Milk lactose protects against porcine group A rotavirus infection

Figures

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  FIGURE 1. Phylogenetic tree of rotavirus VP8* genes of 48 Belgian porcine RVAs and 5 related human RVAs (indicated by a black dot). The phylogenetic tree was constructed based on the VP8* sequences using the Maximum-likelihood method with bootstrap 500. The bootstrap cutoff value for each clade under 70 is not shown. The red marked RVAs were selected to produce recombinant VP8*.

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  FIGURE 2. Characterization of recombinant VP8*. (A) Amplification of target fragments 498 bp from fecal samples, (lane M = standard molecular weight marker). (B) The SDS-PAGE of the GST-VP8* fusion proteins (Lane M = blue prestained protein standard). The 26 kDa band represents free GST.

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  FIGURE 3. Hemagglutination assays (HA) with the different recombinant VP8* proteins. Human red blood cells of type O, A, and B (marked as O, A, and B), and porcine red blood cells (marked as P) were used. GST-VP8* proteins were serially diluted with PBS and mixed with 0.5% of different types of red blood cells. The PBS buffer without protein was used as negative control (NC).

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  FIGURE 4. Glycan array results of VP8* proteins from porcine RVA strains 14R103 (G5P[6]), 13R054 (G5P[7]) and 12R010 (G4P[13]). The glycans from an array library containing 300 glycans, that are recognized by each VP8*, were ranked following the binding strength. The values are the fold increase of the fluorescence signal compared to that of GST (negative control). The β-lactose monomer was marked in yellow, the LewisX monomer was marked in blue, and the cellobiose monomer was marked in green.

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  FIGURE 5. Binding of the recombinant VP8* proteins from the RVA strains 14R103 (G5P[6]), 13R054 (G5P[7]), 12R010 (G4P[13]), 12R046 (G9P[23]) and 12R022 (G2P[27]) to synthetic oligosaccharides. Polyacrylamide (PAA)-biotin conjugates of LewisX, β-lactose, cellobiose and L-Rha [negative control [NC)] were used in the binding assay. The GST-VP8* fusion proteins from RVAs 14R103 (G5P[6]), 13R054 (G5P[7]), 12R010 (G4P[13]), 12R046 (G9P[23]), and 12R022 (G2P[27]) were used in the binding assay to confirm the binding specificity. GST-tagged recombinant proteins and the GST tag alone were tested. Error bars in the panel represent the means ± the SD from triplicate experiments. ***, P < 0.001. *, P < 0.05. ns, P > 0.05.

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  FIGURE 6. The infection of MA104 cells by the porcine RVA strains 13R054 G5P[7] and 12R046 G9P[23] is strongly reduced by β-lactose. (A) The effects of pre-incubation of the virus with β-lactose on the infectivity of the porcine RVAs 13R054 G5P[7] and 12R046 G9P[23] were assessed using an immunofluorescence assay with polyclonal antibodies against the RVA VP6 protein. (B) The infection of MA104 cells with both RVA strains was expressed as percentage of infected cells and compared with the mock-treated, virus-inoculated control. (C) The titration of the supernatant was performed on MA104 cells. All experiments were performed in three independent experiments. Error bars in the panel represent the means ± the SD from triplicate experiments. **, P < 0.01. *, P < 0.05. ns, P > 0.05.

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  FIGURE 7. β-lactose pretreatment decreases the replication of rotavirus in porcine enterocytes. After isolation, the ileum enterocytes were incubated with lactose pretreated RVAs for 12 h. (A) The viability of clusters during incubation was checked with EMA. The percentage of infected clusters (B-13R054 G5P[7] and C-12R046 G9P[23]) and the percentage of infected cells in a single cluster (D-13R054 G5P[7] and E-12R046 G9P[23]) were determined. (A–C) The error bars in the figures represent the means ± the SD from triplicate experiments. (D,E) The error bars in the figures represent the min to max. *P < 0.05. ns, P > 0.05.