdepleted group. (I, J) Concentration of IgM in serum (I) and gut mucus (J) of control and IgM-depleted fish as determined from G and H. Data are representative of at least three independent experiments (means ± SEM). Statistical analysis was performed by unpaired Student’s t test. *p < 0.05, **p < 0.01, and ***p < 0.001.

LMBV-specific IgM markedly decrease cytopathic effect (CPE) caused by LMBV, related to Figure 6.

(A) The CPE in the EPC cells after treatment with serum from the control, 42-DPI-S, and 42-DPI-S-IgMDEP fish. (B) The CPE in the EPC cells after treatment with gut mucus from the control, 42-DPI-S, and 42-DPI-S-IgMDEP fish. Scale bar = 50 μm.

Viral neutralization exerted by purified LMBV-specific sIgM of serum and gut mucus.

(A, B) LMBV-MCP gene copies (Log10) were quantified by qPCR in EPC cells from the purified LMBV-specific sIgM of serum treatment group (A) and the gut mucus group (B) (n = 6). (C, E) The LMBV-MCP protein expression in EPC cells from the treatment with the purified LMBV-specific sIgM of serum group (C) and gut mucus group (E) was evaluated by western blot. (D, F) Relative expression of LMBV-MCP protein in EPC cells from the purified LMBV-specific sIgM of serum treatment group (D) and the gut mucus group (F) was assessed using densitometric analysis of immunoblots (n = 6). (G) The cellular cytopathic effect in the different groups was assessed by the crystal violet stain.

Primers used in this study, related to Figure 2.