KR20130092520A - Novel porcine reproductive respiratory syndrome virus and uses thereof - Google Patents

Abstract

PURPOSE: A novel porcine reproductive respiratory syndrome virus is provided to use for manufacturing an immunogenic composition since the GP5 coding sequence of the virus is different from the existing North America type reference strain and European type reference strain. CONSTITUTION: PRRS-GC-EU 0907 (deposit number KCTC 11854BP) is a novel porcine reproductive respiratory syndrome virus (PRRSV). An immunogenic composition for the prevention or treatment of porcine reproductive and respiratory syndrome (PRRS) contains inactivated PRRS-GC-EU0907 (deposit number KCTC 11854BP). The PRRS-GC-EU 0907 (deposit number KCTC 11854BP) is inactivated by bromoethylamine hydrobromide. A step of injecting the immunogenic composition to mammals except human is included in the method for the prevention or treatment of PRRS.

Description

Novel porcine reproductive respiratory syndrome virus and uses thereof

The present invention is a novel porcine genital respiratory syndrome virus (PRRSV), inactivated porcine genital respiratory syndrome virus (PRRSV) containing immunogenic composition for the prevention or treatment of swine genital respiratory syndrome, and mammalian porcine genital respiratory syndrome using the same It relates to how to prevent or treat.

Porcine reproductive respiratory syndrome virus (PRRSV) is a single-stranded positive sense RNA virus with an envelope. Belongs. PRRSV is a recently-occurred pathogen, causing sows miscarriage, stillbirth, and fetal pneumonia and hypoplasia, lower delivery rates, lower growth rates, and other secondary bacterial infections and respiratory diseases in pigs of all ages. It is an infectious disease that is seriously damaging the pig farming industry. PRRSV, which was first isolated and reported in Korea in 1993, proliferates favorably in macrophages. Its onset forms are genital, respiratory, and mixed, which are primarily transmitted from pigs to pigs, and colostrum antibodies can be transmitted to non-feeding piglets through feces, urine, and milk. It can also be transmitted by needles, insects and air.

At the same time, PRRSV has a wide variety of genetically diverse strains according to its geopolitical location. The two major genotypes are the European type (genotype 1) and the North American type (genotype 2), which exhibit 40% nucleotide differences in terms of the genomic level. The strain that occurs mainly in Korea has been found to be VR2332. According to the results of the study, the North American type PRRSV has been evolving since it spread to Korean pig farms, and it is predicted that the genetic degeneration of PRRSV has occurred due to geopolitical effects.

The total genome size of PRRSV is 15 kb and has 9 open reading frames (ORFs) (ORFs 1a, 1b, 2a, 2b, 3-7) in which lower genomic mRNAs are overlapped with each other. ORF 1a and 1b are replication-related non-structural proteins, and ORF 2-7 constitutes structural proteins as GP2a, GP2b, GP3, GP4, GP5, membrane/matrix (M) protein and nucleocapsid (N) protein. The main structural proteins of these are the N protein, the M protein, and the primary envelope glycoprotein, GP5. Among them, the GP5 (ORF 5) protein is the most abundant protein known to induce neutralizing antibodies in the host upon infection.

As a method of preventing or treating PRRS, a method of administering a live venom vaccine using an attenuated VR2332 strain to pigs has been proposed. However, this method has a disadvantage in that its effectiveness is somewhat low, and infection of PRRSV may not be successfully controlled, and infection by the vaccine strain VR2332 may be induced.

The damage to farms caused by PRRSV infection around the world is not decreasing easily despite a lot of efforts. According to data released by the Korean Pig Veterinarians Association, the total domestic loss due to PRRSV infection was 53.1 billion won as of 2008. The problem is that these enormous damages are occurring despite the enormous use of PRRS imported vaccines. In fact, only two types of vaccines used in Korea are sold: live venom vaccines from multinational companies and dead venom vaccines from domestic companies. In the case of live venom vaccine, there have been many reports on its effectiveness, but due to reports of virus release, there were questions about its safety, and its application in the piglet section was inevitably limited due to the interference effect of maternal transfer antibodies. And, in the case of the Zadok vaccine, only one North American virus was used as the vaccine.Since it is a Zadok vaccine, its safety can be considered to be higher than that of the live venom vaccine, but it is only limited because the frequent genetic mutations of PRRSV have a high effect on the defense function. The use of a type of virus as a vaccine has limitations in providing effective immunity. Moreover, despite the increasing use of these vaccines, the percentage of PRRS-positive farmers per month from 2008 to 2009 seldom declined and remained high.

In the case of PRRSV, it is largely divided into two subtypes, North America and Europe, and a 40% genetic difference is observed between North America and Europe. There is a need for individual strategies for strain. Until 2006, it was known that only North America was infected in Korean pig farms, and a vaccine using the North American virus has been used accordingly. However, after 2006, domestic infections in European states have been confirmed. Moreover, European infection, which was a sporadic small-scale form of infection in 2006, was reported in a joint study between Green Cross Veterinary Medicine and Seoul National University College of Veterinary Medicine in 2010 (Lee et al., Virus genes, 2010, 40). The proportion of infected farms increased rapidly, accounting for half.

Even according to the prior art as described above, there is still a need for a vaccine that can protect against European PRRSV and/or against various North American PRRSV found in Korea.

One embodiment provides a novel porcine genital respiratory syndrome virus (PRRSV).

Another embodiment provides an immunogenic composition for the prevention or treatment of porcine genital respiratory syndrome (PRRS) having high safety and excellent immunogenicity, including the novel porcine reproductive respiratory syndrome virus (PRRSV) inactivated.

Another embodiment provides a method of efficiently preventing or treating PRRS in mammals using the composition.

One embodiment is a novel porcine reproductive respiratory syndrome selected from the group consisting of PRRS-GC-6262, PRRS-GC-4019, PRRS-GC-DM, PRRS-GC-4172, PRRS-GC-EU0907, and combinations thereof. Virus (PRRSV) is provided.

All of these novel PRRSVs are separate states in Korea, and four states, namely, PRRS-GC-6262, PRRS-GC-4019, PRRS-GC-DM, and PRRS-GC-4172, have a North American genotype. On the other hand, PRRS-GC-EU0907 has a European genotype.

The coding nucleotide sequence of these novel PRRSV GP5 is SEQ ID NO: 1 to 5 [PRRS-GC-6262 GP5 coding sequence (VR2332 GP5 reference 1-603): SEQ ID NO: 1, PRRS-GC-4019 GP5 coding sequence (VR2332 GP5 reference) 1-603): SEQ ID NO: 2, PRRS-GC-DM GP5 coding sequence (VR2332 GP5 reference 44-540): SEQ ID NO: 3, PRRS-GC-4172 GP5 coding sequence (VR2332 GP5 reference 44-540): SEQ ID NO: 4 And PRRS-GC-EU0907 GP5 coding sequence (Lelystad GP5 standard 1-605): as shown in SEQ ID NO: 5. In addition, the results of comparing the nucleotide sequence of the GP5 coding sequence of these novel PRRSVs and the GP5 coding sequence of the North American and European standard strains VR2332 and Lelystad are shown in Table 1.

VR2332 PRRSV-GC-6262 PRRSV-GC-4019 PRRSV-GC-DM PRRSV-GC-4172 PRRSV-GC-EU0907 Lelystad VR2332 - 0.844 0.996 0.877 0.883 0.64 0.637 PRRSV-GC-6262 0.844 - 0.844 0.851 0.85 0.616 0.612 PRRSV-GC-4019 0.996 0.844 - 0.874 0.879 0.64 0.637 PRRSV-GC-DM 0.877 0.851 0.874 - 0.931 0.638 0.635 PRRSV-GC-4172 0.883 0.85 0.879 0.931 - 0.627 0.624 PRRSV-GC-EU0907 0.64 0.616 0.64 0.638 0.627 - 0.988 Lelystad 0.637 0.612 0.637 0.635 0.624 0.988 -

Table 1 shows a comparison of the PRRSV of the present invention and the GP5 coding sequence of the North American and European standard strains. In Table 1, the values represent the homology obtained by sequence alignment using CLustal W using Bioedit (North Carolina Univ. NC. USA) software. Since the GP5 part plays an important role in the formation of neutralizing antibodies, the sequence homology of GP5 is an indicator of whether cross-protection is within the possible range and how much genetic mutation has progressed.

As shown in Table 1, compared to the North American standard state VR2332, except that PRRS-GC-4019 shows 99.6% homology, all other four states, namely PRRS-GC-6262, PRRS-GC- DM, PRRS-GC-4172 and PRRS-GC-EU0907 showed 64.0% to 88.3% homology, confirming that they had significant genetic variation with the North American standard strain. These correspond to the strains in which antigenic mutations progressed from the standard strain, and the range of defense against PRRSV can be broadened by using these antigenic mutations in strains as inactivated vaccines.

In addition, compared to the European standard state Lelystad, all other four states, namely PRRS-GC-6262, PRRS-GC-4019, PRRS-GC-DM, except that PRRS-GC-EU0907 shows 98.8% homology. And PRRS-GC-4172 showed 61.2% to 63.7% homology, and it was confirmed that they had a significant genetic variation with the European standard strain.

In addition, compared to each other, PRRS-GC-DM and PRRS-GC-4172 showed 61.6% to 88.3% homology, except that PRRS-GC-DM and PRRS-GC-4172 had 93.1% homology. It was confirmed to have.

Therefore, these five PRRSV strains correspond to the strains in which antigenic mutations progressed between each other and between the standard strains, and by using these antigenic mutations strains as inactivated vaccines, the range of defense against PRRSV can be broadened.

The above new PRRSV states, PRRS-GC-6262, PRRS-GC-4019, PRRS-GC-DM, PRRS-GC-4172 and PRRS-GC-EU0907, as of January 21, 2011, comply with the provisions of Article 7 of the Budapest Treaty. It has been deposited with KCTC (Korean Collection for Type Cultures) under the Korea Institute of Bioscience and Biotechnology, which is an international depository institution, and the accession numbers are KCTC 11852BP, KCTC 11850BP, KCTC 11853BP, KCTC 11851BP, and KCTC 11854BP, respectively.

1 shows a phylogenetic tree based on the homology of the GP5 coding sequence of the novel PRRSV strain and the North American and European PRRSV. As shown in FIG. 1, it can be seen that each of the five new PRRSVs has a different group.

Other embodiments are inactivated PRRS-GC-6262 (accession number KCTC 11852BP), inactivated PRRS-GC-4019 (accession number KCTC 11850BP), inactivated PRRS-GC-DM (accession number KCTC 11853BP), fire For the prevention or treatment of porcine reproductive respiratory syndrome (PRRS) selected from the group consisting of activated PRRS-GC-4172 (accession number KCTC 11851BP), inactivated PRRS-GC-EU0907 (accession number KCTC 11854BP), and combinations thereof Immunogenic compositions are provided.

The composition may be one comprising each or all of the five inactivated PRRSV strains. That is, it may be a combination vaccine containing 5 PRRSVs. In this specification, the term "immunogenic composition" is used interchangeably with the term "vaccine".

The composition may further include a pharmaceutically acceptable carrier or adjuvant. The carrier may include one or more components selected from the group consisting of diluents, buffers, preservatives, excipients, disintegrants, binders, and lubricants. The adjuvant is a substance used to enhance the immunogenicity of an antigen, such as mineral salts such as alum, aluminum hydroxide, aluminum phosphate and calcium phosphate; Surfactants and particulates such as nonionic block polymer surfactants (e.g. cholesterol), virosomes, saponins (e.g. Quil A, QS-21 and GPI-0100), proteosomes, immune stimulation Complex, coclate, quaternary amine (dimethyl dioctadecyl ammonium bromide (DDA)), abridin, vitamin A, vitamin E; Bacterial products such as RIBI adjuvant system (Ribi Inc.), the cell wall backbone of Mycobacterum phlei (Detox®, Muramyl dipeptide (MDP) and Tripeptide (MTP), monophosphoryl lipid A, Bacillus Calmete-Guerin (BCG), heat-labile E. coli enterotoxin, cholera toxin, trehalose dimicolate, CpG oligodeoxynucleotide; cytokines and hormones such as interleukin (IL-1, IL-2, IL-6, IL-12, IL-15, IL-18), granulocyte colony stimulating factor, dehydroepiandrosterone, 1,25-dihydroxy vitamin D3; polyanions such as dextran; poly Acrylic (e.g., polymethylmethacrylate, Carbopol 934P); carriers such as tetanus oxide, diphtheria oxide, cholera toxin B subunit, mutant heat-labile enterotoxin (rmLT) of endotoxin-producing E. coli, heat shock protein ; Oil-in-water emulsions such as AMPHIGEN® (Hydronics, USA); IMS1312N or Montanide Gel, RehydraGel, and water-in-oil emulsions, such as complete and incomplete adjuvants of Freund, among the IMS series from Seppic (France) The content of adjuvant to be added varies depending on the individual's weight, sex, age, severity of disease, viral titer, etc. The composition may also be 10-15% (w/w). , Suitable preservatives such as thimerosal, formaldehyde and/or gentamicin.

Inactivation of the novel PRRSV can be accomplished by a method known in the art. The inactivation may be performed by, for example, subculture in host cells and treatment with a chemical substance such as formaldehyde (or formalin) or bromoethylamine hydrobromide. The inactivation may be performed by immersing the virus bulk in a solution of bromoethylamine hydrobromide (BEA:BrCH ₂ CH ₂ NH _{2 HBr: molecular weight: 204.9 Da).} Here, the virus bulk refers to a state before vaccine production obtained by centrifugation from a virus culture solution prepared for vaccine production. The BEA solution may be 0.0001 to 0.1M, or 0.0005 to 0.01M BEA solution. The inactivation may be by immersing the virus in a BEA solution and leaving it at 30 to 45°C, for example, 37°C with or without stirring. The standing may be allowed to stand for an appropriate time, for example, 10 to 24 hours, for example, 18 hours. In the case of inactivation with formalin, for example, it may be achieved by adding formalin at a concentration of 0.2-0.5%. After performing the inactivation, in order to neutralize the used inactivating agent, sodium thiosulfate may be added to neutralize it. For example, 1M sodium thiosulfate can be added and neutralized by sensitizing by gradually stirring at 37°C for 1 hour and overnight at 4°C.

When the composition is a mixed vaccine containing two or more inactivated viruses, a process of mixing a solution of the inactivated virus is included. The ratio of mixing the culture medium is not limited, but inactivated PRRS-GC-6262 (accession number KCTC 11852BP), inactivated PRRS-GC-4019 (accession number KCTC 11850BP), inactivated PRRS-GC-DM ( Accession number KCTC 11853BP), inactivated PRRS-GC-4172 (accession number KCTC 11851BP) and inactivated PRRS-GC-EU0907 (accession number KCTC 11854BP), for example, in a volume ratio of 1:1.5-3.5:1.5 -3.5:1.5-3.5:1.5-3.5:1.5-3.5, for example, may be mixing 1:2:2:2:2. In addition, immunity enhancing agents such as IMS1312N (Seppic, France) or RehydraGel may be added to enhance the immunity of the individual.

Porcine reproductive respiratory syndrome (PRRS) can include any of a variety of symptoms related to reproductive and respiratory diseases in pigs caused by PRRSV, for example, sow-like acid and fetal pulmonary cyanosis, lower delivery rates, and other secondary It is a symptom caused by a typical bacterial infection.

The immunogenic composition of the present invention may be in any form known in the art, for example, in the form of a solution and an injection. For liquids or injections, it may contain 10-40% propylene glycol, if necessary, and sodium chloride in an amount sufficient to prevent hemolysis (eg, about 1%). The solution or injection may contain any diluent or buffer known in the art. In addition, the immunogenic composition of the present invention may be freeze-dried, stored in a container such as a vial, and prepared immediately before use by adding a carrier, adjuvant, saline, or the like necessary for an injection before use.

The immunogenic composition of the present invention can be administered by any means of administration known in the art. For example, the administration may be administered intravenously, intramuscularly, orally, transdermal, mucosal, intranasal, intratracheal or subcutaneously. The immunogenic composition can be administered systemically or locally.

In the immunogenic composition of the present invention, the mammal may be human, cow, pig, goat, dog, sheep, or the like.

The present invention provides a method of preventing or treating PRRS in a mammal comprising administering the immunogenic composition to a mammal other than a human.

In the method of the present invention, the mammal may be a cow, a pig, a goat, a dog, a sheep, or the like.

In the method of the present invention, the immunogenic composition of the present invention can be administered by any means of administration known in the art. For example, the administration may be administered directly intravenously, intramuscularly, orally, transdermally, mucosally, nasal, intratracheally or subcutaneously. The strain or immunogenic composition can be administered systemically or locally.

In the immunogenic composition or method of the present invention, the immunogenic composition of the present invention may be administered in an effective amount. "Effective amount" means an amount required to alleviate or eliminate symptoms to be treated or prevented, and can be appropriately selected by those skilled in the art. For example, a therapeutically or prophylactically effective amount may comprise 1x10 ^4.5 to 1x10 ^6.5 TCID ₅₀ /ml, for example, 1x10 ^5.0 to 1x10 ^6.0 TCID ₅₀ /ml of the inactivated PRRSV per unit dose. For example, the composition has a virus titer of 1x10 ^6.0 TCID ₅₀ / ml, respectively, inactivated PRRS-GC-6262 (accession number KCTC 11854BP), inactivated PRRS-GC-4019 (accession number KCTC 11850BP), Activated PRRS-GC-DM (accession number KCTC 11853BP), inactivated PRRS-GC-4172 (accession number KCTC 11851BP) and inactivated PRRS-GC-EU0907 (accession number KCTC 11854BP) 10% by volume: 20 vol. %: 20% by volume: 20% by volume: 20% by volume, and 10% by volume is montanide gel ^TM (SEPPIC, Paris, France). montanide gel ^TM (SEPPIC, Paris, France) is a composition consisting of a synthetic polymer and an aqueous gel.

According to the PRRSV strain according to the present invention, it can be used to prepare an immunogenic composition because the GP5 coding sequence is different from that of the conventionally known North American and European standard strains.

According to the immunogenic composition for preventing or treating porcine reproductive respiratory syndrome (PRRS) according to the present invention, it is possible to prevent or treat porcine reproductive respiratory syndrome (PRRS) in an individual. In particular, since the strain showing antigenic variability compared to the standard strain was used, it can protect against a wide range of PRRSV.

According to the method for preventing or treating PRRS in mammals according to the present invention, it is possible to prevent or treat porcine reproductive respiratory syndrome (PRRS) in an individual.

1 shows a phylogenetic tree based on the homology of the GP5 coding sequence of the novel PRRSV strain and the North American and European PRRSV.
2 is a diagram showing the results of measuring the neutralizing antibody titer in the vaccine group and the control group.

Hereinafter, the present invention will be described in detail by examples. However, the present invention is not limited by the following examples.

Example : Preparation of mixed vaccine and confirmation of effectiveness

In this embodiment, PRRS-GC-6262 (accession number KCTC 11852BP), PRRS-GC-4019 (accession number KCTC 11850BP), PRRS-GC-DM (accession number KCTC 11853BP), PRRS-GC-4172 (accession number KCTC 11851BP) ) And PRRS-GC-EU0907 (accession number KCTC 11854BP) were cultured, proliferated and inactivated, respectively, and then an immunogenic composition containing five inactivated viruses was prepared. Next, the effect of the composition on PRRS was confirmed.

(1) Virus used

Viruses used in the combination vaccine were PRRS-GC-6262 (accession number KCTC 11852BP), PRRS-GC-4019 (accession number KCTC 11850BP), PRRS-GC-DM (accession number KCTC 11853BP), PRRS- isolated from domestic farms. GC-4172 (accession number KCTC 11851BP) and PRRS-GC-EU0907 (accession number KCTC 11854BP) were used. These strains were isolated by inoculating cells susceptible to PRRSV with an emulsion of tissues confirmed to be infected at the Veterinary Research Institute of Green Cross Stitch Medicine Co., Ltd.

(2) Of the master seed virus Passage method

(2.1) Culture and maintenance of host cells

The culture and maintenance of MARC-15, which is a host cell of PRRSV, was performed as follows. MARC-145 cells dispersed by digestion with trypsin solution (Trypsin EDTA, Gibco, Cat. No. 15400-054) were suspended in a growth medium, subdivided into a culture vessel, and cultured at 37° C. for 2-3 days, After culturing for 3 days, it was used for passage culture. MARC-145 cell proliferation medium is α-MEM 900 ml, FBS (fetal serum albumin) 100 ml, antibiotic-antimycotics 1% (PAA, Cat.NO. p11-002), non-essential amino acids (NEAA) Consisting of 1%, 1% L-glutamine, and 1% 7% sodium bicarbonate solution, and adjusted to pH 7.0-7.4, MARC-145 cells have high tolerance for PRRSV of MA-104 cells, monkey kidney cells. It is derived from the PRRSV high permissive subpopulation.

(2.2) Virus culture and passage

For the original seed venom, the virus isolated by Green Cross Stitch Medicine Co., Ltd. Veterinary Research Institute was inoculated into cells with a monolayer of about 80% or more, sensitized at 37° C. for 1 to 2 hours, and then cultured for 72 hours. When the cytopathic effect (CPE) was more than 80%, the culture solution was aseptically collected and lyophilized or stored frozen at -80°C. Seed virus was inoculated, cultured and collected in the same manner as the original seed virus, and then freeze-dried or stored frozen at -80°C. The maintenance medium is α-MEM 1000ml, FBS 20ml, antibiotic-antimycotics 1% (PAA, Cat. No. p11-002), non-essential amino acids (NEAA) 1%, L-glutamine 1% and It consists of 1% of a 7% sodium hydrogen carbonate solution.

Viruses produced in bulk were not to be passaged more than 5 generations in the original seed venom. Inoculated, cultured, and harvested in the same manner as the original seed venom, stored frozen at -80°C, and used for vaccine production.

The virus was cultured as follows. After removing the medium from the cell culture vessel in which the monolayer was formed, each virus ^{was inoculated with 10 3} TCID ₅₀ /ml and adsorbed for 1 hour. After 1 hour, the cell monolayer was washed with PBS, and then a maintenance medium was added, followed by stationary culture at 37°C for 3 to 4 days.

Virus harvesting was carried out as follows. The virus-inoculated cells were cultured for 3 to 4 days to harvest the infected cells, centrifuged at 2,500 rpm for 15 minutes, and the precipitated cells were resuspended in PBS to 1/10 of the culture solution, and then freezing and thawing were repeated 3 times. . After complete destruction of these cells, the supernatant was harvested by centrifugation at 2,500 rpm for 15 minutes. Virus titers were PRRS-GC-4019 10 ^6.0 TCID ₅₀ /ml, PRRS-GC-6262 10 ^6.0 TCID ₅₀ /ml, PRRS-GC-4172 10 ^6.0 TCID ₅₀ /ml, PRRS-GC-DM 10 ^6.0 TCID ₅₀ /ml , PRRS-GC-EU0907 10 ^6.0 TCID ₅₀ / ml or more was confirmed.

(3) Vaccine manufacturing

Inactivation of the virus bulk was performed as follows. First, BEA (bromoethylamine hydrobromide: BrCH ₂ CH ₂ NH ₂ HBr, molecular weight 204.9 Da) (Sigma B 9258) was used as an inactivating material. BEA was dissolved in a 0.2N NaOH solution (8 g/L) heated to 37°C to a concentration of 0.1 M (20.49 g of BEA was added to 1 L of 0.2N NaOH solution). It was shaken well for 1 hour in a 37 ℃ constant temperature water bath to complete the BEI (Binary Ethylenimine) solution. 0.1M BEI solution was added to the virus bulk to 1% (diluted 100 times) to finally obtain a concentration of 0.001M (1mM). It was inactivated for about 18 hours while stirring with a magnetic bar at 37°C. After cooling to 4° C., sodium thiosulfate was added to a final concentration of 2 mM to neutralize the action of BEI (2M sodium thiosulfate, i.e. 31.62 g sodium thiosulfate, 100 ml of distilled water). The solution dissolved in was sterilized and diluted 1000 times to be used). A 1% thimerosal solution was added to a final concentration of 0.01%. In this way, each of the five PRRSVs described above was inactivated.

Next, a mixed vaccine was prepared by mixing each of the inactivated PRRSVs and adjuvants. First, the inactivated PRRSV antigen was centrifuged at 3,000 rpm for 30 minutes to obtain a supernatant. Virus titers in the obtained supernatant were all 10 ^6.0 _{TCID 50} /ml or more. The titers are both 10 ^6.0 TCID ₅₀ / ml or more, inactivation of the PRRS-GC-4019, inactivate the PRRS-GC-6262, inactivate the PRRS-GC-DM, inactivation of the PRRS-GC-4172, inactivated PRRS-GC-EU0907 was mixed in a volume ratio of 10: 20: 20: 20, and a 10% volume synthetic polymer gel, montanide gel ^{TM, for 20 minutes while being stirred using a magnetic bar.} Was added. The mixed vaccine obtained by mixing for about 1 hour was dispensed into an appropriate container and sealed.

(4) Vaccine safety test

Eight mice weighing 15 to 20 g, 4 guinea pigs weighing 300 to 350 g, and 2 healthy weaning piglets with negative antibodies to the swine genital respiratory syndrome virus were disclosed. Eight mice were inoculated subcutaneously with 0.5 ml of the vaccine, and 4 guinea pigs were inoculated subcutaneously and intraperitoneally at 2 ml each, and observed for 7 days to determine that it is safe if they survive without abnormalities. Two weaning piglets (2 ml) were observed for 21 days after intramuscular inoculation, and if they survived without abnormalities such as purulent, necrosis, fever, and respiratory symptoms at the injection site, it was judged as safe. Table 2 shows the results of the safety test for experimental animals.

Table 3 shows the results of the safety test for pigs.

In Table 3, * indicates body temperature change, dyspnea, depression, vomiting, swelling at the inoculation site, purulent spot at the inoculation site, and loss of appetite.

(5) Vaccine Potency test

The potency test was carried out according to the following Test 1 or Test 2.

Test 1 :

Ten guinea pigs weighing 300 to 350 g were used. Eight guinea pigs were injected with 1.0 ml of the vaccine intramuscularly, and after 2 weeks, 1.0 ml of the vaccine was inoculated again, and after 2 weeks, blood was collected with 2 control animals to measure neutralizing antibody titers. The test method is as follows. First, the serum was separated, and the serum was unassisted at 56°C for 30 minutes as a medium (α-MEM 1000ml, FBS 20ml, antibiotic-antifungal agent 1% (Antibiotic/Antimycotic, PAA, Cat. No. p11-002), non-essential amino acids) (NEAA) 1%, consisting of 1% L-glutamine and 1% 7% sodium hydrogen carbonate solution) and mixed in equal amounts with _{porcine genital respiratory syndrome virus (300 TCID 50 /0.1ml) at 4℃, 48} After neutralization for more than an hour, 50 µl of a medium containing fresh guinea pig serum (3 to 5%) was added, followed by reaction at 37° C. for 60 minutes. Add 50 µl of a medium (including 3% FBS) containing MARC-145 cells, MA-104 cells, or cells susceptible to porcine genital respiratory syndrome virus and incubate at 37°C for 5 days to observe the cytopathic effect, or Was fixed in 80% acetone, and the antibody titer was measured by indirect fluorescent antibody method (IFA: indirect immunoflorescence assay).

The neutralizing antibody titer against the swine genital respiratory syndrome virus is determined to be positive when 80% or more of the inoculated animals are 8 times or more, and the control group must be negative.

Table 4 shows the results of the titer test in test animals conducted according to Test 1.

The five types used as controls in Table 4 represent a phosphate buffered saline (PBS) solution.

(6) Vaccination and defense against attack vaccination

The pig groups used in the challenge test were assigned as follows. Pigs of 3 weeks of age who did not eat colostrum or had an antibody negative for PRRS virus were assigned 3 heads each as a vaccine-vaccinated group, a vaccine control group, and an inoculation control group.

Inoculation of the vaccine was performed as follows. 3 weeks old vaccine group was inoculated intramuscularly with 1 ml of a vaccine containing 5 inactivated PRRSVs of the present invention, and a second inoculation was performed at 5 weeks of age. At this time, the inoculation control group was inoculated with a medium containing no virus in the same manner.

Field virus inoculation was performed as follows. Four weeks after the last vaccination, when the pigs were 9 weeks old, the field virus isolates were challenged. As the challenge inoculation virus, PRRSV-GC-4019, which was not inactivated as an outdoor isolate, was used, and ^{the isolate having a titer of 10 4} TCID ₅₀ /ml was inoculated with 2 ml each nasally. The vaccine group and the inoculation control group were challenged, respectively, and the vaccine control group was inoculated with the same amount of medium intranasally.

The concentration of antibody in the blood of the challenge-inoculated pigs was measured. First, blood was collected through blood collection at intervals of 2 weeks after the vaccination date. Blood was collected from the heads of 3, 5, 7, 9 and 11 weeks of age, and neutralizing antibodies were measured using a serum neutralization test (SN test).

2 is a diagram showing the results of measuring the neutralizing antibody titer in the vaccine group and the control group. As shown in Fig. 2, it was confirmed that the neutralizing antibody value was increased in the vaccine group.

In addition, after the challenge vaccination, the pigs were observed for clinical symptoms and measured whether the virus was released. Specifically, after the challenge inoculation, body temperature and clinical symptoms were observed at intervals of 2 days. Observations were made for each group of individuals, and any specific matters were immediately recorded. Virus excretion was performed for 14 days after challenge, and the antigen of the virus was detected in the nasal secretions using RT-PCR. RT-PCR uses the total RNA isolated from nasal secretions as a template, and performs a reverse transcription reaction using a random hexamer as a reverse transcription primer, and uses the obtained cDNA as a template, a forward primer (SEQ ID NO: 6) and a reverse primer (SEQ ID NO: PCR was performed using number 7) as a primer.

Tables 5 and 6 show the results of observing clinical symptoms for the vaccine group and the control group, respectively.

From the above results, the immunogenic composition of the present invention has an effect of protecting an individual against a wider range of PRRSV compared to conventionally known vaccines.

Korea Research Institute of Bioscience and Biotechnology KCTC11852BP 20110121 Korea Research Institute of Bioscience and Biotechnology KCTC11850BP 20110121 Korea Research Institute of Bioscience and Biotechnology KCTC11853BP 20110121 Korea Research Institute of Bioscience and Biotechnology KCTC11851BP 20110121 Korea Research Institute of Bioscience and Biotechnology KCTC11854BP 20110121

<110> Green Cross Veterinary Products Co., Ltd. <120> Novel porcine reproductive respiratory syndrome virus and uses thereof <130> PN101552 <150> 11/028215 <151> 2011-03-29 <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 603 <212> DNA <213> PRRS-GC-6262 <400> 1 atgttgggga aatgcttgac cgcgggttgt tgctcgcaat tgcctttttt gtggtgtatc 60 gtgccgttct gttttgctgc gctcgtcaac gccagcagca acagcagctc ccatttacag 120 ttgatatata acttgacgat atgcgagctg aatggcacag agtggttaaa tgaaaagttt 180 ggctgggcag tggaaacgtt cgtcatcttt cctgtgttga ctcatattgt ttcctacggc 240 gccctcacca ccagccatct ccttgacaca gttggtctga tcactgtgtc caccgccgga 300 tattaccatg ggcggtatgt cttgagtagt gtctatgctg tttgcgcctt ggctgcgtta 360 acttgcttcg tcattagatt gacaaaaaac tgcatggctt ggcgctactc gtgtaccaga 420 tacactaact ttcttttgga caccaagggc agactctatc gctggcgatc acccgtcatc 480 atagagaaag ggggtaaggt tgaggttggg ggtcacctaa tcgacctcaa gagagtcgtg 540 cttgatggtt ccgcggcaac ccctataacc aaagtttcgg cggaacaatg gggtcgtccc 600 tag 603 <210> 2 <211> 603 <212> DNA <213> PRRS-GC-4019 <400> 2 atgttggaga aatgcttgac cgcgggctgt tgctcgcaat tgctttcttt gtggtgtatc 60 gtgccgttct gttttgctgt gctcgccaac gccagcaacg acagcagctc ccatctacag 120 ctgatttaca acttgacgct atgtgagctg aatggcacag attggctagc taacaaattt 180 gattgggcag tggagagttt tgtcatcttt cccgttttga ctcacattgt ctcctatggt 240 gccctcacta ccagccattt ccttgacaca gtcgctttag tcactgtgtc taccgccggg 300 tttgttcacg ggcggtatgt cctaagtagc atctacgcgg tctgtgccct ggctgcgttg 360 acttgcttcg tcattaggtt tgcaaagaat tgcatgtcct ggcgctacgc gtgtaccaga 420 tataccaact ttcttctgga cactaagggc agactctatc gttggcggtc gcctgtcatc 480 atagagaaaa ggggcaaagt tgaggtcgaa ggtcatctga tcgacctcaa aagagttgtg 540 cttgatggtt ccgtggcaac ccctataacc agagtttcag cggaacaatg gggtcgtcct 600 tag 603 <210> 3 <211> 540 <212> DNA <213> PRRSV-GC-DM <400> 3 tttttttgtg gtatatcgtg ccgtcctgtt ttgttgcgct cgtcagcgcc agcaacaaca 60 gcagctccaa atttcagttg atttataact tgacactatg cgagctgaac ggcacagact 120 ggctagccag taagtttgat tgggcagttg agaccttcgt catttttccc gtgttgactc 180 acattgtctc ctatggtgcc cttactacta gccatttcct tgacacagct ggtctggcca 240 ctgtgtctac cgccggttat tatcatgggc ggtatgtttt gagtagcatc tacgcggtct 300 gtgccctggc tgcgttgatt tgtttcatca tcaggttcgt gaaaaattgc atgtcttggc 360 gctactcatg taccaggtat accaactttc ttctggacac caagggcaga ctctatcgtt 420 ggcgttcacc tgtcatcata gagaaagggg gtaaagttga ggtcgaaggt catctaatcg 480 atctcaagag agttgtgctt gacggttccg cggcaacccc tgtaaccaaa gtttcagcgg 540 540 <210> 4 <211> 540 <212> DNA <213> PRRSV-GC-4172 <400> 4 tttttttgtg gtgtatcgtg ccgtcctgtt ttgttgcgct cgtcagcgcc aacaacagca 60 gcagctccca tttgcagttg atttataatt tgacgctatg cgagctgaac ggcacagatt 120 ggctagccga caaattttcc tgggcagttg agacttttgt catctttccc gtgttgaccc 180 acatcgtctc ttatggtgcc ctcactacta gccatttcct tgacacagtt ggtctggtca 240 ctgtgtctgc cgccggttat tatcacaggc ggtatgtttt gagtagcatt tacgcggtct 300 gtgccctggc tgcgttaatt tgtttcatca tcaggttcgt gaaaaactgc atgtcctggc 360 gctactcatg taccaggtat accaactttc ttctggacac caagggcaga ctctatcgtt 420 ggcgctcacc tgtcatcata gagagagggg gtaaagttga ggtcgaaggt catctgatcg 480 acctcaagag agttgtgctt gatggttccg cggcaacccc tgtaaccaaa gtttcagcgg 540 540 <210> 5 <211> 605 <212> DNA <213> PRRSV-GC-EU0907 <400> 5 atgagatgtt ctcacaaatt ggggcgtttc ttgactccgc actcttgctt ctggtggttt 60 tttttgctgt gtaccggctt gtcctggtcc tttgccgatg gcaacggcaa cagctcgaca 120 taccaataca tatataactt gacgatatgc gagctgaatg ggaccgactg gttgtccagc 180 cattttggtt gggcagtcga gacctttgtg ttttacccgg ttgccactca tatcctctca 240 ctgggttttc tcacaacaag ccattttttt gacgcgctcg gtctcggcgc tgtatccact 300 gcaggatttg ttggcgggcg gtatgtactc tgcagcgtct acggcgcttg tgctttcgca 360 gcgttcgtat gttttgtcat ccgtgctgct aaaaattgca tggcctgccg ctatgcccgt 420 acccggttta ccaacttcat tgtagacaac cgggggagag ttcatcgatg gaagtctcca 480 atagtggtag aaaaattggg caaagccgaa gtcgacggca acctcgtcac catcaaacat 540 gtcgtcctcg aaggggttaa agctcaaccc ttgacgagga cttcggctga gcaatgggag 600 gccta 605 <210> 6 <211> 20 <212> DNA <213> PRRSV-GC-4019 <400> 6 gtacattctg gcccctgccc 20 <210> 7 <211> 20 <212> DNA <213> PRRSV-GC-4019 <400> 7 gccctaattg aataggtgac 20

Claims (4)

PRRS-GC-EU0907 (Accession No. KCTC 11854BP), a novel porcine respiratory syndrome virus (PRRSV). An immunogenic composition for preventing or treating porcine reproductive and respiratory syndrome (PRRS), comprising inactivated PRRS-GC-EU0907 (Accession No. KCTC 11854BP). 3. The composition of claim 2 wherein the inactivated PRRS-GC-EU0907 (Accession No. KCTC 11854BP) is inactivated by bromoethylamine hydrobromide. A method of preventing or treating PRRS in a mammal, comprising administering to the mammal an immunogenic composition according to any one of claims 2 or 3, excluding a human.

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