KR101694607B1 - An Attenuated Infectious bronchitis virus and An Infectious bronchitis vaccine using the same - Google Patents

Abstract

The present invention relates to attenuated infectious bronchitis virus strains and infectious bronchitis vaccines using the same.

Description

An attenuated infectious bronchitis virus and an infectious bronchitis vaccine using the same,

The present invention relates to attenuated infectious bronchitis virus strains and infectious bronchitis vaccines using the same.

Infectious bronchitis (hereinafter referred to as 'IB') is an acute infectious disease of chickens with a very high spreading power generated by IB virus (Infectious bronchitis virus: hereinafter referred to as IBV). IB is not high in mortality but has a high morbidity rate and causes a decrease in egg production accompanied by a cough, a runny nose, a decrease in weight, a decrease in external and internal quality, as well as a persistent chronic disease due to colicosis accompanied by respiratory sequelae And has caused enormous economic damage to the poultry industry worldwide.

IBV is an RNA virus belonging to the coronavirus, and has a characteristic that the gene constituting the virus particle is easily mutated similarly to the influenza virus. Therefore, very diverse serotype IBVs have been reported worldwide, and it is presumed that several kinds of IBVs including mutated viruses in each of these serotypes are included. The susceptibility of the virus to various serotypes is variable, and the long-term and pathogenicity varies. In addition, cross-reactivity and cross-immunity between these serotypes are not well-established, which is accompanied by many difficulties in the prevention and control of disease.

IB, which is currently prevalent in Korea, can be divided into two types, respiratory type IB, which mainly causes severe respiratory and spawning degradation, and kidney type IB, which is accompanied by a respiratory disease but has more severe nephritis and scattering.

Domestic IB outbreaks were first reported in 1986, and respiratory viruses continued to be prevalent until late 1990s. Effective control of viruses and Sadok oil vaccines made with mesothecous IBV (H120, Ma5) Respectively. However, in spite of the use of vaccine nationwide since 1990, there have been many cases of damages caused by laying hens or laying hens, and in case of broiler chickens It has been reported that kidney type IB, which is accompanied by severe depression, diarrhea, diarrhea, kidney uric acid deposition, and nephritis together with 10% or so of the chicks being raised, has caused serious damage locally until now.

In order to effectively prevent IBD, we developed IB Xodox oil vaccine using KM91 vaccine in 1997 to prevent industrialization of IBD in Korea after effective vaccination despite extensive vaccination. And has been used in domestic market.

The kidney-type IB Xedok oil vaccine has been effective in preventing spawning degradation caused by IB infection in Korea, but it has also effectively inhibited spoilage caused by respiratory IB infection. However, since early 2000, a new serotype from China In the case of the kidney-type IBV (QXIBV strain), the cross-over immunity was lowered, and the Sdox vaccine manufactured by the domestic renal IBV strain (KM91 strain) was inoculated.

In addition, since 2005, the number of new mutant IBVs in Korea has increased, necessitating the development of new vaccines.

[Prior Patent Literature]

Korean Patent Publication No. 1020120133044

Disclosure of Invention Technical Problem [8] The present invention has been made in view of the above problems, and it is an object of the present invention to provide a novel attenuated virulent virulent vaccine.

In order to achieve the above object, the present invention provides an infectious bronchitis virus K40 / 09 CE50 deposited with the deposit number KCTC 12741BP attenuated infectious bronchitis virus K40 / 09 strain.

In one embodiment of the present invention, the infectious bronchitis virus K40 / 09 CE50 is preferably attenuated by high passage of the infectious bronchitis virus K40 / 09 strain, and the high passage is preferably infectious bronchitis virus K40 / 09 It is desirable that the strain is attenuated to 30 or more and 70 or less.

The above 30 or more and 70 or less passages are determined by reference to the passages which are attenuated while improving the safety due to heat resistance. When the passages are over 70 passages, the ability to defend the vaccine is lost, 30 or more and 70 or less.

In another embodiment of the present invention, the virus of the present invention expresses the S1 protein consisting of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

The virus of the present invention is characterized in that it is a virus that can distinguish it from existing vaccine viruses due to the phylogenetic characteristics of the virus using the S1 gene sequence analysis data.

The present invention also provides a vaccine for poultry protection against diseases caused by infectious bronchitis virus infection comprising the infectious bronchitis virus of the present invention and a pharmaceutically acceptable carrier or diluent.

In a preferred embodiment of the present invention, it is preferred that said vaccine further comprises an adjuvant,

In another preferred embodiment of the present invention, the vaccine preferably comprises but is not limited to one or more vaccine components of other pathogens infectious to poultry.

The present invention also provides a method for controlling diseases caused by infectious bronchitis infection in poultry comprising administering the virus of the present invention to an algae.

The present invention also provides a method for producing a vaccine for poultry protection against diseases caused by infectious bronchitis virus infection comprising the step of binding the infectious bronchitis virus of the present invention with a pharmaceutically acceptable carrier or diluent.

The present invention also relates to a method for the treatment of infectious bronchitis virus comprising the steps of: a) inoculating a susceptible substrate with an infectious bronchitis virus K40 / 09 strain; b) propagating the infectious bronchitis virus; c) collecting the infectious bronchitis virus-containing substance; And d) heat treating the collected virus to confirm whether the virulent virulence of the virus is weakened, and repeating steps a) through d) until the virus is weakened. Virus K40 / 09 CE50.

In one embodiment of the invention, the substrate is preferably an embryonated egg,

In another embodiment of the present invention, the repetition is preferably performed 30 to 70 times, but is not limited thereto.

In another embodiment of the present invention, the heat treatment is preferably performed for 5 minutes at 56 DEG C, but is not limited thereto.

The prior art patents disclosed in Korean Patent Laid-open Nos. 1020120133044 and PCT / KR2013 / 007944 are sadox vaccines for the infectious bronchitis virus K40 / 09 strain and the attenuated live vaccine vaccine of the infectious bronchitis virus K40 / 09 of the present invention As a completely different vaccine, Sadox vaccine is generally only involved in humoral immunity, and the virulence vaccine is involved in cell-mediated immunity as well as humoral immunity, thus acting as a more effective vaccine.

In addition, the inventor of the present invention has already developed a vaccine for infectious bronchitis virus K40 / 09 [2012 Poultry Science 91: 89.94, Graduated from Konkuk University, Kim Byung Yun, Cross-Protective Immune Responses Elicited by a Korean Variant of Infectious Bronchitis Virus, 2014 ; (Including 2-4 strains) were all vaccinated as low passages (2-4 strains). As can be seen from Table 2 of the present invention, the vaccine of 56 ° C To 120 minutes, but it does not have the safety for a long time due to the loss of viability in a longer period of time. Thus, it is a side effect which is caused by inadequate refrigeration during transport, which is a problem of a virulent vaccine, and a lack of vaccine.

In contrast, the high passivation attenuated vaccine of the present invention, as shown in Table 2, survives at 56 ° C for 450 minutes or more, and thus can prevent side effects that may be caused by inadequate refrigeration maintenance during transportation, And it is possible to administer it as a virulent vaccine even in tropical regions such as Southeast Asia.

In one embodiment of the present invention, the virus is preferably an infectious bronchitis virus isolated from domestic poultry, but is not limited thereto.

As a preferred starting material in the virus isolation method of the present invention, it can be separated from diseased algae, in particular from the respiratory organs of poultry, or excreta excreted by such algae. It should not be excluded that other organs can be used as a starting material for the isolation of viruses of the present invention.

In another aspect, the invention provides a vaccine for the protection of poultry against clinical symptoms resulting from infectious bronchitis virus infection, comprising the infectious bronchitis virus of the present invention and a pharmaceutically acceptable carrier or diluent.

The infectious bronchitis virus of the present invention may be included in the vaccine as an attenuated form. If the infectious bronchitis virus is in an attenuated form, the nature of the infectious bronchitis virus inducing the abovementioned disease is completely eliminated.

The vaccine of the present invention may be prepared according to conventional methods, which are commonly used, for example, in a commercial attenuated infectious bronchitis virus vaccine. The preparation of veterinary vaccine compositions is described in particular in "Handbunch der Schutzimpfungen in der Tiermedizin" (eds. Mays, A., Verlag Paul Parey, Berlin und Hamburg, Germany, 1984) and "Vaccines or Veterinary Applications" Et al., Butterworth-Heinemann Ltd, 1993).

Briefly, infectious bronchitis virus of the present invention is inoculated in live form to the susceptible substrate, and propagated until the virus is replicated at a predetermined infection specific antigen concentration (mass). Thereafter, the infectious bronchitis virus carrying the substance can be collected and formulated into a pharmaceutical composition having prophylactic activity.

All substrates that can be used for replication of the infectious bronchitis virus defined above can be used to generate a vaccine according to the invention, if necessary after the infectious bronchitis virus has adapted to the substrate.

Alternatively, the infectious bronchitis virus of the present invention may be propagated in embryonated eggs, and then the infectious bronchitis virus material may be collected by a conventional method.

The present invention also provides a vaccine comprising infectious bronchitis virus in an attenuated form against infectious bronchitis infection. The main advantage of an attenuated vaccine is that a high level of protective antibody lasts for a long time. Due to this nature, attenuated vaccines are particularly suitable for vaccination with vertical axis.

In addition, the vaccine optionally comprises a pharmaceutically acceptable carrier or diluent. Such carriers include stabilizers, preservatives and buffers, and suitable stabilizers include, for example, sucrose-phosphate-glutamate-albumin (SPGA), hydrocarbons such as sorbitol, mannitol, starch, sucrose, dextran, glutamate, Or glucose), a protein (e.g., dried milk serum, albumin or casein) or a degradation product thereof. Suitable buffers are, for example, alkali metal phosphates. Suitable preservatives are thimerosal, methiolate, and gentamicin. The diluent is water, aqueous buffer (e.g., buffered saline), alcohol and polyol (e.g., glycerol).

The attenuated vaccine is administered orally or parenterally, preferably orally, by inhalation, by spray, or intramuscularly or subcutaneously.

The vaccine of the present invention comprises an effective amount of an infectious bronchitis virus as an active ingredient, i.e., an amount that immunizes a vaccinated bird or an infectious bronchitis virus substance that will induce immunity in their offspring against an attack by a toxic virus (antigen administration) . Immunity is defined herein as inducing a significantly higher level of protection in a population of birds following vaccination compared to the non-vaccinated population.

Typically, the attenuated vaccine of the present invention can be administered in an amount equivalent to 10 ^2.5 -10 ^6.5 TCID50 per bird and equivalent antigen.

The infectious bronchitis virus vaccine of the present invention can be effectively used in chickens, but other poultry such as turkey, guinea fowl, and quail can also be effectively vaccinated. Chickens include edible chickens, replica livestock, and livestock.

The age of animals receiving a live or attenuated vaccine according to the invention is equal to the age of the animal on which the currently marketed attenuated infectious bronchitis virus vaccine is administered. Vaccination of a parent animal such as a feeder's chickens may be performed with a live attenuated or attenuated vaccine of the present invention, or a mixture of both. The advantages of this type of vaccination program include the direct protection of one day old offspring provided by maternally derived antibodies transmitted vertically to the offspring.

Typical breeder and laying hens vaccination programs include inoculating a live attenuated vaccine at day 1 post-calf axes followed by vaccination at 2, 4, 8, and 10-12 weeks of age.

A typical broiler vaccination program involves inoculating a live attenuated vaccine to the calf's first day of life and then inoculating the vaccine two weeks after birth.

The invention also encompasses vaccine combinations comprising one or more vaccine components of other pathogens that are infected with poultry in addition to the infectious bronchitis virus of the present invention.

Preferably the vaccine component in the vaccine combination is a live attenuated or attenuated form of the pathogen that infects the poultry.

In particular, the present invention provides vaccine combinations wherein all vaccine components of the vaccine combination are attenuated.

The combination of vaccines may include one or more of Newcastle disease virus, influenza virus, avian reovirus, infectious bursal disease virus (IBDV), and fowl adenovirus (FAdV) Lt; / RTI > vaccine strain).

As can be seen from the present invention, the infectious bronchitis vaccine strain K40 / 09 high passage of the present invention is superior to conventional vaccine strains in heat resistance and is one of the biggest disadvantages of the virulent vaccine, Side effects and lack of vaccine activity can be alleviated.

1 is a phylogenetic tree in which the S1 gene of the infectious bronchitis virus,
FIG. 2 is a graph showing the results of the test for contamination of other pathogens.

Hereinafter, the present invention will be described in more detail by way of non-limiting examples. The following examples are provided to illustrate the present invention and the scope of the present invention is not construed as being limited by the following examples.

Example  1: At a domestic outdoor farm Infectiosu Bronchitis K40 / 09 Primary isolation, identification and other pathogen fraud testing

In 2009, kidneys were taken from broiler chickens commissioned from broiler farms in Chungbuk Province, and sterilized phosphate buffer solution (pH 7.2) was added to make a 10% by weight emulsion. This was centrifuged at 3,000 g for 15 minutes to precipitate cells and tissues, and the supernatant was filtered with a 0.45 μm syringe filter. Strain IBV K40 / 09 was finally identified by reverse transcription-polymerase chain reaction (RT-PCR) using an Allantoic Cavity (AC) inoculation method at 11 days of SPF development.

Example  2: New IBV Outdoor separator  My other pathogen fraud

(AIV) and Newcastle Disease Virus (NDV) to determine the presence or absence of other pathogenic agents capable of proliferating in the metaplasias of the fetal canine. PCR) was performed to confirm that there were no other pathogens other than the infectious bronchitis virus isolate (K40 / 09 strain).

Example  3: Through the heat treatment process, Attenuation

In order to use the isolated IBV K40 / 09 strain as a raw monospecific newborn strain, the virulence of the virus was attenuated by continuous passage into 10 - day - old SPF embryos. In a subculture of subcultivated subculture for attenuation, as described by Jackwood et al. (1993), a temperature of 56 ° C was used for the rapid heat-treatment of infectious bronchitis virus, Avian Pathology (June 2010) 39 (3), 227-233 The heat treatment was repeated until the virulence of the virus was weakened to secure the K40 / 09 high passive strain.

Assessment of attenuated viruses was carried out at a particle size of ₅₀ mu m per 1 day old SPF chickens, with IBV10 ^4.5 EID ₅₀ per dose. The evaluation of virus attenuation was performed by spraying the negative control with distilled water, the K40 / 09 low passage strain not subcultured, and the K40 / 09 high passage strain subcultured, respectively, and then comparing the mortality rate with the K40 / 09 high passage Caution The safety was confirmed. (Table 1)

Spray 2 Weeks / Total number (%) K40 / 09 Low Passage 4/20 (20%) K40 / 09 High Passage 0/20 (0%) Negative control group 0/20 (0%)

Table 1 summarizes the results of the safety tests for K40 / 09 high passages at 1 day old chick spray

In order to confirm the heat resistance of the confirmed K40 / 09 high passageway, the existing vaccine hosts IBV K2p172, IBV K40 / 09 Low Passage and IBV K40 / 09 High Passage were reacted at 56 ° C, The viability of the virus at 56 ° C was confirmed by inoculation in the developing field. As a result of the test, low survival rate was not viable after 120 minutes, whereas high survival rate was confirmed up to 450 minutes, and heat resistance of K40 / 09 high passage was confirmed. (Table 2)

virus 56 ℃ Heat treatment time (min) 30 60 120 180 ~ 450 K2
High Passage survival survival survival - - - K40 / 09
Low Passage survival survival survival - - - K40 / 09
High Passage survival survival survival survival survival survival

Table 2 compares the heat resistance of IBV K40 / 09

Example  4: Genome Sequence Analysis

The virus was inoculated into the 11-day-old SPF developmental field and RNA was extracted using 150 μl of the urea membrane solution harvested 5 days later. 10 pmol of deoxynucleotide triphosphates (dNTPs), 20 pmol of Superscript II reverse transcriptase (Invitrogen), 200 units of reverse primer (reverse primer, 5'-GTT TGT ATG TAC TCA TCT GTA AC- At 42 ° C for 60 minutes, and the reverse transcriptase was inactivated by treatment at 70 ° C for 15 minutes.

1 μl of cDNA solution obtained by diluting the obtained cDNA 4 times, 1 μl of 10 × PCR buffer, 0.2 μl of 2.5 mM dNTPs, and 5 μl of each primer (forward: 5 'TAG TGA CCC TTT TGT GTG CAC TAT- (Reverse: 5 'GTT TGT ATG TAC TCA TCT GTA AC-3'; SEQ ID NO: 4), 1 μl of Taq polymerase and 7.2 μl of DW were mixed. (Infection Genetics and Evolution 11 (2011) 678-685)

The PCR product was purified using the above reaction solution, and the base sequence was determined using an ABI3100 automatic base sequence analyzer. The nucleotide sequence of the protein of S1 was obtained and is shown in Sequence Listing 1.

Example  5: IBV K40 / 09 Hi  Passage Caution Defense  And crossing Defense  exam

IBV K40 / 09 High Passage In order to confirm the protection against IBV, IBV H120, IBV K2, and IBV K40 / IBV K40 / 09 Defense of KP91 strain (Korean II subgroup 1), QXIBV strain (Korean II subgroup 2), and K40 / 09 strain (Korean New Genetic Cluster 1), which are the domestic trendy renal viruses of the Low Passage State and IBV K40 / 09 High Passage .

As a test method, vaccine was inoculated into 3 week old SPF chickens by instillation of each vaccine strain with 10 ^3.5 EID ₅₀ and after 3 weeks, each of the attacked viruses was inoculated with 10 ^4.5 EID ₅₀ by inoculation via instillation. Five days after the inoculation, the test system was euthanized, and then the virus was re-isolated from the organ and kidney to evaluate the effect of the virus if the virus was not re-isolated.

As a result, when IBV K40 / 09 high passive strain was inoculated into 3 week old chickens by instillation, the defenses against QXIBV and KM91 strains were superior to those of the existing K2 high passive strain, It was judged that the defense against epidemic IBV was excellent. (Table 3)

vaccine Attack inoculation Re-isolation of virus inoculation Agency kidney system Strain Control group Vaccine group Control group Vaccine group IBV H120 Korean II subgroup 1 KM91 10/10 5/10 8/10 4/10 Korean New Genetic Cluster 1 K40 / 09 10/10 10/10 10/10 9/10 Korean II subgroup 2 QXIBV 10/10 8/10 9/10 8/10 IBV K2
High Passage Korean II subgroup 1 KM91 10/10 4/10 ^* 8/10 0/10 Korean New Genetic Cluster 1 K40 / 09 10/10 5/10 ^* 10/10 1/10 ^*** Korean II subgroup 2 QXIBV 10/10 3/8 ^** 8/10 0/8 ^*** IBV K40 / 09
High Passage Korean II subgroup 1 KM91 10/10 0/10 ^*** 9/10 0/10 ^*** Korean New Genetic Cluster 1 K40 / 09 10/10 0/10 ^*** 10/10 0/10 ^*** Korean II subgroup 2 QXIBV 10/10 1/10 ^*** 10/10 0/10 ^***

Table 3 shows the cross-immunogenicity of IBV K40 / 09 High Passage

^* P < 0.05 by Student's T test

^** P < 0.01 by Student's T test

^*** P <0.001 by Student's T test

Tissue and kidney tissues were collected at the time of re - separation, and the histological examination results (inflammation index) and the ciliary loss index of the organ 5 days after the inoculation were determined. The test results are shown in the following table. In summary, significant tissue lesion reduction and ciliolysis index were observed in comparison with the control group.

In addition, as shown in Table 2, in the case of the high passages, the heat resistance is maintained, and the pathogenicity is decreased when spraying at one day, which is superior to the low pass.

vaccine Attack inoculation Histological examination system Strain Top
Agency Central
Agency bottom
Agency kidney Control group Korean II subgroup 1 KM91 3.0 3.0 3.0 1.7 Korean New Genetic Cluster 1 K40 / 09 2.9 3.0 3.0 1.7 Korean II subgroup 2 QXIBV 3.0 3.0 2.8 1.2 IBV K40 / 09
High Passage Korean II subgroup 1 KM91 2.1 *** 1.3 *** 0.5 *** 2.2 Korean New Genetic Cluster 1 K40 / 09 2.7 1.4 *** 0.5 *** 2.2 Korean II subgroup 2 QXIBV 2.7 1.3 *** 0.9 *** 2.0 IBV K40 / 09
Low Passage Korean II subgroup 1 KM91 0.3 *** 0.0 *** 0.0 *** 2.0 Korean New Genetic Cluster 1 K40 / 09 0.6 *** 0.4 *** 0.0 *** 1.9 Korean II subgroup 2 QXIBV 0.7 *** 0.9 *** 0.7 *** 0.8 **

Table 4 shows the results of histologic examination of IBV K40 / 09 high passages. The lesion criteria (0 = normal, 1 = focal, 2 = multifocal, 3 = diffused)

vaccine Attack inoculation Ciliary loss index system Strain Top
Agency Central
Agency bottom
Agency Control group Korean II subgroup 1 KM91 4.0 4.0 4.0 Korean New Genetic Cluster 1 K40 / 09 4.0 4.0 4.0 Korean II subgroup 2 QXIBV 3.6 3.6 3.0 IBV K40 / 09
High Passage Korean II subgroup 1 KM91 1.8 *** 0.8 *** 0.5 *** Korean New Genetic Cluster 1 K40 / 09 1.4 *** 0.6 *** 0.3 *** Korean II subgroup 2 QXIBV 1.3 *** 0.9 *** 0.3 *** IBV K40 / 09
Low Passage Korean II subgroup 1 KM91 0.6 *** 0.3 *** 0.1 *** Korean New Genetic Cluster 1 K40 / 09 1.4 *** 0.8 *** 0.5 *** Korean II subgroup 2 QXIBV 1.5 *** 0.9 *** 0.4 ***

Table 5 shows the ciliary dysfunction index of the IBV K40 / 09 high passive lesion with a 75% cilia damaged, 3% <75% cilia damaged, )

Korea Biotechnology Research Institute KCTC12741BP 20150113

<110> Konkuk University Industrial Cooperation Corp <120> An Attenuated Infectious bronchitis virus and An Infectious          bronchitis vaccine using the same <130> HY150223 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1634 <212> DNA <213> IBV K40 / 09 high passage <400> 1 gtgaccattt tgtgtgcact atgtagtgca aatttgtttg attctggtaa taattatgtg 60 tactactacc aaagtgcttt tagaccgcca aatgggtggc acctacaagg aggtgcttat 120 gcagtagtta attccactaa ttatactaat aatgccggtt ctgcacatga gtgcactgtt 180 ggtgttatta aggatgttta taatcaaagt gcggcttcca tagctatgac agcacctctt 240 cagggtatgg cttggtctaa ggcacaattc tgtagtgcac actgtaactt ttctgaaatt 300 acagtttttg tcacacattg ttatagtagt ggtagggggt cttgtcctat aacaggcatg 360 attccacgtg atcatattcg tatttctgca atgaaaaatg gttctttatt ttataattta 420 acagttagcg tatctaaata ccctaatttt aaatcttttc aatgtgttaa caacttcaca 480 tctgtttatt taaatggtga tcttgttttt acttccaaca aaactactga tgttacgtca 540 gcaggtgtgt attttaaagc aggtggacct gtaaattata gtattatgaa agaatttaag 600 gttcttgctt actttgttaa tggtacagca caagatgtaa ttttgtgcga caattccccc 660 aagggtttgc tagcttgtca atataacact ggcaattttt cagatggctt ttatcctttt 720 actaatagta ctttagttag ggaaaagttc atcgtctatc gcgaaagtag tgttaatact 780 actctggcgt taactaattt cacttttact aatgtaagta atgcacagcc taatagtggt 840 ggtgttaata cttttcattt atatcaaaca caaacagctc agagtggtta ttataatttt 900 aatttatcat ttctgagtag ttttgtgtat aaagagtctg atttcatgta tgggtcttac 960 catccatcat gtaattttag accagaaact attaataatg gtttatggtt taactcactt 1020 tcaatttcgc ttgcttacgg accactccaa ggtgggtgta agcaatcggt ttttagtagt 1080 agagccactt gttgttatgc ttattcatat aatggtccaa gcgcttgtaa gggtgtttat 1140 gcaggtgagt tgcaacaaag ttttgaatgt ggattgttgg tttatgttac taagagcgat 1200 ggctctcgta tacaaacagc caccgaaccg ccagttataa ttcaacacaa ttataataat 1260 attactttaa atacttgtgt tgattataat atatatggca gaattggcca aggttttatt 1320 actaatgtaa ctgactcatc atctagttat aattatttag cagatgcagg gttggctatt 1380 ttagatacat caggtgccat agacatcttc gttgtacaag gtgaatatgg tcttaattat 1440 tacaaggtta atccctgtga agatgtaaac cagcagtttg tagtttctgg tggtaaatta 1500 gtaggtattc ttacctcaca taatgaaaca ggttctcagc ctcttgagaa tcaattctat 1560 attaaactca ctaaagagac acgtcgtttt agacgttcaa ctagtgaaag tgtaacaagc 1620 tgctcttatg taag 1634 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gtttgtatgt actcatctgt aac 23 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tagtgaccct tttgtgtgca ctat 24 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gtttgtatgt actcatctgt aac 23

Claims (13)

Deposited with accession number KCTC 12741BP, which attenuated the infectious bronchitis virus K40 / 09 strain with high passage, wherein said high passive attenuates infectious bronchitis virus K40 / 09 strain from 30 to less than 70 passages Infectious bronchitis virus characterized by the K40 / 09 CE50. The use according to claim 1, wherein said infectious bronchitis virus K40 / 09 is isolated from domestic poultry, infectious bronchitis virus K40 / 09 CE50. The infectious bronchitis virus according to claim 1, wherein said infectious bronchitis virus K40 / 09 CE50 comprises the nucleotide sequence of SEQ ID NO: 1, and expresses a gene encoding the S1 protein. A vaccine for a disease caused by an infectious bronchitis virus infection,
Deposited with accession number KCTC 12741BP, which attenuated the infectious bronchitis virus K40 / 09 strain with high passage, wherein said high passive attenuates infectious bronchitis virus K40 / 09 strain from 30 to less than 70 passages Infectious bronchitis virus K40 / 09 CE50, characterized by; And
Vaccine against diseases caused by infectious bronchitis virus infections, including a pharmaceutically acceptable carrier or diluent, capable of protecting against KM91 strain, QXIBV strain and K40 / 09 strain, which are pandemic strain viruses in Korea. delete 5. The vaccine of claim 4, wherein the vaccine further comprises an adjuvant. 5. The vaccine of claim 4, wherein the vaccine further comprises at least one vaccine component of another pathogenic organism infectious to the poultry. delete A method for producing a vaccine against a disease caused by an infectious bronchitis virus infection,
Deposited with accession number KCTC 12741BP, which attenuated the infectious bronchitis virus K40 / 09 strain with high passage, wherein said high passive attenuates infectious bronchitis virus K40 / 09 strain from 30 to less than 70 passages Wherein the method comprises the step of binding the infectious bronchitis virus K40 / 09 CE50 with a pharmaceutically acceptable carrier or diluent, characterized in that it has the ability to protect against K39, QXIBV and K40 / A method for producing a vaccine against a disease caused by an infectious bronchitis virus infection. delete a) inoculating an infectious substrate with an infectious bronchitis virus K40 / 09 strain;
b) propagating the infectious bronchitis virus;
c) collecting the infectious bronchitis virus-containing substance; And
and d) heat-treating the collected virus to confirm whether the pathogenicity of the virus is weakened. In the case where the virus is not weakened, the steps a) to d) are repeated until the virus is weakened.
Wherein the repetition is performed from 30 times to 70 times
Production method of infectious bronchitis virus K40 / 09 CE50 deposited with accession number KCTC 12741BP. 12. The method according to claim 11, wherein the substrate is embryonated egg K40 / 09 CE50. delete

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