KR20040092760A - A attenuated porcine epidemic diarrhrea virus, an immunogenic composition comprising the same and a method for detecting the virus - Google Patents

Abstract

PURPOSE: An attenuated porcine epidemic diarrhea virus, an immunogenic composition comprising the same and a method for detecting the same virus are provided. The virus has improved safety, stability, immunogenicity and protection efficiency for a pig. The porcine epidemic diarrhea can be prevented and treated by oral administration of the composition. CONSTITUTION: The attenuated porcine epidemic diarrhea virus(KCCM-10474) is provided. The immunogenic composition comprises the attenuated porcine epidemic diarrhea virus(KCCM-10474) and pharmaceutically acceptable carriers. The method for detecting the same attenuated porcine epidemic diarrhea virus(KCCM-10474) comprises the steps of: (a) providing a sample containing ORF3 DNA derived from the attenuated porcine epidemic diarrhea virus(KCCM-10474); (b) digesting the sample with one or more restriction enzymes selected from Hind III and Xho II, and detecting DNA fragments to obtain RFLP pattern; and (c) comparing the RFLP pattern of the step (b) with the standard RFLP pattern of ORF3 DNA which is obtained by treating the standard sample with the same restriction enzyme and detecting DNA fragments.

Description

Attenuated porcine epidemic diarrhrea virus, an immunogenic composition comprising the same and a method for detecting the virus}

The present invention relates to an attenuated porcine epidemic diarrhea virus, a composition for preventing or treating epidemic diarrhea, and a method for detecting the presence of the epidemic diarrheal virus.

Porcine epidemic diarrhea virus (PEDV) is a coronavirus and(CoronaviridaeAs a member of the family, it is a single-stranded RNA virus with an envelope, causing severe intestinal diarrhea in pigs, leading to severe economic losses in Asia. PEDV related symptoms include diarrhea, anorexia and pyremia.

PEDV consists of the spike (S), membrane (M), small membrane (sM), ORF3 and nucleocapsid (N) genes, all of which were sequenced (Bridgen A et al. , J. Gen. Virol. 1993; 74: 1795-1804; Duarte M et al. , J. Gen. Virol. 1994; 75: 1195-1200). Adaptation of wild-type PEDV to cell culture reduces the toxicity of wild-type viruses, which is known to be associated with the loss of ORF3 products (Bridgen A et al . , Adv. Exp. Med. Biol. 1998; 101: 781-786; Tobler K et al . , Adv. Exp. Med. Biol. 1995; 86: 541-542). It is known that the disease is attenuated when the piglets are inoculated with PEDV serially cultured in Vero cells (Kweon CH et al., Vaccine 1999; 17: 2546-2553).

PEDV attenuated strains have been developed as vaccines to prevent or treat pandemic diarrhea. An example of such an attenuated vaccine is the currently available KPEDV9 strain (KCTC 8641P) (for example, Korea Microorganism Co., Ltd.). Korean Patent Publication No. 1996-023048 discloses KPEDV9. According to the patent document, KPEDV9 isolates the virus from the small intestine and intestine of 6-day-old piglets died of epidemic diarrheal disease, propagates it in Vero cells (ATCC C1008), and continuously passages about 90 times at intervals of 4 to 5 days. As attenuated strain by carrying out, it is safe but induces immunity in pigs inoculated.

However, if the KPEDV9 strain is used as an actual vaccine, it should be administered via muscle. The actual product specification of KPDEV9 is directed to intramuscular injection and does not mention oral administration at all. Compared to intramuscularly administered vaccines, oral vaccines are types of vaccines that stimulate gut-associated Lymphoid Tissue (GALP) in the gut to induce effective immunity. When a vaccine is administered, unlike an intramuscular vaccine in which antibodies and immune cells are activated in an immune organ and delivered to a target organ through blood, the oral vaccine has an advantage in that it is delivered directly to the target organ. However, these oral vaccines, when administered, must be acid resistant to the extent that they are not inactivated by gastric acid and must be resistant to digestive enzymes. The effectiveness of such oral vaccines can be presumed to be usable as oral vaccines when the virus is excreted in excreta released from administered individuals.

The KPEDV9 vaccine is also known to have a low protection rate for piglets when administered to sows. In addition, when the strain was used as a vaccine, there was no analysis method that could be easily detected other than an immunological analysis method, and there was a point that the analysis of the immunization process and the molecular biological research were not efficient.

Accordingly, the present inventors conducted intensive studies to obtain attenuated PEDV, which can be detected by an easier method, as well as immunologically, while improving safety, stability and immunogenicity when used as a vaccine and orally administered. As a result, the present invention has been completed.

Therefore, it is an object of the present invention to provide attenuated swine epidemic diarrheal virus (PEDV) which is enhanced in safety, stability and immunogenicity, can be induced by oral administration, and is easy to detect.

It is also an object of the present invention to provide a composition for the prevention or treatment of epidemic diarrhea disease comprising the attenuated epidemic diarrhea virus.

It is also an object of the present invention to provide a method for easily detecting the attenuated porcine epidemic diarrheal virus.

1 shows PEDV DR13 titers measured at five passage intervals.

Figure 2 is a diagram showing the results of PEDV DR13 via S gene specific RT-PCR.

Figure 3 shows the results of PEDV DR13 identification through ORF3 gene specific RT-PCR.

Figures 4a, 4b and 4c shows the sequencing results of the passages of the PEDV DR13 ORF3 portion aligned with the standard and parent strains.

5 is a result of RFLP analysis of PEDV DR13 ORF3 part by passage using Hind III.

6 is a result of RFLP analysis of PEDV DR13 ORF3 part by passage using Xho II.

Figure 7 shows the results of RFLP analysis of several PEDV strain ORF3 using Hind III.

8 shows the results of RFLP analysis of several PEDV main ORF3 fragments using Xho II.

Figure 9 shows the change in antibody for 6 weeks at weekly intervals in 14-day-old piglets.

The present invention provides swine epidemic diarrhea virus attenuated strains. The virus is preferably PEDV DR13 (Accession No. KCCM-10474).

The virus of the present invention isolates the porcine epidemic diarrhea virus (PEDV) DR13 strain from a pig with porcine epidemic diarrhea (PED), which is up to 100 in Vero cells. It was obtained by successive passages up to times. The virus has a high immunogenicity and no pathogenicity. In addition, when the virus is orally administered to pigs, immunity can be induced, and when administered to sows, piglets have a characteristic of protecting 80% or more.

The present invention also provides a composition for preventing or treating swine pandemic diarrheal disease comprising a therapeutically effective amount of swine pandemic diarrhea virus attenuated strain (Accession No. KCCM-10474) and a pharmaceutically acceptable carrier. The dose of the composition is a pig per person 10 ⁷ ~10 ⁸ TCID _50, preferably 10 ^6.5 ~10 ^7.5 TCID _50. The composition may be in the form of solutions, injections, tablets, and capsules, but is not limited thereto. The composition may be administered twice, orally, in the case of a liquid formulation, preferably in the case of vaccinating sows, the timing of 4 weeks before delivery and 2 weeks before delivery. The pharmaceutically acceptable carrier may be optionally used one or two or more of conventional excipients, disintegrants, binders and lubricants, specifically, as an excipient, microcrystalline cellulose, lactose, low-substituted hydroxycellulose, etc. For example, polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, and the like may be used as binders such as sodium starch glycolate, sodium hydrogen phosphate anhydrous as a disintegrating agent, and magnesium stearate, silicon dioxide, talc, and the like. You can choose to use it.

The present invention also provides a method for detecting the presence of an attenuated swine pandemic diarrheal virus (Accession No. KCCM-10474) comprising the following steps:

(a) providing a sample comprising ORF3 DNA from attenuated swine pandemic diarrheal virus (Accession No. KCCM-10474);

(b) treating the sample with one or more restriction enzymes selected from the group consisting of restriction enzymes Hind III and Xho II to obtain a RFLP pattern by detecting a DNA fragment obtained; And

(c) comparing the RFLP pattern of ORF3 DNA derived from standard swine pandemic diarrheal virus obtained by treating the RFLP pattern with the same restriction enzyme and detecting the DNA fragment obtained.

The method, for example, obtains ORF3 DNA via RT-PCR with RNA of attenuated swine pandemic diarrheal virus (Accession No. KCCM-10474) as a template, and treats the DNA with restriction enzymes Hind III or Xho II to perform electrolysis. It is obtained by activating the RFLP pattern and comparing the obtained RFLP pattern with the RFLP pattern of the standard swine pandemic diarrheal virus to detect the presence of the virus. Here, the reverse transcription reaction is performed using the ORF 3-2 reverse primer of SEQ ID NO: 4, and the ORF3 DNA obtained by PCR using the ORF 3-1 forward and ORF 3-2 reverse primer of SEQ ID NOs: 3 and 4 is about 883 bp fragment. As an RFLP pattern of the virus, when treated with Hind III, the standard virus showed a ladder of about 471, 183, and 179 bp, but for the attenuated virus of the present invention, only two fragments of 650 and 183 bp appeared. In addition, when treated with Xho II, one ladder of about 833 bp of standard virus appears, but two fragments of 563 and 270 bp appear in the attenuated virus of the present invention. "ORF3 DNA" used in the method of the present invention does not simply mean the sequence corresponding to the ORF3 gene, but DNA containing the Hind III or the newly introduced Xho II restriction site removed from the ORF3 gene by serial passage. As a fragment, the sequence adjacent to the ORF3 gene may also be included. RFLP patterns obtained by restriction enzyme treatment and electrophoresis may vary depending on the primer design, which is well known to those skilled in the art because such primer design can be done through conventional DNA manipulation techniques. In the step (b), detection of the fragments is usually carried out by electrophoresis, but can also be performed by other detection means. For example, the fragment may be labeled with fluorescence and then detected by a fluorescence detector.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the present invention is not limited to these Examples.

Example

Continuous Vero cell lines (ATCC, CCL-8) used in the Examples were 5% fetal calf serum, penicillin (100 units / ml), streptomycin (100 μg / ml), and amphotericin B (0.25 μg / ml). Was maintained regularly in α-MEM (α-minimum essential medium) supplemented with). KPEDV9 strain, a cell attenuated PEDV, was provided by a Korean green cross drug company.

Example 1: Isolation and passage of PEDV using cell culture

49 alimentary tract specimens from suckling pigs suspected of having epidemic diarrheal disease (PED) were homogenized in buffer solution (PBS; 0.1M, pH 7.2) to 10% (v / v) emulsion. Made with. The emulsion was mixed vigorously and cleared by centrifugation at 4800 × g for 10 minutes. Supernatants that passed through a 0.2 μm syringe filter (Acrodisk, Gelman) were used for virus isolation from Vero cells. Prior to inoculation of the supernatant, growth media of confluent cells grown in 25 cm ² flasks (Falcon, USA) was removed and washed three times with PBS. 1 ml of supernatant per flask was inoculated into the cells. After 1 hour of adsorption at 37 ° C., the cells were incubated in α-MEM supplemented with 0.02% yeast extract, 0.3% tryptopose phosphate broth, and 2 μg trypsin. As a result, PEDVs were first selected from the 49 samples that cause cytopathic effect (CPE) (cytopahtic effec), and then inoculated into Vero cells to continuously show about 70% to 80% of cytotoxicity effects. Was isolated and named PEDV DR13 strain and used as the parental line for continuous passage.

In addition, the DR13 strain, as well as cytopathic effect, RT-PCR analysis using PEDV-specific primers (SEQ ID NO: 1 and SEQ ID NO: 2) (Kim et al., J. Vet. Diagn. Invest. 2001; 13: 516-520) Through PEDV (about 651bp PCR product generation) was confirmed. The primer targets the S gene of PEDV, which is known to cause pathogenicity, causes infectious gastroenteritis, which is diagnosed with diarrheal disease in swine pigs, and detects and detects TGEV (transmissible gastroenteritis virus) belonging to the same Coronaviridae family as PEDV. It is designed to do that. First, complementary DNA was prepared by reverse transcription using a reverse primer of SEQ ID NO: 2. PCR reactions were carried out by a three-step process in a thermal cycler (Perkin-Elmer, Applied Biosystems, Foster city, USA). Samples were amplified by a program consisting of: incubating at 94 ° C. for 5 minutes; 30 seconds denaturation at 94 ° C; 30 seconds annealing at 55 ° C .; And 5 30 second extensions at 72 ° C .; And, 30 seconds denaturation at 94 ℃; 30 seconds annealing at 53 ° C .; And repeating the 30 second extension at 72 ° C. for 30 times, increasing by 1 second each time. Finally, the sample was held at 72 ° C. for 7 minutes and then cooled to complete the reaction.

Example 2 Attenuation Through Continuous Passage of Separated PEDV

The DR13 strain isolated in Example 1 was serially passaged up to 100 times in Vero cells at 25 cm ² in the same manner as in Example 1. Passaging was performed about 5 days after inoculation. After 72 hours of inoculation, cell denaturation and syncytial formation occurred, followed by cell denaturation effect that caused cell destruction.

1.PEDV Titer Analysis by Passage

The titers of PEDV were measured for each five passages using the Reed and Muench methods (Kusanagi K et al . , J. Vet. Med. Sci. 1992; 54 (2): 303-318). Titer assays were with Vero cells and 96 well microplates. Virus cultures were serially diluted 10-fold with virus propagation medium containing trypsin. Dense Vero cells in microplates were washed three times with PBS and inoculated in five wells at 0.1 ml / well. After 1 hour of adsorption at 37 ° C., the inoculum was removed and washed three times with PBS. Next, 0.1 ml of fresh trypsin containing virus propagation medium was added to each well, and the cells were further incubated at 37 ° C. for 5 days. The 50% tissue culture infective dose (TCID ₅₀ ) is shown as the inverse of the maximum virus dilution factor that exhibits cytopathic effect.

1 is a diagram showing PEDV titers measured at five passage intervals. As shown in FIG. 1, TCID ₅₀ /0.1 ml of the DR13 parent strain was 10 ^2.2 and the titer of the virus was maintained at 10 ^5.0 to 10 ^6.0 after peaking at 55 passages. That is, cell adaptation was performed after 55 passages.

2. Confirmation of PEDV by RT-PCR

In addition, PEDV was identified through S gene-specific RT-PCR and ORF3-specific RT-PCR shown in Example 1 at 0, 1, 30, 60, 90 and 100 passages.

Figure 2 is a diagram showing the result of PEDV confirmation through S gene specific RT-PCR. As shown in FIG. 2, not only KPEDV9 but also attenuated DR13 band S gene specific PCR product (about 651 bp) bands were identified. In Figure 2, M: molecular marker, lane 1: positive control (commercial KPEDV9), lane 2: negative control, lanes 3-8: 0, 1, 30, 60, 90 and 100 passaged DR13 strains.

Figure 3 is a diagram showing the result of PEDV identification through ORF3 gene specific RT-PCR. As shown in FIG. 3, the ORF3 gene-specific PCR product (about 833 bp) band was identified for the attenuated DR13 as well as the positive control (commercial KPEDV9). In Figure 3, M: molecular marker, lane 1: positive control (commercial KPEDV9), lane 2: negative control, lanes 3-11: 0, 1, 30, 60, 70, 75, 85, 90, and 100 times For DR13 weeks.

Here, the ORF3 gene specific RT-PCR is designed based on the published sequences of the S and sM genes, including the ORF3 gene, ORF3-1 (forward) and ORF3-2 (reverse) (SEQ ID NO: 3 and SEQ ID NO: 4) Was used as the primer. The expected PCR product size is about 833 bp. Reverse transcription reaction was done using ORF3-2. PCR reaction was carried out in the same manner as S gene specific PCR described in Example 1.

3. ORF3 Sequencing and RFLP Analysis

(1) ORF3 sequencing

ORF3 gene fragments amplified according to the method described above (3 repeated amplifications each time) were separated by electrophoresis on 1.5% agarose gel, and each PCR product was separated using a QIAquick Gel Extraction Kit (Qiagen, Germany). Purified according to the manual, and then sequenced bidirectionally with FS Dye Primer kit (Perkin-Elmer, Applied Biosystem) using PCR primers (ORF3-1 and ORF3-2), and ABI 373 automated sequencer (Perkin-Elmer). , Applied Biosystem). ORF3 genes of 20, 40, 60, 80 and 100 passages were sequenced and standardized by the BCM search launcher Multiple Sequence Alignment Program (Human Genome Sequencing center, Houston, TX, USA) PEDV CV777 known from Genbank. To the ORF3 sequence (accession number: Z24733).

Figures 4a, 4b and 4c shows the results of sequencing the passages of the PEDV ORF3 portion of the passage line with the sequence of standard strain and parent strain. As shown in Figures 4a, 4b and 4c, the ORF3 sequence (SEQ ID NO: 5) of the 100 passage PEDV DR13 has several deletions or substitutions as compared to the parent strain. Specifically, seven nucleotide sequence variants and two deletions were found. In addition, for the parent DR13, there are two restriction enzyme Hind III (underlined) recognition sites, whereas there is only one Xho II (marked box) recognition site present in the 100 passage PEDV DR13 ORF3 sequence and not in the parent DR13. Newly introduced. This suggests that the attenuated PEDV of the present invention can be easily distinguished from other PEDV strains using the RFLP method. 4A, 4B and 4C, CV777: standard strain, Field: parent DR13 strain, P (20), P (40), P (60), P (80), P (100); PEDV DR13 passed 20, 40, 60, 80 and 100 passages, respectively.

(2) RFLP analysis of ORF3

For passage PEDV DR13, passage 100 PEDV DR13 and 12 PEDV strains previously known by RT-PCR, the ORF3 gene was amplified via RT-PCR and RFLP analysis was performed on the amplified ORF3 fragment (approximately 833 bp). It was.

After performing each type classified PEDV DR13 the RFLP analysis using Hind III and Xho II, Hind was 75 times passaged for III The RFLP pattern of the Hind III begins to vary, RFLP patterns from 90th passage for Xho II This started to run. 5 is a result of RFLP analysis of PEDV DR13 ORF3 part by passage using Hind III. As shown in FIG. 5, 471, 183 and 179 bp fragments were generated at low passage numbers, but two fragments of 650 and 183 bp were generated after 75 passages. The 183 and 179 bp fragments overlap in agarose gel electrophoresis. In Figure 5, M: molecular weight marker, lane 1: commercial KPEDV9, lane 2: negative control (mock-infected), lane 3-10: 0, 1, 30, 60, 70, 75, 85, and 100 times For DR13 weeks.

6 is a result of RFLP analysis of PEDV DR13 ORF3 part by passage using Xho II. As shown in FIG. 6, 833 bp fragments were generated before passage 90, but two fragments of 563 and 270 bp were generated after passage 90. In Figure 6, M: molecular weight marker, lane 1: commercial KPEDV9, lane 2: negative control (mock-infected), lanes 3-11: 0, 1, 30, 60, 70, 75, 85, 90, and 100 This is a sub- DR13 week.

Next, FIG. 7 shows RFLP analysis results of various PEDV main ORF3 parts using Hind III, and FIG. 8 shows RFLP analysis results of various PEDV main ORF3 parts using Xho II. As a result, the 12 known PEDV isolates showed the same RFLP pattern as the low pass PEDV DR13 as shown in FIGS. 5 and 6. 7 and 8, M: molecular weight marker (100 bp ladder), lane 1: DR13 of 100 passages, lane 2: DR13 parent, lane 3: negative control, lanes 4-15: PEDV isolates, respectively, DR11, B409, DR183, B640, DR195, GC2, B719, 1101, KJY1, KJY2, B720, and DH1. As a result, it was confirmed that the attenuated PEDV DR13 of the present invention can be identified through RFLP pattern analysis.

Example 3 Characterization of Attenuated PEDV DR13

1. Examination of pathogenicity in piglets not fed colostrum

One-day-old pigs without colostrum were inoculated with DR13, 75 and 100 passages of DR13, respectively, to test for pathogenicity. The number of piglets used was 5 each group, and 5 ml (10 ^5.8 TCID ₅₀ /0.1 ml) of the virus was administered once through the oral cavity. In the negative control group, 5 ml of α-MEM medium (minimal essential media) was inoculated into 1 day-old pig without colostrum. Feces were collected at 24 hour intervals after virus administration, and S gene-specific RT-PCR described in Example 1 was performed to confirm the discharge of virus and to monitor daily clinical symptoms.

The results are shown in Table 1. As shown in Table 1, all pigs died due to severe diarrhea and dehydration within 3 days in the DR13 group, but still survived in the 75 and 100 passages. Thus, piglets can survive safely even if they are accidentally exposed to the attenuated DR13 virus.

Table 1. DR13 emission test results in pigs that did not eat colostrum

PEDV Note Pig number PEDV detection by RT-PCR according to days after infection One 2 3 4 5 P100 P100-1 + + + + - P100-2 + + + ± - P100-3 + + + ± NT P100-4 + + + - - P100-5 + + + + NT P75 P75-1 + + + + NT P75-2 + + + + NT P75-3 + + + + NT P75-4 + + + + + P75-5 + + + + NT P0 P0-1 + + + ^* Our company P0-2 + + + ^* Our company P0-3 + + ^* Our company P0-4 + + ^* Pesa P0-5 + + ^* Our company Control - -

2. Pathogenicity and Immunogenicity of Attenuated DR13 in 14-Day and Pregnant Sows

(1) examination of pathogenicity

To confirm attenuation, 100 passages DR13 weeks were tested for 14-day-old piglets and pregnant sows. The test was designed as shown in Table 2 below.

Table 2. Pathogenicity and Immunogenicity of Attenuated DR13 at 14-Day and Pregnancy Sows

group virus Quantity of pig administration designation Titer ^a Route Volume (ml) 14-day old piglet P100 ^b 10 ^5.8 4 oral- 5 P75 ^c 10 ^6.0 4 oral- 5 Transcript ^d 10% emulsion ^e 4 oral- 5 Control α-MEM 4 oral- 5 Pregnant sow P100 ^b 10 ^5.8 2520 oral- One

^a TCID ₅₀ /0.1ml ^b Attenuated DR13 (100 passages)

^c Attenuated DR13 (75th Passage) ^d Moju DR13 (0th Passage)

10% of ^e-liquid emulsion Director (viral reverse does not go measure.)

^f α-Minimum essential media

For pathogenicity comparison, the outdoor isolate DR13, prior to cell adaptation, was prepared from the small intestine of piglets. The small intestine was homogenized with PBS. Next, the 10% small intestine emulsion was filtered using 0.8 μm and 0.2 μm microfilters (Satorius, Germany). 1 ml of virus (100 passages) containing 10 ^5.8 TCID ₅₀ /0.1 ml was administered once orally to a total of 2520 pregnant sows from four commercial farms (300, 500, 1200 and 1300, respectively). Clinical symptoms of diarrhea and mortality were observed in pigs and pregnant sows for 10 and 5 days, respectively. In addition, the piglets born were compared with the average size of piglets born from control gestational sows on the farm during the same period.

As a result, piglets and pregnant sows treated with attenuated DR13 did not show anorexia and pyremia symptoms associated with diarrhea and PEDV infection. However, in piglets treated with parental DR13, severe aqueous diarrhea and dehydration occurred in all piglets for 3 days. Viral excretion lasted 7 and 8 days in the attenuated DR13 and parent DR13 groups, respectively. Pregnant sows administered attenuated DR13 (100 passages) showed no clinical symptoms such as diarrhea, anorexia and pyremia. There was no difference in mean size of gestational sows born with attenuated DR13 (100 passages) and piglets born from control sows (average 10.4).

(2) Examination of immunogenicity

In addition, to compare the immune response, serum and colostrum at delivery collected from 14-day-old piglets were measured using indirect enzyme-linked immunosorbent assay (ELISA) (Kweon CH et al., Vaccine 1999; 17: 2546-2553). ). In addition, antibody titers were measured before inoculation with attenuated DR13 (100 passages), two weeks after inoculation, and colostrum. In addition, 10 colostrums were collected from control pigs. Indirect enzyme immunoassay specifically, cell purified PEDV is dissolved in polyethylene glycol (PEG) (molecular weight: 6,000), and dissolved in TEN buffer solution (0.01 M Tris, 0.001 M EDTA, 0.1 M NaCl) and purified by antigen. Used as. Carbonate buffer was added to a 96 well plate, 1-2 μg coated antigen, blocked with 1% skimmed milk for 1 hour at 37 ° C. and then left at 5 ° C. overnight. Porcine gum serum was diluted 1: 400 in phosphate buffer solution for 30 minutes, and secondary antibody (goat HRP conjugated anti-swine IgG) (KPN) was diluted 1: 2,000 and reacted at 37 ° C for 1 hour. I was. Color development was performed using ABTS.

As a result, protective ELISA titers were shown in all inoculated piglets and sows (FIG. 9). Figure 9 shows the change in antibody for 6 weeks at weekly intervals in 14-day-old piglets. As shown in FIG. 9, when the parent DR13 was inoculated, peaks were observed at 2 weeks, and when attenuated strains were inoculated, peaks were observed at 3 weeks. No control for PEDV was detected in control piglets and sows. In addition, high levels of anti-PEDV antibody were detected in colostrum of sows at delivery. In the ELISA, the OD ₄₀₅ value for colostrum of pregnant sows administered attenuated DR13 (100 passages) was 0.479 to 0.718, higher than 0168 to 0.231 of the control group.

Example 4 Comparison of Protective Capability against Inoculation by Oral Administration of Currently Available Vaccine (KPEDV9) and Attenuated PEDV DR13 (100 Pass) Vaccine of the Present Invention.

In this example, in order to compare the protective capacity against the challenge of the currently commercially available KPEDV9 vaccine and the attenuated PEDV DR13 (100 passages) vaccine of the present invention, each vaccine was inoculated into pregnant sows, and the pigs born therefrom 1 The challenge rate was confirmed by the survival challenge.

KPEDV9 vaccine and attenuated PEDV DR13 (100 passages) were administered orally twice a titer of 10 ^5.5 TCID50 / 0.1 ml to 4 and 2 weeks before delivery. Next, 1 ml of pathogenic pig epidemic diarrheal virus outdoor strain was challenged with 5 piglets born from sows after delivery. The open air virus was used to emulsify the small intestine of piglets collected from a farm in which swine epidemic diarrheal disease occurred, to make a 10% emulsion, and to filter using a 0.2 μm filter (see Example 1). Attack vaccination was performed on piglets at 3 days of age, and then recorded clinical symptoms and mortality of piglets for 7 days. The results are shown in Table 3.

Table 3. Survival rate of piglets after oral administration of vaccine to sows

week Route of administration Dosage Virus titer Dosing and recovery period (sows) Number of attacking piglets % Survival up to 10 days of age Attenuated DR13 oral- 1ml 10 ^5.5 TCID ₅₀ /0.1ml 4 times before and 2 weeks before delivery 5 80 KPEDV9 oral- 1ml 10 ^5.5 TCID ₅₀ /0.1ml 4 times before and 2 weeks before delivery 5 20 Control oral- 1ml α-MEM medium 4 times before and 2 weeks before delivery 5 100 Outdoor PEDV oral- 1ml Emulsion - 5 0

As a result, in piglets born from sows vaccinated with KPEDV9 vaccine, 4 animals died due to severe watery diarrhea for challenge. In other words, the survival rate was 20%. On the other hand, in piglets born from sows vaccinated with the attenuated PEDV DR13 (100 passages) of the present invention, only one died. In other words, the survival rate was 80%.

As shown in the above examples, the attenuated PEDV DR13 of the present invention was able to induce immunity safely by oral administration to sows and piglets. In addition, the attenuated PEDV of the present invention was able to protect more than 80% of piglets when administered orally to sows, but only 20% or less of piglets, when commercially available KPEDV9 was administered via muscle to sows, Protected. Conventional KPEDV9 was mainly administered through muscle, but the attenuated PEDV DR13 of the present invention was able to induce an immune effect superior to conventional KPEDV9 even through oral administration. The attenuated PEDV DR13 of the present invention appears to release the same type of virus after oral administration, so that it remains safe, stable in the body and can be distinguished from standard strains (eg, Hind III and Xho II). ), The vaccine containing the attenuated PEDV DR13 can be administered to sows or piglets, and the immunization process can be easily monitored through RFLP analysis, and PEDV vaccine-related molecular biological studies can be facilitated. Among the attenuated PEDV DR13 of the present invention having the above-mentioned advantages, PEDV DR13 strains passaged up to 100 times were deposited at the Korea Microorganism Conservation Center on March 12, 2003 (Accession No. KCCM-10474).

The attenuated PEDV DR13 of the present invention can be effectively used to prepare a vaccine containing attenuated PEDV DR13, which has high safety, stability, immunogenicity and protection efficiency for piglets when administered to sows, and has a restriction enzyme marker. It can also be used effectively to monitor this after vaccination.

The composition for preventing or treating swine pandemic diarrhea comprising attenuated PEDV DR13 of the present invention can effectively prevent or treat swine pandemic diarrheal disease in sows or piglets through oral administration.

According to the method for detecting the attenuated PEDV DR13 of the present invention, it is possible to facilitate the analysis of the immunization process by the vaccine and related molecular biological studies.

<110> PARK, BONGKYUN <120> A attenuated porcine epidemic diarrhrea virus, a immunogenic          composition comprising the same and a method for detecting the          virus <130> GN14098 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PEDV S gene <400> 1 ttctgagtca cgaacagcca 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PEDV S gene <400> 2 catatgcagc ctgctctgaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ORF3-1 forward primer for ORF3 amplification <400> 3 tcctagactt caaccttacg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ORF3-2 reverse primer for ORF3 amplification <400> 4 ggtgacaagt gaagcacaga 20

Claims (3)

Swine pandemic diarrheal virus attenuated strain (Accession No. KCCM-10474). A composition for the prevention or treatment of swine epidemic diarrhea comprising a therapeutically effective amount of swine epidemic diarrhea virus attenuated strain (Accession No. KCCM-10474) and a pharmacologically acceptable carrier. A method for detecting the presence of an attenuated swine pandemic diarrheal virus (Accession No. KCCM-10474) comprising the following steps: (a) providing a sample comprising ORF3 DNA from attenuated swine pandemic diarrheal virus (Accession No. KCCM-10474); (b) treating the sample with one or more restriction enzymes selected from the group consisting of restriction enzymes Hind III and Xho II to obtain a RFLP pattern by detecting a DNA fragment obtained; And (c) comparing the RFLP pattern of ORF3 DNA derived from standard swine pandemic diarrheal virus obtained by treating the RFLP pattern with the same restriction enzyme and detecting the DNA fragment obtained.