African green monkey kidney epithelial (Vero) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) containing 10% fetal bovine serum (FBS) (Sigma, USA) and 1% penicillin-streptomycin (Solarbio, China). The wild-type PEDV strain GX4/2021 (GenBank accession number OP382083) was grown on Vero cells using a maintenance medium, DMEM supplemented with 2 μg/ml of trypsin (Gibco, USA), 0.3% tryptose phosphate broth (TPB) (Sigma, USA), and 1% penicillin-streptomycin. A monoclonal antibody (mAb) against PEDV N protein was produced in mice, and a polyclonal antibody (pAb) against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Proteintech, China, and a mAb against beta actin was purchased from Huabio, China.

The PEDV full-length cDNA is assembled into pBeloBAC11 vector. The restriction sites, namely BamHI, BbvcI, PacI, AvrII, BlpI, ClaI, and SacII, which naturally occurred in the PEDV-GX4/2021 genome, were selected. The synthesis of PEDV cDNA was carried out by Hifair^® AdvanceFast 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology, China). Seven overlapping DNA fragments (A1, A2, B, C1, C2, C3, C4) were amplified by PCR using PrimeSTAR^® Max DNA Polymerase (Takara, China) and the primers were listed in Table 1 . A CMV promoter, a 35-residue poly (A) tail, hepatitis delta virus (HDV) ribozyme sequence and bovine growth hormone (BGH) termination signal were inserted into the PEDV infectious clone. Finally, all the fragments were ligated into the linearized vector by restriction enzyme sites as shown in Figure 1A . The PEDV infectious clone plasmid is named pBAC-PEDV.

The cDNA clone plasmids containing Nluc, RFP, or GFP ( Figure 2A ) were produced with Red recombination using the E. coli strain GS1783. As shown in Figure 2B , the en passant marker cassette containing a short duplication sequence of insertion (ins, the fragment a), I-SceI, and kanamycin (Kana) is amplified by PCR using primers ( Figure 2B II). The universal transfer construct ins-Kana containing the 40-50 bases homologous arms for the PEDV genome was amplified by overlap PCR ( Figure 2B III). The universal transfer ins-Kana was electroporated into the GS1783 E. coli containing the full-length PEDV infectious clone plasmid for the first Red recombination ( Figure 2B IV). The positive colonies in the Red recombination could grow on an LB agar plate containing Kana (30 μg/ml) and chloramphenicol (CAP, 34 μg/ml). Finally, the marker cassette is deleted from the intermediate ( Figure 2 V) by the I-SceI site cleavage through the induction of L-arabinose in vivo for a second red recombination ( Figure 2 VI). The primers used above were described in Table S1 in the Supplementary Material .

In a 6-well culture plate, Vero cells with 70% confluence were co-transfected with 1.8μg full-length cDNA clone plasmid (pBAC-PEDV, pBAC-PEDV-RFP/ORF3, pBAC-PEDV-Nluc/ORF3, or pBAC-PEDV-GFP-ORF3), and 0.2μg pCAGGS-PEDV-N using Lipofectamine™ 3000 reagent (Invitrogen, USA). After 24 hours of transfection, the cell monolayers were washed with phosphate-buffered saline (PBS) and treated with a maintenance medium. The cells were then inoculated at 37°C. The cytopathic effect (CPE) and red or green fluorescence were observed daily with a fluorescence microscope (Leica, Germany).

In a 12-well culture plate, Vero cells were washed twice with PBS and infected with the indicated viruses at a multiplicity of infection (MOI) of 0.001 for 24h. The cell monolayers were fixed with 4% paraformaldehyde for 10 min. After washed with PBS, the cells were permeabilized using 0.1% Triton X-100 and blocked with 2% bovine serum albumin (BSA) in PBS for 30 min at room temperature. The cell monolayers were incubated with mAb PEDV N for 1 h at 37°C and further stained with Dylight 488-labeled goat anti-mouse IgG (H+L) (Abbkine, China). Then the cell nuclei were stained with 2 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) (Solarbio, China) for 10 min. After washing twice with PBS, fluorescent pictures were taken using a fluorescence microscope (Leica, Germany).

The Vero cells were lysed in RIPA Lysis Buffer (Beyotime, China). The cell lysates were mixed with 5× sodium dodecyl sulfate (SDS) loading buffer and heated at 98°C for 5 min. The proteins were separated through 12% SDS-polyacrylamide gels and were subsequently transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk in TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween 20) at room temperature for 2 hours. Then, the membranes were incubated overnight at 4°C with mAb against PEDV N and pAb against GAPDH. The membranes were washed three times with TBST and then were incubated with HRP-conjugated secondary antibodies. Finally, the membranes were visualized using NcmECL Ultra (NCM Biotech, China) by a chemiluminescence imaging system (Tanon, China).

In a 24-well (10⁵ cells/well) plate, Vero cells were infected with recombinant viruses at 0.001 MOIs. After incubation for 2 h, the cell supernatants were removed and a maintenance medium was added to the cell monolayers. The cell culture supernatants were collected at different time points. The virus titers were determined by a TCID₅₀ assay following the Karber method. The viral growth curves were made by using GraphPad Prism software (version 8.0).

In a 12-well culture plate, recombinant viruses were introduced to confluent Vero cells at a multiplicity of infection (MOI) of 0.0001. Following a 2-hour incubation, the cell culture supernatants were aspirated. Subsequently, the cells were washed twice with PBS, and a maintenance medium supplemented with 1% low melting point agarose was applied to the cell monolayers. At 36 hours post-infection (hpi), the cells were fixed and stained using a solution of 20% alcohol and 1% crystal violet in physiological saline.

In a 96-well culture plate, Vero cells were infected with the indicated viruses in triplicate wells. Samples were collected at 3, 6, 9, and 12 hpi, respectively. Following the manufacturer’s instructions, the luciferase activity was then measured with Nano-Glo® Luciferase Assay System (Promega, USA).

PEDV reporter viruses were passaged nine times (P1 to P9) in Vero cells and the viral RNAs of P3 and P9 were extracted. Then the exogenous genes were identified by RT-PCR and DNA sequencing.

After Vero cells and ST cells were treated with the indicated drugs, the cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Yeasen Biotechnology, China). In a 96-well cell plate, the single layers were washed twice with PBS, followed by incubation at different concentrations of drugs or DMSO. CCK-8 solution (10 μL/well) was added to the plate at the indicated time. After incubation for 3 h at 37°C, the absorbance was tested at the 450 nm wavelength.

Total RNA was extracted from the cells using RNA-easy Isolation Reagent (Vazyme Biotech, China), followed by reverse transcription of the RNAs into cDNA using Hifair^® V one-step RT-gDNA digestion SuperMix (Yeasen Biotechnology, China). The primers targeting PEDV N gene, monkey GAPDH, and pig GAPDH were designed in Table 2 . Gene expression was estimated using Hieff^® qPCR SYBR Green Master Mix (Yeasen Biotechnology, China) by a LineGene 9600 system (Bioer Technology, China). Relative expression levels of PEDV N were calculated with the 2^-ΔΔCt method.

The experimental groups were compared to the mock-treated group using one-way ANOVA in GraphPad Prism (version 8.0) software. The levels of statistical significance were defined as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

To construct the PEDV full-length cDNA clone, a set of fragments covering the entire PEDV-GX4/2021 genome were assembled into pBeloBAC11 ( Figure 1A ). The PEDV infectious clone plasmid was named pBAC-PEDV. The CMV promoter was inserted at the 5’ terminus of virus genome, and a 35-residue poly (A) tail was placed at 3’end. The complete cDNA clone plasmid was digested with EcoRI to perform restriction fragment length polymorphism (RLFP) analysis, which produced four specific fragments as expected ( Figure 1B ). Meanwhile, the pBAC-PEDV was detected using PCR with the primers described in Table 1 ( Figure 1C ) and verified by full-length nucleotide sequencing. Taken together, we successfully constructed the PEDV full-length cDNA clone plasmid.

According to the strategy outlined in Figure 3A , the supernatant of the transfected cells was further passaged onto Vero cells to generate rPEDV. In accordance with wild-type PEDV, the expression of PEDV N in rPEDV-infected cells could also be detected by IFA ( Figure 3B ) and western blot ( Figure 3C ). To investigate the growth characteristics of rPEDV in relation to wild type PEDV, the viral growth curves and plaque size were detected. As shown in Figure 3D , the rescued rPEDV showed similar growth kinetic to the wild-type PEDV, with their titers peaking at 24 hpi (10^5.5TCID₅₀/mL). There is also no significant difference between the rPEDV and wild-type PEDV as confirmed by plaque assay ( Figures 3E–G ).

PEDV ORF3 accessory protein is a virporin, which bears a potassium ion channel activity (Wang et al., 2012). It has been reported that reporter genes can replace the non-essential ORF3 gene in PEDV replication (Jengarn et al., 2015). The Red recombination with E. coli strain GS1783 is a convenient tool for manipulating virus infectious clones, so three PEDV reporter virus plasmids were generated using the Red recombination. As shown in Figure 2A , the ORF3 gene was swapped with a RFP gene or Nluc gene to produce ORF3-deleted variants. To achieve the fusion expression of exogenous genes while preserving the ORF3 gene, the GFP gene and the porcine teschovirus 1 (PTV-1) 2A autocleavage peptide (Caì et al., 2018) were inserted upstream of the PEDV ORF3 gene ( Figure 2A ). The positive co-integrate rate of the first Red recombination, identified through colony PCR, exceeded 80% ( Table 3 ). Through twice Red recombination, three reporter virus plasmids were generated and named pBAC-PEDV-RFP/ORF3, pBAC-PEDV-Nluc/ORF3, and pBAC-PEDV-GFP-ORF3, respectively. Then DNA sequencing was performed to verify the first and second recombination of the three resulting plasmids ( Figures 4A, B ). Taken together, three rPEDV reporter virus plasmids were successfully generated with the E. coli strain GS1783.

Similar to the strategy in the recovery of rPEDV, we obtained three rPEDV reporter viruses. To furtherly confirm the rPEDV reporter viruses, the expression of PEDV N in rPEDV reporter virus-infected cells was detected by IFA ( Figure 5A ) and western blot ( Figure 5B ). We further examined the growth kinetics of the rPEDV reporter viruses. The results indicated no significant difference in plaque size and morphology ( Figure 5D ). All the recombinant viruses replicated effectively and reached peak titer at 24 hpi ( Figure 5C ). The rPEDV-GFP-ORF3 displayed similar replication kinetics to the rPEDV, while the highest titers of rPEDV-RFP/ORF3 and rPEDV-Nluc/ORF3 were approximately 0.5 logs lower than that of rPEDV. To detect the stability of exogenous genes, rPEDV reporter viruses were serially cultured in the Vero cells. RT-PCR and DNA sequencing analysis showed that the foreign genes (Nluc, RFP, and GFP) were stable in P3 and P9 stocks in vitro, and the N gene was as control ( Figures 5E-G ). Taken together, three rPEDV reporter viruses were successfully rescued, and the foreign genes possessed the stability up to P9 in Vero cells.

The novel engineered luciferase, Nluc, is 150 times brighter than firefly or Renilla luciferase. It can produce high-intensity luminescence of the glow-type using the synthetic substrate furimazine while maintaining low background activity (Hall et al., 2012; England et al., 2016). To examine the sensitivity of PEDV infection in different cell lines, Vero, ST, and LLC-PK1 cells were infected with rPEDV-Nluc/ORF3. Subsequently, the cell lysates were collected to detect Nluc activity and PEDV N protein expression levels. The Nluc signal was observed in Vero cells at 3 hpi and exhibited a time-dependent increase in comparison to the mock-infected group. In contrast, western blot assay showed that PEDV N protein expression could be detected at 12 hpi ( Figure 6A ). As shown in Figures 6B, C , rPEDV-Nluc/ORF3 could infect both ST and LLC-PK1 cell lines, with the Nluc activity detected early at 6 hpi and PEDV N protein expression observed at 12 hpi. The results indicated that the Nluc reporter virus is more sensitive in quantify features to reflect the viral replication levels than N protein expression, particularly during the early stage of infection.

To assess the viability of using rPEDV-Nluc/ORF3 for antiviral drug screening, we selected three drugs, namely Ganoderma lucidum triterpenoids (GLTs) (Min et al., 1998; Bharadwaj et al., 2019), Bergenin (Yang et al., 2022), and Umifenovir hydrochloride (Arbidol) (Wang et al., 2020). The CCK-8 assay was used to determine the cytotoxic effect of these drugs on Vero cells by measuring cell viability. Results showed there was no significant cytotoxicity on cells when treated with GLTs (up to 80 μg/ml), Bergenin (up to 30 μM), or Arbidol (up to 16 μM) ( Figures 7A–C ). When rPEDV-Nluc/ORF3-infected Vero cells were treated with GLTs ( Figure 7D ), the Nluc activity and expression level of PEDV N protein were dose-dependently inhibited. In comparison, the other two drug treatments (Bergenin and Arbidol) had no significant inhibitory impact on Nluc activity and PEDV N expression in rPEDV-Nluc/ORF3-infected cells ( Figures 7E, F ). These results suggested that GLTs has a significant antiviral effect on rPEDV-Nluc/ORF3 infection. Meanwhile, the antiviral effect of GLTs on rPEDV-Nluc/ORF3 infection in ST cells was also confirmed ( Figures 7G, H ). In addition, the effect of GLTs on wild-type PEDV strain GX4/2021 was further evaluated by qPCR, western blot, and a TCID₅₀ assay. The data showed that the infection of PEDV-GX4/2021 was also inhibited by GLTs in a dose-dependent manner ( Figures 7I, J ). The Bergenin treatment was as control ( Figures 7K, L ). When ST cells was treated with GLTs, the replication of wild-type PEDV was also inhibited ( Figures 7M, N ). Taken together, the treatment with GLTs effectively inhibited PEDV infection in Vero and ST cells.

Generally, viral life cycle of coronaviruses includes entry, viral genome translation, RNA synthesis, viral particle assembly, and release, and each stage is crucial to viral infection (V’kovski et al., 2021). To explore the potential inhibition targets, GLTs was added to the cells at varying concentrations during different stages of the virus life cycle, including “Entry”, “Post entry” and “Full time” ( Figure 8A ) (Chen et al., 2022). Compared to the mock-treated groups, the Nluc activity and the expression level of N protein were suppressed in “Post entry” groups of the rPEDV-Nluc/ORF3-infected Vero cells, especially at a concentration of 80 μg/ml ( Figure 8B ). The replication of GX4/2021 strain was also considerably inhibited by GLTs treatment at the stage of “Post entry” as shown by the qPCR and western blot assay ( Figure 8C ). Moreover, viral titers decreased at the virus “Post entry” stage but not at the virus “Entry” stage when compared to control groups in Vero cells ( Figure 8D ). Similar results were obtained when we repeated the experiments in ST cells ( Figures 8E–G ). Taken together, GLTs mainly inhibit PEDV replication after the viruses enter host cells.