KR20170142369A - Novel H5N9 recombinant influenza virus and vaccine composition comprising the same - Google Patents

Abstract

The present invention relates to avian influenza H5N9 recombinant virus and a vaccine containing the same. The recombinant virus or a vaccine composition comprising the recombinant virus of the present invention has excellent stability, exhibits a significantly higher antibody titer in chickens, and is superior in defense against avian influenza virus.

Description

A novel avian influenza H5N9 recombinant virus and a vaccine composition comprising the same,

To avian influenza H5N9 recombinant viruses and vaccines comprising them.

Influenza viruses are RNA viruses belonging to the family Orthomyxoviridae and consist of subtypes named A, B and C. Influenza A is known to cause infections in humans, horses, pigs, other mammals and various species of poultry and wild birds. Depending on the characteristics of the hemagglutinin hemagglutinin present on the virus surface, H1 to H16 16 species, Nyuraminidase, according to the characteristics of the surface protein represented by the enzyme N1 to N9 nine subtypes are divided into subtypes of H and N-type combination, A type influenza virus, theoretically a total of 144 species Subtypes are present. Among these influenza A strains, the avian influenza H9N2 and H5N1 viruses that cause infections in birds are continuously infecting poultry in the country.

Highly Pathogenic Avian Influenza (HPAI) is an A disease listed in the International Bureau of OIE (OIE), which is classified as a Type 1 livestock infectious disease in Korea. It is also an important disease in the trade of livestock products between countries. Among these highly pathogenic influenza, the H5N1 subtype virus has been reported frequently in poultry infections in Asia, Europe and Africa since 2003.

Recently, in Korea, the incidence of highly pathogenic avian influenza virus (H5N1) in wild winter avian feces and dead birds in 2010, and the highly pathogenic avian influenza in ducks and poultry farms has been the fourth in Korea since 2003, 2006 and 2008 Poultry infections of highly pathogenic avian influenza have occurred.

These highly pathogenic avian influenza pandemics are causing great public health concerns as well as significant economic losses due to high mortality rates in poultry infections. The highly pathogenic avian influenza strain H5N1 has been reported to cause fatal outbreaks in humans, and since 2003, when the first human cases of infection were reported in Hong Kong, .

The number of reported cases of human infections of the highly pathogenic avian influenza type H5N1 was 553 and the death toll was 323, and it was reported that most of HPAI H5N1 occurs in the poultry areas of Vietnam and Indonesia. An effective eradication method is necessary.

In the case of highly pathogenic avian influenza, rapid disinfection policies have been favored and have been dealt with in Korea at the same time. However, the disposal policy has enormous environmental and ethical problems, including enormous rewards, and the disposal policy of the H5N1 influenza A type of avian influenza, such as China, Vietnam and Indonesia, has not been successful. Poultry infections of the highly pathogenic avian influenza of the new clade have been continuously reported in several areas, including In addition, as there is a possibility of poultry through migratory birds of highly pathogenic avian influenza, there is a growing need for the development of vaccines that can be effectively used in such situations.

In fact, in China, Vietnam and Indonesia, vaccination against poultry is being carried out as one of the policies to control highly pathogenic avian influenza. Domestic poultry vaccination against avian influenza is prohibited, but infection and transmission of avian influenza poultry There is a need to develop a vaccine against emergencies in a situation that occurs repeatedly.

To date, the avian influenza vaccine has been developed in Mexico using the inactivated oil vaccine (Intervet Mexico) for the H5N2 serotype and the H5 recombinant fowl pox- (DIVA) vaccine that is developed and used in Italy (Difiventing Infected from Vaccinated Animals; DIVA).

In order to eradicate the highly pathogenic avian influenza, it is necessary to confirm the presence of the virus continuously through monitoring after vaccination. In the case of the vaccine using the same serotype, DIVA can not be applied through the serum, Although DIVA can be applied through serum, it is pointed out that the fluorescent antibody method that discriminates NA type is not suitable for large-scale antibody monitoring test.

As a result of efforts to solve these problems, the present inventors have found that recombinant avian influenza virus having the serotype H5N9 was produced using reverse gene method and that the virus had excellent stability as a vaccine and excellent effect as a vaccine, .

Korean Patent Publication No. 10-2012-0131725 Korean Patent No. 10-1605521

Accordingly, an object of the present invention is to provide a recombinant avian influenza H5N9 virus.

Another object of the present invention is to provide a vaccine composition comprising the virus or an antigen of the virus.

It is another object of the present invention to provide a composition for diagnosing avian influenza virus, a diagnostic kit and a diagnostic method using the virus or the antigen of the virus.

The present invention relates to a hemagglutinin gene (HA) of the avian influenza virus H5 virus represented by the nucleotide sequence of SEQ ID NO: 1; And

The present invention provides a recombinant avian influenza H5N9 virus comprising the neuraminidase (NA) gene of the avian influenza N9-type virus represented by the nucleotide sequence of SEQ ID NO: 2.

Hereinafter, the present invention will be described in detail.

The recombinant avian influenza H5N9 virus according to the present invention comprises a gene encoding a matrix protein, a gene encoding a polymerase subunit A (PA), and a gene encoding a polymerase subunit B1 (polymerase subunit B1, PB1) A gene encoding a polymerase subunit B2, a gene coding for a polymerase subunit B2, and a gene encoding a nonstructural protein, and the gene is selected from the group consisting of A / Puerto Rico / 8/1934 (H1N1).

In the present invention, the hemagglutinin (HA) gene may be obtained by removing MBCS (multiple-basic amino-acid cleavage site). The hemagglutinin gene is derived from A / baikal teal / Korea / 1449/2014 ( H5 N8), and the neuraminidase (NA) gene is derived from ENV-15 (H11N9) .

The recombinant virus of the present invention can be produced by a reverse genetics method, and specific genes can be cloned into expression vectors, respectively. The expression vector may be a pHW2000 vector.

After the cloning, the cells are transfected, the cells are cultured, the supernatant is recovered, and the cells are inoculated into SPF (specific pathogen free) developmental cells to produce viruses. The cells can be obtained from HEK-293T cells .

The recombinant avian influenza H5N9 virus of the present invention may be avian influenza virus of accession number KCTC13027BP.

There can be provided a vaccine composition for avian influenza comprising avian influenza virus or an antigen thereof as an active ingredient or a pharmaceutical composition for preventing or treating an influenza virus infection disease, which comprises the virus of the present invention as an active ingredient.

The vaccine composition of the present invention has excellent stability, exhibits a significantly higher antibody titer in chicken and duck, and is superior in defense against avian influenza virus.

The vaccine of the present invention may be at least one selected from the group consisting of an attenuated virulent virulent vaccine, a sadox vaccine, a subunit vaccine, a synthetic vaccine, and a genetic engineering vaccine,

The antigen may be hemagglutinin (HA) encoded by the nucleotide sequence of SEQ ID NO: 1 or neuraminidase (NA) protein encoded by the nucleotide sequence of SEQ ID NO: 2.

The vaccine composition may be used for the prevention or treatment of common influenza viruses, but preferably can be prevented or treated against avian influenza virus or avian influenza viruses of serotype H5N1, H5N9 and H5N8, H5N8 infection can be prevented or treated.

 The term " live vaccine "means a vaccine comprising live viral active ingredients.

The term "attenuation" refers to artificially weakening the toxicity of living pathogens, which means mutating the genes involved in the essential metabolism of pathogens to induce immunity by stimulating only the immune system without causing disease in the body. The attenuation of the virus can be accomplished by ultraviolet (UV) irradiation, drug treatment, or in-vitro cross-over subculture. Inactivation can also be accomplished by making clear genetic changes, for example by insertion of sequences into viral genomes or specific deletions of viral sequences known to provide toxicity.

The term " Sadox vaccine "is also referred to as inactivated vaccine or killed vaccine, and is a vaccine containing a dead virus. Examples thereof include whole-virus vaccines and split vaccines, which are readily manufacturable by known methods. For example, the whole virus vaccine can be prepared by treating the whole virus with formalin, and the divided vaccine can be prepared by treating the virus with ether to crush and obtain only the envelope.

The term "subunit vaccine" is a vaccine prepared by extracting only an antigen component that can cause an immune function among components of a virus, and can minimize side effects by inducing immune formation only at an antigen site necessary for viral defense.

For example, HA protein and / or NA protein of avian influenza virus can be extracted and used.

The term "synthetic vaccine" may be used by synthesizing only the antigen or antigenic determinant of a virus chemically, or by using a vaccine containing a peptide produced by recombinant DNA technology, such as HA protein and / or NA protein of avian influenza virus.

The term " genetic engineering vaccine "may be a modification or deletion of a specific gene that causes virulence of the virus.

In addition, the influenza vaccine of the present invention can be combined with inactivated cells or antigens used for the production of other vaccines used for preventing diseases other than influenza caused by avian influenza, thereby preventing other diseases including influenza together Mixed or conjugated vaccine.

The term "combined vaccine " refers to a vaccine that uses different antiviral vaccines together, and the term" combination vaccine " refers to a vaccine that uses both a vaccine and a bacterial vaccine.

In addition, the vaccine composition of the present invention may further comprise at least one member selected from the group consisting of a solvent, an adjuvant, and an excipient. Examples of the solvent include physiological saline or distilled water. Examples of the immunity enhancer include Freund's incomplete or complete adjuvant, aluminum hydroxide gel, and vegetable and mineral oil. Examples of excipients include aluminum phosphate, But are not limited to, rocksides or aluminum potassium sulphate (alum), and may further include any of the known materials used in the manufacture of vaccines well known to those skilled in the art.

In the present invention, viruses or antigens may be mixed in a ratio of 10-40: adjuvant 10-60, preferably in a ratio of antigen 30: adjuvant 70.

 The vaccine composition of the present invention can be prepared in oral or parenteral formulations, preferably in the form of a parenteral preparation, injectable solution, and can be administered orally, intraperitoneally, intramuscularly, intraperitoneally, intravenously, subcutaneously, epidural) route of administration.

In another aspect of the present invention, there is provided a method for preventing or treating an influenza virus infection disease by administering the vaccine composition to an individual.

The term "individual" of the present invention refers to all animals, including humans, that are already infected or susceptible to influenza virus. By administering the composition of the present invention to an individual, animals infected with various influenza virus subtypes or variants can be treated.

More specifically, an object is any kind of animal that can spread to an object, such as a canine animal, a weasel and an animal, a cat animal, a wild boar and an animal, a bovine animal, a deer and an animal, a giraffe and an animal, Animals, horses and animals, veins and animals, rhinoceros and animals, rodents and primates and birds, and more preferably, chickens, ducks, turkeys, and humans.

"Influenza virus infection disease" of the present invention is a disease caused by influenza virus infection including, but not limited to, sinusitis, paroxysmal asthma, otitis media, cystic fibrosis, bronchitis, pneumonia, diarrhea and the like.

The term "prophylactic" in the present invention means all actions that inhibit or delay the onset of an influenza virus infection by administration of the composition. The term "treatment" in the present invention means any action that improves or alleviates symptoms due to influenza virus infection by the administration of the composition.

The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and the effective dosage level will vary depending on the species and severity, age, sex, Activity, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including simultaneously used drugs, and other factors well known in the medical arts.

The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The present invention also provides a composition for the diagnosis of influenza virus comprising the H5N9 serotype-type avian influenza virus or an antigen thereof, or an influenza virus diagnostic kit. Also provided is a method of diagnosing an infection of influenza virus comprising the step of treating the composition of the invention with a sample.

The sample includes, but is not limited to, cells, blood, urine, saliva, and tissue that are expected or infected with the influenza virus.

The influenza virus or antigen thereof of the present invention can be used not only for the elimination of influenza viruses in infected or infected cells through an antigen-antibody complex reaction, but also for specifically detecting influenza viruses.

These diagnostic kits include influenza viruses of the present invention as well as tools, reagents and the like commonly used in the art used for immunological analysis. Such tools / reagents include, but are not limited to, suitable carriers, labeling substances capable of generating a detectable signal, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Suitable carriers include, but are not limited to, soluble carriers, e. G., Physiologically acceptable buffers such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, Polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

Antigen-antibody complex formation can be induced by immunofluorescent staining, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, immunohistochemistry, (Complement Fixation Assay), FACS, protein chip, and the like.

Labels that enable qualitative or quantitative measurement of the formation of antigen-antibody complexes include, but are not necessarily limited to, enzymes, minerals, ligands, emitters, microparticles, redox molecules, and radioisotopes . Enzymes that can be used as detection labels include, but are not limited to,? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, A prodrug thereof, a prodrug thereof, a prodrug, a hexose kinase, a GDPase, an RNase, a glucose oxidase and a luciferase, a phosphofructokutase, a phosphoenolpyruvate carboxylase, an aspartate aminotransferase, a phosphoenolpyruvate decarboxylase, ≪ RTI ID = 0.0 > Ratama < / RTI > The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. Emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like.

Fine particles include, but are not limited to, colloidal gold, colored latex, and the like. The redox molecules include, but are not limited to, ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone and the like.

 The vaccine composition of the present invention is excellent in stability, exhibits a significantly higher antibody titer in chicken, and is excellent in defense against avian influenza virus.

FIG. 1 shows a method for producing a recombinant virus using a reverse gene method.
Fig. 2 is a diagram showing a schematic diagram and a cloning site of a pHW2000 vector.
FIG. 3 is a diagram for confirming the proliferation of recombinant virus using a hemagglutination reaction.
FIG. 4 is a view showing that the MBCS region of the HA of the recombinant virus is removed.

Hereinafter, the present invention will be described in detail with reference to the following examples. It should be noted, however, that the present invention is not limited to the following examples.

[ Example  One] H5N9  Production of recombinant avian influenza virus

1. Use gene donor

As shown in Table 1, for the production of H5N9 recombinant vaccine, a total of three influenza virus strains were used.

First, A / Puerto Rico / 8/1934 (H1N1), the prototype of the influenza A virus isolated from influenza patients in the Puerto Rico area, was used as the backbone of the recombinant virus and the major antigenicity of the highly pathogenic influenza A / baikal teal / Korea / 1449/2014 derived from H5N8 isolated from the domestic wild bird feces in 2014 was used for the HA gene, and the NA gene for DIVA vaccine was derived from H11N9 Of the ENV-15 strain.

division Internal protein expression gene Surface protein expression gene rgH5N9-2344 use
gene PB2, PB1, PA,
NP, M, NS HA NA Strain name A / Puerto Rico / 8/1934
(H1N1) A / baikal teal / Korea / 1449/2014 (H5N8) ENV-15
(H11 N9 )

2. Reverse genetics  Preparation of recombinant strains by the method

The process for producing the H5N9 recombinant strain is shown in Fig. First, the HA gene derived from the highly pathogenic influenza with the MBCS removed, the NA gene derived from the self-isolating ENV-15 strain (H11N9), and the six genes (PB2, PB1, PA, NP, M) derived from the A / Puert / Rico / 1934 strain (H1N1) , NS) was cloned into the expression vector pHW2000 vector. Then, 8 plasmids (pHW2000 vector cloned in each of PB2, PB1, PA, HA, NP, NA, M and NS genes) prepared above were prepared in advance in 6 wells so that HEK- After mixing 300ng each, they were mixed with lipofectamine to perform transfection (see FIG. 2)

Thereafter, the cells were cultured at 37 ° C and 5% CO ₂ for 48 hours. Then, trypsin (1 μl / ml) was treated in each well, and the supernatant was recovered after 24 hours. The recovered supernatant was again inoculated with 200 μl of SPF (specific pathogen free) at 10 days of age and incubated at 37 ° C. for 72 hours. The allantoic fluid was recovered and the presence of recombinant virus was confirmed by HA test (See FIG. 3). The identified strain was named rgH5N9-2344 based on the subtype name of the HA gene donor and deposited with the Biotechnology Research Institute on February 24, 2014 and assigned the accession number KCTC13027BP.

[ Example  2] Confirmation of the stability of recombinant avian influenza virus

The safety confirmation test of the recombinant strain was carried out to confirm that the recombinant virus prepared in Example 1 could be produced in the BSL-2 research facility.

1. Confirmation of elimination of highly pathogenic factor of recombinant virus

To confirm the removal of the multibasic amino acid sequence of the recombinant virus, the RNA of the recombinant virus was amplified by RT-PCR and sequenced. The results are shown in Fig.

As shown in FIG. 4, it was confirmed that the removal of the polybasic amino acid region of the HA gene was confirmed to be eliminated.

2. Of recombinant virus MBCS  Confirm reacquisition possibility

Also, in order to examine the possibility of regeneration of multibasic amino acid expression gene region (MBCS) in the transfection of the recombinant virus using the strain of the present invention, repeated passage experiments using 10-day-old SPF developmental cells were performed.

First, the recombinant virus was inoculated into the 10 - day - old SPF developmental cell and cultured for 72 hours at 37 ° C. The viability of the embryo was confirmed during incubation, and the presence of virus was confirmed by HA test after 72 hours. This process was repeated 5 times, and the death of the fetus due to the virus infection did not occur in the 5 repeated passages.

Sequence analysis of the HA gene of the recombinant virus obtained after 5 consecutive developmental strains was confirmed by the same method as described above, and it was confirmed that the multibasic amino acid expression gene region (MBCS) was not regenerated.

3. Confirmation of strain safety in laboratory animals using recombinant virus

Using the rgH5N9-2344 strain of the present invention, 10 recombinant viruses were inoculated into the nasal cavity at a concentration of 10 ^6.5 EID50 / 100 μl using 10 6-week-old BALB / C mice that had never been exposed to avian influenza virus and were observed for 14 days. As a result, it was confirmed that they survived healthy without mortality during the observation period.

4. Identification of strain safety in algae using recombinant virus

Using the rgH5N9-2344 strain of the present invention, 10 recombinant viruses were inoculated into the nasal cavity with 10 ^6.5 EID50 / 100 μl using 10 6-week-old chickens that had never been exposed to avian influenza virus and were observed for 14 days. As a result, it was confirmed that they survived healthy without mortality during the observation period.

5. Verification of vaccine safety in birds using recombinant virus

In order to confirm the safety of the inactivated vaccine prepared using the strain rgH5N9-2344 of the present invention, an oil emulsion vaccine was prepared. Each strain was reacted with formalin 0.2% (v / v%) at room temperature for 18 hours, and then mixed with oil adjuvant to produce emulsion. Two 5-week-old chickens that had never been exposed to avian influenza virus were used, and two doses of vaccine (1 ml) were inoculated into the muscle and observed for 14 days.

As a result, it was confirmed that there was no mortality and clinical symptoms during the observation period and survived well.

6. Conclusion

Through the above experimental results, it was confirmed that the MBCS region in the HA gene of the recombinant virus was completely removed as intended at the time of production, and it was also confirmed that the problem of returning to the pathogenic state that might occur during the vaccine manufacturing process could be avoided.

In addition, the results of the safety test of recombinant viruses using mouse and avian influenza antibody-negative chickens confirmed that the pathogenicity was completely eliminated, thus confirming that the viruses can be safely handled in the BSL-2 facility for producing a low-pathogenic influenza virus vaccine.

[ Example  3] Confirmation of effectiveness of the recombinant virus of the present invention as a vaccine.

1. Preparation of vaccine using the recombinant virus of the present invention

In order to confirm the effect of the recombinant virus vaccine of the present invention, the rgH5N9-2344 strain of the present invention was inoculated into the allantoic fluid of a 10-day old fetal bovine egg and after 72 hours, the urine membrane fluid was collected and subjected to a hemagglutinin assay to confirm the presence of virus. The collected virus was inactivated for 24 hours at 4 ℃ in 0.2% (v / v) formalin and used as an antigen. ISA 70 VG (SEPPIC, France) and W / O emulsion were prepared at a ratio (w / w) of 30 (antigen): 70 (adjuvant) and stored at 4 ° C for 24 hours. One milligram of vaccine used (0.5 ml) contained more than 256 HA of antigen,

2. Identification of the immunogenicity of the vaccine of the present invention.

In order to confirm the immunogenicity of the vaccine according to the present invention, 3-week-old SPF chicken 18 (Namduk) was used as a control axis, and sterilized PBS was used as a control. A / baikal teal / Korea / 1449/2014) H5N8 virus was used.

First, according to the method of use, 0.5 mL of the test drug or control agent was inoculated into the dorsal neck of the SPF chickens at 3 weeks of age, blood was collected from the jugular vein 2 or 3 weeks after the vaccination, Respectively. Separated sera were tested for haemagglutinin test (HA) using antigens (rgH5N9-2344) conjugated with the vaccine.

chicken vaccination G1 Inoculate countermeasures G2

3. Vaccine infection and clinical symptoms of vaccine of the present invention

After 3 weeks of vaccination, 10 ^6.3 EID50 / 200ul / bird was injected into both sides of the specimen shaft. Clinical symptoms and mortality were recorded daily for 14 days. After 14 days of viral injection, blood was collected from the jugular vein and the serum was separated. Hemagglutinin test (Hemagglutinin test) was performed using the antigens in the separated serum. Virus outbreak test Attack Inoculation Samples released to oral and total excreta on days 3, 5, 7, and 9 after virus infection were harvested and RNA was extracted to detect virus in collected samples. Real-time RT-PCR was used to determine the presence and amount of the viral gene.

4. After 3 weeks of vaccination, serum and infection in blood after 2 weeks , Serum in blood back New objects and Infected object  Distinction.

 After 3 weeks of vaccination, the serum in the blood and 2 weeks after the infection were tested using N1-competitive ELISA (positive cut-off value> 0.6)

5. Confirm the results

The above results are shown in Tables 3 to 6.

The antibody titers in the blood for the same antigen at 2 and 3 weeks after vaccination in chickens were found to be significantly higher than those of the vaccine administered with the control (see Table 3)

In addition, all of the chickens (100%) were able to protect against viral infection, and only 11.1% of the subjects receiving the control drug survived (see Table 4)

In addition, the group vaccinated with chicken developed lower virus outbreaks after infection compared to the control group (see Table 5). On days 3, 5, 7 and 9 after viral infection, oral pharyngeal / total excretory virus Discharge degree)

In addition, the average ELISA value at 3 weeks after vaccination in chicken was 0.152, which was confirmed to be negative in N1-cELISA after rgH5N9-2344 vaccine, and the average ELISA value at 1 week after infection was 0.786, Reference).

As a result, it was confirmed that the rgH5N9-2344 vaccine of the present invention can prevent the virus from being released at the same time as the clone 2.3.4.4 H5N8 infection occurred in the chicken in 2014, and also the vaccine can be inhibited by using the N1-cELISA It was confirmed that the individual can be distinguished from the infected individual.

Vine species Chicken (Log ₁₀ (HI titer) SD) vaccine rgH5N9-2344 Treaty after 2 weeks 7.9 0.6 0 0 After 3 weeks 10.2 0.8 0 0

Vine species Chicken (survival / total number (survival rate)) vaccine rgH5N9-2344 Treaty Before infection 9/9 9/9 After infection 9/9 (100%) 1/9 (11.1%)

Number of virus outbreaks / total number (average virus release amount (Log ₁₀ (EID ₅₀ / ml)) 3 days 5 days 7 days 9th 11th Oral pharyngeal Total dung Oral pharyngeal Total dung Oral pharyngeal Total dung Oral pharyngeal Total dung Oral pharyngeal Total dung rgH5N9-2344 3/9
(0.4) 0/9
(0.2) 2/9
(1.1) 3/9
(1.3) 0/9 0/9 0/9 0/9 0/9 0/9 Treaty 9/9
(2.9) 9/9
(1.7) 9/9
(4.4) 9/9
(3.9) 6/6
(4.1) 6/6
(3.7) 1/1
(1.8) 0/1 0/1 1/1
(1.2)

One 2 3 4 5 6 7 8 9 chicken After 3 weeks of vaccination 0.074
(voice) 0.188
(voice) 0.121
(voice) 0.085
(voice) 0.201
(voice) 0.305
(voice) 0.019
(voice) 0.042
(voice) 0.258
(voice) Two weeks after infection 0.858
(positivity) 0.708
(positivity) 0.864
(positivity) 0.763
(positivity) 0.803
(positivity) 0.786
(positivity) 0.779
(positivity) 0.726
(positivity) 0.786
(positivity)

Korea Biotechnology Research Institute KCTC13027BP 20160524

<110> konkuk university idustry cooperation <120> Novel H5N9 recombinant influenza virus and vaccine composition          comprising the same <130> P-1 <160> 8 <170> KoPatentin 3.0 <210> 1 <211> 1766 <212> DNA <213> Artificial Sequence <220> <223> HA sequence <400> 1 agcaaagcat gggttcaatc tgtcaaaatg gagaaaatag tgcttcttct tgcagtggtt 60 agccttgtta aaagtgatca gatttgcatt ggttaccatg caaacaactc aacaaagcag 120 gttgacacga taatggaaaa aaacgtcact gttacacatg cccaagacat actggaaaag 180 acacacaacg ggaagctctg cgatcttaat ggagtgaagc ccctgattct aaaggattgt 240 agcgtagctg ggtggctcct tggaaatcca atgtgcgacg agttcatcag ggtgccggaa 300 tggtcttaca tcgtggagag ggctaaccca gccaacgacc tctgttaccc agggaccctc 360 aatgactatg aggaactgaa acacctattg agcagaataa atcattttga gaaaactctg 420 atcatcccca agagttcttg gcccaatcat gaaacatcat taggggtgag cgcagcatgt 480 ccataccagg gagcatcctc atttttcaga aatgtggtat ggctcatcaa aaagaacgat 540 gcatacccga caataaagat aagctacaat aataccaatc gggaagatct tttgatactg 600 tgggggattc atcattccaa caatgcagca gagcagacaa atctctataa aaacccagac 660 acttatgttt ccgttgggac atcaacatta aaccagagat tggtgccaaa aatagctact 720 agatcccaag taaacgggca acgtggaaga atggatttct tctggacaat tttaaaaccg 780 aatgatgcaa tccactttga gagtaatgga aatttcattg ctccagaata tgcctacaaa 840 attgtcaaga aaggggactc aacaattatg aaaagtgaag tggagtatgg ccactgcaac 900 accaaatgtc aaactccaat aggggcgata aactctagca tgccattcca caatatacac 960 cctctcacca tcggggaatg ccccaaatac gtgaagtcaa acaaattagt ccttgcgact 1020 gggctcagaa atagtcctct aagagaaaaa agaggactat ttggagctat agcagggttt 1080 atagagggag gatggcaggg aatggtagac ggttggtatg ggtaccacca tagcaatgag 1140 caggggagtg ggtacgctgc agacaaagaa tccacccaaa aggcaataga tggagttacc 1200 aataaggtca actcaatcat tgacaaaatg aacactcaat ttgaggccgt tggaagggaa 1260 tttaataact tagaaaggag aatagagaat ttaaacaaga aaatggaaga cggattccta 1320 gatgtctgga cttataatgc tgaactttta gttctcatgg aaaatgagag aactctagat 1380 ttccatgact caaatgtcaa gaacctttac gacaaagtcc gactacagct tagggataat 1440 gcaaaggagc tgggtaatgg ttgtttcgag ttctatcaca aatgtgataa cgaatgtatg 1500 gaaagcgtaa gaaatgggac gtatgactac cctaagtatt cagaagaagc aagattaaaa 1560 agagaagaaa taagcggagt gaaattagaa tcaataggaa cttaccaaat actgtcaatt 1620 tattcaacag tggcgagttc cctagcactg gcaatcatag tggctggtct atctttatgg 1680 atgtgctcta atgggtcgct acaatgcaga atttgcatct aaatttgtga gctcagattg 1740 taattaaaaa cacccttgtt tctact 1766 <210> 2 <211> 1463 <212> DNA <213> Artificial Sequence <220> <223> N9 sequence <400> 2 gggagcaaag cagggtgaaa atgaatccaa atcggaagat tctatgcact tcagccactg 60 ctctcgtaat aggcacaatc gcagtactca taggaatagc aaatctaggg ttgaatatag 120 gactgcatct aaaaccgagc tgcaattgct cacactcaca acctgaagca accaacacaa 180 gccaaacaat aataaacaac tatcacaatg aaacaaatat cacccaaata agcaacacca 240 acattcaaat agaagagaga gcaagcagga gtttcaataa cttaactaaa gggctctgta 300 ctataaattc atggcacata tacgggaaag acaatgcaat aagaattgga gagaactcag 360 atgttttagt cacaagagaa ccctatgttt catgcgaccc agatgaatgc aggttctatg 420 ctctcagcca aggaacaaca atcagaggga aacactcaaa tggaacaata cacgataggt 480 cccagtatcg cgccctgata agctggccac tatcatcacc gcccacagta tacaacagcg 540 gggtggaatg cattgggtgg tcaagcacta gttgccatga tggtaaatcc aggatgtcaa 600 tatgcatatc aggaccaaac aacaatgcat ctgcagtagt atggtacaac agaagacctg 660 ttgcagaaat taacacatgg gcccgaaaca tactaagaac acaggaatct gaatgtgtat 720 gccacaacgg tgtatgccca gtagtgttca cagatgggtc tgccactgga cctgcagaca 780 caagaatata ttatttcaaa gagggaaaaa tattgaaatg ggaatctctg actggaactg 840 ccaagcatat tgaggaatgc tcatgttacg gggaacaaac aggaattacc tgcacatgta 900 gagacaattg gcagggctca aatagaccag taattcaaat agatccagtg gcaatgacac 960 acactagtca gtatatatgc agtcctgttc tcacagacaa tccccgacca aatgacccaa 1020 atgtaggtaa gtgtaacgat ccttatccag gtaataataa caatggagtc aaagggttct 1080 catacctgga tggggttaac acgtggctag ggaggacaat aagcacagct ttgaggtctg 1140 gatatgagat gctaaaagtg ccaaatgcat tgacagatga tagatcaaag cccattcaag 1200 gtcaaacaat tgtattaaac actgactgga gtggttacag tggatctttc atggactatt 1260 gggctgaggg ggactgctat cgagcgtgtt tttacgtgga gttaatacgt ggaagaccca 1320 aggaggataa agtgtggtgg accagcaata gtatagtatc gatgtgttcc agtacagaat 1380 tcctgggaca atggaactgg cctgatgggg ctaaaataga gtacttcctc taagatacaa 1440 aaaacaccct tgtttctact aat 1463 <210> 3 <211> 1027 <212> DNA <213> Artificial Sequence <220> <223> M sequence <400> 3 agcgaaagca ggtagatatt gaaagatgag tcttctaacc gaggtcgaaa cgtacgtact 60 ctctatcatc ccgtcaggcc ccctcaaagc cgagatcgca cagagacttg aagatgtctt 120 tgcagggaag aacactgatc ttgaggttct catggaatgg ctaaagacaa gaccaatcct 180 gtcacctctg actaagggga ttttaggatt tgtgttcacg ctcaccgtgc ccagtgagcg 240 aggactgcag cgtagacgct ttgtccaaaa tgcccttaat gggaacgggg atccaaataa 300 catggacaaa gcagttaaac tgtataggaa gctcaagagg gagataacat tccatggggc 360 caaagaaatc tcactcagtt attctgctgg tgcacttgcc agttgtatgg gcctcatata 420 caacaggatg ggggctgtga ccactgaagt ggcatttggc ctggtatgtg caacctgtga 480 acagattgct gactcccagc atcggtctca taggcaaatg gtgacaacaa ccaatccact 540 aatcagacat gagaacagaa tggttttagc cagcactaca gctaaggcta tggagcaaat 600 ggctggatcg agtgagcaag cagcagaggc catggaggtt gctagtcagg ctagacaaat 660 ggtgcaagcg atgagaacca ttgggactca tcctagctcc agtgctggtc tgaaaaatga 720 tcttcttgaa aatttgcagg cctatcagaa acgaatgggg gtgcagatgc aacggttcaa 780 gtgatcctct cgctattgcc gcaaatatca ttgggatctt gcacttgaca ttgtggattc 840 ttgatcgtct ttttttcaaa tgcatttacc gtcgctttaa atacggactg aaaggagggc 900 cttctacgga aggagtgcca aagtctatga gggaagaata tcgaaaggaa cagcagagtg 960 ctgtggatgc tgacgatggt cattttgtca gcatagagct ggagtaaaaa actaccttgt 1020 ttctact 1027 <210> 4 <211> 1565 <212> DNA <213> Artificial Sequence <220> <223> NP sequence <400> 4 agcaaaagca gggtagataa tcactcactg agtgacatca aaatcatggc gtcccaaggc 60 accaaacggt cttacgaaca gatggagact gatggagaac gccagaatgc cactgaaatc 120 agagcatccg tcggaaaaat gattggtgga attggacgat tctacatcca aatgtgcacc 180 gaactcaaac tcagtgatta tgagggacgg ttgatccaaa acagcttaac aatagagaga 240 atggtgctct ctgcttttga cgaaaggaga aataaatacc tggaagaaca tcccagtgcg 300 gggaaagatc ctaagaaaac tggaggacct atatacagga gagtaaacgg aaagtggatg 360 agagaactca tcctttatga caaagaagaa ataaggcgaa tctggcgcca agctaataat 420 ggtgacgatg caacggctgg tctgactcac atgatgatct ggcattccaa tttgaatgat 480 gcaacttatc agaggacaag agctcttgtt cgcaccggaa tggatcccag gatgtgctct 540 ctgatgcaag gttcaactct ccctaggagg tctggagccg caggtgctgc agtcaaagga 600 gttggaacaa tggtgatgga attggtcagg atgatcaaac gtgggatcaa tgatcggaac 660 ttctggaggg gtgagaatgg acgaaaaaca agaattgctt atgaaagaat gtgcaacatt 720 ctcaaaggga aatttcaaac tgctgcacaa aaagcaatga tggatcaagt gagagagagc 780 cggaacccag ggaatgctga gttcgaagat ctcacttttc tagcacggtc tgcactcata 840 ttgagagggt cggttgctca caagtcctgc ctgcctgcct gtgtgtatgg acctgccgta 900 gccagtgggt acgactttga aagagaggga tactctctag tcggaataga ccctttcaga 960 ctgcttcaaa acagccaagt gtacagccta atcagaccaa atgagaatcc agcacacaag 1020 agtcaactgg tgtggatggc atgccattct gccgcatttg aagatctaag agtattaagc 1080 ttcatcaaag ggacgaaggt gctcccaaga gggaagcttt ccactagagg agttcaaatt 1140 gcttccaatg aaaatatgga gactatggaa tcaagtacac ttgaactgag aagcaggtac 1200 tgggccataa ggaccagaag tggaggaaac accaatcaac agagggcatc tgcgggccaa 1260 atcagcatac aacctacgtt ctcagtacaga agaaatctcc cttttgacag aacaaccatt 1320 atggcagcat tcaatgggaa tacagaggga agaacatctg acatgaggac cgaaatcata 1380 aggatgatgg aaagtgcaag accagaagat gtgtctttcc aggggcgggg agtcttcgag 1440 ctctcggacg aaaaggcagc gagcccgatc gtgccttcct ttgacatgag taatgaagga 1500 tcttatttct tcggagacaa tgcagaggag tacgacaatt aaagaaaaat acccttgttt 1560 ctact 1565 <210> 5 <211> 890 <212> DNA <213> Artificial Sequence <220> <223> NS sequence <400> 5 agcaaaagca gggtgacaaa gacataatgg atccaaacac tgtgtcaagc tttcaggtag 60 attgctttct ttggcatgtc cgcaaacgag ttgcagacca agaactaggt gatgccccat 120 tccttgatcg gcttcgccga gatcagaaat ccctaagagg aaggggcagc accctcggtc 180 tggacatcga gacagccaca cgtgctggaa agcagatagt ggagcggatt ctgaaagaag 240 aatccgatga ggcacttaaa atgaccatgg cctctgtacc tgcgtcgcgt tacctaactg 300 acatgactct tgaggaaatg tcaagggact ggtccatgct catacccaag cagaaagtgg 360 caggccctct ttgtatcaga atggaccagg cgatcatgga taagaacatc atactgaaag 420 cgaacttcag tgtgattttt gaccggctgg agactctaat attgctaagg gctttcaccg 480 aagagggagc aattgttggc gaaatttcac cattgccttc tcttccagga catactgctg 540 aggatgtcaa aaatgcagtt ggagtcctca tcgggggact tgaatggaat gataacacag 600 ttcgagtctc tgaaactcta cagagattcg cttggagaag cagtaatgag aatgggagac 660 ctccactcac tccaaaacag aaacgagaaa tggcgggaac aattaggtca gaagtttgaa 720 gaaataagat ggttgattga agaagtgaga cacaaactga agataacaga gaatagtttt 780 gagcaaataa catttatgca agccttacat ctattgcttg aagtggagca agagataaga 840 actttctcgt ttcagcttat ttaataataa aaaacaccct tgtttctact 890 <210> 6 <211> 2233 <212> DNA <213> Artificial Sequence <220> <223> PA sequence <400> 6 agcgaaagca ggtactgatc caaaatggaa gattttgtgc gacaatgctt caatccgatg 60 attgtcgagc ttgcggaaaa aacaatgaaa gagtatgggg aggacctgaa aatcgaaaca 120 aacaaatttg cagcaatatg cactcacttg gaagtatgct tcatgtattc agattttcac 180 ttcatcaatg agcaaggcga gtcaataatc gtagaacttg gtgatccaaa tgcacttttg 240 aagcacagat ttgaaataat cgagggaaga gatcgcacaa tggcctggac agtagtaaac 300 agtatttgca acactacagg ggctgagaaa ccaaagtttc taccagattt gtatgattac 360 aaggagaata gattcatcga aattggagta acaaggagag aagttcacat atactatctg 420 gaaaaggcca ataaaattaa atctgagaaa acacacatcc acattttctc gttcactggg 480 gaagaaatgg ccacaaaggc agactacact ctcgatgaag aaagcagggc taggatcaaa 540 accagactat tcaccataag acaagaaatg gccagcagag gcctctggga ttcctttcgt 600 cagtccgaga gaggagaaga gacaattgaa gaaaggtttg aaatcacagg aacaatgcgc 660 aagcttgccg accaaagtct cccgccgaac ttctccagcc ttgaaaattt tagagcctat 720 gtggatggat tcgaaccgaa cggctacatt gagggcaagc tgtctcaaat gtccaaagaa 780 gtaaatgcta gaattgaacc ttttttgaaa acaacaccac gaccacttag acttccgaat 840 gggcctccct gttctcagcg gtccaaattc ctgctgatgg atgccttaaa attaagcatt 900 gaggacccaa gtcatgaagg agagggaata ccgctatatg atgcaatcaa atgcatgaga 960 acattctttg gatggaagga acccaatgtt gttaaaccac acgaaaaggg aataaatcca 1020 aattatcttc tgtcatggaa gcaagtactg gcagaactgc aggacattga gaatgaggag 1080 aaaattccaa agactaaaaa tatgaagaaa acaagtcagc taaagtgggc acttggtgag 1140 aacatggcac cagaaaaggt agactttgac gactgtaaag atgtaggtga tttgaagcaa 1200 tatgatagtg atgaaccaga attgaggtcg ctagcaagtt ggattcagaa tgagtttaac 1260 aaggcatgcg aactgacaga ttcaagctgg atagagctcg atgagattgg agaagatgtg 1320 gctccaattg aacacattgc aagcatgaga aggaattatt tcacatcaga ggtgtctcac 1380 tgcagagcca cagaatacat aatgaagggg gtgtacatca atactgcctt gcttaatgca 1440 tcttgtgcag caatggatga tttccaatta attccaatga taagcaagtg tagaactaag 1500 gagggaaggc gaaagaccaa cttgtatggt ttcatcataa aaggaagatc ccacttaagg 1560 aatgacaccg acgtggtaaa ctttgtgagc atggagtttt ctctcactga cccaagactt 1620 gaaccacata aatgggagaa gtactgtgtt cttgagatag gagatatgct tataagaagt 1680 gccataggcc aggtttcaag gcccatgttc ttgtatgtga gaacaaatgg aacctcaaaa 1740 attaaaatga aatggggaat ggagatgagg cgttgcctcc tccagtcact tcaacaaatt 1800 gagagtatga ttgaagctga gtcctctgtc aaagagaaag acatgaccaa agagttcttt 1860 gagaacaaat cagaaacatg gcccattgga gagtccccca aaggagtgga ggaaagttcc 1920 attgggaagg tctgcaggac tttattagca aagtcggtat tcaacagctt gtatgcatct 1980 ccacaactag aaggattttc agctgaatca agaaaactgc ttcttatcgt tcaggctctt 2040 agggacaacc ttgaacctgg gacctttgat cttggggggc tatatgaagc aattgaggag 2100 tgcctgatta atgatccctg ggttttgctt aatgcttctt ggttcaactc cttccttaca 2160 catgcattga gttagttgtg gcagtgctac tatttgctat ccatactgtc caaaaaagta 2220 ccttgtttct act 2233 <210> 7 <211> 2341 <212> DNA <213> Artificial Sequence <220> <223> PB1 sequence <400> 7 agcgaaagca ggcaaaccat ttgaatggat gtcaatccga ccttactttt cttaaaagtg 60 ccagcacaaa atgctataag cacaactttc ccttatactg gagaccctcc ttacagccat 120 gggacaggaa caggatacac catggatact gtcaacagga cacatcagta ctcagaaaag 180 ggaagatgga caacaaacac cgaaactgga gcaccgcaac tcaacccgat tgatgggcca 240 ctgccagaag acaatgaacc aagtggttat gcccaaacag attgtgtatt ggaagcaatg 300 gctttccttg aggaatccca tcctggtatt tttgaaaact cgtgtattga aacgatggag 360 gttgttcagc aaacacgagt agacaagctg acacaaggcc gacagaccta tgactggact 420 ctaaatagaa accaacctgc tgcaacagca ttggccaaca caatagaagt gttcagatca 480 aatggcctca cggccaatga gtctggaagg ctcatagact tccttaagga tgtaatggag 540 tcaatgaaaa aagaagaaat ggggatcaca actcattttc agagaaagag acgggtgaga 600 gacaatatga ctaagaaaat gataacacag agaacaatag gtaaaaagaa gcagagattg 660 aacaaaagga gttatctaat tagagcattg accctgaaca caatgaccaa agatgctgag 720 agagggaagc taaaacggag agcaattgca accccaggga tgcaaataag ggggtttgta 780 tactttgttg agacactggc aaggagtata tgtgagaaac ttgaacaatc agggttgcca 840 gtggaggca atgagaagaa agcaaagttg gcaaatgttg taaggaagat gatgaccaat 900 tctcaggaca ccgaactttc tttcaccatc actggagata acaccaaatg gaacgaaaat 960 cagaatcctc ggatgttttt ggccatgatc acatatatga caagaaatca gcccgaatgg 1020 ttcagaaatg ttctaagtat tgctccaata atgttctcaa acaaaatggc gagactggga 1080 aaagggtata tgtttgagag caagagtatg aaacttagaa ctcaaatacc tgcagaaatg 1140 ctagcaagca tcgatttgaa atatttcaat gattcaacaa gaaagaagat tgaaaaaatc 1200 cgaccgctct taatagaggg gactgcatca ttgagccctg gaatgatgat gggcatgttc 1260 aatatgttaa gcactgtatt aggcgtctcc atcctgaatc ttggacaaaa gagatacacc 1320 aagactactt actggtggga tggtcttcaa tcctctgacg attttgctct gattgtgaat 1380 gcacccaatc atgaagggat tcaagccgga gtcgacaggt tttatcgaac ctgtaagcta 1440 cttggaatca atatgagcaa gaaaaagtct tacataaaca gaacaggtac atttgaattc 1500 acaagttttt tctatcgtta tgggtttgtt gccaatttca gcatggagct ccccagtttt 1560 ggggtgtctg ggatcaacga gtcagcggac atgagtattg gagttactgt catcaaaaac 1620 aatatgataa acaatgatct tggtccagca acagctcaaa tggcccttca gttgttcatc 1680 aaagattaca ggtacacgta ccgatgccat agaggtgaca cacaaataca aacccgaaga 1740 tcatttgaaa taaagaaact gtgggagcaa acccgttcca aagctggact gctggtctcc 1800 gacggaggcc caaatttata caacattaga aatctccaca ttcctgaagt ctgcctaaaa 1860 tgggaattga tggatgagga ttaccagggg cgtttatgca acccactgaa cccatttgtc 1920 agccataaag aaattgaatc aatgaacaat gcagtgatga tgccagcaca tggtccagcc 1980 aaaaacatgg agtatgatgc tgttgcaaca acacactcct ggatccccaa aagaaatcga 2040 tccatcttga atacaagtca aagaggagta cttgaagatg aacaaatgta ccaaaggtgc 2100 tgcaatttat ttgaaaaatt cttccccagc agttcataca gaagaccagt cgggatatcc 2160 agtatggtgg aggctatggt ttccagagcc cgaattgatg cacggattga tttcgaatct 2220 ggaaggataa agaaagaaga gttcactgag atcatgaaga tctgttccac cattgaagag 2280 ctcagacggc aaaaatagtg aatttagctt gtccttcatg aaaaaatgcc ttgtttctac 2340 t 2341 <210> 8 <211> 2341 <212> DNA <213> Artificial Sequence <220> <223> PB2 sequence <400> 8 agcgaaagca ggtcaattat attcaatatg gaaagaataa aagaactaag aaatctaatg 60 tcgcagtctc gcacccgcga gatactcaca aaaaccaccg tggaccatat ggccataatc 120 aagaagtaca catcaggaag acaggagaag aacccagcac ttaggatgaa atggatgatg 180 gcaatgaaat atccaattac agcagacaag aggataacgg aaatgattcc tgagagaaat 240 gagcaaggac aaactttatg gagtaaaatg aatgatgcag gatcagaccg agtgatggta 300 tcacctctgg ctgtgacatg gtggaatagg aatggaccaa taacaaatac agttcattat 360 ccaaaaatct acaaaactta ttttgaaaga gtcgaaaggc taaagcatgg aacctttggc 420 cctgtccatt ttagaaacca agtcaaaata cgtcggagag ttgacataaa tcctggtcat 480 gcagatctca gtgccaagga ggcacaggat gtaatcatgg aagttgtttt ccctaacgaa 540 gtgggagcca ggatactaac atcggaatcg caactaacga taaccaaaga gaagaaagaa 600 gaactccagg attgcaaaat ttctcctttg atggttgcat acatgttgga gagagaactg 660 gtccgcaaaa cgagattcct cccagtggct ggtggaacaa gcagtgtgta cattgaagtg 720 ttgcatttga ctcaaggaac atgctgggaa cagatgtata ctccaggagg ggaagtgagg 780 aatgatgatg ttgatcaaag cttgattatt gctgctagga acatagtgag aagagctgca 840 gtatcagcag atccactagc atctttattg gagatgtgcc acagcacaca gattggtgga 900 attaggatgg tagacatcct taggcagaac ccaacagaag agcaagccgt ggatatatgc 960 aaggctgcaa tgggactgag aattagctca tccttcagtt ttggtggatt cacatttaag 1020 agaacaagcg gatcatcagt caagagagag gaagaggtgc ttacgggcaa tcttcaaaca 1080 ttgaagataa gagtgcatga gggatatgaa gagttcacaa tggttgggag aagagcaaca 1140 gccatactca gaaaagcaac caggagattg attcagctga tagtgagtgg gagagacgaa 1200 cagtcgattg ccgaagcaat aattgtggcc atggtatttt cacaagagga ttgtatgata 1260 aaagcagtca gaggtgatct gaatttcgtc aatagggcga atcagcgatt gaatcctatg 1320 catcaacttt taagacattt tcagaaggat gcgaaagtgc tttttcaaaa ttggggagtt 1380 gaacctatcg acaatgtgat gggaatgatt gggatattgc cagacatgac tccaagcatc 1440 gagatgtcaa tgagaggagt gagaatcagc aaaatgggtg tagatgagta ctccagcacg 1500 gagagggtag tggtgagcat tgaccgtttt ttgagaatcc gggaccaacg aggaaatgta 1560 ctactgtctc ccgaggaggt cagtgaaaca cagggaacag agaaactgac aataacttac 1620 tcatcgtcaa tgatgtggga gattaatggt cctgaatcag tgttggtcaa tacctatcaa 1680 tggatcatca gaaactggga aactgttaaa attcagtggt cccagaaccc tacaatgcta 1740 tacaataaaa tggaatttga accatttcag tctttagtac ctaaggccat tagaggccaa 1800 tacagtgggt ttgtaagaac tctgttccaa caaatgaggg atgtgcttgg gacatttgat 1860 accgcacaga taataaaact tcttcccttc gcagccgctc caccaaagca aagtagaatg 1920 cagttctcct catttactgt gaatgtgagg ggatcaggaa tgagaatact tgtaaggggc 1980 aattctcctg tattcaacta taacaaggcc acgaagagac tcacagttct cggaaaggat 2040 gctggcactt taactgaaga cccagatgaa ggcacagctg gagtggagtc cgctgttctg 2100 aggggattcc tcattctggg caaagaagac aagagatatg ggccagcact aagcatcaat 2160 gaactgagca accttgcgaa aggagagaag gctaatgtgc taattgggca aggagacgtg 2220 gtgttggtaa tgaaacggaa acgggactct agcatactta ctgacagcca gacagcgacc 2280 aaaagaattc ggatggccat caattagtgt cgaatagttt aaaaacgacc ttgtttctac 2340 t 2341

Claims (13)

Hemagglutinin gene (HA) of avian influenza virus H5 virus represented by the nucleotide sequence of SEQ ID NO: 1; And
A recombinant avian influenza H5N9 virus comprising the neuraminidase (NA) gene of avian influenza N9 type virus represented by the nucleotide sequence of SEQ ID NO: 2. The recombinant avian influenza virus according to claim 1, wherein the recombinant avian influenza virus H5N9 comprises a gene encoding a matrix protein, a gene encoding a polymerase subunit A (PA), a polymerase subunit B1 (PB1) A gene encoding a polymerase subunit B2, a gene encoding a polymerase subunit B2, and a gene encoding a nonstructural protein, and the gene is selected from the group consisting of A / Recombinant avian influenza H5N9 virus, which is derived from Puerto Rico / 8/1934 (H1N1). 2. The recombinant avian influenza virus H5N9 virus according to claim 1, wherein the hemagglutinin (HA) gene has been removed from MBCS (multiple-basic amino-acid cleavage site). The method of claim 1, wherein the hemagglutinin genes are A / baikal teal / Korea / 1449 /2014 will originate from the (H 5 N8), neuraminidase (Neuraminidase; NA) gene, ENV-15 (H11N9) , The recombinant avian influenza virus H5N9. The recombinant avian influenza virus H5N9 according to claim 1, wherein said recombinant avian influenza H5N9 virus is avian influenza virus of deposit number KCTC13027BP. A vaccine composition for avian influenza comprising the avian influenza virus or its antigen as an active ingredient comprising the virus of claim 1 as an active ingredient. 7. The vaccine composition of claim 6, wherein the vaccine composition is at least one selected from the group consisting of attenuated live virulence vaccine, sadox vaccine, subunit vaccine, synthetic vaccine and genetic engineering vaccine. The antigen according to claim 6, wherein the antigen is a hemagglutinin (HA) protein encoded by the nucleotide sequence of SEQ ID NO: 1 or a neuraminidase (NA) protein encoded by the nucleotide sequence of SEQ ID NO: 2 Lt; / RTI &gt; 7. The vaccine composition of claim 6, wherein the composition further comprises an adjuvant. A composition for the diagnosis of avian influenza virus comprising the recombinant avian influenza H5N9 virus of any one of claims 1 to 5 or an antigen thereof. 6. A kit for the diagnosis of avian influenza virus comprising the recombinant avian influenza H5N9 virus according to any one of claims 1 to 5 or an antigen thereof. A method for diagnosing an infection of avian influenza virus comprising the step of treating the composition of claim 10 to a sample. 13. The method of claim 12, wherein the sample is at least one selected from the group consisting of cells, blood, urine, saliva, and tissue that are expected or infected with avian influenza virus.

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