IPEC-J2 and African green monkey kidney cells (Vero) were both cultured in DMEM medium containing 10% fetal bovine serum, 1% double antibody in a 37°C, 5% CO₂ cell culture incubator. The cells mentioned above were all pre-preserved in the laboratory. The PEDV-LJX variant was kindly presented by Guangliang Liu, a researcher from Lanzhou Veterinary Research Institute. Recombinant human EGF from Gibco (Grand Island, NY, USA) at 10 ng/mL. The tyrosinase inhibitor AG1478 was purchased from Apexbio (Houston, TX, USA) and used at a dose of 30 μM.

IPEC-J2 cells were seeded in 60 mm dishes. An experimental group and a control group were set up, and each group was replicated three times independently. When cells grew to 90%, PEDV virus fluid (MOI = 0.1), or an optimal dose of EGFR modulator, was inoculated at 2 h and 72 h, respectively. The proteins were extracted using Beyotime’s cell membrane and plasma protein extraction kits, added with 6 × Loading Buffer (TransGen Biotech, China), denatured at 100°C for 10 min, cooled, and stored at −20°C before use.

Equal amounts of protein samples were separated by SDS-PAGE gel electro-phoresis and transferred to polyvinylidene fluoride membranes. The membranes was blocked with 5% skimmed milk powder diluted by TBST. After incubation overnight at 4°C with the appropriate primary antibody, the membrane was washed three times with 1 × TBST for 10 min/time and then incubated with goat anti-rabbit secondary antibody or goat anti-mouse secondary antibody (Proteintech, Rosemont, IL, USA) for 90 min on a shaker at room temperature. Images of the immunoreactive proteins on the membrane were chromogenic using the FX5 imaging system (VILBER, Marne-la-Vallée cedex 3, France). The grayscale values were analyzed. The phospho-EGFR (Tyr1068) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies recognizing EGFR, SLC9A3 (NHE3), β-Actin, β-Tubulin (Rabbit Antibody), 6 × His (His-Tag), and FLAG® tag were purchased from Proteintech. The phospho-extracellular kinase (ERK)1/2 (Thr202 + Tyr204) antibody was purchased from Beijing BoaoSen Biotechnology Co. (Beijing, China).

After IPEC-J2 cells had grown to spread all over the cell bottles, pre-treatment of cells with 10 ng/mL of EGF or 30 μM of AG1478 in 2 mL, and then inoculated with 10⁵ TCID₅₀/mL PEDV-LJX strain, and both virus-infected and blank control groups were established. After incubation with the virus for 1.5 h, the cells were freeze-thawed three times and then centrifuged at 12000 × g for 10 min. The supernatant was collected into centrifuge tubes, and the TCID₅₀ of each group of PEDV was determined in Vero cells according to the Reed-Muench method.

Sequences of the coding regions of the PEDV S and EGFR genes registered in GenBank according to the NCBI, PEDV S1 (637–2,127 bp) was selected and constructed into vector pTT5 as a Flag tag fusion. The fragment of EGFR encoding the extracellular region (139–1,473 bp) was constructed into vector pTT5 as a His tag fusion. The above gene fragments were synthesized, and the recombinant plasmids were constructed, and sequenced by Wuhan GeneCreate Biological Engineering Co., Ltd. (Wuhan, China). Splicing HEK-293 T cells into 60 mm dishes, and when the cells grew to about 80% confluence, they were transfected with the constructed eukaryotic expression vector plasmids of PEDV S1 and EGFR extracellular region. At 48 h after transfection, samples were prepared for immunoprecipitation and the target bands were detected using western blotting.

IPEC-J2 cells were inoculated with 1 × 10⁴ cells per well in a confocal dish (medium without antibiotics), and when the cells reached 80% growth, the cells were washed 3 times with PBS. After 6 h of plasmid transfection, the AG1478 treatment solution was slowly added from the edge of the confocal dish, mixed well and incubated at 37°C with 5% CO₂ for 24 h. The pEGFP-NHE3 DMSO treatment group was treated with the same dose of DMSO for 24 h. The pEGFP-NHE3 EGF treatment group was replaced with serum-free phenol red-free DMEM after 6 h of plasmid transfection. After the above groups had reached the treatment time point, they were washed twice with PBS and replaced with phenol red-free maintenance medium for FRAP assay. A Zeiss LSM 800 laser confocal microscope with a 63 × oil objective (numerical aperture of 1.4) was used to observe and photograph the cell samples, and the ROI was used to observe the real-time fluorescence intensity of the three areas. Finally, the ZEN (blue) software was used to export the data and to compare the fluorescence intensity of the bleached areas throughout the process. When the test is completed for all groups, use ZEN (blue) software to export the data and analyze the fluorescence intensity of the bleached area throughout the process (Yang Z. et al., 2018).

Nine 3-day-old lactating Rongchang piglets were randomly divided into Control, PEDV-infected and Tenapanor groups, with three piglets in each group. 10 mL of saline was administered to the Mock group, 10 mL of 1 × 10⁶ TCID₅₀/mL of PEDV LJX strain was administered orally to the PEDV-infected group, and 10 mL of Tenapanor was administered to the Tenapanor group. After inoculation, clinical signs, such as diarrhea, were assessed daily. After significant diarrhea developed in the PEDV group, piglets from both groups were uniformly dissected to observe intestinal lesions.

All animal experiments were approved by the Southwestern University Institutional Animal Care and Use Committee (animal protocol approval number: CQLA-2021-0122). The National Institutes of Health guidelines for the performance of animal experiments were followed.

All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Inc., La Jolla, CA, USA). All data are expressed as the mean ± SD or standard error of the mean (SEM) of three separate repetitions. ANOVA and t-tests were used to analyze significant differences in p values (p * < 0.05; p ** < 0.01; p *** < 0.001).

To determine whether NHE3 is associated with piglet diarrhea, piglets in the control group received 10 mL of saline orally, PEDV-infected groups were orally infected with the PEDV-LJX strain, and the NHE3 inhibitor-fed piglets received Tenapanor at a concentration of 15 mg/kg. The anatomical results showed that control piglets had normal, dry, and formed stools (Figure 1A). However, the PEDV-infected piglets and the NHE3 inhibitor-fed piglets showed dilute watery diarrhea and their gastrointestinal tracts were thinning with transparency, and swollen (Figures 1B,C). These studies demonstrate that reducing the activity of the transporter protein NHE3 in piglet small intestinal epithelial cells leads to severe diarrhea and rapid dehydration in piglets.

To further investigate the relationship between PEDV and NHE3, we detected the NHE3 protein expression levels at different titers of PEDV by Western blot. The results showed that when PEDV was infected at MOI = 1.0 or 0.5 for 2 h, the level of NHE3 decreased significantly at MOI = 0.1 (Figures 2A,C). 72 h after PEDV infection, the level of NHE3 decreased significantly at MOI = 0.1, 0.5 and 1 (Figures 2B,C). These results indicated that NHE3 levels decreased in a dose-dependent manner according to PEDV titers. The higher of PEDV titer, the more pronounced of the decrease in NHE3 levels.

To verify that PEDV infection activates EGFR, activation of the EGFR was evaluated by detecting the level of phosphorylated EGFR (p-EGFR) at different time points after PEDV infection of IPEC-J2 cells. The Western-blot results (Figure 3) indicated that the level of p-EGFR increased significantly during the pre-PEDV infection stage (10, 30, and 60 min) compared with the control group. In the advanced stages of PEDV infection (24, 48 h), p-EGFR levels remained upregulated but not significantly, then significantly higher at 72 h (0.01 < p < 0.05). These findings showed that PEDV infection rapidly activates EGFR, contributing to its phosphorylation and increased its expression.

The S protein of PEDV is a type I transmembrane glycoprotein consisting of two structural domains, S1 and S2, responsible for binding and fusion with the host cell. EGFR exists as a dimer on the cell membrane surface and the functional region is divided into an extracellular receptor binding region, a transmembrane region and an intracellular kinase structural domain (Figure 4B). To investigate the interaction between the S protein and EGFR, the PEDV S gene (637 bp-2127 bp) (Figure 4A) and the EGFR gene (139-1473 bp) were ligated onto the ptt5 vector (Figure 4B) with different tags (flag and his) to identify the size by PCR through Wuhan GeneCreat (Figures 4C,D). Immunoprecipitation was used to test whether the PEDV S1 structural domain could directly interact with the extracellular region of EGFR. Lysates from HEK 293 T cells co-transfected with pTT5-PEDV S1 and pTT5-EGFR plasmids were subjected to western-blot after Co-IP to detect the results. The results confirmed the interaction of the PEDV S1 structural domain with the extracellular region of EGFR (Figure 4E), indicating that EGFR mediates PEDV invasion through binding of the extracellular receptor binding region to the PEDV S1 structural domain.

Based on the fact that PEDV infection activates EGFR, we investigated whether altering the phosphorylation level of EGFR would affect PEDV invasion. In this experiment, EGFR phosphorylation was regulated by EGF (a specific activator of EGFR) and AG1478 (a specific inhibitor of EGFR). It has been shown that EGFR phosphorylation can be induced by treating cells with 10 ng/mL of EGF, and previous studies in our laboratory have shown that 30 μM of AG1478 has the best inhibitory effect on EGFR phosphorylation and it is not toxic to IPEC-J2 cells (Yang Z. et al., 2018). The effect of EGFR modulators on phosphorylated EGFR levels was examined using western blotting, which showed that the optimal duration of action was 15 min for EGF (Figures 5A,B) and 24 h for AG1478 (Figures 5C,D). IPEC-J2 cells were pretreated with the optimal dose and duration of the EGFR modulators determined above, the expression of PEDV N protein was detected by western blotting and the viral titer was determined by TCID₅₀ after 1 h and 2 h of PEDV incubation. The results indicated that PEDV infection increased and EGFR phosphorylation levels were elevated after EGF pretreatment (Figures 5E–G,K), and was significantly higher at 1 h of PEDV infection (0.01 < p < 0.05), indicating that activation of EGFR promotes PEDV infection. In contrast, PEDV infection as well as EGFR phosphorylation levels was significantly reduced after 24 h of AG1478 pretreatment (0.01 < p < 0.05), indicating that inhibition of EGFR activity reduced PEDV infection (Figures 5H–J,L).

To investigate whether there is a direct correlation between EGFR and changes in NHE3, the level of NHE3 was detected after modulating EGFR activity. Western blotting showed that total NHE3 protein levels remained essentially unchanged for the first 60 min after EGFR activation using EGF, and surface NHE3 levels were slightly upregulated in the first 30 min, but decreased at both 24 h and 72 h, in which the decrease at 72 h was significant (Figures 6A–D). Total NHE3 levels and surface NHE3 levels were upregulated for 48 h after EGFR inhibition using AG1478, but were slightly downregulated at 72 h (Figures 6E–H). The above results proved that there is a correlation between EGFR activity and NHE3 levels, and when EGFR activity is inhibited, NHE3 protein levels are upregulated.

To investigate whether PEDV can regulate the level of NHE3 through the EGFR/ERK signaling pathways, PEDV was used to infect IPEC-J2 cells after activation of EGFR, and the levels of key signaling factors in the EGFR/ERK signaling pathway, and the level of NHE3 was examined. The results showed that EGFR was activated after PEDV infected cells for 2 h and 72 h, there was a significant increase in the level of p-EGFR (Figures 7A,B), and the change in level of p-ERK was basically consistent with that of p-EGFR (Figures 7A,C). However, the NHE3 level was downregulated at both 2 h and 72 h of PEDV infection after activation of EGFR activity (0.01 < p < 0.05), and the level was lower than that of the PEDV infection alone group (Figures 7A,D). PEDV infection at 2 h and 72 h after inhibition of EGFR activity continued to elevate p-EGFR levels. However, the elevated levels of p-EGFR were significantly higher in the PEDV-infected group than in the group with inhibition of EGFR activity (Figures 7E,F). Changes in the levels of p-ERK were largely consistent with those of p-EGFR and were also elevated (Figures 7E,G). NHE3 was downregulated after PEDV infection at both 2 h and 72 h compared with that of control group after inhibition of EGFR activity (Figures 7E,H), which was significant at 72 h (0.01 < p < 0.05). However, its level of downregulation was less significant than that in the PEDV-infected group, with a significant difference at 72 h (0.01 < p < 0.05). The above results suggest that PEDV infection leads to notable upregulation of p-EGFR and p-ERK following activation of EGFR activity in IPEC-J2 cells, and that ERK is regulated by EGFR following PEDV infection. This demonstrates that the effect of PEDV infection on NHE3 is regulated by the EGFR/ERK signaling pathway. Activation of EGFR downregulated NHE3 levels more significantly, while inhibition of EGFR activity somewhat attenuated the downregulation of NHE3 in IPEC-J2 induced by PEDV infection.

To better observe the dynamic changes in NHE3 induced by PEDV processing, the impacts of modulating EGFR activity on the mobility of NHE3 across the plasma membrane of IPEC-J2 cells after PEDV infection was examined using FRAP. The fluorescence intensity of the bleached areas in all groups decreased significantly after bleaching and started to recover again with time, indicating that the NHE3 fluorescent molecules remained mobile after PEDV infection (Figures 8A, 9A). The pEGFP-NHE3 group (Control) showed stronger recovery of fluorescence intensity than all PEDV-infected groups, while the pEGFP-NHE3 EGF + PEDV group had the weakest recovery of bleached areas post-bleaching. The fluorescence intensity of the anchored bleached area before and during recovery was analyzed for each group using ZEN (blue) software, which was used to detect the fluorescence recovery rates and dynamic fraction (Mobile fraction, Mf) of each group.

The results showed that the NHE3 fluorescence recovery rate was significantly lower in the EGF-treated group compared with that in the pEGFP-NHE3 group. The fluorescence recovery rate in the AG1478-treated group was higher than that in the DMSO group in the first 6 min and lower than that in the DMSO group after 6 min (Figures 8A,B). However, the NHE3 fluorescence recovery rate in all three groups was lower than the pEGFP-NHE3 group (Figure 8C). This indicated that NHE3 fluorescent bleaching recovery rate was relatively reduced after activation of EGFR, and inhibition of EGFR activity upregulated the fluorescence recovery rate of NHE3. Compared with that in the control group, NHE3 fluorescent bleaching recovery rate in all PEDV-infected groups was weaker at each time point (Figures 9A–C). After activating EGFR, infection with PEDV induced a more noticeable decline in the NHE3 fluorescence recovery rate at 72 h, while inhibiting EGFR activity followed by infection with PEDV relatively upregulated the fluorescence recovery rate of NHE3, and the effect was better at 2 h (Figure 9D). The above results show that compared with the control group, PEDV infection of IPEC-J2 cells reduced the fluidity of NHE3 on the plasma membrane. When EGFR was activated, PEDV infection further reduced the fluidity of NHE3, whereas infection with PEDV after inhibition of EGFR activity could enhance the fluidity of NHE3, indicating that during PEDV infection, the stronger the activity of EGFR on the IPEC-J2 cell membrane, the weaker the fluidity of NHE3, i.e., negative feedback by EGFR regulates the fluidity of NHE3 on the plasma membrane.