The PEDV YN15 (GenBank accession No. KT021228.1), GD (GenBank accession No. KU985230), CV777 (GenBank accession No. AF353511.1) and DR13 (GenBank accession No. JQ023161) strains were preserved in our laboratory. Vero CCL81 (African green monkey kidney) cells, ST (Swine testis) cells and LLC-PK1 (Porcine kidney) cells were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 8% fetal bovine serum (FBS, Suero, Industria Argentina) and incubated at 37°CC with 5% CO₂. Reed-Muench method was used to calculate the 50% tissue culture infectious doses (TCID₅₀) to determine the virus titer. Cinchonine, 3-Methyladenine (3MA), Tunicamycin and 4-Phenylbutyric acid (4-PBA) were purchased from MedChemExpress (MCE) and dissolved in DMSO for preservation. The β-actin polyclonal antibody, LC3 polyclonal antibody and HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) were purchased from ABclonal Technology (China, Wuhan). The Alexa 488-labeled anti-mouse antibody was purchased from Antgene. Monoclonal antibody (MAb) against the PEDV N protein and MAb against the PEDV S were developed in our laboratory.

The cytotoxicity of cinchonine was evaluated in Vero CCL81 cells, ST cells and LLC-PK1 cells by using Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology, China). The cells seeded in 96-well cell culture plate were treated with increasing concentrations of the cinchonine ranging from 0 to 200 μM. The treated cells were then incubated for 48 h at 37°C in 5% CO₂. After incubation, the medium was removed, and the cells were washed with PBS three times. Thereafter, 110 μL CCK8 solution (10 μL CCK8 regent in 100 μL DMEM) was added to the wells and then incubated for 2 h at 37°C with 5% CO₂ in the dark. The optical density (OD) value was measured at the wavelength of 450 nm with a microplate reader (Victor NIVO 3S, USA). The percentage of cell viability was calculated by using the following formula: Cell viability= [As – Ab]/[Ac – Ab]. As: the OD₄₅₀ value of the cinchonine treated wells (wells with cinchonine treated cells and CCK8 solution); Ab: the OD₄₅₀ value of the mock wells (wells with CCK8 solution and DMEM); Ac: the OD₄₅₀ value of the wells without cinchonine (wells with cells and CCK8 solution only).

Total RNA was extracted from Vero CCL81 cells by using a Simply P Total RNA Extraction Kit (Bioflux, China) and reverse transcribed into cDNA using HiScript^® II Q RT SuperMix (Vazyme, China), following the manufacturer’s instructions. The quantitative real-time PCR assay for quantifying the PEDV genome was carried out by using the following primer and probe sequences: PEDV forward primer: 5’- CGTACAGGTAAGTCAATTAC-3’; PEDV reverse primer: 5’-GATGAAGCATTGACTGAA-3’; and PEDV Taq-Man^® probe: FAM-TTCGTCACAGTCGCCAAGG-TAMRA. Quantitative real-time PCR was performed using Hieff UNICON^® qPCR TaqMan Probe Master Mix (YENSEN, China). Each reaction was performed in triplicate.

The cells grown on the 12-well plate were lysed with 100 μL RIPA lysis buffer (Beyotime Biotechnology, China) on ice for 30 min. After adding the loading buffer, the cell lysates were boiled for 10 min and then centrifuged at 12,000 r/min for 5 min at 4°C. The protein extract was loaded on SDS-PAGE and then transferred onto a polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk for 2 h at room temperature. After blocking, the membranes were incubated with primary antibodies overnight at 4°C, followed by being washed three times with TBST. Then the membranes were incubated with HRP-linked secondary antibody for 2 h at room temperature. The target protein was exposed with an Omni-ECL™ Pico Light Chemiluminescence Kit (EpiZyme, China).

Vero CCL81 cells in 96-well culture plate were inoculated with serial 10-fold dilutions of virus. After inoculation, the plates were incubated at 37°C with 5% CO₂ until complete cytopathic effect (CPE) was completed. For the YN15 strain, the wells with syncytium formation, and the specific cytopathic effect caused by YN15, were classified as PEDV-positive. As for the DR13 strain, IFA staining was performed and those with specific staining were considered to be PEDV-positive. Eventually, the PEDV titration was calculated by using 50% tissue culture infectious doses (TCID₅₀) following the Reed–Muench method (Wang et al., 2020).

Vero CCL81 cells in 48-well culture plate were washed with PBS for three times and then fixed with 4% paraformaldehyde for 15 min at room temperature. Vero CCL81 cells were then permeated with ice cold methanol, and blocked in PBS containing 5% bovine serum albumin (BSA) for 30 min at 37°C. After blocking, the cells were incubated with anti-PEDV S protein antibodies diluted in PBS for 1 h at 37°C. Thereafter, the cells were incubated with Alexa 488-labeled anti-mouse antibody for 30 min at 37°C. After washing with PBS, the cells were visualized using a Nikon inverted fluorescence microscope.

Vero CCL81 cells in 24-well culture plate were washed with PBS for three times and infected with the PEDV at a MOI of 0.01. Then, 100 μM cinchonine was added into infected cells at the different time periods after PEDV infection: 0-10, 0-2, 2-4, 4-6, 6-8 and 8-10 h, and the cultures were thereafter incubated for 24 h at 37°C.

Vero CCL81 cells were seeded in 24-well plate, cultured for 48 h, and then infected with PEDV (MOI of 0.01). Serially diluted cinchonine was then added to the culture medium of the cells (final concentrations used were 20, 40, 60, 80 and 100 μM), and the cultures were incubated for 24 h at 37°C. The well without any infection was used as the positive control and DMSO was used as the negative control.

The statistical analysis was accomplished by GraphPad Prism 6.0. Statistical differences with two test groups were analyzed using Student’s t-test. Asterisks indicated significance as follows:

* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

To determine the potential antiviral activities of cinchonine against PEDV, we first used the CCK8 assay to evaluate the safe concentrations of cinchonine that can be used against Vero CCL81 cells, ST cells and LLC-PK1 cells. The cells were treated with different concentrations of the cinchonine ranging from 0 to 200 μM for 48 h, respectively. The results of CCK8 assay showed that no apparent cytotoxicity of cinchonine were observed on Vero CCL81 cells, ST cells and LLC-PK1 cells at a concentration of less than or equal to 200 μM ( Figures 1A-C ).

To explore the effect of different doses of cinchonine on the proliferation of the PEDV, Vero CCL81 cells were treated with cinchonine at a dose of 20, 40, 60, 80 or 100 μM or DMSO, and then infected with PEDV (MOI of 0.01), respectively. At 24 h post-infection, the cells were harvested and thereafter used for further experiments. To analyze the inhibitory effect of cinchonine on PEDV DR13 infection, the mRNA level of PEDV in cells and their supernatants were measured by quantitative real-time PCR. The results showed that cinchonine treatment significantly decreased PEDV mRNA levels in a dose-dependent manner ( Figure 2A ), thereby indicating that cinchonine can effectively inhibit PEDV replication. Meanwhile, the TCID₅₀ results demonstrated that the titer of PEDV DR13 was significantly reduced as compared to the controls ( Figure 2B ). To further analyze the effect of cinchonine on PEDV protein synthesis, Western Blot assay was performed to detect the level of PEDV N protein in infected cells. As shown in Figure 2C , as the concentration of cinchonine increased, the expression level of N protein decreased gradually in infected cells, and moreover cinchonine at a dose of 80 μM or 100 μM nearly blocked the PEDV N protein synthesis completely. Finally, we applied IFA to determine the potential inhibitory effect of cinchonine on the number of PEDV infected cells. As a result, the PEDV specific immunofluorescence gradually reduced in infected Vero CCL81 cells ( Figure 2D ), thereby clearly suggesting that the antiviral effects of cinchonine exhibited a concentration-dependent response. To further verify that cinchonine displayed anti-PEDV effects, CV777, YN15 and GD were used to examine the antiviral effects of cinchonine, excluding the specificity of PEDV DR13 strain. As shown in Figures 2E-G , cinchonine could substantially reduce the mRNA level of PEDV infected cells in a dose-dependent manner. In addition, cinchonine had similar inhibitory effects against PEDV in LLC-PK1 cells ( Figure 2H ). Based on the above experimental results of cinchonine against PEDV, the anti-PEDV activity of cinchonine was confirmed.

The life cycle of PEDV includes adsorption, penetration, replication, assembly, and release (Fehr and Perlman, 2015). In order to in-depth explore the detailed mechanism of action of cinchonine in inhibiting PEDV infection, the time-of-addition assay was adopted to evaluate the potential stage of PEDV infection that was specifically affected by cinchonine. As shown in Figure 3A , vero CCL81 cells were exposed to PEDV and then treated with cinchonine (100 μM) for the indicated periods of time (0-10, 0-2, 2-4, 4-6, 6-8 and 8-10 h). At 24 h post-infection, the samples were collected for TCID₅₀ assay and the quantitative real-time PCR. As shown in Figures 3B, C , the virus titers and mRNA levels were significantly reduced in the presence of cinchonine between 0-10 h. In contrast, cinchonine exhibited the strongest anti-PEDV activity when it was added at 2 h, thereby demonstrating that cinchonine may primarily affect PEDV adsorption and invasion to cells. In conclusion, these results indicated that cinchonine may exhibit a repressive inhibitory effect during the early stages of PEDV infection.

To elucidate the anti-PEDV activity of cinchonine in vitro, we further performed experiments to explore its underlying antiviral mechanism. We have convincingly demonstrated from the above findings that cinchonine could significantly attenuate the replication of PEDV on Vero CCL81 and LLC-PK1 cells. Existing studies have confirmed that rapamycin could also inhibit PEDV infection in porcine intestinal epithelial cells by inducing autophagy (Ko et al., 2017), so we hypothesized whether inhibitory actions of cinchonine on PEDV infection may also be related to autophagy. The autophagy protein LC3, which is involved in the formation of autophagosomal vacuoles, is widely used as an autophagosomal marker protein to monitor autophagy. When the autophagy occurs, LC3-I can combine with phosphatidyl ethanolamine (PE) to form LC3-II, which is located on the autophagosome double-layered membrane. Accordingly, autophagy can be monitored by measurement of the expression of LC3-II and conversion of LC3-I to LC3-II proteins (Rusmini et al., 2019). Vero CCL81 cells, ST cells and LLC-PK1 cells were treated with 100 μM cinchonine for 24 h respectively, and the protein expression of LC3 was examined by Western Blot. The results showed that there was a remarkable increase in LC3-II protein level and the conversion of LC3-I to LC3-II was also markedly increased as compared to the negative control group ( Figures 4A-C ). These findings indicated that cinchonine may regulate intracellular autophagy in Vero CCL81, ST and LLC-PK1 cells.

A number of previous studies have indicated that ER stress inducers can display significant antiviral activities against PEDV (Wang et al., 2014). Interestingly, as cinchonine has been reported to activate ER stress through increasing the expression of GRP78/CHOP in human liver cancer cells especially in HeLa and A549 cells (Qi et al., 2017), we further identified whether cinchonine could stimulate ER stress in Vero CCL81 cells, ST cells and LLC-PK1 cells. Glucose regulating protein 78 (GRP78) is the primary protein that can regulate the refolding or degradation mechanism of misfolded proteins in ER (Elfiky et al., 2020). It is main player involved in the regulation of the unfold protein response (UPR) in ER and its upregulation can act as a potential marker of ER stress (Sun et al., 2021). Vero CCL81 cells, ST cells and LLC-PK1 cells were exposed to cinchonine for 24 h, and the expression of GRP78 protein was detected by Western Blot analysis. It was found that compared with the positive control (Tu, which is a mixture of antibiotic that can inhibit N-linked glycosylation which causes accumulation of unfold proteins and activates ER stress, cinchonine treatment group did not lead to an increase the expression of GRP78 protein effectively, thus demonstrating that cinchonine could not induce ER stress in Vero CCL81 cells, ST cells and LLC-PK1 cells ( Figures 5A-C ). The above results suggested that cinchonine might activate ER stress only in a cell-specific manner and is not a universal ER stress activator.

Since cinchonine could induce autophagy, we further analyzed the potential effect of cinchonine on PEDV infection in the presence of 3MA. 3MA is a PI3K inhibitor, which is widely used to inhibit autophagy via its inhibitory effect on class III PI3K (Miller et al., 2010). Vero CCL81 cells were pretreated with 3MA for 2 h and then infected with PEDV in the presence or absence of 3MA for 24 h. As shown in Figure 6A , the expression of LC3-II protein was markedly reduced in 3MA treatment group as compared with the 3MA untreated group. In addition, when autophagy was inhibited, we observed that the numbers of PEDV infected cells ( Figure 6D ) and mRNA levels of PEDV ( Figure 6B ) were significantly attenuated in the presence of cinchonine and that the infection of PEDV was rescued in the presence of 3MA. These results revealed that autophagy inhibitor 3MA could significantly alleviate the reduction of PEDV infection caused by cinchonine.

It has been reported that cinchonine could activate ER stress, so we next analyzed the effect of cinchonine on PEDV replication in the presence of ER stress inhibitor. We pretreated Vero CCL81 cells with 4-Phenylbutyric acid (4-PBA), which has been reported to relieve ER stress by inhibiting the phosphorylation of eIF2α (Ozcan et al., 2006; Ozcan et al., 2008). As shown in Figures 6C, E , no significant changes in the numbers of PEDV infected cells and the mRNA levels of PEDV were observed compared with the group treated with cinchonine without 4-PBA, thereby indicating that 4-PBA was not able to effectively mitigate PEDV infection inhibition by cinchonine.

Collectively, these findings supported our hypothesis that autophagy induced by cinchonine was primarily responsible for the inhibition of PEDV infection.