# Patent

## Title
Canine influenza virus and vaccine therefore

## Abstract

Novel influenza viruses A/Canine/Korea/01/07 (H3N2), A/Canine/Korea/02/07 (H3N2) and A/Canine/Korea/03/07 (H3N2) are disclosed. A vaccine composition comprising at least one of the viruses, a method for preventing or treating diseases resulting from influenza virus infection by administering the vaccine composition, and an assay kit for detecting the viruses are also disclosed.

## Background
This application is U.S. National Phase of International Application PCT/KR2007/005789, filed Nov. 16, 2007 designating the U.S., which claims priority to Korean Patent Application No. 10-2007-0109535, filed Oct. 30, 2007. The International Application has not been published as of the filing date.
TECHNICAL FIELD
The present invention relates to novel influenza viruses A/Canine/Korea/01/07 (H3N2), A/Canine/Korea/02/07 (H3N2) and A/Canine/Korea/03/07 (H3N2), a vaccine composition comprising at least one of the viruses as an active ingredient, a method for preventing or treating diseases resulting from influenza virus infection, and an assay kit for detecting the viruses.
BACKGROUND ART
Influenza, caused by Influenza A virus of the family Orthomyxoviridae, is the most economically important disease in humans, pigs, horses, and fowls.
Influenza A viruses are further classified based on the characteristics of two surface proteins known as hemagglutinin (H) and neuraminidase (N). Influenza A virus is expressed as a combination of the H (hemagglutinin) subtype and N (neuraminidase) type one (e.g., H9N2). There are 16 different H subtypes and 9 N subtypes, resulting in a total of 144 different possible combinations of H and N subtypes of influenza A viruses.
Influenza is a zoonosis. The type A viruses are the most virulent human pathogens among the three influenza types, and cause the most severe disease. In addition, they are highly apt to mutate and can be readily transmitted from one species to another, causing pandemics. Accordingly, the breakout of pandemic influenza is emerging as a great problem to be solved. Furthermore, there are several reports that influenza viruses are infecting new species that have heretofore been known to be resistant to virus infection.
Canine influenza refers to new varieties of Influenza virus A that cause influenza in canines. Because of the lack of previous exposure to this virus, dogs have no natural immunity to this virus. Therefore, all species and ages are susceptible to this virus. Dogs with canine influenza may suffer from acute pneumonia, showing the symptoms of a severe cough, a high fever and rhinorrhea.
A highly contagious influenza virus was found to have been the cause of Greyhound race dog fatalities from a respiratory illness at a Florida racetrack in 2004. Then, as outbreaks thereof were reported in Texas, Alabama, Arkansas and other states in the U.S.A., canine influenza was regarded as a new epidemic in dogs. An epidemiological survey showed the virus, isolated from a dog with canine influenza, was almost identical to the equine influenza virus H3N8, indicating the creation of canine influenza as a result of transmission from horses to dogs. There are reports of the equine influenza virus H3N8 causing hemorrhagic pneumonia in racing dogs and of isolation of the human influenza virus H3N8 from dogs. However, sufficient serological and virological evidence must be found for canine influenza.
In addition, there have been reported cases of the outbreak of avian influenza in canines. It is inferred that the epidemiological mechanism of the transmission of influenza from birds to dogs has two routes: one is by feeding dogs with uncooked birds carrying influenza, such as ducks, chickens, etc.; and the other main way that the influenza virus is spread is from infected dogs to normal dogs in respiratory droplets of coughs and sneezes. As such, it is inferred that canine influenza is established after infected dogs are exposed to new environments and brought into contact with normal dogs. It is important to prevent canine influenza because canine influenza viruses may cause secondary infection with various mortalities. There is no vaccine available for dogs at this time.
DISCLOSURE
Technical Problem
Leading to the present invention, intensive and thorough research into the production of the influenza virus in canines, conducted by the present inventors on the above-mentioned background, resulted in the finding that influenza viruses from some dogs in Korea were A serotype variants, which are different from previous influenza viruses and, although belonging to an avian cluster, showed interspecies transmission among birds and dogs through virological, serological, pathological and phylogenetic analysis. Further, a highly stable vaccine against these viruses has been successfully developed.
Technical Solution
It is an object of the present invention to provide a novel H3N2 serotype canine influenza virus.
It is another object of the present invention to provide a nucleotide sequence encoding a protein constituent of the influenza virus.
It is a further object of the present invention to provide a vaccine composition against the novel virus.
It is still a further object of the present invention to provide an assay kit for detecting H3N2 serotype influenza viruses, comprising the virus of the present invention or an antigenic determinant thereof.



DESCRIPTION OF DRAWINGS
FIG. 1 shows the entire amino acid sequence encoded by an NA gene of A/Canine/Korea/01/07 (H3N2) influenza virus in comparison with those of A/Dove/Korea/S11/03 (H3N2), A/Duck/Korea/S7/03 (H3N2) and A/Chicken/Korea/S6/03 (H3N2) influenza viruses.
FIG. 2 shows the entire amino acid sequence encoded by an HA gene of A/Equine/Jilin/1/1989 (H3N8) influenza virus in comparison with those of A/Canine/Korea/01/07 (H3N2), A/Dove/Korea/S11/03 (H3N2), A/Duck/Korea/S7/03 (H3N2) and A/Chicken/Korea/S6/03 (H3N2) influenza viruses.
FIG. 3 shows a partial amino acid sequence encoded by an HA gene of A/Equine/Jilin/1/1989 (H3N8) influenza virus in comparison with those of A/Canine/Korea/01/07 (H3N2), A/Canine/Korea/02/07 (H3N2), A/Canine/Korea/03/07 (H3N2), A/Dove/Korea/S11/03 (H3N2), A/Duck/Korea/S7/03 (H3N2) and A/Chicken/Korea/S6/03 (H3N2) influenza viruses.
FIG. 4 shows a phylogenetic tree for HA gene, rooted with A/Canine/Korea/01/07 (H3N2) influenza virus.
FIG. 5 shows a phylogenetic tree for NA gene, rooted with A/Canine/Korea/01/07 (H3N2) influenza virus.
FIG. 6 is a graph showing changes in body temperature, antibody titer and viral progeny production over one week in animals immunized with A/Canine/Korea/01/07 (H3N2) influenza and animals not immunized therewith.
FIG. 7 shows histopathological lesions in the organs and lungs of animals immunized with A/Canine/Korea/01/07 (H3N2) influenza virus (immunized group B, D and F) and animals not immunized therewith (control, A and C): (A) the pseudostratified columnar epithelium lining in a normal organ of the control group 9 days after aggressive inoculation (400-fold magnification); (B) necrotic organ (n), squamous metaplasia (s), and epithelial hyperplasia and chronic inflammation on connective tissue (C) in the immunized group 9 days after aggressive inoculation (400-fold magnification); (C) normal alveoli of the control group 3 days after aggressive inoculation (200-fold magnification); (D) severe diffuse necrotizing bronchitis and purulent bronchiolitis in the bronchial lumen of the immunized group 3 days after aggressive group (100-fold magnification); (E) severe necrotizing bronchiolitis in the immunized group 6 days after aggressive inoculation (filled with separated necrotizing cells and neutrophils, and mild or moderate chronic inflammation observed around the bronchiole (200-fold magnification); (F) severe necrotizing alveolitis in the immunized group 9 days after aggressive inoculation (necrotizing cell infiltration in the alveolar duct (ad) and alveolus (a) (200-fold magnification) H&E stained).



Best Mode 
In accordance with an aspect thereof, the present invention pertains to novel H3N2 serotype canine influenza viruses.
The novel H3N2 serotype canine influenza viruses according to the present invention have a hemagglutinin (HA) protein represented by the amino acid sequence of SEQ ID NO. 10 or an amino acid sequence sharing 95% or higher homology therewith.
When compared to the previously known equine influenza virus H3N8, the canine influenza virus of the present invention was found to have a very characteristic change in amino acid sequence, as analyzed for the entire neuraminidase (NA) amino acid sequence (FIG. 1) and the entire hemagglutinin (HA) amino acid sequence (FIG. 2). Particularly, the HA amino acid sequences of A/Canine/Korea/01/07 (H3N2), A/Canine/Korea/02/07 (H3N2) and A/Canine/Korea/03/07 (H3N2) are characteristically altered to have N (Asparagine) at position 27, I (Isoleucine) at position 127, T (Threonine) at position 142, T (Threonine) at position 176, N (Asparagine) at position 188, S (Serine) at position 209, I (Isoleucine) at position 212 and I (Isoleucine) at position 252 (FIG. 3). The amino acid sequence sharing at least 95% homology with the amino acid sequence of SEQ ID NO. 10 contains the same amino acid residue as the amino acid sequence of SEQ ID NO. 10 at least one of positions 97, 127, 142, 176, 188, 209, 212 and 252.
Also, the novel H3N2 serotype canine influenza viruses according to the present invention have a neuraminidase (NA) protein, represented by the amino acid sequence of SEQ ID NO. 12, or an amino acid sequence sharing 95% or higher homology therewith.
In addition, the novel canine influenza viruses in accordance with the present invention may further comprise a protein selected from among a non-structural protein (NS), encoded by the nucleotide sequence of SEQ ID NO. 3, a matrix protein (M) encoded by the nucleotide sequence of SEQ ID NO. 4, a nucleoprotein (NP) encoded by the nucleotide sequence of SEQ ID NO. 5, a polymerase (PA) encoded by the nucleotide sequence of SEQ ID NO. 6, a polymerase protein 2 (PB2) encoded by the nucleotide sequence of SEQ ID NO. 7, a polymerase protein 1 (PB1) encoded by the nucleotide sequence of SEQ ID NO. 8, and combinations thereof.
As used for hemagglutinin or neuraminidase herein, the term “homology” is intended to refer to similarity to a wild-type amino acid sequence. The hemagglutinin and the neuraminidase, expressed in the influenza viruses of the present invention, shares 90% or higher, preferably 95% or higher, more preferably 98% or higher, and most preferably 99% or higher homology with the amino acid sequences of SEQ ID NOS. 10 and 12, respectively. In general, a protein homologue has the same active site as the prototype thereof. Homology comparison between amino acid sequences can be conducted using the naked eye or using software. Homology between two or more amino acid sequences can be calculated and expressed as percentages using commercially available software.
The canine influenza viruses of the present invention comprise A/Canine/Korea/01/07 (H3N2), A/Canine/Korea/02/07 (H3N2) and A/Canine/Korea/03/07 (H3N2).
The proteins of the A/Canine/Korea/01/07 (H3N2) influenza virus show 95.5-98.9% homology with those of the avian influenza virus. For example, the A/Canine/Korea/01/07 (H3N2) influenza virus of the present invention shares the highest homology with A/Dove/Korea/S11/03 (H3N2) with regard to HA (Hemagglutinin) and NA (Neuraminidase) genes and with A/Chicken/Nanchang/7-010/2000 (H3N6) with regard to an NS (non-structural) gene. As for genes of PB1 (polymerase basic protein 1), PB2, PA (polymerase), NP (nucleoprotein) and M (matrix), they showed high homologies with avian influenza viruses found in Hong Kong, Japan, and China. The A/Canine/Korea/01/07 (H3N2) influenza virus was deposited at Korean Collection for Type Cultures (KCTC) of Korean Research Institute of Bioscience and Biotechnology, located in 52 Oun-dong, Yusong-Ku, Daejeon, South Korea 305-333 on Sep. 19, 2007, with accession number KCTC 11205BP.
A/Canine/Korea/01/07 (H3N2) influenza virus has a hemagglutinin (HA) gene comprising the nucleotide sequence of SEQ ID NO. 1 and a neuraminidase (NA) gene comprising the nucleotide sequence of SEQ ID NO. 2. The entire nucleotide sequence of HA is given, along with the entire amino acid sequence thereof, in SEQ ID NO. 9, while the entire nucleotide sequence of NA is given, along with the entire amino acid sequence thereof, in SEQ ID NO. 11. In addition, the nucleotide sequence of SEQ ID NO. 3 is contained in the NS gene, the nucleotide of SEQ ID NO. 4 is contained in the M gene, the nucleotide sequence of SEQ ID NO. 5 is contained in the NP gene, the nucleotide sequence of SEQ ID NO. 6 is contained in the PA gene, the nucleotide sequence of SEQ ID NO. 7 is contained in the PB2 gene, and the nucleotide sequence of SEQ ID NO. 8 is contained in the PB1 gene.
The A/Canine/Korea/02/07 (H3N2) influenza virus of the present invention has an HA gene comprising the nucleotide sequence of SEQ ID NO. 13 and an NA gene comprising the nucleotide sequence of SEQ ID NO. 14. This virus was found to be substantially the same as the A/Canine/Korea/01/07 (H3N2) influenza, as the HA and NA nucleotide sequences and amino acid sequences were analyzed to share 98% homology between the two viruses. The A/Canine/Korea/02/07 (H3N2) influenza virus was deposited in Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Bioscience and Biotechnology, located in 52 Oun-dong, Yusong-Ku, Daejeon, South Korea 305-333 on Sep. 19, 2007, with accession number KCTC 11206BP.
The A/Canine/Korea/03/07 (H3N2) influenza virus has an HA gene comprising the nucleotide sequence of SEQ ID NO. 1 and an NA gene comprising the nucleotide sequence of SEQ ID NO. 16. This virus was identified to be substantially the same as the A/Canine/Korea/02/07 (H3N2) influenza virus and the A/Canine/Korea/03/07 (H3N2) influenza virus, as the HA and NA nucleotide sequences and amino acid sequences were analyzed to share 99% homology between the A/Canine/Korea/03/07 (H3N2) influenza virus and the A/Canine/Korea/01/07 (H3N2) influenza virus and 98% homology between the A/Canine/Korea/03/07 (H3N2) influenza virus and the A/Canine/Korea/02/07 (H3N2). The A/Canine/Korea/03/07 (H3N2) influenza virus was deposited in Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Bioscience and Biotechnology, located in 52 Oun-dong, Yusong-Ku, Daejeon, South Korea 305-333 on Sep. 19, 2007, with accession number KCTC 11207BP.
The canine influenza viruses according to the present invention, isolated from the nasal cavity of Korean dogs, have the phylogenetic relationship shown in the phylogenetic diagrams of FIGS. 4 and 5. The phylogenetic trees of FIGS. 4 and 5, both based on the A/Canine/Korea/01/07 (H3N2) influenza virus, were constructed for the HA gene and the NA gene. As seen in these phylogenetic trees of the HA and NA genes, the influenza viruses of the present invention, along with avian influenza viruses, form a cluster which is different from the cluster to which the H3N8 viruses isolated from horses and dogs belong.
When used to infect dogs, the canine influenza viruses according to the present invention showed pathogenicity, causing fever and pneumonia, and thus are epidemic viruses in dogs in Korea. When administered with vaccines against the canine influenza viruses of the present invention, dogs were, for the most part, found to have immunity to the viruses and to suppress the propagation and generation of viruses therethrough.
In accordance with another aspect thereof, the present invention pertains to a gene encoding a hemagglutinin (HA) protein which has the amino acid sequence of SEQ ID NO. 10 or an amino acid sequence sharing 95% or higher homology therewith. Preferably, the gene has the nucleotide sequence of SEQ ID NO. 9.
Also, the present invention pertains to a gene encoding a neuraminidase (NA) protein which has the amino acid sequence of SEQ ID NO. 12 or an amino acid sequence sharing 95% or higher homology therewith. Preferably, the gene has the nucleotide sequence of SEQ ID NO. 11.
In accordance with a further aspect thereof, the present invention pertains to a vaccine composition which can provide immunity to canine influenza viruses.
Preferably, the vaccine composition of the present invention comprises canine influenza virus or an antigen thereof as an active ingredient. The canine influenza virus for use in the vaccine composition is selected from among A/Canine/Korea/01/07 (H3N2), A/Canine/Korea/02/07 (H3N2), A/Canine/Korea/03/07 (H3N2) and combinations thereof.
The antigen useful in the present invention refers to an antigenic part of the constituents of the virus, which causes an immune response, and may comprise a hemagglutinin (HA) protein having an amino acid sequence sharing 90% or higher homology, and preferably 95% or higher homology, with the amino acid sequence of SEQ ID NO. 10 or a fragment thereof. The amino acid sequence sharing at least 95% homology with the amino acid sequence of SEQ ID NO. 10 contains the same amino acid residue at least one of positions 97, 127, 142, 176, 188, 209, 212 and 252 as in the amino acid sequence of SEQ ID NO. 10.
Alternatively, the antigen may comprise a neuraminidase (NA) protein having an amino acid sequence sharing 95% or higher homology with the amino acid sequence of SEQ ID NO. 12, or a fragment thereof.
The vaccine according to the present invention may include an attenuated live or killed vaccine, a subunit vaccine, a synthetic vaccine, and a genetically engineered vaccine, with preference for a live vaccine due to the ability thereof to induce an effective immune response.
As used herein, the term “live vaccine” refers to a vaccine prepared from a virus that has been attenuated but can still replicate the cells of the host organism. The term “attenuation”, as used herein, is intended to mean artificial reduction in the toxicity of pathogens by mutating a gene involved in the essential metabolism of the pathogen in such a manner that it loses pathogenicity, but retains antigenicity. Generally, attenuation is achieved through UV radiation, chemical treatment, or in vitro sequential high-order subculture. An explicit genetic alteration, such as the deletion of a specific nucleotide in a sequence known to provide toxicity or the insertion of a nucleotide into a viral genome, may also result in attenuation.
As used herein, the term “killed vaccine”, also called an inactivated vaccine, refers to a suspension of killed virus used as an antigen to produce immunity. Examples of killed vaccines include whole-virus vaccines and split vaccines. Killed vaccine can be easily produced using known methods. For example, a whole-virus vaccine can be obtained by treating a virus with formalin. Split vaccines are prepared from virus envelopes after treatment with ether.
The term “subunit vaccine” refers to a vaccine composed of a purified antigenic determinant that is separated from the virulent organism by extraction. It is less likely to cause adverse reactions than the whole-virus vaccine. For example, an HA protein and/or an NA protein, extracted from the canine influenza virus, may be used to prepare a subunit vaccine.
By the term “synthetic vaccine” is meant a vaccine consisting mainly of chemically synthetic or genetically engineered antigens, antigenic determinants or peptides. For example, the HA protein and/or the NA protein of the canine influenza virus may be synthesized for use as a vaccine.
A genetically engineered vaccine may be free of a specific gene which is responsible for pathogenicity or may contain a modified gene.
In addition, the influenza vaccine of the present invention may be used in combination with other inactivated organisms or antigens to prepare a mixed or complex vaccine against various diseases including influenza. The term “mixed vaccine”, as used herein, is intended to refer to a vaccine prepared from a viral mixture of the canine influenza virus of the present invention and at least one different virus. The term “complex vaccine” means a vaccine prepared from a virus and bacteria. For example, the canine influenza viruses of the present invention may be mixed or combined with canine parainfluenza virus, canine distemper virus, canine adenovirus, and/or Bordetella bronchiseptica.
The canine influenza virus vaccine according to the present invention may be prepared using a method comprising: (a) injecting the canine influenza virus of the present invention into an embryonated egg and proliferating the virus therein; (b) treating a chorioallantoic fluid from the embryonated egg with formalin, BPL (betapropiolactone) or BEI (binary ethyleneimine); and harvesting the inactivated virus from the chemically treated chorioallantoic fluid.
In step (a), the canine influenza virus is injected into an embryonated egg 9-11 days old and incubated at 30 to 40° C. for 24 to 72 hours. In step (b), a chorioallantoic fluid is obtained from the incubated egg using a conventional method, treated with 0.005-0.2 (v/w) % of formalin, BEI or BPL and incubated at a low temperature to inactivate the virus. In step (c), the inactivated virus was harvested from the chorioallantoic fluid treated with formalin, BEI or BPL by centrifugation or filtration. Then, the virus is adsorbed onto aluminum hydroxide gel. This method may comprise well-known techniques, or may be modified into more readily practicable versions.
Also, the vaccine composition of the present invention may further comprise a medium, an adjuvant, and/or an excipient. Physiological saline or distilled water may be used as a medium. Examples of the adjuvant useful in the vaccine composition include a Freund's incomplete or complete adjuvant, aluminum hydroxide gel, vegetable or mineral oil, etc. Examples of the excipient include aluminum phosphate, aluminum hydroxide, and aluminum potassium sulfate, but are not limited thereto. In practice, all materials known for use in vaccine preparation to those skilled in the art may be applicable to the vaccine composition of the present invention.
Preferably, the vaccine composition of the present invention may comprise the canine influenza virus in an amount of 25 HAU (hemagglutination unit). When the canine influenza virus is used in an amount less than 25 HAU, the vaccine cannot induce antibody production effectively. On the other hand, an amount exceeding 25 HAU may be uneconomical.
The vaccine composition according to the present invention may be prepared into oral dosage forms or non-oral dosage forms. Preferable are non-oral dosage forms which can be administered through intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, or epidural routes.
The vaccine composition according to the present invention is applicable to all subjects that are susceptible to A/Canine/Korea/01/07 (H3N2), A/Canine/Korea/02/07 (H3N2) or A/Canine/Korea/03/07 (H3N2) and act as hosts, including humans, pets, poultry and birds, such as dogs, pigs, chicken, ducks, turkeys, etc.
In accordance with a further aspect thereof, the present invention pertains to a method for the prevention and treatment of influenza virus-related diseases, comprising the administration of the vaccine composition to individuals at risk.
As used herein, the term “influenza virus-related disease” is intended to refer to a disease which results from infection with influenza virus. Examples thereof include paranasal sinusitis, spasmodic asthma, tympanitis, cystic fibrosis, bronchitis, pneumonia, diarrhea, etc. (Pitkaranta and Hayden, 1998. Ann. Med.), but are not limited thereto.
The term “individuals”, as used herein, is intended to refer to all vertebrates, including humans, which are already infected with, or can be infected with, influenza viruses. By the administration with the vaccine composition of the present invention, the diseases can be effectively prevented and treated. For example, humans infected with various subtypes or variants of influenza viruses can be treated with the vaccine composition of the present invention. In addition, chickens or pigs may be immunized with the vaccine composition in order to take precautions against influenza. The vaccine composition of the present invention may be administered in combination with conventional therapeutics against influenza virus-related diseases.
As used herein, the term “prevention” means all actions for inhibiting influenza virus infection or delaying influenza outbreak through the administration of the vaccine composition according to the present invention. The term “treatment” means all actions by which symptoms resulting from influenza virus infection are relieved or take a turn for the better through administration with the vaccine composition according to the present invention.
In a pharmaceutically effective amount, the vaccine composition according to the present invention is administered. The term “pharmaceutically effective amount”, as used herein, is intended to refer to an amount that is useful to treat influenza virus-related diseases at reasonable ratios of beneficence to danger for medicinal therapy. Dosages of the compound of the present invention depend on the kind and severity of diseases, activity of the drug, sensitivity to the drug, frequency and time period of administration, administration routes, excretion rates, and factors well known in the art including, for example, concurrently used drugs, etc. The vaccine composition of the present invention may be used as a single medicine or in combination with other medicines concurrently or sequentially, and may be administered in single dosages or multiple dosages. Taking into account the elements mentioned above, it is important to determine the dosage that elicits maximum therapeutic effects without undesirable side effects, which is easy for those skilled in the art.
In accordance with a further aspect thereof, the present invention pertains to an assay kit for detecting H3N2 serotype influenza viruses, comprising the influenza virus of the present invention or an antigenic determinant thereof.
Through an antigen-antibody complex reaction, the influenza virus of the present invention or an antigenic determinant thereof is useful in the specific detection of influenza viruses as well as the extermination of influenza viruses in cells infected therewith.
This assay kit comprises tools/reagents generally used in the immunological field as well as the influenza virus of the present invention. Examples of the tools/reagents include suitable carriers, labels producing detectable signals, solubilizers, detergents, buffers, stabilizers, etc., but are not limited thereto. When the labels are enzymes, the kit may further comprise substrates for analyzing enzymatic activity and reaction terminators. Examples of suitable carriers include, but are not limited to, soluble carriers, such as physiologically acceptable buffers well known in the art, e.g., PBS, insoluble carriers, such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resin, crosslinking dextran, polysaccharide, etc., magnetic microparticles, such as metal-coated latex, paper, glass, metal, agarose, and combinations thereof.
As for an assay for the formation of antigen-antibody complexes, examples thereof include immunohistochemical staining, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation assay, immunodiffusion assay, complement fixation assay, FACS, and protein chip, but are not limited thereto.
Labels for allowing the qualitative or quantitative analysis of the antigen-antibody complex are exemplified by enzymes, fluorescents, ligands, luminescents, microparticles, redox molecules and radioisotopes, but are not limited thereby. Examples of enzymes useful as detectable labels include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholine esterase, glucose oxidase, a combination of hexokinase and GDPase, RNase, a combination of glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decarboxylase, and β-lactamase, but are not limited thereto. Illustrative, non-limitative examples of the fluorescents useful in the present invention include fluorescein, isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocynanin, o-phthaldehyde, and fluorescamine. A biotin derivative may be used as the ligand, but does not limit the scope of the present invention. Acridinium ester, luciferin, and luciferase are useful as luminescents, but this list is not intended to limit the scope of the present invention. Illustrative, non-limiting microparticles include colloidal gold, coated latex, etc. As redox molecules useful in the present invention, there are ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4W(CN)8, [Os(bpy)3]2+, [RU(bpy)3]2+, and [MO(CN)8]4−, which are given for illustrative purposes only, but are not intended to limit the scope of the present invention. Examples of radioisotopes useful in the present invention include 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re, but are not limited thereto.
Mode for Invention 
A better understanding of the present invention may be obtained through the following examples, which are set forth to illustrate, but are not to be construed as the limit of the present invention.
EXAMPLE 1
Specimen Sampling and Virus Isolation
Specimens were sampled from dogs which were treated in veterinary hospitals located in Kyeonggi-Do, Korea: One Miniature Schnauzer dog five years old suffered from rhinorrhea for three days and sneezing for two days and then recovered from the flu; One Cocker Spaniel dog three years old suffered from fever, cough, rhinorrhea and inappetence and finally died: One Yorkshire terrier dog and two Jindo dogs suffered from severe cough, fever and rhinorrhea and died 2 days after hospitalization.
All of these animals were identified to be infected with influenza virus type A as analyzed by a Rapid Kit, purchased from Anigen, and RT-PCR. Other pathogens were not detected from the dogs.
The specimens (nasal secretions) from the animals were inoculated into 11 day old eggs, after which chorioallantoic fluid was sampled therefrom. The fluid was found to aggregate chicken erythrocytes. The viruses isolated from the animals were serologically identified as an H3N2 serotype. These viruses were named A/Canine/Korea/01/07 (H3N2), A/Canine/Korea/02/07 (H3N2) and A/Canine/Korea/03/07 (H3N2) and deposited in Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Bioscience and Biotechnology, located in 52 Oun-dong, Yusong-Ku, Daejeon, South Korea on Sep. 19, 2007, with Accession Numbers KCTC 11205BP, KCTC 11206BP and KCTC 11207BP, respectively.
EXAMPLE 2
Genetic Characteristics of Isolated Viruses
Genetic characteristics of the viruses isolated in Example 1 were determined through gene analysis. Total influenza virus RNA isolated from the chorioallantoic fluid using Trizol LS was used as a template for RT-PCR using random hexamer primers, followed by PCR using primers shown in Table 1. Primer sequences for amplifying H3, N2, PB1, PB2, PA, NP, M and NS genes were designed using a modified Primer 3 program (Whitehead Institute/MT Center for Genome Research).
cDNA (2 μl) was mixed with a reagent mix {2.5 μl, 10× Taq DNA polymerase buffer, MgCl2 1.5 mM, dNTPs (2.5 mM/μl) 2.0 μl, each primer (10 pmol) 1 μl, Taq DNA polymerase (Promega, USA) 1 μl)} and the final volume was adjusted to 25 μl with distilled water to prepare a PCR mixture. PCR started by denaturation at 94° C. for 10 min, and was performed with 32 cycles of denaturing at 96° C. for 30 sec, annealing at 53° C. for 30 sec, and extending at 72° C. for 2 min, followed by extension at 72° C. for 10 min. PCR was terminated at 4° C. The PCR product thus obtained was analyzed by electrophoresis on 1.5% agarose gel containing ethidium bromide. The sequence data thus obtained were analyzed with Bioedit software.







TABLE 1







PCR Primer Sequences









Target

PCR


Genes
Primer Sequence(5′>3′)
Products





H3
CARATTGARGTGACHAATGC(SEQ ID NO 15)
720 bp



GGTGCATCTGAYCTCATTA(SEQ ID NO 16)


 


N2
TGTTCCGTTTCATTTGGGAA(SEQ ID NO 17)
477 bp



CCAACAAGCCCTGAACACAC(SEQ ID NO 18)


 


PB1
AAAGTGCCAGCACAAAATGC(SEQ ID NO 19)
764 bp



TTCTCACAGATGCTCCTCGC(SEQ ID NO 20)


 


PB2
TCATGGAGGTCGTTTTTCCA(SEQ ID NO 21)
661 bp



TGAATCAGCCTTCTGGTTGC(SEQ ID NO 22)


 


PA
GAAGTGAGCGCCAAAATTGA(SEQ ID NO 23)
477 bp



CTCTGGCTCATCGCTGTCAT(SEQ ID NO 24)


 


NP
ACGGTCTGCACTCATCCTGA(SEQ ID NO 25)
602 bp



GCCCCTGGAAAGACACATCT(SEQ ID NO 26)


 


M
AACATTCCATGGGGCTAAGG(SEQ ID NO 27)
456 bp



CGGCAATAACGAGAGGATCA(SEQ ID NO 28)


 


NS
GACTGGTTCATGCTCATGCC(SEQ ID NO 29)
844 bp



GAGAGAGTGAAGGTCCCCCA(SEQ ID NO 30)









Eight gene segments from A/Canine/Korea/01/07 (H3N2) were base sequenced and compared with genes of GeneBank (SEQ ID NOS. 1 to 12). The viruses according to the present invention were found to have 95.5 to 98.9% homology to previously known avian influenza viruses (Table 2). Particularly, the viruses according to the present invention shared the highest homology with S11, which was isolated in Korea, in terms of the HA and NA genes, and with a strain isolated from chickens in China in terms of the NS gene. As for PB1, PB2, PA, NP and M genes, high homologies were detected between the viruses of the present invention and avian influenza viruses isolated in Hong Kong, Japan and China. HA and NA genes were found to have 98%-99% homology among the viruses of the present invention, A/Canine/Korea/01/07, A/Canine/Korea/02/07 and A/Canine/Korea/03/07, which indicates that they are substantially the same.







TABLE 2







Comparison for Gene Homology of Canine Influenza Viruses















Influenza






Highly 
Virus Type 






Homologous
of RNA 
Homology
Rgst'n


Viruses
Gene
Viruses
Segment
(%)
Nos.





A/Canine/
HA
A/Chicken/
Avian
96.6
AY862607


Korea/01/07

Korea/







S6/03(H3N2)






NA
A/Dove/Korea/
Avian
97.4
AY862644




S11/03(H3N2)






PB1
A/Duck/
Avian
98.9
EF061124




Yangzhou/







02/2005(H8N4)






PB2
A/Duck/
Avian
97.6
AY585523




Zhejiang/







11/2000(H5N1)






PA
A/Duck/
Avian
95.9
AB286878




Hokkaido/







120/2001(H6N2)






NP
A/Duck/
Avian
95.5
AF156406




Hong Kong/







Y439/97(H9N2)






M
A/Duck/Jiang
Avian
97.5
EF597295




Xi/1850/







2005(H5N2)






NS
A/Chicken/
Avian
97.5
AT180648




Nanchang/







7-010/







2000(H3N6)









EXAMPLE 3
Phylogeny of Isolated Viruses
The position of A/Canine/Korea/01/07 in a phylogenetic tree was determined using a clustal alignment algorithm and MEGALIGN software (DNASTAR, Madison, Wis.). In a point of view of HA and NA genes, the virus of the present invention was identified as belonging to a cluster different from the cluster of previously isolated H3N8 viruses from horses and dogs, and showed a very close genetic relationship with H3N2 viruses isolated in Korea (FIGS. 4 and 5).
EXAMPLE 4
Assay of Isolated Viruses for Pathogenicity
In order to examine the pathogenicity thereof, A/Canine/Korea/01/07 (H3N2) was inoculated into dogs.
Ten beagles 10 weeks old were divided into a test group of 7 and a control group of 3. The seven beagles in the test group were administered intranasally and orally with the isolated virus (2 ml) having an HA titer of 1:64 (106.9 EID50/0.1 ml) while the three beagles in the control group were administered intranasally and orally with pathogen-free PBS (phosphate buffer saline, 2 ml), followed by monitoring clinical symptoms for 7 days. The discharge of virus through excretions and rhinorrhea was monitored using RT-PCR for 10 days starting from the day of inoculation. A serological study was conducted using a competitive ELISA Animal Genetics Inc. Korea) with a recombinant NP (nucleoprotein) serving as an antigen. Serum samples were also analyzed in order to detect antibodies to the recombinant NP, as recommended by OIE. Two beagles of the test group and one beagle of the control group were subjected to euthanasia with 1 ml of xylazine at 3, 6 and 9 days each after the inoculation and autopsied in order to observe pathologic lesions.
From day 2 to day 7 after the inoculation, the beagles were observed to suffer from clinical symptoms including sneezing and rhinorrhea. Rectal temperature was maintained at 39° C. in the beagles of the control group throughout the experiment, but increased to 40.14° C. on average in the beagles of the test group 24 after the inoculation (FIG. 6).
Serological tests were negative for the viruses in all of the experimental dogs before the inoculation, and remained negative in the beagles in the test group during the experiment. ELISA showed a far higher percentage inhibition in the test group than in the control group 6 days after the inoculation, indicating that antibodies were produced. Interestingly, the inoculated beagles were found to have an HI titer of 1:80 8 days after inoculation.
The virus was found in the nasal secretion from the inoculated beagles for 6 days after the inoculation, but was not detected in the excretion. Typically, canine influenza virus started to be discharged from dogs 1 day after infection and peaked with a titer of 106.0 EID50/0.1 ml 4 days after infection.
It was found that histopathological lesions were limited to the lungs. Histologically, serious necrotic lesions were discovered in the upper respiratory tract (bronchi) and the lower respiratory tract (bronchiole and alveolar). Bronchiolitis and bronchitis, although somewhat different in the extent thereof, occurred in all of the inoculated beagles (FIG. 7).
Consequently, the isolated virus was identified to be pathogenic in dogs, causing an increase in body temperature and pneumonia. In addition, it was found that viruses were discharged for 6 days.
EXAMPLE 5
Vaccine Preparation
The newly isolated canine influenza virus A/Canine/Korea/01/07 (H3N2) was seeded into chorioallantoic membranes of embryonated eggs 10 days old. Three days later, a chorioallantoic fluid was sampled as a virus bulk. To this virus bulk was added 0.2% formalin, followed by incubation at room temperature for 24 hours for inactivation thereof. The virus bulk was determined to be inactivated when no viral progeny were detected after the virus bulk had been re-inoculated into the embryonated eggs. The inactivated virus bulk was concentrated into 25 HAU or higher. This bulk was mixed at a ratio of 7:3 with aluminum hydroxide gel by stirring at 10,000 rpm for 10 min. Following a negative test for viruses, the mixture was used as a vaccine.
EXAMPLE 6
Aggressive Inoculation Following Vaccination
The prepared vaccine was subcutaneously injected at a dose of 0.5 ml into ten beagles, each 10 weeks old, and additionally injected in the same manner three weeks later. Two weeks after the secondary injection, the beagles were aggressively inoculated at a dose of 2 ml through an oral or intranasal route, with the isolated virus A/Canine/Korea/01/07 (H3N2), having an HA titer of 1:64 (106.9EID50/0.1 ml). As a control, three beagles were inoculated with PBS before the aggressive inoculation. The experimental animals were monitored for body temperature, virus production, clinical symptoms and antibody titer during the experiment.
Even after inoculation, the vaccinated beagles exhibited no changes in body, discharged no viral progeny, and showed no clinical symptoms. In contrast, the control was increased in body temperature for one week after the inoculation (Table 3). PCR showed that viral progeny was discharged from all three beagles in the control group, but was not discharged from any of them 6 days after inoculation (Table 4). Also, the control group was observed to suffer from clinical symptoms, including rhinorrhea and cough, such as kennel cough or wet productive cough. As for antibody titer, it started to increase with regard to the ELISA antibody to nucleoprotein and the HI antibody to hemagglutinin over the experimental time period starting 7 days after the aggressive inoculation in the vaccinated beagles (Table 5). However, the control started to increase in the titer of antibodies to nucleoprotein and hemagglutinin starting 7 days after the aggressive inoculation.
Therefore, the vaccinated beagles were found to have a defense against the aggressive inoculation, indicating that the vaccine composition of the present invention is useful as a vaccine against the influenza virus.







TABLE 3







Body Temperature in Vaccinated and Unvaccinated Animals


After Aggressive Inoculation


(Booster on Day 21, Aggressive Inoculation on Day 35)









Days After
Vaccinated
Control


Vaccination
Group
(Unvaccinated)












0
37.8 ± 0.1
38.2 ± 0.2


7
37.6 ± 0.2
37.8 ± 0.1


14
38.4 ± 0.1
38.0 ± 0.2


21
38.2 ± 0.1
37.6 ± 0.1


28
38.1 ± 0.2
38.3 ± 0.1


35
38.3 ± 0.2
37.6 ± 0.1


36
40.2 ± 0.3
38.3 ± 0.2


37
40.6 ± 0.1
38.0 ± 0.3


38
39.84 ± 0.2 
38.0 ± 0.2


39
39.3 ± 0.1
37.6 ± 0.1


40
38.9 ± 0.2
38.3 ± 0.1


41
38.7 ± 0.1
37.6 ± 0.1


42
38.6 ± 0.1
38.0 ± 0.1


49
38.0 ± 0.1
38.0 ± 0.1





* No. of PCR positive/No. of PCR Tested













TABLE 4







Virus Discharge from Vaccinated and Unvaccinated Animals


After Aggressive Inoculation


(Booster on Day 21, Aggressive Inoculation on Day 35)









Days After
Vaccinated
Control


Vaccination
Group
(Unvaccinated)












0
 0/10*
0/3


7
0/10
0/3


14
0/10
0/3


21
0/10
0/3


28
0/10
0/3


35
0/10
0/3


36
0/10
3/3


37
0/10
3/3


38
0/10
3/3


39
0/10
3/3


40
0/10
2/3


41
0/10
0/3


42
0/10
0/3


49
0/10
0/3





*PI value positive













TABLE 5







Antibody Titer in Vaccinated and Unvaccinated Animals After


Aggressive Inoculation (Booster on Day 21, Aggressive Inoculation


on Day 35)












Vaccinated

Control



Days After
Group

(Unvaccinated)











Vaccination
ELISA*
HI
ELISA
HI














0
14
<10
12
<10


7
89
10
20
<10


14
87
40
14
<10


21
96
40
19
<10


28
89
80
26
<10


35
94
80
18
<10


42
92
80
97
80


49
98
160
92
160









INDUSTRIAL APPLICABILITY
As described hitherto, the present invention provides novel canine influenza viruses and a vaccine against the same. Capable of inducing effective immunity against the canine influenza virus, the vaccine is useful in the prevention and treatment of influenza virus-related diseases in dogs and individuals secondarily infected from dogs.
Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

## Claims

1. An H3N2 serotype influenza virus, comprising:
a hemagglutinin (HA) protein comprising amino acid sequence of SEQ ID NO. 10 or an amino acid sequence sharing at least 98% homology with the amino acid sequence of SEQ ID NO. 10; and
a neuraminidase (NA) protein comprising amino acid sequence of SEQ ID NO. 12 or an amino acid sequence sharing at least 98% homology with the amino acid sequence of SEQ ID NO. 12.

2. The H3N2 serotype influenza virus according to claim 1, wherein the amino acid sequence sharing at least 98% homology with the amino acid sequence of SEQ ID NO. 10 contains the same amino acid residue as the amino acid sequence of SEQ ID NO. 10 at least one of positions 97, 127, 142, 176, 188, 209, 212 and 252.

3. The H3N2 serotype influenza virus according to claim 1, having accession number KCTC 11205BP.

4. The H3N2 serotype influenza virus according to claim 1, having accession number KCTC 11206BP.

5. The H3N2 serotype influenza virus according to claim 1, having accession number KCTC 11207BP.

6. An influenza virus vaccine composition, comprising the H3N2 virus of claim 1 acid sequence of SEQ ID NO. 10 or 12—has been deleted and—, wherein the influenza virus is attenuated—inserted therefor.

7. The influenza virus vaccine composition according to claim 6, further comprising aluminum hydroxide gel or oil as an adjuvant.

8. The influenza virus vaccine composition according to claim 6, wherein said vaccine composition further comprises canine parainfluenza virus, canine distemper virus, canine adenovirus or Bordetella bronchiseptica, wherein the viruses and bacteria in the vaccine composition are attenuated.

9. The influenza virus vaccine composition according to claim 6, comprising the influenza virus in an amount of 25 HAU or higher.

10. A method of preventing or treating diseases in canine resulting from influenza virus infection, the method comprising administering the influenza virus vaccine composition of claim 6 to canine in need of such administration.

11. An assay kit for detecting an H3N2 serotype influenza virus, the kit comprising the virus of claim 1.

12. The H3N2 serotype influenza virus according to claim 1, comprising a hemagglutinin (HA) nucleotide comprising the nucleotide sequence of SEQ ID. 9 or a nucleotide sequence sharing at least 98% homology with the nucleotide sequence of SEQ ID NO. 9, and a neuraminidase (NA) nucleotide comprising the nucleotide sequence of SEQ ID. 11 or a nucleotide sequence sharing at least 98% homology with the nucleotide sequence of SEQ ID NO. 11.

13. An influenza virus immunogenic composition, comprising the H3N2 virus of claim 1 or an antigenic determinant thereof, wherein the antigenic determinant is selected from the group consisting of a hemagglutinin (HA) protein comprising the amino acid sequence of SEQ ID NO. 10, a neuraminidase (NA) protein comprising the amino acid sequence of SEQ ID NO. 12, and an amino acid sequence sharing at least 98% homology with the amino acid sequence of SEQ ID NO. 10 or 12.

14. A method of inducing an immune response in canine, the method comprising administering the immunogenic composition of claim 13 to a canine.