Thirty small intestinal homogenates from pigs diagnosed with PED based on clinical signs were selected for virus isolation at the Jeju and Gangwon-do Veterinary Service Laboratory. Small intestinal tissues were used to generate a 10% homogenate in Dulbecco's modified Eagle's medium (DMEM) containing 2.0 µg/mL crystalized trypsin (Sigma-Aldrich, USA). After preparing the 10% homogenate, the samples were centrifuged at 4,000 × g for 15 min at 4℃. The supernatant was filtered through a 0.45 µm membrane (Millipore, USA) and used for virus isolation.

The Vero cells (ATCC CCL-81 and ATCC CCL-1586) used for virus isolation were cultured in DMEM supplemented with antibiotics (100 IU/mL penicillin, 10 µg/mL streptomycin, and 0.25 µg/mL amphotericin B) and 10% heat-inactivated fetal bovine serum (Gibco BRL, USA). The cells were cultured in 24-well plates at 37℃ in a 5% CO₂ incubator. Confluent Vero cells were washed twice with phosphate buffered saline (PBS, pH 7.2) and inoculated with 100 µL of the final samples for 1 h at 37℃. After adding 200 µL DMEM, the 24-well plates were incubated for 2 h at 37℃ in a CO₂ incubator. After incubation, the samples were discarded, and 1 mL fresh DMEM containing 1 µg/mL crystalized trypsin was added to each well. The plates were incubated at 37℃ in 5% CO₂ for 5 days. When cytopathic effects (CPEs) were detected in the cells, the supernatant was harvested for further passage and inoculated into newly prepared Vero cells for the second passage. If no CPEs were observed 5 days after harvesting the supernatant, the Vero cells were fixed in 80% cold acetone and subjected to an immunofluorescence assay (IFA). If CPEs were not detected, and the IFA results were negative after three passages, the sample was considered negative for the virus.

Viral titration was conducted in 96-well microplates by using 10-fold serial dilutions, and CPEs were observed under a microscope at 5 days post-inoculation (DPI). The viral titers were calculated according to the Reed and Muench method and expressed as the 50% tissue culture infectious dose (TCID₅₀/mL).

Vero cells were plated and infected with the QIAP1401 strain in 24- or 96-well plates and fixed in cold acetone (−20℃) for 20 min. After three successive washes with PBS (pH 7.2), the cells were reacted with a 7G7 mouse monoclonal antibody (APQA, Korea) specific to PEDV for 45 min at 37℃ and then stained with a 100× dilution of fluorescein isothiocyanate-conjugated goat-anti-mouse IgG + IgM (KPL Laboratories, USA). After washing the plates with PBS, the Vero cells were air-dried and examined at 200× under a fluorescence microscope (Nikon, Japan). Samples showing specific fluorescence in the cytoplasm were considered positive.

Viral RNA extraction from 200 µL of the small intestinal homogenate samples was performed by using a viral RNA isolation kit (iNtRON Biotechnology, Korea). Viral RNA was eluted using 50 µL of elution buffer, and the eluted RNA was subjected to conventional reverse transcription-polymerase chain reaction (RT-PCR) using the i-TGEV/PEDV detection kit (iNtRON Biotechnology, Korea) and following the manufacturer's instructions. The PCR products were visualized via 1.8% agarose gel electrophoresis and ethidium bromide staining. Samples showing a 525 bp band were considered positive.

A commercial rapid detection kit for detecting PEDV was applied to 30 intestinal homogenate samples and two PED isolates according to the manufacturer's instructions (Bionotes, Korea). Briefly, a 10% suspension of each intestinal sample was prepared, and a 100 µL aliquot of the sample was applied to the sample well. The results were read 5 min after applying the sample, and the appearance of two lines (test line and control line) was considered positive. The formation of a single line in the control area was considered a negative result.

Vero cells infected with the QIAP1401 isolate were harvested 36 h post-inoculation and were frozen and thawed three times. After centrifugation at 4,000 × g for 30 min to remove cell debris, the viral supernatant was treated with 10% polyethylene glycol (MW 8000; Sigma-Aldrich, USA) and 0.5 M NaCl and precipitated overnight at 4℃. After precipitation, the pellet was resuspended in GTNT buffer (20 mM glycine, 100 mM Tris-Cl, 100 mM NaCl, and 1 mM ethylenediaminetetraacetic acid; pH 7.6) at 1% of the original volume. The suspension was layered on top of a 20% to 50% sucrose solution and centrifuged at 100,000 × g for 3 h in a SW-41 rotor (Beckman, USA). The band between the 20% and 30% sucrose layers was collected and dialyzed with PBS solution to eliminate the residual sucrose overnight at 4℃. One drop of the purified PEDV was placed on Formvar-coated grids and stained negatively with 1% uranyl acetate. The PEDV particles were examined under an electron microscopy (EM) (Hitachi 7100; Hitachi, Japan).

Viral RNA was extracted from the QIAP1401 and QIAP1402 isolates by using an RNA extraction kit (Qiagen, Germany) according to the manufacturer's instructions. The extracted RNA was eluted in 50 µL RNase- and DNase-free water. RT-PCR was carried out with specific primers to amplify the ORF3, S, M, and N regions of PEDV (Table 1). The RT-PCR was performed in a reaction mixture containing 10 µL denatured RNA, 1 µL each primer (50 pmol), 10 µL 5× buffer (12.5 mM MgCl₂), 2 µL dNTP mix, 2 µL enzyme mix (reverse transcriptase and Taq polymerase), and 24 µL distilled water (Qiagen). The cycling profile consisted of cDNA synthesis at 42℃ for 30 min, followed by 35 cycles of 95℃ for 45 sec, 50℃ for 45 sec, and 72℃ for 1 min, with a final extension at 72℃ for 5 min. The PCR products were visualized via 1.8% agarose gel electrophoresis and ethidium bromide staining. All PCR products purified using the gel extraction kit were ligated into the pGEM-T Easy vector system (Promega, USA) according to the manufacturer's protocol. Plasmid DNA was isolated from amplified Escherichia coli (DH5α), and recombinant plasmids were identified by EcoRI enzyme digestion (Bioneer, Korea). The sequences of the purified plasmids were analyzed by using the MJ Research PTC-225 Peltier Thermal Cycler and ABI PRISM BigDye Terminator Cycle Sequencing kits with AmpliTaq DNA polymerase (Applied Biosystems, USA), according to the manufacturers' protocols. Single-pass sequencing was performed for each template by using universal primers (e.g., SP6 and T7). The fluorescently labeled fragments were purified from the unincorporated terminators by applying an ethanol precipitation protocol. The samples were resuspended in distilled water and subjected to electrophoresis using the ABI 3730xl sequencer (Applied Biosystems). Both DNA strands were sequenced to verify the sequences.

Phylogenetic analysis of the nucleotide sequences from the two PEDV isolates and 25 other PEDV strains obtained from the GenBank database (National Center for Biotechnology Information [NCBI], USA) was performed. Multiple sequence alignments and sequence similarity calculations were performed by using DNASTAR (DNASTAR, USA) and DNASIS software (Hitachi Solution, USA), respectively. Phylogenic analyses were conducted by applying the neighbor-joining method using MEGA version 7.0.18 software [9]. The bootstrap method with 1,000 replicates was applied to determine the reliability of the inferred phylogenetic tree.

Group 1 consisted of four 3-day-old piglets, previously non-vaccinated for PEDV, purchased from a commercial pig farm that was PEDV- and maternal antibody-free. All pigs were housed at 32℃ in a pig isolator. Two pigs were orally administered a 1 mL dose of 10^3.0 TCID₅₀/mL QIAP1401-P70 via 5 mL pipette, and the remaining two pigs received no treatment. Group 2 comprised seven 11-week-old growing pigs seronegative for PEDV. The pigs were orally administered 1 mL 10^5.0 TCID₅₀/mL QIAP1401-P70 via 5 mL pipette. All pigs were monitored daily for clinical signs of PEDV infection, such as watery diarrhea and dehydration. Clinical or RT-PCR scores were divided into four grades: 0, 1, 2, and 3. Stool samples were collected daily from all pigs using cotton-tipped swabs and were subjected to the rapid detection and RT-PCR kits to detect the PEDV antigen. The four piglets in group 1 were necropsied. Small intestinal tissues collected from each pig were fixed in 10% formalin and embedded in paraffin according to standard procedures. The deparaffinized intestinal tissue sections were stained with hematoxylin and eosin for histopathologic examination. A PEDV-N specific monoclonal antibody was used for immunohistochemistry (IHC) assay. The antigen was detected by using an avidin-biotin kit (Ventana Medical System, USA) and an automatic immunohistochemical stainer (Ventana Medical System). Epithelial cells within intestinal samples that turned brown were considered positive. The pigs in group 2 were observed for 3 weeks post-inoculation, but were not necropsied.

Among 30 intestinal homogenate samples, 26 and 22 tested positive in the RT-PCR and rapid detection kit assessments, respectively. Virus isolation was attempted using the 30 intestinal homogenates in Vero cells. Two PEDV isolates, designated QIAP1401 and QIAP1402, grew successfully in Vero cells inoculated with small intestinal samples from piglets from the Jeju-do pig farms (collected 26 May 2014). As shown in Fig. 1, clear CPEs, characterized by cell fusion and large syncytia, were observed in the QIAP1401 and QIAP1402 isolates after the second passage. The supernatants from the two isolates were analyzed by the rapid detection kit and were positive for PEDV presence. No difference in CPEs was observed between the QIAP1401 and QIAP1402 isolates. The two PEDV isolates were further passaged serially to confirm efficient propagation. Distinct CPEs were usually observed in Vero cells infected with each isolate within 30 h post-inoculation, and large cells containing many nuclei and floating cells were observed over time. After fixing the cells with cold acetone and staining with the 7G7 monoclonal antibody, specific fluorescence was detected in the cytoplasm of Vero cells infected with QIAP1401 and QIAP1402 (Fig. 2). The QIAP1401 strain was serially passaged 70 times in Vero cells, and the viral titers of QIAP1401-P10, -P40, and -P70 cultured in roller bottles were 10^6.5, 10^6.8, and 10^7.0 TCID₅₀/mL, respectively (panel A in Fig. 2). The QIAP1401-P10, -P40 and -P70 viral titers peaked at 10^5.7, 10^6.0, and 10^6.7 TCID₅₀/mL, respectively, at 36 h post-inoculation (panel B in Fig. 2). We concluded that the QIAP1401 strain adapted to the Vero cells because the titer increased continuously until passage 70.

PEDV particles from the QIAP1401 strain purified by gradient sucrose density centrifugation were examined by EM. As shown in Fig. 3, the viral particles were 80 to 120 nm in diameter with crown-shaped projections, indicating a morphological appearance typical of Coronaviridae.

A 4161 bp sequence of the S gene from the two PEDV isolates was identified, and the amino acid sequence was deduced. The nucleotide sequence of the QIAP1401 strain was deposited in GenBank (NCBI) under accession number KX793713. The nucleotide sequences from the two Korean PEDV isolates were compared with those from 25 PEDV strains retrieved from GenBank to analyze the epidemiological relationships among PEDVs. A phylogenetic tree based on the nucleotide sequences of the S gene revealed that the Korean PEDV strains isolated in 2014 belong to the G2 group, with the QIAP1401 and QIAP1402 strains most closely related to the USA strain designated USA/Minnesota271/2014 (Fig. 4). The similarities of the PEDV S gene nucleotide sequences were 99.6% to 99.7% among the Korean isolates obtained after 2013 and 93.0% to 96.6% among QIAP1401 and the Korean PEDV isolates obtained before 2011. Alignment of the deduced amino acid sequences showed 99.4% to 99.7% similarity among QIAP1401 and the Korean PEDV strains obtained after 2013.

The complete S, ORF3, M, and N genes were sequenced after passages 3, 20, 40, and 60, respectively. As shown in Table 2, QIAP1401 after 60 passages revealed 12 amino acid changes compared with the original QIAP1401 strain. Among the four structural proteins, the S protein had the highest mutation rate of 58.3% (7/12). No deletions were detected in the four QIAP1401 structural proteins after 60 passages (data not shown).

Two piglets were orally administered the QIAP1401-P70 strain, and another two piglets were housed together with the inoculated piglets in a pig isolator for direct contact exposure. All piglets started to exhibit clinical PED signs, such as vomiting and diarrhea, at 1 DPI and proceeded to have severe watery diarrhea at 2 DPI (panel A in Fig. 5). One of the control piglets died of dehydration at 4 DPI, and the remaining three piglets died of dehydration at 5 and 6 DPI. All feces obtained from the four piglets at 1 to 6 DPI were positive for PEDV as determined by both RT-PCR and the rapid detection kit (panel A in Fig. 5). The dead piglets were necropsied on the day they died and showed shortened and fused small intestinal villi on microscopic examination (panel A in Fig. 6). In addition, the PEDV antigen was detected in the jejunal epithelial cells of atrophied villi by IHC (panel B in Fig. 6).

Seven growing, 11-week-old, PEDV-seronegative pigs were administered the same PEDV strain and housed in a compartment. As shown in Fig. 5B, the clinical signs began with mild diarrhea at 2 DPI. The pigs exhibited the most severe clinical signs at 4 DPI and returned to normal with no clinical symptoms at 11 DPI. The PEDV antigen was first detected in feces by RT-PCR at 1 DPI, and the greatest antigen level was detected at 3 and 4 DPI. The PEDV antigen in feces was not detected at 11 DPI in growing pigs, all of whom survived administration of QIAP1401-P70.