Antiviral Activity of Marine Bacterium Paraliobacillus zengyii Against Enterovirus 71 In Vitro and In Vivo

Figures

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  Figure 1. P. zengyii downregulates EV71 replication in multiple cell lines. (A) EV71 RNA levels relative to those of GAPDH were detected via RT–qPCR. (B) The virus titer was determined via a TCID50 assay. (C) Intracellular EV71 VP1 was examined by IFA. Multiple cell lines, including HT-29, Caco2, and RD cells, were used. All the cell lines were pretreated with or without P. zengyii for 24 h and then infected with EV71 for 24 h. The nuclei were labeled with DAPI, and images were taken using fluorescence microscopy (ECHO Revolve). The scale bars represent 170 μm. The data are shown as the means ± SDs of three independent experiments. One-way ANOVA and two-tailed unpaired Student’s t tests were used. ns: not significant; * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 2. P. zengyii enhances the production of IFN-β in HT-29 cells. (A,C) IFN-β mRNA levels were measured via RT–qPCR and normalized to GAPDH levels after 6 and 12 h of treatment. (B,D) IFN-β protein levels were measured by ELISA after 24 h of treatment. HT-29 cells were pretreated with or without P. zengyii (MOI of 100) for 24 h and then infected with EV71 or transfected with poly(I:C) (0.5 μg/mL) for 6, 12, or 24 h. The control represents untreated HT-29 cells. The data are shown as the means ± SDs of three independent experiments. Two-tailed unpaired Student’s t tests were used. ns: not significant; * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 3. The establishment of the EV71 infection mouse model. (A) A schematic diagram of EV71 serial passaging from passages zero to five (P0-P5). Magnification on the images is 10×. (B) A schematic description of the intraperitoneal injection of EV71-P5 or DMEM into 14-day-old BALB/c mice (n = 5). (C) Changes in the body weights of the mice were recorded for 5 days after EV71-P5 infection, and the changes in body weight over time are expressed as a percentage of the original weight. (D) EV71-P5-infected mice presented hindlimb paralysis (black arrow). (E) The viral genomes detected by qPCR in mice hindlimb muscles, spleens, and ileums. The thin line indicates the internal reference gene GAPDH, and the thick line indicates EV71 VP1. (F) IHC analysis indicated that the viral antigen was diffusely distributed in the hindlimb muscles, spleens, and ileums (black arrow). The scale bars indicated 20 μm. Two-tailed unpaired Student’s t tests were used. ** p < 0.01.

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  Figure 4. P. zengyii reduces EV71 infection in suckling mice. (A) A schematic representation of the animal experimental setup. Fourteen-day-old suckling mice were infected with 108 TCID50 of EV71 and treated with P. zengyii or PBS from 2 days before infection until 3 days after infection. (B) Body weight was measured daily after EV71 infection and normalized to the corresponding initial weight. (C) The viral RNA levels in the hindlimb muscle, ileum, and spleen tissues of the infected mice were analyzed by RT-qPCR. (D) The viral titers of the hindlimb muscle, ileum, and spleen tissues of the infected mice were assessed via a TCID50 assay. (E) The HE staining of the hindlimb muscles. The scale bars indicate 100 μm. (F) The detection of the EV71 VP1 antigen in the mice hindlimb muscles, ileums, and spleens using IHC. The scale bars indicate 20 μm. GAPDH was used as an internal control for RT–qPCR. Two-tailed unpaired Student’s t tests were used. ns: not significant; ** p < 0.01, *** p < 0.001.

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  Figure 5. P. zengyii upregulates ISG expression in EV71-infected suckling mice. (A) A schematic diagram showing the spleen transcriptome analysis. (B) The PCA plot of the spleen transcriptome from the RNA sequencing analysis. (C) A volcano plot showing the upregulated (red), downregulated (blue) genes, and nonsignificant (gray) genes in the spleen tissues of EV71-infected mice treated with P. zengyii compared with those of the untreated mice. Fold change (FC) > 1.5; p value < 0.05. (D) A heatmap of some expressed ISGs. (E) The RT–qPCR expression of Ifit1, Oas3, Ifitm1, Oasl2, Oas2, and Mx1 in the spleens of EV71-infected mice treated with or without P. zengyii. GAPDH was used as an internal control for RT–qPCR. Two-tailed unpaired Student’s t tests were used. * p < 0.05, ** p < 0.01.