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. 2022 Nov 10:13:1002349.
doi: 10.3389/fmicb.2022.1002349. eCollection 2022.

Differences in cytokines expression between Vero cells and IPEC-J2 cells infected with porcine epidemic diarrhea virus

Chen Yuan  1 , Lidan Sun  1 , Ligong Chen  1 , Limin Li  1 , Zuojun Yao  1 , Yawen Wang  1 , Haiyong Guo  2 , Tanqing Li  1 , Qinye Song  1
Affiliations

Differences in cytokines expression between Vero cells and IPEC-J2 cells infected with porcine epidemic diarrhea virus

Chen Yuan et al. Front Microbiol. .

Abstract

Porcine epidemic diarrhea virus (PEDV) primarily infects suckling piglets and causes severe economic losses to the swine industry. Cytokines, as part of the innate immune response, are important in PEDV infection. The cytokines secreted by cell infection models in vitro might reflect true response to viral infection of target cells in vivo. Vero cells and IPEC-J2 are commonly used as an in vitro model to investigate PEDV infection. However, it is not clear which type of cells is more beneficial to the study of PEDV. In our study, firstly, Vero cells and IPEC-J2 were successfully infected with PEDV virulent strains (HBQY2016) and attenuated vaccine strains (CV777) and were capable of supporting virus replication and progeny release. Moreover, cytokine differences expression by Vero cells and IPEC-J2 cells infected with two PEDV strains were analyzed. Compared with IPEC-J2 cells, only the mRNA levels of TGF-β, MIP-1β and MCP-1 were detected in Vero cells. ELISA assay indicated that compared to the control group, the PEDV-infected group had significantly induced expression levels of IL-1β, MIP-1β, MCP-1, IL-8, and CXCL10 in IPEC-J2 cells, while only secretion level of IL-1β, MIP-1β and IL-8 in Vero cells were higher in PEDV infected group. Finally, cytokines change of piglets infected PEDV-HBQY2016 strains were detected by cDNA microarray, and similar to those of IPEC-J2 cells infected PEDV. Collectively, these data determined that the IPEC-J2 could be more suitable used as a cell model for studying PEDV infection in vitro compared with Vero cells, based on the close approximation of cytokine expression profile to in vivo target cells.

Keywords: IPEC-J2 cells; PEDV; Vero cells; cytokines; difference.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Replication of porcine epidemic diarrhea virus (PEDV) in Vero cells. (A) The viral titer of PEDV-HBQY2016 or -CV777 strain was determined with Vero cells with the results expressed as TCID50/mL at 72 h post infection (hpi). (B) The copies (Log10) of PEDV-HBQY2016 or PEDV-CV777 strain in Vero cells at different time points was evaluated by RT-qPCR. (C) Immunofluorescence staining of Vero cells infected with PEDV-HBQY2016 or -CV777 strain at different time points.
Figure 2
Figure 2
Replication of PEDV in IPEC-J2 cells. (A) The viral titer of PEDV-HBQY2016 or -CV777 strain was determined with IPEC-J2 cells with the results expressed as TCID50/mL at 72 h post infection. (B) The copies (Log10) of PEDV-HBQY2016 or PEDV-CV777 strain in IPEC-J2 cells at different time points was evaluated by RT-qPCR. (C) Immunofluorescence staining of IPEC-J2 cells infected with PEDV-HBQY2016 or -CV777 strain at different time points.
Figure 3
Figure 3
Cytokines expression in Vero cells infected with PEDV. (A) Changes of gene copies of IL-1β, IL-6, TGF-β, MIP-1β, MCP-1, IL-8, and CXCL-10 gene expression in Vero cells after PEDV infection. (B) Protein concentration of the cytokines secreted by PEDV-infected Vero cells. The supernatant of Vero cells was collected at 24 h after infection with 0.1 MOI PEDV, and the cytokine secretion level was detected by ELISA. *Represents a significant difference relative to the control group (p < 0.05), **Represents an extremely significant difference relative to the control group (p < 0.01).
Figure 4
Figure 4
Cytokines expression in IPEC-J2 cells infected with PEDV. (A) Changes of gene copies of IL-1β, IL-6, TGF-β, MIP-1β, MCP-1, IL-8, and CXCL-10 gene expression in IPEC-J2 cells after PEDV infection. (B) Protein concentration of the cytokines secreted by PEDV-infected IPEC-J2 cells. The supernatant of IPEC-J2 cells was collected at 24 h after infection with 0.1 MOI PEDV, and the cytokine secretion level was detected by ELISA. *Represents a significant difference relative to the control group (p < 0.05), **Represents an extremely significant difference relative to the control group (p < 0.01).
Figure 5
Figure 5
Expression levels of cytokines in ileum of piglets orally inoculated with PEDV. (A) The volcano drawing of ileum tissues plot of cDNA microarray. g1 represents ileum tissue of piglets in control group. g2 represents ileum tissue of piglets in PEDV infected group. The ordinate is the -lg (p-value) value. The abscissa is log2 (Fold Change). (B) Ileum of heat map of cDNA microarray. K, Negative control piglet; I, PEDV-infected piglet. (C) The expression levels of the cytokines gene copies in ileum of the PEDV-HBQY2016 infected and the negative control group were quantitatively determined via qRT-PCR. (D) Comparison of fluorescent quantitative PCR and gene chip results in ileum of piglets. *Represents a significant difference relative to the control group (p < 0.05), **Represents an extremely significant difference relative to the control group (p < 0.01).
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