Study On The Mechanism Of Three Natural Polysaccharides Against SVCV Infection In Zebrafish

Intensive fish culture may cause outbreak of fish diseases.The economic losses caused by aquatic diseases are about 10 billion yuan each year in China.Cyprinids comprise major cultured fish species in China,and play an important role in boosting the economy and providing high-quality protein for human.Spring viraemia of carp virus(SVCV)is a critical pathogen of cyprinids.At present,there is no effective treatment available.Functional feed additives have significant effect on improving fish non-specific immunity and are effective for preventing fish viral diseases.In this study,we focused on three kinds of natural polysaccharides,i.e.Astragalus polysaccharide(APS),yeast ?-glucan and Lactobacillus rhamnose GG extracellular polysaccharide(LGG EPS),and investigated the protection effect of dietary supplementation of APS,yeast ?-glucan and LGG EPS against SVCV infection in zebrafish.Furthermore,we have investigated the expression of interferon signaling genes,the level of autophagy and the modulation of intestinal microbiota induced by three kind of natural polysaccharides to elucidate the mechanism of antiviral activity.This study will provide the reference for the application of three kinds of polysaccharides in aquaculture.The results are as follows:1 The effect of dietary APS on zebrafish against SVCV infectionZebrafish were fed control diet and the experimental diets containing 0.01% or0.02% APS for three weeks.Zebrafish fed with 0.01% APS showed higher survival rate after SVCV challenge.Accordingly,the expression of antiviral genes in the spleen was increased at four days post infection.Zebrafish fed with both 0.01% and0.02% APS increased weight gain and decreased the feed conversion ratio.The intestinal expression of anti-inflammatory cytokine il-10,tjp1 b,and occludin1 was significantly up-regulated in 0.01% APS group compared with control.Furthermore,0.01% APS decreased the content of MDA and increased total antioxidant capacity in zebrafish intestine,and the relative expression of sod,gst and gpxa were improved.Total bacteria and the abundance of Fusobacteria and Firmicutes in the intestinal microbiota was increased in 0.01% and 0.02% APS groups compared with control.In contrast to 0.01% supplementation,0.02% APS compromised liver health,and exerted no positive effects on antiviral immunity.2 The effect of dietary yeast ?-glucan on zebrafish against SVCV infectionZebrafish fed with 0.025% yeast ?-glucan diet displayed a higher survival rate than control group after SVCV challenge.Meanwhile,the expression of genes related to interferon(IFN)antiviral immune pathway(ifn?1,ifn?2,ifn?3,irf3,irf7,mavs,mxb,and mxc)in zebrafish spleen post SVCV infection was significantly increased.Moreover,yeast ?-glucan can inhibit the proliferation of SVCV in ZF4 cells at 5 ?g/ m L.Yeast ?-glucan did not affect viral adhesion and cell viability in ZF4 cells,and showed no effect on type ? interferon signaling activation.Myd88 was knocked down by si RNA to confirm that the antiviral capability of yeast?-glucan was independent of My D88 signaling pathway.We further investigated the effect of yeast ?-glucan on autophagy level post SVCV infection.The antiviral effect of yeast ?-glucan in ZF4 cells was eliminated by using the specific inhibitor of autophagy.0.025% yeast ?-glucan also changed the composition of zebrafish intestinal microbiota,the abundance of Proteobacteria significantly decreased,but the abundance of Fusobacteria and Firmicutes significantly increased,and the abundance of Cetobacterium also significantly increased.Germ-free zebrafish colonized by ?-glucan-associated microbiota showed enhanced resistance against SVCV infectioin compared with control microbiota colonized counterparts.Consistently,C.somerae inhibited the replication of SVCV in germ-free zebrafish.The polysaccharide of C.somerae also showed antiviral activity.3 The effect of dietary LGG EPS on zebrafish against SVCV infection0.5% and 1% LGG EPS supplementation significantly increased the survival rate of zebrafish post SVCV infection.The expression of interferon(IFN)signaling genes in the spleen(ifn?1,ifn?2,ifn?3,irf3,irf7,mavs,mxb,and mxc)were significantly increased in 1% LGG EPS group after SVCV challenge.LGG EPS can inhibit the replication of SVCV in ZF4 cells at 5 ?g/ml.LGG EPS did not affect viral adhesion and cell viability in ZF4 cells,however LGG EPS can significantly enhance the expression of genes in type ? interferon signaling pathways.The antiviral effect of LGG EPS in ZF4 cells was eliminated by si RNA-mediated knockdown of tbk1 gene,suggesting that the antiviral capability of LGG EPS was dependent on type ? interferon signaling pathway.Moreover,1% LGG EPS has modulated the composition of intestinal microbiota of zebrafish.The abundance of Proteobacteria significantly decreased,while the abundance of Firmicutes significantly increased.Germ-free zebrafish colonized by LGG-EPS-associated microbiota showed enhanced resistance against SVCV infectioin compared with control microbiota colonized counterparts.The supernatant of the LGG EPS intestinal microbiota cultured in vitro showed antiviral activity,and the active ingredient was predicted to be protein.In conclusion,APS,yeast ?-glucan and LGG EPS enhanced the antiviral immunity of zebrafish.The yeast ?-glucan protected against SVCV infection by enhancing autophagy in ZF4 cell line,while the Lactobacillus rhamnosus GG EPS activated type ? interferon signaling pathway against SVCV infection.The intestinal microbiota contributed to the antiviral effects of yeast ?-glucan and LGG EPS in zebrafish.Cetobacterium somerae polysaccharide and a protein produced by LGG EPS induced microbiota also showed antiviral effect on zebrafish.Together,the three polysaccharides showed application prospects as feed additives against virus infection in cultured fish.What is more,the intestinal microbiota of fish will provide more probiotics and prebiotics against virus infection.