KR101490292B1 - Novel H3N8 equine influenza virus - Google Patents

Abstract

The present invention relates to a H3N8 horse influenza virus isolated from horses (Accession No. KCTC 12158BP). The H3N8 influenza virus (Accession No. KCTC 12158BP) isolated from horses according to the present invention is a novel influenza virus with naturally occurring deletion of an aspartate protein (NS) gene site known as an effector site of an IFN inhibitor, It is possible to produce a naturally attenuated H3N8 horse influenza virus vaccine.

Description

Novel H3N8 equine influenza virus < RTI ID = 0.0 >

The present invention relates to H3N8 horse influenza virus (Accession No. KCTC 1218BP) isolated from horses.

Influenza (Influenza) is a disease caused by influenza virus type A of Orthomyxoviridae, which is an economically important disease in people, pigs, horses and birds. The serotype of influenza A is classified according to the type of two proteins, hemagglutinin and neuraminidase, and is indicated by H serotype and N serotype (eg H9N2). There are 16 serotypes of H avian influenza virus, 9 serotypes of N serotypes, and 144 serotypes of avian influenza virus.

As influenza is a common infectious disease, it is highly variable and has the potential to be transmitted directly from one species to another, and addressing the global spread of highly pathogenic influenza is becoming a big challenge.

Equine influenza virus (EI) is a highly contagious virus in the horses and is known to cause endemic diseases in Europe, North America, South America, North Africa and Asia. Two subspecies of the equine influenza viruses reported so far are H7N7 and H3N8. The first reported influenza virus, H7N7 (A / Equine / Prague / 1/56), was isolated from the US in 1956. The H3N8 horse influenza virus has been isolated from horses showing respiratory illnesses in 1963 and is known to cause highly contagious respiratory illnesses. The H3N8 virus can be classified into A / equine / Newmarket / 2/93 and A / equine / Suffolk / 89 and U.S.A., and American type can be divided into Argentina, Kentucky and Florida subgroups. The Florida subgroup clade1 includes influenza A / equine / Wisconsin / 03, and the Florida subgroup clade 2 includes A / eq / Newmarket / 5/03.

The H3N8 endophthalmitis virus was derived from horse, but it was confirmed in 2004 that it could induce respiratory disease in greyhound dogs in the United States, and it was confirmed to be infectious in dogs.

As such, influenza viruses have a possibility of being transmitted from one species to another species due to mutation, and therefore there is a need for identification of various influenza viruses and research on vaccines using them.

Korean Patent Application No. 10-2007-0109635

The inventors of the present invention confirmed that the isolated virus was a new H3N8 influenza virus while isolating and studying viruses from domestic horses and completed the present invention.

It is an object of the present invention to provide a novel H3N8 horse influenza virus (Accession No. KCTC 12158BP) isolated from horses.

In order to achieve the above object, the present invention provides H3N8 horse influenza virus (Accession No. KCTC 12158BP) isolated from horse.

The H3N8 influenza virus (Accession No. KCTC 12158BP) isolated from horses according to the present invention is a novel influenza virus with naturally occurring deletion of an aspartate protein (NS) gene site known as an effector site of an IFN inhibitor, It is possible to produce a naturally attenuated H3N8 horse influenza virus vaccine.

1 shows the deletion (a) of the non-cariogenic protein (NS) gene region and the deletion region (genes at positions 351 to 373) of the non-cariogenic protein (NS) gene of H3N8 influenza virus (Accession No. KCTC 12158BP) b to e).
2A to 2H are diagrams showing the results of a phylogenetic analysis through nucleotide sequence analysis of H3N8 influenza virus (Accession No. KCTC 12158BP) (2a: hemagglutinin (HA), 2b: matrix protein (M) (NA), 2d: nucleoprotein (NP), 2e: acetopic composition protein (NS), 2f: polymerase protein (PA), 2g: polymerase protein 1 (PB2)
FIG. 3 is a graph showing the weight change of a mouse inoculated with H3N8 influenza virus (Accession No. KCTC 12158BP).

The present invention provides a novel H3N8 horse influenza virus (Accession No. KCTC 12158BP) isolated from horses.

The H3N8 influenza virus (Accession No. KCTC 12158BP) of the present invention is characterized in that the 351 to 373 regions of the nonsynthetic protein (NS) gene are deleted, and the non-active protein (NS) gene has the nucleotide sequence of SEQ ID NO: .

The H3N8 virus (Accession No. KCTC 12158BP) of the present invention comprises a hemagglutinin (HA) gene comprising the nucleotide sequence of SEQ ID NO: 1, a matrix protein (M) gene comprising the nucleotide sequence of SEQ ID NO: 2, A nuclear protein (NP) gene comprising the nucleotide sequence of SEQ ID NO: 4, a gene encoding a polymerase protein (PA) gene comprising the nucleotide sequence of SEQ ID NO: 6, A polymerase protein 1 (PB1) gene comprising the nucleotide sequence of SEQ ID NO: 7, and a polymerase protein 2 (PB2) gene comprising the nucleotide sequence of SEQ ID NO: 8.

The H3N8 is a virus having the hemagglutinin subtype 3 and the neuraminidase type 8, and such subtypes are serologically analyzed for hemagglutinin (HA) and neuramidase (NA) Can be based.

The hemagglutinin (HA) is a virion surface glycoprotein that attaches the virus to its receptor on the host cell and fuses the viral envelope with the membrane of the intracellular cyst to initiate the infection process. It is also the most important virion component in the stimulation and formation of protective antibodies, and the amino acid sequence of hemagglutinin (HA), the position of its N-glycosylation site, is determined by the viral genome.

The nonconjugated protein (NS) is a region commonly found in common influenza viruses and can produce a highly destructive nonconjugated protein (NS) in cells infected with influenza virus, which is the site of action of an IFN inhibitor.

The H3N8 equine influenza virus (Accession No. KCTC 12158BP) identifies horses, animals or canine animals that are indicative of clinical signs of respiratory disease and can be obtained from samples of oral or nasal secretions or from respiratory or visceral tissue of horses and animals or dogs The resulting sample is obtained, and the sample can be analyzed and separated. The isolated H3N8 equine influenza virus (Accession No. KCTC 12158BP) can be isolated, purified, cultured, grown, produced, concentrated and adapted to embryonated eggs or dog cells or horse cells and subcultured, It can be identified as H3N8 virus.

The homology of the present invention can be performed using a comparison program that is visually easy to purchase, with a degree of identity shared by the polynucleotide or polypeptide sequence. Commercially available computer programs can calculate homology between two or more sequences as a percentage, and homology (%) can be calculated for adjacent sequences.

Intranasal administration of the present invention refers to the introduction of a component, such as a vaccine, into the body of an individual through the nose or by the nose, and includes the transport of the component, primarily through the nasal mucosa.

The present invention also provides a vaccine composition comprising the H3N8 equine influenza virus (Accession No. KCTC 12158BP).

The vaccine of the present invention further comprises a pharmacologically acceptable carrier or diluent. Suitable carriers for vaccines are known to those skilled in the art and include, but are not limited to, proteins, sugars, and the like. Such carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous carrier include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable organic ester such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer ' s dextrose, dextrose and sodium chloride, lactic acid treatment linger or fixed oils. The intravenous carrier includes electrolyte supplements, liquids and nutritional supplements based on, for example, Ringer ' s Dextrose. Preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents, inert gases and the like may be additionally present. Preferred preservatives include formalin, thimerosal, neomycin, polyamicin B and amphotericin B.

In addition, the vaccine composition may further comprise an adjuvant.

The adjuvant includes any absorption-promoting agent which refers to a compound or mixture that promotes an immune response and / or promotes the rate of absorption after inoculation. Acceptable adjuvants include, but are not limited to, Freund's complete adjuvant, Freund's incomplete adjuvant, saponin, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oils or hydrocarbon emulsions, , But not limited to, di-nitrophenol, and the like. The preferred adjuvant is the METASTIM adjuvant of Fort Dodge Animal Health.

The dosage of the vaccine composition for the purpose of the present invention is preferably 0.1 ml to 5 ml per 1 kg of body weight (based on a viral titer of 107.0 EID50 / ml).

The vaccine composition of the present invention may be administered by a method known to those skilled in the art, for example, muscle, subcutaneous, intraperitoneal, intravenous, oral, dermal, or ocularly as needed.

In addition, the present invention provides a diagnostic kit for H3N8 horse influenza virus comprising the above H3N8 horse influenza virus (Accession No. KCTC 12158BP) or an antigen thereof.

The present invention also provides a method for detecting H3N9 endophosphorus virus in an infected or infected cell through an antigen-antibody reaction with H3N8 equine influenza virus (Accession No. KCTC 12158BP) or antigen thereof.

The diagnostic kit may be prepared using a method commonly used in the art.

These diagnostic kits include H3N8 equine influenza virus (Accession No. KCTC 12158BP) as well as tools, reagents and the like commonly used in the art for use in 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.

Immunoprecipitation Assay, Immunodiffusion Assay, Complement Fixation Assay (ELISA), Western Blotting, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay Complement Fixation Assay), FACS, protein chip, and the like, but the present invention is not limited thereto.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example  One. H3N8  Isolation of horse influenza virus

Nasal secretion was obtained from 88 horses, and the virus was collected from the nasal cavity and transferred to the medium. The obtained nasal secretion was centrifuged and stored at -80 ° C. After re-thawing, 100 μl of nasal secretion was inoculated into eggs of 11-day-old chicken. 3-4 days after inoculation. Low - speed centrifugation was used to purify the urokinase, and the virus was isolated therefrom. The isolated virus was found to be H3N8 virus and named as A / equine / Kyonggi / SW1 / 2011 / H3N8 and deposited with KCTC 12158BP on March 14, 2012 under the accession number KCTC 12158BP of the Korea Research Institute of Bioscience and Biotechnology.

Example  2. Sequence analysis of isolated influenza virus

To analyze the genetic characteristics of the isolated H3N8 equine influenza virus (Accession No. KCTC 12158BP), the gene of the virus was amplified by a method known in the art (Hoffmann et al., 2001) and sequenced. Viral RNA was extracted from the intestinal fluid of chicken eggs infected with viruses isolated using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Each fragment of the viral gene was amplified using a OneStep RT-PCR Kit (Qiagen, Valencia, Calif., USA) using a universal primer. (NS) gene, hemaglutinin (HA) gene, matrix protein (M) gene, neuramidase (NA) gene, nuclear protein (hereinafter referred to as NP ) Gene, a polymerase protein (hereinafter referred to as PA) gene, a polymerase protein 1 (hereinafter referred to as PB1) gene, and a polymerase protein 2 (hereinafter referred to as PB2) Each amplified gene fragment was purified using QIAquick Gel Extraction Kit (Qiagen, Valencia, Calif., USA) and analyzed by Sequencing with Cosmogenetech (Seoul, Republic of Korea). All of the obtained sequences were registered in GenBank.

The nucleotide sequence of the H3N8 horse influenza virus (Accession No. KCTC 12158BP) was compared with the sequence registered in GenBank and the homology was analyzed. The full-length sequence of the isolated endoplasmic reticulum virus was homologous with the Japanese influenza virus type A. The results are shown in Table 1.

virus gene A virus having high homology Homology (%) A / equine / Kyonggi / SW1 / 2011 / H3N8 HA A / equine / Kanazawa / 1/2007 / H3N8 99 NA A / equine / Kanazawa / 1/2007 / H3N8 99 M A / equine / Tottori / 1/07 / (H3N8) virus 99 NP A / equine / Tottori / 1/07 / (H3N8) virus 99 NS A / equine / Tottori / 1/07 / (H3N8) 96 PA A / equine / Tottori / 1/07 (H3N8) 99 PB1 A / equine / Tottori / 1/07 (H3N8) 99 PB2 A / equine / Tottori / 1/07 (H3N8) 99

As shown in Table 1, the isolated HA and NA genes showed 99% high homology with A / equine / Kanazawa / 1/2007 / H3N8 virus, and the M and NP genes were A / equine / Tottori / 1 / 07 / (H3N8) virus. In addition, the NS gene showed 96% homology with A / equine / Tottori / 1/07 / (H3N8) virus and PA, PB1 and PB2 genes showed 99% homology with A / equine / Tottori / %. ≪ / RTI > Also, as a result of gene comparison analysis, it was confirmed that 23 pairs of base pairs were deleted in the NS gene of the isolated influenza virus.

The deletion site of the non-cariogenic protein (NS) gene of H3N8 influenza virus (Accession No. KCTC 12158BP) is shown in FIG.

As shown in Fig. 1, it was confirmed that the NS gene deleted the 351 to 373 region of the base sequence. The NS gene is the site of action of the IFN inhibitor, and the synthesis of destructive NS proteins has been reported in fatal influenza virus-infected bacteria. Thus, the newly isolated H3N8 equine influenza virus (Accession No. KCTC 12158BP) was found to be a virus that could be used as an attenuated virus vaccine due to deletion of NS gene locus naturally.

Example  3. Analysis of phylogenetic analysis of isolated influenza virus

The nucleotide sequences of the H3N8 horse influenza virus (Accession No. KCTC 12158BP) gene fragment were analyzed using the BioEdit program with ClustalW (Thompson et al., 1994) and the virus profile was generated using the Phylip program (Pversion 3.67). The entire coding region of all gene fragments was used for virus system analysis.

The results are shown in Figs. 2A to 2H.

As shown in Figs. 2A to 2H, the systematic analysis of the full-length nucleotide sequence of all genes confirmed the evolutionary position of the isolated H3N8 horse influenza virus (Accession No. KCTC 12158BP). The results of the phylogenetic analysis indicated that the isolated H3N8 influenza virus (Accession No. KCTC 12158BP) belonged to the florida sub lineage clade 1.

Example  4. Results of animal experiments using isolated horse influenza virus

To confirm the virulence of the isolated H3N8 equine influenza virus (Accession No. KCTC 12158BP), mice were inoculated with virus. All animal experiments were conducted at Korea Biotechnology Research Institute with biosafety level 2+ (BSL2 +). 6-week-old BALB / c mice received from KAT (Korea Animal Technology, Pyeongtaek, South Korea) were used in the experiment. Sixteen mice received 10 ⁶ (EID50; 50% egg infectious doses), and 10 control mice were inoculated with PBS (Phosphate Buffered Saline). As shown in Table 2, three mice of the infected mouse group and two mice of the control group were sacrificed on the fifth and seventh days of inoculation, respectively, and the viral titers of lung, brain, heart, kidney, liver and spleen were measured. Ten mice in the infected group and six mice in the control group observed changes in body weight, survival rate, and motor ability during 14 days after the inoculation. Blood samples were collected prior to infection, and blood was collected again 14 days after inoculation to assess serum conversion.

N (= number of males) Experimental Method Infected group 6 3 5 days after infection, the virus was titrated 3 Measurement of sacrifice virus titers 7 days after infection 10 10 2-week weight change, survival rate, exercise capacity observation Control group 4 2 Measurement of sacrifice virus titers 5 days after infection 2 Measurement of sacrifice virus titers 7 days after infection 6 6 2-week weight change, survival rate, exercise capacity observation

The results are shown in Fig.

As shown in FIG. 3, it was confirmed that the weight of the mice inoculated with the virus was reduced as compared with the control group, and the pathogenicity of the H3N8 horse influenza virus (Accession No. KCTC 12158BP) was confirmed.

Korea Biotechnology Research Institute KCTC12138BP 20120314

<110> Korea Research Institute of Bioscience and Biotechnology          OPTIFARM SOLUTION CO., LTD. <120> Novel H3N8 equine influenza virus <130> 1.6p <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 1698 <212> DNA <213> H3N8 equine influenza virus <400> 1 atgacgacaa ccattatttt gatactactg acccattggg cttacagtca aaacccaatc 60 agtggcaaca acacagccac attgtgtatg ggacaccatg cagtagcaaa tggaacattg 120 gtgaaaacaa taagtgatga tcaaattgag gtgacaaatg ctacagaatt agttcagagc 180 atttcaatgg ggaaaatatg caacaactca tatagaattc tagatggaag gaattgcaca 240 ttaatagatg caatgctagg agacccccac tgtgacgcct ttcagtatga gaattgggac 300 ctctttatag aaagaagcag cgctttcagc aattgctacc catatgacat ccctgactat 360 gcatcgctcc gatccattgt agcatcctca ggaacattgg aattcacagc agagggattc 420 acatggacagta gtgtcactca aaacggaaga agtggagcct gcaaaagggg atcagccgat 480 agtttcttta gccgactgaa ttggctaaca aaatccggaa gctcttactc cacattgaat 540 gtgacaatgc ctaacaataa aaatttcgac aagctataca tctgggggat tcatcacccg 600 agctcaaatg aagagcagac aaaattgtac atccaagaat caggacgagt aacagtctca 660 acaaaaagaa gtcaacaaac aataatccct aacatcggat ctagaccgtg ggtcagaggt 720 caatcaggta ggataagcat atactggacc attgtaaaac ctggagatat cctaatgata 780 aacagtaatg gcaacttagt tgcaccgcgg ggatatttta aattgaaaac agggaaaagc 840 tctgtaatga gatcagatgt acccatagac atttgtgtgt ctgaatgtat tacaccaaat 900 ggaagcatct ccaacgacaa gccattccaa aatgtgaaca aagttacata tggaaaatgc 960 cccaagtata ttaggcaaaa cactttaaag cttgccactg ggatgaggaa tgtaccagaa 1020 aagcaaatca gaggaatctt tggggcaata gcgggattca tcgaaaacgg ctgggaagga 1080 atggttgatg ggtggtatgg gttccgatat caaaactctg aaggaacagg gcaagctgca 1140 gatctaaaga gcactcaagc agccatcgac cagattaatg gaaagttaaa cagagtgatt 1200 gaaagaacca atgagaaatt ccatcaaata gagaaggaat tctcagaagt agaagaaaga 1260 attcaggact tggagaaata tgtagaagac accaaaatag acctatggtc ctacaatgca 1320 gaattgctgg tggctttaga aaatcaacat acaattgact taacagatgc agaaatgaac 1380 aaattatttg agaagactag acgccagtta agagaaaacg cagaagacat gggaggtgga 1440 tgtttcaaaa tttaccacaa atgtgataat gcatgcattg gatcaataag aaatgggaca 1500 tatgaccatt acatatacag agatgaagca ttaaacaacc gatttcagat caaaggtgta 1560 gagttgaaat caggctataa agattggata ctgtggattt cattcgccat atcatgcttc 1620 ttagtttgcg ttgttctatt gggtttcatt atgtgggctt gccaaaaagg caacatcaga 1680 tgcaacattt gcatatga 1698 <210> 2 <211> 759 <212> DNA <213> H3N8 equine influenza virus <400> 2 atgagtcttc taaccgaggt cgaaacgtac gttctctcta tcataccatc aggccccctc 60 aaagccgaga tcgcgcggag acttgaagat gtctttgcag ggaggaacgc cgatcttgag 120 gcactcatgg aatggctaaa gacaagacca atcctgtcac ctctaactaa agggatttta 180 ggatttgtat tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240 caaaatgccc ttagtggaaa cggagatcca aacaacatgg acagagcagt aaaactgtac 300 aggaagctta aaagagaaat aacattccat ggggcaaaag aggtggcact cagctattcc 360 actggtgcac tagccagctg catgggactc atatacaaca gaatgggaac tgttacaacc 420 gaagtggcat ttggcctggt atgcgccaca tgtgaacaga ttgctgattc ccagcatcga 480 tctcacaggc agatggtgac aacaacaaac ccattaatca gacatgaaaa cagaatggta 540 ttagccagta ccacggctaa agccatggaa cagatggcag gatcaagtga gcaggcagca 600 gaggccatgg aggttgctag tagggctagg cagatggtac aggcaatgag aaccattggg 660 acccacccta gctccagtgc cggtttgaaa gatgatctcc ttgaaaactt acaggcctac 720 cagaaacgga tgggagtgca aatgcagcga ttcaagtga 759 <210> 3 <211> 1391 <212> DNA <213> H3N8 equine influenza virus <400> 3 atggcaattg gatttgcatc attggggata ttaatcatta atgtcattct ccatgtagtc 60 agcattatag taacagtact gaccctcaat aacaatagaa cagatctgaa ctgcaaaggg 120 acgatcataa aagagtacaa tgaaacagta agagtagaaa aaattactca atggtataat 180 accagtacaa ttaaatacat agagagacct tcaaatgaat actacatgaa caacactgaa 240 ccactttgtg aggcccaagg ctttgcacca ttttccaaag ataatggaat acgaattggg 300 tcgagaggcc atgtttttgt gataagagaa ccttttgtat catgttcgcc ctcagaatgt 360 agaacctttt tcctcacaca gggctcatac tcaatgacaa acattctaac ggcacaataa 420 aggaccgaag tccgtatagg actttgatga gtgtcaaaat agggcagtca cctaatgtat 480 atcaagctag gtttgaatcg gtggcatggt cagcaacagc atgccatgat ggaaaaaaat 540 ggatgacagt tggagtcaca gggcccgaca atcaagcaat tgcagtagtg aactatggag 600 gtgttccggt tgatattatt aattcatggg cagggaatat tttaagaacc caagaatcat 660 catgcacctg cattaaagga gactgttatt gggtaatgac tgatggaccg gcgaataggc 720 aagctaaata taggatattc aaagcaaaag atggaagagt aattggacag actgatataa 780 atttcaatgg gggacacata gaggagtgtt cttgttaccc caatgaaggg aaggtggaat 840 gcatatgcag ggacaattgg actggaacaa atagaccaat tttggtaata tcttctgatc 900 tatcatacac agttggatat ttgtgtgctg gcattcccac tgacactcct aggggagagg 960 atagtcaatt cacaggctca tgtacaaacc ctttgggaaa taaaggatac ggtgtaaaag 1020 gtttcggatt tcgacaagga actgacgtat gggccggaag gacaattagt aggacttcaa 1080 gatcaggatt cgaaataata aaaatcagga atggttggac acagaacagt aaagaccaaa 1140 taaggaggca agtgattatc gatgacccaa attggtcagg atatagcggt tctttcacat 1200 tgccggttga actaacaaaa aagggatgtt tggtcccctg tttctgggtt gaaatgatta 1260 gaggtaaacc tgaagaaaca acaatatgga cctctagcag ctccattgtg atgtgtggag 1320 tagatcataa aattgccagt tggtcatggc acgatggagc tattcttccc tttgacatcg 1380 ataagatgta a 1391 <210> 4 <211> 1497 <212> DNA <213> H3N8 equine influenza virus <400> 4 atggcgtctc aaggcaccaa acgatcctat gaacagatgg aaactgatgg ggaacgccag 60 aatgcaactg aaatcagagc atctgtcgga aggatggtgg gaggaatcgg ccggttttat 120 gttcagatgt gtactgagct taaactaaac gaccatgaag ggcgactgat tcagaacagc 180 ataacaatag aaaggatggt actttcggca ttcgacgaaa gaagaaacaa gtatctcgag 240 gagcatccca gtgctgggaa agaccctaag aaaacaggag gcccgatata cagaaggaaa 300 gatgggaaat ggatgaggga actcatcctc tatgataaag aagaaatcat gagaatctgg 360 cgtcaggcca acaatggtga agacgctact gctggtctta ctcatatgat gatctggcac 420 tccaatctca atgacaccac ataccaaaga acaagggctc ttgttcgaac tgggatggat 480 cccagaatgt gctctctgat gcaaggctca accctcccac ggagatctgg agcagctggt 540 gctgcaataa aaggtgttgg aacaatggta atggaactca tcagaatgat caaacgcgga 600 ataaatgatc ggaatttctg gagaggtgaa aatggtcgaa gaaccagaat tgcttatgaa 660 agaatgtgca atatcctcaa agggaaattt cagacagcag cacaacgggc tatgatggac 720 caggtgaggg aaggtcgcaa tcctggaaac gctgagattg aggatctcat tttcttggca 780 cgatcagcac ttattttgag aggatcagta gcccataaat catgcctacc tgcctgtgtt 840 tatggccttg cagtaaccag tgggtatgac tttgagaagg aaggatactc tcttgttgga 900 attgatcctt tcaaactact ccagaacagt caaattttca gtctaatcag accaaaagaa 960 aacccagcac acaagagcca gttggtgtgg atggcatgcc attctgcagc atttgaagac 1020 atgagagttt taaatttcat tagaggaacc aaagtaatcc caagaggaca gttaacaacc 1080 agaggagttc aaatagcttc aaatgaaaac atggagacaa tagattcttg cacacttgaa 1140 ctgagaagca aatattgggc aataaggacc agaagcggag gaaacaccag tcaacagaga 1200 gcatctgcag gacagataag tgtgcaacct actttctcag tacagaggaa tcttcccttt 1260 gagagagcaa ccattatggc tgcattcact ggtaacaccg aagggaggac ttccgacatg 1320 agaacggaaa tcataaggat gatggaaaat gccaaatcag aagatgtgtc tttccagggg 1380 cggggagtct tcgagctctc ggacgaaaag gcaacgaacc cgatcgtgcc ttcctttgac 1440 atgagcaatg aagggtctta tttcttcgga gacaatgctg aggagtttga cagttaa 1497 <210> 5 <211> 637 <212> DNA <213> H3N8 equine influenza virus <400> 5 atggattcca acactgtgtc aagctttcag gtagactgtt ttctttggca tgtccgcaaa 60 cgattcgcag accaagaatt gggtgatgcc ccattccttg accggcttcg ccgagaccag 120 aagtccctaa ggggaagagg cagcactctt ggtctggaca tcgaaacagc cactcatgca 180 ggaaagcaga tagtggagca gattctggaa aaggaatcag atgaggcact taaaatgacc 240 attgcctctg ttcctacttc acgctactta actgacatga ctcttggtga gatgtcaaga 300 gactggttca tgctcatgcc caagcataag aatggaccag gcaatcatgg ataagaacat 360 catacttaaa gcaaacttta gtgtgatttt cgaaaggctg gaaacactaa tactacttag 420 agccttcacc gaagaaggag cagtcgttgg cgaaatttca ccattacctt ctcttccagg 480 acatactaat gaggatgtca aaaatgcaat tggggtcctc atcggaggac ttaaatggaa 540 tgataatacg gttagaatct ctgaaactct acagagattc gcttggagaa gcagtcatga 600 gaatgggaga ccttcattcc cttcaaagca gaaatga 637 <210> 6 <211> 2159 <212> DNA <213> H3N8 equine influenza virus <400> 6 atggaagact ttgtgcgaca gtgcttcaat ccaatgatcg tcgagcttgc ggaaaaggca 60 atgaaagaat atggagagga cccaaaaatc gaaacaaaca aatttgcagc aatatgcact 120 cacttggaag tctgcttcat gtactcggat ttccacttta ttaatgaact gggtgagtca 180 gtggtcatag agtctggtga cccaaatgct cttttgaaac acagatttga aatcattgag 240 gggagagatc gaacaatggc atggacagta gtaaacagca tctgcaacac cacaagagct 300 gaaaaaccta aatttcttcc agacttatac gactataagg agaacagatt tgttgaaatt 360 ggtgtgacaa ggagagaagt tcacatatac tacctggaga aggccaacaa aataaagtct 420 gagaaaacac acatccacat tttctcattt acaggagagg aaatggctac aaaagcggac 480 tatactcttg atgaagagag tagagccaga atcaagacca ggctattcac tataagacaa 540 gt; attgaagaaa gatttgaaat cacagggacg atgcgcaagc ttgccaatta cagtctccca 660 ccgaacttct ccagccttga aaattttaga gtctatgtgg atggattcga accgaacggc 720 tgcattgaga gtaagctttc tcaaatgtcc aaagaagtaa atgccagaat cgaatcattt 780 tcaaagacaa caccccgacc actcaaaatg ccaggtggtc caccctgcca tcagcgatct 840 aaattcttgc taatggatgc tctgaaactg agcattgagg acccaagtca cgagggagag 900 ggaataccac tatatgatgc aatcaaatgc atgaaaactt tctttggatg gaaagagccc 960 agtattgtta aaccacatga aaagggtata aacccgaact atctccaaac ttggaagcaa 1020 gtattagaag aaatacaaga ccttgagaac gaagaaagga cccccaagac caagaatatg 1080 aaaaaaacaa gccaattgaa atgggcacta ggtgaaaata tggcaccaga gaaagtggat 1140 tttgaggatt gtaaagacat cagtgattta aaacagtatg acagtgatga gccagaaaca 1200 aggtctcttg caagttggat tcaaaatgag ttcaacaaag cttgtgagct gacagattca 1260 agctggatag agctcgatga aattggggag gatgtcgccc caatagaata cattgcgagc 1320 atgaggagaa attattttac tgctgagatt tcccattgta gagcaacaga atatataatg 1380 aaaggagtgt acgtcaacac cgctctactc aatgcatcct gtgctgcaat ggatgaattt 1440 caattaattc cgatgataag taaatgcagg accaaagaag ggagaaggaa aacaaattta 1500 tatggattca tagtaaaggg aaggtcccat ttaagaaatg atactgacgt ggtgaacttt 1560 gtaagtatgg aattttctct cactgatcca agatttgagc cgcacaaatg ggaaaaatac 1620 tgcgttctag aaattggaga catgcttcta agaactgctg taggtcaagt gtcaagaccc 1680 atgtattgg atgtaaggac aaatggaacc tctaaaatta aaatgaaatg gggaatggaa 1740 atgaggcgct gcctccttca gtctctgcaa cagattgaaa gcatgatcga agctgagtcc 1800 tcagtcaaag aaaaggacat gaccaaagaa ttttttgaga acaaatcaga gacatggcct 1860 ataggagagt cccccaaagg agtggaagag ggctcaatcg ggaaggtttg caggacctta 1920 ttagcaaaat ctgtgtttaa cagtttgtat gcatctccac aactggaagg gttttcagct 1980 gaatctagga aattacttct catcgttcag gctcttaggg ataacctgga acctggaacc 2040 tttgatattg aggggttata tgaatcaatt gaggagtgcc tgattaatga tccctgggtt 2100 ttgcttaatg catcttggtt caactccttc cttacacatg cactgaagta gttgtggca 2159 <210> 7 <211> 2274 <212> DNA <213> H3N8 equine influenza virus <400> 7 atggatgtca atccgactct acttttctta aaggtgcaag cacaaaatgc tataagcaca 60 acattccctt atactggaga tcctccctac agtcatggaa cagggacagg atacaccatg 120 gatactgtca acagaacaca ccaatattca gaaaaaggga aatggacaac aaacactgag 180 attggagcac cacaacttaa tccaatcgat ggaccacttc ctgaagacaa tgaaccaagt 240 gggtacgccc aaacagattg tgtattggaa gcaatggctt tacttgaaga atcccatccc 300 gggatctttg aaaattcgtg tcttgaaacg atggaggtga ttcagcaaac aagagtggac 360 aaactaacac aaggccgaca aacttatgat tggaccttga ataggaatca acctgccgca 420 acagcacttg ctaatacgat tgaagtattc agatcaaatg gtctgacttc caatgaatcg 480 gggagattga tggacttcct caaagatgtc atggagtcca tgaacaagga agaaatggaa 540 ataacaacac actttcaacg gaagagaaga gtaagagaca acatgacaaa gagaatggta 600 acacagagaa ccatagggaa gaaaaaacaa cggttaaaca gaaagagcta tctaatcaga 660 acattaaccc taaacacaat gaccaaggac gctgagagag ggaaattgaa acgacgagca 720 atcgctaccc cagggatgca gataagaggg tttgtatact ttgttgaaac tttagcccga 780 agaatatgtg aaaagcttga acaatcagga ttgccagttg gcggtaatga gaaaaaggcc 840 aaactggcta atgtcgtcag aaaaatgatg actaattccc aagacactga actctccttc 900 accatcactg gggacaatac caaatggaat gaaaatcaga acccacgcat attccttgca 960 atgatcacat acataactag aaaccagcca gaatggttc gaaatgttct aagcattgca 1020 ccgattatgt tctcaaataa aatggcaaga cttgggaaag gatatatgtt tgaaagcaaa 1080 agtatgaaat tgagaactca aataccagca gaaatgctag caagcattga cctgaaatat 1140 ttcaatgatt caacaaaaaa gaaaattgaa aagatacgac cacttctggt tgacgggact 1200 gcttcactaa gtcctggcat gatgatggga atgttcaaca tgttgagcac tgtgctgggt 1260 gtatccatat taaacctggg ccagaggaaa tacacaaaga ccacatactg gtgggatggt 1320 ctgcaatcat ccgatgactt tgctttgata gtgaatgcgc ctaatcatga ggggatacaa 1380 gctggagtag acagattcta tagaacttgc aaactggtcg ggatcaacat gagcaaaaag 1440 aagtcctaca taaatagaac tggaacattc gaattcacaa gctttttcta ccggtatggt 1500 tttgtagcca atttcagcat ggaactaccc agttttggag tttccggaat aaatgaatct 1560 gcagacatga gcattggagt gacagtcatc aaaaacaaca tgataaataa tgacctcggt 1620 cctgccacag cacagatggc actccaactc ttcattaagg attatcggta cacataccgg 1680 tgccatagag gtgataccca gatacaaacc aggagatctt ttgagttgaa gaaactgtgg 1740 gaacagactc gatcaaagac tggtctactg gtatcagatg ggggtccaaa cctatataac 1800 atcagaaacc tacacatccc ggaagtctgt ttaaaatggg agctaatgga tgaagattat 1860 agggggaggc tatgcaatcc attgaatcct ttcgttagtc acaaagaaat tgaatcagtc 1920 aacagtgcag tagtaatgcc tgcgcatggc cctgccaaaa gcatggagta tgatgctgtt 1980 gccacaacac attcttggat ccccaagagg aaccggtcca tattgaacac aagccaaagg 2040 ggaatactcg aagatgagca gatgtatcag aaatgctgca acctgtttga aaaattcttc 2100 cccagcagct catacagaag accggttggg atttctagta tggttgaggc catggtgtcc 2160 agggcccgca ttgatgcacg aattgacttc gaatctggac ggataaagaa ggatgagttc 2220 gctgagatca tgaagatctg ttccaccatt gaagagctca gacggcaaaa atag 2274 <210> 8 <211> 2250 <212> DNA <213> H3N8 equine influenza virus <400> 8 atgttacaat cccgcacccg cgagatacta acaaaaacca ctgtggacca catggccata 60 atcaagaaat acacatcggg aagacaagag aagaaccctg cacttaggat gaaatggatg 120 atggcaatga aatacccaat tacagcagat aagaggataa tggagatgat tcctgagaga 180 aatgaacagg gacaaaccct ttggaacaaa acgaatgatg ctggctcaga ccgcgtaatg 240 gtatcacctc tggcagtgac atggtggaat aggaacggac caacaacaag cacaattcat 300 tatccaaaag tctacaaaac ttattttgaa aaggttgaaa gattgaaaca cggaaccttt 360 ggccccgttc attttaggaa tcaagtcaag ataagacgaa gagttgatgt aaaccctggt 420 cacgcggacc tcagtgccaa agaagcacaa gatgtaatca tggaagttgt tttcccaaat 480 gaagtgggag ccagaattct aacatcggaa tcacaactaa caataaccaa agagaaaaag 540 gaagaacttc aggactgcaa aattgctccc ttgatggtag catacatgct agaaagagag 600 ttggtccgaa aaacaaggtt cctcccagta gcaggcggaa caagcagtgt atacattgaa 660 gtgttgcatc tgactcaggg aacatgctgg gagcaaatgt acaccccagg aggagaagtt 720 agaaacgatg atattgatca aagtttaatt attgcagcac ggaacatagt gagaagagca 780 acagtatcag cagatccact agcatcccta ctggaaatgt gccacagtac acagattggt 840 ggaataagga tggtagacat ccttaagcat aatccaacag aggaacaagc tgtggatata 900 tgcaaagcag caatgggatt gagaattagc tcatcattca gctttggtgg attcaccttc 960 aaaagaacaa gtggatcatc agtcaagaga gaagaagaaa tgcttacggg caaccttcaa 1020 acattgaaaa taagagtgca tgagggctat gaagaattca caatggttgg aagaagagca 1080 acagccattc tcagaaaggc aaccagaaga ttgattcaat tgatagtaag tgggagagat 1140 gaacaatcaa ttgctgaagc aataattgta gccatggtgt tttcgcaaga agattgcatg 1200 ataaaagcag ttcgaggcga tttgaacttt gttaatagag caaatcagcg tttgaacccc 1260 atgcatcaac tcttgaggca tttccaaaaa gatgcaaaag tgcttttcca aaattgggga 1320 attgaaccca tcgacaatgt aatggggatg attggaatat tgcctgacat gaccccaagc 1380 accgagatgt cattgagagg agtgagagtc agcaaaatgg gagtggatga gtactccagc 1440 actgagagag tggtggtgag cattgaccgt tttttaagag ttcgggatca aaggggaaac 1500 atactactgt cccctgaaga agtcagtgaa acacaaggaa cggaaaagct gacaataatt 1560 tattcgtcat caatgatgtg ggagattaat ggtcccgaat cagtgttggt caatacttat 1620 caatggatca tcaggaactg ggaaattgtc aaaattcagt ggtcacagga ccccacaata 1680 ttatacaata ggatagaatt tgagccattc caatccctgg tccctagggc caccagaagc 1740 caatacagcg gtttcgtaag aatcctgttt cagcaaatgc gagatgtact tggaacattt 1800 gatactgctc aaataataaa actcctccct tttgccgctg ctcctccgga acagagtagg 1860 atgcagttct cttctttgac tgttaatgta agaggttcgg gaatgaggat acttgtaaga 1920 ggcaattccc cagtgttcaa ctacaataaa gccaccaaaa ggctcacagt cctcggaaag 1980 gatgcaggtg cgcttactga ggacccagat gaaggtacgg ctggagtaga atctgctgtt 2040 ctaagagggt ttctcatttt gggtaaagaa aacaagagat atggcccagc actaagcatc 2100 aatgaactaa gcaaacttgc aaaaggggag aaagccaatg tactaattgg gcaaggggac 2160 gtagtgttgg taatgaaacg gaaacgtgac tctagcatac ttactgacag ccagacagcg 2220 accaaaagga ttcggatggc catcaattag 2250

Claims (8)

H3N8 horse influenza virus isolated from horses (Accession No. KCTC 12158BP).
2. The H3N8 strain of influenza virus according to claim 1, wherein the virus comprises the nucleotide sequence of SEQ ID NO: 5, wherein the 351 to 373 regions of the non-constitutive protein (NS) gene are deleted.
delete The virus according to claim 1, wherein the virus comprises a hemagglutinin (HA) gene consisting of the nucleotide sequence of SEQ ID NO: 1, a matrix protein (M) gene consisting of the nucleotide sequence of SEQ ID NO: 2, (NP) gene comprising the nucleotide sequence of SEQ ID NO: 4, a polymerase protein (PA) gene comprising the nucleotide sequence of SEQ ID NO: 6, a polymerase protein comprising the nucleotide sequence of SEQ ID NO: 1 (PB1) gene, and a polymerase protein 2 (PB2) gene consisting of the nucleotide sequence of SEQ ID NO: 8 (accession number KCTC 12158BP).
18. A vaccine composition comprising the H3N8 horse influenza virus of claim 1 (Accession No. KCTC 12158BP).
A diagnostic kit for H3N8 equine influenza virus according to claim 1 (Accession No. KCTC 12158BP) or H3N8 horse influenza virus comprising the antigen.
Characterized in that it detects H3N8 equine influenza virus (Accession No. KCTC 12158BP) in the infected or infected cells through an antigen-antibody reaction with the H3N8 horse influenza virus (Accession No. KCTC 12158BP) or its antigen of claim 1, Detection method of horse influenza virus (Accession No. KCTC 12158BP).
The method according to claim 7, wherein the antigen-antibody reaction is assayed by a method selected from the group consisting of tissue immunostaining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation assay, Wherein the method is carried out by using at least one method selected from the group consisting of Immunodiffusion Assay, Complement Fixation Assay, FACS and protein chip assay. The H3N8 influenza virus (Accession No. KCTC 12158BP).

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