Diet-supplemented probiotic Bacillus velezensis protects against viral infection via the lipopeptide surfactin in zebrafish

Background: Probiotic nutrition has the potential to improve health and prevents diseases, and probiotics such as Bacillus spp. may protect against viral infection. However, the in vivo antiviral mechanism has not yet been fully determined. Objective: We aimed to elucidate the antiviral mechanism of Bacillus velezensis strain by comparing the effect of wild type and isogenic mutant in a zebrafish model. Methods: Zebrafish (60 ± 3 mg) were fed with control diet or diet supplemented B. velelzensis T23, and were challenged with spring viremia of carp virus (SVCV). T23 mutants deficient in the production of lipopeptides and/or polyketides were constructed by two-step replacement recombination. Zebrafish were fed with diet supplemented with wild type or mutant followed by SVCV infection. Germ free zebrafish were mono-colonized with T23 or its mutant, and were infected by SVCV. When appropriate, type I interferon (IFN) receptors or myeloid differentiation factor 88 (Myd88) were knocked down by vivo-morpholino. ZF4 (zebrafish embryonic fibroblast like cell line) were treated with bacterial fractions of T23 or surfactin, and were infected with SVCV. Results: Bacillus velezensis T23 improved survival of zebrafish after SVCV challenge (P < 0.05) by 250%, and inhibited viral replication in germ-free zebrafish (P < 0.05). Genes involved in biosynthesis of secondary metabolites were knocked out in T23, and the antiviral function of Δsfp and ΔsrfAA mutants deficient in the production of surfactin was abrogated both in vitro and in vivo. T23 enhanced type I IFN antiviral immunity post SVCV infection (P < 0.05). Knockdown of type I IFN receptors or Myd88 in gnotobiotic zebrafish did not influence the antiviral effect of T23. Finally, surfactin can exert antiviral effect by directly inhibiting the infectivity of SVCV. Conclusion: Probiotic Bacillus velezensis T23 protected against viral infection in zebrafish. The antiviral effect of B. velezensis T23 was mainly mediated by direct inhibition of surfactin on virus, and was not attributable to type I IFN antiviral immunity.