WO2016099226A1 - Novel porcine epidemic diarrhea virus and use thereof - Google Patents

Abstract

The present invention relates to a novel porcine epidemic diarrhea virus (PEDV) Korean isolate. The present invention relates to a porcine epidemic diarrhea (PED) vaccine composition containing, as an active ingredient, the virus, a culture of the virus, or an antigen therefor. In addition, the present invention relates to a pharmaceutical composition for preventing or treating porcine epidemic diarrhea (PED), containing, as an active ingredient, the virus, a culture of the virus, or an antigen therefor. In addition, the present invention relates to a composition for diagnosing porcine epidemic diarrhea (PED), containing, as an active ingredient, the virus, a culture of the virus, an antigen therefor, or an agent capable of detecting the virus. In addition, the present invention relates to a pharmaceutical composition for preventing or treating porcine epidemic diarrhea (PED), containing an inhibitor of growth of the virus.

Description

Novel swine epidemic virus and its use

The present invention relates to a novel Porcine Epidemic Diarrhea Virus (PEDV) Korean isolate.

The present invention relates to a porcine epidemic diarrhea (PED) vaccine composition comprising the virus, the culture of the virus or an antigen thereof as an active ingredient.

The present invention also relates to a pharmaceutical composition for preventing or treating porcine epidemic diarrhea (PED) comprising the virus, the culture of the virus or an antigen thereof as an active ingredient.

The present invention also relates to a composition for diagnosing porcine epidemic diarrhea (PED) comprising the virus, the culture of the virus or its antigen, or an agent capable of detecting the same as an active ingredient.

The present invention also relates to a pharmaceutical composition for preventing or treating Porcine Epidemic Diarrhea (PED), comprising the virus growth inhibitor.

Porcine epidemic diarrhea virus (PED virus) causes acute enterocolitis and watery diarrhea in piglets, especially in young piglets, with a 100% mortality rate.

The disease was first reported in the United Kingdom in 1971 and quickly spread to pig farms in Europe. In the 1990s, PEDs became rare in Europe and were associated with weaning diarrhea in adult pigs. PED was circulated in Asia in 1982 and subsequently caused significant economic losses to the Asian pig industry (Chen et al., 2008; Kweon et al., 1993; Li et al., 2012; Puranaveja et al., 2009; Takahashi et al., 1983).

In May 2013, the PED outbreaked in the United States and spread worldwide in Canada and Mexico, causing massive killing of new pigs and huge economic concerns (Mole, 2013; Stevenson et al., 2013 Vlasova et al. ., 2014).

PED virus (PEDV), a pathogenic inducer of PED, was identified as the corona virus in 1978, and belongs to the genus Nidovirales, Coronaviridae, and Alphacoronavirus (Lai et al., 2007; Pensaert and de Bouck, 1978; Saif et al., 2012).

PEDV is an enveloped virus that contains about 28 kb of single-stranded positive sense RNA genome with a 5 'cap and a 3' poly A tail (Pensaert and de Bouck, 1978; Saif et al., 2012). Spike proteins are the major envelope glycoproteins of virions, which interact with cellular receptors to mediate viral entry and neutralization of antibodies in natural hosts (Jackwood et al. , 2001; Lai et al., 2007; Lee et al., 2010). Accordingly, PEDV S glycoprotein is a viral gene that determines the genetic relevance of isolates of PEDV, or is suitable for diagnostic assay development and vaccine development (Chen et al., 2014; Gerber et al., 2014; Lee et. al., 2010; Lee and Lee, 2014; Oh et al., 2014).

The epidemic of PED was confirmed in Korea in 1992 (Kweon et al., 1993).

However, retrospective studies showed that PEDV already existed in early 1987 (Park and Lee, 1997).

PED occurred every year and caused huge economic losses to the Korean pig industry by early 2010. During the period 2010-2011, after several occurrences of foot-and-mouth disease (FMD), infection of PEDV in Korea was sporadic.

However, starting in November 2013, the PED epidemic reappeared in Korea and swept over 40% of hog farmers (Lee and Lee, 2014; Lee et al., 2014a, b). Although live or dead bacteria against PEDs are commercially available in Korea, the ongoing prevalence of PEDs indicates the low efficiency of such vaccines. These results arise from genetic and antigenic differences in the S protein between vaccine and field week (Lee et al., 2010; Oh et al., 2014; Lee and Lee, 2014). Accordingly, the absence of effective vaccines requires the development of the next generation of vaccines that can control PED.

Identification of PEDV in cell culture is essential for the study of vaccines or PEDV for PED prevention. However, the identification of PEDV is very difficult, and even the identified virus was unable to maintain its infectivity in several cell passages (Chen et al., 2014). To date, only two reports of Korean PEDV cultured up to 20 passages were genetically different from outdoor PEDV (Kweon et al., 1999; Song et al., 2003).

Therefore, it is urgent to develop a next generation vaccine using the current outdoor wine. However, it is difficult to isolate and cultivate outdoor strain PED virus in laboratory, and it is difficult to develop vaccine using field strain.

Therefore, the inventor of the present invention isolated the PED virus field strain currently in the country for the first time in the swine flu diarrhea-positive fecal specimens, and confirmed the in vitro characteristics, including the culture method for this field strain, viral gene analysis and in vivo pathogenic characteristics The present invention has been completed.

Accordingly, one aspect of the present invention is to provide a novel Porcine Epidemic Diarrhea Virus (PEDV) Korean isolate.

In addition, an aspect of the present invention is to provide a Porcine Epidemic Diarrhea (PED) vaccine composition comprising the virus, the culture of the virus or an antigen thereof as an active ingredient.

In addition, an aspect of the present invention is to provide a pharmaceutical composition for preventing or treating swine epidemic diarrhea (PED) comprising the virus, the culture of the virus or an antigen thereof as an active ingredient.

In addition, another aspect of the present invention is to provide a composition for diagnosing Porcine Epidemic Diarrhea (PED) comprising the virus, the culture of the virus or an antigen thereof as an active ingredient.

Another aspect of the present invention is to provide a pharmaceutical composition for preventing or treating Porcine Epidemic Diarrhea (PED), comprising the virus growth inhibitor.

One aspect of the invention provides a novel Porcine Epidemic Diarrhea Virus (PEDV) Korean isolate.

Hereinafter, the present invention will be described in detail.

The present inventors classified the PED virus which was first prevalent in 2014 from porcine diarrhea-positive feces and obtained the mutated virus induced by such PEDV and subculture, and identified in vitro characteristics and in vivo pathogenic characteristics and sequence thereof. It was.

Accordingly, the present invention provides a swine epidemic diarrhea virus (PEDV) Korean isolate comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having a homology of 98% or more and comprising a spike protein having an equivalent immunogenicity. to provide.

As used herein, the term "porcine epidemic diarrhea virus" (PEDV) belongs to the family of coronaviridae (coronaviridae) has a single stranded RNA genome (genome) of about 28Kb in length, the three major constituent proteins spike protein (180-220KDa) ) And membrane protein or envelope protein (27-32 KDa) and nucleocapsid protein (55-58 KDa) (Shenyang 2007). It has a structure similar to that of the same species SARS coronavirus and is cultured in African green monkey kidney cells with trypsin added (Kim 2003; Stadler 2003; Vol. 2009).

As used herein, the term "Spike protein" is a major constituent protein of PEDV, and has a biologically important function of recognizing target cells and fusing viruses and cellular membranes (Chang 2002).

Porcine Epidemic Diarrhea Virus (PEDV) Korean isolates of the present invention may comprise an amino acid sequence of SEQ ID NO: 1, or may comprise an amino acid sequence exhibiting at least 98% homology with the amino acid sequence. The isolate has an amino acid sequence of SEQ ID NO: 1 and preferably 98%, 98,5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% It may include an amino acid showing homology of. In addition, the amino acid sequence showing 98% or more homology with the amino acid sequence of SEQ ID NO: 1 preferably shows an immunogenicity equivalent to or corresponding to the amino acid sequence of SEQ ID NO: 1.

The amino acid sequence having at least 98% homology may be an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 15, 22, 28, 34, and 40.

In addition, the isolate is preferably an ORF (open reading frame) 1a / 1b of SEQ ID NO: 2, ORF 3 of SEQ ID NO: 3, envelope protein of SEQ ID NO: 4, membrane protein of SEQ ID NO: 5 (membrane protein) And, and may further comprise one or more amino acid sequence selected from the group consisting of the nucleocapsid protein of SEQ ID NO: 6 or an amino acid sequence having at least 98% homology thereto. More preferably, the separation strain may be composed of the amino acid sequence of SEQ ID NO. Most preferably, the isolate may be one deposited with accession number KCTC12736BP. The present inventors deposited the KNU-141112 virus, a domestic isolate of the swine epidemic diarrhea virus, at the Korea Biotechnology Research Institute Microbial Resource Center, located in Ueun-dong, Yuseong-gu, Daejeon, Korea on December 18, 2014. KCTC12736BP was given.

In addition, the isolate is a spike protein of SEQ ID NO: 8, ORF (open reading frame) 1a / 1b of SEQ ID NO: 9, ORF 3 of SEQ ID NO: 10, envelope protein of SEQ ID NO: 11, membrane protein of SEQ ID NO: 12 (membrane protein), and at least one amino acid sequence selected from the group consisting of a nucleocapsid protein of SEQ ID NO: 13, preferably the isolate is composed of the amino acid sequence of SEQ ID NO: 14 Can be.

In addition, the isolate is a spike protein of SEQ ID NO: 15, ORF (open reading frame) 1a / 1b of SEQ ID NO: 16, ORF 3 of SEQ ID NO: 17, envelope protein of SEQ ID NO: 18, membrane protein of SEQ ID NO: 19 (membrane protein), and one or more amino acid sequences selected from the group consisting of the nucleocapsid protein of SEQ ID NO: 20, preferably, the isolate consists of the amino acid sequence of SEQ ID NO: 21 Can be.

In addition, the isolate is a spike protein of SEQ ID NO: 22, ORF 3 of SEQ ID NO: 23, envelope protein of SEQ ID NO: 24, membrane protein of SEQ ID NO: 25, and neurocapside protein of SEQ ID NO: 26 (nucleocapsid protein) may include one or more amino acid sequences selected from the group consisting of, preferably, the isolate may include the amino acid sequence of SEQ ID NO: 27.

In addition, the isolate is a spike protein of SEQ ID NO: 28, ORF 3 of SEQ ID NO: 29, envelope protein of SEQ ID NO: 30, membrane protein of SEQ ID NO: 31, neurocapside protein of SEQ ID NO: 32 (nucleocapsid protein) may include one or more amino acid sequences selected from the group consisting of, preferably, the isolate may comprise the amino acid sequence of SEQ ID NO: 33.

In addition, the isolate has a spike protein of SEQ ID NO: 34, ORF 3 of SEQ ID NO: 35, an envelope protein of SEQ ID NO: 36, a membrane protein of SEQ ID NO: 37, and a neurocapside protein of SEQ ID NO: 38 (nucleocapsid protein) may include one or more amino acid sequences selected from the group consisting of, preferably, the isolate may include the amino acid sequence of SEQ ID NO: 39.

In addition, the isolate is a spike protein of SEQ ID NO: 40, ORF 3 of SEQ ID NO: 41, envelope protein of SEQ ID NO: 42, membrane protein of SEQ ID NO: 43, and neurocapside protein of SEQ ID NO: 44 (nucleocapsid protein) may include one or more amino acid sequences selected from the group consisting of, preferably, the isolate may include amino acids of SEQ ID NO: 45.

The sequence number information is shown in Table 1 below.

Amino acid name	SEQ ID NO:	Amino acid name	SEQ ID NO:
KNU-141112-feces spike protein	One	KNU-141112-P3spike protein	22
KNU-141112-feces ORF1a / 1b	2	KNU-141112-P3ORF3	23
KNU-141112-feces ORF3	3	KNU-141112-P3envelope protein	24
KNU-141112-feces envelope protein	4	KNU-141112-P3membrane protein	25
KNU-141112-feces membrane protein	5	KNU-141112-P3nucleocapsid protein	26
KNU-141112-feces nucleocapsid protein	6	KNU-141112-P3complete cds	27
KNU-141112-feces complete genome	7	KNU-141112-P4 spike protein	28
KNU-141112-p5 spike protein	8	KNU-141112-P4 ORF3	29
KNU-141112- p5 ORF1a / 1b	9	KNU-141112-P4 envelope protein	30
KNU-141112- p5 ORF3	10	KNU-141112-P4 membrane protein	31
KNU-141112- p5 envelope protein	11	KNU-141112-P4 nucleocapsid protein	32
KNU-141112- p5 membrane protein	12	KNU-141112-P4 complete cds	33
KNU-141112- p5 nucleocapsid protein	13	KNU-141112-P20 spike protein	34
KNU-141112- p5 complete genome	14	KNU-141112-P20 ORF3	35
KNU-141112-p10 spike protein	15	KNU-141112-P20 envelope protein	36
KNU-141112- p10 ORF1a / 1b	16	KNU-141112-P20 membrane protein	37
KNU-141112- p10 ORF3	17	KNU-141112-P20 nucleocapsid protein	38
KNU-141112- p10 envelope protein	18	KNU-141112-P20 complete cds	39
KNU-141112- p10 membrane protein	19	KNU-141112-P30 spike protein	40
KNU-141112- p10 nucleocapsid protein	20	KNU-141112-P30 ORF3	41
KNU-141112- p10 complete genome	21	KNU-141112-P30 envelope protein	42
		KNU-141112-P30 membrane protein	43
		KNU-141112-P30 nucleocapsid protein	44
		KNU-141112-P30 complete cds	45

In addition, an aspect of the present invention provides a Porcine Epidemic Diarrhea (PED) vaccine composition comprising the virus, the culture of the virus or an antigen thereof as an active ingredient.

As used herein, the term “vaccine” refers to a biological agent containing an antigen that immunizes a living body, and refers to an immunogen or antigenic substance that immunizes the living body by injection or oral administration to a human or animal to prevent infection. . In vivo immunization is largely divided into autoimmunity, in which immunity is automatically obtained after infection by pathogens, and passive immunity obtained by an externally injected vaccine. While autoimmunity is characterized by a long period of generation of antibodies related to immunity and continuous immunity, passive immunization with vaccines acts immediately to treat infectious diseases, but has a disadvantage of poor sustainability.

As used herein, the term "immunogen" or "antigenic substance" is composed of a peptide derived from the virus, a polypeptide, a lactic acid bacterium expressing the polypeptide, a protein, a lactic acid bacterium expressing the protein, an oligonucleotide, a polynucleotide, and a recombinant virus. It may be any one selected from the group. In specific examples, the antigenic material may be in the form of an inactivated whole or partial viral preparation, or in the form of an antigen molecule obtained by conventional protein purification, genetic engineering techniques or chemical synthesis.

The content of the antigen may be 1 to 10% by weight relative to the total weight of the vaccine composition, preferably 5% by weight or less.

Vaccine compositions according to the invention are stabilizers, emulsifiers, aluminum hydroxide, aluminum phosphate, pH adjusters, surfactants, liposomes, iscom adjuvants, synthetic glycopeptides, extenders, carboxypolymethylenes, bacterial cell walls, derivatives of bacterial cell walls, Bacterial vaccine, animal poxvirus protein, subviral particle adjuvant, cholera toxin, N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) -propanediamine, monophosphoryl lipid A, dimethyldioctadecyl-ammonium bromide and mixtures thereof may be further contained in at least one second adjuvant.

In addition, the vaccine composition of the present invention may comprise a veterinary acceptable carrier. As used herein, the term "veterinarily acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvant, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorptive delay agents and the like. Carriers, excipients, and diluents that may be included in the composition for vaccines include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the vaccine composition of the present invention can be used in the form of oral dosage forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injectable solutions, respectively, according to conventional methods. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used can be prepared. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, shoe, etc., in the lecithin-like emulsifier. Sucrose or lactose, gelatin and the like can be mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc may also be used. As a liquid preparation for oral administration, suspending agents, liquid solutions, emulsions, syrups, etc. may be used, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations. As the non-aqueous preparation and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used.

The vaccine can be injected into a subject in various forms. "Injection" can be performed by any one method selected from the group consisting of subcutaneous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, nasal administration, oral administration, transdermal administration and oral administration.

In the present invention, the term "injection" or "administration" may vary depending on the age, sex, and weight of the subject to be administered, and the dose of the vaccine also depends on the route of administration, the degree of disease, sex, weight, age, and the like. The immunoadjuvant for the vaccine of the present invention can be used together when administering vaccines by parenteral, mucosal (oral and nasal) and transdermal routes.

Vaccine compositions of the present invention may include one or more adjuvants and the like to improve or enhance an immune response. Suitable auxiliaries include compositions consisting of mineral oils or vegetable oils such as peptides, aluminum hydroxides, aluminum phosphates, aluminum oxides and Marcol 52 and one or more emulsifiers or surface actives such as lysolececitin, polyvalent cations, polyvalent anions and the like. .

The vaccine composition of the present invention may further comprise one or more immunostimulants, preferably cholera toxin (CT), aluminum hydroxide, carbopol, mineral oil or Biodegradable Oil may be included.

The term “immune enhancer” in the present invention generally refers to any substance that increases the humoral and / or cellular immune response to the antigen. Traditional vaccines consist of unprocessed preparations of dead pathogenic microorganisms, while impurities associated with cultures of pathogenic microorganisms can act as an adjuvant to enhance the immune response, but homogeneous preparations of purified protein subunits can be used as antigens for vaccination. In use, the immunity triggered by such antigens is insufficient, requiring the addition of some foreign substance as an adjuvant. By using an adjuvant, smaller doses of antigen may be required to stimulate an immune response, thereby reducing the cost of vaccine production. These adjuvants are classified according to their raw materials (minerals, bacteria, plants) and components (emulsion suspensions). Commercially available adjuvants include aluminum hydroxide, carbopol, mineral oil (mineral oil) or biodegradable oil.

In addition, the vaccine may be live or dead vaccine.

Live vaccine or live vaccine means a virus that shows the absence or reduction of clinical symptoms of the disease when administered to an animal by weakening the pathogenicity of a bacterium or virus, and the attenuated strain of the present invention is a method known in the art, for example, The virus can be isolated through passage of the virus.

Bacillus vaccine or deadly poisoned vaccine is inactivated pathogenicity without losing immunogenicity by heating bacteria or virus (56 ~ 60 ℃, 30 ~ 60 minutes) or chemicals such as formalin or phenol. Inactivation of the virus may be by methods known in the art.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating swine epidemic diarrhea (PED) comprising the virus, the culture of the virus or an antigen thereof as an active ingredient.

As used herein, the term "prevention" refers to any action that inhibits or delays the PEDV infection by administration of the virus, a culture of the virus, or a composition containing the antigen as an active ingredient.

In the present invention, the term "treatment" refers to any action in which the symptoms of infection of PEDV are improved or benefited by administration of the culture of the virus or a composition containing the antigen as an active ingredient.

The pharmaceutical composition can be used, for example, as a feed additive.

The feed of the present invention may be properly configured by those skilled in the art in various forms of composition known in the art as long as the feed additive according to the present invention as an active ingredient, preferably 20% protein, fat, ether extract 4.5 %, Fat, acid hydrolysis 5.4%, crude fiber 4.7%, ash 6%, calcium 0.80% and phosphate 0.62%.

In addition, an aspect of the present invention provides a composition for diagnosing swine epidemic diarrhea (Porcine Epidemic Diarrhea, PED) comprising the virus, the culture of the virus or an antigen or an agent capable of detecting the same as an active ingredient.

The virus, the culture of the virus or the agent capable of detecting the antigen thereof may preferably be an antibody specific for the virus, the culture of the virus or the antigen thereof.

In the present invention, the term "antibody" is a substance produced by the stimulation of the antigen in the immune system, also called immunoglobulin, and specifically binds to a specific antigen, floating the lymph and blood, causing an antigen-antibody reaction. While antibodies exhibit specificity for specific antigens, immunoglobulins include both antibodies and antibody like substances lacking antigen specificity. The latter polypeptide is produced at low levels, for example in the lymphatic system, and at elevated levels by myeloma. In the present invention, the PEDV itself, the spike protein, or may be an epitope included therein.

The antibody may be a monoclonal or polyclonal antibody.

In addition, an aspect of the present invention provides a pharmaceutical composition for preventing or treating Porcine Epidemic Diarrhea (PED), comprising the virus growth inhibitor.

Inhibitors of growth of the virus include: siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), ribozyme, DNAzyme, peptide nucleic acids against the virus, the culture of the virus or antigens thereof. ), Antisense oligonucleotides, antibodies, aptamers, natural extracts, chemicals, and the like. Preferably it may be an antibody.

In the present invention, isolated PED virus outdoor strain currently in the first in Korea in the swine-like diarrhea-positive fecal specimen, the outdoor strain shows the pathogenicity of the typical PEDV, based on this, can be applied to a variety of vaccine compositions, therapeutics, diagnostics, etc. It is expected to be.

Figure 1 shows the cytopathology and IFA results of KNU-141112 PEDV isolate infected with Vero cells. Vero cells were gastric-infected or infected with PEDV KNU-141112-P5 and KNU-141112-P10 isolates. PEDV-specific CPE was observed daily and photographed using an inverted microscope at a magnification of 200 × at 24 hpi (first panel). For immunostaining, infected cells were fixed at 24 hpi, incubated with Mab for N protein, and incubated with Alexa green-conjugated goat anti-mouse secondary antibody (second panel). Cells were then counterstained with DAPI (third panel) and identified by fluorescence spectroscopy at 200 × magnification.

Figure 2 shows the growth kinetics of KNU-141112 in Vero cells.

(A) Virus titers at selected passages. Vero cells were infected with PEDV KNU-141112 recovered from the indicated passage numbers. At 24 or 48 hpi, viral supernatants were collected and virus titers were determined.

(B) Figure 1 shows the proliferation curve of KNU-141112. Vero cells were independently infected with PEDV KNU-141112-P10, -P20, and -P30. At the indicated time points after infection, the culture supernatants were recovered and virus titers were determined. TCID ₅₀ (50% tissue culture infectious dose) was calculated. Results are expressed as mean values in three wells, with error bars representing standard deviation.

Fig. 3 shows the titers of virus neutralizing antibodies in the serum of guinea pigs inoculated with PEDV KNU-141112 isolates. Guinea pigs were immunized twice with subcutaneous administration of inactivated KNU-141112-P10. Blood samples were collected before immunization and two weeks after the second immunization, virus neutralization assays were performed using homologous (KNU-141112; original) and heterologous (SM98-1; rhombus) viruses. Neutralizing antibody titers of each of the infected animals were plotted in log 2 values. Values are presented as the average of two independent experiments, with error bars representing standard deviations.

Figure 4 shows the microscopic and macroscopic observations of the intestine of the lower limbs inoculated with KNU-141112 in Korean PEDV strain. (A) The small intestine at 2 DPI of infected pigs shows a thin, transparent barrier and an expanded stomach filled with curds.

(B and C) Hematoxylin and eosin stained jejunum in 3DPI of KNU-141112-inoculated pigs (100 × and 200 × magnifications, respectively).

(D) IHC analysis (100 × magnification) of factory tissue manipulation isolated from pigs at 3 DPI.

(E and F) Immunofluorescence staining of small intestine sections at 3 or 4 DPI of pigs inoculated with KNU-141112. Sections were counterstained with DAPI and confirmed by fluorescence microscopy at 400 × magnification.

Figure 5 shows the phylogenetic analysis results based on the nucleotide sequence of the spike gene (A) and the full-length genome of the PEDV strain. Tentative similar sites and complete genome sequences of spike proteins of TGEV were included as outgroups within each panel. Multi-sequencing alignments were performed using the ClustalX program, and phylogenetic trees were prepared using the neighbor-joining method with the aligned nucleotide sequences. The number of branches represents a bootstrap value of at least 50% of 1000 replicates. The name, country, and year of isolation, GeneBank accession number, and genogroups and subgroups are shown. PEDV isolates identified in the present invention are shown in a circle. Scale bar means nucleotide substitution at each position.

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

Example  1. Samples and Methods

1.1. Cells, Clinical Samples, Viruses and Antibodies

Vero cells (ATCC CCL-81) were incubated in α-MEM containing 5% FBS (Invitrogen) and antibiotic-antimycotic solutions (100 ×; Invitrogen) and maintained at 37 ° C. in a 5% CO 2 incubator. Seven small intestinal crushes and 50 stool samples positively identified by RT-PCR using the i- TGE / PED Detection Kit (iNtRON Biotechnology, Seongnam, South Korea) were selected for virus isolation experiments. Small intestine mills were prepared as a 10% (wt / vol) suspension in PBS by running MagNA Lyser (Roche Diagnostics, Mannheim, Germany) three times at 7,000 rpm for 15 seconds. Fecal samples were diluted with 10% (wt / vol) suspension in PBS. The suspension was then stirred and centrifuged with 4,500 × g (Hanil Centrifuge FLETA5, Incheon, South Korea) for 10 minutes. Supernatants were filtered through a 0.22-μm syringe filter (Millipore, Billerica, Mass.) And stored at −80 ° C. until used as inoculum of virus isolates.

Virus isolates of PEDV were applied to Vero cells. Specifically, confluent Vero cells grown in 6-well plates were washed with PBS and seeded with 400 μl sample containing 10 μg / ml trypsin (USB, Cleveland, OH). After incubation at 37 ° C. for 1 hour, 2 ml of virus growth medium [antibiotic-antifungal solution, 0.3% TPB (Sigma, St. Louis, MO), 0.02% yeast extract (Difco, Detroit, MI), 10 mM HEPES ( Invitrogen), and α-MEM supplemented with 5 μg / ml trypsin. Inoculated cells were maintained at 37 ° C. under 5% CO 2 and CPE (cytopathic effects) were observed daily. When 70% CPE appeared, the inoculated cells were thawed three times. The culture supernatant was then collected and centrifuged at 400 × g for 10 minutes. Purified supernatants were aliquoted and stored at −80 ° C. as “P1 (passage 1)” until used.

100 millimeter diameter tissue culture dishes were used for multiple passages of separation.

If the cells inoculated for 7 days did not show CPE, the plate was frozen and thawed three times, the supernatant was recovered using centrifugation, and seeded into fresh Vero cells for the next passage. If the results of CPE and RT-PCR were negative after six blind passages, the virus isolate was considered negative. PEDV N protein-specific monoclonal antibody (MAb) was obtained from ChoogAng Vaccine Laboratory (CAVAC; Daejeon, South Korea).

1.2 Virus Titer  Virus titration

In the presence of trypsin, KNU-141112 virus stock of each passage was infected with Vero cells as described above. Culture supernatants were collected after 24 or 48 hours postinfection (hpi) when 70% CPE was generally exerted. For growth cohort experiments, supernatants were collected from virus infected cells of each selected passage at different time points (6, 12, 24, 36, and 48 hpi) and stored at -80 ° C.

Virus titers were measured three times per dilution using a 10-fold dilution of the sample on a 96-well plate, which measures the amount of virus required to form 50% CPE of infected Vero cells, Reed-Muench Calculated as TCID 50 per ml using the method (Reed and Muench, 1938). PEDV titers were also determined by plaque assay, Vero cells, and plaque-forming units (PFU) per ml.

1.3 Immunofluorescence Assay  ( IFA , Immunofluorescence  assay)

Vero cells grown on microscope coverslides placed on 6-well tissue culture places were infected or infected with PEDV at 0.1 multiplicity of infection (MOI). Virus-infected cells were then propagated up to 24 hpi, fixed at room temperature for 4 minutes with 4% paraformaldehyde and permeabilized for 10 minutes at room temperature with 0.2% Triton 1 X-100 in PBS. Cells were blocked with 1% BSA in PBS for 30 minutes at room temperature and then incubated with N-specific Mab for 2 hours. After 5 washes with PBS, cells were incubated with goat anti-mouse secondary antibody conjugated with Alexa Fluor 488 (Invitrogen) for 1 hour at RT followed by DAPI (4?, 6-diamidino-2-phenylindole, Sigma). Counterstained. Cover slides were mounted onto microscope glass slides in mounting buffer and cell staining was visualized using a fluorescent Leica DM IL LED microscope (Leica, Wetzlar, Germany).

1.4. Quantitative Real-Time RT- PCR

Virus RNA was extracted using i- TGE / PED Detection Kit from virus isolates or fecal cells prepared as described above. Quantitative real-time RT-PCR was performed using One Step SYBR PrimeScript RT-PCR Kit (TaKaRa, Otsu, Japan). The reaction was performed using a Thermal Cycler Dice Real Time System (TaKaRa), and the results were analyzed using the system described in Sagong and Lee, 2011.

1.5 Immunization of Guinea Pigs

Eight guinea pigs were randomly allocated to the inoculation group (n = 6) and the control group (n = 2). Six guinea pigs were immunized subcutaneously with 0.5 ml of binary ethylenimine (BEI) -inactivated KNU-141112-P10 virus in the presence of Freund's complete adjuvant (Sigma), followed by a two week interval. And boosted once with an emulsion of freshly prepared inactivated virus and Freund's complete adjuvant. Two gastric-inoculated guinea pigs were dosed and boosted with cell culture medium in the presence of each adjuvant. Pre-immune serum was collected before the start of immunization and antiserum was collected 2 weeks after the last boost.

1.6 Serum Neutralization

The presence of PEDV-specific neutralizing antibodies in serum samples collected from all groups of guinea pigs was determined by serum neutralization experiments using PEDV isolate KNU-141112 or vaccine SM98-1 in 96-well microtiter plates. . Specifically, Vero cells were grown for 1 day in 2 × 10 ⁴ / well in 96-well tissue culture plates. KNU-141112-P10 virus stock was diluted with serum free α-MEM to 200 TCID in 50 μl volume. Subsequently, the diluted virus was mixed with 50 μl of 2-fold step diluent of individually inactivated serum in 96-well plates. The mixture was inoculated into Vero cells and incubated at 37 ° C. for 1 hour. The mixture was removed and the cells washed with PBS five times and kept in virus growth medium at 37 ° C. in a 5% CO 2 incubator for two days. For serum neutralization experiments with vaccine strains, PEDV strain SM98-1 propagated in the presence of trypsin was diluted with serum-free α-MEM to make 200 PFU in 50 μl volume. The virus diluted as described above in a 96-well plate was then mixed with each serum and incubated at 37 ° C. for 1 hour. Subsequently, about 1 × 10 ⁴ Vero cells in 100 μl of α-MEM containing 5% FBS were added to each well and the mixture was maintained in a CO ₂ incubator at 37 ° C. for 3 days. Neutralization titers are calculated as the reciprocal of the highest serum dilution that inhibits virus specific CPE in all duplicate wells.

1.7 Swine Infection Experiment

In vivo pig experiments were conducted at the ImproAH Animal Facility according to the guidelines of the Animal Experimental Ethics Committee. A total of eight 7-day-old suckling pigs were purchased that were not identified or vaccinated with PEDV. All animals were found to be free of PEDV as well as antibodies to transmissible gastroenteritis virus (TGEV) and porcine reproductive and respiratory syndrome virus. Pigs were randomly divided into three experimental groups and reared in two separate rooms: in room 1 the PEDV-vaccinated group ( n = 4) and the contact control ( n = 2), in the room 2 the gastric-vaccinated control group ( n = 2). After a day of acclimation, one pig of the PEDV-vaccinated group was orally administered with a 1 ml dose of 10 ³ TCID50 / ml KNU-141112-P10 virus. Two pigs were bred in the same room and exposed to the virus by direct contact with the inoculated pigs. Gastric-vaccinated pigs were administered with cell culture medium in placebo.

The clinical symptoms of diarrhea and mortality were monitored daily. Bowel samples from all groups were inoculated and collected daily with a 16 inch swab and RT-PCR and real-time RT-PCR using the i- TGE / PED Detection Kit were performed. PEDV-inoculated pigs were necropsied daily (1, 2, 3, and 4 days) after challenge for post necropsy, whereas all pigs in contact and control were post-challenge around post autopsy for post necropsy. Euthanized on. Visceral tissue samples (<3 mm thick) collected from each pig were fixed with 10% formalin at room temperature for 24 hours and embedded in paraffin. Tissue fixed and embedded in paraffin was cut to 5-8 μm thick on a microtome (Leica), suspended in a water bath at 40 ° C. containing distilled water, and then moved onto a glass slide. Tissues were deparaffinized in xylene for 5 minutes and washed with decreasing concentrations of ethanol at 100%, 95%, 90%, 80%, and 70% for 3 minutes respectively. Deparaffinized intestinal tissue sections were stained with hematoxylin and eosin (sigma) and subjected to immunostaining assay using PEDV N-specific Mab.

1.8. Immunohistostaining ( Immunohistochemistry )

Tissue sections embedded in paraffin were deparaffinized, treated with 0.01 M citrate buffer (pH 6.0), placed in a microwave for 5 minutes, cooled at room temperature for 20 minutes, and incubated with 0.3% hydrogen peroxide in DW for 20 minutes. Endogenous peroxidase was blocked. After washing three times with PBS, sections were blocked with normal horse serum (VECTASTAIN ABC Kit; Vector Laboratories, Burlingame, Calif.) And incubated with N-specific Mab for 1 hour at room temperature. After washing with PBS, the sample was reacted with the equine anti-mouse secondary antibody (VECTASTAIN ABC Kit) for 45 minutes at room temperature, incubated with the avidin-biotin peroxidase complex (VECTASTAIN ABC Kit) for 45 minutes, and DAB substrate Kit (Vector Laboratories). ) Expression. The slides were then counterstained with hematoxylin, dried, washed with xylene, then mounted on a microscope glass slide in the mounting buffer and tissue staining visualized under a microscope.

1.9. Nucleotide Sequencing

The complete genome sequence of the original fecal sample and its isolates of P5 and P10 were determined using next-generation sequencing (MGS) techniques. Total RNA from fecal and P5 and P10 isolates was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and used as a template for amplifying cDNA fragments.

Ten overlapping cDNA fragments were generated, collected in equimolar amounts to cover the entire genome of each sample, followed by the Ion Torrent Personal Genome Machine (PGM) Sequencer System (Life Technologies, Carlsbad, Calif.) And 316 v2 sequencing chip (Life Technologies NGS was performed using Single-nucleotide variants (SNVs) were analyzed using CLC Genomic Workbench version 7.0 (CLC Bio, Cambridge, Mass.) And individual NGS reads were analyzed in the PEDV reference KOR / KNU-1305 / 2013 (Genbank accession). no.KJ662670).

The 5 'and 3' ends of the feces and genomes of the isolates of P5 and P10 were determined using rapid amplification of cDNA end (RACE).

Full-length genomic nucleotide sequences of KNU-141112-feces, KNU-141112-P5, and KNU-141112-P10 were deposited in GenBank with accession numbers of KR873431, KR873434, and KR873435, respectively. S glycoprotein gene sequence of virus isolates was determined by a typical Sanger method. Two overlapping cDNA fragments covering the S gene of each isolate were amplified by RT-PCR. Individual cDNA amplifiers (gellic) were gel-purified, cloned into pGEM-T easy (Promega, Madison, Wis.) And sequenced in both directions using vector-specific T7 and SP6 primers and S gene-specific primers. It was.

In addition, the complete structural gene sequence of the virus isolate (KNU-141112-P3, KNU-141112-P4, KNU-141112-P20, and KNU-141112-P30) of the selected passage was determined by the Sanger method, and shown in FIG. The accession number was deposited in the GenBank database.

1.10 Phylogenetic analysis

Sequence alignments and phylogenetic analyzes were performed independently on 42 fully sequenced S gene sequences and 25 complete genomes of PEDV isolates. Multi-sequence alignments were generated using the ClustalX 2.0 program (Thompson et al., 1997) and the percentage of nucleotide sequence divergences was measured with the same software.

Openings were constructed from aligned nucleotide or amino acid sequences using a neighbor-joining method, followed by bootstrap analysis using replicates, whereby each internal node of the phylogenetic tree ( The percent confidence value on the internal node) was determined. All phylogenetic drawings were generated using Mega 4.0 software (Tamura et al., 2007).

1.11 Statistical Analysis

Student's t test was used for all statistical analyzes and p-values less than 0.05 were considered significant.

Example  2. Results

2.1 Virus isolation and in vitro specificity

We sought to isolate PEDV on Vero cells from a PCR-positive clinical sample containing 50 feces and 7 intestinal mills. Among them, one PEDV isolate named KNU-141112 was successfully isolated from feces of naturally infected pigs from a farm located in Gyeongsangbuk-do, which was obtained around September 29, 2014.

In addition, the present inventors deposited the KNU-141112 virus, a domestic isolate of the swine epidemic diarrhea virus, at the Korea Biotechnology Research Institute microbial resource center located in Eeun-dong, Yuseong-gu, Daejeon, Korea on December 18, 2014. Accession number KCTC12736BP was assigned.

KNU-141112 virus exhibited clear CPE, typical of PEDV infection, from passage 3 (P3) to cell fusion, syncytium and detachment in infected Vero cells.

Subsequently, it was confirmed that this isolate was effectively cultured and maintained in cell culture. Accordingly, isolated PEDV strain KNU-141112 was serially passaged for 30 total passages in Vero cells (P1 to P30). The time point for CPE initiation was 24 hpi, so that significant CPE was observed in the first 2 productive passages (P3 and P4) within 48 hpi. In subsequent passages, branched CPE appeared at 12 hpi and was pronounced at 24 hpi. Virus proliferation was confirmed by detecting PEDV antigen by IFA using PEDV N protein-specific Mab. Distinct staining was distributed in the cytoplasm of typical syncytial cells. In contrast, no gastric-inoculated Vero cells showed CPE and N-specific staining. Examples of CPEs in selected passages and related IFA images are shown in FIG. 1.

The level of viral genome in selected passages was measured by real-time RT-PCR and the mean cycle threshold (Ct) value was determined to be 16.7 in the range of 15.3 (P10) to 18.7 (P5).

Infectious titers up to P5 of the isolate were determined in the range of 10 ^5.1 to 10 ^6.1 TCID ₅₀ / ml, and in subsequent passages were determined to be about 10 ⁷ TCID ₅₀ / ml. From pass 10 the peak virus titer reached 10 ^7.8 TCID ₅₀ / ml (corresponding to 10 ^7.5 PFU / ml) (FIG. 2A).

In addition, growth kinetics experiments showed that KNU-141112 replicates quickly and effectively in Vero cells and reaches a maximum titer> 10 ⁷ TCID ₅₀ / ml at 24 dpi.

2.2. In guinea pigs KNU -141112 Antibody Reactions

Antisera of guinea pigs before preimmune (pre-immune) guinea pigs were collected and tested for neutralization activity against final boost and isolated KNU-141112 or vaccine strains 2 weeks after the last boost.

As shown in FIG. 3, guinea pig antiserum was very effective in suppressing KNU-141112 infection and had an average neutralizing antibody (NA) titer of 1: 112. On the other hand, relatively low dilution of antiserum completely protected Vero cells from SM98-1 infection, with an average NA titer of 1:37. In contrast, serologous or unimmunized sera exhibit neutralizing activity for each week.

Taken together, these data indicate that isolated KNU-141112 can elicit potential antibody responses in immunized animals. However, while antiserum strongly recognized allogeneic outdoor isolates, it was ineffective against heterologous PEDV vaccine strains, suggesting antigenic variation between the vaccine strain and the field PEDVs.

2.3. KNU - 141112Infective  Pathogenic

Four pigs were challenged orally with the KNU-141112 virus, and two control animals were inoculated with apostasy culture medium. Two pigs were raised with pigs inoculated in the same room for direct contact exposure. Clinical signs were recorded daily and fecal swabs were collected before and after inoculation during this experiment. After the acclimation period, all pigs were active and showed normal fecal concentrations. PEDV-challenged pigs showed clinical signs including lethargy and diarrhea at 1 DPI (day post-inoculation), and severe watery diarrhea followed by vomiting. PEDV-related mortality occurred from pigs inoculated at 1DPI. Contact pigs bred with the inoculated group showed diarrhea and vomiting at 2 DPI, and the progress of clinical signs was similar to the inoculated animals. In addition, as determined by RT-PCR, all inoculated and contacted pigs at 1 or 2 DPI were positive for PEDV, with mean Ct values of 18.7 (range 16.4-21.0) and 20.1 (range 15.4-23.1), respectively. PEDV was excreted in feces. Negative control pigs were active with normal feces and feces with PEDV were not detected during the experimental period.

One pig died of PEDV, followed by necropsy at 1 DPI, and the remaining inoculated pigs were then randomly selected for necropsy. All contact and control animals were euthanized for postmortem examination. All inoculated or naive contact pigs showed macroscopic typical PED-like lesions. The small intestine was swollen, yellow rapeseed was accumulated, and the wall was thin and transparent due to villi atrophy (FIG. 4, Panel A). The stomach was also swollen and filled with a blunt, undigested fluid. In contrast, other intestinal organs were normal. Microscopic observations of the gut coincident with viral growthitis have developed in all inoculated and contacted pigs, which include the vacuoles of the small intestine and the reduction and fusion of the small intestinal villi (Fig. 4, Panels B and C). Similar micro lesions were observed continuously in challenged and contacted pigs up to 4 DPI. Moreover, IFA and IHC staining showed that viral antigens were distinctly detected in the cytoplasm of epithelial cells on atrophied villi in all fragments of the small intestine (FIG. 4, Panels D to F). These results are comparable to those of pigs infected naturally or experimentally with malignant PEDV strains (Debouck et al., 1981; Jung et al., 2014; Madson et al., 2014; Stevenson et al, 2013). No microscopic or macroscopic intestinal lesions were observed in the negative control pigs during the experimental period.

2.3. KNU Specific genome of -141112

Whole genome sequence data of circular fecal samples (KNU-141112-feces) and cell culture-passed KNU-141112-P5 and -P10 viruses were determined using NGS techniques.

The full-length genome of KOR / KNU-1305 / 2013 PEDV was used as an initial reference for each NGS read, and individual complete genome sequences were successfully obtained by collection of each NGS read. In addition, the 5 'and 3' ends of this genome were sequenced with RACE. All three genomes were 28,038 nucleotides in length except for the 3'poly (A) tail, 292-nt 5 'UTR, 20345-nt ORF1a / 1b (nt 293-12601 for 1a, and nt 12601 for 1b). To 20637), 4161-nt S gene (nt 20634 to 24794), 675-nt ORF3 (nt 24794 to 25468), 231-nt E gene (nt 25449 to 25679), 681-nt cell membrane (membrane, M) gene ( nt 25687 to 26367), 1326-nt N genes (nt 26379 to 27704), and 334-nt 3 'UTR, showing typical genomic structures of conventionally sequenced PEDV strains. The slippery heptamer sequence, TTTAAAC, followed by a stem-loop structure, appeared at the 3 'end of ORF1a in the PEDV genome, corresponding to a potential signal of ribosomal frame shift during translation to produce pp 1ab. .

The complete canonical sequences of all three viruses were compared and the results are shown in Table 2 below.

Comparison of KNU-141112-feces, KNU-141112-P5 shows one different nt at position 21756, which results in a change in one amino acid (aa) in the S protein (Lhe to Phe). In contrast, KNU-141112-P10 obtained two additional nt changes of 21448 and 24492, resulting in two aa mutations on the S protein.

The full-length S gene of KNU-141112 virus of circular feces and selected passages (P3, P4, P5, P10, P20, and P30) was sequenced using conventional sequencing methods.

The S protein sequences of KNU-141112-feces, -P5, and -P10 were completely identical to those determined by NGS. One nt change in the S-gene of KNU-141112-P5 occurred from passage 3 (KNU-141112-P3). A total of three nt mutations, identified at 10 passages compared to feces, persisted up to passage 30 (KNU-141112-30).

In addition, in comparison of KNU-141112-P10, KNU-141112-P20 was able to detect two independent mutations at positions 24869 and 25656, which were amino acids of ORF3 (Tyr in Asp) and E (Ser in Pro), respectively. Causing a change, which was maintained up to passage 30.

Taken together, these results indicate that PEDV isolated KNU-141112 is genetically stable in cell passage culture.

The entire genome sequence of the PEDV determined in the present invention was compared with three conventional domestic or eight foreign PEDV strains which can be identified in Genebank, and these nucleotide homology data are shown in Table 3 below.

In addition, it was compared with three other domestic strains or eight foreign PEDV strains which can be identified in the conventional Genebank for the PEDV spike protein determined in the present invention, and these nucleotide homology data are shown in Table 4 below.

KNU-141112 isolates (feces, 4 P5, and P10) exhibited 8 to 46 nt differences at the genome level with domestic reexpressed virus strains KOR-KNU-1305 and US strain, IN17846 and MN, resulting in the highest nucleotide homology. (99.9%).

All three viruses were genetically isolated from the Korean vaccine strains SM98-1 and DR-13, and the original CV777 strain, and exhibited relatively low nucleotide homology in the range of 96.3% to 96.8%.

According to the worldwide alignment of PEDV strains, single-nucleotide insertions between 20204 and 20205 have been conventionally identified in one Chinese AH2012 strain and three US strains, resulting in a shortening of the pp 1ab protein in length (Chen et al., 2014 ). However, all three PEDV dogs do not include this insertion and this result was confirmed by Sanger sequencing method.

Both parts of the published PEDV strain were used for phylogenetic analysis, as the S gene and S1 moiety were suitable sites for analyzing the genetic association between different PEDV isolates. Conventional results are the same (Lee et al., 2010; Lee and Lee, 2014; Lee et al., 2014a; Lee et al., 2014b), and the full-length S gene-based phylogenetic tree is evident in two clusters of global PEDV strains. Genogroup 1 (G1; classical) and genogroup 2 (G2; field epidemic). Each genogroup can be subdivided into subgroups 1a, 1b, 2a, and 2b (Fig. 5A). PEDV viruses passaged to all circular feces and up to 30 passages were all recently classified as subgroup 2b, similar to domestic field isolates, and clustered closest to the emerging US strains in adjacent lineages of the same subgroupso. Subsequent phylogenetic analysis using the S1 moiety showed the same grouping structure as the S gene-based phylogenetic tree.

In addition, phylogenetic analysis based on the whole genome sequence demonstrated that KNU-141112 was recently classified into the same cluster as domestic and US strains (FIG. 5B).

[Accession number]

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC12736BP

Deposit Date: 20141218

Claims (23)

1. Porcine Epidemic Diarrhea Virus (PEDV) Korea isolate comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having a homology of 98% or more and having an equivalent immunogenicity.
2. The method according to claim 1, wherein the amino acid sequence having at least 98% homology is an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 15, 22, 28, 34, and 40, Porcine Epidemic Diarrhea Virus , PEDV) Republic of Korea.
3. The method of claim 1, wherein the isolate is ORF (open reading frame) 1a / 1b of SEQ ID NO: 2, ORF 3 of SEQ ID NO: 3, envelope protein of SEQ ID NO: 4, membrane protein of SEQ ID NO: 5 , And further comprising at least one amino acid sequence selected from the group consisting of a nucleocapsid protein of SEQ ID NO: 6 or an amino acid sequence having at least 98% homology therewith, Porcine Epidemic Diarrhea Virus, PEDV) South Korea.
4. The isolate of claim 3, wherein the isolate is composed of the amino acid sequence of SEQ ID NO: 7. Porcine Epidemic Diarrhea Virus (PEDV).
5. The isolate of claim 3, wherein the isolate has been deposited with accession number KCTC12736BP, Porcine Epidemic Diarrhea Virus (PEDV).
6. The method of claim 1, wherein the isolate is a spike protein of SEQ ID NO: 8, ORF (open reading frame) 1a / 1b of SEQ ID NO: 9, ORF 3 of SEQ ID NO: 10, envelope protein of SEQ ID NO: 11, SEQ ID NO: 12 Porcine Epidemic Diarrhea Virus (PEDV), which comprises at least one amino acid sequence selected from the group consisting of a membrane protein, and a nucleocapsid protein of SEQ ID NO: 13. Segregation Note.
7. The isolate of claim 6, wherein the isolate consists of the amino acid sequence of SEQ ID NO: 14, Porcine Epidemic Diarrhea Virus (PEDV).
8. The method of claim 1, wherein the isolate is a spike protein of SEQ ID NO: 15, ORF (open reading frame) 1a / 1b of SEQ ID NO: 16, ORF 3 of SEQ ID NO: 17, envelope protein of SEQ ID NO: 18, SEQ ID NO: 19 Porcine Epidemic Diarrhea Virus (PEDV) Korea, which comprises at least one amino acid sequence selected from the group consisting of a membrane protein of protein and a nucleocapsid protein of SEQ ID NO: 20 .
9. The isolate of claim 8, wherein the isolate is composed of the amino acid sequence of SEQ ID NO: 21. Porcine Epidemic Diarrhea Virus (PEDV) Korea isolate.
10. The method of claim 1, wherein the isolate is a spike protein of SEQ ID NO: 22, ORF 3 of SEQ ID NO: 23, envelope protein of SEQ ID NO: 24, membrane protein of SEQ ID NO: 25, and neuro of SEQ ID NO: 26 Porcine epidemic diarrhea virus (PEDV) South Korea isolate comprising at least one amino acid sequence selected from the group consisting of a nucleocapsid protein (nucleocapsid protein).
11. The isolate of claim 10, wherein the isolate comprises the amino acid sequence of SEQ ID NO: 27. Porcine Epidemic Diarrhea Virus (PEDV) Korea isolate.
12. The method of claim 1, wherein the isolate is a spike protein of SEQ ID NO: 28, ORF 3 of SEQ ID NO: 29, envelope protein of SEQ ID NO: 30, membrane protein of SEQ ID NO: 31, neurons of SEQ ID NO: 32 Porcine epidemic diarrhea virus (PEDV) South Korea isolate comprising at least one amino acid sequence selected from the group consisting of a nucleocapsid protein (nucleocapsid protein).
13. The isolate of claim 12, wherein the isolate comprises the amino acid sequence of SEQ ID 33. Porcine Epidemic Diarrhea Virus (PEDV).
14. The method of claim 1, wherein the isolate is a spike protein of SEQ ID NO: 34, ORF 3 of SEQ ID NO: 35, envelope protein of SEQ ID NO: 36, membrane protein of SEQ ID NO: 37, and neuro of SEQ ID NO: 38 Porcine epidemic diarrhea virus (PEDV) South Korea isolate comprising at least one amino acid sequence selected from the group consisting of a nucleocapsid protein (nucleocapsid protein).
15. The isolate of claim 14, wherein the isolate comprises the amino acid sequence of SEQ ID NO: 39. Porcine Epidemic Diarrhea Virus (PEDV) Korea isolate.
16. The method of claim 1, wherein the isolate is a spike protein of SEQ ID NO: 40, ORF 3 of SEQ ID NO: 41, envelope protein of SEQ ID NO: 42, membrane protein of SEQ ID NO: 43, and neuro of SEQ ID NO: 44 Porcine epidemic diarrhea virus (PEDV) South Korea isolate comprising at least one amino acid sequence selected from the group consisting of a nucleocapsid protein (nucleocapsid protein).
17. The isolate of claim 16, wherein the isolate comprises the amino acid sequence of SEQ ID NO: 45. Porcine Epidemic Diarrhea Virus (PEDV) Korea isolate.
18. Porcine epidemic diarrhea (PED) vaccine composition comprising the virus of any one of claims 1 to 17, a culture of the virus or an antigen thereof as an active ingredient.
19. The vaccine composition of claim 18, wherein the vaccine is a live vaccine or a dead vaccine.
20. A pharmaceutical composition for preventing or treating swine epidemic diarrhea (PED) comprising the virus of any one of claims 1 to 17, a culture of the virus or an antigen thereof as an active ingredient.
21. 18. A composition for diagnosing swine epidemic diarrhea (Porcine Epidemic Diarrhea, PED) comprising the virus of any one of claims 1 to 17, a culture of the virus or an antigen thereof as an active ingredient.
22. A composition for diagnosing swine epidemic diarrhea (Porcine Epidemic Diarrhea, PED) comprising the virus of any one of claims 1 to 17, a culture of the virus or an agent capable of detecting the antigen thereof as an active ingredient.
23. A pharmaceutical composition for preventing or treating porcine epidemic diarrhea (PED), comprising the virus growth inhibitor of claim 1.

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