WO2014163257A1

WO2014163257A1 - Novel infectious bursal disease (ibd) virus k7 strain and vaccine against infectious bursal disease using same

Info

Publication number: WO2014163257A1
Application number: PCT/KR2013/007945
Authority: WO
Inventors: 송창선; 최강석
Original assignee: 건국대학교기술지주 주식회사
Priority date: 2013-04-03
Filing date: 2013-09-03
Publication date: 2014-10-09
Also published as: KR101511712B1; KR20140120533A

Abstract

The present invention relates to a novel infectious bursal disease (IBD) virus K7 strain and a vaccine against an infectious bursal disease using the same.

Description

New Infectious Fang Disease Virus Ｂ7 week and infectious Fang disease vaccine using the same

The present invention relates to a novel infectious F cystic virus IBD K7 strain and an infectious F cystic vaccine using the same.

This disease, called infectious bursal disease (IBD) or Gumboro disease in chickens, is an infectious disease caused by IBD virus (IBDV). IBDV belongs to the family Birnaviridae. The genome consists of two strands of double-stranded A and B RNA, segment A contains VP5, VP2, VP4 and VP3 genes, and segment B contains the VP1 gene. IBDV has two serotypes, type 1 and type 2, which can be classified by viral neutralization (VN) tests. Serotype 1 virus has been shown to be pathogenic for chickens, whereas serotype 2 IBDV causes subacute disease only in turkey. Infectious F cystic disease is an important target that causes infection of the F sac (Bursa of Fabricius) cells to cause immunosuppression. Infection of chickens at 3-6 weeks of age leads to clinically infectious F-cysts with high morbidity and mortality, and if infected before 3 weeks of age, rapid immunodepression occurs due to the rapid destruction of lymphocytes in the antibody-producing F-cysts. Immunity against various vaccines is reduced and resistance to infectious diseases is weakened. Therefore, diseases that were not observed due to uncontrollable or latent infections in normal chickens show clinical symptoms or more severe clinical symptoms in chickens infected with infectious F cystic disease, causing enormous economic losses to the poultry industry.

Since the disease was first reported by Cosgrove in 1962, it has been found worldwide. In 1979, it was confirmed that more than 50% of chickens were infected by a team of animal hygiene research institutes. Then, in 1987, the very first very virulent IBD appeared in Europe, causing serious illness. In 1992, it was first reported in 9-week-old laying hens in Seonghwan, Chungnam. The farms at that time were reported to have a high mortality rate of more than 30% with severe clinical symptoms. Unlike the existing classic type, the highly toxic infectious F cyst causes high mortality even after 3 weeks or 6 weeks of age, and the disease progresses rapidly due to atrophy of inflammatory reactions such as swelling or hyperemia in the F organ, which is an immune organ. .

Currently, open field infectious F cysts have few classic or variant infectious F cysts and virtually all are toxic infectious F cysts (79%). Infectious F-cytopathic live vaccines used at home and abroad to prevent illness are severely toxic (M; mild) and intermediate (I; intermediate) depending on their ability to overcome virulence and maternal antibody levels. It is classified as intermediate plus (I +), which has a strong virulence of intermediate and intermediate poison vaccines that can overcome maternal antibodies in order to prevent early infectious infectious F cystic disease. .

* Based on the data of the Animal Drug Association, the annual sales rate of infectious F-cystic vaccine vaccine strain by strain is medium-plus vaccine (74-79% of the total), intermediate-type vaccine (16-22%), and attenuated drug (4 ~ 9%). This may indicate a preference for the use of intermediate poisoning plus vaccines that can overcome maternally-transduced antibodies more quickly to prevent premature infection of highly infectious F-cystic disease. (Figure 2) Medium Poison Plus Vaccine Produces a Strong Immune Response That Effectively Prevents Infectious F-Cysts, but Strong Vaccine Responds to Similar Symptoms of Infectious F-Cysts It is known. (Rautenschlein S et al., Protective efficacy of intermediate and intermediate plus infectious bursal disease virus (IBDV) vaccines against very virulent IBDV in commercial broilers, Avian Dis. 2005 Jun; 49 (2): 231-7)

Currently, the intermediate poisonous plus vaccine strains used in Korea form a strong immunity against infectious F cystic disease but can cause severe damage to F organ, which is an immune organ, and thus lower resistance to other diseases. Compared to virulence, low virulence and development of a vaccine with higher immunogenicity than an intermediate infectious F follicular vaccine are urgently needed.

As a related patent, Korean Patent Publication No. 1020110116315 describes a novel non-pathogenic infectious FDR virus (BP-IBDVac) having excellent vaccine efficacy against infectious FDR disease, an infectious FBD disease vaccine comprising the same, and a method of preparing the same.

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems and the need for the above, and an object of the present invention is to provide a novel infectious F cystic virus.

Another object of the present invention is to provide a novel infectious F cystic vaccine.

In order to achieve the above object, the present invention provides infectious F cystic virus IBD K7 strain deposited with accession number KCTC 12376BP.

The strain was deposited on March 11, 2013 with the accession number KCTC 12376BP to the Gene Bank of Korea Biotechnology Institute (Yeoseong-gu, Daejeon, Korea) as an international depositing institution.

In one embodiment, the virus is preferably isolated from domestic broilers, and the segments A and B of the virus preferably have the nucleotide sequences shown in SEQ ID NOs: 1 and 2, respectively, but are not limited thereto.

In one embodiment segment A of the virus has amino acid sequences set forth in SEQ ID NOs: 3 (VP 5) and 4 (VP2-VP4-VP3) and segment B has amino acid sequences set forth in SEQ ID NO: 5 (VP 1) , 45, 74, 133 in SEQ ID NO: 3, 222, 242, 256, 279, 284, 294, 299, 451, 680, 715, and 751 in SEQ ID NO: 4 , 981, 1005 residues, and 4, 13, 61, 145, 146, 147, 242, 287, 390, 393, 508, 511, 562 in SEQ ID NO: 5 Times, 687, 695th residue is preferably different from other strains, but is not limited thereto.

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

In one embodiment, it is preferred that the virus is in live form, it is preferred that the vaccine further comprises an adjuvant, and that the vaccine further comprises one or more vaccine components of other pathogens infectious to poultry. One is not limited thereto.

In another aspect, the present invention comprises the steps of a) inoculating a substrate susceptible to infection with an infectious F cystic virus as defined in any one of claims 1 to 3; b) propagating said infectious F cystic virus; And c) provides a method for producing an infectious F-Cyst virus comprising the step of collecting the infectious F-Cyst virus-containing material.

In one embodiment, the substrate is one cell selected from the group consisting of embryonic liver cells (CEL), chicken embryo fibroblasts (CEF), chicken kidney cells (CK), and Vero cell lines. But is not limited thereto.

The present invention also provides a method of controlling a disease caused from an infectious F cystic infection in poultry comprising administering the vaccine of the present invention to birds.

Vaccines of the invention containing live virus are prepared and marketed in the form of lyophilized or (frozen) suspensions. The vaccine further comprises a diluent or pharmaceutically acceptable carrier commonly used in the composition. Carriers include stabilizers, preservatives and buffers. Suitable stabilizers are, for example, SPGA, hydrocarbons (eg sorbitol, mannitol, starch, sucrose, dextran, glutamate, or glucose), proteins (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. Dilutions are water, aqueous buffers (eg buffered saline), alcohols and polyols (eg glycerol).

If necessary, the live vaccine of the present invention may contain an adjuvant. Examples of suitable compounds and compositions with adjuvant activity are the same as mentioned below for the preparation of inactivated vaccines.

The live vaccines of the present invention can be administered, for example, by intramuscular or subcutaneous injection, but the live vaccines are preferably administered by low-cost mass administration techniques commonly used for infectious F cystic virus vaccination. This technique includes drinking water and spray vaccination.

Another method for the administration of live vaccines includes in ovo administration, eye drop administration and beak dipping administration.

The vaccine of the invention is an active ingredient in an amount effective to immunize an infectious F follicular virus substance, ie an infectious F follicular virus substance that will induce immunity in a vaccinated bird or their offspring against an attack by an toxic virus (antigen administration). Include. Immunity is defined herein to induce a significantly higher level of protection in avian populations after vaccination compared to the non-vaccinated group.

Typically, the live vaccine of the present invention can be administered at a dose of 10 ² -10 ⁹ TCID 50, preferably 10 ² -10 ⁶ TCID 50 per bird.

The infectious F cystic virus vaccine of the present invention can be used effectively in chickens, but other poultry such as turkey, guinea fowl and quail can also be effectively vaccinated. Chickens include edible chickens, cloned cattle, and laying eggs.

The age of the animals receiving the live vaccine according to the present invention is the same as the age of the animals receiving the currently available live infectious F cystic virus vaccine. For example, edible chickens are directly vaccinated with the live vaccine of the present invention from day 1 of birth. Vaccination of parental livestock, such as breeders of edible chickens, can be performed with the live vaccine of the present invention. The advantages of this type of vaccine program include the direct protection of one day old offspring provided by antibodies from the maternal line that are delivered vertically to the offspring. A typical breeder's vaccination program includes two to three weeks old livestock inoculated with live, weakened vaccines followed by 14 to 18 weeks old inactivated vaccines.

In the present invention, it is possible to effectively protect poultry from infectious F cystic disease by providing a novel infectious F cystic virus, a vaccine comprising the same, and a method for preparing the same.

1 is classified by virulence type of infectious F cystic virus isolated in Korea <Source: Agriculture, Forestry and Fisheries Quarantine and Inspection Headquarters>

Figure 2 is a graph showing the sales ratio of the infectious F cyst live vaccine type in Korea,

3-7 is another pathogen contamination test photograph,

8 is a systematic analysis of the VP2 hypervariable region

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as limited by the following examples.

Example 1: Isolation of infectious bursal disease virus IBD K7 strain , identification of other pathogens

In 2007, F bags were collected from broilers from Chungnam broiler farms and sterile phosphate buffer solution (pH7.2) was added to make 10% by weight emulsion. Cells and tissues were precipitated by centrifugation at 3,000 g for 15 minutes, and the supernatant was filtered with a 0.45 μm syringe filter. IBD K7 strain was finally identified by reverse transcriptase polymerase chain reaction (RT-PCR) and isolated by chorioallantoic membrane (CAM) inoculation of 11-day-old SPF embryonated eggs.

Example 2: Other Pathogen Negative Tests in New IBDV Field Isolates

Infectious bronchitis virus (IBV), Newcastle disease (NDV), Fowl adeno virus to check for the presence of other pathogens capable of proliferation in the ureteral fluid of the chick embryo , FAdV), Chicken infectious anemia virus (CIAV), Mycoplasma, and reverse transcription polymerase chain reaction (RT-PCR) and polymerase chain reaction (PCR) were carried out It was confirmed that there was no other pathogen other than (IBD K7).

Example 3: Genome Sequencing

RNA was extracted by inoculating the 11-day-old SPF embryonated eggs with 150 μl of the urinary membrane solution harvested 5 days later. 1 ㎍ of RNA extracted above, random hexamer 10 pmol, deoxynucleotide triphosphates (dNTPs) 20pmol, Superscript II reverse transcriptase (Invitrogen) at a concentration of 200 units and mixed at 42 ℃ for 60 minutes, the reverse transcriptase is 70 ℃ Inactivated by treatment for 15 minutes at.

The cDNA thus obtained was mixed with 1 µl of 4-fold diluted cDNA solution, 1 µl of 10x PCR buffer, 0.2 µl of 2.5 mM dNTPs, 2 µl of each primer, 1 µl of Taq polymerase and 7.2 µl of DW. (I. Lojkic et al., Sequence Analysis of Both Genome Segments of Three Croatian Infectious Bursal Disease Field Viruses, AVIAN DISEASES 52: 513-519, 2008)

The PCR product obtained using the reaction solution was purified, and the nucleotide sequence was determined using an ABI3100 autobase sequencer. As a result, the genome sequence of IBD K7 (Segment A base of IBD K7 virus and Segment B base of IBD K7 virus represented by Segment A and Segment B) is SEQ ID NO: 1, respectively. And 2) to obtain the amino acid sequence of the proteins VP1, VP5-VP2-VP4-VP3.

Table 1

Table 1 shows the sequence and position of the primer set used in the Segment A base analysis

TABLE 2

Table 2 is a table showing the nucleotide sequence and position of the primer set used in Segment B nucleotide analysis

Example 4: Molecular Biology of IBD K7 Virus

The entire gene of IBD K7 virus was compared with domestic and foreign infectious F cystic virus registered on GenBank using BLAST search (www.ncbi.nlm.nih.gov/blast) and Bioedit program. As a result, IBD K7 showed relatively higher homology to the attenuated virus than to the virulent virus (Table 3).

In addition, the VP2 hypervariable region analysis applied to phylogenetic analysis revealed that it is a new type that does not belong to any existing type. In the case of pathogenic motifs of protective protein VP2, IBD K7 is a very unique antigen with both toxic and attenuated viral motifs. However, for other structural proteins other than VP2 protein, IBD K7 had a typical attenuated viral motif (Tables 4 and 5).

In conclusion, IBD K7 is a novel recombinant IBD virus compared to existing viruses with some of the virogenic viral antigen motif.

TABLE 3

Table 3 shows overall gene homology with IBD K7 and conventional IBDV strain.

[Revision under Rule 26 01.10.2013]
Table 4 [Table 4]

Table 4 shows the comparative analysis of VP5, VP2, VP4 and VP3 of IBD K7 and vaccine and field strains.

[Revision under Rule 26 01.10.2013]
Table 5 [Table 5]

Table 5 shows the comparative analysis of IBD K7, VP1 and vaccine strains.

Example 5 Pathogenicity Experiments of IBD K7 Strain

To investigate the possibility of vaccine strain, IBD K7 strain at 3 weeks of age of SPF chicken and Winterfield strain and D78 strain of IBDV vaccine were administered orally at 103 ELD50 / dose to 8 chickens in each group. The rest was euthanized and subjected to body weight and BB-rate [(F-bag weight (g) / weight (g)) × 1000] and antibody response test. As a result, in Winterfield strain group and IBD K7 strain group, each of them died within 4 days, and the remaining groups survived for 7 days. As a result of comparing body weight and BB-rate, it was found to have intermediate pathogenicity between intermediate poisoning and intermediate poisoning. The ELISA titer of serum isolated from 4 and 7 days after vaccination showed that the immune response was somewhat lower than that of medium poisoning plus. It was found to be low but stronger than medium poisons (Tables 6 and 7).

Table 6

Table 6 shows body weight and BB rate after 4 and 7 days of IBD K7 strain, medium poison plus, medium poison vaccination

TABLE 7

Table 7 shows ELISA titers 4 and 7 days after IBD K7 strain, intermediate poison plus, intermediate poison vaccination

Example 6: Immunogenicity and Protective Activity in SPF Chickens

To determine the efficacy of IBD K7 as a live vaccine, three-week-old SPF chickens were orally vaccinated with IBD K7 strain and the existing vaccine strain Winterfield strain and D78 at 10 ³ ELD ₅₀ per dose, and 2 weeks after vaccination. The highly infectious F cynovirus was challenged by instillation of 10 ⁵ ELD ₅₀ per dose. The control group was also challenged with the same virus in the same manner. Five days after inoculation, clinical symptoms such as depression, feather inversion, and mortality were observed.

After 2 weeks of vaccination, the ELISA antibody and the clinical symptoms showed better immunogenicity than the mid-dose vaccine D78 and no significant difference from the mid-dose plus vaccine Winterfield strain inoculation group. Table 8

Table 8

Table 8 is a table showing the protective capacity and immunogenicity against toxic IBDV challenge in IBD K7 strain vaccine chicken.

Claims

Infectious F cystic virus IBD K7, deposited under accession number KCTC 12376BP.
The strain of infectious F cystic virus IBD K7 according to claim 1, wherein the virus is isolated from domestic broilers.
The infectious F cystic virus IBD K7 strain according to claim 1, wherein the segments A and B of the virus have the nucleotide sequences shown in SEQ ID NOs: 1 and 2, respectively.
The method of claim 1, wherein Sigma A of the virus has the amino acid sequence of SEQ ID NO: 3 and 4, Segment B has the amino acid sequence of SEQ ID NO: 5, 45, 74, 133 in SEQ ID NO: In SEQ ID NO: 4, 222, 242, 256, 279, 284, 294, 299, 451, 680, 715, 751, 981, 1005 residues, and in SEQ ID NO: 5 Residues 4, 13, 61, 145, 146, 147, 242, 287, 390, 393, 508, 511, 562, 687 and 695 were different from other strains. Infectious F cystic virus IBD K7 strain, characterized by a difference.
A vaccine for poultry protection against a disease resulting from an infectious follicular virus infection comprising the virus of any one of claims 1 to 4 and a pharmaceutically acceptable carrier or diluent.
6. The vaccine of claim 5 wherein said infectious F cystic virus is in live form.
6. The vaccine of claim 5 wherein said vaccine further comprises an adjuvant.
The vaccine of claim 5, wherein said vaccine further comprises one or more vaccine components of other pathogens infectious to poultry.
a) inoculating a substrate susceptible to infection with the infectious F cystic virus as defined in any one of claims 1 to 4;

b) propagating said infectious F cystic virus; And

c) A method for producing an infectious FLA virus comprising the step of collecting the infectious F disease virus-containing material.
The method of claim 9, wherein the substrate is one cell selected from the group consisting of embryonic liver cells (CEL), chicken embryo fibroblasts (CEF), chicken kidney cells (CK), and Vero cell line. A method for producing an infectious F cystic virus, characterized in that.
A method for controlling a disease resulting from an infectious F cystic infection in poultry comprising administering the vaccine of claim 5 to a bird.