Fig 2: Bmp8a increases Tbk1–Irf3–Ifn antiviral signaling.a, b, e, f Expression of irf3, irf7, and tbk1 mRNA after transfected with 2 µg bmp8a or empty vector in ZFL (a, b) or EPC (e, f) cells. The cells were collected at 36 h (a, e) or 48 h (b, f) post-transfection. c, d, g, h Expression of irf3, irf7, and tbk1 mRNA after transfected with 2 µg bmp8a or empty vector in ZFL (c, d) or EPC (g, h) cells for 24 h, followed by infection with GCRV for another 24 h (c, g) or 36 h (d, h). i–l Expression of irf3, irf7, and tbk1 mRNA after bmp8a knockdown in ZFL cells. The cells were collected at 36 h (i) and 48 h (j) post-knockdown or at 24 h (k) and 36 h (l) post-infected with GCRV. m, o Immunoblot analysis of phosphorylated (p-) Tbk1 and Irf3 after transfected with 2 µg bmp8a or empty vector in ZFL (m) or EPC (o) cells. The cells were collected at 36 or 48 h post-transfection for Immunoblot analysis. n, p Immunoblot analysis of phosphorylated (p-) Tbk1 and Irf3 after transfected with 2 µg bmp8a or empty vector in ZFL (n) or EPC (p) cells for 24 h, followed by infection with GCRV for another 24 or 36 h. q, r Immunoblot analysis of phosphorylated (p-) TBK1 and IRF3 after bmp8a knockdown in ZFL cells. The cells were collected at 36 and 48 h post-knockdown or at 24 and 36 h post-infected with GCRV. s–u EPC cells were cotransfected with IFN-f1pro-luc (200 ng, s), IFN-f3pro-luc (200 ng, t) or EPC IFNpro-luc (200 ng, u), and bmp8a (100 ng) together with each of the dominant negative plasmids including tbk1–K38M (100 ng), irf3DN (100 ng) and irf7DN (100 ng). At 48 h post-transfection, the cells were collected for luciferase assays. Renilla luciferase was used as the internal control. v–y Expression of irf3, irf7, and tbk1 mRNA in the liver, kidney, intestine, and spleen from WT or bmp8a-/- zebrafish injected i.p. with 50 µl of GCRV (108TCID50 per ml). The expression of zebrafish actb1 or EPC actin was used as an internal control for the qRT-PCR. Data were from three independent experiments and were analyzed by Student’s t-test (two-tailed) for comparison of two groups or one-way ANOVA followed by Games–Howell post hoc tests for comparison of multiple groups. All data were presented as mean ± SD (**p < 0.01, ***p < 0.001).

Fig 3: Effects of Sparassis latifolia neutral polysaccharides SLNP western blot results on protein expression. (A), TLR4 (B), TRAF6 (C), JNK (D), p-JNK (E), ERK (F), p-ERK (G), p38 (H), p-p38 (I), and IRF3 (J) in RAW264.7 macrophage. Compared with blank control group, *p < 0.05, **p < 0.01; Compared with LPS control group, ?p < 0.05, ??p < 0.01.

Fig 4: Schematic illustration of Bmp8a serving as positive regulator in the antiviral immune.Upon virus infection, the transcriptions of bmp8a are activated through the Jak-Stat1 pathway. The Bmp8a binds to BMP type I receptor Alk6a, promoting phosphorylation of Tbk1 and Irf3 to induce the expression of Ifn through p38 MAPK pathway.

Fig 5: Alk6a is involved in the antiviral innate immune responses.a, b Expression of alk2, alk3, alk6a, bmpr2a, bmpr2b, actr2a, and actr2b mRNA in ZFL cells stimulated with poly(I:C) (2 µg/ml, a) or GCRV (5 × 104TCID50 per ml, b) for 48 h. c–e Expression of ifnf1 (c) and ifnf3 (d) mRNA in ZFL cells or EPC ifn (e) in EPC cells which were transfected with 2 µg of alk2, alk3, alk6a, bmpr2a, bmpr2b, actr2a, actr2b or empty vector for 48 h. f, h Expression of irf3, irf7, tbk1, ifn (or ifnf1 and ifnf3), and mx mRNA after transfected with 2 µg of pcDNA3.1-alk6a or empty vector in ZFL (f) or EPC (h) cells for 48 h. g, i Expression of irf3, irf7, tbk1, ifn (or ifnf1 and ifnf3), and mx mRNA after transfected with 2 µg of of pcDNA3.1-alk6a or empty vector in ZFL (g) or EPC (i) cells for 24 h, followed by infection with GCRV for another 36 h. j Schematic drawing of the alk6a-?GS mutation that the GS domain of Alk6a was deleted. k, m Expression of irf3, irf7, tbk1, ifn (or ifnf1 and ifnf3), and mx mRNA after transfected with 2 µg pcDNA3.1-alk6a-?GS or empty vector in ZFL (k) or EPC (m) cells for 48 h. l, n Expression of irf3, irf7, tbk1, ifn (or ifnf1 and ifnf3), and mx mRNA after transfected with 2 µg pcDNA3.1-alk6a-?GS or empty vector in ZFL (l) or EPC (n) cells for 24 h, followed by infection with GCRV for another 36 h. The expression of zebrafish actb1 or EPC actin was used as an internal control for the qRT-PCR. Data were from three independent experiments and were analyzed by Student’s t-test (two-tailed) for comparison of two groups or one-way ANOVA followed by Games–Howell posthoc tests for comparison of multiple groups. All data were presented as mean ± SD (*p < 0.05, **p < 0.01, and *** p < 0.001, ns means no significant difference).