KR20120082796A - Influenza virus capable of infecting cat and use thereof - Google Patents

Abstract

PURPOSE: An influenza virus with infectivity to Felis catus is provided to effectively detect anti-influenza virus H3N2 antibody in a sample. CONSTITUTION: An influenza virus is A/feline/Korea/01/2010 (H3N2)(deposit number KCTC 11849BP), A/canine/Korea/01/07 (H3N2) (deposit number KCTC 11205BP), A/canine/Korea/02/07 (H3N2) (deposit number KCTC 11206BP), or A/canine/Korea/03/07 (H3N2) (deposit number KCTC 11207BP). A method for detecting anti-cat influenza virus H3N2 antibody in a sample isolated from Felis catus comprises: a step of contacting the H3N2 antigen with the sample; a step of detecting the presence of a complex of cat influenza virus H3N2 antigen and anti-cat influenza virus H3N2 antigen; and a step of determining the sample is anti-cat influenza virus H3N2 antigen in case of the presence of the complex.

Description

Influenza virus capable of infecting cats and use thereof

The present invention relates to influenza viruses infectious to cats and methods of using the same.

Influenza is a disease caused by influenza virus type A of orthomyxoviridae and is an economically important disease in humans, pigs, horses, birds and the like. The serotypes of influenza A are classified according to two types of proteins, hemagglutinin and neuraminidase, and there are H serotypes and N serotypes. The serotypes of influenza viruses are indicated by their respective H and N serotypes (e.g., H9N2). Avian influenza viruses have 16 H serotypes and 9 N serotypes, which are arithmetically influenza viruses. There are 144 serotypes of.

Influenza is a common infectious disease, and the influenza virus is highly mutant and has the potential to spread directly from one species to another, making it a challenge to address the worldwide spread of highly pathogenic influenza. However, influenza virus transmission to an entirely new species that is unrelated has been reported but not uncommon.

On the other hand, canine influenza virus is a new virus and is susceptible to dogs because dogs are not currently vaccinated or naturally immunized. Canine influenza virus causing acute respiratory disease has clinical symptoms such as severe cough, fever and nasal secretions.

Dog influenza was first reported in 2004 at the Greyhound Dog Raceway in Florida, USA. Since then, influenza outbreaks have occurred in Texas, Alabama and Arkansas in the United States. Epidemiological studies of canine influenza in the United States have shown that it is nearly identical to equine influenza H3N8, suggesting that influenza transfer from horses to dogs may have resulted in the production of a new canine influenza virus. Although there have been reports of hemorrhagic pneumonia caused by horse influenza H3N8 and human influenza H3N2 isolated from dogs, the serological and virological evidence of influenza from dogs has not been fully provided.

In addition, an example of avian influenza propagation to dogs has been reported, and the mechanism by which avian influenza propagates to dogs can be estimated to have two dynamics. One is the possibility of viral transmission by feeding raw by-products of ducks and chickens from dog farms, and the other by direct contact or respiratory transmission of dogs infected with normal and avian influenza. . It is speculated that the infected dogs would be exposed to other environments and come into contact with other dogs to help establish the influenza.

Despite this prior art, no influenza virus, such as canine influenza virus, that can infect cats has been reported.

The inventors have found for the first time that the influenza virus can infect cats and thus cause cats with severe respiratory diseases and have completed the present invention.

One aspect is to provide a method for diagnosing influenza virus H3N2 infection in a cat.

Another aspect is to provide a method of detecting anti-influenza virus H3N2 antibodies in a sample derived from cats.

Another aspect is to provide a method for detecting influenza virus H3N2 antigen in a cat derived sample.

Another aspect is to provide a method of searching for a composition for preventing or treating influenza virus H3N2 infection in a cat.

Another aspect is to provide a kit for detecting influenza virus H3N2 in a sample.

Another aspect is to provide novel influenza virus H3N2 A / feline / Korea / 01/2010 (H3N2) and antibodies binding thereto.

Another aspect is to provide a composition and a method for preventing or treating influenza virus H3N2 infection in a cat.

One aspect of the present invention provides a method for diagnosing influenza virus H3N2 infection in a cat, comprising detecting influenza virus H3N2 in a sample isolated from the cat.

The cat may be a cat suspected of being infected with influenza virus H3N2. The cat may be, for example, a cat suffering from severe respiratory disease. The sample may be any one separated from the cat. For example, the sample may be blood, serum or plasma. The influenza virus H3N2 can infect a cat and includes any H3N2 type. The inventors have surprisingly found that the influenza virus, conventionally known as canine influenza virus H3N2, can also infect cats. Thus, the cat influenza virus H3N2 is used in the sense containing the dog influenza virus H3N2. For example, the feline influenza virus is A / feline / Korea / 01/2010 (H3N2) (Accession No. KCTC 11849BP), A / canine / Korea / 01/07 (H3N2) (Accession No. KCTC 11205BP), A / canine / Korea / 02/07 (H3N2) (Accession No. KCTC 11206BP), or A / canine / Korea / 03/07 (H3N2) (Accession No. KCTC 11207BP).

In the detecting step, a virus detection method known in the art may be used. For example, it may be using an antigen antibody reaction or using a nucleic acid amplification reaction. The detection may be to detect influenza virus H3N2 directly or indirectly to detect its presence. Direct detection may include the direct isolation of influenza virus H3N2, and indirect detection may include the detection of the antigen of influenza virus H3N2 and the antibody binding thereto, or the genomic sequence, mRNA or product expressed therefrom of influenza virus H3N2. It involves doing. The method comprises, for example, detecting an antibody that binds to influenza virus H3N2 antigen or influenza virus H3N2 antigen in a sample isolated from a cat; And when it is detected that there is an antibody binding to the influenza virus H3N2 antigen or influenza virus H3N2 antigen, the cat is determined to be infected with influenza virus H3N2, and the antibody that binds to the influenza virus H3N2 antigen or influenza virus H3N2 antigen If it is detected that it does not exist, it may include determining that the cat is not infected with influenza virus H3N2.

Another aspect of the present invention provides a method for treating an influenza virus H3N2 antigen with a sample isolated from a cat; Detecting the presence of a complex of the influenza virus H3N2 antigen and an anti-influenza virus H3N2 antigen antibody, and if the complex is detected, the sample is determined to contain an anti-influenza virus H3N2 antibody and the complex Is determined to be absent, the sample provides a method of detecting anti-influenza virus H3N2 antibody in a sample isolated from a cat, comprising determining that the sample does not comprise anti-influenza virus H3N2 antibody.

The method includes contacting the influenza virus H3N2 antigen with a sample isolated from the cat. The contact can be carried out by mixing and incubating the sample with the influenza virus H3N2 antigen in a liquid medium. The liquid medium includes anything that can provide a medium for antigen-antibody reactions without destabilizing the influenza virus H3N2 antigen and antibody. For example, the liquid medium includes a buffer, such as a PBS buffer. The contact can be used conditions known in the art to form a complex of antigen and antibody. For example, the contact can be carried out at 4 to 37 ℃. In addition, the contact can be carried out with or without stirring.

The influenza virus H3N2 antigen may be immobilized on a solid substrate. The solid substrate may have one or more shapes selected from well, plate and bead shapes. The well may be a micro well. The cat may be a cat suspected of being infected with influenza virus H3N2. The cat may be, for example, a cat suffering from severe respiratory disease. The sample may be any one separated from the cat. For example, the sample can be cat blood, serum or plasma.

The method includes detecting the presence of a complex of the influenza virus H3N2 antigen and anti-influenza virus H3N2 antibody. The detection may be an antigen-antibody complex detection method known in the art. The detection may be made by, for example, an electrical method, a spectroscopic method, or a combination thereof. In addition, the detection may be made by a known ELISA analysis process. Anti-cat anti-influenza virus H3N2 antibodies labeled with a detectable label can be made by binding to the antibody and detecting a signal from the label of the bound product. The detectable label can be a fluorescent label, an electrical label, a radioactive material, an enzyme having the activity of converting the substrate into a spectroscopically detectable material, or a combination thereof. The enzyme may comprise one or more selected from alkaline phosphatase, horseradish peroxidase and beta-galactosidase.

The method determines that the sample comprises an anti-influenza virus H3N2 antibody when the complex is detected, and wherein the sample detects the anti-influenza virus H3N2 antibody when the complex is detected. Determining not to include. The influenza virus H3N2 is A / feline / Korea / 01/2010 (H3N2) (Accession No. KCTC 11849BP), A / canine / Korea / 01/07 (H3N2) (Accession No. KCTC 11205BP), A / canine / Korea / 02 / 07 (H3N2) (Accession No. KCTC 11206BP) or A / canine / Korea / 03/07 (H3N2) (Accession No. KCTC 11207BP).

Another aspect of the invention provides a method of administering influenza virus H3N2 to a non-human mammal animal; And separating the antibody which binds to the protein from the animal.

The method comprises administering a protein as described above to a non-human mammalian animal. The animal may be an animal used for antibody production in the art. For example, it can be a cat, mouse, rabbit, sheep, pig or cow. Such administration can be used administration methods known in the art for producing antibodies in an animal. For example, the animals may be administered with an amount sufficient to induce an immune response, followed by boost administration two weeks later.

The method may comprise the step of isolating an antibody that binds to the protein from the animal. The separation can be used for antibody separation methods known in the art. For example, affinity chromatography using the protein as a ligand or an antibody that specifically binds to the antibody may be used.

Another aspect of the present invention provides a method of preparing a kit comprising contacting an anti-influenza virus H3N2 antibody with a sample isolated from a cat; Detecting a complex of the antibody with an influenza virus H3N2 antigen; And when the complex is detected to be present, the sample is determined to contain influenza virus H3N2 antigen, and when the complex is detected to be absent, the sample is determined to be free of influenza virus H3N2 antigen. It provides a method for detecting influenza virus H3N2 antigen in a sample, comprising the step.

The method comprises contacting an anti-influenza virus H3N2 antibody with a sample isolated from a cat. Anti-influenza virus H3N2 antibodies may be prepared according to the methods described above. The contact may be performed by mixing and incubating the sample with the anti-influenza virus H3N2 antibody in a liquid medium. The liquid medium includes anything that can provide a medium for antigen-antibody reactions without destabilizing the anti-influenza virus H3N2 antibody and the sample. For example, the liquid medium includes a buffer, such as a PBS buffer. The contact can be used conditions known in the art to form an antigen and a complex of antigens. For example, the contact can be carried out at 4 to 37 ℃. In addition, the contact can be carried out with or without stirring.

The antibody may be immobilized on a solid substrate. The solid substrate may have one or more shapes selected from well, plate and bead shapes. The well may be a micro well. The sample may include any sample derived from a cat. For example, the sample can be cat blood, serum or plasma.

The method comprises detecting the presence of a complex of the antibody and influenza virus H3N2 antigen. The detection may be an antigen-antibody complex detection method known in the art. The detection may be made by, for example, an electrical method, a spectroscopic method, or a combination thereof. In addition, the detection may be made by a known ELISA analysis process. Anti-influenza virus H3N2 labeled with a detectable label can be made by binding the antigen and detecting a signal from the label of the bound product. The detectable label can be a fluorescent label, an electrical label, a radioactive material, an enzyme having the activity of converting the substrate into a spectroscopically detectable material, or a combination thereof. The enzyme may comprise one or more selected from alkaline phosphatase, horseradish peroxidase and beta-galactosidase.

The method determines that the sample contains an anti-influenza virus H3N2 antigen when the complex is detected, and wherein the sample determines the anti-influenza virus H3N2 antigen when the complex is detected. Determining not to include. The influenza virus H3N2 is A / feline / Korea / 01/2010 (H3N2) (Accession No. KCTC 11849BP), A / canine / Korea / 01/07 (H3N2) (Accession No. KCTC 11205BP), A / canine / Korea / 02 / 07 (H3N2) (Accession No. KCTC 11206BP) or A / canine / Korea / 03/07 (H3N2) (Accession No. KCTC 11207BP).

Another aspect of the invention is a method of searching for a composition for preventing or treating influenza virus H3N2 infection, comprising using the influenza virus H3N2 antigen to search for a composition for preventing or treating influenza virus H3N2 infection in a cat. .

The influenza virus H3N2 antigen may be an influenza virus H3N2 virus particle itself or a surface protein thereof. The search attenuates the influenza virus H3N2 virus itself to search for an attenuated influenza virus H3N2 virus vaccine that is immunogenic to the cat and not lethal to cats for use as a vaccine, or a subunit of the influenza virus H3N2 virus. The cat may have an immunogen to detect whether it is effective in preventing or treating infection with the influenza virus H3N2 virus. The search process may use methods known to those skilled in the art related to attenuated viruses or subunit vaccines of viruses. The attenuation includes attenuation by treatment of chemicals such as formalin, ultraviolet treatment or passaging. The attenuation includes a complete loss of the virus's ability to propagate, ie a deadly poison vaccine. The influenza virus H3N2 is A / feline / Korea / 01/2010 (H3N2) (Accession No. KCTC 11849BP), A / canine / Korea / 01/07 (H3N2) (Accession No. KCTC 11205BP), A / canine / Korea / 02 / 07 (H3N2) (Accession No. KCTC 11206BP) or A / canine / Korea / 03/07 (H3N2) (Accession No. KCTC 11207BP).

Another aspect of the invention provides a kit for detecting feline influenza virus, comprising influenza virus H3N2 or an antibody that binds to influenza virus H3N2. The kit may further include a reagent capable of detecting a complex of the influenza virus H3N2 and an anti-influenza virus H3N2 antibody. The reagent may be an antibody that binds to an anti-influenza virus H3N2 antibody or an anti-influenza virus H3N2 antibody labeled with a detectable label. The detectable label can be a fluorescent label, an electrical label, a radioactive material, an enzyme having the activity of converting the substrate into a spectroscopically detectable material, or a combination thereof. The enzyme may include alkaline phosphatase, horseradish peroxidase and beta-galactosidase. The influenza virus H3N2 is A / feline / Korea / 01/2010 (H3N2) (Accession No. KCTC 11849BP), A / canine / Korea / 01/07 (H3N2) (Accession No. KCTC 11205BP), A / canine / Korea / 02 / 07 (H3N2) (Accession No. KCTC 11206BP) or A / canine / Korea / 03/07 (H3N2) (Accession No. KCTC 11207BP). The kit may comprise one or more selected from the group consisting of standard controls, diluents and buffers.

Another aspect of the present invention provides a kit for detecting influenza virus H3N2 in a sample comprising the antibody obtained according to the antibody production method.

Another aspect of the invention provides a novel feline influenza virus A / feline / Korea / 01/2010 (H3N2) (Accession No. KCTC 11849BP) capable of infecting cats.

Another aspect of the invention provides antibodies that bind to novel feline influenza virus A / feline / Korea / 01/2010 (H3N2) (Accession No. KCTC 11849BP) capable of infecting cats.

Another aspect of the invention provides a composition for the prevention or treatment of influenza virus H3N2 infection in a cat, comprising attenuated influenza virus H3N2 or an immunogenic subunit of influenza virus H3N2.

The influenza virus H3N2 is A / feline / Korea / 01/2010 (H3N2) (Accession No. KCTC 11849BP), A / canine / Korea / 01/07 (H3N2) (Accession No. KCTC 11205BP), A / canine / Korea / 02 / 07 (H3N2) (Accession No. KCTC 11206BP) or A / canine / Korea / 03/07 (H3N2) (Accession No. KCTC 11207BP). The attenuation may be performed by a method known in the art. The attenuated influenza virus H3N2 may be attenuated by chemical treatment, for example, formalin treatment, or attenuated by ultraviolet irradiation or passage. In the present invention, "attenuation" refers to a toxicity that is not fatal enough to induce an immune response in a cat but can be used as a vaccine in a cat. The attenuation includes not only the virus's ability to proliferate but also its complete loss. The "subunit" refers to a part of the virus, not the entire virus. Such subunits include proteins, for example individual proteins such as HA or NA, as well as complexes thereof. The complex may be, for example, obtained by grinding and obtaining only an envelope by treating ether with a virus particle.

Attenuated viruses include, for example, (a) propagating by injecting feline influenza virus H3N2, such as canine influenza virus H3N2, into a hatching egg; (b) obtaining the intestinal umbilical fluid from the embryonated egg and treating it with formalin, betapropiolactone (BPL) or binary ethylenimine (BEI); And (c) obtaining an inactivated virus from the treated intestinal urea solution. Step (a) is injecting feline influenza virus H3N2, for example, canine influenza virus H3N2 into hatching eggs of 9 to 11 days of age, and hatching eggs to incubate the virus for 24 to 72 hours at 30 to 40 ° C. Can be. Step (b) may be to obtain the intestinal umbilical fluid in a conventional manner, in which formalin, BEI or BPL is added at a final concentration of 0.005 to 0.2 (v / w)% and left at low temperature to inactivate the virus. Step (c) may be to obtain the inactivated virus through centrifugation or filtration of the formalin, BEI or BPL treated ileum. The virus obtained can be adsorbed onto alumina hydroxide gel.

The composition may comprise a veterinary acceptable carrier known in the art. The carrier may be selected from the group consisting of a solvent, an adjuvant, and an excipient. The solvent may be a buffer such as saline, distilled water or PBS. The adjuvant may be selected from the group consisting of Freund's complete or incomplete adjuvant, aluminum hydroxide gel and oil. The excipient may be aluminum phosphate, aluminum hydroxide or aluminum potassium sulfate (alum).

The composition may include influenza virus H3N2 virus as 2 ⁶ HAU (hemagglutination unit).

The composition may be prepared in oral or parenteral preparation. Examples of parenteral preparations include injection solutions. The composition may be one having a formulation that can be administered by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal or epidural route. In the present invention, "feline" refers to an animal belonging to the Felidae family, for example, felinae, pantherinae, machairodontinae ), And animals belonging to the proailurinae subfamily. The animal can be, for example, Felis catus belonging to the felinae subfamily.

Another aspect of the invention provides a method of preventing or treating influenza virus H3N2 infection in a cat, comprising administering the composition to the cat.

The cat may be a cat infected with or suspected of having an influenza virus. The cat may exhibit influenza virus symptoms. For example, it may be a symptom selected from sinusitis, paroxysmal asthma, otitis media, cystic fibrosis, bronchitis, pneumonia and diarrhea.

As used herein, the term "prevention" means to inhibit influenza virus infection or to delay the onset by administration of the composition. As used herein, the term "treatment" means any action that improves or advantageously alters the symptoms caused by influenza virus infection by 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, with an effective dose level of the individual type and severity, age, sex, type of virus infected, drug Activity, sensitivity to drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

In this method, administration may be administered orally or parenterally. For example, the composition can be administered by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal or epidural routes.

According to the method for diagnosing the influenza virus (CIV) H3N2 infection in a cat of the present invention, it is possible to efficiently diagnose whether the cat is infected with influenza virus H3N2.

According to the method for detecting the anti-influenza virus H3N2 antibody in a sample derived from the cat of the present invention, the anti-influenza virus H3N2 antibody in the sample derived from the cat can be detected efficiently.

According to the method for detecting influenza virus H3N2 antigen in a sample derived from a cat of the present invention, influenza virus H3N2 antigen in a sample derived from a cat can be detected efficiently.

According to the method for searching for a composition for preventing or treating influenza virus H3N2 infection in a cat of the present invention, it is possible to efficiently search for a composition for preventing or treating influenza virus H3N2 infection in a cat.

According to the kit for detecting influenza virus H3N2 in a sample of the present invention, it can be used to detect influenza virus H3N2 in a sample.

According to the novel influenza virus A / feline / Korea / 01/2010 (H3N2) (Accession No. KCTC 11849BP) of the present invention and the antibody binding thereto, it can be used for applications related to influenza virus H3N2 infection in cats.

According to the composition for preventing or treating influenza virus H3N2 infection in a cat of the present invention, it is possible to prevent or treat influenza virus H3N2 infection in a cat.

1 is a diagram showing the RT-PCR results of isolate A / feline / Korea / 01/2010 (H3N2).
2 and 3 are diagrams showing a generational position of A / feline / Korea / 01/2010 (H3N2).
4 is a diagram showing rectal temperature and virus titer with days post infection.
5 shows clinical symptoms after infection.
Figure 6 shows the change in rectal body temperature with time after challenge inoculation.
7 is a diagram showing virus release through nasal juice over time after challenge inoculation.
FIG. 8 shows antibody titers in cats and guinea pigs according to dilution multiples of vaccine. FIG.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

In early 2010, we isolated the canine-origin cat H3N2 influenza virus from a dead cat with a severe respiratory disease. Experimentally, we infected domestic cats with isolated influenza virus H3N2 (A / feline / Korea / 01/2010 (H3N2)) to investigate whether the virus causes respiratory infections and diseases in cats again. . The infected animals all had fever, nasal virus shedding and severe pulmonary lesions. This finding indicates that isolated feline influenza virus is infected, pathogenic, and spread in cats.

1. Materials and Methods

(1) History of occurrence

In December 2009 and January 2010, an explosion of flu-like severe respiratory disease was identified in cat and dog kennels in Seoul, Korea. A total of 200 dogs and 50 cats suffered from this disease. After respiratory symptoms, dogs showed 25% mortality, while cats showed 100% morbidity and respiratory symptoms such as tachypnea, dyspnea, and lethargy. After about 40% mortality. A veterinary autopsy examined a cat with severe pneumonia, characterized by reddish dark consolidation of about 30-70% of the total lung, accompanied by pulmonary edema in five cats. .

In five dead cats with severe respiratory disease, influenza virus infection was confirmed, and 60% (3/5) were infected with influenza virus by the antigen detection Rapid kit (Bionote, Korea). The virus was isolated in 20% (1/5) of embryonated chicken eggs. The sample used for confirmation used the frozen sample.

(2) Separator  ( isolate ) Detection and Characterization

For detection of influenza virus antigens in lung specimens and nasal swabs, a detection experiment is carried out using a commercially available Rapid Kit (Bionote, Korea) according to the manufacturer's instructions and using lung and nasal swabs as samples. And nucleocapsid protein (NP) of influenza A virus was detected.

The analysis revealed a positive signal in three cats. Virus isolation was performed in 11 day old embryonated eggs and subtyping was performed by RT-PCR.

Sequencing of the 8 gene segments from the isolate was performed. The sequence of the isolated viral gene was analyzed using Bioedit software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Phylogenetic trees were generated using the Molecular Evolutionary Genetics Analysis (MEGA version 4.1) program (http://www.megasoftware.net) together with the ClustalX alignment algorithm.

(3) A / to your home cat feline Of Korea / 01/2010 ( H3N2 Experimental Infection of Viruses

All animal experiments were in accordance with Korean law. Animal care and treatment was in accordance with the guidelines established by the Green Cross Veterinary Products Institutional Animal Care and Use Committee.

Five four-month-old conventional Korean shorthair cats were assigned to two treatment groups (infected group [n = 3] and non-infected group [n = 2]). Each cat group was placed in a separate isolator under negative pressure in a separation facility of Green Cross Medicine (Yongin, Korea). At the start of the experiment all cats were seronegative against influenza virus. A / feline / Korea / 01/2010 (H3N2) (Accession No. KCTC 11849BP) virus isolated from dogs with severe respiratory disease was used as inoculum.

Infected cats were inoculated intranasally with 1 ml of phosphate-buffered saline (PBS) containing 10 ^6.5 egg infectious dose (EID ₅₀ ), whereas non-infected cats were inoculated with only 1 ml of PBS. It became. After inoculation, rectal temperature and clinical symptoms were observed. Nasal swab samples were collected on days post-inoculation (dpi) at 0, 2, 4, and 7 inoculations to examine shedding viral titers using real-time RT-PCR. At 7 dpi, all cats were sacrificed for viral antigen detection experiments by pathological and immunochemical methods. Serum antibody titers against influenza A nucleoprotin were tested using a commercially available ELISA kit (Bionote, South Korea).

(4) nucleotide sequence

The sequences of the eight viral gene segments of influenza virus isolated from house cats are as follows.

Hemagglutinin (HA), neuraminidase (NA), matrix protein (M), PA (polymerase PA: PA) of A / feline / Korea / 01/2010 (H3N2), PB1 (polymerase PB1: PB1), PB2 (polymerase PB2: PB2), NP (nucleoprotein: NP), and NS genes (Non-structural proteins: NS) each have a nucleotide sequence of SEQ ID NOs: 1-8.

Example  1: Virus Separation from Field Samples

Influenza virus was isolated from hatching eggs inoculated with lung suspension of dead cats. The isolate was identified as subtype H3N2 type by RT-PCR (FIG. 1). 1 is a diagram showing the RT-PCR results of the isolate. In FIG. 1E, it was confirmed that the virus detection experiment was performed using a rapid diagnosis kit for influenza virus antigen diagnosis. In F, RT-PCR was performed using primers specific for H3 and N2 genes of influenza H3. And the band corresponding to N2 was confirmed.

By PCR methods and commercially available antigen detection kits (Bionote, South Korea), lung specimens were cat feline calicivirus, herpesvirus, feline infectious peritonitis virus (FIPV), And other viral agents, including feline immunodeficiency virus (FIV). However, telra chevron kisep to VOR in the waste sample urticae (Bordetella bronchiseptica ) was confirmed with co-infection.

This isolated strain was named A / feline / Korea / 01/2010 (H3N2). The new A / feline / Korea / 01/2010 (H3N2) stock was deposited on January 21, 2011 with KCTC (Korean Collection for Type Cultures) under the Korea Deposit Research Institute, an international depository institution under Article 7 of the Budapest Treaty. The accession number is KCTC 11849BP.

Example 2 A / feline / Korea / 01/2010 ( H3N2 ) Virus base sequence and developmental system diagram

From the cat influenza isolates, the 8 gene segments (H3, N2, M, NS, NP, PB1, PB2 and PA) were completely sequenced and homologous sequences were retrieved from GenBank. Overall, the base sequence of the cat isolate was 98.0% to 99.8% homologous to all 8 gene segments of H3N2 canine influenza virus (CIV) (data not shown). The influenza isolate was named A / feline / Korea / 01/2010 (H3N2).

As a result of developmental analysis, the HA and NA gene segments of A / feline / Korea / 01/2010 (H3N2) were clustered in a manner similar to that of CIV H3N2 detected in Korea in 2007-2010 (FIGS. 2 and 3). 2 and 3 are diagrams showing a generational position of A / feline / Korea / 01/2010 (H3N2). Similarly, internal genes (PB2, PB1, PA, NP, M and NS) were also closely related to those of CIV H3N2 detected in Korea (data not shown).

Example  3: house From cat  Pathogenicity of Influenza Viruses

Clinical symptoms such as sneezing, coughing, abdominal breathing and nasal discharges were observed in cats inoculated with the isolated influenza virus H3N2 at 4-7 dpi. Inoculated cats were fevered at 2-7 dpi (working temperature 39.50-39.90 ° C). Peak mean rectal temperature (39.7 ± 0.2 ° C.) was observed at 4 dpi (FIG. 4). 4 is a diagram showing rectal temperature and virus titer with days post infection.

Fever and clinical symptoms were not observed in the two cats of the uninfected group. The gross and pathological findings of the influenza virus-infected cats at necropsy were severe reddish-tan consolidation and suppurative necrotizing bronchopneumonia (FIGS. 5A-5C). . 5 shows clinical symptoms after infection. In Figure 5, A is a photograph of lung macroscopic lesions 7 days after infection with influenza virus H3N2, it was able to identify a particularly hard red sclerosis in the mesenchyme. B was photographed of lung tissue lesions 7 days after infection with feline influenza virus H3N2, which showed severe purulent necrotic bronchial pneumonia. C is a photograph of lung tissue lesions 7 days after infection with feline influenza virus H3N2, and moderate purulent necrotic alveolitis was observed. Alveolar walls turned into type II pneumocytes. D detected influenza virus antigens in the lungs of cats 7 days after infection with feline influenza virus H3N2 by immunohistochemical staining (see arrow).

Immunohistochemical analysis revealed a large amount of influenza type A virus (NP) antigen in infected lungs (FIG. 5D). Virus release through nasal juice was examined at 2, 4, and 7 dpi by real-time RT-PCR. In infected cats, the virus release titers through mean nasal juice were 10 ^3.79 , 10 ^3.69 , and 10 ^2.79 EID ₅₀ / ml at 2, 4, and 7 dpi, respectively. Virus release titers were highest at 2 dpi, but no viral release was observed in the uninfected group. According to nucleoprotin-specific ELISA, all cats in the infected group were seropositive at 10 dpi, while all cats in the uninfected group were negative serum.

The results of the above examples indicate that whole CIV H3N2 is infected and propagated in house cats raised in animal breeding sites in Korea. During development, cats had 100% morbidity and 40% mortality. Experimentally house cats infected with influenza virus H3N2 have reproduced clinical symptoms such as fever, nasal virus shedding and severe pneumonia, as observed in natural cases. This is the first indication that cats are susceptible to influenza virus H3N2 infection.

Example  4: From cat  Influenza virus H3N2  Preparation of vaccine for the prevention or treatment of infection

In this example, the A / feline / Korea / 01/2010 (H3N2) strain was inactivated to prepare an immunogenic composition.

(1) production of bulk

Virus A / feline / Korea / 01/2010 (H3N2) strain isolated by the Green Veterinary Medicine Co., Ltd. (Korea) Veterinary Research Institute was inoculated into 11-day-old hatching eggs and incubated at 37 ° C. for 72 hours. After incubation, the culture solution was aseptically taken from the embryonated eggs and lyophilized or stored at -80 ° C.

(2) bulk inactivation

The virus bulk prepared in (1) was inactivated by treatment in 0.1 (v / w)% formalin solution for 24 hours at room temperature.

In order to confirm the inactivation, after the completion of inactivation, a part of the stock solution was collected, placed in a sterile dialysis membrane, and placed in phosphate buffered saline in a cold room at 4 to 5 ° C. for one night to remove the inactivating agent. Next, the cells were inoculated with MDCK cells and 11-day-old embryonated eggs and cultured at 37 ° C. for 7 days, and the cell denaturation effect and the cell culture supernatant and the ileum erythrocyte aggregation were observed. Cohesiveness was not recognized.

(3) mixture of antigen and adjuvant

The supernatant was harvested by centrifuging the cat influenza antigen inactivated in (2) at 3,000 rpm for 30 minutes. Rehydragel ^™ LV (General Chemical Inc.) was added to the antigen in the collected embryonated egg broth while stirring using a magnetic bar for 20 minutes to 10 (w / v)%. Rehydragel ^™ LV is an aluminum hydroxide gel that has long been used as an adjuvant in veterinary vaccines. It is a highly active protein adsorbent specifically formulated for use as a liquid adjuvant. It also had a low oxide content and carefully controlled Al ₂ O ₃ content and protein binding capacity. In addition, Rehydragel ^™ LV has a low viscosity to make it easier to handle and handle. The raw material of the prepared vaccine is as follows.

ingredient content Feline Influenza Virus
A / feline / Korea / 01/2010 (H3N2) 27 HA or more 90 (w / v)% Rehydragel ^TM LV 10 (w / v)%

It was confirmed in the following examples whether the vaccine thus prepared has a protective effect in cats.

(4) bottle

Mix for about 1 hour, dispense into an appropriate volume of container and seal.

Example  4: feline influenza virus H3N2  Confirmation of the protective effect of the vaccine against challenge vaccination

(1) Test method

(1.1) Evaluation of efficacy of feline influenza vaccine through challenge

The efficacy against challenge after cat vaccination was evaluated. Fifteen cats (10 weeks of age) were published, six cats were inoculated with the test vaccine one or two minutes and boosted two weeks later. Nine cats were inoculated with PBS instead of vaccination as a control. Two vaccines and 9 non-vaccine groups were used for the challenge vaccine (A / feline / Korea / 01/2010 (H3N2) with a titer of 10 ^6.9 TCID ₅₀ /0.1 ml), which was used for the pathogenicity test 2 weeks after the booster dose. Six cats were inoculated 2 ml each through the nasal cavity. The remaining three of the non-vaccine groups were not challenged. After inoculation, the body temperature and virus release patterns were checked for 10 days, and antibody titer was measured before challenge.

(1.2) Immunogenicity Test

Cats were inoculated with the vaccine prepared in Example 3.

Specifically, to examine the cat influenza antigen and antibody negative cats to determine the antibody formation ability, 58 cats of 10-30 weeks of age were selected and two vaccine vaccines were administered two times at intervals of two weeks. In addition, for comparison of antibody titers with experimental animals, 15 vaccines of cats and guinea pigs were inoculated with vaccines prepared using 2-fold concentrated bulk and 2-fold diluted virus bulk.

Two weeks after the second inoculation, all 58 inoculation groups were collected and serum was separated and inactivated at 56 ° C. for 30 minutes. Antibody titer was measured by hemagglutination inhibitor (HI).

(2) test result

(2.1) Evaluation of efficacy of feline influenza vaccine through challenge

The results of clinical symptoms after challenge were as follows. Cats in the vaccinated group had a runny nose and cough until 2-7 days after challenge (DPC). The body temperature of non-vaccine group I animals began to increase 24 hours after the inoculation (mean rectal temperature = 39.80 ° C) (FIG. 6), and the temperature increase continued until DPC 5. Rectal body temperature of the vaccine and control groups was maintained below 39 ℃ throughout the test period. Significant differences from controls were observed from 2 to 7 days after inoculation (p <0.05). Figure 6 shows the change in rectal body temperature with time after challenge inoculation. In Figure 6, the control group was not challenged in the non-vaccinated group.

The results of measuring virus release are as follows. Vaccine group cats started viral release from DPC 1 to nasal cavity and continued until DPC 4. The maximum discharge amount was 10 ^2.5 EID ₅₀ / ml at DPC 2-4. Cats from the non-vaccinated group began viral excretion from DPC 1 to the nasal cavity and continued to DPC 10. Maximum emissions were 10 ^6.5 EID ₅₀ / ml at DPC 4. Virus release in the vaccine group was significantly lower than that in the non-vaccine group (p <0.05). No virus was detected in the cats of the control group. 7 is a diagram showing virus release through nasal juice over time after challenge inoculation.

The analysis results for antibody titers are as follows. Two weeks after the second inoculation, the mean hemagglutination inhibitor (HI) antibody titer was 80 in the vaccine group, 1.67 in the non-vaccine group and 3.33 in the control group. Table 2 shows HI antibody titers of each test group two weeks after the second inoculation.

Antibodies Antibodies Antibodies Vaccine group 1 80 Non-vaccine group 1 0 Control group 1 10 Vaccine group 2 160 Non-vaccine group 2 0 Control 2 0 Vaccine group 3 80 Non-vaccine group 3 10 Control group 3 0 Vaccine group 4 80 Non-vaccine group 4 0 Vaccine group 5 40 Non-vaccine group 5 0 Vaccine group 6 40 Non-vaccine group 6 0 Average 80 Average 1.67 Average 3.33

(2.2) From cat  Immunogenicity Check

As a result of immunogenicity in cats, 77.3 HI titers were confirmed when the two vaccines were inoculated as shown in Table 3 (n = 58). In the inoculation test of cats conducted above, if the HI titer was 40.0 or higher, defense against the pathogenic virus was recognized. Referring to Table 3 and FIG. 8, if the cat was recognized 40 times or more (72 HI titers) in guinea pigs, the protective effect was also in the cat. Can be admitted. Table 3 shows the immunogenicity comparison in cats and guinea pigs according to virus concentration.

cat Guinea pig Double Concentrated Vaccine (256 HAU, n = 15) 128 112 1 vaccine (128 HAU, n = 58) 77.3 72 2-fold dilution vaccine (64 HAU, n = 15) 26 24.6 Control group (n = 3) below 10 below 10

FIG. 8 shows antibody titers in cats and guinea pigs according to dilution multiples of vaccine. FIG.

Korea Biotechnology Research Institute KCTC11849BP 20110121

<110> GREEN CROSS VETERINARY PRODUCTS CO., LTD <120> Canine influenza virus capable of infecting cat and use <130> PN092742 <160> 8 <170> Kopatentin 1.71 <210> 1 <211> 1701 <212> DNA <213> A / feline / Korea / 01/2010 (H3N2) <400> 1 atgaaaactg ttattgcttt aagctgcatt ttctgcctgg cttttggtca gaatcttcca 60 ggaaatgaaa ataatgctgc aacactatgc ctgggacatc atgcagtgcc gaacgggaca 120 atagtgaaaa ctatcacaga cgatcaaatt gaggtgacca acgccaccga gctagtccaa 180 aactcctcaa cagggaaaat atgcaacaat ccccacaaga ttcttgatgg gagggactgc 240 acactaatag atgccctact aggggacccg cactgtgacg tcttccaaaa tgagacatgg 300 gacctttttg tggaacgaag caatgctttt agcaattgct acccttatga tgtaccagac 360 tatgcatccc ttcgatccat agttgcatca tcaggcacat tggagttcat cactgaaggt 420 ttcacttggg caggagtaac tcaaaatgga ggaagcggtg cttgtaaaag 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 ccaaacattg gatctagacc cttggtaagg 720 ggccaatctg gcagaataag cgtatattgg acaatagtca aacctggaga cgtactggta 780 ataaacagta atggaaacct aatcgctcct cgaggctact tcaaaatgca cattgggaaa 840 agctcaataa tgagatcaga tgcgcctatt ggcacctgca tttccgaatg tatcactccg 900 aacgggagca tccccaatga aaagcccttc caaaatgtaa acaagatcac atacggagca 960 tgtcccaaat atgttaagca aaacaccttg aaactggcaa caggaatgcg gaatgtccct 1020 gagaggcaaa ccagaggcct gttcggcgca atagcaggct tcatagaaaa tggatgggaa 1080 gggatggtag acggttggta tggcttcagg caccaaaatt ccgaaggtac aggacaagca 1140 gcagacctta aaagcactca ggcagccatt gaccagatta atgggaaatt gaacagagtg 1200 attgaaaaaa cgaatgagaa gttccatcaa attgaaaagg agttttccga agtagaaggg 1260 aggattcaag gccttgagag atacgttgaa gacacaaaag tagatctttg gtcttacaat 1320 gccgagcttc ttgttgcttt agaaaaccag aacacaattg 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 caaagactgg atcttgtgga tttcctttgc catatcatgc 1620 tttttgcttt gtgttgtctt gctgggtttc attatgtggg cctgccagag aggcaacatt 1680 aggtgcaaca tttgcatttg a 1701 <210> 2 <211> 1410 <212> DNA <213> A / feline / Korea / 01/2010 (H3N2) <400> 2 atgaacccaa atcagaagat aatagcaata ggctctgtct ctctaaccat tgcaacagta 60 tgtttcctct tgcagattgc catcctagca acaactgtga cactgtactt caagcaaaat 120 gaatgcaaaa tcccctcgaa cagtcaagta gtgccatgta aaccaatcat aatagaaagg 180 aacataacag aggtagtata tttgaataat actaccatag aaaaagaaat ttgttccgta 240 gtgctaggat acaggaactg gtcgaaaccg cagtgtcaaa ttacaggatt tgctcctttc 300 tccaaggaca actcaatccg actctccgct ggtggggaca tttgggtaac aagggaacct 360 tatgtgtcat gcgaccccag caaatgttat 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 atggtctcaa 660 aatatcctca gaactcaaga gtcagaatgc gtttgcatca 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 atacaccaag gaatgatgat agctctagca gcagtaactg cagggatcct 1020 aataatgaga gagggaatcc aggagtgaga gggtgggctt ttgataatga gaatgacgtt 1080 tggatgggga ggacaatcag caaagatttg cgctcaggtt atgagacttt caaggtcatt 1140 ggtggctgga ccactgctaa ttccaagtca caggtcaata gacaagtcat agttgacaat 1200 aataactggt ctggttattc tggtattttc tccgttgaag gcaaaagctg tgttaatagg 1260 tgtttttatg tggagttgat aaggggaggg ccacaagaga ctagagtacg gtggacttca 1320 aatagcattg tcgtattttg tggtacttct ggtacctatg gaacaggctc atggcctgat 1380 ggggcgaata tcaacttcat gcctatataa 1410 <210> 3 <211> 982 <212> DNA <213> A / feline / Korea / 01/2010 (H3N2) <400> 3 atgagtcttc taaccgaggt cgaaacgtac gttctctcta tcgtcccatc aggccccctc 60 aaagccgaga tcgcgcagag acttgaagat gtctttgcag ggaaaaacac cgatctcgag 120 gctctcatgg aatggctaaa gacaagacca atcctgtcac ctttaactaa ggggatttta 180 ggatttgtgt tcacgctcac ccgtgcccag tgagcgagga ctgcagcgta gacgctttgt 240 ccaaaatgcc ctaaatggga atggagaccc aaacaacatg gacagggcag tcaaactgta 300 caggaagttg aagagagaga taacattcca tggggctaaa gaagttgcac tcagctactc 360 aaccggcgca cttgccagtt gtatgggtct catatacaac aggatgggaa cagtgaccac 420 agaagtggct tttggcctag tgtgtgccac ctgtgagcag attgctgatt cacagcatcg 480 gtcccacagg cagatggtaa ctaccaccaa cccactaatc aggcatgaaa acaggatggt 540 gctagccagc accacagcta aggctatgga gcagatggct gggtcgagtg agcaggcagc 600 ggaagccatg gaggttgcca gtcaggctgg gcagatggtg caagcaatga ggacaattgg 660 aactcaccct agctccagtg ccggtctgaa agatgatctt cttgaaaact tgcaggccta 720 ccagaaacga atgggagtgc agatgcagcg atttaagtga tcctctcgtt attgccgcaa 780 gtatcattgg gatcttgcac ttgatattgt ggattcttga tcgtcttttc ttcaaatgta 840 tttatcgtcg ccttaaatac ggtttgaaaa gagggccttc tacggaagga gtgcctgagt 900 ctatgaggga agagtatcgg caggaacagc agagtgctgt ggtgttgacg atggtcattt 960 tgtcaacata gagctggagt aa 982 <210> 4 <211> 1792 <212> DNA <213> A / feline / Korea / 01/2010 (H3N2) <400> 4 atggaagact ttgtgcgaca atgcttcaat ccaatgattg tcgagcttgc ggaaaaggca 60 atgaaagagt atggggaaga tccaaaaatc gaaaccaaca aattcgctgc aatatgcaca 120 catttggaag tctgtttcat gtattcggat ttccacttca ttgacgaacg gggcgaatca 180 ataattgtag aatatggtga cccaaatgca ttactgaaac atcggtttga gataattgag 240 gggagagacc gaacaatggc atggacagtg gtgaatagca tctgcaacac cacaggagtc 300 gagaagccta aatttctccc tgatttgtat gattacaagg agaaccgatt cattgaaatt 360 ggagtgacac ggagggaggt ccacatatac tacctagaaa aagccaacaa aataaaatct 420 gagaagacac acatccatat tttttcattc actggagaag agatggccac caaagcggac 480 tatactcttg atgaagagag ccgagcaaga ataaaaacca gactgttcac aataaggcaa 540 gaaatggcca gcagggggct atgggattcc tttcgtcagt ccgaaagagg cgaagagaca 600 attgaagaaa gatttgaaat cgcaggaacc atgcgcaggc ttgccgccca aagtctccca 660 ccgaacttct ccagccttga aaattttaga gcctatgtga atggatttga accgaacgga 720 tgcattgatg gcaagctttc tcaaatgtca aaagaagtga acgccagaat tgaaccattt 780 ttgaagacaa caccacgccc cctcagatta cctgatgggc ctccctgcac ccaacggtca 840 aaattcttgc tgatggatgc tctgaaacta agcattgaag acccgagtca tgagggggag 900 gggataccgc tatacgatgc gatcaaatgc atgaagacat ttttcggctg gaaagagccc 960 aacataatca aaccacatga gaaaggcata aaccccaatt atctcttggc ttggaaacag 1020 gtgctagcag agctccagga tattgaaaat gaggagaaaa tcccaaagac aaagaatatg 1080 aagaaaacaa gccaactaaa atgggtactt ggtgaaaata tggcaccaga aaaagtggac 1140 tttgaggatt gcaaggatgt tagcgaccta aaacaatatg atagtgatga gccagagcca 1200 gatcgctagc aagctggatt cagagtgaat tcaataaggc atgtgaattg accgattcga 1260 gctggataga acttgatgag ataggagaag atattgcccc aattgagcac attgcaagca 1320 agaggaggaa ctattttacc gcggaagtgt cccattgcag ggctactgaa tacataatga 1380 agggagtgta cataaacaca gctttgctca atgcatcttg tgcagccatg gatgacttcc 1440 aactgattcc aatgataagc aaatgcagga caaaagaagg gagacggaaa acaaacctgt 1500 atgggttcat tataaaagga aggtctcact tgaggaatga cactgacgtg gttaattttg 1560 tgagtatgga gttctctctt actgacccga ggctggaacc acacaaatgg gaaaagtact 1620 gtgttcttga aataggagat atgctcctga ggactgcaat aggtcaagtg tcaaggccca 1680 tgtttctgta tgttagaacc aatggaacct ccaagatcaa gatgaaatgg ggtatggaaa 1740 tgaggcgctg ccttctccaa tctctccaac aaattgagag tatgattgag gc 1792 <210> 5 <211> 1229 <212> DNA <213> A / feline / Korea / 01/2010 (H3N2) <400> 5 agcgaaaggc cgcaaaccat ttgaatggat gtcaatccga ctttactttt cttgaaagtg 60 ccagcacaaa atgctataag tactacattc ccttatactg gagatcctcc atacagccat 120 ggaacaggga caggatacac catggacaca gtcaatagaa cacatcaata ctcagaaaaa 180 ggaaaatgga caacaaacac agagactgga gcaccccaac tcaatccaat tgatggacca 240 ctacccgagg ataatgagcc aagcggatat gcacaaacag attgtgtgtt ggaagcaatg 300 gctttccttg aagagtccca cccagggatc tttgaaaact catgtattga aacgatggaa 360 gttgttcagc aaacaagagt ggacaaattg acccaaggtc gccagaccta tgactggaca 420 ttgaatagaa accagccggc tgcaactgct ttggccaata ctatagaggt cttcagatcg 480 aacggtctaa cagccaatga atcgggaaga ctaatagatt tccttaagga tgtaatggaa 540 tcaatggaca aagaagagat ggagataaca acacatttcc agagaaaaag aagagtaagg 600 gacaacatga ccaagaaaat gatcacacag aggacaatag ggaagaaaaa gcagaggctg 660 aacaagagga gctacgtaat aagagcactg acattgaaca caatgaccaa ggatgcagaa 720 agaggcaaat tgaagagacg ggcaattgca acacccggga tgcagatcag agggttcgtg 780 tactttgttg aaacattagc aaggagcatc tgtgagaaac ttgagcagtc tggactccct 840 gttggaggga atgaaaagaa ggctaaatta gcaaatgtcg taagaaagat gatgactaac 900 tcacaagaca cagagctttc ctttacaatt actggagaca ataccaaatg gaatgagaat 960 caaaatcctc ggatgtttct ggcaatgata acatacatca caagaaacca acctgaatgg 1020 tttagaaatg tcttgagcat tgcccctata atgttctcaa gcaaaatggc aagattagga 1080 aaaggataca tgttcgagag taagaacatg aaactacgaa cacaaatacc agcagaaatg 1140 cttgcaaaca ttaatctgaa atacttcaat gaatcaacaa gaaagaaaat cgagaaaata 1200 agacctctct taacagatgg cacagcctc 1229 <210> 6 <211> 2280 <212> DNA <213> A / feline / Korea / 01/2010 (H3N2) <400> 6 atggagagaa taaaagaatt aagagatcta atgtcgcagt cccgcactcg cgagatacta 60 acaaaaacca ctgtggacca tatggccata atcaagaaat acacatcagg aagacaagag 120 aagaaccctg ctctcagaat gaaatggatg atggcaatga aatatccaat cacagcagac 180 aagagaataa tggagatgat tcctggaagg 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 aaaattgtaa gatagctcct 600 ttaatggtgg catacatgtt ggaaagagaa ctggtccgca aaaccaggtt cctaccggta 660 gcaggcggaa caagcagtgt gtacattgag gtattgcatt tgacacaagg gacctgctgg 720 gaacagatgt acactccagg cggagaagtg agaaatgacg atgttgacca gagtctgatc 780 atcgccgcca gaaacattgt taggagagca acggtatcag cggatccact ggcatcactg 840 ctggagatgt gccacagcac acaaattggt gggacaagga tggtggacat ccttaggcaa 900 aatccaactg aggaacaagc tgtggatata tgcaaagcag caatgggctt gaggatcagt 960 tcatccttta gctttggagg cttcactttc aaaagaacaa atgggtcatc catcaagaag 1020 gaagaaggag tactcacagg 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 gacgcaaaag tgctgtttca gaattggggg attgaaccta ttgataatgt catggggatg 1380 atcggaatat tacctgacat gactccaagc acagagatgt cactgagagg agtaagagtt 1440 agtaaaatgg gagtagatga atattccagc actgaaagag tggttgtaag tattgatcgt 1500 ttcttacggg ttcgagatca gcgggggaac atactcttat ctcccgaaga ggtcagcgaa 1560 acacaaggaa ccgagaaatt gacaataaca tactcatcat caatgatgtg ggagatcaac 1620 ggtcctgagt cagtgcttgt taacacctat caatggatca tcagaaattg ggagaccgtg 1680 aagattcaat ggtctcaaga ccccacggta ctgtacaata agatggagtt tgaaccgttc 1740 cgatccttgg tacccaaagc tgccagaggt caatacagtg gatttgtgag agcactattc 1800 caacaaatgc gtgacgtact ggggacattt gatactgtcc agataataaa gctgctacca 1860 tttgcagcag ccccaccaga gcagagcaga atgcagtttt cttctttgac tgtgaatgtg 1920 agaggctcag gaatgagaat actcgtaagg ggcaattccc ctgtgttcaa ctacaataag 1980 gcaaccaaaa ggcttaccgt tcttggtaaa gacgcaggtg cattaacgga ggatccagat 2040 gagggaactg ccggagtgga atctgcagta ctgaggggat tcctaatttt aggcaaagag 2100 gacaaaagat acggaccagc attaagcatc aatgaactga gcaatcttgc gaaaggggag 2160 aaagctaatg tgctgatagg acaaggagac gtggtgttgg taatgaaacg gaaacgggac 2220 tctagcatac ttactgacag ccagacagcg accaaaagaa ttcggatggc catcaattag 2280                                                                         2280 <210> 7 <211> 1497 <212> DNA <213> A / feline / Korea / 01/2010 (H3N2) <400> 7 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 ccgaagaaga 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 tgcttgtgtg 840 tacggacttg ccgtggccca gtggatatga ttttgagaga gaagggtact ctctggttgg 900 aatagatcct ttccgtctgc ttcaaaacag ccaggtcttc agtctcatta gaccaaatga 960 gaatccagca cataagagtc agttagtgtg gatggcatgc cattctgcag catttgaggc 1020 ctaagagtct caagtttcat cagaggaaca agggtaattc caagaggaca actatccacc 1080 agaggagttc aaattgcttc aaatgagaac atggaaaaaa tagaccccag tactcttgaa 1140 ctgagaagca gatattgggc tataagaacc aggagtggag ggaacaccaa ccaacagaga 1200 acatctgcag gacaaatcag tgtacagcca actttctcgg tacagagaaa tattcccttc 1260 gagcgagctg ccattatggc aacattcaca gggaatactg agggcagaac atctgacatg 1320 cggactgaaa tcataagaat gatggaaagt gccaaaccag aagatgtgtc tttccagggg 1380 cggggagtct tcgagctctc ggacgaaaag gcaacgaagc cgatcgtgcc ttcctttgac 1440 atgagtaacg aaggatctta tttcttcgga gacaatgcag aggagtatga caattaa 1497 <210> 8 <211> 601 <212> DNA <213> A / feline / Korea / 01/2010 (H3N2) <400> 8 cagatagtgg agtggattct agaggaagaa tccgataagg cacttaaaat gaccattgct 60 tcagttccgg cttcacgcta cctaactgat atgacacttg aagaaatgtc aagagactgg 120 ttcatgctca tgccaaagca aaaagtggca gggtcccttt gcatcaaaat ggaccaggca 180 ataatggata aaaacatcac attgaaagca aacttcagtg tgatttttga gcgactggaa 240 actctaatac tacttagagc tttcacaaac gaaggagcaa ttgtgggaga aatttcaccg 300 ttaccttctc ttccaggaca tactgataag gatgtcaaaa atgcaattgg gatcctcatc 360 ggaggacttg aatggaatga taacacagtt cgagtctctg aaactctaca gagattcgct 420 tggagaagca gtgatgagaa tgggagacct tcactccctc caaagcagaa acggaaaatg 480 gcgagaacaa ttgagtcaga aatttgagga aataaggtgg ctgatcgaag aagtacggca 540 tagattgaag attacggaaa acagctttga acaaataaca tttatgcaag ccttacaact 600 a 601

Claims (20)

A method for diagnosing influenza virus H3N2 infection in a cat, comprising detecting feline influenza virus H3N2 in a sample isolated from the cat. The method of claim 1, wherein the influenza virus is A / feline / Korea / 01/2010 (H3N2) (Accession No. KCTC 11849BP), A / canine / Korea / 01/07 (H3N2) (Accession No. KCTC 11205BP), A / canine / Korea / 02/07 (H3N2) (Accession No. KCTC 11206BP), or A / canine / Korea / 03/07 (H3N2) (Accession No. KCTC 11207BP). The method of claim 1, wherein said detecting detects feline influenza virus H3N2 antigen or an antibody binding thereto. Contacting the feline influenza virus H3N2 antigen with a sample isolated from the cat;
Detecting the presence of a complex of said feline influenza virus H3N2 antigen and an anti-cat influenza virus H3N2 antigen; and
The sample is determined to contain an anti-cat influenza virus H3N2 antibody when the complex is detected and the sample comprises an anti-cat influenza virus H3N2 antibody when it is detected that the complex is not present. A method for detecting an anti-cat influenza virus H3N2 antibody in a sample isolated from a cat, comprising determining to not. The method of claim 4, wherein the feline influenza virus H3N2 antigen is the feline influenza virus H3N2 virus particle itself or a surface protein thereof. The method of claim 5, wherein the surface protein is immobilized on a solid substrate. The method of claim 4, wherein the sample is cat blood, serum or plasma. Contacting the anti-cat influenza virus H3N2 antibody with a sample isolated from the cat;
Detecting a complex of the antibody with feline influenza virus H3N2 antigen; And
The sample is determined to contain the feline influenza virus H3N2 antigen when the complex is detected, and the sample is determined to not contain the feline influenza virus H3N2 antigen when the complex is detected to be absent. A method for detecting feline influenza virus H3N2 antigen in a sample isolated from the cat, comprising the step of. A method of searching for a composition for preventing or treating feline influenza virus H3N2 infection, comprising using a feline influenza virus H3N2 antigen to search for a composition for preventing or treating feline influenza virus H3N2 infection in a cat. The method of claim 9, wherein the feline influenza virus H3N2 antigen is the feline influenza virus H3N2 virus particle itself or a surface protein thereof. The method of claim 9, wherein the feline influenza virus H3N2 is A / feline / Korea / 01/2010 (H3N2) (Accession No. KCTC 11849BP), A / canine / Korea / 01/07 (H3N2) (Accession No. KCTC 11205BP), A / canine / Korea / 02/07 (H3N2) (Accession No. KCTC 11206BP) or A / canine / Korea / 03/07 (H3N2) (Accession No. KCTC 11207BP). A kit for detecting feline influenza, comprising an antibody that binds feline influenza virus H3N2. The method of claim 12, wherein the feline influenza virus H3N2 is A / feline / Korea / 01/2010 (H3N2) (Accession No. KCTC 11849BP), A / canine / Korea / 01/07 (H3N2) (Accession No. KCTC 11205BP), A / canine / Korea / 02/07 (H3N2) (Accession No. KCTC 11206BP) or A / canine / Korea / 03/07 (H3N2) (Accession No. KCTC 11207BP). Novel influenza virus A / feline / Korea / 01/2010 (H3N2) (accession number KCTC 11849BP) that can infect cats. The antibody which binds to A / feline / Korea / 01/2010 (H3N2) (Accession No. KCTC 11849BP) of claim 14. A composition for preventing or treating feline influenza virus H3N2 infection in a cat, comprising an immunogenic subunit of attenuated feline influenza virus H3N2 or feline influenza virus H3N2. The method of claim 16, wherein the feline influenza virus H3N2 is A / feline / Korea / 01/2010 (H3N2) (Accession No. KCTC 11849BP), A / canine / Korea / 01/07 (H3N2) (Accession No. KCTC 11205BP), A / canine / Korea / 02/07 (H3N2) (Accession No. KCTC 11206BP) or A / canine / Korea / 03/07 (H3N2) (Accession No. KCTC 11207BP). The composition of claim 17, wherein the attenuated feline influenza virus H3N2 is attenuated by formalin treatment or attenuated by passage culture. 18. The composition of claim 17, wherein the subunit is the outer surface protein of feline influenza virus H3N2. 20. A method for preventing or treating feline influenza virus H3N2 infection in a cat, comprising administering to the cat the composition of any one of claims 16-19.

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