KR100468037B1 - New infectious bronchitis viruses and vaccines for preventing infectious bronchitis using the same - Google Patents

Abstract

The present invention relates to a novel chicken infectious bronchitis virus (hereinafter referred to as "IBV") suitable as a vaccine for preventing chicken infectious bronchitis (hereinafter referred to as "IB") and an IBV vaccine using the same. More specifically, it relates to novel IBV field isolates with broad immunogenicity, to attenuated IBV attenuated strains, and to vaccines against IBV infection using them. The novel IBV outdoor isolate according to the present invention is for the production of deadly poisoned vaccine and the attenuated IBV attenuated strain is for the production of lively poisoned vaccine. It will greatly contribute to the prevention of chicken infectious bronchitis.

Description

Novel infectious bronchitis viruses and vaccines for preventing infectious bronchitis using the same

The present invention relates to a novel chicken infectious bronchitis virus (hereinafter referred to as "IBV") suitable as a vaccine for preventing chicken infectious bronchitis and an IBV vaccine using the same, and more particularly to a broad range of immunogenicity. The present invention relates to a novel IBV field isolate showing an attenuated strain, an attenuated IBV attenuated strain, and an IBV infection preventive vaccine using the same.

Infectious bronchitis (hereinafter referred to as "IB") is an acute infectious disease of chickens caused by IBV, and is one of the major productivity-lowering diseases that cause a lot of economic damage to the poultry industry as well as domestically. In Korea, chickens infected with IB generally cause three major symptoms. The first is a condition that causes respiratory symptoms such as a runny nose or cough due to a young chick, which sometimes causes secondary respiratory bacterial infections and causes mortality with high morbidity. The second is a condition that damages the spawning organs and lowers egg production and egg quality and egg quality. The third is a condition that causes severe damage to the kidneys, causing many deaths.

IBV is a virus belonging to the Coronaviridae coronavirus, and it has been experimentally confirmed that mutations occur due to recombination between different IBVs, and there is a possibility that new serotype IBVs will continue to emerge. In addition, it is estimated that there will be dozens of kinds including each of these serotypes, including mutated mutant viruses. In addition, since the cross-reaction between the serotypes and cross-immunity is not good, it is difficult to prevent the variant IBV.

There are two types of IBV serotypes that have been reported worldwide so far: Massachusetts type and variant IBV called 793B or 4/91. In addition, IBVs that are popular in countries around the world, including Korea, can be said to be a virus that is spread by country or region.

In the United States, Massachusetts-type IBs became popular since 1950, and Connecticut-type IBs became popular since 1957. Since then, a wide variety of serotypes of IB, including Arkansas, Florida, Delaware, and Georgia Mutant strains, have become popular. Currently in the United States, based on Massachusetts-type vaccines that show a wide range of defense against various serotypes, Connecticut and Arkansas-type vaccines that are popular in each region of the United States are being developed and used in parallel.

In the UK, eight serotypes of IBV, including UK6 / 82, have been isolated and reported. Before Massachusetts IB vaccine was identified in 1991, only the Massachusetts type IB vaccine was used, but now type 793 / B. IB vaccines are being used in parallel.

In Australia, a new IB epidemic prevails, which is quite different from the IB prevalent in the US and Western European countries. In other words, IBs designated as serotypes A (Vac1), B (Vic S), C, D, E, F, G, H, I, J, K, L, M, O, etc. are in epidemic, and double serum Two types of vaccines, Vac 1 and Vic S, derived from types A and B are used. Australian-based IBVs divide epidemic IBVs in Australia into two genetic groups, Group I (kidney type) and Group II (respiratory type), based on the virus's S1 genetic analysis.

In Korea, the first IB report was confirmed in 1986, and live and dead venom oil vaccines made with Massachusetts type IBV were used to prevent damage. At that time, damage caused by IB infection was mainly caused by respiratory symptoms and spawning damage accompanied by malformed eggs. Massachusetts type IB vaccination effectively prevented the damage caused by outdoor IB infection. However, despite the use of Massachusetts type IB vaccines nationwide since 1990, various cases of low laying losses have been reported in laying hens and broilers, and in broiler chicks, about 10% mortality and severe nephritis have been reported in broilers. The accompanying variant IB has occurred and damage continues to this day.

Meanwhile, Korean Patent Application No. 2000-0019033 discloses a Marek virus type 2 (MDV2) transfer vector containing the S1 gene of chicken infectious bronchitis virus (IBV), and a recombinant Marek virus transformed with the same, and a method of manufacturing the same. Is starting.

However, the patent is a basic technology for the development of the Marekvirus vector expressing the IBV S1 protein, but the immunogenicity of the IBV S1 protein expressed in the recombinant virus has been demonstrated. Many additional studies for commercialization, such as the review of local immunity in the figure, economic analysis with existing vaccines in the development of commercial vaccines, etc. remain a problem to be supported. In addition, the Marek virus expressing the IBV S1 protein has a disadvantage in that its overall immunogenicity is lower than that of the existing venom and live venom vaccines manufactured using the IB virus itself. Thus, in order to effectively prevent domestic variant IB, the existing IB vaccine production method This is excellent and more realistic.

IBV is easily present in the mutant type, and cross-protection between the mutant virus is not so good, it is required to use the vaccine strain to match the characteristics of the virus spread by country or region. Currently, the vaccine used in Korea uses vaccine isolates from the United States, Western Europe, etc., so it is considered to be limited to defend against various types of mutant IBVs that are popular in Korea. Therefore, there is an urgent need for the development of a vaccine suitable for the domestic situation.

In the present invention, as a result of research to develop an effective IB vaccine that can minimize the damage of the mutant infection that is currently in vogue in Korea and overcome and overcome the disadvantages of the existing IB vaccine, A novel IBV outdoor isolate for the production of deadly poisoned vaccines with broad immunogenicity against mutant IBV was selected, and its pathogenicity was attenuated by egg laying method to confirm safety and efficacy as a vaccine against live venom vaccine in host animals. The present invention has been completed based thereon.

Therefore, an object of the present invention is to provide an IBV outdoor isolate for the production of a prophylactic vaccine effective against various types of mutant IBV in Korea.

Another object of the present invention is to attenuate the pathogenicity of the IBV outdoor isolate, to provide an IBV apothecary for the production of an effective vaccine against IBV.

It is another object of the present invention to provide an effective preventive vaccine against IBV using the IBV outdoor isolate and attenuated strain.

It is another object of the present invention to provide a biological agent such as a prophylactic vaccine, diagnostic kit, etc., which is effective against IBV containing the S1 protein of IBV field isolate and attenuated strain or gene sequence thereof.

In order to achieve the above object, the novel IBV outdoor isolate of the present invention is an infectious bronchitis virus (IBV) K2 low passage (IBV), which is a variant IBV outdoor isolate causing kidney lesions showing extensive immunogenicity against IBV. passage) (KCTC 10105BP).

Infectious bronchitis virus of the present invention to attenuate the chicken infectious bronchitis virus (IBV) K2 low passage (KCTC 10105BP) to achieve another object of the present invention: IBV) K2 high passage (KCTC 10106BP).

Vaccine of the present invention for achieving another object of the present invention is a deadly poisoned vaccine that inactivated the IBV K2 low passage, chicken infectious bronchitis virus attenuated the IBV K2 low passage (Infectious bronchitis) virus: IBV) K2 high passage live vaccine, and a biologic including a prophylactic vaccine using the genes and proteins of the virus.

Figure 1 is an electrophoresis picture of the genotype of the novel IBV outdoor isolate according to the present invention by IBV gene classification method using RT-PCR method.

Figure 2 is a phylogenetic diagram based on the novel IBV virus S1 nucleotide sequence analysis data according to the present invention.

Looking at the present invention in more detail as follows.

As mentioned above, the serotypes of IBVs that are popular all over the world are estimated to be dozens or more, and when the serotypes are different, most of the cases are not recognized cross-protective ability. However, among the IBVs that are prevalent in the open air, it is reported that one type of serotype is not perfect but exhibits significant cross-protection against IBV of several other serotypes. It is mainly used for the development of IB vaccines. Therefore, in recent years, when a new type of IBV is isolated, a genotype or genogroup investigation, or whether an existing IB vaccine can protect against a new type of IBV, rather than attempting to classify serotypes, may be necessary. IBV classification is preferred as a method of determining protectotype.

1. Selection and Identification of New IBV Outdoor Separators

In the present invention, in order to select a novel IBV strain exhibiting a wide range of immunogenicity and cross-protective ability, the genotype and genetic group of strains mainly distributed in Korea were first identified through a genetic classification method using RT-PCR (see FIG. 1). ).

Since 1986, when IB first occurred in Korea, 60 genotypes of IBV isolates isolated from Korea were examined by genotyping of the virus by IBV gene classification using RT-PCR. The genetic group of the virus was examined based on the S1 protein gene analysis data of domestically popular IBV species (see Figure 2).

The IBV S1 protein contains neutralizing epitopes and serotype-specific epitopes, and is known to play an important role in determining the type of virus, such as its neutralization capacity and its serotypes. It is commonly used in protein production research. (Vol., Avian Diseases, 37, 194-202, 1993; Songdeung, Journal of General Virology, 79, 719-723, 1998)

In the present invention, the selection of IBV showing a wide range of immunogenicity among the major domestic genotypes was made by examining the cross-protection ability with IBV representing six domestic genotypes, not all kinds of IBV isolates show a broad cross-immunogenicity In the present invention, the IBV K2 low passage selected as a vaccine for the production of deadly vaccines is classified as a novel virus, which shows IBV showing a wide range of immunogenicity against IBV representing the six domestic genotypes shown in FIG. It was confirmed by the present invention. Extensive immunogenicity of domestically mutated IBV was confirmed by inoculating a novel IBV field isolate according to the present invention into a specific pathogen-free (SPF) chick and attacking with a virus representative of various types of IBV genotypes isolated in Korea. It was confirmed by examining the defense ability afterwards. The selected IBV for the killed vaccine is thus named IBV K2 low passage.

2. IBV K2 low passage isolation and culture method

Separation of IBV field isolates was performed after aseptic sampling of organs or kidneys of suspected IB infection, 20% emulsion with sterile PBS, centrifugation at 3,000 rpm for 20 minutes, and supernatant taken to infiltrate the 10-day-old SPF oviduct. After inoculation and incubation at 37 ° C. for 72 hours, the intestinal ureum fluid is collected aseptically. The collected intestinal urea fluid was determined by the method of Song et al. (Avian Diseases, 42, 92-100, 1998) to determine the presence of IBV in the intestinal ureum fluid, and the method of song and others (Avian Pathology, 27, 409-416, 1998). ), The genotype of IBV is determined.

3. Preparation of deadly vaccine vaccine using IBV K2 low passage

The inactivation of the IBV K2 low passage was treated with intestinal urea solution containing IBV cultured in SPF oocytes by adding 0.2% formalin for 15 hours at 37 ° C. and then inactivating at 4 ° C. for 1 week. Alternatively, 0.01M BEI (Binary ethylenimine) was treated in the intestinal urea solution for 3 hours at 37 ° C or 0.2% B-PL (Beta-propiolacton) in the intestinal urea solution for 3 hours at 25 ° C.

4. Passage and incubation method of IBV K2 high passage

The virus was attenuated by virtue of serial passage in 10-day-old SPF fetuses for use as live virulent strains of the IBV field isolates selected as the vaccine for the preparation of the deadly-sinked vaccine.

10 days old SPF eggs are inoculated with IBV K2 low passages into the intestinal lumen and incubated at 37 ° C. for 30 hours, followed by aseptic collection of the intestinal sera. The collected intestinal urea fluid is inoculated again into the intestinal urea for 10 days of age SPF eggs and incubated for 30 hours at 37 ℃ and aseptic collection of urea. This process is repeated until the virus's original pathogenicity is attenuated.

The novel IBV attenuated strains according to the present invention are fully attenuated in pathogenicity to the respiratory and renal kidneys of the mother strain, and have a wide range of immunogenicity against IBV representative of two domestic genetic groups (respiratory type and renal type). It was confirmed by the present invention that the IBV. IBV for the production of safe live vaccines that have been attenuated in this way is termed IBV K2 high passage, which is almost identical to the parental strain before attenuation in several aspects, including morphological and viral properties, IBV K2 high passages, which are attenuated for the production of live vaccines, are also classified as novel viruses because they do not show new properties, but rather protect against IBV infection.

5. Preparation of live vaccines using IBV K2 high passage

The IBV K2 high passages were multiplied in 10 day-old fetuses to prepare vaccines.

The degree of attenuation was determined by pathogenicity in 1 day-old SPF chicks in Table 3 and organ-specific tissue affinity and tissue lesions in 1-day-old SPF chicks in Tables 4 and 5 below. The IBV K2 high passage did not kill the chicks at all when inoculated into a 1-day-old SPF chick, and the proliferation rate in tissues of the organs, kidneys, cecum tonsils, lungs, and bursas of the chicks was increased. Significantly lower than In addition, histopathological lesions on organs and kidneys were reduced to the same level as the existing Massachusetts type IB live vaccine.

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 (eg, sorbitol, mannitol, starch, sucrose, dextran, glutamate, Or glucose), protein (eg, dried milk serum, albumin or casein) or degradation products thereof. Suitable buffers are, for example, alkali metal phosphates. Suitable preservatives are thimerosal, methiolate, gentamicin. The diluents are water, aqueous buffers (eg buffered saline), alcohols and polyols (eg glycerol).

Figure 1 is a picture showing the 6 IBV genotypes of the domestic and representative foreign strains, the novel IBV screened in the present invention in the venom and live vaccine vaccine is selected from the renal IBV belonging to genotype III genotype III genotype of six popular domestic genotypes It can be called an IBV. Therefore, the novel IBV according to the present invention shows the same RFLP pattern as the genotype III of FIG. 1 when the genotype of the virus is classified by the IBV gene classification technique using the RT-PCR method. It was confirmed in the present invention that it can be used as an easy feature for differentiation from the vaccine strain.

Based on the S1 protein sequencing data of the virus, the novel IBV field isolates selected from the present invention are shown in FIG. 2, as shown in FIG. 2, the Korean 1 group (respiratory type genetic group) and the Korean 2 group (renal genetic code). K2 / 01 (K2 low passage), a renal IBV belonging to the Korean 2 group of Korean IBV genetic groups divided into two genetic groups, and the like. It can be confirmed that the IB genotype.

These IBVs were deposited in the Korean Collection for Type Cultures (KCTC), an international depository institution, on November 27, 2001.The IBV K2 low passages are KCTC 10105BP, and the IBV K2 high passages. Has been assigned the accession number of KCTC 10106BP.

The IBV K2 low passage and IBV K2 high passage are good proliferative and capable of mass proliferation in 10-day-old fetuses, and are suitable for vaccine production, and RFLP gene using IBV S1 gene amplification. The virus can be distinguished from the existing vaccine virus due to the phylogenetic characteristics of the virus using the classification technique and IBV S1 gene sequence analysis data.

Meanwhile, as described above, since the S1 protein of chicken infectious bronchitis virus includes a neutralizing epitope of the virus and a serotype-specific epitope, a recombinant vector vaccine containing the S1 protein of the novel IB virus or a base sequence of the gene thereof is also infected with IBV. It is expected to be effective for prevention. In addition, the S1 protein of the IB virus or a nucleotide sequence of the gene thereof may be applied to a diagnostic kit and used to determine whether the IBV infection is present.

Hereinafter, the present invention will be described in more detail with reference to the following Examples, but the scope of the present invention is not limited to the following Examples.

Example 1: Genotype and Pathogenicity Study of Domestic Trendy IBV

In order to investigate the genotype of the domestic IBV, two primers, BES1F1 and BES1R1, which specifically react the IBV S1 gene to IBV, were used to amplify a gene fragment corresponding to about 1.7 kb. The genotype of the virus was confirmed by examining the pattern of restriction fragment length polymorphism (RFLP), which is developed by electrophoresis on 1% agarose gel after treatment with Hae III. Gene sequencing, RT-PCR, and restriction enzyme analysis conditions of primers used in RT-PCR were performed according to the method of Song et al. (1998) (Avian Pathology, 27, 409-416, 1998).

As shown in FIG. 1, genotypic analysis of domestic IBV was performed in Korea. From 1986 to now, 60 genotypes of IBV isolates isolated from Korea were attempted to classify genotypes of viruses by IBV gene classification using RT-PCR. Results Sixty genotypes of IBV collected in Korea over the past 15 years were classified into six genotypes, genotypes I, II, III, IV, V, and VI. Among the six genotypes, genotype III was found to be the genotype III of the 60 isolates that attempted genotyping. Since 1990, it has been continuously separated from domestic outdoor poultry farms. It is considered a genotype.

In addition, IBVs belonging to the genotype III, the main genotype among these six genotypes, were identified as renal IBV, which causes not only severe uric acid deposition but also mortality of up to 50% when inoculated into a specific pathogen-free (SPF) 1-day-old chick. On the other hand, IBVs belonging to five other genotypes have been identified as respiratory type IBVs that cause only respiratory symptoms in 1-day-old chicks. IBV isolates were examined by topical inoculation of ₅₀ IBV 10 ^4.5 EID (egg infectious dose) per chick, except for the control group, with respect to the 1-day-old SPF chicks of major IBVs representing the six genotypes of IBV in Korea. This is shown in Table 1 below.

Pathogenicity of 1-day-old SPF Chicks of IBV Domestic Isolates Representing Six IBV Genotypes Classified by RFLP Method Genotype IBV Isolation Inoculation number % Mortality Respiratory symptoms Nephritis / Uric Acid Deposition I M41 10 0 +++ + I RB86 10 0 +++ - II K12 / 01 10 0 +++ - II B4 10 0 +++ - III K2 / 01 10 30 ++ ++ III K91 / 01 10 30 ++ +++ III K604 / 97 10 50 ++ +++ III KM91 10 50 ++ +++ IV EY95 10 0 +++ - V EJ95 10 0 +++ - VI K71 / 01 10 0 +++ - VI K620 / 97 10 0 +++ - Control 10 0 - -

1) Respiratory symptoms: +++ (water respiratory, accompanied by shortness of breath), ++ (more than 30% respiratory, no shortness of breath),-(less than 30% respiratory)

2) Nephritis / Uric acid Deposition: Only dead chicks cause symptoms, +++ Nephritis is severe, ++ Nephritis is slight,-Nephritis is mild

The novel IBV outdoor isolate according to the present invention can be found to belong to genotype III, which is a domestic genotype genotype by the RFLP method (see FIG. 1). It can be found in the kidney type IBV.

Example 2: Phylogenetic Characterization of Domestic Trendy IBV

The IBV S1 gene fragment corresponding to about 1.7 kb obtained in Example 1 was examined using the ABI 377 gene sequencing device, and the gene sequence was examined. Phylogenetic correlations with IBV separated by region were analyzed (see FIG. 2). The novel IBV S1 gene sequences of the present invention analyzed above are shown in SEQ ID NO: 1 for IBV K2 low passage and SEQ ID NO: 2 for IBV K2 high passage. For the phylogenetic correlation analysis of the virus, 38 IBV S1 gene sequencing data were used, including 18 strains of IBV isolates and 20 strains of IBV isolates (Wang et al ., Virus Research, 49, 139-145, 1994). It was.

Phylogenetic correlation of domestic IBV is shown in Figure 2, 18 domestically isolated IBV from 1986 to present based on the S1 protein gene sequencing data of the virus and Korean 1 and It was divided into two genetic groups, including two groups, and identified as a new IBV genetic group that is completely different from the viruses isolated from other regions outside Korea.

In addition, among the six domestic genotypes identified by the RFLP method in Example 1, 11 renal IBVs belonging to genotype III, the major genotypes, were classified as belonging to the Korean 2 group, and 7 belonging to the other 5 genotypes. Respiratory type IBVs of the species are all classified as Korean 1 except RB86 strain (Massachusetts group: Mass group). Thus, domestic epidemic IBVs are respiratory type IBV based on the S1 protein gene sequence analysis of the virus. The Korean 1 group (Respiratory Genetic Group), which includes the Korean and the Korean 2 Group (Kidney Genetic Group), which contains renal IBVs, were identified as two broad genetic groups.

The novel IBV field isolate according to the present invention can be confirmed to belong to Korean 2 group (kidney genetic group) based on the S1 protein sequencing data of the virus.

Test Example 1: Immunogenicity of Domestic Mutant IBV of IBV K2 Low Passage for Preparation of Poison Vaccine

An IBV K2 low passage selected for the production of a deadly vaccine was vaccinated in 3 drops of IBV 10 ^3.0 EID ₅₀ per chick, except for the control group, to the 3 week old SPF chicken as a host animal, and Example 3 three weeks after vaccination. Six types of IBVs, which are representative of major domestic IBV genetic groups identified in and 2, were challenged by inoculation with IBV 10 ^4.5 EID ₅₀ per chick, and the protective immune effects were investigated. The protective immunity effect was evaluated as having a protective effect if the virus was not re-isolated by re-isolating the challenge virus from the kidney and organ 5 days after challenge. In addition, the inoculation may be a conventional administration method such as drinking water, spray inoculation. The final immunogenicity test results are shown in Table 2 below.

Protective Effect of the IBV K2 Low Passage Inoculation System against IBV Domestic Isolation of the IBV Genetic Isolates Vaccination Ivb Attack IBV Genetic Group Attack IBV Genotype Attack Vaccine IBV Re-Isolation of Vaccination Virus / Vaccination kidney Agency Control Vaccine group Control Vaccine group IBV K2 low passage Mass I M41 2/10 0/10 10/10 2/10 Korean 1 II K12 / 01 2/10 0/10 10/10 1/10 Korean 1 VI K71 / 01 0/10 1/10 9/10 0/10 Korean 2 III K2 / 01 10/10 1/10 10/10 1/10 Korean 2 III K90 / 01 10/10 1/10 10/10 0/10 Korean 2 III KM91 10/10 0/10 10/10 0/10

Test Example 2: Pathogenicity of IBV K2 high passage for preparing attenuated live vaccine

The virus-induced pathogenicity was attenuated by successive passages of 70 or more passages in 10-day-old SPF embryos to use the IBV K2 low passage selected for the production of a deadly vaccine. To test the safety of 1-day-old chicks of IBV K2 high passage for the production of attenuated live vaccines, IBV attenuated K2 high passages were administered to 1-day-old SPF chicks with IBV 10 per chick except for the control group. ^{4.5 The} clinical symptoms and mortality were assessed for 2 weeks after challenge with inoculation by EID ₅₀ . In addition, the inoculation may be a conventional administration method such as drinking water, spray inoculation. The results are shown in Table 3 below.

Pathogenicity for 1-day-old SPF chicks with attenuated IBV K2 high passage virus Inoculation number % Mortality Respiratory symptoms Nephritis / Uric Acid Deposition K2 low passage 10 30 ++ ++ K2 high passage 10 0 - - Control 10 0 - -

1) Respiratory symptoms: ++ (more than 30% respiratory, no difficulty breathing),-(less than 30% respiratory)

2) Nephritis / Uric acid Deposition: Only dead chicks cause symptoms, ++ some nephritis,-mild nephritis

Test Example 3: Tissue affinity and histopathology of IBV K2 high passage for attenuated live venom vaccine

Tissue affinity and histopathology were examined for 1 day old chicks of IBV K2 high passage for attenuated live vaccine. Tissue affinity of each organ of the chicks 1 week and 2 weeks after the inoculation of the IBV attenuated strain K2 high passage (IbV 10 ^4.5 EID ₅₀ per chicken chick) to the day-to-day SPF chicks except the control group Tissue lesions were examined separately. In addition, the inoculation may be a conventional administration method such as drinking water, spray inoculation. The results are shown in Tables 4 and 5, respectively.

Tissue affinity when inoculated into 1 day old SPF chicks of attenuated IBV K2 high passage virus Virus Separation / Vaccination Agency kidney Cecum one-way lungs Bursa K2 low passage 10/10 8/10 8/10 9/10 10/10 K2 high passage 8/10 0/10 1/10 3/10 0/10 K71 / 01 6/10 0/10 1/10 0/10 1/10 Control 0/10 0/10 0/10 0/10 0/10

1) Virus isolation: 1 week after challenge vaccination virus re-separation / vaccination

Long-term histopathologic lesions of 1-day-old SPF chicks with attenuated IBV K2 high passage virus Tracheal lesion Kidney lesions Island Epithelial cell proliferation Epithelial cell necrosis Exudate Congestion / bleeding Epithelial cell necrosis Tubules Interstitial nephritis play K2 low passage 2.2 1.4 1.8 0.6 0.6 0.8 0 2.4 1.8 K2 high passage 1.2 0.6 0.8 0.2 0.8 0.6 0 0.4 0.6 H120 1.6 0 0.6 0.2 0.4 0.4 0 1.0 0 Ma5 1.6 0.8 0.8 0 0.2 0.6 0 1.2 0.8 Control 0.6 0 0 0 0.4 0.6 0 0.8 0.4

Tracheal / renal lesions: 0-normal, 1-focal, 2-extensively focal, 3-multifocal, 4-diffuse

2) H120, Ma5-Massachusetts type IB live vaccine

Test Example 4: Immunogenicity of IBV K2 high passage for preparing attenuated live vaccine

IBV K2 high passages selected for the production of live vaccines were vaccinated to 3 week old SPF chickens as host animals by instillation of IBV 10 ^3.0 EID ₅₀ per chick, except for the control group, and 3 weeks after vaccination. Mutant IBV representing two domestic genetic groups (Respiratory and Renal Genetic Groups) classified based on the S1 protein gene sequencing data of the virus at 3 weeks after vaccination. We investigated the effect of defensive immunity after inoculation by IBV 10 ^4.5 EID ₅₀ per allowance. The protective immune effect was measured by re-separation of challenge virus from kidney and organ 5 days after challenge. In addition, the inoculation may be a conventional administration method such as drinking water, spray inoculation. The results are shown in Table 6 below.

Protective Effect of H120 and IBV K2 High Passage Vaccination System against IBV Isolation of Korean Isolates Vaccination Ivb Attack IBV Genetic Group Attack IBV Genotype Attack Vaccine IBV Re-Isolation of Vaccination Virus / Vaccination kidney Agency Control Vaccine group Control Vaccine group H120 Korean 1 VI K71 / 01 1/10 0/10 9/10 5/10 Korean 2 III K2 / 01 (K2 low passage) 10/10 7/10 10/10 3/10 IBV K2 high passage Korean 1 VI K71 / 01 0/10 1/10 9/10 0/10 Korean 2 III K2 / 01 (K2 low passage) 10/10 1/10 10/10 1/10

Chicken infectious bronchitis virus according to the present invention as a vaccine for the effective prevention of domestic variant IB supplementing the shortcomings of the existing vaccine using the isolated IBV as a vaccine strain, will greatly contribute to the prevention of domestic chicken infectious bronchitis.

<110> KONKUK UNIVERSITY <120> New infectious bronchitis viruses and vaccines for preventing infectious bronchitis using the same <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 1611 <212> DNA <213> S1 gene of the Infectious Bronchitis Virus K2 low passage <400> 1 atgttgggga agttactgtt tttagtgacc attttgtgtg cactatgtag tgcaaatttg 60 gataataatt atgtgtacta ctaccaaagt gcttttagac ctccaaatgg ttggcactta 120 caagggggtg cttatgcagt agtgaattct actaatcata ctaataatgc cggaggtgca 180 agtgagtgca ctgttggtgt tattaaggat gtttataatc aaagtgcggc ttctatagct 240 atgacagcac ctcttcaggg tatggcttgg tctaagtcac aattttgtag tgcacactgt 300 aacttttctg aaattacagt ctttgtcaca cattgttata gtagcggtat ggggtcttgt 360 cctataacag gcttgattcc acagaatcat attcgttttt ccgcaatgaa aaatggttct 420 ttattttata atttaacagt tagcgtatct aaatacccta attttaaatc ttttcaatgt 480 gttaacaact tcacatctgt ttatttaaat ggtgatcttg tttttacttc caacttaact 540 actgatg cgtcagcagg tgtgtatttt aaagcaggtg gacctgtaac ttataatatt 600 atgaaagaat ttaaggttct tgcttatttt gttaatggta ctgtgcaaga tgtaattctg 660 tgtgatgaca caccgagagg cttgctagca tgtcaatata atactggcaa tttctcagat 720 ggtttttatc ctttcactaa tagtagttta gttaagcaaa ggtttattgt ttatcgtgag 780 agtagtgtta atactactct tactttaacc aatttcactt ttcataatga aactaatgcc 840 tcgcctaatt caggtggtgt caattccatt tcaacttatc aaacacaaac agctcagagt 900 ggttattata attttaattt atcatttctg agtagttttg tgtataaaga gtctaatttc 960 atgtatgggt cttaccatcc atcatgtaat tttagactag aaactattaa taatgattta 1020 tggtttaatt cactttcaat ttcgcttgct tacggaccac ttcaaggtgg gtgtaagcgg 1080 tcggttttta gtagtaaagc cacttgttgt tatgcttatt catataatgg tccacgcgct 1140 tgtaagggtc tttacgcaag tgatttgagt caaagttttg aatgtggatt gttggtttat 1200 gttactaaga gcgatggctc tcgtatacaa acagccaccg aaccaccagt tataactcaa 1260 cacaattata ataatattac tttaaatact tgtgttgatt ataatatata tggcagaatt 1320 ggccgaggtt ttattactaa tgtaactgac tcatcttcta ggtataatta tttagcagat 1380 gcagggttgg ctattttaga tacatcaggt gccatagaca tcttcgttgt acaaagtgaa 1440 tatggtctta attattacga ggttaatccc tgtgaagatg taaaccagca gtttgtagtt 1500 tctggtggta aattagtagg tattcttacc tcacgtaatg aaacaggttc tcagcctctt 1560 gagaatcaat tctatattaa actcactaaa gggacacgtc gttttagacg t 1611 <210> 2 <211> 537 <212> PRT <213> S1 protein of the Infectious Bronchitis Virus K2 low passage <400> 2 Met Leu Gly Lys Leu Leu Phe Leu Val Thr Ile Leu Cys Ala Leu Cys 1 5 10 15 Ser Ala Asn Leu Asp Asn Asn Tyr Val Tyr Tyr Tyr Gln Ser Ala Phe 20 25 30 Arg Pro Pro Asn Gly Trp His Leu Gln Gly Gly Ala Tyr Ala Val Val 35 40 45 Asn Ser Thr Asn His Thr Asn Asn Ala Gly Gly Ala Ser Glu Cys Thr 50 55 60 Val Gly Val Ile Lys Asp Val Tyr Asn Gln Ser Ala Ala Ser Ile Ala 65 70 75 80 Met Thr Ala Pro Leu Gln Gly Met Ala Trp Ser Lys Ser Gln Phe Cys 85 90 95 Ser Ala His Cys Asn Phe Ser Glu Ile Thr Val Phe Val Thr His Cys 100 105 110 Tyr Ser Ser Gly Met Gly Ser Cys Pro Ile Thr Gly Leu Ile Pro Gln 115 120 125 Asn His Ile Arg Phe Ser Ala Met Lys Asn Gly Ser Leu Phe Tyr Asn 130 135 140 Leu Thr Val Ser Val Ser Lys Tyr Pro Asn Phe Lys Ser Phe Gln Cys 145 150 155 160 Val Asn Asn Phe Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Phe Thr 165 170 175 Ser Asn Lys Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys Ala 180 185 190 Gly Gly Pro Val Thr Tyr Asn Ile Met Lys Glu Phe Lys Val Leu Ala 195 200 205 Tyr Phe Val Asn Gly Thr Val Gln Asp Val Ile Leu Cys Asp Asp Thr 210 215 220 Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser Asp 225 230 235 240 Gly Phe Tyr Pro Phe Thr Asn Ser Ser Leu Val Lys Gln Arg Phe Ile 245 250 255 Val Tyr Arg Glu Ser Ser Val Asn Thr Thr Leu Thr Leu Thr Asn Phe 260 265 270 Thr Phe His Asn Glu Thr Asn Ala Ser Pro Asn Ser Gly Gly Val Asn 275 280 285 Ser Ile Ser Thr Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr Tyr Asn 290 295 300 Phe Asn Leu Ser Phe Leu Ser Ser Phe Val Tyr Lys Glu Ser Asn Phe 305 310 315 320 Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu Thr Ile 325 330 335 Asn Asn Asp Leu Trp Phe Asn Ser Leu Ser Ile Ser Leu Ala Tyr Gly 340 345 350 Pro Leu Gln Gly Gly Cys Lys Arg Ser Val Phe Ser Ser Lys Ala Thr 355 360 365 Cys Cys Tyr Ala Tyr Ser Tyr Asn Gly Pro Arg Ala Cys Lys Gly Leu 370 375 380 Tyr Ala Ser Asp Leu Ser Gln Ser Phe Glu Cys Gly Leu Leu Val Tyr 385 390 395 400 Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Ala Thr Glu Pro Pro 405 410 415 Val Ile Thr Gln His Asn Tyr Asn Asn Ile Thr Leu Asn Thr Cys Val 420 425 430 Asp Tyr Asn Ile Tyr Gly Arg Ile Gly Arg Gly Phe Ile Thr Asn Val 435 440 445 Thr Asp Ser Ser Ser Arg Tyr Asn Tyr Leu Ala Asp Ala Gly Leu Ala 450 455 460 Ile Le u Asp Thr Ser Gly Ala Ile Asp Ile Phe Val Val Gln Ser Glu 465 470 475 480 Tyr Gly Leu Asn Tyr Tyr Glu Val Asn Pro Cys Glu Asp Val Asn Gln 485 490 495 Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg 500 505 510 Asn Glu Thr Gly Ser Gln Pro Leu Glu Asn Gln Phe Tyr Ile Lys Leu 515 520 525 Thr Lys Gly Thr Arg Arg Phe Arg Arg 530 535 <210> 3 <211> 1611 <212> DNA < 213> S1 gene of the Infectious Bronchitis Virus K2 high passage <400> 3 atgttgggga agttactgtt tttagtgacc actttgtgtg cactatgtag tgcaaatttg 60 gataataatt atgtgtacta ctaccaaagt gcttttagac ctccaaatgg ttggcactta 120 caagggggtg cttatgcagt agtgaattct actaatcata ctaataatgc cggaggtgca 180 agtgagtgca ctgttggtgt tattaaggat gtttataatc aaagtgcggc ttctatagct 240 atgacagcac ctcttc aggg tatggcttgg tctaagtcac aattttgtag tgcacactgt 300 aacttttctg aaattacagt ctttgtcaca cattgttata gtagcggtgt ggggtcttgt 360 cctataacag gcttgattcc acagaattat attcgtattt ccgcaatgaa aaatggttct 420 ttattttata atttaacagt tagcgtatcg aaatacccta attttaaatc ttttcaatgt 480 gttaacaact tcacatctgt ttatttaaat ggtgatcttg tttttacttc caacaaaact 540 actgatgtta cgtcagcagg tgtgtatttt aaagcaggtg gacctgtaac ttataatatt 600 atgaaagaat ttaaggttct tgcttatttt gttaatggta ctgtgcaaga tgtaattctg 660 tgtgatgaca caccgagagg cttgctagca tgtcaatata atactggcaa tttctcagat 720 ggtttttatc ctttcactaa tagtagttta gttaagcaaa ggtttattgt ttatcgtgag 780 agtagtgtta atactactct tactttaacc aatttcactt ttcataatga aactaatgcc 840 tcgcctaatt caggtggtgt caattccatt tcaacttatc aaacacaaac agctccgagt 900 ggttattata attttaattt atcatttctg agtagttttg tgtataaaga gtctaatttc 960 atgtatgggt cttaccatcc atcatgtaat tttagactag aaactattaa taatgattta 1020 tggtttaatt cactt tcaat ttcgcttgct tacggaccac ttcaaggtgg gtgtaagcag 1080 tcggttttta gtagtaaagc cacttgttgt tatgcttatt catataatgg tccacgcgct 1140 tgtaagggtg tttacgcaag tgagttgagt caaagttttg aatgtggatt gttggtttat 1200 gttactaaga gcgatggctc tcgtatacaa acagccaccg aaccaccagt tataactcaa 1260 cacaattata ataatattac tttaaatact tgtgttgatt ataacatata tggcagaatt 1320 ggccgaggtt ttattactaa tgtaactgac tcatcatcta ggtataatta tttagcagat 1380 gcagggttgg ctattttaga tacatcaggt gccatagaca tcttcgttgt acaaagtgaa 1440 tatggtctta attattacga ggtaaatccc tgtgaagatg taaaccagca gtttgtagtt 1500 tctggtggta aattagtagg tattcttacc tcacgtaatg aaacaggttc tcagcctctt 1560 gagaatcaat tctatattaa actcactaaa gagacacgtc gttttagacg t 1611 <210> 41211 Gly Lys Leu Leu Phe Leu Val Thr Thr Leu Cys Ala Leu Cys 1 5 10 15 Se r Ala Asn Leu Asp Asn Asn Tyr Val Tyr Tyr Tyr Gln Ser Ala Phe 20 25 30 Arg Pro Pro Asn Gly Trp His Leu Gln Gly Gly Ala Tyr Ala Val Val 35 40 45 Asn Ser Thr Asn His Thr Asn Asn Ala Gly Ala Ser Glu Cys Thr 50 55 60 Val Gly Val Ile Lys Asp Val Tyr Asn Gln Ser Ala Ala Ser Ile Ala 65 70 75 80 Met Thr Ala Pro Leu Gln Gly Met Ala Trp Ser Lys Ser Gln Phe Cys 85 90 95 Ser Ala His Cys Asn Phe Ser Glu Ile Thr Val Phe Val Thr His Cys 100 105 110 Tyr Ser Ser Gly Val Gly Ser Cys Pro Ile Thr Gly Leu Ile Pro Gln 115 120 125 Asn Tyr Ile Arg Ile Ser Ala Met Lys Asn Gly Ser Leu Phe Tyr Asn 130 135 140 Leu Thr Val Ser Val Ser Lys Tyr Pro Asn Phe Ly s Ser Phe Gln Cys 145 150 155 160 Val Asn Asn Phe Thr Ser Val Tyr Leu Asn Gly Asp Leu Val Phe Thr 165 170 175 Ser Asn Lys Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe Lys Ala 180 185 190 Gly Gly Pro Val Thr Tyr Asn Ile Met Lys Glu Phe Lys Val Leu Ala 195 200 205 Tyr Phe Val Asn Gly Thr Val Gln Asp Val Ile Leu Cys Asp Asp Thr 210 215 220 Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe Ser Asp 225 230 235 240 Gly Phe Tyr Pro Phe Thr Asn Ser Ser Leu Val Lys Gln Arg Phe Ile 245 250 255 Val Tyr Arg Glu Ser Ser Val Asn Thr Thr Leu Thr Leu Thr Asn Phe 260 265 270 Thr Phe His Asn Glu Thr Asn Ala Ser Pro Asn Ser Gly Gly Val Asn 275 280 285 Ser Ile Ser Thr Tyr Gln Thr Gln Thr Ala Pro Ser Gly Tyr Tyr Asn 290 295 300 Phe Asn Leu Ser Phe Leu Ser Ser Phe Val Tyr Lys Glu Ser Asn Phe 305 310 315 320 Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu Thr Ile 325 330 335 Asn Asn Asp Leu Trp Phe Asn Ser Leu Ser Ile Ser Leu Ala Tyr Gly 340 345 350 Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Ser Lys Ala Thr 355 360 365 Cys Cys Tyr Ala Tyr Ser Tyr Asn Gly Pro Arg Ala Cys Lys Gly Val 370 375 380 Tyr Ala Ser Glu Leu Ser Gln Ser Phe Glu Cys Gly Leu Leu Val Tyr 385 390 395 400 Val Thr Lys Ser Asp Gly Ser Arg Ile Gln Thr Ala Thr Glu Pro Pro 405 41 0 415 Val Ile Thr Gln His Asn Tyr Asn Asn Ile Thr Leu Asn Thr Cys Val 420 425 430 Asp Tyr Asn Ile Tyr Gly Arg Ile Gly Arg Gly Phe Ile Thr Asn Val 435 440 445 Thr Asp Ser Ser Ser Arg Tyr Asn Tyr Leu Ala Asp Ala Gly Leu Ala 450 455 460 Ile Leu Asp Thr Ser Gly Ala Ile Asp Ile Phe Val Val Gln Ser Glu 465 470 475 480 Tyr Gly Leu Asn Tyr Tyr Glu Val Asn Pro Cys Glu Asp Val Asn Gln 485 490 495 Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr Ser Arg 500 505 510 Asn Glu Thr Gly Ser Gln Pro Leu Glu Asn Gln Phe Tyr Ile Lys Leu 515 520 525 Thr Lys Glu Thr Arg Arg Phe Arg Arg 530 535

Claims (8)

Infectious bronchitis virus, characterized in that it is a variant IBV outdoor isolate that causes kidney lesions deposited with KCTC (Korean Collection for Type Cultures) under the Korea Biotechnology Institute on November 27, 2001 with accession number KCTC 10105BP. : IBV) K2 low passage. Chickens characterized by attenuating the chicken infectious bronchitis virus K2 low passage (KCTC 10105BP) according to claim 1 deposited with KCTC under the Korea Biotechnology Institute on November 27, 2001 with accession number KCTC 10106BP. Infectious bronchitis virus (IBV) K2 high passage. Killing vaccine, characterized in that the chicken infectious bronchitis virus (IBV) K2 low passage (KCTC 10105BP) according to claim 1 inactivated to contain a pharmaceutically effective amount thereof. The live vaccine according to claim 2, which contains a pharmaceutically effective amount of the chicken infectious bronchitis virus (IBV) K2 high passage (KCTC 10106BP). The vaccine for chicken infectious bronchitis virus according to claim 3 or 4, wherein the vaccine further comprises a pharmaceutically acceptable carrier or diluent. A biological agent comprising the S1 protein or base sequence according to SEQ ID NO: 1 of the Chicken Infectious bronchitis virus (IBV) K2 low passage (KCTC 10105BP) according to claim 1. A biological agent comprising the S1 protein or base sequence according to SEQ ID NO: 2 of the chicken infectious bronchitis virus (IBV) K2 high passage (KCTC 10106BP) according to claim 2. The biological agent according to claim 6 or 7, wherein the biological agent is a recombinant vector vaccine or a diagnostic kit.