Levan produced by probiotic Bacillus subtilis CU1 inhibits human norovirus GII.4 replication in zebrafish via high-avidity binding

Figure 1. Effect of CES from B. subtilis CU1, B. subtilis R0179, and L. plantarum 299V on TV infecting LLC-MK2 cells. The CPE were scored with positive control as the reference (CPE score of 100). For pre-treatment groups (A), CES were added to the cell cultures and incubated for 1 h before the inoculation of viruses; For co-treatment groups (B), the CES were incubated with TV for 1 h before inoculating the mixtures to the cells; For post-treatment groups (C), the viruses were inoculated to the cell cultures and incubated for 1 h before the addition of CES. The CPE was scored after 72 h. Each value was presented as mean ± standard deviation (n = 8) in panel. * p < 0.05 compared to positive control.  Figure 2. The inhibition of hNoV replication in zebrafish of exopolysaccharides from B. subtilis CU1 was mainly attributed to the interaction with virus capsid protein rather than innate immunity regulation. (A) CES from B. subtilis CU1 but not B. subtilis R0179, and L. plantarum 299V was able to inhibit hNoV GII.4[P16] infecting zebrafish larvae. Virus was incubated with CES for 1 h before injecting the mixture on 3 dpf (co-treatment) and detected on 5 dpf. (B) Significant inhibition was also observed when applying CES-CU1 and hNoV GII.4[P16] mixture to zebrafish embryo on 0 dpf (co-treatment) and detected on 3 dpf and 5 dpf. In comparison, pre-treatment (CES-CU1 and virus were injected on 0 dpf and 3 dpf respectively) and post-treatment (virus and CES-CU1 were injected on 0 dpf and 3 dpf respectively) did not induce any antiviral effect. (C) A purification of CES-CU1 generating PES-CU1 further improved its antiviral effect. PES-CU1 was effective on hNoV GII.4[P16] but not GII.2[P16] or GII.17[P31] in inhibiting virus replication in zebrafish embryo (0 dpf) with a co-treatment set-up. (D) The antiviral effect of CES-CU1 and PES-CU1 on hNoV GII.4 might be due to a binding blocking effect, as CES and PES from CU1 was able to significantly reduce the binding signals of hNoV GII.4 VLPs, but not from hNoV GII.2 VLPs or hNoV GII.17 VLPs, in a saliva-binding ELISA. (E) Effect of PES-CU1 on the expression of type I interferon response activated genes after 1, 6, 24, 48, 72 and 120 h post injection (hpi). 4 mg/mL CES or PES were used for all of the above tests. *p < 0.05, ** p < 0.01, *** p < 0.001. (F) The UV spectrum confirmed the purity of the freeze-dried form of PES-CU1, showing a single absorbance peak at 190 nm (polysaccharides) and no detectable peak at 280 nm (protein) while CES-CU1 showed both peaks at 190 nm and 280 nm. Figure 3. (A) Anion exchange chromatography identified three components of PES-CU1, with PES-CU1-0 as the dominant component in comparison with PES-CU1-1 and PES-CU1-5. (B) Single peak in HPSEC chromatography showed homogeneity of PES-CU1-0. (C) SEM image of PES-CU1-0. (D-F) Antiviral validation of PES-CU1-0. (D) Among the three components of PES-CU1, PES-CU1-0 contributed most significantly to the binding blocking effects against hNoV VLP GII.4, compared to PES-CU1-1 and PES-CU1-5, in a saliva-binding ELISA. 10 mg/mL of PES-CU1-0, 1.5 mg/ mL of PES-CU1-1 and 1.8 mg/ mL of PES-CU1-5, corresponding to the area ratio in anion exchange chromatography were used. (E) PES-CU1-0 exhibited comparable inhibition activity against hNoV GII.4[P16] to that of PES-CU1 in zebrafish embryos. Virus was incubated with PES-CU1/PES-CU1-0 for 1 h before injecting the mixture on 3 dpf (co-treatment) and detected on 5 dpf. (F) PES-CU1-0 showed stronger direct binding to hNoV V P particles than PES-CU1 in BLI assay. The binding signal was presented as fitted 1:2 bivalent model with standard deviation. *p < 0.05, ** p < 0.01, *** p < 0.001 compared to positive control. Figure 4. PES-CU1-0 was identified as levan. (A) PES-CU1-0 was consisted of fructose units in the HPLC chromatograms in comparison with mixed sugar standards. (B) PES-CU1-0 had typical characteristic of fructan in FTIR spectrum. (C-G) PES-CU1-0 was identified as levan composed of β-(2,6)-linked Fruf in 1D and 2D NMR spectra. Figure 5. B. subtilis CU1 fermented carrot juice conferred antiviral potential. Fermented 80 ℃-preheated carrot juice (J-80-F) exhibited blocking binding effects on hNoV VLP GII.4 in saliva-binding ELISA, whereas the unfermented sample showed no effect (A). Similarly, the polysaccharides extracted from fermented 80 ℃-preheated carrot juice (PS-80-F) exhibited inhibition on hNoV GII.4[P16] replication in zebrafish embryo (0 dpf) with a co-treatment set-up, whereas the unfermented sample showed no effect (B). *p < 0.05, ** p < 0.01, *** p < 0.001 compared to positive control. The yield of EPS in J-80-F was initially optimized by individually varying the carbon source, fermentation time, and inoculum size using in OVAT assay (C). Student-Newman-Keuls analysis was used for comparing the mean value. Data was presented as mean values ± standard deviation. p < 0.05, b < a. The maximum yield of EPS was generated by RSM (D).