KR101626798B1 - Novel H3N2 influenza virus and vaccine composition compirising the same - Google Patents

Abstract

The present invention relates to a novel H3N2 serotype influenza virus and a vaccine composition comprising the same. The virus is highly pathogenic, replicable and infectious in a host, and can be used as a vaccine and a diagnostic kit for various influenza virus infections. In addition, the infectivity of influenza virus can be studied using the virus.

Description

Novel H3N2 serotype influenza viruses and vaccine compositions comprising same H3N2 influenza virus and vaccine composition compiling the same

The present invention relates to a novel H3N2 serotype influenza virus and a vaccine composition comprising the same.

Influenza (Influenza) is a disease caused by influenza virus type A of Orthomyxoviridae and is an economically important disease in people, pigs, horses and birds. Thus, as influenza is a common infectious disease, influenza viruses are highly variable and have the potential to spread from one species to another, thus addressing the global spread of highly pathogenic influenza is becoming a major challenge. Serologic types of highly pathogenic influenza A are classified according to the types of hemagglutinin and neuraminidase, and there are H serotype and N serotype. The serotypes of influenza viruses are represented by H serotype and N serotype, respectively. Especially, there are 16 serotypes of H serotype of avian influenza virus, 9 serotypes of N serotype and the sera of influenza virus There are 144 types.

Meanwhile, influenza A / H1N1 (pH1N1), a pandemic influenza virus, has been worldwide prevalent in 2009. The virus was also isolated from other animals such as cats, cheetahs, dogs, turkeys and skunks. The contagious nature of influenza viruses beyond these barriers has led to the emergence of novel recombinant viruses, such as the serotype H3N2 swine influenza virus variant, which contains the pH1N1 virus matrix (M) matrix in pigs and contains the M gene of the pH1N1 virus It is believed that the virus can affect humans.

On the other hand, it was reported that influenza A (H1N1) was first reported in 2004 at the Greyhound dog breeding ground in Florida, USA. Since then, the outbreak of influenza in Texas, Alabama, and Arkansas has been recognized as a new epidemic of the disease. According to epidemiological studies of influenza in the United States, it is almost identical to the H3N8 strain of horse infections, suggesting that new influenza viruses would have been produced by the transfer of influenza from horses to dogs. In addition, the influenza virus infection in Korea was reported in Korea in 2008, and the sequence analysis of the virus confirmed that it was caused by the virus byan avian H3N2 influenza A virus. Since then, H3N2 serotype influenza virus has been continuously reported in Korea and China. In particular, H3N2 serotype influenza viruses from Korean cats and H3N2 viruses with 98% to 99.8% homology in all gene regions Influenza-like viruses were isolated. In particular, the H3N1 serotype influenza virus is thought to have been generated through the reassortment event between the H3N2 serotype influenza virus and the pH1N1 virus. The genetically hemagglutinin gene locus of the virus was derived from the H3N2 influenza virus and the other gene was derived from the pH1N1 virus. Thus, the H3N2 influenza virus has evolved continuously through a mechanism of antigenic drift and shift. As described above, the mechanism by which influenza of other animals is transmitted to a dog can be dynamically estimated in two ways. One is the possibility that the virus can spread by feeding the uncooked ducks and chickens by dogs to dog farms and the other is the possibility of direct contact or respiratory propagation of dogs infected with normal dogs and other animal influenza Possibility. It is speculated that the infected dog would have been exposed to other environments and contacted with other dogs to help establish the influenza virus. Because of the potential for secondary infections and the varying mortality rates of these influenza viruses, prevention with vaccines is important. However, the development of influenza virus vaccines worldwide is currently insignificant, and it is very difficult to manufacture influenza vaccines due to the constant evolution of viruses.

Therefore, the present inventors isolated a H3N2 serotype infectious influenza virus (A / canine / Korea / mv01 / 2012 (H3N2), accession number: KCTC 12441BP) having a matrix gene of pH1N1 virus while conducting studies on influenza virus And confirming replication ability, pathogenicity and infectivity in dogs and ferrets of the virus, thus completing the present invention.

Accordingly, an object of the present invention is to provide an influenza virus of the H3N2 serotype, which comprises a matrix gene represented by the nucleotide sequence of SEQ ID NO: 7 in which a homology with the matrix of the pH1N1 virus is homologous.

Another object of the present invention is to provide a vaccine composition for an influenza virus comprising the virus or an antigen thereof as an active ingredient, and a method of preventing or treating an influenza virus infection using the composition.

Another object of the present invention is to provide an influenza virus diagnostic kit and a diagnostic method comprising the virus or its antigen as an active ingredient.

The present invention provides an influenza virus of the H3N2 serotype comprising a gene encoding a matrix (M) matrix protein represented by the nucleotide sequence of SEQ ID NO: 7.

Hereinafter, the present invention will be described in detail.

The influenza virus of the H3N2 serotype according to the present invention comprises a gene (PB2) encoding the polymerase protein 2 represented by the nucleotide sequence of SEQ ID NO: 1, a gene encoding the polymerase protein 1 represented by the nucleotide sequence of SEQ ID NO: 2 PB1), a gene (PA) encoding a polymerase protein represented by the nucleotide sequence of SEQ ID NO: 3, a gene (HA) encoding hemagglutinin represented by the nucleotide sequence of SEQ ID NO: 4, a nucleotide sequence of SEQ ID NO: (NP), a gene encoding a neuramidase represented by the nucleotide sequence of SEQ ID NO: 6, and a gene encoding an nonsucopenic protein represented by the nucleotide sequence of SEQ ID NO: (NS). ≪ / RTI >

The gene coding for the matrix protein of the present invention is 99% or more homologous to a gene encoding a matrix protein of influenza virus of H1N1 serotype. PB2, PB1, PA, HA, NP, NA and NS genes Is characterized by having 99% or more homology with the gene of the influenza virus disclosed in GeneBank.

The virus of the present invention includes A / canine / Korea / mv01 / 2012 (H3N2), and the virus is homologous to an influenza virus, so it can be classified as an influenza virus. The virus was isolated from the nasal secretions of dogs, and its gene was analyzed. As a result, it was confirmed that the matrix gene was derived from the matrix gene of pH1N1 strain and the other genes were homologous with the influenza virus. Accordingly, the above virus was deposited with the Biological Resource Center of the Institute of Bioscience and Biotechnology on Jul. 5, 2013 and received the deposit number KCTC 12441BP.

In the case of the virus of the present invention, it was confirmed that the virus of the present invention was inoculated into dogs and pereotes, and its pathogenicity, infectivity and reproductive ability were high. Especially, it was confirmed that the virus was spread by intergalactic contacts but was not transmitted to the air Reference)

In another aspect of the present invention, there is provided a vaccine composition for influenza virus comprising the influenza virus H3N2 serotype or an antigen thereof as an active ingredient, or a pharmaceutical composition for preventing or treating an influenza virus infection disease.

The vaccine composition may be for the prevention or treatment of general influenza virus, but preferably it can be prevented or treated against H3N2 serotype influenza virus or H1N1 serotype influenza virus.

In the present invention, "antigen" is an antigenic component capable of inducing an immunological function among constituents of a virus, and is preferably a protein expressed by a virus. Thus, the antigen of the present invention comprises the polymerase protein 2 encoded by the nucleotide sequence of SEQ ID NO: 1, the polymerase protein 1 encoded by the nucleotide sequence of SEQ ID NO: 2, the polymerase protein encoded by the nucleotide sequence of SEQ ID NO: 3, 4, the nucleotide sequence encoded by the nucleotide sequence of SEQ ID NO: 5, the nucleotide sequence encoded by the nucleotide sequence of SEQ ID NO: 6, the matrix protein encoded by SEQ ID NO: 7, and the nucleotide sequence of SEQ ID NO: , And the antigens can be easily prepared from the nucleotide sequences of SEQ ID NOS: 1 to 8 of the present invention by methods well known in the art.

The vaccine may be an attenuated live vaccine, a sadox vaccine, a subunit vaccine, a synthetic vaccine or a genetic enginerring vaccine, but is preferably a live vaccine that induces an effective immune response .

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 achieved by ultraviolet (UV) irradiation, drug treatment, or in vitro high-order serial passaging. 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 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 a vaccine containing a peptide produced by recombinant DNA technology, such as HA protein and / or NA protein of 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 used to prevent other diseases including influenza by mixing with inactivated cells or antigens used in the production of other vaccines used for preventing diseases other than influenza in dogs 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.

The vaccine composition of the present invention is not, but may include an influenza virus with 2 ⁵ HAU (hemagglutination unit, hemagglutination units), limited to this.

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, eidural) 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, it is not possible to treat animals infected with various influenza virus subtypes or variants. 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, It is preferable to be an animal, a horse and an animal, a vein and an animal, a rhinoceros animal, a rodent and a primate and a bird, more preferably a dog, a pig, a chicken, a duck, a pereote, a turkey and a human being.

"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 H3N2 serotype-type 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. Radioactive isotopes include, but are not limited to, ³ H, ¹⁴ C, ³² P, ³⁶ Cl, ⁵¹ Cr, ⁵⁶ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re.

The H3N2 serotype influenza virus of the present invention has high virulence, reproducibility and infectivity in the host, and can be used as a vaccine and a diagnostic kit for various influenza virus infections. In addition, the infectivity of influenza virus can be studied using the virus.

FIG. 1 is a graph showing changes in body temperature with time in dogs inoculated with the virus of the present invention or dogs in direct contact with the dogs (IN: group inoculated with virus, DC: group in direct contact with virus infected dog, NC: Control group)
FIG. 2 is a graph showing changes in virus titer according to time in dogs inoculated with the virus of the present invention or upper respiratory tracts in direct contact with the dogs.
3 is a diagram showing lungs and organs inoculated with the virus of the present invention or lungs and organs in direct contact with the dog (A: negative control lung, B: lungs inoculated with virus, C: direct contact with virus- E: Lungs inoculated with the virus; F: Lungs in direct contact with the virus-infected dog)
FIG. 4 is a graph showing changes in body temperature with time of the isolated pearl in a space away from the pearl infected with the virus of the present invention, the pearl directly in contact with the pearl, and the virus infected pearl (IN: DC: group with direct contact with virus infected dogs, AE: airborne exposure; NC: negative control group).
FIG. 5 is a graph showing a change in body weight of a pellet inoculated with the virus of the present invention, a pellet in direct contact with the pellet, and a pellet isolated in a space away from the virus-infected pellet.
FIG. 6 is a graph showing changes in virus titer with time in the upper respiratory tract of the isolated pearl in a space away from the pearl infected with the virus of the present invention, the pearl directly in contact with the pearl, and the virus infected pearl.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example  1] H3N2  Identification of serotype of influenza virus

Influenza - Eight dog nasal specimens with similar symptoms (dog nasal secretions collected with a cotton swab) were collected at a veterinary clinic. The samples were analyzed using a commercial influenza virus antigen kit (Bionote, Hwasung, Gyeonggi, South Korea). Three out of eight specimens were positive for influenza A antigen. One of the three specimens was positive for influenza A virus and the positive sample was collected on a 10-day-old 10-month-old egg of Webster R. et al (Manual on Animal Influenza Diagnosis and Surveillance. World Health Organization) day-old embryonated chicken eggs), and the serotype was found to be H3N2 virus. The virus was designated as A / canine / Korea / mv01 / 2012 (H3N2), deposited with the Korean Agency for Bioscience and Biotechnology as of July 5, 2013 and assigned the deposit number KCTC 12441BP.

[ Example  2] H3N2  Genetic characteristics of serotype influenza virus

To identify the genetic characteristics of the virus isolated in Example 1, each gene was analyzed for the virus. Amplification and sequencing of the gene was performed as described in Hoffmann E. et. (Universal primer set for the full-length amplification of all influenza viruses. Archives of virology. 2001 Dec; 146 (12): 2275-89). More specifically, virus RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, Calif.). Then, RT-PCR was performed using a one-step RT-PCR kit (Qiagen) using primer sets commonly used in the art in order to amplify the respective viral gene fragments. Amplified portions of the gene were purified using QIAquick Gel Extraction Kit (Qiagen), followed by commercial sequencing (Cosmo genetech, Seoul, South Korea). The full-length nucleotide sequence of the isolated eight gene parts of the virus of the present invention was registered in GenBank and the sequence numbers of KF155142 to KF155149 (KF155142: PB2 (gene name), polymerase PB2 (protein); KF155143: PB1, polymerase PB1; KF155144 KF155145: NA, neuraminidase; M (M1 and M2), matrix proteins 1 and 2; KF155149: NA, neuraminidase; PA, polymerase PA and PA-X protein; KF155145: HA, hemagglutinin 1 and 2; KF155146: NP; NEP and NS1, nuclear export protein and nonstructural protein 1). The above sequences are shown in Sequence Numbers 1 to 8 in this order. In addition, the whole of the viral diarrhea according to the present invention was sequenced and compared with the sequence of other influenza A registered in GeneBank, and it was confirmed that the virus had a homology of 99.5% or more except for the matrix (M) gene.

The results of analyzing the homology of the virus gene of the present invention with the genes of other known influenza viruses are shown in Table 1 below.

gene  A virus having high homology orgin / subtype Homology (%) Registration Number SEQ ID NO: PB2 A / Canine / Korea / 01/2007 (H3N2) Canine / H3N2 99.9 JX163253 One PB1 A / Canine / Korea / GCVP01 / 2007 (H3N2) Canine / H3N2 100 FJ560889 2 PA A / Canine / Korea / GCVP01 / 2007 (H3N2) Canine / H3N2 100 FJ560888 3 HA A / Canine / Korea / 01/2007 (H3N2) Canine / H3N2 99.5 JX163256 4 NP A / Canine / Korea / GCVP01 / 2007 (H3N2) Canine / H3N2 99.9 FJ560887 5 NA A / Canine / Guangdong / 02/2007 (H3N2) Canine / H3N2 99.8 GU433363 6 M A / Korea / S1 / 2009 (H1N1) Pandemic / H1N1 99.7 CY069622 7 NS A / Canine / Korea / GCVP01 / 2007 (H3N2) Canine / H3N2 100 FJ560886 8

As shown in Table 1, except for the M gene, the other genes are 99.5% or more homologous with other H3N2 that is currently popular in China and Korea, but the M gene shows 99.7% homology with the pH1N1 virus Respectively. The above results indicate that the influenza virus of the present invention is produced by a small genetic change between the influenza virus H3N2 serotype and the pH1N1 virus.

[ Example  3] from H3N2  The pathogenicity of the serotype of influenza virus, Replication ability  And infectious confirmation

1. Inoculation of the virus of the present invention into dogs,

To confirm the replication potential, pathogenicity and intraspecies transmissibility of the viruses of the present invention, dogs were infected with the virus of the present invention. More specifically, all animal experiments were conducted at the Biosafety Level 2-plus facility within the Biotechnology Institute under the permission of the Institutional Animal Care and Use Committee (IACUC). Two 9-week-old beagle dogs (Bridge Animal Inc. Seoul, Korea) were anesthetized with Zoletil® (virvac, Carros France) and inoculated with 0.5 mL of 10 ^7.75 EID ₅₀ / mL virus. Negative-control beagle dogs were inoculated with PBS (phosphate-buffered saline). After inoculation, dog body temperature was checked according to the elapsed time. In order to confirm the infectivity caused by the direct contact of the virus of the present invention, two beagle dogs infected with the virus and two uninfected beagle dogs were contacted together, and the body temperature was checked over time. The results are shown in Fig.

As shown in Fig. 1, it was confirmed that high fever occurred in two infected dogs, and each body temperature was confirmed to be 40.1 캜 and 39.7 캜.

2. virus Potency  Confirm

In order to confirm the replicability of the virus in the host, the virus was transferred from the nasal secretion of infected dogs to the virus of the upper respiratory tract using a chicken embryo at various time points, ). In order to confirm the infectivity due to the direct contact of the virus of the present invention, two virus beagle dogs and two uninfected beagle dogs were contacted together, and then the virus titer was measured over time. The results are shown in Fig.

As shown in FIG. 2, 7.3 and 7.9 log EID ₅₀ / ml were observed in the dogs directly inoculated with two viruses, respectively.

In addition, dogs that were in direct contact with the infected dog showed lower nasal mucosa, constriction and lower fever (38.9 ° C and 38.8 ° C) than the virus-infected dog and lower virus titers in the upper respiratory trac ( , 5.6 and 5.5 log EID ₅₀ / mL).

3. Histopathological lesion confirmation

The dog was euthanized, and the pathological conditions of the lungs and bronchi were examined. The lungs and bronchi were cut and fixed in 10% neutral-buffered formalin. The fixed tissue was paraffinized and sagittal sections of each tissue (3-um thickness) were obtained. The resulting sections were stained with H & E (Hematoxylin and eosin). The results are shown in Fig.

As shown in Fig. 3, it was confirmed that lesions were present in the lungs of the dogs inoculated with the virus. And had suppurative bronchopneumonia characterized by pulmonary necrosis (Fig. 3B). Further, in the dogs, necrosis of the protoepithelial cells and suppurative necrotizing tracheitis showing the infiltration of mononuclear cells in the submucosa of the mucosa (see FIG. 3E) were confirmed.

It has also been shown that weak interstitial pneumonia characterized by interstitial infiltration and proliferation of mononuclear cells in lungs in direct contact with the virus-infected dog (see Figure 3C), but no pathological changes in the organs (See Figure 3F).

In contrast, no pathological changes were observed in the lungs or organs of the negative control group (see FIG. 3A and FIG. 3D).

4. Serological analysis

In order to reaffirm the replication ability and the transforming ability of the virus of the present invention, an HI (hemagglutination inhibition) experiment using an ELISA kit (Bionote) using an NP (Nucleoprotein) as an antigen and chicken erythrocytes (Or before direct contact) and after inoculation (or after direct contact).

Results were expressed as Percent Inhibition (PI) values. The PI value was obtained by the following formula (1) (if the PI value is 50 or more, the sample is classified as positive, and if the PI value is less than 50, the sample is classified as negative). The results are shown in Table 2 below.

[ Equation  One]

PI = (1- (OD _sample / mean OD _{negative control} ]) x 100

group
ELISA PI value HI activity 0 dpi 10 dpi 0 dpi 10 dpi Inoculation group 0.02 90.36 <10 640 Inoculation group 13.84 96.65 <10 320 Direct contact group 10.59 47.11 <10 10 Direct contact group 8.21 59.47 <10 40 PBS 3.14 2.31 <10 <10

As shown in Table 2, it was confirmed that serologic conversion (PI value:> 90, HI titer:> 320) was observed after inoculation of the virus of the present invention.

In addition, serologic conversion was observed in dogs directly in contact with virus-infected dogs (PI values: 10.59 to 47.11 and 8.21 to 59.47).

5. Conclusion

The above results confirmed that the virus of the present invention is able to replicate efficiently when the virus is infected by a dog, causes typical respiratory disease symptoms including pathological lesions of the respiratory tract, and is transmitted through direct contact. In addition, it was confirmed that the symptom was alleviated somewhat as compared with direct inoculation when direct contact was transmitted.

[ Example  4] Pearl ( ferret )in H3N2  The pathogenicity of the serotype of influenza virus, Replication ability  And infectious confirmation

One. Pearl  Body temperature change, weight change and virus Potency  Identification (direct inoculation, direct contact and aerial exposure)

Two birds of 12-week-old Ferret (Bridge Animal Inc. Seoul, Korea) were inoculated with the virus of the present invention into the nasal cavity in the same manner as in Example 3. Negative-control pearl was inoculated with PBS.

In order to confirm that the virus of the present invention is transmitted by direct contact, the virus-infected pelot and the uninfected two pelot were directly contacted with each other and isolated.

In addition, in order to confirm whether the virus of the present invention is transmitted by airborne exposure, two pellets infected with the virus and one infected with the virus were isolated from each other and spaced about 10 cm apart.

The body temperature change of the pellet was shown in FIG. 4, and the body weight change was shown in FIG. 5 after the inoculation, direct contact, and air exposure. The change in the titer of the virus is shown in Fig.

As shown in FIG. 4 to FIG. 6, it was confirmed that typical symptoms of respiratory diseases such as runny nose and high fever appeared in the pellets after the inoculation of the virus. Especially, the body temperatures were 40.5 ° C. and 40.2 ° C., respectively. In addition, significant weight loss was observed in infected pearl, and viral titer was increased to 6.2 and 5.8 log EID ₅₀ / mL in the upper respiratory tract.

In addition, pelots that were in direct contact with the virus-infected pearl showed symptoms of high fever and runny nose, significant weight loss, and increased viral titer in the upper respiratory tract to 6.2 and 5.9 log EID ₅₀ / mL .

However, in the case of air exposure, it was confirmed that no infection occurred.

2. Serological analysis

Serological changes were confirmed in the pellet in the same manner as in Example 3-4. The results are shown in Table 3 below.

group
ELISA PI value HI activity 0 dpi 10 dpi 14 dpi 0 dpi 10 dpi 14 dpi Inoculation group 11.35 97.29 97.37 <10 320 640 Inoculation group 0.12 97.65 97.79 <10 640 640 Direct contact group 18.15 20.8 96.81 <10 20 320 Direct contact group 6.54 93.83 97.88 <10 160 640 Aerial exposure 5.78 13.6 11.69 <10 <10 <10 Aerial exposure 0.26 2.54 1.08 <10 <10 <10 PBS Inoculation 0.36 1.48 0.24 <10 <10 <10

As shown in Table 3, in the case of the pellet directly inoculated with the virus and the pellet in direct contact with the pellet infected with the virus, the change in the PI value of the ELISA and the change in the HI titer were significant, In the case of the ferrets (aerial exposed group), it was confirmed that there was no change in ELISA PI value and HI titer.

3. Conclusion

Taken together, these results confirm that the virus of the present invention is infected, replicated and causes typical respiratory disease symptoms. It was confirmed that the individuals in direct contact with the virus and the infected pearl showed the same symptoms and virus proliferation as the infected individuals, but did not infect the air.

Korea Biotechnology Research Institute KCTC12441BP 20130705

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel H3N2 influenza virus and vaccine composition compirising          the same <130> P-134 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 2280 <212> DNA <213> It is PB2 gene of Influenza A virus (A / canine / Korea / MV1 / 2012 (H3N2)) <400> 1 atggaaagaa taaaagaatt aagagatcta atgtcgcagt cccgcactcg cgagatacta 60 acaaaaacca ctgtggacca tatggccata atcaagaaat acacatcagg aagacaagag 120 aagaaccctg ctctcagaat gaaatggatg atggcaatga aatatccaat cacagcagac 180 aagagaataa tggagatgat tcctgaaagg aatgaacaag ggcagatgct ttggagtaag 240 acaagtgacg ctggatcgga cagggtgatg gtgtctcccc tagctgtaac ctggtggaac 300 aggaatgggc cgacgacaag tacagtccac tatccaaagg tttacaaaac atactttgag 360 aaggttgaaa ggttaaaaca tggaaccttc ggtcccgttc atttccgaaa ccaagttaaa 420 atacgccgcc gagttgatac aaatccgggc catgcagatc tcagtgctaa agaagcacaa 480 gatgtcatca tggaggtcgt ttttccaaat gaagtgggag ctagaatatt gacatcagag 540 tcgcaattga caataacaaa agagaagaag gaagagctcc aagattgtaa gattgctcct 600 ttaatggtgg catacatgtt ggaaagagaa ctggtccgca aaaccaggtt cctaccggta 660 gcaggcggaa caagcagtgt gtacattgag gtattgcatt tgacacaagg gacctgctgg 720 gaacagatgt acactccagg cggagaagtg agaaatgacg atgttgacca gagtttgatc 780 atcgccgcca gaaacattgt taggagagca acagtatcag cggatccact ggcatcactg 840 ctggagatgt gccacagcac acaaattggt gggataagga tggtggacat ccttaggcaa 900 aatccaactg aggaacaagc tgtggatata tgcaaagcag caatgggttt gaggatcagt 960 tcatccttta gctttggagg cttcactttc aaaagaacaa gtgggtcatc cgtcaagaag 1020 gaagaggaag tgctcacagg aaacctccaa acattgaaaa taagagtaca tgaggggtat 1080 gaggaattca caatagttgg gcggagagca acagctatcc taaggaaagc aactagaagg 1140 ctgattcagt tgatagtaag tggaagagac gaacaatcaa tcgctgaagc aatcattgta 1200 gcaatggtgt tctcacagga ggattgcatg ataaaggcag tccgaggcga tctgaatttc 1260 gtaaacagag caaaccaaag attaaacccc atgcatcaac tcctgagaca cttccaaaaa 1320 gatgcaaaag tgctgtttca gaattggggg attgaaccta ttgataatgt catggggatg 1380 atcggaatat tacctgacat gactccaagc acagagatgt cactgagagg agtaagagtt 1440 agtaaaatgg gagtagatga atattccagc actgaaagag tggttgtaag tattgatcgt 1500 ttcttacggg ttcgagatca tcgggggaac gtactcttat ctcccgaaga ggtcagcgaa 1560 acacaaggaa ccgagaaatt gacaataaca tacttatcat caatgatgtg ggagatcaac 1620 ggtcctgagt cagtgcttgt taacacctat caatggatca tcagaaattg ggagaccgtg 1680 aagattcaat ggtctcaaga ccccacggta ctgtacaata agatggagtt tgaaccgttc 1740 caatccttgg tacccaaagc tgccagaggt caatacagtg gatttgtgag aacactattc 1800 caacaaatgc gtgatgtact ggggacattt gatactgtcc agataataaa gctgctacca 1860 tttgcagcag ccccaccaga gcagagcaga atgcagtttt cttctttgac tgtgaatgtg 1920 agaggctcag gaatgagaat actcgtaagg ggcaattccc ctgtgttcaa ctacaataag 1980 gcaccaaaa ggcttaccgt tcttggaaag gacgcaggtg cattaacaga ggatccagat 2040 gagggaactg ccggagtgga atctgcagta ctgaggggat tcctaatttt aggcaaagag 2100 gacaaaagat atggaccagc attaagcatc aatgaactga gcaatcttgc gaaaggggag 2160 aaagctaatg tgctgatagg gcaaggagac gtggtgttgg taatgaaacg gaaacgggac 2220 tctagcatac ttactgacag ccagacagcg accaaaagaa ttcggatggc catcaattag 2280                                                                         2280 <210> 2 <211> 2274 <212> DNA <213> It is PB1 gene of Influenza A virus (A / canine / Korea / MV1 / 2012 (H3N2)) <400> 2 atggatgtca atccgacttt acttttcttg aaagtgccag cgcaaaatgc tataagtact 60 acattccctt atactggaga tcctccatac agccatggaa cagggacagg atacaccatg 120 gacacagtca atagaacaca tcaatactca gagaaaggga aatggacaac aaacacagag 180 actggagcac cccaactcaa tccaattgat ggaccactac ccgaggataa tgagccaagc 240 ggatatgcac aaacagattg tgtgttggaa gcaatggctt tccttgaaga gtcccaccca 300 gggatctttg aaaactcatg tattgaaacg atggaagttg ttcagcaaac aagagtggac 360 aaattgaccc aaggtcgcca gacctatgac tggacattga atagaaacca gccggctgca 420 actgctttgg ccaatactat agaggtcttc agatcgaacg gtctaacagc caatgaatcg 480 ggaagactaa tagatttcct taaggatgta atggaatcaa tggacaaaga agagatggag 540 ataacaacac atttccagag aaaaagaaga gtaagggaca acatgaccaa gaaaatggtc 600 acacagagga caatagggaa gaaaaagcag aggctgaaca agaggagcta cgtaataaga 660 gcactgacat tgaacacaat gaccaaggat gcagaaagag gcaaattgaa gaggcgggca 720 attgcaacac ccgggatgca gatcagaggg ttcgtgtact ttgttgaaac attagcgagg 780 agcatctgtg agaaacttga gcagtctgga ctccctgttg gagggaatga gaagaaggct 840 aaattagcaa atgtcgtaag aaagatgatg actaactcac aagacacaga gctttccttt 900 acaattactg gagacaatac caaatggaat gagaatcaga atcctcggat gtttctggca 960 atgataacat acatcacaag aaaccaacct gaatggttta gaaatgtctt gagcattgcc 1020 cctataatgt tctcaaacaa aatggcaaga ttaggaaaag gatacatgtt cgagagtaag 1080 aacatgaaac tacggacaca aataccagca gaaatgcttg caaacattaa tctgaaatac 1140 ttcaacgaat caacaagaaa gaaaatcgag aaaataagac ctctcttaac agatggcaca 1200 gcctcactga gccctggaat gatgatgggc atgttcaaca tgctgagtac agtcttagga 1260 gtctcaatct tgaatcttgg acaaaagagg tataccaaaa ccacatactg gtgggatgga 1320 ctccaatcct ccgatgattt cgccctcata gtgaatgcac cgaatcatga ggggatacaa 1380 gcaggagtgg ataggttcta taggacctgc aaactggttg ggatcaatat gagcaaaaag 1440 aagtcttaca taaatcggac aggaacattt gagttcacaa gctttttcta ccgctatgga 1500 tttgtagcca acttcagtat ggaactgccc agctttggag tgtccggggt taacgaatcg 1560 gctgacatga gcattggagt tacggtgata aagaacaata tgataaacaa cgacctggga 1620 ccagcaacag ctcagatggc ccttcagcta ttcatcaaag actacagata cacatatcgg 1680 tgtcacaggg gtgatacaca aatccagaca agaagatcat tcgaactgaa gaagctgtgg 1740 gaacagaccc gttcaaaggc aggactgttg gtttcagatg gaggaccaaa cctatacaac 1800 atccggaatc tccacatccc cgaggtctgc ctgaagtggg aattaatgga tgaagattac 1860 cagggtagac tgtgtaatcc tctgaaccca tttgtcagtc ataaggaaat tgagtccgta 1920 aacaatgctg tggtaatgcc agctcatggc ccagccaaga gcatggaata tgatgctgtt 1980 gcgactacac actcatggat ccctaagagg aaccgttcca ttctcaatac cagccaaagg 2040 ggaattcttg aggacgaaca gatgtaccag aagtgttgca atctattcga gaaatttttc 2100 cctagtagtt catacaggag gccggttgga atttccagta tggtggaagc catggtgtct 2160 agggcccgaa ttgatgcgcg aattgacttc gagtctggaa ggattaagaa agaagagttt 2220 gctgagatcg taaagatctg ttccaccatt gaagagctca gacggcaaaa atag 2274 <210> 3 <211> 2151 <212> DNA <213> it is PA gene of Influenza A virus (A / canine / Korea / MV1 / 2012 (H3N2)) <400> 3 atggaagact ttgtgcgaca atgcttcaat ccaatgattg tcgagcttgc ggaaaaggca 60 atgaaagagt atggggaaga tccaaaaatc gaaaccaaca aattcgctgc aatatgcaca 120 catttggaag tctgtttcat gtattcggat ttccacttca ttgacgaacg gggcgaatca 180 ataattgtag aatatggtga tccaaatgca ttactgaaac accggtttga gataattgag 240 ggaagagacc gaacaatggc atggacagtg gtgaatagca tctgcaacac cacaggagtc 300 gagaagccta aatttctccc tgatttgtat gattacaagg agaaccgatt cattgaaatt 360 ggagtgacac ggagggaggt ccacatatac tacctagaaa aagccaacaa aataaaatct 420 gagaagacac acatccatat tttttcattc actggagagg agatggccac caaagcggac 480 tatactcttg atgaagagag ccgagcaaga ataaaaacca ggctgttcac aataaggcaa 540 gaaatggcca gcagggggct atgggattcc tttcgtcagt ccgaaagagg cgaagagaca 600 attgaagaaa gatttgaaat cgcaggaacc atgcgcaggc ttgccgacca aagtctccca 660 ccgaacttct ccagctttga aaattttaga gcctatgtga atggatttga accgaacgga 720 tgcattgagg gcaagctttc tcaaatgtca aaagaagtga acgccagaat tgaaccattt 780 ttgaagacaa caccacgccc tctcagatta cctgatgggc ctccctgcac ccaacggtca 840 aaattcttgc tgatggatgc tctgaaacta agcattgaag acccgagtca tgagggggag 900 gggataccgc tatacgatgc gatcaaatgc atgaagacat ttttcggctg gaaagagccc 960 aacataacca aaccacatga gaaaggcata aaccccaatt atctcttggc ttggaaacag 1020 gtgctagcag agctccagaa tattgaaaat gaggagaaaa tcccaaagac aaagaatatg 1080 aagaaaacaa gccaattaaa atgggtactt ggtgaaaata tggcaccaga aaaagtggac 1140 tttgaggatt gcaaggatgt tagcgaccta aaacaatatg atagtgatga gccagagccc 1200 agatcgctag caagctggat tcagagtgaa ttcaataagg catgtgaatt gaccgattcg 1260 agctggatag aacttgatga gataggagaa gatattgccc caattgagca cattgcaagc 1320 aagaggagga actattttac cgcggaagtg tcccattgca gggctactga atacataatg 1380 aagggagtgt acataaacac agctttgctc aatgcatctt gtgcagccat ggatgacttc 1440 caactgattc caatgataag caaatgtagg acaaaagaag ggagacggaa aacaaacctg 1500 tatgggttca ttataaaagg aaggtctcac ttgaggaatg acactgacgt ggttaatttt 1560 gtgagtatgg agttctctct tactgacccg aggctggaac cacacaaatg ggaaaagtac 1620 tgtgttcttg aaataggaga tatgctcctg aggactgcaa taggccaagt gtcaaggccc 1680 atgtttctgt atgttagaac caatggaacc tccaagatca agatgaaatg gggtatggaa 1740 atgaggcgct gccttcttca atctctccaa cagattgaga gtatgattga ggcagagtct 1800 tctgttaaag aaaaagacat gaccaaagaa ttctttgaga acagatcgga aacatggcca 1860 attggagagt cacccaaggg agtggaagaa ggctctattg ggaaagtgtg cagaacctta 1920 ttggcaaaat ccgtattcaa cagtctatat gcatctccac aactcgaggg attttcagca 1980 gaatcgagga aattgcttct cattgttcag gcacttaggg acaacctgga acctggaacc 2040 ttcgatcttg gagggctata tgaagcaatt gaggagtgcc tgattaatga tccctgggtt 2100 ttgcttaatg catcttggtt caactccttc ctcatacatg cactgaaata g 2151 <210> 4 <211> 1701 <212> DNA <213> It is an HA gene of Influenza A virus (A / canine / Korea / MV1 / 2012 (H3N2)) <400> 4 atgaaaactg ttattgcttt aagctacatt ttctgcctgg cttttggtca gaatcttcca 60 ggaaatgaaa ataatgctgc aacactatgc ctgggacatc atgcagtgcc gaacgggaca 120 atagtgaaaa ctatcacaga cgatcaaatt gaggtgacca acgccaccga gctagtccaa 180 aactcctcaa cagggaaaat atgcaacaat ccccacaaga ttcttgatgg gagagactgc 240 acactaatag atgccctact aggggacccg cactgtgatg tcttccaaaa tgagacatgg 300 gacctttttg tggaacgaag caatgctttt agcaattgtt acccttatga tgtaccagac 360 tatgcatccc ttcgatccat agttgcatca tcaggcacat tggagttcat cactgaaggt 420 ttcacttggg caggagtaac tcaaaatgga ggaagcggtg cttgcaaaag gggacctgct 480 aatggtttct tcagtagatt gaattggtta actaagtcag gaaatacata tccagtgttg 540 aatgtgacta tgccaaacaa taacaatttc gacaaattat acatttgggg agttcatcac 600 ccaagcacta atcaagaaca aaccagcctg tatattcagg cctcaggaag agtcacagtc 660 tctaccagga gaagccaaca gaccataatc ccatacattg gatctagacc cttggtaagg 720 ggccaatctg gcagaataag cgtatattgg acaatagtca aacctggaga cgtactggta 780 ataaacagta atggaaacct aatcgctcct cgaggctact tcaaaatgcg cattgggaaa 840 agctcaataa tgagatcaga tgcacctatt gacacctgca tttccgaatg tatcactccg 900 aacgggagca tccccaatga aaagcccttc caaaatgtaa acaagatcac atacggagca 960 tgtcccaaat atgttaagca aaacaccttg aaactggcaa caggaatgcg gaatgtccct 1020 gagaggcaaa ccagaggcct gttcggcgca atagcaggtt tcatagaaaa tggatgggaa 1080 gggatggtag acggttggta tggcttcagg caccaaaatt ccgaaggtac aggacaagca 1140 gcagacctta aaagcactca ggcagccatt gaccagatta atgggaaact gaacagagtg 1200 attgaaaaaa cgaatgagaa gttccatcaa atcgaaaagg agttttccga agtagaaggg 1260 aggattcaag accttgagag atacgttgaa gacacaaaag tagatctttg gtcttacaat 1320 gccgagcttc ttgttgcttt agaaaaccag aacataattg atttaactga ttcagaaatg 1380 aacaaattgt ttgaaaagac taggaggcaa ttgagggaaa atgctgaaga catgggcaat 1440 ggctgcttca agatatacca caagtgtgac aatgcttgca tagaatcgat tagaaacgga 1500 acttatgacc ataacatata tagagatgag gcagtgaaca atcggttcca gatcaaaggt 1560 gttgagctaa agtctggata caaagactgt atcttgtgga tttcctttgc catatcatgc 1620 tttttgcttt gtgttgtctt gctgggtttc attatgtggg cctgccagag aggcaacatt 1680 aggtgcaaca tttgcatttg a 1701 <210> 5 <211> 1497 <212> DNA <213> It is NP gene of Influenza A virus (A / canine / Korea / MV1 / 2012 (H3N2)) <400> 5 atggcgtctc aaggcaccaa acgatcgtat gaacagatgg aaactggtgg agaacgccag 60 aatgctaccg agatcagagc atctgttggg cgaatggttg gtggcattgg gaggttttac 120 atacagatgt gcaccgaact caaactcagc gaccatgaag gaaggctgat ccagaacagc 180 ataacaatag agagaatggt tctctctgca tttgatgaaa ggaggaacaa atacctggag 240 gaacatccca gtgcagggaa agacccgaag aaaactggag gtccaattta ccgaaggaga 300 gacgggaaat gggtgagaga gctggttctg tatgacaaag aagagatcag aagaatttgg 360 cgtcaggcga acggcgggga agatgcaact gctggtctca ctcatctaat gatctggcat 420 tccaacctaa atgatgccac ataccagaga acaagagctc tcgtgcgtac tgggttggat 480 cccaggatgt gctctctgat gcaaggatca actctaccga ggagatcagg agctgctggt 540 gcagcagtaa agggagtcgg gacaatggtg atggaactaa ttcggatgat aaagcgaggg 600 attaatgatc ggaatttctg gagaggcgaa aacggacgga gaacaagaat tgcatatgag 660 agaatgtgca acatcctcaa agggaaattc caaacagcag cacaaagagc aatgatggat 720 caggtgcgag aaagcagaaa tcctggaaat gctgaaattg aagatctcat ctttctggca 780 cggtctgcac tcatcttgag aggatcagtg gcccataagt cctgcttgcc tgcttgtgtt 840 tacggacttg ccgtggccag tggatatgat tttgagagag aagggtactc tctggttgga 900 atagatcctt tccgtctgct tcaaaacagc caggtcttca gtctcattag accaaatgag 960 aatccagcac ataagagtca gttggtgtgg atggcatgcc attctgcagc atttgaggac 1020 ctaagagtct caagtttcat cagaggaaca agagtaattc caagaggaca actatccacc 1080 agaggagttc aaattgcttc aaatgagaac atggaaaaaa tagactccag tactcttgaa 1140 ctgagaagca gatattgggc tataagaacc aggagtggag ggaacaccaa ccaacagaga 1200 gcatctgcag gacaaatcag tgtacagcca actttctcgg tacagagaaa tattcccttc 1260 gagcgagcta ccattatggc aacattcaca gggaatactg agggcagaac atctgacatg 1320 cggactgaaa tcataagaat gatggaaagt gccaaaccag aagatgtgtc tttccagggg 1380 cggggagtct tcgagctctc ggacgaaaag gcaacgaagc cgatcgtgcc ttcctttgac 1440 atgagtaacg agggatctta tttcttcgga gacaatgcag aggagtatga caattaa 1497 <210> 6 <211> 1410 <212> DNA <213> It is NA gene of Influenza A virus (A / canine / Korea / MV1 / 2012 (H3N2)) <400> 6 atgaacccaa atcagaagat aatagcaata ggctctgtct ctctaaccat tgcaacagta 60 tgtttcctct tgcagattgc catcctagca acaactgtga cactgcactt caagcaaaat 120 gaatgcaaca tcccctcgaa cagtcaagta gtgccatgta aaccaatcat aatagaaagg 180 aacataacag aggtagtata tttgaataat actaccatag aaaaagaaat ttgttccata 240 gtgctagaat acaggaactg gtcgaaaccg cagtgtcaaa ttacaggatt tgctcctttc 300 tccaaggaca actcaatccg actctccgct ggtggggaca tttgggtaac aagggaacct 360 tatgtgtcat gcgaccccag caaatgttac cagtttgcac ttgggcaggg gaccacgctg 420 aacaataaac actcaaacgg cacaatacat gataggatct ctcatcgaac tcttttaatg 480 aatgagttgg gtgttccgtt tcatttggga accaaacaag tgtgcatagc atggtccagt 540 tcaagttgtc acgatgggaa agcatggtta catgtttgtg tcactgggga tgatagaaat 600 gcgactgcta gtttcgttta taatggaatg cttgttgaca gtattggttc atggtctcga 660 aatatcctca gaactcagga gtcagaatgc gtttgcatta atggaacttg tacagtagta 720 atgactgatg gaagtgcatc aggaagggct gatactagaa tactattcat cagagagggg 780 aaaattgtcc atattagccc attgtcaggg agtgctcaac atatagagga atgttcctgt 840 tatcctcgat atccaaatgt tagatgtgtt tgcagagaca attggaaggg ctctaatagg 900 cccgttatag atataaatat ggcagattat agcatcgatt ccagttatgt gtgttcagga 960 cttgttggcg acacaccaag gaatgatgat agctctagca gcagcaactg cagggatcct 1020 aataatgaga gagggaatcc aggagtgaaa gggtgggctt ttgataatga gaatgacgtt 1080 tggatgggga ggacaatcag caaagatttg cgctcaggtt atgagacttt caaggtcatt 1140 ggtggctgga ccactgctaa ttccaagtta caggtcaata gacaagtcat agttgacaat 1200 aataactggt ctggttattc tggtattttc tccgttgaag gcaaaagctg tgttaatagg 1260 tgtttttatg tggagttgat aagaggaggg ccacaagaga ctagagtatg gtggacttca 1320 aatagcattg tcgtattttg tggtacttct ggtacctatg gaacaggctc atggcctgat 1380 ggggcgaata tcaacttcat gcctatataa 1410 <210> 7 <211> 982 <212> DNA <213> It is M gene of Influenza A virus (A / canine / Korea / MV1 / 2012 (H3N2)) <400> 7 atgagtcttc taaccgaggt cgaaacgtac gttctttcta tcatcccgtc aggccccctc 60 aaagccgaga tcgcgcagag actggaaagt gtctttgcag gaaagaacac agatcttgag 120 gctctcatgg aatggctaaa gacaagacca atcttgtcac ctctgactaa ggggatttta 180 ggatttgtgt tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240 caaaatgccc taaatgggaa tggggacccg aacaacatgg atagagcagt taaactatac 300 aagaagctca aaagagaaat aacgttccat ggggccaagg aggtgtcact aagctattca 360 actggtgcac ttgccagttg catgggcctc atatacaaca ggatgggaac agtgaccaca 420 gaagctgctt ttggtctagt gtgtgccact tgtgaacaga ttgctgattc acagcatcgg 480 tctcacagac aaatggctac taccaccaat ccactaatca ggcatgaaaa cagaatggtg 540 ctggctagca ctacggcaaa ggctatggaa cagatggctg gatcgagtga acaggcagca 600 gaggccatgg aggttgctaa tcagactagg cagatggtac atgcaatgag aactattggg 660 actcatccta gctccagtgc tggtctgaaa gatgaccttc ttgaaaattt gcaggcctac 720 cagaagcgaa tgggagtgca gatgcagcga ttcaagtgat cctctcgtca ttgcagcaaa 780 tatcattggg atcttgcacc tgatattgtg gattactgat cgtctttttt tcaaatgtat 840 ttatcgtcgc tttaaatacg gtttgaaaag agggccttct acggaaggag tgcctgagtc 900 catgagggaa gaatatcaac aggaacagca gagtgctgtg gatgttgacg atggtcattt 960 tgtcaacata gagctagagt aa 982 <210> 8 <211> 838 <212> DNA <213> It is NS gene of Influenza A virus (A / canine / Korea / MV1 / 2012 (H3N2)) <400> 8 atggactcca acaccgtgtc aagctttcag gtagactgct ttctttggca tgtccgcaaa 60 cgatttgcag accaagaact gggtgatgcc ccattccttg atcggcttcg ccgagatcag 120 aagtccctaa aaggaagagg cagcaccctt ggtctggaca tcgaaacagc tactcgtata 180 ggaaagcaga tagtggagtg gattctggag gaagaatccg ataaggcact taaaatgacc 240 attgcttcag ttccggcttc acgctaccta actgatatga ctcttgaaga aatgtcaaga 300 gactggttca tgctcatgcc aaagcagaaa gtggcaggtt ccctttgcat caaaatggac 360 caggcaataa tggataaaaa catcacattg aaagcaaact tcagtgtgat ttttgagcga 420 ctggaaaccc taatactact tagagctttc acaaacgaag gagcaattgt gggagaaatt 480 tcaccgttac cttctcttcc aggacatact gataaggatg tcaaaaatgc aattggggtc 540 ctcatcggag gactggaatg gaatgataac acagttcgag tctctgaaac tctacagaga 600 ttcgcttgga gaagcagtga tgagaatggg agaccttcac tccctccaaa gcagaaacgg 660 aaaatggcga gaacaattga gtcagaaatt tgaggaaata aggtggctga tcgaagaagt 720 acggcataga ttgaagatta cggaaaacag ctttgaacaa ataacattta tgcaagcctt 780 acaactattg cttgaggtgg agcaggagat aagaactttc tcgtttcagc ttatttaa 838

Claims (15)

An influenza virus of the H3N2 serotype comprising a gene encoding a matrix (M) matrix protein represented by the nucleotide sequence of SEQ ID NO: 7. 2. The method according to claim 1, wherein the virus comprises a gene (PB2) encoding the polymerase protein 2 represented by the nucleotide sequence of SEQ ID NO: 1, a gene (PB1) encoding the polymerase protein 1 represented by the nucleotide sequence of SEQ ID NO: A gene (PA) encoding a polymerase protein represented by the nucleotide sequence of SEQ ID NO: 3, a gene (HA) encoding hemagglutinin represented by the nucleotide sequence of SEQ ID NO: 4, a nucleotide sequence of SEQ ID NO: (NP), a gene encoding a neuramidase represented by the nucleotide sequence of SEQ ID NO: 6, and a gene (NS) encoding an acetylation protein represented by the nucleotide sequence of SEQ ID NO: The influenza virus of the H3N2 serotype additionally comprising at least one member selected from the group consisting of: 2. The method according to claim 1, wherein the gene encoding the matrix protein is 99% or more homologous to a gene encoding a matrix protein of A / Korea / S1 / 2009 (H1N1) influenza virus. Influenza virus. 3. The method according to claim 2, wherein the gene (PB2) encoding the polymerase protein 2 represented by the nucleotide sequence of SEQ ID NO: 1 and the gene (HA) encoding the hemagglutinin represented by the nucleotide sequence of SEQ ID NO: / Canine / Korea / 01/2007 (H3N2) having 99% or more homology with serotype influenza virus,
A gene (PB1) encoding a polymerase protein 1 represented by the nucleotide sequence of SEQ ID NO: 2, a gene (PA) encoding a polymerase protein represented by the nucleotide sequence of SEQ ID NO: 3, a gene The gene (NS) encoding the nucleoprotein-encoding protein represented by the nucleotide sequence of SEQ ID NO: 8 and the gene (NS) encoding the nonsucopenic protein of the nucleotide sequence of SEQ ID NO: % Or more homology,
The gene (NA) encoding the neuramidase represented by the nucleotide sequence of SEQ ID NO: 6 is characterized by having 99% or more homology with the influenza virus of serotype A / Canine / Guangdong / 02/2007 (H3N2) Influenza virus of the H3N2 serotype. The influenza virus according to claim 1, wherein said virus is an influenza virus of accession number KCTC 12441BP. A vaccine composition for an influenza virus of the H3N2 serotype comprising the influenza virus of the H3N2 serotype of any one of claims 1 to 5 as an active ingredient. delete delete The vaccine composition according to claim 6, wherein the vaccine is at least one selected from the group consisting of attenuated live vaccine and live vaccine. delete delete A composition for influenza virus diagnosis comprising an influenza virus of the H3N2 serotype of any one of claims 1 to 5. An influenza virus diagnostic kit comprising influenza virus of the H3N2 serotype of any one of claims 1 to 5. A method for providing information on the diagnosis of an infection of an influenza virus, comprising the step of treating the composition of claim 12 to a sample. 15. The method according to claim 14, wherein the sample is at least one selected from the group consisting of cells expected to be infected with influenza virus, infected cells, blood, urine, saliva, and tissue. How to.

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