Innate Immune Evasion of Porcine Epidemic Diarrhea Virus Through miRNA‐193a‐5p/IL22/pBD1 Pathway in Intestinal Epithelium

Figures

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  Figure 1. Isolation and identification of PEDV strain FJ2022. (A) PCR identification for PEDV (lane M: 600 DNA marker; lane 1–5: random site detection of diseased materials). (B) PCR amplification for virus in diseased materials (lane M: 600 DNA marker; lane a: TGEV primer; lane b: PoRV primer; lane c: CSFV primer; lane d: PRV primer; lane e: PDCoV primer; lane f: PCV2 primer; lane g: ASFV primer; lane h: PRRSV primer). (C) Phylogenetic tree of PEDV S genes. The phylogenetic analysis by MEGA 7.0 software program. (D) IPECs were incubated with PEDV N protein monoclonal antibody Cy3-labeled goat antimouse IgG. Nuclei were stained by DAPI (40×). (E) PCR amplification of PEDV for different generations of cells on the diseased material (lane M: 600 DNA marker; lane 1–3: first to third generation of cells on the diseased material). (F) The third generation of viruses infected IPECs for 12, 24, and 48 h. (G) TEM image of PEDV FJ2022 virus. Scale bar is 20 nm.

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  Figure 2. Artificial infection of piglets with PEDV strain FJND-2022. (A) The antibody and antigen of PEDV double negative suckling piglets were artificially infected with PEDV FJ2022 for 5 days (oral administration), and the mock group received DMEM orally. (B) Images of infected and uninfected piglets dissected after 5 days. (C) PEDV M gene amplification in the intestine of infected piglets. (D) H and E staining of the duodenum and jejunum. The scale bar is 100 µm.

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  Figure 3. Exosomes-derived from IPECs infected with PEDV. (A) TEM images of exosomes derived from PEDV-infected IPECs and mock groups. (B) Western blotting was performed using the primary antibody of exosomal markers CD9, calnexin, and TSG101.

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  Figure 4. Different expression profile of microRNA in exosomes from IPECs. (A) PHRED quality scores for reads in the PEDV infection and mock groups. (B) Volcano diagram of differential expression of miRNAs. (C) GO functional classification statistics of differentially expressed miRNA target genes. (D) KEGG pathway classification statistics of differentially expressed miRNA target genes. The mRNA levels of ssc-let-7a (E), miR-30d (F), miR-193a-5p (G), miR-10a-3p (H), and ssc-miR-98 (I) were determined by RT-qPCR.

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  Figure 5. PEDV downregulates miR-193a-5p in IPECs to evade the antiviral effect of IL22/pBP1. At 48 h postinfection with PEDV (A), the mRNA levels of miR-193a-5p (B), IL22 (C), and pBD-1 (D) in IPECs were determined by RT-qPCR. Then at 48 h postinfection with PEDV in the small intestine epithelium of piglets (E), the mRNA levels of miR-193a-5p (F), IL22 (G), and pBD-1 (H) were detected by RT-qPCR. (I) IL22/pBD-1 protein levels were examined by Western blotting in PEDV-infected piglets or IPEC J2. (J) Image J quantification of Western blot band intensities. The a and b bars in each panel without a common superscript letter were signifcantly different (p < 0.05).

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  Figure 6. PEDV downregulated miR-193a-5P/IL22/PBD1 pathway to promote its multiplication. Effect of miR-193a-5p mimic on mRNA expression of PEDV (A), miR-193a-5p (B), IL22 (C), and pBD-1 (D) in IPECs. Effect of miR-193a-5p inhibitor on mRNA expression of PEDV (E), miR-193a-5p (F), IL22 (G), and pBD-1 (H) in IPECs. Effect of small interfering IL22 on mRNA expression of PEDV (I), miR-193a-5p (J), IL22 (K), and pBD-1 (L) in IPECs. The a, b, c, and d bars in each panel without a common superscript letter were signifcantly different (p < 0.05).

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  Figure 7. Co-IP analysis of IL22 and pBD1 in small intestine of piglet.

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  Figure 8. Scheme of PEDV downregulates miR-193a-5p in exosomes from IPECs to immunologic escape from IL22/β-defensin 1 antivirus (by Figdraw).