Korean Journal of
Veterinary Service

Porcine epidemic diarrhea (PED) is a highly contagious acute viral digestive disease that affects pigs of all ages, but is more lethal in suckling piglets. PEDV mainly proliferates in the cytoplasm of small intestine villous epithelial cells of piglets, causing degeneration and necrosis of villous epithelium, atrophy, and shedding of villi, resulting in malabsorption, and depletion of electrolytes and dehydration due to continuous watery diarrhea, leading to dehydration of piglets (Debouck and Pensaert, 1980; Horzinek et al, 1988; Beall et al, 2016). Since the mortality rate is almost 100%, outbreaks cause serious economic damage to the swine industry (Sun et al, 2012).

Porcine epidemic diarrhea virus (PEDV), the causative agent of PED, is a virus belonging to the nidoviral order coronaviridae family alphacoronavirus genus (Pensaert and de Bouck, 1978; Cavanagh, 1997; Song and Park, 2012; Huang et al, 2013; Song et al, 2015). It is a single-stranded positive-sense RNA virus. The genome is length 28 kb long with a 5 cap and a 3’polyadenylated tail. It has 7 Open Reading Frame (ORF) (Kocherhans et al, 2001; Pensaert and Martelli, 2016). The two large ORFs 1a and 1b the genome coding for nonstructural proteins (nsps). ORF1a contains proteases and ORF1b contains RNA-dependent RNA polymerase, exonuclease, endoribonuclease, and methyltransferase (Su et al, 2016). The remaining ORFs in the 3’terminal region code for four major structural proteins, and a single accessory gene ORF3 (non-structure proteins) (Duarte et al, 1994). The Spike protein of PEDV is a type I membrane glycoprotein composed of 1383 amino acids. Spike protein can be divided into S1 (1∼735 aa) and S2 (736∼1383 aa) domains based on its homology with S proteins of another coronavirus (Sturman and Holmes, 1984; Jackwood et al, 2001), and PEDV S glycoprotein is known to play roles interacting with the cellular receptor to mediate viral entry and inducing neutralizing antibodies in the natural host (Godet et al, 1994; Chang et al, 2002; Bosch et al, 2003; Sato et al, 2011). The Membrane protein of coronavirus is the most abundant component among the viral envelope proteins and plays important roles in virus assembly by interacting with S and N proteins (Klumperman et al, 1994; Opstelten et al, 1995; Nguyen and Hogue, 1997; de Haan et al, 2000). The N proteins of interact with viral genomic RNA. This protein is useful for the diagnosis of PEDV infection (Vennema et al, 1996; de Haan et al, 1999; McBride et al, 2014). The small envelope E protein plays the role of ion channel affecting the release of virtue us, and is responsible for the assembly of the virion with M protein (Baudoux et al, 1998). The product of ORF3, the only accessory gene in PEDV, influences virus production and virulence (Song et al, 2003; Wang et al, 2012).

Porcine epidemic diarrhea virus (PEDV) was first isolated in Belgium 1970s, coronavirus-like strain CV777. It was named PEDV in 1982 (Wood, 1977; Pensaert and de Bouck, 1978). During the 1970s and 1980s, the virus spread throughout Europe. Since the 1990s, PED cases have become rare in Europe. Infected suckling piglets showed only mild clinical signs. In Korea, it was first reported in 1992, and caused a lot of damage to pig farms (Kweon et al, 1993; Li et al, 2012; Stevenson et al, 2013; Lee, 2015; Zhang et al, 2015). In the 1990s, Asian pig farming countries suffered damage from PED, and it was becoming endemic. PEDV of this period, which was mainly a problem in Asia, is classified as a phylogenetically G1 type. In late 2010, PEDV spread rapidly in China, and in 2013 there was an outbreak of PED in the United States. The virus is highly virulence and contagious, and when infected, it causes watery diarrhea, vomiting, and dehydration, which is especially lethal to suckling piglets. The PED virus that was prevalent at this time is genetically classified as G2 type. The G2 type PED virus spread rapidly worldwide, including Canada, Mexico, and Asia. Even now, it is causing huge economic losses to the pork industry.

One of the most effective ways to prevent PED is vaccination. The most recommended method in Korea is L (live)-K (killed)-K (killed) or L-L-K-K method. The killed vaccines are produced through inactivation after culturing the viruses. It is safe and does not cause disease. However, the duration of immunity is short and the cellular immune response is weak, so adjuvants are required. Live vaccines are made by attenuating the virulence of the live viruses. A rapid immune response occurs and the duration of immunity is long. The ability to form antibodies is good, but virulence could be reverted. Because of these advantages and disadvantages, a method of priming with a live vaccine and then boosting with a killed vaccine is currently used.

Most commercial strains currently in use are live or killed vaccines based on G1 type strains. However, the virus prevalent in farms is the G2 type virus, and it is impossible to completely protect it with existing vaccines due to genetic differences. PEDV, as an RNA virus, has been mutated compared to previous viruses, and the mutation of PEDV has raised questions about the protective effect of the previously used G1 type vaccines.

In this study, suckling piglets were inoculated with the G2 type PED vaccine (HSGP strain) and safety and virulence reversion of the vaccine strain were evaluated through in vivo passage. In addition, a group of non-vaccinated pigs was included to confirm the transmission of the vaccine virus to negative piglets.

PEDV HSGP strain

Porcine epidemic diarrhea HSGP is a genotype G2b strain that is a mixture of the HS strain and SGP-M1 strain. The HS strain was isolated from the porcine epidemic diarrhea that occurred at a farm in HongSeong, Chungcheongnam-do in 2017, while the SGP-M1 strain was isolated from Gimpo Farm in Gyeonggi-do in 2018. The attenuated live vaccine candidate has a common deletion part of the spike gene sequence. Vero cells (ATCC CCL-81) were cultured in ɑ-MEM (minimum essential medium) with TBP (tryptose phosphate broth solution), YES (yeast extract solution), 2.5% trypsin. As a result of sequencing, the HS strain had a deletion of the 55th to 56th amino acid corresponding to the s1 domain in the standard strain of Colorado 2013, and the SGP-M1 strain had a deletion of the 1380th to 1386th amino acid in the s2 domain. HSGP was isolated and propagated in Vero cells (Fig. 1).

Figure 1. The structure of HSGP STRAIN. The SGP-M1 strain is a form in which 6 base sequences are deleted in the 164∼169 BP region of the S1 domain in the standard strain (USA/Colorado/ 2013/KF272920), and the HS strain is a form in which the 55∼56th amino acid in the S1 domain is deleted.

Experimental design and inoculation

Animal study

Conventional four-passage reversion to virulence tests was performed using 14 suckling piglets of porcine epidemic diarrhea antibody negative. The first passage experiment is divided into two groups. one group is the vaccination group (n=3) and the other group is not vaccinated (n=2), but was raised in the same room to check the spread of the virus by the vaccination group. All pigs have euthanized 7 days post-inoculation (dpi) and necropsied. In the second passage experiment, the intestinal contents obtained from the first passage were inoculated into three piglets to observe clinical symptoms and collect samples (Fig. 2). The same experiment was repeated for passage 2 to passage 4. All animal experiments were approved by the Jeonbuk National University Institutional Animal Care and Use Committee (JBNU2021–0100). The animals were fed with milk substitute 3 times a day. Room temperature was 23 to 24°C and the humidity was 60%. Drinking water was supplied ad libitum.

Figure 2. Summary of animal experiment.

Inoculation

In this study, PED-free, 5-day-old piglets were used for the entire experiment. In the first passage experiment, the vaccination group was orally inoculated with a 5 mL dose (10^6.0TCID₅₀/mL) of the HSGP strain, and the non-vaccinated control group received the same dose of 1X phosphate-buffered saline (PBS; Biosesang, Seongnam, South Korea). Intestinal contents obtained in the first passage experiment were prepared as a homogenous suspension with PBS and 1:10. In the second passage, 5 mL each of the intestinal contents was orally inoculated into PED-free, 5-day-old piglets (n=3). The same experiment was repeated for passage 2 to passage 4.

Observation and sample collection

In this study, fecal samples were collected before inoculation and at 1 to 7 dpi. The small intestine was collected by euthanasia 7 dpi, and a necropsy was performed. A swab of the same size was used so that the same amount of sample could be collected and stored at −80°C. Piglets were monitored daily for clinical signs of diarrhea, scored on a scale of 1 to 4 as follows: 1 (Pasty), 2 (Semi-liquid), 3 (Liquid), 4 (Profuse liquid). Intestines and intestinal contents were collected at 7 dpi and stored at −80°C. All pigs were weighed at 0 and 7 dpi.

Quantitative real-time RT-PCR

In this study, viral loads in fecal swabs and intestines of pigs were quantified by real-time polymerase chain reaction (RT-qPCR) using a Prime-Q PEDV/TGEV Detection Kit (Genetbio, Cat. No. ADP-1200Q) according to the manufacturer’s instructions. PCR was performed on a BID-RAD fast real-time PCR system (BIO-RAD. C1000 Touch). To calculate the amount of PEDV in the sample, CT values were converted to virus titers (TCID₅₀/mL). A PEDV isolate with a known infectivity titer was 10-fold serially diluted to generate a standard curve. The cDNA synthesis was performed at 50°C for 20 min, pre-denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec during which denaturation and annealing/extension.

Histopathology and immunohistochemistry

In this study, the intestines stored in a 5% formalin solution were paraffin-embedded to make slides. Slides were stained with hematoxylin & eosin, and small intestinal epithelial cell changes and villi were observed under an optical microscope (Olympus BX53, Japan). Immunohistochemical staining was performed using an antibody specific to porcine epidemic diarrhea virus to confirm the degree of antigen distribution in intestinal tissue. The degree of intestinal lesion formation and the distribution of antigens by site was scored by dividing into a total of 4 stages including negative through relative comparison (0: none, 1: mild or few, 2: moderate or some, 3: severe or many).

Statistical analysis

Graphical presentations and statistical analysis of data were prepared using GraphPad Prism 9.0.0. Data were collected from sequence GenBank, and a phylogenetic tree was prepared using MEGA11.

Results in the first passage of the vaccine virus

In this study, fecal swabs were collected from 0 to 7 dpi and analyzed by PEDV qRT-PCR. From the 3 dpi, the virus titer was observed as an average of 10^1.28TCID₅₀/mL (N=3), and a maximum of 10^3.3TCID₅₀/mL was observed from the 4 to 6 dpi. No virus was detected until the end of the experiment in the non-vaccinated control group (n=2) to determine whether or not the infection was caused by the viral shedding from the vaccinated pigs (Fig. 3, 4).

Figure 3. The average fecal PEDV titer in each passage experiment.

Figure 4. The average intestinal PEDV titer in each passage experiment.

Clinical symptoms were observed daily after HSGP strain inoculation. In two piglets of the inoculation group, diarrhea in the form of pasty stools was observed from day 4 post-inoculation, and it was observed that the symptoms of diarrhea liquid progressed on day 5 post-inoculation and recovered on day 7 post-inoculation. Diarrhea was not observed in the non-vaccinated group (Fig. 5).

Figure 5. Fecal score analysis table for each passage experiment.

Histopathological examination results showed that in the inoculated group, no modification in small intestinal epithelial cells or atrophy of villi was observed. As a result of IHC, a few PED-positive cells was observed in one pig. In the non-vaccinated group, small intestinal epithelial cell modification or villous atrophy was not observed, and no viral antigen was detected in the IHC examination (Table 1).

Table 1 . Analysis of histopathology and immunohistochemistry in the small intestine of pigs used in passage experiments.

Passage no.	Animal no.	Histopatholgy		Presence of PEDV antigens (IHC)
		Degeneration of epithelial cells	Villous atrophy
Passage 1 (Non-inoculation)	1	−	−	−
	2	−	−	−
Passage 1 (PED HSGP)	3	−	−	−
	4	−	−	−
	5	−	−	+
Passage 2	6	+	+	+++
	7	−	−	++
	8	−	−	−
Passage 3	9	++	+++	−
	10	−	−	−
	11	++	+++	−
Passage 4	12	−	−	−
	13	−	−	−
	14	−	−	−

Results in the second passage of the vaccine virus

In this study, piglets were orally inoculated with intestinal contents collected at necropsy in the passage 1 experiment. Fecal swabs were collected from 0 to 7 dpi and analyzed by PEDV qRT-PCR. At 2 dpi, the virus titer was 10^3.32TCID₅₀/mL, and from 3 to 4 dpi, it decreased to 10^2.14TCID₅₀/mL. At 5 dpi, the virus titer increased to 10^3.68TCID₅₀/mL. The intestinal viral titer observed at necropsy after the passage 2 experiment was 10^6.03TCID₅₀/mL (Fig. 3, 4).

Clinical symptoms were observed daily after inoculation of intestinal contents obtained after the passage 1. In two piglets (#6, #7), diarrhea in the form of pasty stools was observed from days 2 to 5 post-inoculation, but it showed recovery from day 6 post-inoculation, and in other piglets (#8), semi-liquid diarrhea was observed from day 2 post-inoculation and liquid stool was observed from day 3 post-inoculation (Fig. 5).

The average weight gain analysis result showed that two piglets (#6, #7) increased weight by 0.2 kg and 0.5 kg, respectively, and one piglet (#8) did not gain weight. The average weight gain was 0.233 kg, which decreased compared to passage 1 (Fig. 6).

Figure 6. The average weight gain in each passage experiment.

In histopathology, mild intestinal epithelial cell transformation and villous atrophy were observed in one piglet, and no lesions were observed in other piglets. As a result of IHC, PEDV antigen was observed on all villi in two piglets (#6 and #7) (Fig. 7, Table 1).

Figure 7. (A) Passage 1 negative, (B) passage 1 inoculation, (C) passage 2, (D) passage 3, (E) passage 4. Histopathology and immunohistochemistry of small intestines in pigs used in passage experiments. (Magnification: HE_×100, IHC_×200).

Results in the third passage of the vaccine virus

In this study, piglets were orally inoculated with intestinal contents collected at necropsy in the passage 2 experiment. Fecal swabs were collected from 0 to 7 dpi and analyzed by PEDV qRT-PCR. From day 1 post-inoculation, the virus was observed at 10^2.25TCID₅₀/mL. From days 2 to 4 post-inoculation, a high virus titer of 10^6.68, 10^6.81, and 10^6.4TCID₅₀/mL was observed. However, on day 5 post-inoculation, it decreased to 10^5.31 and 10^5.05TCID₅₀/mL. The intestinal viral titer observed at necropsy after the passage 3 experiment was 10^2.41TCID₅₀/mL, and the virus titer was reduced rapidly (Fig. 3, 4).

Clinical symptoms were observed daily after inoculation of intestinal contents obtained after the passage 3 experiment. In one piglet (#9), diarrhea in the form of liquid stools was observed from day 1 post-inoculation. However, semi-liquid diarrhea was observed on day 3 post-inoculation, pasty diarrhea was observed on day 6 post-inoculation, and the symptoms gradually alleviated. One piglet (#10) showed semi-liquid diarrhea on day 1 post-inoculation, pasty stool on day 3 post-inoculation, and no diarrhea was observed on day 7 post-inoculation. No diarrhea was observed in piglets (#11), (Fig. 5).

In histopathology, moderate intestinal epithelial cell transformation and severe villus atrophy were observed in some piglets (#9, #11). The piglet (#10), which had no clinical symptoms, showed normal opinion without lesions. As a result of the IHC test, no antigen was detected in the intestinal tissue. Additional tests revealed that coccidia were detected in the damaged epithelial cells. Coccidia is judged to be the cause of intestinal epithelial cell transformation and villous atrophy (Fig. 7, Table 1).

Results in the fourth passage of the vaccine virus

In this study, piglets were orally inoculated with intestinal contents collected at necropsy in the passage 3 experiment. Fecal swabs were collected from 0 to 7 dpi and analyzed by PEDV qRT-PCR. In the passage 4 experiment, no virus was detected in the fecal swab and intestinal tissue (Fig. 3, 4).

Clinical symptoms were observed daily after inoculation of intestinal contents obtained after the passage 3 experiment. Pasty diarrhea was observed on days 1 and 2 post-inoculation, and thereafter, no clinical symptoms appeared. Even though no viral antigen was detected, diarrhea symptoms are predicted to be caused by feeding substitute milk (Fig. 5).

The average weight gain analysis result showed that two piglets (#13, #14) increased weight by 0.3 kg and 0.5 kg, respectively, and one piglet (#12) did not gain weight. The average weight gain was 0.2666 kg, which increased compared to passage 3 (Fig. 6).

In histopathology, no change in epithelial cells of the small intestine or atrophy of villi was observed in all piglets. As a result of the IHC test, no antigen was detected in the intestinal tissue (Fig. 7, Table 1).

Since 2013, severe PED outbreaks have occurred nationwide, and it is still affecting a lot of pig farms (Kim et al, 2015). Vaccination is essential to prevent PED. Since existing G1 type vaccines do not perfectly protect against the new G2 type viruses, a few of attenuated G2 type vaccines have been developed. Although attenuated vaccines are better in inducing both humoral and cellular immune responses as mimicking natural infections, there is still risks of developing disease or reversion to virulence. Therefore, the safety and virulence reversion were evaluated for the newly invented G2 vaccine strain in the current study.

As a result of the first passsgae experiment, the virus was not detected in the control group, so it was concluded that the vaccine virus cannot transmit to negative pigs. As the serial passage progressed, virus infection occurred faster and higher levels from passage 1 to passage 3, but no virus was detected in passage 4. Based on the results of average weight gain and clinical symptoms, it was confirmed that viral virulence was not recovered even after serial passages. Virus titer in the small intestines peaked at passage 2, but undetectable at passage 4. Clinical symptom (fecal score) peaked at passage 2, but gradually alleviated until passage 4.

In the small intestine of pigs infected with PEDV, atrophy of intestinal villi and shedding of epithelial cells are histologically observed, resulting in decreased absorption function in the small intestine and severe malabsorption diarrhea and dehydration. Visually, the small intestine is swollen and contains yellow watery contents. In the small intestines of pigs infected with PEDV in passage 2-passage 3, atrophy of intestinal villi and shedding of epithelial cells are histologically observed. But viral antigen was detected in the small intestines only in passage 2. The reason is that infection occurred at the time of inoculation, but the virus disappeared before sufficient virus proliferation occurred. The modification and atrophy of small intestinal villous epithelial cells were determined to be caused by coccidia after further examination (Stevenson et al, 2013).

In another previous study (Jang et al, 2019), five consecutive passage experiments were conducted to evaluate the safety of virulence reversion. As a result of the experiment, no significant clinical symptoms were observed during the five serial passage experiments, and no small intestine epithelial cell deformation or villus atrophy was observed in the small intestine based on histopathological examination results. And through sequence analysis for each passage, it was confirmed that no mutation occurred at the S-ORF3-E-M N site. Differences in the experimental results will require additional verification of virus mutation and vaccine safety through additional experiments. Based on this study, we concluded that the evaluation of virulence reversion and safety of attenuated vaccines through serial passage in the target animal could be a useful method for assessing the safety of the attenuated virus.

In this study, 5-day-old piglets were inoculated with the PED attenuated vaccine strain (HSGP strain), and passage experiments were performed four consecutive times. In the passage 1, the virus was not transmitted to the negative pigs raised together with the vaccinated pigs, and the virus was not detected. The growth rate and diarrhea symptoms improved until the fourth passage, and no piglets died during the entire evaluation period. Based on these results, it was concluded that the PED attenuated vaccine strain virus is safe and has a low risk of virulence reversion.

This work was supported by the Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through the Animal Disease Management Technology Advancement Programs (122005-02) funded by the Ministry of Agriculture, Food and Rural Affair (MAFRA) in the Republic of Korea.

No potential conflict of interest relevant to this article was reported.