Vero cells were obtained from ATCC (ATCC number: CCL-81) (USA) and cultured in Dulbecco's modified eagle medium (DMEM) (Hyclone, USA), supplemented with 10% fetal bovine serum (FBS) (BOVOGEN, Australia), 100 U/mL penicillin, and 100 U/mL streptomycin in 37°C with 5% CO₂ incubator. The maintenance medium for PEAV propagation was DMEM supplemented with 10 μg/mL trypsin (Gibco, USA) and cultured under the conditions described above. The PEAV GDS04 strain was isolated from piglets with severe diarrhea in our laboratory (8).

Virus propagation was performed as previously described (8). Briefly, Vero cells were seeded into T175 flasks and cultured for 90% confluence. One mL of PEAV GDS04 strain together with 50 mL of maintenance medium were added to flasks after the cell monolayers were washed three times with sterile pH 7.4 1 × phosphate buffered saline (PBS). The virus-inoculated cells were cultured continuously at 37°C in 5% CO₂ to observe the cytopathic effect (CPE). Around one day post-infection (d.p.i.), >80% CPE was evidently observed in the inoculated cell monolayers; the flasks were then twice frozen at −80°C and thawed. The cells and supernatants were harvested together and stored at −80°C until subsequent viral titers' determination. Virus titers were calculated using the Reed-Muench method (9) and expressed as TCID₅₀ per milliliter.

The animal study was approved by the Institutional Animal Care and Use Committee of the Sun Yat-sen University (Guangdong, China) and animals were treated in accordance with the regulations and guidelines of this committee. Thirty-three-day-old healthy conventional weaned piglets, crossbred of Duroc × Landrace × Native pigs of Guangdong of China, were procured from Wen' s Foodstuffs Group Co., Ltd. (Guangdong, China). All pigs were housed in the vivarium under standard environmental conditions and maintained in our animal facility with food and water ad libitum for a minimum of 7 days before the experimentation.

PEAV-Coated were prepared using centrifugal granulation- fluidized bed coating technology as described previously, with some modifications (10). Briefly, we added the commercial sucrose microspheres (450 g) (Anhui Sunhere Pharmaceutical Excipients Co., Ltd, China) into a centrifugal granulator (Shenzhen Xinyite Science and Technology Co., Ltd, China), and adjusted the solid rotation disc' s speed to 27 × g to keep the vorticity of the microspheres. We also adjusted the supply air velocity and the exhaust air velocity to 326 × g and 666 × g, respectively. We subsequently set the machine to spray pure water (80–100 mL) to blend the powders into the microspheres at 0.02 × g for 30 min by a tangential spray nozzle. About 136 g new powders were constantly added into the microspheres. Then the final product was dried at 37°C for 1 h under the same air quantity. Subsequently, the dried PEAV-loaded microspheres smaller than 0.7 mm were screened by sieving, and then filled into a fluidized bed apparatus with a bottom spray configuration (Shenzhen Xinyite Science and Technology Co., Ltd., China). We used the magnetic stirrer to mix enteric coating suspensions (Weight gain 25%) with 6% Hydroxypropyl methyl cellulose phthalate (HPMCP) (Shanghai Yunhong Chemical Co., Ltd., China), and then PEAV-loaded microspheres were continuously sprayed with the enteric coating suspensions at 0.25 mL/min through a spray nozzle at the bottom of the fluidized bed apparatus. Of note, the relative bed temperature needed to be kept at 29°C to avoid an agglomeration of the microspheres during the coating process. After all the enteric coating suspensions were sprayed out, the products needed to dry for an additional 30 min.

According to the method described by Kashappa-Goud H. Desai and Steven P. Schwendeman (11), we used a Hitachi S-3400N scanning electron microscope (Hitachi, Japan) to examine the surface morphology of microspheres by taking SEM images. Briefly, we fixed the microspheres on a brass stub with a double-sided adhesive tape and then coated them with ~3–5 nm electrically conductive gold for 100 s at 40 W in a vacuum. Then, the excitation voltage was set as 8–10 kV to take the images of the microsphere surface. One hundred randomly selected samples of PEAV-Coated were selected to measure the diameter with a Vernier caliper (Guangzhou Heyue Biotechnology Co., Ltd., China) to determine the size distribution of the PEAV-Coated. In addition, 100 samples of PEAV-Coated or sucrose microspheres were randomly selected and measured with Electronic scales (Sartorius Group, Germany) to calculate the weight gain of the single microsphere and to analyze the weight gain of the single PEAV-Coated.

In line with previous studies (12), we used simulated gastric fluid and simulated intestinal fluid to determine the acid resistance and enteric solubility of PEAV-Coated in vitro. Briefly, PEAV-Coated were successively incubated in pH 1.2 simulated gastric fluid prepared with 2 g/L NaCl, 3.2 g/L porcine pepsin, 0.7% HCl, and pH 6.8 simulated intestinal fluid prepared with 6.8 g/L NaH₂PO₄, 7.7% 0.2 N NaOH, and 10 g/L pancreatin at 37°C for 2 h, and freeze-dried powders containing the PEAV GDS04 strain were treated under the same conditions. The TCID₅₀ assay as described above was used to analyze the acid resistance of PEAV-Coated after the simulated gastric fluid treatment.

To analyze the acid resistance and enteric solubility of PEAV-Coated in vivo, weaned piglets were orally inoculated with these microspheres to assess virus shedding according to previous studies with some modifications (10). Briefly, 18 conventionally weaned piglets were randomly divided into three groups with six piglets in each, and were housed in three separate rooms. On day 0, weaned piglets in group one were orally challenged with 20 g of PEAV-Coated containing a total of 2 × 10⁵ TCID₅₀ of the PEAV GDS04 strain (1 gram of microspheres contained 1 × 10⁴ TCID₅₀ of PEAV GDS04 strain). Weaned piglets in groups two and three as controls were orally inoculated with 20 mL of maintenance medium or 20 mL of maintenance medium containing a total of 2 × 10⁵ TCID₅₀ of the PEAV GDS04 strain (1 milliliter of medium contained 1 × 10⁴ TCID₅₀ of PEAV GDS04 strain), respectively. After challenge, rectal swabs were collected from each piglet on day 0, 2, 4, 6, and 8 after the challenge to assess PEAV virus shedding with real-time PCR as described below.

Twenty-four conventionally weaned piglets, negative of the major porcine enteric viruses including PDCoV, PEDV, TGEV, PRoV, and PEAV by testing the rectal swabs on day −1 as previously described (13), were randomly divided into two groups with 12 piglets in each and were housed in two separate rooms. On day 0, weaned piglets in one group were orally challenged with 100 g/head of PEAV-Coated containing a total of 1 × 10⁶ TCID₅₀ of the PEAV GDS04 strain (1 gram of microspheres contained 1 × 10⁴ TCID₅₀ of PEAV GDS04 strain) for 3 days and weaned piglets in another group were orally inoculated with 100 g/head sucrose microspheres for 3 days and served as uninfected controls. After infection, clinical signs of vomiting, diarrhea, and lethargy were observed daily in each piglet. In addition, the diarrhea severity of each piglet was scored daily according to the previous criteria (14): 0 = normal, 1 = soft (cowpie), 2 = liquid with some solid content, 3 = watery with no solid content.

Rectal swabs were collected from each piglet before inoculation and then every day until 7 d.p.i. and were homogenized in 1 mL sterile pH 7.4 1 × PBS immediately after collection. Six of the challenged piglets and six of the negative control piglets were randomly selected from each group and humanely sacrificed for necropsy at 3 d.p.i., and the remaining weaned piglets were necropsied at 7 d.p.i. At necropsy, the fresh Peyer's patches from ileum were collected for analysis of the antiviral molecules with real-time RT-PCR and the fresh jejunum were also analyzed with histopathology and immunohistochemistry.

RNA extraction and RT were performed as previously described with some modifications (8). Briefly, viral RNA was extracted from the rectal swab fluids from each piglet by using an RNeasy kit (Magen, China) according to the manufacturer' instruction. Two μg of viral RNA was converted to cDNA by using an RT-PCR kit (TaKaRa, Dalian). Primers for the nucleocapsid (n) gene of PEAV (sense: 5′-CTGACTGTTGTTGAGGTTAC-3′; antisense: 5′-TCTGCCAAAGCTTGTTTAAC-3′), and probe (5′-FAM-TCACAGTCTCGTTCTCGCAATCA-TARMA-3′) were designed as previously described (15) and synthesized by Invitrogen Company (Shanghai, China). The real-time PCR assay was performed on an Applied Biosystem 7500 instrument (Life Technologies, USA) with a 20-μL volume containing 1 μL of cDNA, 10 μL of Thunderbird Probe qPCR Mix, 0.04 μL 50 × Rox reference dye (TOYOBO, Shanghai), 0.2 μmol/L of probe, and a 0.3 μmol/L of each gene-specific primer. The PCR program was as follows: 95°C for 30 s; 45 cycles of 95°C for 5 s, 62°C for 30 s. The n gene was amplified by using the specific primers (sense: 5′-CCGCTCGAGATGGCAACTGTTAATTGG-3′; antisense: 5′-CGCGGATCCCGATTAATAATCTCATCCAC-3′) that were designed according to the sequence of PEAV strain GDS04 (GenBank, Accession no: MF167434.1), and the PCR products were ligated with the pEGFP-N1 vector (Clontech, USA) by using a PCR cloning kit (NEB, USA), and then the 10-fold serially diluted known plasmid concentration was used as the template to construct a real-time PCR standard curve in each plate. The quantity of PEAV viral RNA in rectal swabs was calculated based on the cycle threshold (Ct) values for the standard curve.

To analyze antiviral molecular changes in the piglets with PEAV infection, equal quantities (1 g) of Peyer's patches were homogenized in sterile pH 7.4 1 × PBS, and 200 μL of the supernatant was used for RNA extraction by using an RNeasy kit (Magen, China) following the manufacturer's instruction. Two μg of total RNA was converted to cDNA by using an RT-PCR kit (TaKaRa, Dalian). The specific primers for porcine IFN-α (sense: 5′-TCTCATGCACCAGAGCCA-3′; antisense: 5′-CCTGGACCACAGAAGGGA-3′), IFN-β (sense: 5′-AGTGCATCCTCCAAATCGCT-3′; antisense: 5′-GCTCATGGAAAGAGCTGTGGT-3′), PKR (sense: 5′-AAAGCGGACAAGTCGAAAGG-3′; antisense: 5′-TCCACTTCATTTCCATAGTCTTCTGA-3′), OAS (sense: 5′-GAGCTGCAGCGAGACTTCCT-3′; antisense: 5′-TGCTTGACAAGGCGGATGA-3′), Mx1 (sense: 5′-GGCGTGGGAATCAGTCATG-3′; antisense: 5′-AGGAAGGTCTATGAGGGTCAGATCT-3′), and glyceraldehydes-3-phosphate dehydrogenase (GAPDH; sense: 5′-CCTTCCGTGTCCCTACTGCCAAC-3′; antisense: 5′-GACGCCTGCTTCACCACCTTCT-3′) were designed as previously described (16, 17) and synthesized by Sangon Company (Shanghai, China). The real-time PCR assay was performed on an Applied Biosystem 7500 instrument (Life Technologies, USA) with a 20-μL volume containing 1 μL of cDNA, 10 μL of 2 × SYBR green Premix Ex Taq (TaKaRa, Dalian), and 0.4 μM of each gene-specific primer. The amplification conditions were referring to previous publications (16) and were as follows: 95°C for 30 s; then 40 cycles of 95°C for 3 s, 60°C for 30 s; and 1 cycle of 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s, 60°C for 15 s. A melt curve for the PCR products was obtained to determine the specificity of the amplification at the final step. The antiviral molecules expressions were calculated relative to the expression of the reference gene GAPDH and presented as the change (n-fold) relative to the control samples.

Histological and Immunohistochemical staining were performed as previously described with some modifications (8). Briefly, at necropsy, the jejunum tissue samples of the piglets from the challenged and control groups were separated, routinely fixed in 10% formalin, embedded, sectioned, and stained with hematoxylin and eosin (H&E); the slides were then examined and analyzed with conventional light microscopy. Five-μm sections of formalin-fixed paraffin-embedded tissues were mounted onto positively charged glass slides. Slides were air dried at 60 °C for 120 min prior to deparaffinization. Slides were then rinsed and incubated with target retrieval solution (Servicebio, China). The sections were incubated with PEAV (GenBank, Accession no: MF167434.1) specific mouse antisera (Wen's Foodstuffs Group Co., Ltd, China) (1:400) as the primary antibody for 12 h at 4 °C after to block with 1% BSA (Solarbio, China). They were then incubated with peroxidase-labeled goat anti-mouse IgG secondary antibody (Dako, Denmark) (1:200) for 50 min at room temperature prior to visualization with a 3, 3′-diaminobenzidine (DAB) chromogen kit (Dako, Denmark). In addition, hematoxylin was used for counterstaining. Jejunum tissue samples from uninfected piglets were used as a negative control.

Statistical comparisons were performed using GraphPad Prism software. The significance of the differences between the treatment group and controls in the mRNA expressions [TCID₅₀, antiviral molecules (IFN-α/β, OAS, PKR, and Mx1)] was determined by the ANOVA and Mann-Whitney accordingly.

Viruses by oral feeding failed to infect the body due to the labile components that were degraded by the acid pH of the stomach (18). To successfully infect the animals, we used centrifugal granulation technology to load the freeze-dried powders containing the PEAV GDS04 strain onto sucrose microspheres (Figure 1A). To protect PEAV from low pH while facilitating controlled virion release in the intestine, pH-resistant enteric coating (HPMCP) was loaded on the PEAV-loaded microspheres using a fluidized bed coating apparatus after a drying process (Figure 1B). The final products of the manufacturing process yielded the coated mini-spheres, with a faint-yellow surface (Figure 1C).

To observe the encapsulation effect of PEAV in the microspheres, we used a scanning electron microscope to examine the morphology of the microspheres. As shown in Figure 2B, a scanning electron micrograph revealed the microparticles containing PEAV characterized by their pill shape, rough surface, lack of porosity, and relatively uniformed size. In addition, compared to sucrose microspheres, PEAV-Coated were around 1.3 times larger (Figure 2A). Further, we found that more than 95% of microspheres were in the size range of 700–800 μm in diameter (Figure 2C). In addition, single PEAV-Coated weight gained 60% more than single sucrose microspheres (Figure 2D).

Considering that viruses by oral feeding overcoming the acid pH of the stomach is the key to infection, we examined the acid resistance and enteric solubility of PEAV-Coated in vitro and in vivo. As shown in Figures 3A,B, we found that the shape of PEAV-Coated was unaffected after simulated gastric fluid treatment, but it could dissolve in simulated intestinal fluid. In addition, compared to the microspheres before treatment, virus titers in microspheres after the simulated gastric fluid treatment dropped slightly (Figure 3C). To further determine whether PEAV-Coated could resist gastric acid in vivo, we fed weaned piglets with PEAV-Coated and the control groups with DMEM without mixing with sucrose microspheres or PEAV without mixing with sucrose microspheres. As shown in Figure 3D, in half of the weaned piglets orally inoculated with PEAV-Coated PEAV RNA could be detected in fecal swabs at the sixth day after the challenge. In contrast, no PEAV RNA were detected in DMEM and PEAV-inoculated weaned piglets.

In order to determine whether PEAV-Coated could infect weaned piglets, 33-day old conventionally weaned piglets were orally infected with PEAV-Coated at a dose of 1 × 10⁶ TCID₅₀/100 g/head. Compared to the negative control, watery diarrhea was observed in all weaned piglets inoculated with PEAV-Coated from 1 d.p.i. to 7 d.p.i. (Figure 4A). We further examined the viral shedding by real-time PCR in fecal swabs collected from inoculated-piglets from 1 d.p.i. to 7 d.p.i. As shown in Figure 4B, PEAV RNA was detected in PEAV-Coated-challenged piglets, while no PEAV RNA was detected in sucrose microspheres-challenged piglets during the study. Taken together, these results suggest that PEAV-Coated could infect weaned piglets to cause diarrhea in vivo.

Type I interferon (IFN-α/β) plays an important role in innate immune response, which prompted us to examine the effect of PEAV on IFN-α and IFN-β. We found that PEAV could inhibit the mRNA expressions of IFN-α (p < 0.05) and IFN-β (p < 0.05) in Peyer's patches from PEAV-Coated-challenged weaned piglets at 7 d.p.i. (Figure 5). It was reported that IFN-stimulated genes (ISGs) could be induced after IFNs production (19). We further examined the mRNA expressions of ISGs in Peyer's patches from weaned piglets infected with PEAV-Coated at 3 d.p.i. and 7 d.p.i. As shown in Figure 6, we found that PEAV could inhibit the mRNA expressions of OAS (p < 0.05), Mx1 (p < 0.05), and PKR (p < 0.01 or p < 0.05) in Peyer's patches, indicating that PEAV could overcome an antiviral response to infect pigs.

Pathology tests were conducted to determine the histological changes in the jejunum of weaned piglets infected with the PEAV-Coated. Compare to the negative control (Supplemental Figures 1A,C), the typical histological lesions characterized by intestinal villus detachment due to injury of intestinal epithelial cells were observed in intestinal villus from piglets that were necropsied at 3 d.p.i. and 7 d.p.i. (Supplemental Figures 1B,D). Consistent with the histopathological results, PEAV antigens were detected in the villous enterocytes of jejunum collected from PEAV-Coated-challenged piglets that were necropsied at 3 d.p.i. and 7 d.p.i. (Supplemental Figures 1F,H), but no PEAV antigen in the negative control was detected by immunohistochemical analysis (Supplemental Figures 1E,G), indicating that PEAV-Coated could cause intestinal lesions in weaned piglets.