Porcine epidemic diarrhea virus (PEDV) causes diarrhea of pigs age-independently and death of young piglets, resulting in economic loss of porcine industry. We have screened 333 natural oriental herbal medicines to search for new antiviral candidates against PEDV. We found that two herbal extracts, KIOM 198 and KIOM 124, contain significant anti-PED viral effect. KIOM 198 and KIOM 124 were identified as
Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea, dehydration, vomiting, and high mortality in the piglets [
Traditionally, natural oriental herbal medicines have been used for relieving and curing many kinds of symptoms arisen from viral infection including cold, flu, and other virus-related diseases [
Several compounds including Mizoribine, Deoxynojirimycin, and Ribavirin are available commercially as antiviral drugs. A lot of reports demonstrated that they have the inhibitory effect on various viruses, including cytomegalovirus (CMV) [
Traditionally,
In the present study, we first demonstrate the water extract of
Vero cells (African green monkey kidney cell line; ATCC CCR-81) and ST-cells (pig testis cell line; ATCC CRL-1746) were purchased from KCLB, Korean Cell Line Bank (Seoul, Republic of Korea) and maintained in alpha-minimum essential medium (Hyclone, Logan, UT) with 5% fetal bovine serum and 100 U/mL of Penicillin and Streptomycin at 37°C with 5% CO2. Two strains of PED, KPEDV-9 and sm98 and other subtype TGE viruses were obtained from National Veterinary Research and Quarantine Service in Korea.
The Korean traditional herbal medicines including 333 single medicinal herbal extract were obtained from Yeongcheon Oriental Herbal Market (Yeongcheon, Korea) and verified by Professor Ki Hwan Bae at the College of Pharmacy, Chungnam National University. Fifty grams of each herb were placed in 1000 mL of water and boiled for 3 h at 115°C using medical heating plate (Gyeongseo Extractor Cosmos-600, Incheon, Korea). After boiling until final volume of extract reaches 100 mL, the solution was filtered using standard testing sieves (150
Vero cells were seeded in 96 wells with complete confluency. Herbal extract containing KIOM 198 or KIOM 124 was 20-fold diluted (1.5 mg/mL) and added to the Vero cells preincubated with viruses for 1 h. The mixture was further incubated for 48 h or 72 h until cytopathic effect (CPE) formation.
Vero cells seeded with complete confluent condition were infected with PED virus at a multiplicity of infection (MOI) of 0.1. KIOM 198 or KIOM 124 extract (1.5 mg/mL) was added to the cells infected with PED virus and incubated for 72 h or 96 h. The supernatants containing virus were harvested and used for determination of virus yield. Plaque assay was used for comparison of virus propagation yield with or without KIOM 198 or KIOM 124 extract. Plaque assay was performed as below. The supernatants harvested were serially diluted up to 106 and added to Vero cells seeded in 6 well (
Virus-containing supernatants were collected from the cells infected with virus (0.01 or 0.1 MOI) with or without KIOM 198 or KIOM 124 at 0, 24 h, 40 h, and 48 h postinfection. Total RNA was isolated using viral Gene-spin Viral DNA/RNA Extraction Kit (European Biotech Network, Belgium) and used for cDNA synthesis using iScript Reverse transcriptase (iNtRON Biotech, Daejeon, Korea) according to manufacturer’s instruction. Real-time PCR using Bio-Rad iQ5 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was performed by subjecting the reaction mixtures to initial denaturation at 94°C for 3 min, followed by 40 cycles of 94°C for 20 sec, 65°C for 20 sec, and 72°C for 30 sec. The primer sequences specific for Nucleocapsid gene of PED virus are used for PCR and as follows: 5′-CGCAAAGACTGAACCCACTAATTT-3′ for forward, 5′-TTGCCTCTGTTGTTACTTGGAGAT-3′ for reverse [
To evaluate toxicity of natural herbal extract on Vero cells, lactate dehydrogenase (LDH) assay was performed according to manufacturer’s recommendation (Roche, Mannheim, Germany). The level of LDH released into the media is used as a marker of dead cells. Cells seeded at 96 wells (
The procedures used in this study were conducted in accordance with the Guidelines for Animal Experimentation of the Institutional Animal Care and Use Committee (IACUC, KFDA, 2004). Two germ-free piglets per group were dieted with or without 198 for 4 days and were orally infected with 10 LD50 PED viruses. KIOM 198 extract was added up to 0.6% of diet. After 24 h and 48 h of virus inoculation and at necropsy, the feces were collected for determination of viral yield. After 24 h, the feces of control or KIOM 198-dieted piglets were visually observed. Necropsy was conducted to check the intestine of piglet infected with virus in the presence and absence of KIOM 198.
The standard compounds of quercetin and icariin were purchased from Sigma Co. (USA) and Korea Food and Drug Administration (KFDA), respectively. Purity (%) of all standard compounds was above 98.0%. The powder of
Screening of herbal extract containing antiviral activity against PED virus. 333 herbal extracts were evaluated for the inhibitory effect on PED viral production and 27 herbal extracts having anti-PED viral activity were selected. Antiviral activity was examined by plaque assay and determined as a PFU (plaque forming unit). KIOM 124 and KIOM 198 exhibited strong inhibitory effect on PED viral propagation.
Extract number | PEDV-PFU | PEDV log PFU | Fold inhibition |
---|---|---|---|
30 | 713.1 | 2.853 | 410 |
32 | 7391 | 3.869 | 40 |
43 | 2488 | 3.396 | 118 |
44 | 1736 | 3.24 | 168 |
48 | 2962 | 3.472 | 99 |
49 | 20570 | 4.313 | 14 |
51 | 85600 | 4.932 | 3 |
55 | 1084 | 3.035 | 270 |
56 | 2174 | 3.337 | 135 |
57 | 2678 | 3.428 | 109 |
58 | 1817 | 3.259 | 161 |
60 | 6042 | 3.781 | 48 |
71 | 256.8 | 2.41 | 1139 |
77 | 749.5 | 2.875 | 390 |
78 | 47.48 | 1.676 | 6160 |
|
|
|
|
130 | 133.9 | 2.127 | 2184 |
143 | 179100 | 5.253 | 2 |
|
|
|
|
208 | 2563 | 3.409 | 114 |
223 | 58240 | 4.765 | 5 |
232 | 2212 | 3.345 | 132 |
274 | 344.2 | 2.537 | 850 |
297 | 431900 | 5.635 | 1 |
324 | 3582 | 3.554 | 82 |
325 | 734200 | 5.866 | No inhibition |
326 | 165600 | 5.219 | 2 |
Positive control (No treatment) | 292500 | 5.466 |
|
To search for novel herbal extract containing antiviral activity against PED virus, we screened 333 different oriental herbal medicines. Vero cells were infected with 1 MOI of PED for 1 h, and each herbal extract was 20-fold diluted and then added to the mixture. At 72 h or 96 h post-incubation, each supernatant having virus was collected and total viral yields were measured as plaque forming unit (PFU) using qRT-PCR [
KIOM 124 and KIOM 198 were more investigated to confirm the antiviral activity. Using Vero cells infected with virus at an MOI of 0.1, KIOM 124 and 198 further added and incubated for 48 h or 72 h. The control cells infected by virus exhibited partial CPE at 48 h postinfection and full CPE at 72 h postinfection (Figure
The antiviral activities of KIOM 124 and KIOM 198 on PED virus. The Vero cells were infected with 0.1 MOI of PEDV for 1 h and followed by adding 20-fold diluted herbal extract containing KIOM 124 (a) or KIOM 198 (b), and further incubated for 48 h or 72 h.
Next, we evaluated the antiviral activities of KIOM 198 and KIOM 124 using plaque assay. The cells preinfected with virus at an MOI of 0.1 were treated with KIOM 124 or KIOM 198 and incubated for 18 h and 24 h. From the supernatant harvested at each time-point, the viral titer was determined by plaque assay. Figure
Effect of KIOM 124 and KIOM 198 on viral propagation. Cells infected with 0.1 MOI of virus were further incubated with 20-fold diluted KIOM 124 or KIOM 198 herbal extract. At 18 h, 24 h, and 48 h postinfection, each supernatant was collected and serially diluted for titration. Total viral titers were determined as a plaque forming unit (PFU). (a) Time-course kinetics of virus replicated in the presence or absence of KIOM 124 or KIOM 198 herbal extract. (b) The cells and plaques stained with crystal violet in the presence of KIOM 124 or KIOM 198.
Since KIOM 198 contains antiviral activity on KPEDV, it is of interest to investigate whether KIOM 198 could inhibit the activity of other subtype of coronavirus. To examine broad inhibitory activity on other corona virus by KIOM 198, other subtype sm98 and TGE viruses were used for antiviral activity test. When cells were infected with sm98 virus, CPE was observed from 24 h postinfection. At 48 h or 72 h postinfection, most cells were lysed by viral propagation (Figure
Antiviral effect of KIOM 198 on corona virus other subtype sm98 virus and TGE virus. (a) The Vero cells were infected with sm98 virus at an MOI of 0.01 for 1 h and added with 20-fold diluted KIOM 198 extract, and further incubated for 24 h, 48 h, and 72 h. (b) TGE virus at an MOI of 0.01 was used to infect ST-cells and incubated with KIOM 198 for 24 h.
The antiviral activity test showed that KIOM 198 has a remarkable ability to inhibit coronavirus propagation. Next, we investigated the toxicity of KIOM 198 using lactate dehydrogenase (LDH) assay in Vero cells and MTT assay in mouse primary liver cells. Vero cells were lysed, mixed with 20 fold-diluted herbal extract, and the activity was measured using substrate. Figure
(a) Cytotoxicity of herbal extract containing antiviral activity in Vero cells. The 27 different herbal extracts were evaluated to examine toxicity on the cells. (b) Cytotoxicity of KIOM 198 on mouse primary liver cells.
To prove the inhibitory activity of KIOM 198 against PED virus, we established disease model in the piglet. Piglets were infected with 10 LD50 PED viruses after 4 days feeding with milk and KIOM 198. At 24 h postinfection, the piglets fed with KIOM 198 had normal feces, whereas control piglets had diarrhea (Figure
Effect of KIOM 198 on pigs infected by PED virus. The germ-free piglets were orally injected PED virus after 4 days diet with or without KIOM 198. KIOM 198 extract was added up to 0.6% of diet. After 24 h, the feces of control or KIOM 198-dieted piglets were observed (a). Necropsy was performed to check the intestine of piglet infected with virus in the presence and absence of KIOM 198 (b). After 24 h and 48 h of virus inoculation and at necropsy, the feces were collected to determine viral propagation yield (c).
The main component profile of KIOM 198 was analyzed using HPLC. As shown in Figure
Analysis of the component profile of KIOM 198 by RP-HPLC-DAD (run time: 70 min, mobile phase A: water 99.9% + TFA 0.1%, B: Acetonitrile 100%; 10% B (0–5 min), 10–40% B (5–60 min), and 40% (60–70 min), injection: 10
In this study, we have represented the antiviral activity of KIOM 198, water extract of
KIOM 198 exhibited antiviral activity on not only PED virus but other corona virus like sm98 and TGE virus. We examined whether antiviral agents available commercially, such as Deoxynojirimycin, Mizoribine, and Ribavirin, could inhibit PED viral growth. Each compound at concentration of 100
Importantly, the cytotoxicity of antiviral reagents should be considered prior to use. Traditional oriental herbal medicines have been used for human being for a long time and any severe side-effect after dose did not reported. KIOM 198 showed the least toxicity among 27 antiviral candidates extracted from herbal medicines and also did not have any significant toxicity on normal primary cells.
We analyzed KIOM 198 using HPLC and detected several peaks including two known compounds, icariin and quercetin. Recent reports demonstrated that Quercetin 7-rhamnoside reduces PED viral replication [
Finally, we have demonstrated that KIOM 198, water extract of
Won-Kyung Cho and Hyunil Kim contributed equally in this work.
All authors have no conflict of interests.
This work was supported by Grant K10050 awarded to Korea Institute of Oriental Medicine (KIOM) from Ministry of Education, Science and Technology (MEST), Republic of Korea.