2.5. Animal Experiment
Thirty-seven, six-day-old, Large White × Duroc, crossbred, fecal PEDV and TGEV shedding-negative suckling piglets were purchased from a conventional pig farm devoid of G2b PEDV infection history based on the negative result of our long-term surveillance of serum antibody and colostrum against PEDV in this pig farm. These piglets from different sows were fed with artificial milk and were randomly assigned to five groups, acclimated for one day, and then inoculated orally with indicative recombinant viruses at a dose of 2 mL of 0.5 × 102 TCID50/mL or PI-medium, respectively. Clinical signs and weight gain were recorded daily. Fecal consistency was monitored daily and scored visually as: 0 = normal, 1 = loose, 2 = semi-fluid, and 3 = watery as previously described. Calculation of average daily weight gain was only performed on piglets that were not humanly euthanized. The formula used for calculation is as follows: Weight gained/ surviving period.

2.5.1. Quantification of PEDV Fecal Viral Shedding
Methods to quantify fecal PEDV viral shedding has been described previously [19]. Briefly, fecal samples collected from rectal swabs were resuspended in DPBS and then subjected to automated nucleic acid extraction using Cador Pathogen 96 QIAcube HT Kit with QIAcube (Qiagen Inc., Hilden, Germany) according to the manufacturer’s instructions. Complementary DNA was synthesized via reverse transcription using QuantiNova™ Reverse Transcription kit (Qiagen Inc., Hilden, Germany) and proceeded to quantitative real-time PCR analysis using the primer-probe set published previously on a CFX96 Thermal Cycler (Bio-Rad, Hercules, CA, USA). The thermal profile comprised an initial denaturation at 95 °C for 2 min and then 45 cycles of 95 °C for 15 s followed by 60 °C for 15 s. The detection limit of the assay was determined by generating standard curves from serial 10-fold dilutions of known amounts of in vitro transcribed RNA followed by reverse transcription and real-time PCR quantification as described above. The detection limit was calculated as 4.8 log10 RNA copies per mL.

2.5.2. Histopathology and Immunohistochemistry
At three days post-inoculation, three pigs from each virus-treated group and one pig from mock group were humanely euthanized by electrocution followed by exsanguination for histopathological and immunohistochemical assessments, as described previously [22]. Duodenum, jejunum, ileum, cecum, colon, rectum and mesenteric lymph nodes were collected, formalin-fixed, paraffin-embedded, sectioned at 4 μm, and stained routinely with hematoxylin and eosin (H&E) for morphometric analysis by assessing the ratio of villi height to crypt depth blindly by one veterinary pathologist.
Immunohistochemistry was performed to evaluate the distribution of PEDV antigen. Briefly, formalin-fixed paraffin-embedded tissues were sectioned at 4 μm, deparaffined in xylene, rehydrated in serially diluted ethanol, and proceeded to epitope retrieval with the Trilogy antigen retrieval system (Cell Marque, Rocklin, CA, USA). After being washed three times with Tris-buffered saline plus 0.1% Tween 20 (TBST), tissue slides were treated with 3% hydrogen peroxidase (KYB, New Taipei City, Taiwan) and 10% normal goat serum (Dako, Carpinteria, CA, USA) to block the endogenous peroxidase activity and non-specific signals, respectively. For antigen detection, an in-house anti-PEDV N antibody, DE-1, at a dilution of 1:1000 in 10% normal goat serum was applied to the slides for 1 h at RT followed by three times wash with TBST. The first antibodies were then captured using the polyclonal anti-rabbit/mouse immunoglobulin, EnVision-DAB+ system (Agilent Technologies, Santa Clara, CA, USA) at RT for 1 h and color was developed afterward with 3, 3′-diaminobenzidine (DAB) chromogen (Agilent Technologies, Santa Clara, CA, USA). The slides were counterstained with hematoxylin (MUTO, Tokyo, Japan), mounted in Entellan (Merck, Darmstadt, Germany) and cover slipped. Positive signals were visualized under an inverted light microscope (Nikon, Tokyo, Japan).