Infectious bursal disease virus suppresses humoral immunity via blocking antibody affinity maturation in chickens

Figure 1. IBDV infection inhibits the affinity maturation of antibodies against viral antigens (A) Schematic diagram illustrates the timeline of viral infection and serum collection. Blue arrows indicate primary and secondary infections with IBDV Lx, and red arrows indicate primary and secondary infections with NDV LaSota. Serum samples were collected at 14 days post-primary infection and 4 days post-secondary infection (32 days post-primary infection). (B–E) Antibody affinity constants against IBDV VP2 or NDV F protein analyzed by indirect ELISA. Saturation binding curves were generated using serial dilutions of the coating antigen (IBDV VP2 or NDV F protein), with the x axis representing antigen concentration (c, in 10−6 mol/L). Affinity constants (Ka) were calculated from the curves. Nine-day-old SPF chickens were inoculated with NDV LaSota (106 TCID50, 200 μL, B and C) or IBDV Lx (106 TCID50, 200 μL, D and E) via nasal and ocular drops (n = 6/group). Four weeks post initial infection, chickens were challenged with homologous viral strains. Serum samples were collected 14 days post-initial viral infection (B and C) and 4 days post-secondary infection (D and E). Ka1 and Ka2 denote…

Figure 2. IBDV infection inhibits the affinity maturation of anti-NDV LaSota antibodies following vaccination (A) Schematics illustrate the timeline of vaccination and serum collection. Red arrows indicate immunization with NDV LaSota vaccine, blue arrows indicate infection with IBDV Lx, and the “IBDV Lx + NDV LaSota” group received both treatments at the indicated time points. (B–D) Antibody affinity constants against NDV analyzed by indirect ELISA. Nine-day-old SPF chickens were randomly divided into four groups (n = 15/group): NDV LaSota vaccination alone (106 EID50, 100 μL/bird; B), IBDV Lx (106 TCID50, 200 μL/bird) and NDV LaSota (106 EID50, 100 μL/bird) co-inoculation (C), IBDV Lx infection (106 TCID50, 200 μL/bird) followed by NDV LaSota vaccination 4 days post-infection (D), and PBS control. Three weeks post-initial vaccination, groups B-D received NDV LaSota boosters. Sera were collected 4 days post-booster for anti-NDV antibody affinity analysis. (E) Comparison of anti-NDV antibody affinity constants determined by indirect ELISA (B–D). The data represent pooled samples from six replicates.

Figure 3. IBDV infection reduces HI titers and splenic IgM+ B-cell proportion post-NDV vaccination (A) HI titers of antibody against NDV LaSota were determined by hemagglutination inhibition assay. (B and C) Quantification of IgM+ and IgY+ splenic B lymphocytes by flow cytometry at 4 days post-infection. Splenic lymphocytes were isolated and dual-stained with IgM/IgY and Bu-1b antibodies. Data are representative of six independent biological replicates and are expressed as mean ± SD. Comparisons between groups were conducted using Student’s t test. ∗∗∗, p < 0.001 and ∗, p < 0.05.

Figure 4. IBDV infection reduces splenic IgM+ B-cell proportion post-NDV reinfection (A and B) Splenic lymphocytes isolated 4 dpi from virus-inoculated chickens were triple-stained with anti-IgM-FITC, IgY-PE, and Bu-1b-AF647 antibodies for flow cytometry analysis. (A) Representative flow cytometry plots. (B) Statistical analysis of IgM+ and IgY+ B-cell percentages. Data represent mean ± SD from six independent replicates. Comparisons between groups were conducted using Student’s t test. ∗∗∗, p < 0.001; ∗∗, p < 0.01; and ∗, p < 0.05.

Figure 5. IBDV infection upregulates the expression of Th1 cytokines and cytidine deaminase (AID) in DT40 cells (A–D) The mRNA expression of cytokines and AID in DT40 cells with or without NDV or NDV-IBDV infection. DT40 cells were mock-infected, infected with NDV LaSota, or co-infected with NDV LaSota and IBDV Lx (MOI = 0.5). At different time points (6, 12, and 24 h) post-infection, cells were harvested and examined for the mRNA expression of IL2 (A), IFNG (B), IL4 (C), and AID (D) by RT-PCR. Data are presented as mean ± SD from three independent replicates. Comparisons between groups were conducted using one-way ANOVA. ∗, p < 0.05; nd, p > 0.05.

Figure 6. IBDV infection upregulates splenic Th1 cytokines and AID expression in IBDV-NDV coinfected chickens (A–D) Transcript levels of IL2 (A), IFNG (B), IL4 (C), and AID (D) in splenic tissue were analyzed by RT-qPCR 12 h post-NDV LaSota revaccination. mRNA expression changes were normalized to mock-infected controls. Data are presented as mean ± SD from three independent replicates. Comparisons between groups were conducted using Student’s t test. ∗∗∗, p < 0.001.

Figure 7. Effect of IBDV viral proteins and dsRNA on Th1 cytokine and AID expression in DT40 cells (A) IBDV viral protein expression analysis by western blot. DT40 cells were transfected with 5 μg of pRK5-Flag, pRK5-VP1, pRK5-VP2, pRK5-VP3, pRK5-VP4, or pRK5-VP5. Cells were harvested 24 h post-electroporation, and lysates were probed with anti-Flag and anti-GAPDH antibodies. Endogenous GAPDH served as the loading control. (B–D) qRT-PCR analysis of IL2 (B), IFNG (C), and AID (D) mRNA levels in DT40 cells transfected with 5 μg pRK5-Flag, pRK5-VP1-VP5 plasmids, or poly(I:C). Total RNA was extracted 24 h post-transfection. Data are presented as mean ± SD from three independent replicates. Comparisons between groups were conducted using one-way ANOVA. ∗∗∗, p < 0.001.