A Korean PEDV strain, QIAP1401, isolated in our laboratory was propagated on African green monkey kidney (Vero) cells (ATCC CCL-81, Manassas, VA, USA), as described previously [17], and was used after 70 passages (QIAP1401-p70). In brief, for viral infection, confluent monolayers of Vero cells were washed with phosphate-buffered saline (PBS) and inoculated with the PEDV isolate at a 0.1 multiplicity of infection in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing 1 µg/mL crystallized trypsin (Sigma, St Louis, MO, USA). After incubation for 1 hour at 37℃ in an atmosphere of 5% CO₂, the culture medium was replaced to remove extracellular virions. The PEDV-infected cells were maintained at 37℃ in 5% CO₂ and monitored daily for a cytopathic effect (CPE) using a microscope. Upon observation of a CPE, the culture flask was subjected to three rounds of freezing and thawing, and clarified viral fluid was collected by centrifugation at 1,000×g for 10 minutes. Serial passaging of the virus was routinely conducted in six-well plates using the sequential limit dilution culture method. After each passage, an aliquot of the virus was stored at −70℃ until use. PEDV QIAP1401-p11, which was passaged 11 times after isolation, was used as the challenge strain.

Viral RNA was extracted from 200 µL aliquots of QIAP1401-p70 using the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The complete genome of the virus was sequenced at an external facility (Macrogen Inc., Seoul, Korea) by a next-generation sequencing technique. The whole genome sequence was compared with those of the following PEDV reference strains isolated in the United States: USA/Iowa303/2014 (GenBank No. KR265827), USA/Minnesota250/2014 (KR265776), and OH1414 (KJ408801). Multiple-sequence alignments were generated using Clustal X 2.0 [18], and the results were visualized using ESPript 3.0 [19].

PEDV QIAP1401-p70 was chemically inactivated using binary ethyleneimine (BEI). In brief, 10^7.0 TCID₅₀/mL virus were obtained from Vero cells cultured in roller bottles. After three rounds of freezing and thawing, the collected viral fluid was clarified and then inactivated by addition of 1 M BEI to a final concentration of 2 mM at 37℃ overnight. The remaining BEI was neutralized by addition of 20% sodium thiosulfate. Virus inactivation was verified by the absence of viral growth in Vero cell cultures.

Experimental vaccines were prepared by mixing BEI-inactivated PEDV QIAP1401-p70 with a commercial adjuvant according to the manufacturers' recommendations. The adjuvants used were Montanide IMS 1313 N VG PR (IMS 1313, Seppic, Paris, France), Montanide GEL (IMS gel), Montanide ISA 201 VG (ISA 201), and Montanide ISA 206 VG (ISA 206). ISA201 and ISA206 are oil-based multiphasic-emulsion adjuvants, and IMS1313 (nanoparticle-based) and IMSgel (synthetic polymer-based) are aqueous adjuvants. Vaccine formulations using IMS1313 and IMSgel were prepared by adding the adjuvants to BEI-inactivated viral fluid to final adjuvant concentrations of 30% and 10% (v/v), followed by vortexing at room temperature for 15 minutes. Vaccine formulations using ISA201 and ISA206 were prepared by emulsifying inactivated viral fluid with an equal volume of adjuvant using a homogenizer with a stirrer-propeller blade at 500 rpm for 10 minutes at 32℃ in a water bath. After cooling at 20℃ for 1 hour, formation of water/oil/water (W/O/W) multiple emulsions was confirmed by microscopy. All adjuvanted vaccines contained 0.01% of thimerosal to prevent growth of microorganisms, and were aseptically transferred to sterile vaccine vials and stored at 4℃ before use.

Twenty growing pigs (6 weeks old) were purchased from a PEDV-free pig farm and housed at the laboratory animal facility of the Animal and Plant Quarantine Agency in accordance with Institutional Animal Care and Use Committee protocols (ethics approval number: 2017-573). Before the start of the experiment, all pigs were verified as serologically negative for PEDV. After 1 week of acclimatization, pigs were randomly divided into four vaccination groups of four pigs each and comingled in two separate rooms for the duration of the experiment. Pigs in the vaccination groups were immunized twice intramuscularly at a 2-week interval with 2 mL of the experimental PEDV vaccine. The four remaining contact pigs were not vaccinated and were used as controls. At 42 days after the first vaccination, all pigs were challenged orally with PEDV QIAP1401-11p (1 mL 10^4.0 TCID₅₀/mL). Clinical signs of PEDV infection were observed daily for 15 days, and the clinical score was determined based on diarrhea severity: 0, no clinical signs, healthy; 1, moderate diarrhea; and 2, severe diarrhea, vomiting and dehydration. Fecal samples were collected daily for the duration of the challenge to monitor viral shedding in feces. Serum samples for immunoserological analysis were collected at 0, 14, and 28 days post-vaccination (dpv) and 15 days post-challenge (dpc), i.e., 43 dpv.

Serum-neutralizing antibody titers were determined by virus neutralization (VN) test in 96-well cell culture plates. Briefly, serum samples collected from the pigs after vaccination and challenge were diluted twofold (1:4 to 1:512) in serum-free DMEM containing 1 µg/mL trypsin and mixed with an equal volume of a virus suspension containing 100–200 TCID₅₀/0.1 mL PEDV. After incubation for 1 hour at 37℃, 200 µL of the virus-serum mixture were added to confluent Vero cell monolayers and incubated for 1 hour at 37℃ in 5% CO₂. Thereafter, the cultures were washed three times with PBS containing 1 µg/mL trypsin and replenished with viral growth medium. After incubation for 48 hours, VN titers were established as reciprocal values of the highest serum dilution that inhibited PEDV-specific CPE.

Serum PEDV-specific antibody levels were measured by indirect enzyme-linked immunosorbent assay (I-ELISA). The commercial PEDV N protein-based ID Screen PEDV Indirect Screening test (IDvet, Montpellier, France) was used, and IELISA was conducted according to the manufacturer's instructions. Optical densities at 450 nm were measured using an enzyme-linked immunosorbent assay (ELISA) plate reader.

Viral RNA was extracted from fecal samples using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. Following extraction, 5 µL viral RNA template were added to 20 µL real-time reverse transcription polymerase chain reaction (rRT-PCR) master mix from the PowerChek PEDV Real-Time RT-PCR kit (Kogenbiotech, Seoul, Korea). The reactions were run in presence/absence mode on the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, Middlesex, MA, USA) at 50℃ for 30 minutes, 95℃ for 10 minutes, and then 40 cycles of 95℃ for 15 seconds and 60℃ for 1 minute. A FAM-labeled probe was used to detect the PEDV, and a VIC-labeled probe was used to detect the internal control. The results were analyzed by the StepOne Plus v2.3 software (Applied Biosystems).

The results, except for the clinical data, are expressed as mean±standard deviation. Statistical analysis was performed using SPSS statistics ver. 21.0 (IBM Corp., Armonk, NY, USA). In the ELISA and VN test, differences between the experimental groups were analyzed by unpaired Student's t test and one-way ANOVA, followed by Tukey's post hoc test. A chi-square or Fisher exact test was employed to compare viral shedding in feces between experimental groups at each time point. A p-value of <0.05 was considered to indicate statistical significance.

The complete genome of the QIAP1401-p70 isolate, comprising 27,920 nucleotides, was determined by next-generation sequencing. The whole genome of the PEDV USA/Minnesota250/2014 strain was used as a reference for sequencing because of its close genetic relationship to QIAP1401 isolates. To assess whether the PEDV QIAP1401 variant had emerged after sequential passaging, the genome sequence was compared with that of U.S. PEDV strains. The PEDV QIAP1401-p70 isolate had > 99.4% sequence homology to the USA/Iowa303/2014, USA/Minnesota250/2014 and OH1414 U.S. PEDV strains. However, we identified 42 amino acid (aa) mutations, including a 25 aa deletion, in the ORF1a gene of the QIAP1401-p70 isolate (Fig. 1A). To determine at which passage the deletion arose, viral RNA isolated from each passage was screened by reverse transcription polymerase chain reaction using ORF1a-specific PCR primers. The results showed that the deletion mutant emerged after 41 passages (Fig. 1B). The deletion mutation detected at passage 37 was present during the following three passages. Moreover, viral stock at passages 37 to 40 contained both parental and mutant viruses.

All growing pigs were apparently healthy and had no clinical symptoms before oral challenge. The protective efficacy of the experimental vaccines against challenge with virulent homologous virus (10^4.0 TCID₅₀/mL PEDV QIAP1401-p11) was determined by the absence of clinical signs and decreased viral shedding during the 15-day observation period. Viral shedding in feces was determined using a commercial rRT-PCR kit. All pigs in the non-vaccinated control (NVC) group exhibited early signs of disease, typically mild diarrhea and loss of appetite (mean clinical score ≤1.0) after 2 dpc and experienced severe watery diarrhea with vomiting thereafter (mean clinical score ≤1.75) (Fig. 2A). The IMS1313p<and IMSgel-adjuvanted vaccine groups exhibited a similar clinical presentation to that of the NVC group. In contrast, the ISA206-adjuvanted vaccine group exhibited weaker and delayed clinical signs, mainly mild diarrhea (mean clinical score ≤1.0). In the ISA201 vaccine group, clinical disease progression was slow, and clinical severity was relatively weak, compared with the NVC group, but watery diarrhea eventually developed.

Viral shedding was defined as the presence of PEDV RNA as detected by rRT-PCR. PEDV shedding in feces in all groups was generally accompanied by clinical signs of disease (Fig. 2B). PEDV RNA was first detected in one of four pigs in the IMS1313, IMSgel, and NVC groups at 3 dpc. The duration of viral shedding differed among the groups. In the ISA206 and ISA201 groups, viral shedding in feces continued for 3 and 6 days, respectively. In contrast, in the other vaccinated groups and in the NVC group, viral shedding in feces was observed until the end of the challenge experiment. The viral shedding rates differed significantly among the groups (number of pigs with PEDV/total number of fecal samples) at both 6 and 9 dpc (p<0.05).

The serum antibody response induced by the experimental vaccines was assessed by I-ELISA and VN. All of the experimental vaccines induced a significant PEDV-specific serum IgG response to PEDV. The mean IgG levels in all vaccine groups, but not the NVC group, increased continuously after the first and second vaccinations. At 14 and 28 dpv, the mean IgG levels in all vaccine groups were significantly higher than those in the NVC group (p<0.01, unpaired Student's t test) (Fig. 3A). The mean IgG levels were significantly higher in the ISA201- and ISA206-adjuvanted vaccine groups than in the IMS1313- and IMSgel-adjuvanted vaccine groups at 14 and 28 dpv (p<0.05, Tukey's post hoc test) (Fig. 3B), indicating that the ISA201 and ISA206 adjuvants induced the strongest PEDV-specific IgG response in growing pigs.

VN titers also increased continuously in all vaccine groups after the first and second vaccinations and were significantly higher than those in the NVC group (p<0.01, unpaired Student's t test) (Fig. 3C). Although the maximum VN titers in all vaccine groups were detected at 28 dpv, there was no significant difference among the values (p<0.05, Tukey's post hoc test). In contrast, at 14 dpv, the mean VN titer of the ISA201 group (5.0 log₂ VN titer) was significantly higher than those of the IMS1313 (3.25 log₂ VN titer) and IMS gel (3.5 log₂ VN titer) groups. The ISA206 group also exhibited a higher mean VN titer (5.5 log₂ VN titer) than those of the IMS1313 and IMSgel groups, while the mean VN titers of the ISA206 and ISA201 groups were comparable (p<0.05, Tukey's post hoc test) (Fig. 3D). These findings indicated that the ISA201 and ISA206 adjuvants induce a virus-neutralizing antibody response more rapidly than do the IMS1313 and IMSgel adjuvants.