Influenza viruses able to infect canids, uses thereof

0001Cross-reference of related applications This application is a partial continuation of US Patent Application No. 11 / 409,416 filed on April 21, 2006; and this application is US Patent Application No. 60 / 728,449 filed on October 19, 2005. No. 60 / 754,881 filed on December 29, 2005, No. 60 / 759,162 filed on January 14, 2006, No. 60 / 761,451 filed on January 23, 2006, and 2006. Claiming priority over No. 60 / 779,080 filed on March 3, 2014, the disclosure of each application is a brief overview, detailed description of the invention, examples, claims, abstracts, figures, tables, The entire body, including the nucleic acid sequence, amino acid sequence and figures, is incorporated herein by reference.

0002Background of the invention "Kennel cough," or infectious tracheobronchitis (ITB), is an acute infectious respiratory infection in dogs, primarily characterized by cough (Ford et al., 1998). Dog ITB is considered one of the most prevalent canine infectious respiratory diseases in the world, and outbreaks reach epidemics when dogs are bred in a densely populated environment such as a kennel. Sometimes. Most outbreaks result from direct dog-to-dog contact or aerosolization of respiratory secretions (Ford et al., 1998). Clinical signs are caused by infection with one or a combination of bacterial and viral substances that colonize the epithelium of the upper and lower respiratory tract. Canine distemper virus (CPiV) and Bordetella bronchiseptica (Bordetella bronchiseptica) are the most common organisms isolated from infected dogs, but canine distemper virus (CDV) and canine distemper virus (CDV) and type 1 and type 2 dogs. Several other viruses, such as adenovirus (CAV-1, CAV-2), as well as Streptococcus Bacteria such as sp.), Pasteurella multocoda and Escherichia coli can influence the clinical course and consequences (Ford et al., 1998). Outbreaks with high prevalence occur extremely efficiently and rapidly in overcrowded populations, but complications of respiratory infections and mortality are rare. Although life-threatening secondary bacterial pneumonia can develop, the majority of ITB cases are self-limited and recover without any treatment (Ford et al., 1998).

0003In July 1992, a presumed "kennel cough" respiratory infection broke out on multiple greyhound trucks in New England, Florida, West Virginia, Wisconsin, Kansas, Colorado, Oklahoma and Arizona. According to veterinarians, the majority of infected dogs recovered with a mild cough, but more than dozens of Greyhounds developed acute hemorrhagic pneumonia and subsequently died suddenly (Greyhound Daily News, 1999).

0004From the end of 1998 to the beginning of 1999, multiple "Kennel cough" outbreaks occurred in racing greyhound kennels across the country, resulting in forced weeks of all racing greyhound trucks and quarantine in the United States. Closed in (Greyhound Daily News, 1999). On one track in Florida (Palm Beach Kennel Club), coughing was recorded in nearly 40% of the dog population per day (personal communication from Dr. William Duggar). Similar to the 1992 outbreak, most greyhounds recovered from coughing, but 10 Florida dogs died of hemorrhagic pneumonia syndrome, which is not characteristic of "Kennel cough" (Putnam, 1999).

0005From March to April 2003, another "Kennel Cough" pandemic broke out on Greyhound trucks in the eastern United States. The outbreak is believed to have originated in four truck kennels in Florida, where dog racing and quarantine were suspended for almost three weeks. Nearly 25% of dogs were infected on trucks in West Palm Beach, and nearly 50% of 1,400 dogs in Derby Lane in St. Petersburg developed cough. Again, most dogs recovered, but several dogs died from respiratory infections. The estimated economic impact of a respiratory pandemic on the Derby Lane truck alone was $ 2 million.

0006There are no previously reported reports demonstrating the etiology or clinical pathology of the "Kennel cough" epidemic in the 1992, 1998-1999, or 2003 racing Greyhound kennels. The infection is presumed to be due to CPiV and / or Bordetella bronchiseptica, the two major causes of Kennilkov. Fatal hemorrhagic pneumonia reported in some coughing dogs is due to infection with β-hemolytic streptococcus (Streptococcus equi subspecies zooepidemicus), according to unproven communications such as the website. , "Canine streptococcal toxin shock" syndrome.

0007Transmission of the virus from one host species to another is an important feature of influenza virus ecology and epidemiology (Webster, 1998). Two basic mechanisms for interspecific transmission of influenza virus are possible (Webster et al., 1992; Lipatov et al., 2004). One is the direct transmission of essentially unchanged virus from one animal species to another. Examples of this mechanism include recent human infections with the H5N1 subtype of avian influenza virus (Subbarao et al., 1998; Peiris et al., 2004; It also includes the 1918 outbreak known as the Spanish flu, Guan et al., 2004) (Reid et al., 2004). The second mechanism is the result of the segmented nature of the influenza genome. When a virus co-infects a host from a different animal species, the segmented viral genes can reassemble and recombination capable of infecting another animal species can occur. For example, in 1957 and 1968, a new virus formed by reassortment between avian and human influenza viruses caused an outbreak of human influenza (Webster et al., 1992; Lipatov et al., 2004; Kawaoka et al.). , 1989).

0008Most direct transmission of the unchanged influenza virus from a natural host animal species to another is a terminal event due to the inability of persistent transmission between individuals of the new animal species. Many virus-host interactions are required for replication and horizontal gene transfer, providing a strong barrier to the persistence of influenza virus in new hosts (Webby et al., 2004). Therefore, the establishment of new host-specific strains of influenza virus is rare and occurs only in poultry, pigs, horses and humans (Webster et al., 1992; Lipatov et al., 2004).

0009Due to the seriousness of influenza infection, there is a need for methods for diagnosing, preventing and treating influenza virus infection.

0010A brief overview of the invention The present invention relates to an isolated influenza virus that can infect canines and cause respiratory disease in canines. The present invention also relates to compositions and methods for inducing an immune response against the influenza virus of the present invention. The present invention also relates to compositions and methods for identifying the viruses of the invention and diagnosing the transmission of the viruses of the invention to animals.

0011One aspect of the invention relates to vaccines and methods for protecting dogs from canine influenza, kits containing such vaccines, and methods for using such vaccines. This protection includes prevention of one or more (typically two or more) of influenza and / or its symptoms, reduction of its risk, delay of its onset, reduction of its spread, relief, suppression, And / or include eradication. The vaccines, kits and methods of the present invention are generally considered suitable for use with respect to dogs. Dogs include wild, zoo and domestic dogs such as wolves, coyotes and foxes. Dogs also include domestic dogs, such as pure and / or hybrid companion dogs, show dogs, working dogs, sheep dogs, hunting dogs, guard dogs, police dogs, race dogs, and / or laboratory dogs. Is done.

0012The present invention also aims in part to methods for protecting dogs from influenza virus infection (ie, preventing influenza virus infection, reducing its risk, delaying, suppressing, ameliorating, or eradicating its onset). The method comprises administering a therapeutically effective amount of a vaccine comprising at least one equine influenza virus antigen, at least one H3 influenza virus antigen, and / or at least one H7 influenza virus antigen.

0013The present invention also aims in part in methods for protecting dogs from respiratory lesions (ie, preventing respiratory lesions, reducing their risk, delaying, suppressing, ameliorating, or eradicating their onset). The method comprises administering to the dog a therapeutically effective amount of a vaccine comprising at least one equine influenza virus antigen, at least one H3 influenza virus antigen, and / or at least one H7 influenza virus antigen.

0014The present invention protects dogs from having canine influenza virus in nasal or oral secretions caused by canine influenza virus infection (ie, prevention of canine influenza virus in nasal or oral secretions, reduction of its risk, Methods for delaying, suppressing, relieving or eradicating its onset) are also partly aimed at. The method comprises administering to the dog a therapeutically effective amount of a vaccine comprising at least one equine influenza virus antigen, at least one H3 influenza virus antigen, and / or at least one H7 influenza virus antigen.

0015The present invention also partially aims at a canine influenza vaccine. In some embodiments, for example, the vaccine comprises a therapeutically effective amount of at least one equine influenza virus antigen, at least one H3 influenza virus antigen, and / or at least one H7 influenza virus antigen.

0016The present invention also partially aims at a kit for protecting a dog from influenza virus infection. The kit contains a therapeutically effective amount of a vaccine containing at least one equine influenza virus antigen, at least one H3 influenza virus antigen, and / or at least one H7 influenza virus antigen. In addition, the kit contains at least one of the following: A device for administering a vaccine to dogs, Pharmaceutically acceptable excipients that support the administration of the vaccine to dogs, A pharmaceutically acceptable excipient that promotes a dog's immune response to a vaccine, Food taken with the vaccine by dogs and / or A treat taken by dogs at the same time as the vaccine.

0017Further benefits of the inventor's invention will become apparent to those skilled in the art by reading this specification.

0018<figref num="1A">Shows the phylogenetic association of the hemagglutinin gene. FIG. 1A shows a phylogenetic tree of HA genes obtained from representative dog, human, avian, porcine and horse isolates, including A / budgerigar / hokkaido / 1/77 (H4) as an outgroup. The phylogenetic tree is estimated from the nucleotide sequence by the neighbor-joining method and shows a bootstrap analysis value of 90%. Bars indicate the number of nucleotide changes per unit length of horizontal branches.</figref><figref num="1B">Shows the phylogenetic association of the hemagglutinin gene. FIG. 1B shows a phylogenetic tree of the canine influenza virus HA gene along with modern and previous equine HA genes using A / duck / Ukraine / 63 (H3) as an outgroup. The phylogenetic tree is estimated from the nucleotide sequence by the neighbor-joining method and shows a bootstrap analysis value of 90%. Bars indicate the number of nucleotide changes per unit length of horizontal branches.</figref><figref num="2">The immunohistochemical detection of influenza H3 antigen in the lung is shown. Lung tissue sections were examined with a mouse monoclonal antibody against H3 hemagglutinin and binding was detected by immunoperoxidase reaction (brown precipitate). FIG. 2A shows the bronchial epithelium obtained from a greyhound with spontaneous disease. Viral H3 antigens were detected in the cytoplasm of bronchial epithelial cells and in macrophages in the airway and alveolar cavities. FIG. 2B shows the bronchial epithelium derived from dogs 5 days after inoculation with A / dog / Florida / 43/2004 (H3N8). The viral H3 antigen was detected in the cytoplasm of bronchial epithelial cells. The memory bar is 66 μm.</figref><figref num="3">It shows the characteristic histological changes in the bronchi of Greyhound who died of hemorrhagic pneumonia associated with influenza infection. The tissue is stained with H &amp; E. Upper: Normal bronchi with ciliated epithelial cells, mucous cells and basal cells. Bottom: Greyhound bronchi with spontaneous influenza. Necrosis and erosion are seen in bronchial ciliated epithelial cells. The memory bar is 100 μm.</figref><figref num="4">Shows the phylogenetic association of the H3 hemagglutinin gene. FIG. 4A shows the phylogenetic tree of the canine influenza virus HA gene as well as the modern and previous equine HA genes. FIG. 4B shows the phylogenetic tree of canine influenza virus HA protein as well as modern and previous equine HA. The phylogenetic tree is estimated from the gene or amino acid sequence by the neighbor-joining method and shows a bootstrap analysis value of 80%. Bars indicate the number of amino acid changes per unit length of horizontal branches.</figref><figref num="5">The influenza virus H3 protein in the epithelial cells of the bronchi and bronchial glands in the lungs of dogs that died of pneumonia associated with influenza virus infection is shown. Upper: Erosion of bronchial ciliated epithelial cells in the bronchi. Tissues were stained with H &amp; E. Bottom: Influenza virus H3 protein in the cytoplasm of epithelial cells of the bronchi (left) and bronchial glands (right). Tissues were stained with a monoclonal antibody against influenza H3 detected by the immunoperoxidase reaction (brown precipitate) and counterstained with hematoxylin.</figref><figref num="6A">An amplification plot of H3 and matrix genes obtained from amplification of transcribed RNA standards in vitro diluted 10-fold is shown.</figref><figref num="6B">An amplification plot of H3 and matrix genes obtained from amplification of transcribed RNA standards in vitro diluted 10-fold is shown.</figref><figref num="6C">Standard curves for H3 and matrix genes created by plotting the logarithm of RNA concentration at the start against the threshold cycle (Ct) obtained from each dilution.</figref><figref num="6D">Standard curves for H3 and matrix genes created by plotting the logarithm of RNA concentration at the start against the threshold cycle (Ct) obtained from each dilution.</figref><figref num="7">Shows that Directigen Flu A susceptibility was tested using a 10-fold serially diluted virus stock containing A / Wyoming / 3/2003 and A / Dog / FL / 242/2003. Purple triangles indicate positive results.</figref>

0019Detailed disclosure of the invention The present invention relates to an isolated influenza virus that can infect canines and cause respiratory illness. In one embodiment, the influenza viruses of the invention are SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78, or any of its functional and / or immunogenic fragments or variants Includes a polynucleotide encoding a protein with the indicated amino acid sequence. In a specific embodiment, the polynucleotides are SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, Includes the nucleotide sequence shown in 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77, or any fragment or variant thereof. The influenza virus of the present invention is the HA subtype of H1, H2, H3, H4, H5, H6, H7, H8, and H9, H10, H11, H12, H13, H14, H15 or H16, or N1, N2, N3. , N4, N5, N6, N7, N8 or N9 NA subtypes can be included. In a specific embodiment, the influenza virus of the present invention is an H3 subtype. The virus can be isolated from infected dogs and cultured in cells or eggs according to the methods disclosed herein. In one exemplary embodiment, the influenza virus is influenza A virus.

0020The present invention also relates to polynucleotides containing whole or part of the gene of the influenza virus of the present invention, or genomic segments. In one embodiment, the polynucleotides of the invention are hemagglutinin (HA) gene, neurominidase (NA) gene, nuclear protein (NP) gene, matrix protein (MA or M) gene, polymerase basic (PB) protein gene, polymerase acidity. Includes (PA) protein genes, non-structured (NS) protein genes, or functional fragments or variants of any of these genes. In one specific embodiment, the polynucleotide of the invention comprises the hemagglutinin (HA) gene or a functional fragment or variant thereof. In a further embodiment, the HA gene encodes a hemagglutinin protein having one or more of the following: serine at position 83; leucine at position 222; threonine at position 328; and / or with respect to the amino acid sequence of the equine H3 common sequence: Threonine in 483rd place. In one embodiment, the HA gene encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 16, 32, 62, or 78, or a functional and / or immunogenic fragment or variant thereof. In a specific embodiment, the HA gene comprises the nucleotide sequence set forth in SEQ ID NO: 15, 31, 61 or 77.

0021In one embodiment, the polynucleotides of the invention are SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78, or any of its functional and / or immunogenic fragments or variants Encodes a polypeptide having the indicated amino acid sequence. In specific embodiments, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, The polynucleotides encoding the amino acid sequences shown in 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 are SEQ ID NOs: 1, 3, 5, 7, respectively. 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, Nucleotide sequence shown in 73, 75 or 77, or SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34 , 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78 functional and / or immunogenic fragments or variants. Contains the array to code. Therefore, the present invention relates to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55. , 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77 nucleotide sequence, or SEQ ID NO:: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55, 57, 59, 61, 63, Concerning a polynucleotide sequence comprising a fragment of any of 65, 67, 69, 71, 73, 75 or 77 or a variant comprising a degenerate variant. In a further specific embodiment, the polynucleotides of the invention can include: nucleotides 1-2271 of SEQ ID NO: 3; nucleotides 1-2148 of SEQ ID NO: 5; Nucleotide sequence: 1 to 1494 nucleotides of SEQ ID NO: 9; 1 to 1410 nucleotides of SEQ ID NO: 11; 1 to 756 nucleotides of SEQ ID NO: 13; 1 to 1695 nucleotides of SEQ ID NO: 15; SEQ ID NO: 19 1 to 2271 nucleotides; SEQ ID NO: 21 to 1 to 2148 nucleotides; SEQ ID NO: 23 to 1 to 657 nucleotides; SEQ ID NO: 25 to 1 to 1494 nucleotides; SEQ ID NO: 29 to 1 to 756 nucleotides SEQ ID NO: 31-1 to 1695 nucleotides; SEQ ID NO: 47 1-2277 nucleotides; SEQ ID NO: 49 1-2271 nucleotides; SEQ ID NO: 51 1-2148 nucleotides; SEQ ID NO: 53 1 to 690 nucleotides; SEQ ID NO: 55 to 1 to 1494 nucleotides; SEQ ID NO: 57 to 1 to 1410 nucleotides; SEQ ID NO: 59 to 1 to 756 nucleotides; SEQ ID NO: 61 to 1 to 1695 nucleotides; SEQ ID NO: 63 1-2277 nucleotides; SEQ ID NO: 65 1-2271 nucleotides; SEQ ID NO: 67 1-2148 nucleotides; SEQ ID NO: 69 1-2690 nucleotides; SEQ ID NO: 71-1 ~ 1494 nucleotides; SEQ ID NO: 73-1 ~ 1410 nucleotides; SEQ ID NO: 75 1 ~ 756 nucleotides; and SEQ ID NO:: 77 1 to 1695 nucleotides. In addition, the nucleotide and amino acid sequences of viral polynucleotide and polypeptide sequences considered within the scope of the present invention can be found in GenBank at accession numbers DQ124147 to DQ124161 and DQ124190, the disclosure of which is incorporated herein by reference. It has been deposited.

0022The present invention also relates to polypeptides encoded by the polynucleotides of the influenza viruses of the present invention. The present invention also relates to functional and / or immunogenic fragments and variants of this polypeptide. Intended polypeptides include HA proteins, NA proteins, NS proteins, nucleoproteins, polymerase basic proteins, polymerase acidic proteins and matrix proteins of the influenza viruses of the invention. In one exemplary embodiment, the polypeptides of the invention have SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78, or any of its functional and / or immunogenic fragments or variants. It has the amino acid sequence shown in.

0023The present invention also relates to a polynucleotide expression construct comprising the polynucleotide sequence of the present invention. In one embodiment, the expression constructs of the invention are SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34. , 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78, or any of its functional and / or immunogenic fragments or variants. Includes a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in. In specific embodiments, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, The polynucleotides encoding the amino acid sequences shown in 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 are SEQ ID NOs: 1, 3, 5, 7, respectively. 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, Nucleotide sequence shown in 73, 75 or 77, or SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34 , 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 encoding any functional and / or immunogenic fragment or variant Contains the sequence to be. Therefore, the present invention relates to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55. , 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77 nucleotide sequence, or SEQ ID NO:: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55, 57, 59, 61, 63, Includes a polynucleotide sequence comprising a fragment of any of 65, 67, 69, 71, 73, 75 or 77 or a variant comprising a degenerate variant. In a preferred embodiment, the expression construct of the invention corresponds to overexpression of the functionally bound polynucleotide of the invention.

0024Expression constructs of the invention generally include regulatory elements that are functional in the target host cell in which the expression construct is to be expressed. Thus, those skilled in the art can select regulatory elements for use in, for example, human host cells, mammalian host cells, insect host cells, yeast host cells, bacterial host cells and plant host cells. In one embodiment, the regulatory element is an element that is functional in dog cells. Regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers and polyadenylation elements. As used herein, the term "expression construct" refers to a combination of nucleic acid sequences that corresponds to the transcription of a functionally bound nucleic acid sequence. As used herein, the term "functionally coupled" refers to the proximalities of the components described that are in a relationship that allows them to function in their intended manner. In general, functionally connected components are in close proximity.

0025Expression constructs of the invention can include promoter sequences that functionally bind to the polynucleotide sequences encoding the polypeptides of the invention. Promoters can be incorporated into polynucleotides using standard techniques known in the art. Many replicas of the promoter or many promoters can be used in the expression constructs of the present invention. In a preferred embodiment, the promoter can be located at a distance from the transcription start site in an expression construct that is approximately identical to the distance from the transcription start site in its native genetic environment. Some variation at this distance is possible without substantially reducing promoter activity. Transcription initiation sites are typically included in the expression construct. Preferably, the promoter associated with the expression construct of the invention corresponds to the overexpression of the functionally bound polynucleotide of the invention.

0026Promoters for use with the expression constructs of the invention in eukaryotic cells can be of viral or cellular origin. Viral promoters include, but are not limited to, the cytomegalovirus (CMV) gene promoter, the SV40 early or late promoter, or the Raus sarcoma virus (RSV) gene promoter. Promoters of cell origin include, but are not limited to, the desmin gene promoter and the actin gene promoter. Promoters suitable for use with the expression constructs of the invention in yeast cells include the 3-phosphoglycerate kinase promoter, the glyceraldehyde-3-phosphate dehydrogenase promoter, the metallothioneine promoter, the alcohol dehydrogenase-2 promoter and the hexokinase promoter. However, it is not limited to these.

0027If the expression construct must be donated or introduced into plant cells, see, for example, the cauliflower mosaic virus (CaMV) 35S (enhanced CaMV 35S promoter) (see, eg, US Pat. No. 5,106,739 and An, 1997). )) Or plant viral promoters such as the CaMV 19S promoter can be used. Other promoters that can be used as expression constructs in plants include, for example, the prolifera promoter, the Ap3 promoter, the heat shock promoter, and A. tumefaciens (A. tumefaciens) T-DNA 1'-or 2'-promoter, polygalacturonase promoter, petunia-derived calconsynthase A (CHS-A) promoter, tobacco PR-1a promoter, ubiquitin promoter, actin promoter, alcA gene promoter, pin2 Promoter (Xu et al., 1993), Corn WipI Promoter, Corn trpA Promoter (US Pat. No. 5,625,136), Corn CDPK Gene Promoter, and RUBISCO The SSU promoter (US Pat. No. 5,034,322) can also be used. U.S. Patent Application No. 6,455,760 or U.S. Patent No. 6,696,623 disclosed or published U.S. Patent Application No. 20040078841; U.S. Patent Application No. 20040067506; U.S. Patent Application No. 20040019934; U.S. Patent Application No. 20030177536; U.S. Patent Application No. Root-specific promoters, such as any promoter sequence disclosed in 20030084486; or US Patent Application No. 200404123349, can be used with the expression constructs of the invention. Constitutive promoters (such as CaMV, ubiquitin, actin or NOS promoters), expression-regulating promoters, and inducible promoters (such as unexpected promoters that can be induced by heat, light, hormones or chemicals) are also polynucleotide expression constructs of the invention. Considered for use with. For example, tissue-specific promoters such as the tomato E8 promoter (accession number: AF515784; Good et al., (1994)) such as the fruit-specific promoter can also be used. Seed-specific promoters such as the glycinin gene (eg, soybean) and other derived promoters can also be used.

0028For expression in prokaryotic systems, the expression constructs of the present invention include, for example, the alkaline phosphatase promoter, tryptophan (trp) promoter, lambda P._LIt can include promoters such as promoters, β-lactamase promoters, lactose promoters, phoA promoters, T3 promoters, T7 promoters, or tac promoters (de Boer et al., 1983).

0029The expression construct of the present invention may optionally include a transcription termination sequence, a translation termination sequence, a sequence encoding a signal peptide and / or an enhancer element. The transcription termination region can typically be obtained from the 3'untranslated region of the eukaryotic or viral sequence. The transcription termination sequence may be located downstream of the coding sequence to prepare for effective termination. The signal peptide sequence is a short amino acid sequence typically located at the amino terminus of the protein to a wide range of post-translational intracellular destinations, from specific organelle compartments to protein sites of action and the extracellular environment. Controls the migration of functionally bound mature polypeptides. Targeting the gene product to a cell and / or extracellular destination of interest through the use of a functionally bound signal peptide sequence is conceivable for use with the polypeptides of the invention. Classic enhancers are cis-regulatory elements that enhance gene transcription and can also be included in expression constructs. Classic enhancer elements are known in the art and CaMV Includes, but is not limited to, 35S enhancer elements, cytomegalovirus (CMV) early promoter enhancer elements and SV40 enhancer elements. Intron-mediated enhancer elements that enhance gene expression are also known in the art. These elements must be within the transcription region and are orientation dependent.

0030DNA sequences that induce polyadenylation of mRNA transcribed from the expression construct can also be included in the expression construct, including, but not limited to, octopin synthase or noparin synthase signals.

0031The expression construct can also include, for example, one or more dominant selectable marker genes, including genes encoding antibiotic resistance and / or herbicide resistance for selecting transformed cells. Antibiotic resistance genes can provide resistance to one or more of the following antibiotics: hyglomycin, kanamycin, bleomycin, G418, streptomycin, paromomycin, neomycin and spectinomycin. Kanamycin resistance can be provided by neomycin phosphotransferase (NPT II). Herbicide resistance genes may provide resistance to phosphinosricin acetyltransferase or glyphosate. Other markers used to screen for cell transformation include β-glucuronidase (GUS), β-galactosidase, luciferase, noparin synthase, chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), or enhanced. Includes, but is not limited to, GFP (Yang et al., 1996).

0032The present invention also relates to a polynucleotide vector comprising the polynucleotide sequence of the present invention encoding the polypeptide of the present invention. Unique restriction enzyme sites can be included at the 5'and 3'ends of expression constructs or polynucleotides of the invention that allow insertion into polynucleotide vectors. As used herein, the term "vector" is a plasmid, cosmid, chromosome, phage that is replicable when bound to the appropriate regulatory region and is capable of transferring polynucleotide sequences between cells. , Refers to any genetic element, including viruses and the like. The vector contains a nucleotide sequence that allows the vector to replicate within a particular host cell. Many vectors are available for expression and / or cloning, including, but not limited to, pBR322, pUC series, M13 series, pGEM series, and pBLUESCRIPT vectors (Stratagene, La Jolla, CA and). Promega, Madison, WI).

0033The present invention also relates to oligonucleotide probes and primers, such as polymerase chain reaction (PCR) primers, which can hybridize to the coding or non-coding sequences of the polynucleotides of the invention. The oligonucleotide probe of the present invention can be used in a method for detecting an influenza virus nucleic acid sequence. The oligonucleotide primers of the present invention can be used in PCR and other methods involving nucleic acid amplification. In a preferred embodiment, the probes or primers of the invention can hybridize to the polynucleotides of the invention under stringent conditions. The probes and primers of the present invention can optionally include detectable label or reporter molecules such as fluorescent molecules, enzymes, radioactive molecules and the like. The probes and primers of the present invention can be of any suitable length in the method or assay used. Typically, the probes and primers of the invention are 10 to 500 or more nucleotides in length. 10-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, or 101 or longer nucleotide length probes And primers are considered to be within the scope of the present invention. In one embodiment, the probes and primers are 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, The length of any 29 or 30 nucleotides. The probes and primers of the present invention may have complete (100%) nucleotide sequence identity with the polynucleotide sequence, otherwise sequence identity may be less than 100%. For example, the sequence identity between a probe or primer and a sequence is 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, or the probe or primer is the book. For the nucleotide sequence of the polynucleotide of the invention It can be any other percentage of sequence identity as long as it can hybridize under stringent conditions. The exemplary probes and primers of the invention are SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, Includes any of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, or any functional fragment of SEQ ID NO: 35-46 or one having the nucleotide sequence shown in the variant.

0034As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide" refer to single- or double-stranded deoxyribonucleotides, ribonucleotides, or mixed polymers of deoxyribonucleotides and ribonucleotides. Includes known analogs of natural nucleotides that can function in a manner similar to natural nucleotides, unless otherwise limited. Polynucleotide sequences include RNA that can be translated into proteins and DNA strand sequences that can be transcribed into RNA strands. Complementary sequences of any nucleic acid, polynucleotide or oligonucleotide of the invention are also considered within the scope of the invention. Polynucleotide sequences further include full-length sequences and shorter sequences derived from full-length sequences. The present invention also includes polynucleotides that are complementary in sequence to the polynucleotides disclosed herein. The polynucleotides and polypeptides of the invention can be provided as purified or isolated forms.

0035Due to the degeneracy of the genetic code, a variety of different polynucleotide sequences can encode the polypeptides of the invention. A table showing all possible triplet codons (U also represents T) and the amino acids encoded by each codon are described in Lewin (1985). Moreover, it is well within the skill of one of ordinary skill in the art to produce alternative polynucleotide sequences encoding identical or essentially identical polypeptides of the invention. These degenerate variants and alternative polynucleotide sequences are within the scope of the present invention. As used herein, references to "essentially identical" sequences do not materially alter the functionality and / or immunogenic activity of the polypeptides encoded by the polynucleotides of the invention. Refers to a sequence encoding an amino acid substitution, deletion, addition or insertion.

0036The present invention also relates to variants of the polynucleotides of the invention encoding the polypeptides of the invention. Mutant sequences include sequences in which one or more nucleotides of the sequence have been substituted, deleted and / or inserted. Nucleotides that can be substituted with respect to the natural nucleotides of DNA include, but are not limited to, inosin, 5-fluorouracil, 5-bromouracil, hypoxanthine, 1-methylguanine, 5-methylcytosine and tritylated bases. Have. The sugar molecules of the nucleotides in the sequence can be further modified to include, but are not limited to, arabinose, xylulose and hexose. In addition, the nucleotides adenine, cytosine, guanine, thymine and uracil bases can be modified with acetyl, methyl and / or thio groups. Sequences containing nucleotide substitutions, deletions and / or insertions can be prepared and tested using standard techniques known in the art.

0037Substitutions of amino acids other than those specifically exemplified or naturally contained in the polypeptides of the invention are also considered to be within the scope of the invention. For example, a non-natural amino acid can be replaced with an amino acid of the polypeptide as long as the polypeptide having the substituted amino acid maintains substantially the same functional activity as the polypeptide in which the amino acid is not substituted. Examples of unnatural amino acids include ornithine, citrulin, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, γ- Aminobutyric acid, ε-aminohexanoic acid, 6-aminohexanoic acid, 2-aminoisobutyric acid, 3-aminopropionic acid, norleucine, norvaline, sarcosin, homocitrulin, cysteine acid, τ-butylglycine, τ-butylalanine, phenylglycine , Cyclohexylalanine, β-alanine, fluoroamino acid, β-methylamino acid, C-methylamino acid, N-methylamino acid and other designer amino acids acids), and common amino acid analogs, but are not limited to these. Non-natural amino acids also include amino acids with derivative-formed side chains. In addition, any amino acid in the protein may be D-type (right-handed) or L-type (left-handed). Allelic variants of the protein sequences of the polypeptides of the invention are also included within the scope of the invention.

0038Amino acids can generally be classified into the following classes: non-polar, uncharged polar, basic and acidic. A conservative substitution in which a polypeptide of the invention having one class of amino acids is replaced by another amino acid of the same class remains substantially identical to a polypeptide in which the substituted polypeptide has not undergone substitution. As long as it retains its functional activity, it is within the scope of the present invention. Polynucleotides encoding polypeptides having one or more amino acid substitutions within the sequence are considered within the scope of the invention. Table 11 below shows a list of amino acid examples belonging to each class. Table 12 shows the abbreviations for amino acids in one-letter notation.

0039Fragments and variants of the influenza virus polypeptides of the invention are made using standard methods known in the art and are functional or immunogenic using standard methods known in the art. You can find out about. For example, in testing fragments and / or variants of the neuraminidase polypeptide of the invention, enzyme activity can be measured. Thus, one of ordinary skill in the art will readily create and test fragments and variants of the polypeptides of the invention, the fragments or variants retaining activity compared to full-length or non-mutant polypeptides. You can find out if you have one.

0040Polynucleotides and polypeptides considered to be within the scope of the invention may also be defined in terms of a higher specific range of identity and / or similarity to the sequences of the invention specifically exemplified herein. it can. Sequence identity is typically higher than 60%, preferably higher than 75%, more preferably higher than 80%, even more preferably higher than 90%, higher than 95%. obtain. Sequence identity and / or similarity is 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, as compared to the sequences exemplified herein. 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, It can be 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%. Unless otherwise noted, the sequence identity and / or percent similarity of the two sequences as used herein is based on the Karlin and Altschul algorithms modified by Karlin and Altschul (1993) (1990). It can be obtained by using. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990). To obtain a sequence with the desired percent sequence identity, a BLAST search can be performed using NBLAST with a score of 100 and a word length of 12. Gapped BLAST can be used as described in Altschul et al. (1997) to obtain gap alignment for comparison. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) may be used. See the NCBI / NIH website.

0041The present invention provides sequences that are sufficiently homologous to the polynucleotide sequences exemplified herein for hybridization with their sequences under standard stringent conditions and standard methods (Maniatis et al., 1982). Polynucleotide molecules with are also intended. As used herein, the "stringent" condition in hybridization is typically a DNA hybrid in hybridization 6 × SSPE, 5 × Denhardt solution, 0.1% SDS, 0.1 mg / ml denatured DNA. Refers to conditions that are carried out overnight at 20 to 25 C below the melting point (Tm) of. The melting point Tm is given by the following equation (Beltz et al., 1983): Tm = 81.5 C + 16.6 Log [Na +] +0.41 (% G + C) -0.61 (% formamide) -600 / base pair double-stranded length.

0042Cleaning is typically done as follows: (1) 2 times for 15 minutes each at room temperature at 1 x SSPE, 0.1% SDS (low stringent cleaning) (2) Once at 0.2 x SSPE, 0.1% SDS with Tm-20C for 15 minutes (medium stringent washing).

0043The present invention also relates to viral proteins and peptides encoded by the genes of the influenza viruses of the present invention. In one embodiment, the viral protein is a mature HA protein. In a specific embodiment, the mature HA protein comprises one or more of: serine at position 82; leucine at position 221; threonine at position 327; and / or threonine at position 482. In one exemplary embodiment, the mature HA protein has the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 34, or a functional and / or immunogenic fragment or variant of SEQ ID NO: 33 or SEQ ID NO: 34. Have. In another embodiment, the viral protein is an NA protein, NS protein, PB protein, PA protein or MA protein. The viral proteins and peptides of the invention can be used to produce antibodies that specifically bind to the protein or peptide. The viral proteins and peptides of the present invention can also be used as immunogens and as vaccine compositions.

0044The invention also relates to compositions and methods for eliciting an immune response against influenza viruses that can infect susceptible host animals and cause respiratory disease. The present invention can be used to elicit an immune response against any subtype of influenza virus in susceptible host animals. For example, influenza viruses are H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16 HA subtypes, and N1, N2, N3, N4. , N5, N6, N7, N8 or N9 NA subtypes. In one embodiment the HA subtype is H3 or H5. In a further embodiment, the NA subtype is N7 or N8. In a specific embodiment, the immune response is elicited against the subtype H3N8 influenza virus. In one embodiment, the host animal is Canidae. Canines include wild, zoo and domestic dogs such as wolves, coyotes and foxes. Dogs also include domestic dogs, such as pure and / or hybrid companion dogs, show dogs, working dogs, sheep dogs, hunting dogs, guard dogs, police dogs, race dogs, and / or laboratory dogs. Is done. In one specific embodiment, the host animal is a domesticated dog, such as a greyhound. In one embodiment, the animal is administered an effective amount of an immunogenic composition of the invention sufficient to elicit an immune response against the influenza virus of the invention. The immune response can be a humoral and / or cell-mediated immune response. In one specific embodiment, the immune response is a defensive immune response that can prevent or minimize viral infection in an immunized host animal for a period of time after immunization. Accordingly, the invention also relates to vaccine compositions and methods capable of providing vaccinated animals with a protective immune response against the viruses of the invention.

0045As described herein, the vaccines or immunogenic compositions of the invention are cell-free complete viruses, including attenuated or inactivated viruses, or subvirion particles (parts of viral lipids that have been treated with virions). All removed "split vaccines"), viral proteins (including individual proteins and high molecular weight complexes of many proteins), polypeptides, and parts of the virus containing the peptides, as well as virus-infected cell lines. , Or any combination thereof. Vaccines or immunogenic compositions containing virus-infected cell lines may contain many cell lines, each infected with a different virus line.

0046In one embodiment of the invention, the dog is one of one or more inactivated (ie, killed) and / or attenuated live influenza virus vaccines, or influenza virus antigens derived from one or more virus isolates. It can be immunized with a vaccine containing one or many. In one embodiment, the influenza virus is a canine influenza virus. In another embodiment, the influenza virus is at least about 90%, or at least about 95%, or at least about 96%, or 97%, or 98%, or 99%, or 99%, or it of the canine influenza virus polypeptide. Equine influenza virus that encodes or expresses a polypeptide with a higher amino acid sequence identity. In one embodiment, the influenza antigen used in the vaccine of the invention has at least about 96% sequence identity with the HA and / or NA antigens of canine influenza virus.

0047An example of an inactivated vaccine is EQUICINE II , which is marketed as a liquid vaccine by Intervet Inc. (Millsboro, DE, USA). EQUICINE II is Carbopol (ie) inactivated A / Pennsylvania / 63 influenza virus ("A / Pa / 63") and A / horse / Kentucky / 93 influenza virus ("A / KY / 93"). , HAVLOGEN® (Intervet Inc.)). More specifically, the dose of EQUICINE II is 10 inactivated A / Pa / 63.^6.0 EID₅₀, Inactivated A / KY / 93 10^6.3 EID₅₀, 0.25% carbopol based on volume, contains enough PBS to make a total volume of 1 ml.

0048Another example of an inactivated vaccine is equine influenza virus A / horse / Ohio / 03 ("Ohio 03"). In some embodiments, such vaccines include CARBIGEN , an emulsified polymer-based adjuvant commercially available from MVP Laboratories, Inc. (Ralston, NE). In such vaccines, the unit of administration typically comprises at least about 250 HA units of virus, about 250 to about 12,500 HA units of virus, or about 1000 to about 6200 HA units of virus. The recommended concentration of CARBIGEN is from about 5 to about 30% (based on mass).

0049An example of a live attenuated vaccine is the modified raw horse / Kentucky / 91 ("A / KY / 91") influenza in the dosage form of a lyophilized vaccine that can be redissolved in water. In some embodiments, this redislysis is performed with sufficient vaccine grade water to bring the vaccine dose to a total fluid volume of 1 ml. Such vaccine aspects are discussed, for example, in US Pat. Nos. 6,436,408, 6,398,774 and 6,177,082, all of which are incorporated herein by reference. When redissolved, the dose of such vaccine would be, for example, 10 per ml.^7.2 TCID₅₀Includes A / KY / 91, 0.015 g NZ AMINE AS per ml, 0.0025 g gelatin per ml, and 0.04 g D lactose per ml. NZ AMINE AS is a purified source of amino acids and peptides produced by enzymatic hydrolysis of casein. NZ AMINE AS is sold by Kerry Bio-Science (Norwich, NY, USA).

0050In a preferred embodiment, the vaccine comprises an H3 influenza antigen having at least about 93% homology to Florida / 43/2004 in the HA coding sequence, eg, horse / Newmarket / 79 strains. Preferred homology is at least about 96%, such as horse / Alaska / 1/91 and horse / Santiago / 85 strains. In subsequent examples, Horse / Kentucky / 91, Horse-2 / Kentucky / 93, Horse-1 / Pennsylvania / 63 and Horse Ohio / 03 influenza antigens were incorporated into the vaccine. Preferred vaccines also include vaccines containing Horse / Wisconsin / 03, Horse / Kentucky / 02, Horse / Kentucky / 93 and Horse / Newmarket 2/93. In subsequent examples, the H3N8 virus was used. However, it is considered that other H3 influenza viruses can be used according to the present invention.

0051Live attenuated vaccines can be prepared in a convenient way. Such methods generally include, for example, modification of pathogenic strains by passage in vitro, low temperature adaptation, modification of biological pathogenicity by genetic engineering, preparation of chimeras, insertion of antigens into viral vectors, non-pathogenic wilderness. Including stock selection and so on.

0052In some embodiments, the attenuated live virus strain is induced by continuous passage of wild-type virus through cell cultures, laboratory animals, non-host animals or chicken eggs. Accumulation of gene mutations during such passages typically leads to a gradual loss of pathogenicity of the organism to the original host.

0053In some embodiments, live attenuated virus strains are prepared by co-infection with attenuated mutant and pathogenic viruses in tolerant cells. The resulting desired recombinant virus has the safety of an attenuated virus that carries the gene encoding the protective antigen from the pathogenic virus.

0054In some embodiments, live attenuated virus strains are prepared by cold adaptation. Cold-adapted viruses have the advantage of replicating only at the temperatures found in the upper respiratory tract. A method for producing a cold-adapted equine influenza virus is described in US Pat. No. 6,177,082. The resulting desirable cold-adapted virus confers one or more of the following phenotypes: cold adaptation, temperature sensitivity, dominant interference and / or attenuation.

0055In some embodiments, the live attenuated virus strain is used to convert a pathogenic virus into a non-pathogenic or less pathogenic virus relative to the original virus while retaining the protective properties of the original virus. Prepared by molecular methods such as point mutation, deletion or insertion.

0056In some embodiments, live attenuated viruses are prepared by cloning candidate genes for protective antigens into the genomes of non-pathogenic or hypopathogenic viruses or other organisms.

0057Inactivated (ie, "killed") virus vaccines can be prepared by inactivating the virus using a convenient method. Typically, such vaccines include excipients capable of promoting an immune response, as well as other excipients customarily used in vaccines. For example, in subsequent examples, EQUICINE II includes HAVLOGEN . Inactivation of the virus inactivates the virus in order to incapacitate the replication ability of the virus, for example, formalin, β-propiolactone ("BPL"), bromoethylamine ("BEA"), and binary ethyleneimine ("BEA"). It can be achieved by treatment with "BEI") or by non-chemical methods (eg, heat, freeze / thaw, or ultrasonic treatment).

0058In subsequent examples, horse / Ohio / 03 was used as the stimulating virus. It is known to have approximately 99% homology with the Florida / 43/04 isolate and has been shown to induce symptoms of infection and serum conversion in dogs. Example 18 illustrates the efficacy of the horse influenza vaccine in dogs and inhibits hemagglutination (or "HI" or "HI" or "HI" in dogs vaccinated with the inactivated Ohio 03 antigen in a vaccine composition comprising CARBIGEN adjuvant. HAI ") Indicates the titer. Table 29 shows the titers before, after vaccination, after the second vaccination, and after stimulation. The results show that there is little or no increase in HI titers at each stage after vaccination in vaccinated dogs, and in controls. Table 30 illustrates the clinical signs, virus isolation, and histopathological results from the same study. Stimulated animals did not show clinical signs, viral shedding, or clear histopathology, but positive HI titers (Table 29) show significant antibody titers in immunized animals.

0059It should be noted that other H3 influenza virus antigen vaccines are also included in the present invention. Those described in this specification and subsequent examples are shown to illustrate the present invention and preferred embodiments thereof, and are not intended to limit the scope of the claimed invention.

0060It should also be noted that influenza antigens other than the H3 influenza virus antigen can be used in accordance with the present invention. Such antigens include, for example, the horse / PA / 63 antigen, which is the horse A1 subtype (H7N7). It is conceivable that one or more of such antigens may be used with or without one or more H3 influenza antigens.

0061Generally, the vaccine is administered in a therapeutically effective amount. A "therapeutically effective amount" is an amount sufficient to elicit a protective response against the target virus in canine patients. Typically, administration causes it to prevent one or more (typically two or more) of influenza or its symptoms, reduce its risk, delay its onset, and spread it. It is "therapeutically effective" when it is reduced, relieved, suppressed or eradicated. Typical influenza symptoms include, for example, fever (typically 103.0 in dogs).^○F; 39.4 ° C), cough, sneezing, histopathological lesions, ocular secretions, nasal secretions, vomiting, diarrhea, depression, weight loss, dry vomiting, hemoptysis, and / or audible rales. Is done. Other often more severe symptoms include, for example, bleeding in the lungs, mediastinum or pleural space; tracheitis; bronchitis; bronchopneumonia; supportive bronchopneumonia; and / or favor in the epithelial lining of the lungs and airway lumen. Infiltration of mediastinal and / or macrophages is included.

0062The vaccine may be administered as part of a combination therapy, i.e., a therapy that includes the administration of one or more additional active substances, adjuvants, therapies, etc. in addition to the vaccine itself. In this case, it is recognized that the amount of vaccine that constitutes a "therapeutically effective" amount may be less than the amount of vaccine that would constitute an "effective" amount if the vaccine were administered alone. It must be. Other therapies include, for example, antivirals, analgesics, antipyretics, sputum, antiinflammatorys, antihistamines, antibiotics to treat bacterial infections caused by influenza virus infections, rest, and / or infusions. It may include therapies known in the art. In some embodiments, the vaccine of the invention is administered in combination with a pertussis vaccine, an adenovirus vaccine, and / or a parainfluenza virus vaccine.

0063In some embodiments, for example, a typical dose of live attenuated vaccine is at least about 10.³pfu / dog, more typically about 10³~ About 10⁹pfu / dog. In the present invention, "pfu" means "plaque forming unit". In some embodiments, the typical dose of live attenuated vaccine is at least about 10³ TCID₅₀/ Dog, more typically about 10³~ About 10⁹ TCID₅₀/ A dog. In some embodiments, the typical dose of live attenuated vaccine is at least about 10.³ EID₅₀/ Dog, more typically about 10³~ About 10⁹ EID₅₀/ A dog. In some embodiments, the typical dose of the killed vaccine is at least about 40 HA units, typically about 40 to about 10,000 HA units, and more typically about 500 to about 6200 HA units. .. In some embodiments, the dose is from about 6100 to about 6200 HA units.

0064In some preferred embodiments, the vaccine makes the live attenuated vaccine at least about 10 more than the immunogenic amount.^0.5 pfu / dog Contains at high concentrations. In some preferred embodiments, the vaccine makes the live attenuated vaccine at least about 10 more than the immunogenic amount.^0.5 TCID₅₀/ Dog Contains in high concentration. In some preferred embodiments, the vaccine makes the live attenuated vaccine at least about 10 more than the immunogenic amount.^0.5 EID₅₀/ Dog Contains at high concentration.

0065The immunogenicity can be determined experimentally by a stimulus dose titration test method generally known in the art. Such techniques typically involve vaccination of many dogs with different doses of vaccine, followed by stimulating the dogs with a pathogenic virus to determine the lowest protective dose.

0066Factors that influence a preferred dosing regimen include, for example, the type of subject (eg, species and breed), age, weight, gender, diet, activity, lung size, and condition; route of administration; specific vaccine used. Profile of efficacy, safety, and immune duration; whether a delivery system is used; and whether the vaccine is administered as part of a drug and / or vaccine combination. Therefore, the doses actually used can vary in a particular animal and can therefore deviate from the typical doses shown above. Such dosage adjustment decisions are generally within the skill of one of ordinary skill in the art using simple methods. It should be further noted that live attenuated viruses are generally self-propagating and therefore the specific amount of such virus administered is not always definitive.

0067It is possible that the vaccine may be given to canine patients once, or, as an alternative, twice or more over the days, weeks, months or years. In some embodiments, the vaccine is administered at least twice. In some such embodiments, for example, the vaccine is administered twice and the second dose (eg, booster) is administered at least about 2 weeks after the first dose. In some embodiments, the vaccine is administered twice, with the second dose being administered within 8 weeks after the first dose. In some embodiments, the second dose is about 2 to about 4 years after the first dose, about 2 to about 8 weeks after the first dose, or about 3 to about 4 weeks after the first dose. Be administered. In some embodiments, the second dose is administered approximately 4 weeks after the first dose. In the above embodiments, the first and subsequent doses may vary, for example, in amount and / or dosage form. However, the doses are often the same in terms of amount and dosage form. When only a single dose is administered, the amount of vaccine at that dose alone generally includes the therapeutically effective amount of vaccine. However, if more than one dose is administered, the combined dose of vaccine may include a therapeutically effective amount.

0068In some embodiments, the vaccine is administered before the canine recipient becomes infected with influenza. In such embodiments, the vaccine is used, for example, to prevent the symptoms of influenza or one or more (typically two or more) influenza, reduce its risk, or delay its onset. Can be administered.

0069In some embodiments, the vaccine is administered after the canine recipient has been infected with influenza. In such embodiments, the vaccine may alleviate, suppress or eradicate, for example, influenza or one or more (typically two or more) influenza symptoms.

0070The preferred composition of the vaccine depends, for example, on whether the vaccine is an inactivated vaccine, a live attenuated vaccine, or both. It also depends on the method of vaccine administration. The vaccine contains one or more of the usual pharmaceutically acceptable carriers, adjuvants, other immune response promoters, and / or vehicles (collectively referred to as "excipients"). Is possible. Such excipients are generally selected to be compatible with the active ingredient in the vaccine. The use of excipients is generally known to those of skill in the art.

0071The term "pharmaceutically acceptable" is used adjective to mean that a modified noun is suitable for use in a pharmaceutical product. For example, when used to indicate an excipient in a pharmaceutical vaccine, it is compatible with the excipient in the other components of the composition and is inconveniently harmful to the intended recipient dog. Not considered.

0072The vaccine can be administered by conventional methods, including, for example, mucosal administration (such as intranasal, oral, intratracheal and ophthalmic) and parenteral administration. Mucosal administration is often particularly advantageous for live attenuated vaccines. Parenteral administration is often particularly advantageous for inactivated vaccines.

0073Mucosal vaccines can be in liquid dosage forms such as, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. Suitable excipients for such vaccines include, for example, water, physiological saline, dextrose, glycerol, lactose, sucrose, starch powder, alkanoic acid cellulose esters, cellulose alkyl esters, talc, stearate, stearate. Included are inert diluents widely used in the art such as sodium and calcium salts of magnesium, magnesium oxide, phosphoric acid and sulfuric acid, gelatin, gum arabic, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol. Excipients can also include various wetting agents, emulsifying agents, suspending agents, flavors (eg, sweeteners), and / or aromatics.

0074The oral mucosal vaccine may be tableted or encapsulated, for example, for convenient administration. Such capsules or tablets can include sustained release formulations. For capsules, tablets and pills, the dosage form can also include sodium citrate or a buffer such as carbonate or bicarbonate or calcium. Tablets and pills can also be prepared using enteric coatings.

0075It is conceivable that the vaccine could be administered via drinking water and / or feed in canine patients. It is further conceivable that the vaccine could be given in the form of a feast or toy.

0076"Parental administration" includes subcutaneous injection, submucosal injection, intravenous injection, intramuscular injection, intrathoracic injection, transdermal injection and infusion. Injectable preparations (eg, sterile injectable aqueous or oily suspensions) are made with suitable excipients such as vehicles, solvents, dispersions, wetting agents, emulsifying substances and / or suspended substances. It can be formulated according to a known technique. These typically include, for example, water, saline, dextrose, glycerol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, 1,3-butanediol, Ringer's solution, isotonic sodium chloride. Liquids, non-irritating non-volatile oils (eg synthetic mono or diglycerides), fatty acids (eg oleic acid), dimethylacetamides, surfactants (eg ionic and nonionic detergents), propylene glycol, and / or polyethylene Contains glycol. Excipients may also include small amounts of other auxiliary substances such as pH buffers.

0077The vaccine may contain one or more excipients that enhance the immune response of the canine patient, which may include antibody response, cellular response, or both, thereby enhancing the effectiveness of the vaccine. The use of such excipients (or "administrators") can be particularly beneficial when using inactivated vaccines. The adjuvant can be a substance that has a direct (eg, cytokine or Calmette-Gelan bacillus ("BCG")) or indirect effect (liposomes) on cells of the immune system of a canine patient. Examples of often suitable adjuvants include oils (eg mineral oils), metal salts (eg aluminum hydroxide or aluminum phosphate), bacterial components (eg bacterial liposomes, Freund's adjuvant, and / or MDP), Plant components (eg Quil A) and / or one or more substances with carrier effects (eg bentnite, latex particles, liposomes, and / or Quil) A, ISCOM) is included. As mentioned above, the adjuvant further comprises, for example, CARBIGEN and Carbopol. It should be recognized that the present invention includes both an adjuvant-containing vaccine and a vaccine that does not contain any adjuvant.

0078It is conceivable that the vaccine may be lyophilized for storage (or otherwise reduced in volume of the liquid) and subsequently redissolved in the liquid before or during administration. Such redissolution can be performed, for example, with vaccine grade water.

0079The present invention further includes kits suitable for use in practicing the above methods. The kit contains a dosage form containing the vaccine described above. The kit also includes at least one additional ingredient, and typically instructions for using the vaccine with the additional ingredient. Additional ingredients may be, for example, one or more additional ingredients that can be mixed with the vaccine before or during administration (eg, one or more of the above excipients, feeds, and / or treats). Can be. Additional ingredients may, as another option (or additionally), include one or more devices for administering the vaccine to canine patients. Such a device can be, for example, a syringe, inhaler, nebulizer, pipette, forceps, or any medically acceptable delivery medium. In some embodiments, the device is suitable for subcutaneous administration of the vaccine. In some embodiments, the device is suitable for intranasal administration of the vaccine.

0080Other excipients and modes of administration known in the art of medical agents or biologics may be used.

0081The vaccine or immunogenic composition of the present invention may contain, for example, genes encoding the HA protein, NA protein, nucleoprotein, polymerase basic protein, polymerase acidic protein and / or matrix protein of the influenza virus of the present invention. It also includes constructs based on the variant virus vector. It is intended that any suitable viral vector that can be used to make recombinant vectors / viral constructs will be used in accordance with the present invention. For example, viral vectors derived from adenovirus, avibox, herpesvirus, vaccinia, canarypox, entomopox, butapox, westnilevirus, and others known in the art are used with the compositions and methods of the invention. be able to. Recombinant polynucleotide vectors that encode and express components can be constructed using standard genetic engineering techniques in the art. In addition, the various vaccine compositions described herein may be used separately or in combination with each other. For example, primary immunization of an animal may use a recombinant vector-based construct with single or multiple lineage components, followed by a vaccine composition comprising an inactivated virus or an inactivated virus-infected cell line. Secondary booster immunization may be performed using a substance. Other immunization protocols using the vaccine compositions of the present invention will be apparent to those skilled in the art and are considered to be within the scope of the present invention.

0082The present invention relates to a rear sortant virus comprising at least one gene or genomic segment of the influenza virus of the invention and the rest of a viral gene or genomic segment derived from a different influenza virus of the invention or an influenza virus other than the virus of the invention. Also related. The rear sortant virus can be produced by the genetic rear sortant of the nucleic acid of the influenza virus of the present invention as a donor and the nucleic acid of the influenza virus as a recipient, and the subsequent selection of the rear sortant virus containing the nucleic acid of the donor virus. it can. Methods for producing and isolating the rear sortant virus are well known in the art (Fields et al., 1996). In one embodiment, the rear sortant virus of the present invention comprises a gene or genomic segment of a human, bird, porcine or equine influenza virus. The rear sortant virus of the present invention may include any combination of nucleic acids derived from the donor and recipient influenza viruses, as long as the rear sortant virus contains at least one gene or genomic segment from the donor influenza virus of the invention. .. In one embodiment, the recipient influenza virus can be an equine influenza virus.

0083Natural, recombinant or synthetic polypeptides of viral proteins, and peptide fragments thereof, can also be used as vaccine compositions according to the methods of the invention. In one embodiment, the vaccine composition comprises a canine influenza virus polynucleotide or polypeptide. In one embodiment, the vaccine composition has SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, The amino acid sequence shown in any of 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78, or a functional and / or immunogenic fragment thereof. Includes a polynucleotide encoding a polypeptide having a variant. In specific embodiments, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, The polynucleotides encoding the amino acid sequences shown in 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 are SEQ ID NOs: 1, 3, 5, 7, respectively. 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, Nucleotide sequence shown in 73, 75 or 77, or SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34 , 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 encoding any functional and / or immunogenic fragment or variant Contains the sequence to be. In a further specific embodiment, the polynucleotides of the invention can include: nucleotides 1-2271 of SEQ ID NO: 3; nucleotides 1-2148 of SEQ ID NO: 5; nucleotides 1-657 of SEQ ID NO: 7; Number: 9 nucleotides 1-1494; SEQ ID NO:: 11 nucleotides 1 to 1410; SEQ ID NO: 13 nucleotides 1 to 756; SEQ ID NO: 15 nucleotides 1 to 1695; SEQ ID NO: 19 nucleotides 1 to 2271; SEQ ID NO: 21 nucleotides 1 to 2148; SEQ ID NO:: 23 nucleotides 1 to 657; SEQ ID NO: 25 nucleotides 1 to 1494; SEQ ID NO: 29 nucleotides 1 to 756; SEQ ID NO: 31 nucleotides 1 to 1695; SEQ ID NO: 47 nucleotides 1 to 2277; SEQ ID NO:: 49 Nucleotides 1-2271; SEQ ID NOs: 51 Nucleotides 1-2148; SEQ ID NOs: 53 Nucleotides 1-690; SEQ ID NOs: 55 Nucleotides 1-1494; SEQ ID NOs: 57 Nucleotides 1-1410; SEQ ID NOs:: 59 nucleotides 1 to 756; SEQ ID NO: 61 nucleotides 1 to 1695; SEQ ID NO: 63 nucleotides 1 to 2277; SEQ ID NO: 65 nucleotides 1 to 2271; SEQ ID NO: 67 nucleotides 1 to 2148; SEQ ID NO:: 69 nucleotides 1 to 690; SEQ ID NO: 71 nucleotides 1 to 1494; SEQ ID NO: 73 nucleotides 1 to 1410; SEQ ID NO: 75 nucleotides 1 to 756; and SEQ ID NO: 77 nucleotides 1 to 1695. In another embodiment, the vaccine composition has the SEQ ID NO:: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, Includes a polypeptide having the amino acid sequence set forth in any of 62, 64, 66, 68, 70, 72, 74, 76 or 78, or a functional and / or immunogenic fragment or variant thereof. In a further embodiment, the vaccine composition comprises at least about 90% or at least about 95%, or at least about 96%, or 97%, or 98%, or 99 of the polynucleotide or polypeptide of the equine influenza polynucleotide or polypeptide. Includes equine influenza virus polynucleotides or polypeptides with% or higher sequence identity. In one embodiment, viral polypeptides derived from many strains can be mixed in the vaccine composition and used to vaccinate the host animal. For example, a polypeptide based on a viral HA protein from at least two different strains of the influenza virus of the invention can be mixed in a vaccine. The polypeptide can be homologous to one lineage, or may include a "hybrid" or "chimeric" polypeptide derived by ligation or binding of a polypeptide whose amino acid sequence is derived from at least two different lines. Procedures for preparing viral polypeptides are well known in the art. For example, viral polypeptides and peptides can be synthesized using solid phase synthesis (Merrifield, 1963). Viral polypeptides and peptides express viral proteins or polynucleotide molecules encoding the peptides in host cells such as bacterial, yeast or mammalian cell lines, and the expressed proteins are standard in the art. It can also be prepared using recombinant DNA technology that is purified using technology. Viral polypeptides derived from many strains can be mixed in the vaccine composition and used for vaccination of host animals. For example, a polypeptide based on a viral HA protein from at least two different strains of the influenza virus of the invention can be mixed in a vaccine. The polypeptide can be homologous to one lineage, or may include a "hybrid" or "chimeric" polypeptide derived by ligation or binding of a polypeptide whose amino acid sequence is derived from at least two different lines. Procedures for preparing viral polypeptides are well known in the art. For example, viral polypeptides and peptides can be synthesized using solid phase synthesis (Merrifield, 1963). Viral polypeptides and peptides express viral proteins or polynucleotide molecules encoding the peptides in host cells such as bacterial, yeast or mammalian cell lines, and the expressed proteins are standard in the art. It can also be prepared using recombinant DNA technology that is purified using technology. Viral polypeptides derived from many strains can be mixed in the vaccine composition and used for vaccination of host animals. For example, a polypeptide based on a viral HA protein from at least two different strains of the influenza virus of the invention can be mixed in a vaccine. The polypeptide can be homologous to one lineage, or may include a "hybrid" or "chimeric" polypeptide derived by ligation or binding of a polypeptide whose amino acid sequence is derived from at least two different lines. Procedures for preparing viral polypeptides are well known in the art. For example, viral polypeptides and peptides can be synthesized using solid phase synthesis (Merrifield, 1963). Viral polypeptides and peptides express viral proteins or polynucleotide molecules encoding the peptides in host cells such as bacterial, yeast or mammalian cell lines, and the expressed proteins are standard in the art. It can also be prepared using recombinant DNA technology that is purified using technology.

0084The vaccine composition of the present invention also includes a naked nucleic acid composition. In one embodiment, the nucleic acid may comprise a nucleotide sequence encoding the HA and / or NA protein of the influenza virus of the invention. Methods for nucleic acid vaccination are known in the art and are disclosed, for example, in US Pat. No. 6,063,385 and US Pat. No. 6,472,375. The nucleic acid can be a plasmid or dosage form of a gene expression cassette. In one embodiment, the nucleic acid is provided encapsulated in liposomes administered to the animal.

0085Vaccine compositions and immunogens such as polypeptides and nucleic acids that can be used in accordance with the present invention can be provided with pharmaceutically acceptable carriers or diluents. The compounds and compositions useful in the present invention may be formulated according to known methods for preparing pharmaceutically useful compositions. The formulation is described in detail in many sources well known and readily available to those skilled in the art. For example, EW Remington's Pharmaceutical Science, Easton Pennsylvania, by Martin, Mack Publishing Company, 19th edition, 1995 describes the formulation that can be used in association with the present invention. In general, the compositions of the invention are formulated to combine an effective amount of immunogen with a suitable carrier to facilitate effective administration of the composition. Also, the compositions used in this method can be in various dosage forms. These include solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquids or suspensions, suppositories, injectable and insoluble liquids, and sprays. The preferred dosage form depends on the intended mode of administration and therapeutic application. The composition preferably also comprises conventional pharmaceutically acceptable carriers and diluents known to those of skill in the art. Examples of carriers or diluents for use with this peptide mimetic include water, physiological saline, oils containing mineral oils, ethanol, dimethylsulfoxide, gelatin, cyclodextran, magnesium stearate, dextrose, cellulose, sugars, carbonates. Includes, but is not limited to, calcium, glycerol, alumina, starch, and corresponding carriers and diluents, or any mixture thereof. The immunogen preparation of the present invention may contain a suspending substance, a protective agent, a lubricant, a buffer, a preservative and a stabilizer. To prepare for administration of such doses for the desired therapeutic administration, the pharmaceutical compositions of the present invention are conveniently based on the weight of the immunogen based on the weight of the entire composition containing the carrier or diluent. Includes about 0.1% to 45%, especially 1 to 15%.

0086The vaccines and immunogenic compositions of the present invention can be prepared by procedures well known in the art. For example, a vaccine or immunogen is typically prepared as an injectable agent, such as a liquid solution or suspension. The vaccine or immunogen is administered in an amount that is therapeutically effective and immunogenic in a method and recipient that is compatible with the dosage product. One of ordinary skill in the art can easily determine the optimal dose and pattern of administration for a particular vaccine or immunogen.

0087The peptides and / or polypeptides of the invention can also be provided in the form of multiantigenic peptide (MAP) constructs. The preparation of the MAP construct is described in Tam (1988). The MAP construct uses a core matrix of lysine residues from which multiple replicas of the immunogen are synthesized (Posnett et al., 1988). Many MAP constructs, each containing the same or different immunogens, can be prepared and administered as a vaccine composition according to the methods of the invention. In one embodiment, the MAP construct is provided and / or administered with one or more adjuvants. The influenza polypeptides of the invention can also be prepared and administered as macromolecular protein structures containing one or more polypeptides. Published US patent application US2005 / 0009008 discloses a method for producing virus-like particles as a vaccine for influenza virus.

0088According to the methods of the invention, the vaccines and immunogenic compositions described herein are susceptible in effective amounts and methods for inducing protective immunity against subsequent viral stimulation or infection of the host. It is administered to the host, typically a canine, more typically a domesticated dog. In one embodiment, the host animal is Canidae. Dogs include wild, zoo and domestic dogs such as wolves, coyotes and foxes. Dogs also include domestic dogs, such as pure and / or hybrid companion dogs, show dogs, working dogs, sheep dogs, hunting dogs, guard dogs, police dogs, race dogs, and / or laboratory dogs. Is done. In one specific embodiment, the host animal is a domesticated dog, such as a greyhound. Vaccines or immunogens are typically administered parenterally, for example by injection either subcutaneously, intraperitoneally or intramuscularly. Other suitable modes of administration include oral or nasal administration. Vaccines or immunogens are usually given to animals at least twice, one week or more between each dose. However, other dosing regimens for initial and booster administration of vaccines or immunogens may be intended and depend on the physician's judgment and the particular host animal to be treated.

0089The virus and virus-infected cells in the vaccine preparation may be inactivated or attenuated using methods known in the art. For example, complete viruses and infected cells are paraformaldehyde, formalin, β-propiolactone (BPL), bromoethylamine (BEA), binary ethyleneimine (BEI), phenol, UV light, high temperature, freeze-thaw, sonication (ultrasonic). It can be inactivated or attenuated by exposure to (including treatment) and the like. The amount of cell-free complete virus in the vaccine dose ranges from about 0.1 mg to about 5 mg, and more usually can be about 0.2 mg to about 2 mg. The dose of vaccine product containing the virus-infected cell line is typically about 10 per dose.⁶About 10 from pieces⁸Individual cells, more usually about 5 x 10 per dose⁶Approximately 7.5 x 10 from pieces⁷Contains individual cells. The amount of protein or peptide immunogen in a dose for an animal depends on the size, age, etc. of the animal to which the dose is administered, from about 0.1 μg to 10000 μg, or from about 1 μg to 5000 μg, or from about 10 μg. It can vary from 1000 μg, or from about 25 μg to 750 μg, or from about 50 μg to 500 μg, or from 100 μg to 250 μg.

0090The immunogenic or vaccine compositions of the invention, such as viruses or virus-infected cells or viral proteins or peptides, may typically be mixed with an adjuvant immediately prior to administration. The adjuvants intended for use in vaccine formulations include threonyl muramyl dipeptide (MDP) (Byars et al., 1987), saponins, Cornebacterium parvum, Freund complete and Freund incomplete adjuvants. Includes aluminum, or any mixture thereof. Various other adjuvants suitable for use with the methods and vaccines of the present invention, such as alum, are known in the art and are intended for use with the present invention.

0091The present invention also relates to antibodies that specifically bind to the proteins or peptides of the invention. Antibodies of the invention include monoclonal and polyclonal antibody compositions. Preferably, the antibody of the invention is a monoclonal antibody. The entire antibody and its antigen-binding fragments are intended in the present invention. So, for example, Fab for a fragment bound to the appropriate antibody.₂, Fab and Fv antibody fragments are included. Antibodies of the invention may be labeled with a detectable molecule such as a fluorescent molecule (eg, fluorescein or enzyme).

0092The present invention also relates to methods and compositions for the detection and identification of the influenza virus of the present invention, as well as the diagnosis of infection of the influenza virus of the present invention in animals. The methods of the invention include the detection of the presence of canine influenza in biological samples of animal origin. Detection of canine influenza in samples is useful for diagnosing canine influenza in animals. In addition, this information may provide the ability to determine the prognosis of an animal based on the characteristic amount of canine influenza present over a period of time, supporting the selection of substances and treatments for treatment in the animal. , Can assist in the monitoring of treatment. This method also provides the ability to prove the absence of canine influenza in the animal under test.

0093The ability to detect canine influenza in animals allows the assessment of canine influenza pandemics at different geographic locations. Since this information allows for earlier detection, infected animals can be quarantined to limit the spread of the disease, allowing early intervention of treatment options. In addition, having this valid information provides medical personnel with directions to prepare for the treatment of many diseased animals, including the assembly of medical supplies and, if effective, vaccines. be able to.

0094In one embodiment, the method of the invention involves taking a biological sample from a test animal such as a dog. The biological sample can be any biological material including cells, tissues, coat, whole blood, serum, plasma, papillary aspirate, lung lavage fluid, cerebrospinal fluid, saliva, sweat and tears.

0095Animal test samples can be taken from animals suspected of carrying the canine influenza virus, whether or not the animal develops influenza symptoms. Control samples can also be provided or collected from animals known to be free of canine influenza. Further controls may be provided to confirm that the reagents in the assay positively detect influenza A virus, for example to suppress false positive and false negative results.

0096In addition to detecting the presence or absence of canine influenza in biological samples, the detection methods used in the present invention include changes in nucleic acid sequences, environment, drug administration, genetic manipulation or mutation, damage, dietary changes, aging, etc. Alternatively, mutations in canine influenza virus that can be attributed to any other characteristic of the animal can also be detected. Mutations may also make canine influenza A resistant to previously effective drugs or allow infection and transmission of the virus in different animal species, namely humans. For example, the avian influenza A virus has been shown to infect other animals and humans.

0097In one embodiment for detecting influenza virus in animals, diagnosis is facilitated by the collection of high quality samples, rapid transport to laboratories, and proper storage prior to testing in the laboratory. Viruses are best detected in samples containing infected cells and secretions. In one embodiment, specimens for direct detection of viral antigens and / or for nucleic acid and / or virus isolation in cell cultures are taken 3 days immediately after the onset of clinical manifestations. Many types of specimens are suitable for diagnosing viral infections of the upper respiratory tract, including, but not limited to, nasal swabs, nasopharyngeal swabs, nasopharyngeal aspirates, nasal lavage fluids and pharyngeal swabs. In addition to swabs, tissue or serum samples may be taken and open procedures can be performed.

0098In one embodiment, respiratory specimens are taken and transported in 1-5 ml virus transport medium. There are many commercially available media sufficient to recover various viruses. Clinical specimens are added to transport medium. Nasal or nasopharyngeal swabs may also be transported in virus transport medium. One example of transport medium is calf extract 10 gm and bovine albumin fraction V 2 gm, added to sterile distilled water up to 400 m. Antibiotics such as 0.8 ml of gentamicin sulfate solution (50 mg / ml) and 3.2 ml of amphoterin B (250 μg / ml) can also be added. The medium is preferably sterilized by filtration. Nasal lavage fluids such as sterile saline (0.85% NaCl) can also be used to collect respiratory virus specimens.

0099In one embodiment, the serum is collected from 1-5 ml of whole blood collected from an acute phase animal immediately after the onset of clinical manifestations, preferably within 7 days. For example, a convalescent serum sample may be collected, such as about 14 days after the onset of symptoms. Serum specimens may be useful in detecting antibodies against respiratory viruses in neutralization tests.

0100In some cases, samples can be taken from individual animals over a period of time (eg, once a day, once a week, once a month, once every six months, or once a year). Taking many samples from individual animals over a period of time confirms the results from early detection and / or reveals responsiveness or resistance to specific treatments, eg, selected therapeutic agents. Can be used.

0101The methods of the invention can be used to detect the presence of one or more pathological substances in a test sample derived from an animal and the amount of each pathological substance. Detects pathological substances including, but not limited to, enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence (IFA) testing, hemagglutination and hemagglutination inhibition (HI) assays, and antibody assays including Western blots. You may use any method for doing so. Known cell culture methods can also be used. Positive cultures may be further identified using immunofluorescence of cell cultures or HI assay of cell culture medium (supernatant).

0102In addition, methods for detecting nucleic acids (DNA or RNA) or proteins can be used. Such methods include, but are not limited to, polymerase chain reaction (PCR) and reverse transcriptase (RT) PCR and real-time tests, as well as quantitative nuclease protection assays. Test kits for performing these assays are commercially available. For example, QIAGEN (Valencia, CA) sells a one-step RT-PCR kit and a viral RNA extraction kit.

0103In one embodiment, the method uses antibodies specific for the virus or viral protein of the invention. In one specific embodiment, an antibody specific for the HA protein of the virus of the invention is used. In another embodiment, an antibody specific for the NP protein of the virus of the invention is used. A suitable sample, such as from the nasal cavity or nasopharyngeal region, is obtained from the animal from which the virus or viral protein is isolated. Subsequently, viral components are screened for binding of antibodies specific for proteins such as HA or NP of the viruses of the invention. In another embodiment, a serum sample (or a sample containing other antibodies) is obtained from the animal and the serum is screened for the presence of antibodies that bind to the proteins of the viruses of the invention. For example, if the plate wall has an HA and / or NP protein that binds to the wall, or a peptide fragment thereof, the ELISA method can be performed. The plate wall is then contacted with serum or antibody from the test animal. The presence of an antibody that specifically binds to the HA and / or NP protein in the animal indicates that the test animal is or has been infected with the influenza virus of the invention.

0104In one embodiment, the presence of a pathological substance is detected by examining the presence or absence of antibodies to the substance in a biological sample. It may take a period of time (eg, months) after an animal is infected before the antibody can be detected by a blood test. Once formed, antibodies usually persist for years, even after successful treatment of the disease. Detection of antibodies against canine influenza A cannot indicate when the infection was recent or past.

0105Antibody tests can also be performed on liquids. Antibody assays include enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence (IFA), and Western blotting. Preferably, antibody testing is performed using a number of assays, such as ELISA or IFA followed by Western blot. The antibody assay can be performed in a two-step process using either an ELISA or IFA assay followed by Western blot assay. ELISA is considered a more reliable and accurate assay than IFA, but IFA may be used when ELISA is not effective. Western blotting (a more specific test) can also be performed on all animals, especially those that are positive or borderline positive (uncertain) by ELISA or IFA.

0106Other antibody-based tests that can be used to detect influenza virus include hemagglutination inhibition tests. Hemagglutination activity is detectable in animal-derived biological samples using chicken or turkey erythrocytes, as described by (Burleson et al., 1992) and Kendal et al., 1982. In one embodiment, the influenza or HA protein or peptide of the invention is contacted with a test sample containing serum or antibody. Subsequently, red blood cells (RBC) derived from animals such as birds are added. In the presence of antibodies to HA, RBC does not aggregate. In the absence of antibodies to HA, RBC aggregates in the presence of HA. Modifications and modifications to standard hemagglutination inhibition assays are known in the art and are considered within the scope of the present invention.

0107Animal infections can also be examined by isolation of the virus from a sample such as a nasal or nasopharyngeal swab. Virus isolation may be performed using standard methods including cell culture and egg inoculation.

0108In a further embodiment, a nucleic acid-based assay can be used for the detection of the virus of the invention. In one embodiment, a nucleic acid sample is obtained from an animal and if the nucleic acid contains a sequence specific for the influenza virus of the invention, the nucleic acid is subjected to PCR with primers that produce amplification products. In a specific embodiment, RT-PCR is used in the assay of the virus of interest. In one exemplary embodiment, real-time RT-PCR is used to assay for the influenza virus of the invention. PCR, RT-PCR and real-time PCR methods are known in the art and are known in the art: US Pat. No. 4,683,202; US Pat. No. 4,683,195; US Pat. No. 4,800,159; US Pat. No. 4,965,188; US Pat. No. 5,994,056; US Pat. It is disclosed in Nos. 6,814,934, as well as in Saiki et al. (1985); Sambrook et al. (1989); Lee et al. (1993); and Livak et al. (1995). In one embodiment, the PCR assay method uses oligonucleotides specific for the influenza matrix (MA) gene and / or the HA gene. The amplification products may be sequenced to determine if the amplification products have the sequence of the influenza virus of the invention. Other nucleic acid-based assays can be used to detect and diagnose viral infections by the viruses of the invention, and such assays are considered within the scope of the invention. In one embodiment, the nucleic acid-containing sample is subjected to PCR-based amplification using forward and reverse primers that are specific for the viral polynucleotide or gene sequence. If the nucleic acid in the sample is RNA, RT-PCR may be performed. For real-time PCR, use detectable probes with primers.

0109Primer sets specific for the hemagglutinin (HA) gene of many circulating influenza viruses are known and are under ongoing development. The influenza virus genome is a single-stranded RNA and a DNA copy (cDNA) must be made using reverse transcriptase (RT) polymerase. For example, amplification of the RNA genome using RT-PCR typically requires a pair of influenza A subtypes and known HA sequences and oligonucleotide primers designed based on neuraminidase (NM) -1. This primer can be selected to specifically amplify RNA of only one viral subtype. DNA produced with subtype-specific primers can be further analyzed by molecular genetic techniques such as sequencing. This test is preferably performed with a positive control, or the product is confirmed by sequencing and comparison with known sequences. The absence of a target PCR product (ie, a "negative" result) may not rule out the presence of the virus. Results are subsequently obtained within hours of clinical swab or infected cell culture. PCR and RT-PCR of influenza virus A have been reported by Fouchier et al., 2000 and Maertzdorf et al., 2004.

0110The present invention also relates to a method for screening a compound or drug having antiviral activity against the virus of the present invention. In one embodiment, cells infected with the virus of the invention are contacted with a test compound or agent. Subsequently, the viral load or virus activity after contact is measured. Compounds or agents exhibiting antiviral activity can be selected for further evaluation.

0111The present invention also relates to isolated cells infected with the influenza virus of the present invention. In one embodiment, the cell is a canine cell, such as a canine renal epithelial cell.

0112The present invention also relates to cells transformed with the polynucleotides of the invention encoding the polypeptides of the invention. Preferably the polynucleotide sequence is provided in the expression construct of the present invention. More preferably, the expression construct prepares for intracellular overexpression of the functionally bound polynucleotide of the invention. In one embodiment, the cells have SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50. , 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78, or a sequence encoding the amino acid sequence shown in any of its functional fragments or variants. Transformed with the polynucleotide sequence containing. In a specific embodiment, the cells have SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, respectively. , 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77, SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, A polynucleotide encoding the amino acid sequence shown in 76 or 78, or SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, Characterized by a sequence encoding any functional fragment or variant of 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 Be converted. Therefore, the present invention relates to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, 53, 55. , 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77 nucleotide sequence, or SEQ ID NO::

0113The transformed cells can be, for example, eukaryotic cells of plant cells containing protoplasts, or the transformed cells can be prokaryotic cells of bacterial cells such as, for example, E. coli or Bacillus subtilis (B. subtilis). Animal cells include human cells, mammalian cells, especially dog cells, avian cells and insect cells. Plant cells include, but are not limited to, dicotyledonous, monocotyledonous and coniferous cells.

0114The present invention also relates to plants, including transgenic plants that express and produce the viral proteins or polypeptides of the invention. Plants, plant tissues and plant cells transformed with the polynucleotides of the invention or crossed to contain the polynucleotides of the invention are intended by the invention. Preferably, the polynucleotides of the invention are overexpressed in plants, plant tissues or plant cells. A plant can be used to produce the influenza vaccine composition of the invention and the vaccine can be administered through ingestion of this plant (see, eg, US Pat. No. 5,484,719 and US Pat. No. 6,136,320). I want).

0115The present invention also relates to a kit for detecting a virus by the virus of the present invention or diagnosing an infection. In one embodiment, the kit comprises an antibody of the invention that specifically binds to the influenza virus of the invention, or an antigenic portion thereof. In another embodiment, the kit comprises one or more polypeptides or peptides of the invention. In a specific embodiment, the polypeptide has SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 33, 34, 48, 50. , 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78, or any of the functional and / or immunogenic fragments or variants thereof. Has an array. In a further aspect, the kit comprises one or more polynucleotides or oligonucleotides of the invention. In a specific embodiment, the polynucleotides are SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 47, 49, 51, It has the nucleotide sequence shown in 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77, or any fragment or variant thereof. The kit optionally comprises one or more antibodies, a regulatory polypeptide or peptide, and / or a regulatory polynucleotide or oligonucleotide. The antibodies, polypeptides, peptides, polynucleotides, and / or oligonucleotides of the kit may be provided in suitable containers or packages.

0116The application also relates to the use of mongrel dogs as a model for influenza virus infection and pathogenesis. In one embodiment, the mongrel dog is inoculated with an influenza virus, such as the canine influenza virus of the invention. Optionally, the dog may be administered a therapeutic substance after inoculation. Dogs may have been administered a composition to elicit an immune response against influenza virus prior to inoculation with the virus. Tissue, blood, serum and other biological samples can be obtained prior to and / or after inoculation and include, but are not limited to, PCR, RT-PCR, nucleic acid sequencing, and immunohistochemistry. The presence of viruses and tissue pathogens can be investigated using methods known in.

0117The canine influenza virus strains (named "A / Dog / Florida / 43/2004" and "A / Dog / Florida / 242/2003") were released on October 9, 2006 in the American Type Culture Collection (ATCC), PO Box. Deposited at 1549, Manassas, VA 20108<u style="single">(The deposit numbers are "PTA-7914" and "PTA-7915", respectively)</u>.. Canine influenza virus strains (named "Dog / Jax / 05" and "Dog / Miami / 05") were released in October 2006.<u style="single">17</u>Deposited on the American Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108<u style="single">(The deposit numbers are "PTA-7941" and "PTA-7940", respectively)</u>.. The virus strain is obtained in culture under 37 CFR 1.14 and 35 USC 122 by persons qualified by the Commissioner of Patents and Trademarks during the pending period of this patent application. Deposited under conditions that ensure that it is done. Deposits may be provided if required by foreign patent law in the country in which this application or an equivalent of its descendant application is filed. However, it should be understood that the availability of deposits does not permit the practice of the present invention in the deviation of patent rights granted by administrative measures.

0118In addition, the virus deposit shall be preserved and published in accordance with the provisions of the Budapest Treaty on the Deposit of Microorganisms, i.e. at least 5 years after the latest request for the distribution of samples of the deposit, and in any case the date of deposit. Preserved with all precautions necessary to remain viable and free of contamination for at least 30 years from or during the exerciseable period of any patent that may issue the disclosure of the culture. The Depositary acknowledges that due to the terms of the Deposit, it is obliged to replace the Deposit if the Depositary is unable to sell the sample upon request. All restrictions on the openness of this culture deposit will be immutably removed as soon as the patent disclosing it is granted.

0119Table 57 shows the canine influenza virus identified as A / Canine / Florida / 43/2004 (Ca / Fla / 43/04) and hemagglutinin in H3N8 horse isolates and canine / Florida / 242/2003 isolates (ie, " It shows similarities in the amino acid sequences encoded by the genes for HA "), neuraminidase (ie" NA ") and nucleoprotein (NP).

0120Any element of any aspect disclosed herein can be combined with any other element or aspect disclosed herein, and such a combination is considered particularly within the scope of the invention. Be done.

0121All patents, patent applications, provisional applications, and publications referred to or cited herein are in their entirety by reference, including all figures and tables, unless they conflict with the express disclosure of the specification. Is incorporated.

<p num="0122">Materials and Methods of Examples 1-6<u style="single">Blood and nasal swab collection from greyhounds</u> Acute and convalescent blood samples were taken by jugular puncture from clinically ill or normal greyhounds in racing kennels with respiratory disease outbreaks. Convalescent samples were taken 4-12 weeks after the acute sample. Serum was collected and stored at -80 ° C. Nasal swabs were harvested and placed in activated charcoal-added Amies transport medium (Becton Dickinson Biosciences) until submission for bacterial isolation.</p><p num="0123"><u style="single">Greyhound Postmortem Inspection</u> Five out of eight greyhounds that died in the January 2004 Florida truck outbreak underwent a thorough postmortem examination at the University of Florida College of Veterinary Medicine (UF CVM) Anatomic Pathology Service. Postmortem examination of another dog was performed at a private veterinary clinic and the tissue was submitted to UF CVM for histopathological diagnosis. Tissues are fixed in 10% neutral buffered formalin, embedded in paraffin and 5 μm sections stained with hematoxylin eosin for histopathological diagnosis or immunohistochemical as described below. Processed for inspection. Unfixed tissue was submitted to bacterial culture and also stored at -80 ° C.</p><p num="0124"><u style="single">Serological test for canine viral respiratory pathogens</u> Paired acute and convalescent serum samples were sent to the Animal Health Diagnostic Laboratory (AHDL) at the Cornell University College of Veterinary Medicine for serum neutralization testing against canine distemper virus, type 2 adenovirus and parainfluenza virus. Antibody titers are expressed as the final dilution of serum that inhibits viral infection of cell cultures. Serum conversion, defined as a 4-fold increase in antibody titer between acute and convalescent samples, indicated viral infection. No serum conversion to these viral pathogens was detected.</p><p num="0125"><u style="single">Microbial testing for canine bacterial respiratory pathogens</u> Paired nasal swabs and postmortem tissue were sent to the UF CVM Diagnostic Clinical Microbiology / Parasitology / Serology Service for bacterial isolation and identification. Samples are non-selective medium and Bordetella (Regan-Lowe; The cells were cultured in selective media of Remel) and Mycoplasma (Remel). All cultures were maintained for 21 days before reporting no growth. Nasal swabs from several greyhounds were also sent to the Department of Diagnostic Medicine / Pathobiology at Kansas State University College of Veterinary Medicine for bacterial culture. Bordetella bronchiseptica was isolated from the nasal cavity of one of the 70 dogs that were clinically diseased and tested, and the genus Mycoplasma was recovered from the nasal cavity of 33 dogs. Was done. Pasteurella multocida was widely recovered from the nasal cavity of dogs with purulent nasal secretions. Two of the dogs that died in the January 2004 outbreak had some growth in the lungs after death, and one had some growth in E. coli and Streptococcus canis. Another dog is Pseudomonas (Pseudomonas) Aeruginosa) and some growth of yeast were observed. Bordetella bronchiseptica and mycoplasma were not isolated from the trachea or lungs of dead dogs.</p><p num="0126"><u style="single">Isolation of virus from post-mortem tissue</u> Frozen tissue was thawed and homogenized with 0.5% bovine serum albumin (BSA) and 10-fold volume of antibiotic-enriched Minimal Essential Medium (MEM). Solid debris was removed by centrifugation and the supernatant was inoculated into cultured cells or 10-day-old hatched chicken eggs. Tissue homogenates from deceased greyhounds were inoculated into various cell cultures that supported the replication of a wide range of viral pathogens. Cell cultures include Vero (African green monkey renal epithelial cells, ATCC No. CCL-81), A-72 (Canine tumor fibroblasts, CRL-1542), HRT-18 (Human rectal epithelial cells, CRL-11663). , MDCK (Canine Renal Epithelial Cell, CCL-34), Canine Primary Renal Epithelial Cell (AHDL, Cornell University), Canine Primary Pulmonary Epithelial Cell (AHDL), and Bovine Primary Testis Cell (AHDL). MDCK and HRT cells were cultured in 2.5 μg / mL TPCK-treated trypsin (Sigma) -enriched MEM, and the remaining cell lines were cultured in 10% fetal bovine serum and antibiotic-enriched MEM. The cells are 25 cm²5% CO using a flask₂The cells were cultured at 37 ° C in a humidified atmosphere. The control culture was inoculated with enhanced MEM. The culture was observed for morphological changes once daily and collected 5 days after inoculation. The recovered fluid and cells were clarified by centrifugation and inoculated into fresh cells as described in the first inoculation; two blind passages were performed. Hemagglutination activity of the clarified supernatant was measured using chicken or turkey erythrocytes as described (Burleson et al., 1992; Kendal et al., 1982). For virus isolation using chicken embryos, 0.1 mL of tissue homogenate was inoculated into the allantois sac and incubated at 35 ° C for 48 hours. After two blind passages, the hemagglutination activity of the allantois cavity fluid was measured as described (Burleson et al., 1992; Kendal et al., 1982).</p><p num="0127"><u style="single">RT-PCR, nucleotide sequencing, and phylogenetic analysis</u> Total RNA was extracted from tissue culture supernatant or allantois cavity fluid using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Total RNA (10 ng) was reverse transcribed into cDNA using a one-step RT-PCR kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. PCR amplification of the coding regions of the eight influenza virus genes in the cDNA was performed as previously reported (Klimov et al., 1992a) using a generic gene-specific primer set. The resulting DNA unit replication sequence was used as a template for automatic sequencing on the Applied Biosystems 3100 automatic DNA sequencer using the cycle sequencing diterminator chemistry (ABI). The nucleotide sequence is GCG Package (copyright), version 10.0 (Accelyrs) (Womble, It was analyzed using 2000). Phylogeny Inference Package (copyright) version 3.5 was used to estimate phylogeny from nucleotide sequences and to calculate boot straps (Felsenstein, 1989). The phylogenetic tree is PAUP 4.0 beta compared to the phylogenetic tree obtained by the neighbor-joining method using the Tamura-Nei gamma model executed in the MEGA (copyright) program (Kumar et al., 2004). Confirmed by the program (Sinauer Associates).</p><p num="0128"><u style="single">Experimental inoculation of dogs</u> Four 6-month-old specific-pathogen-free beagle dogs [(2 males and 2 females (Liberty Research)] were used. Physical tests, as well as baseline blood tests including detailed blood cell / type measurements, serum chemistry. Test panels and urinalysis revealed that the animals were healthy. The animals were group-fed in a BSL 2-enhanced facility accredited by the Laboratory Animal Care Certification Association. Baseline rectal temperature Recorded twice daily for 7 days. Dogs were anesthetized by intravenous infusion of propofol (Diprivan®, Zeneca Pharmaceuticals, 0.4 mg / kg body weight ~ effect) in preparation for intubation of the intratracheal tube. 10 A / dog / Florida / 43/2004 (dog / FL / 04) (H3N8) virus in each dog^6.6Half Tissue Culture Infection Dose (TCID)₅₀) Was inoculated, half was administered to the end of the trachea via an endotracheal tube, and the other half was administered to the deep nasal airway via a catheter. Physical examination and rectal temperature recording were performed twice daily for 14 days after inoculation (pi). Blood samples (4 mL) were collected by jugular vein puncture on days pi0, 3, 5, 7, 10 and 14. Nasal and pharyngeal specimens were taken from each dog on pi0-5, 7, 10 and 14 days using a polyester swab (Fisher Scientific). Swabs were collected in virus transport medium (Remel) and stored at -80 ° C. Two dogs (one male and one female) were euthanized by intravenous inoculation of Beuthanasia-D® solution (1 mL / 5 kg body weight; Schering-Plough Animal Health Corp) on pi5, and the remaining two dogs were euthanized. On the 14th day, he was euthanized for post-mortem examination. The tissue for histological examination was processed as described above. Tissues for virus culture were stored at -80 ° C. This exam is for the University of Florida Institutional Animal Care and Use Approved by the Committee.</p><p num="0129"><u style="single">Viral shedding from experimentally inoculated dogs</u> Serial dilutions of swab extracts prepared by lung homogenate and clarification of swab transport medium by centrifugation were prepared with 0.5% BSA and antibiotic-enriched MEM. The plaque assay was performed as previously reported (Burleson et al., 1992) using monolayer MDCK cells in a 6-well tissue culture plate. The inoculated monolayer was overlaid with fortified MEM containing 0.8% agarose and 1.5 μg / mL of TPCK-trypsin. Cells are fixed and prior to staining with crystal violet, 5% CO₂The cells were cultured at 37 ° C for 72 hours in a humidified atmosphere containing. Virus concentration was expressed as plaque forming units (PFU) per gram of tissue or per swab.</p><p num="0130"><u style="single">Immunohistochemistry</u> Deparaffinized and rehydrated 5 μm lung tissue sections from Greyhound and Beagle dogs were placed on a Bond-Rite slide glass (Richard-Allan Scientific, Kalamazoo, MI) and subjected to Reagentase K (Dako Cytomation, Carpenteria, CA). , Peroxidase blocking reagent (Dako® En Vision Peroxidase Kit, Dako Corp.). Sections are monoclonal antibodies against canine distemper virus (VMRD, Inc.), type 2 canine adenovirus (VMRD, Inc.), canine parainfluenza virus (VMRD, Inc.), or influenza A H3 (Chemicon International, Inc.). Incubated for 2 hours at room temperature with 1: 500 dilution of. Controls include identical sections and mouse IgG (1 mg / mL, Serotec, Inc.) incubation, and incubation of monoclonal antibodies and normal canine lung sections were included. After treatment with the primary antibody, sections were incubated with secondary immunoperoxidase and peroxidase substrate reagents (Dako® EnVision® Peroxidase Kit, Dako Corp.) according to the manufacturer's instructions. Sections were counterstained with hematoxylin, treated with clarifying agent # 2 and Bluing reagent (Richard-Allan Scientific, Kalamazoo, MI), dehydrated, and covered with Permount (ProSciTech).</p><p num="0131"><u style="single">Hemagglutination inhibition (HI) test</u> Serum samples were incubated with receptor-destroying enzyme (RDE, Denka) (1 volume of serum: 3 volumes of RDE) prior to heat inactivation for 60 minutes at 56 ° C. Influenza A / dog / FL / 04 (H3N8) virus was propagated using MDCK cells at 37 ° C for 36-48 hours. The supernatant of the virus culture was collected, clarified by centrifugation and stored at -80 ° C. The HI assay was performed as previously reported (Kendal et al., 1982). That is, 4 hemagglutination units of virus in 25 μl were added to a serially diluted equal volume of serum in a microtiter well and incubated for 30 minutes at room temperature. Equal amounts of 0.5% v / v turkey erythrocytes were added and the hemagglutination titer was visually determined 30 minutes later. The endpoint HI titer was defined as the final dilution of serum that completely inhibits hemagglutination. Serum conversion was defined as a 4-fold increase in HI titer between pairs of acute and convalescent samples. Serum positivity in a single sample was defined as 1: 32 HI antibody titer.</p><p num="0132"><u style="single">Micro Neutralization (MN) Test</u> Neutralization of the serum antibody response to A / dog / FL / 04 (H3N8) was detected by the previously reported MN assay (Rowe et al., 1999). However, canine serum was RDE-treated as described above prior to the assay. The endpoint power is 100 TCID₅₀Was defined as the maximum dilution of serum that neutralizes the virus by 50%. Serum conversion was defined as a 4-fold increase in MN titer between pairs of acute and convalescent samples. Serum positivity in a single sample was defined as a MN titer of 1: 80.</p><p num="0133"> The following is an example exemplifying the procedure for practicing the present invention. These examples should not be considered as limitations. Unless otherwise noted, all percentages are based on weight and all solvent mixture proportions are based on volume.</p><p num="0134"><u style="single">Example 1</u> In January 2004, a respiratory disease outbreak broke out in two kennels on a truck in Florida and 22 racing greyhounds in local breeding farms that supply dogs to these kennels. .. There were about 60 dogs in each kennel building and about 300 dogs in the breeding yard. The pandemic occurred for 6 days, after which no new cases were identified. 14 of the 22 dogs showed a fever of 39.5-41.5 ° C, coughed for 10-14 days and eventually recovered. Of the remaining eight dogs, six apparently healthy dogs died unexpectedly, with bleeding from the mouth and nose. The other two dogs were euthanized within 24 hours of the onset of oral and nasal bleeding due to rapid exacerbations. Both of these dogs had a fever of 41 ° C. Four out of eight deaths occurred in the kennel facility and four in the breeding yard. Fifty percent of deaths occurred on the third day of the pandemic. Twenty-two dogs were 17 to 4 years old, but 73% were 17 to 33 months old.</p><p num="0135"> Two clinical syndromes were observed: a relatively mild illness (14 dogs) characterized by an early fever and a subsequent 10-14 day cough, subsequent recovery, or extremely with respiratory bleeding. Sudden death (8 dogs, mortality rate) 36%). Postmortem examinations were performed on 6 of 8 fatal cases. All dogs showed excessive bleeding in the lungs, mediastinum and pleural space. Histological examination of the respiratory tract showed tracheitis, bronchiolitis, bronchiolitis, and purulent bronchopneumonia in addition to pulmonary hemorrhage (Fig. 3). Infiltration of neutrophils and macrophages was found in the epithelial lining and airway cavity of these tissues. Lung homogenates prepared from these dogs were inoculated into various cell lines of monkeys, humans, bovines and dogs for virus culture. Lung homogenate in one dog caused cytopathic effects in Madin-Darby canine renal epithelial cells (MDCK) cultured in the presence of trypsin, and the supernatant of the cell culture aggregated chicken erythrocytes. Preliminary evidence of influenza A virus was obtained by commercially available ELISA for detection of influenza A and B virus nucleoproteins, as well as PCR analysis using primers specific for the influenza A virus matrix gene. In addition, hemagglutination activity is equine influenza A It was inhibited by reference antisera for the H3 subtype, but not by antisera specific for the H1-H11 and H13 subtypes of human influenza A (Table 3). To elucidate the molecular properties of the virus, we examined the nucleotide sequences of eight RNA segments of the viral genome. By sequence comparison and phylogenetic analysis with known influenza virus genes, the eight genes in the canine isolate are very similar to those derived from modern equine influenza A (H3N8) virus, with 96-97% sequence identity. Has sex (Fig. 1A, Table 4). In contrast, representative genes from avian, porcine and human influenza A isolates showed 94% identity with canine isolates (Table 4). These data identified the canine isolate A / canine / Florida / 43/2004 (dog / FL / 04) as influenza A H3N8, which is closely associated with modern strains of equine influenza virus. Since all genes in the canine isolate were derived from equine influenza virus, we conclude that the entire genome of equine influenza virus was transmitted to dogs.</p><p num="0136"><u style="single">Example 2</u> To investigate the role of canine / FL / 04 virus in greyhound clinical and pathological observations, we performed immunohistochemical staining (IHC) of lung tissue with a monoclonal antibody against influenza A H3. Carried out. Viral H3 antigens were consistently detected in the cytoplasm of bronchial and bronchiolic epithelial cells, bronchial glandular epithelial cells, and macrophages were detected in the airway and alveolar cavities (Fig. 2A). These data support the diagnosis of lung infection with the H3 subtype influenza virus in many dogs.</p><p num="0137"><u style="single">Example 3</u> To investigate the involvement of canine / FL / 04-like viruses in the etiology of respiratory disease epidemics, we investigated 11 diseased dogs and asymptomatic dogs by hemagglutination inhibition (HI) and microneutralization (MN). Acute and convalescent paired sera collected from 16 contact dogs were analyzed. Serum conversion, defined as a 4-fold increase in antibody titer against dogs / FL / 04 from acute to convalescent, occurred in 8 of 11 (73%) sick dogs in both assays (Table). 1). Serum conversion occurred in 6 of 16 (38%) asymptomatic contact dogs by HI assay and 8 of 16 (50%) by MN assay showed serum conversion (Table 1). Serum conversion data demonstrated that canine canine / FL / 04-like virus infection was temporally consistent with the onset of respiratory disease in most animals.</p><p num="0138"> Three months after the pandemic, a single serum sample was taken from an additional 46 asymptomatic dogs kept with the sick dog. Of these, 43 (93%) were serum positive in both assays. For the entire 73-dog population tested, 93%, including 82% of sick dogs (9 of 11) and 95% of healthy contact dogs (59 of 62), were seropositive in both assays. High sero-epidemiology in dogs with no history of respiratory disease indicates that most infections with canine influenza virus are subclinical, suggesting efficient transmission of the virus in dogs. It is unclear whether subclinical infections contribute to the transmission of the virus.</p><p num="0139"><u style="single">Example 4</u> To better understand the ability of the dog / FL / 04 virus to infect dogs, 10 in 4 6-month-old pure beagle dogs^6.6Half Tissue Culture Infection Dose (TCID)₅₀) Was inoculated by the intratracheal or intranasal route, respectively. All dogs developed fever (rectal temperature 39 ° C) during the first 2 days after inoculation (pi), but none showed respiratory symptoms such as cough or nasal discharge throughout the 14-day observation period. It was. Viral shedding was examined by quantification of the virus in nasal and oropharyngeal swabs. Only 2 out of 4 dogs shed a detectable amount of virus. One dog sheds virus on pi 1 and 2 days (1.0-2.5 log per swab)₁₀ PFU) and other dogs shed the virus for 4 consecutive days after inoculation (1.4-4.5 log per swab)₁₀ PFU). pi Postmortem examination of two dogs on day 5 showed necrotizing proliferative tracheitis, bronchiolitis and bronchiolitis, similar to those found in spontaneous disease in Greyhound, but with pulmonary hemorrhage or bronchopneumonia. Was not recognized. Viral H3 antigens were detected by IHC in the cytoplasm of epithelial cells of the bronchi, bronchioles and bronchial glands (Fig. 2B). The infectious virus was recovered from the lung tissue of one dog. Postmortem examination of the remaining two animals on pi14 confirmed minimal histological changes in respiratory tissue, absence of viral H3 antigen by IHC, and no recovery of virus from lung homogenates. Serum conversion in these latter two dogs was detected in the MN assay on day pi7, with antibody titers further increased 2-3-fold by day 14. These results show that the febrile response, the presence of viral antigens and infectious viruses in the lung parenchyma, histopathological findings typical of influenza, and susceptibility of dogs to canine / FL / 04 infection, as shown by serum conversion. Was established. The failure to reproduce severe illness and mortality in experimentally inoculated Beagle dogs is not surprising given that the large population of naturally infected greyhounds was asymptomatic.</p><p num="0140"><u style="single">Example 5</u> HI and MN assays were used on preserved sera from 65 racing greyhounds to determine if the canine / FL / 04-like influenza virus had spread to the greyhound population in Florida prior to the January 2004 pandemic. The presence of antibodies against dog / FL / 04 was investigated. No detectable antibody was found in 33 dogs sampled between 1996 and 1999. Of the 32 samples sampled between 2000 and 2003, 9 were seropositive in both assays-1 in 2000, 2 in 2002, and 6 in 2003 (Table 5). .. Serum-positive dogs were bred in Florida trucks involved in an outbreak of respiratory disease of unknown etiology from 1999 to 2003, and the dog / FL / 04-like virus was the causative agent of these outbreaks. Suggest that there is a possibility. To further investigate this possibility, we examined conservative tissue taken from Greyhound, who died of hemorrhagic bronchopneumonia in March 2003. When MDCK cells and chicken embryos were inoculated with lung homogenate from one dog, the H3N8 influenza virus was recovered and named A / dog / Florida / 242/2003 (dog / FL / 03). Sequence analysis of the complete genome of dog / FL / 03 showed&gt; 99% identity to dog / FL / 04 (Table 4), and the dog / FL / 04-like virus had infected Greyhound before 2004. Show that.</p><p num="0141"><u style="single">Example 6</u> From June to August 2004, thousands of racing greyhounds were outbreaks of respiratory disease on 14 trucks in Florida, Texas, Alabama, Arkansas, West Virginia and Kansas.</p><p num="0142"> Responsible persons for some of these trucks estimated that at least 80% of their dog population had clinical disease. Most dogs showed clinical signs of fever ( 39 ° C) and cough similar to those during the January 2004 outbreak, but many also presented with mucous purulent nasal discharge. Many deaths were reported, but no accurate mortality rate was sought.</p><p num="0143"> We collected a pair of acute and convalescent sera from 94 dogs housed in four Florida trucks: 56% of these dogs had antibody titers against dog / FL / 04. Was increased 4-fold, and 100% were serum-positive (Table 6). Convalescent sera from 29 dogs in West Virginia and Kansas also had antibodies to dog / FL / 04. We isolated the influenza A (H3N8) virus from the lungs of a greyhound that died of hemorrhagic bronchopneumonia on a truck in Texas. Sequencing the entire genome of this isolate, named A / Dog / Texas / 1/12004 (Dog / TX / 04), showed 99% identity with Dog / FL / 04 (Table 4). ). Substantial serological evidence of fatal dog cases during the 13-month period and isolation of three closely related influenza viruses from geographically distinct locations, as well as widespread infection in racing greyhounds. Suggests a persistent spread of canine / FL / 04-like virus in the canine population.</p><p num="0144"> Phylogenetic analysis of the HA genes in canine / FL / 03, canine / FL / 04 and canine / TX / 04 revealed that they formed a monophyletic group, and the robust bootstraps were in 2002. And this confirms that it is clearly different from the modern H3 gene of the horse virus isolated in 2003 (Fig. 1B). Phylogenetic analysis of the other seven genomic segments and paired nucleotide sequence comparisons confirm that the canine gene was isolated as a different subline most closely related to the equine virus lineage (data not shown, table). Four). Clustering of canine influenza viruses as a monophyletic group isolated from equine influenza is also supported by the presence of four sine amino acid changes in HA (Table 2). Together with the 2003 and 2004 serological results, these data are consistent with a single transmission of the virus from horses to dogs and subsequent horizontal transmission of the virus within the greyhound population. However, repeated introduction of endemic strains of influenza virus from unidentified possession species, although unlikely, cannot be formally ruled out.</p><p num="0145"> Viral HA is an important determinant of the host animal specificity of influenza virus (Suzuki et al., 2000). To identify residues within HA that may be associated with adaptation to the canine host, we compared the deduced amino acid sequence of canine HA with that of modern horse virus. The mature HA common amino acid sequences in horses and dogs are distinguished by four amino acid changes: N83S, W222L, I328T and N483T (see Table 2). Amino acid deletions are found in canine viruses compared to the common horse sequence. Therefore, the 7th amino acid in the HA horse sequence is the 6th position in the HA dog sequence, the 29th amino acid in the HA horse sequence is the 28th position in the HA dog sequence, and the 83rd amino acid in the HA horse sequence is the HA dog sequence. Then it is 82nd place. Thus, the four substituted amino acids are at positions 82, 221, 327 and 482 of the amino acid sequences set forth in SEQ ID NO: 33 and SEQ ID NO: 34. Substitution of asparagine at position 83 of the common sequence with serine is a change of unknown functional importance, as various polar residues are found in H3 molecules from other animal species. H3 The strictly conserved isoleucine at position 328 of the common sequence near the HA cleavage site has been replaced by threonine. The central role of host protease cleavage in pathogens suggests that this change deserves further testing. The substitution of tryptophan at position 222 of the common sequence with leucine is very striking because it is a non-conservative change adjacent to the sialic acid binding pocket that can regulate receptor function (Weis et al., 1988). Interestingly, leucine at position 222 is typically found in the H4, H8, H9 and H12 HA subtypes (Nobusawa et al., 1991; Kovacova et al., 2002), so dog H3. Not specific to HA. Replacing leucine is virus-specific in mammalian hosts, as porcine H4 subtype infections (Karasin et al., 2000) and human and porcine H9 subtype virus infections (Peiris et al., 1999) have been reported. It can be sex and even more compatible. Substitution of asparagine with threonine at position 483 of the common sequence resulted in the loss of glycosylation sites in the HA2 subunits conserved in all HA subtypes (Wagner et al., 2002). Although the importance of these amino acid changes in HA in the adaptation of horse virus to dogs remains unclear, similar amino acid changes have been observed in relation to interspecific transmission (Vines et al.). , 1998; Matrosovich et al., 2000). Tables 19 to 25 show the differences between the other influenza virus proteins of the present invention and the amino acids of the horse common sequence.</p><p num="0146"> The source of the equine influenza virus that first infected racing greyhounds remains speculative. Greyhound stadium kennels are not located near horses or racetracks, and contact with greyhounds and horses shedding viruses is not enough to explain many outbreaks in different states in 2004. It is suggested that it is sufficient. A possible source of exposure to the horse virus is feeding horse meat to greyhounds, which are supplemented with raw meat from a canning plant that processes carcasses, including horses that may carry influenza. Will be done. Preliminary examples of this mechanism of infection include reports of interspecific transmission of the H5N1 avian influenza virus to pigs and zoo felines fed infected chicken carcasses (Webster, 1998; Keawcharoen et al., Et al. 2004; Kuiken et al., 2004). While this is a viable route for the initial introduction of equine influenza into dogs, it does not explain the many recent influenza pandemics in thousands of dogs in different states. Our experimental inoculation studies have demonstrated the presence of the virus in the nasal passages and oropharynx of dogs, even at moderate titers. Nonetheless, these results indicate that viral shedding is possible and that dog-to-dog viral transmission through large aerosols, mediators or direct mucosal contact may be involved in livestock epidemiology of the disease. There is.</p><p num="0147"> Interspecific transmission of complete mammalian influenza virus to unrelated mammalian species is a rare event. Previous studies have provided limited serological or virological evidence of transient transmission of the human influenza A (H3N2) virus to dogs-but not both (Nikitin et al., 1972). Year; Kilbourne et al., 1975; Chang et al., 1976; Houser et al., 1980). However, there was no evidence of sustained circulation in dog hosts. Although direct transmission of swine flu virus from pigs to humans has been well documented (Dacso et al., 1984; Kimura et al., 1998; Patriarca et al., 1984; Top et al., 1977), the human host of swine flu virus. There is no evidence of adaptation to. In this report, we provide virological, serological and molecular evidence for interspecific transmission of the complete equine influenza A (H3N8) virus to another mammalian species, the dog. Inherent amino acid substitutions in canine virus HA, as well as serological confirmation of canine infection in many states of the United States, suggest adaptation of the virus to canine hosts. Since dogs are important companion animals to humans, these findings are meaningful to public health; dogs may provide a new source of new influenza A virus transmission to humans.</p><p num="0148"> (Table 1) Antigen-antibody reaction to A / dog / Florida / 43/04 (H3N8)<img id="000002" he="40" wi="155" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />^a The number of dogs with clinical signs of the disease.^{b b} The number of asymptomatic dogs kept in contact with clinically ill dogs.^c A / Canine / Florida / 43/2004 Hemagglutination inhibition (HI) assay using virus.^d A / Canine / Florida / 43/2004 Microneutralization (MN) Assay Using Virus^e Percentage of dogs with at least 4-fold increased antibody titers in paired acute and convalescent sera.^f Percentage of dogs with positive antibody titers (HI antibody titer 32: MN antibody titer 80) in convalescent sera.^g Geometric mean of antibody titers in convalescent sera.</p><p num="0149"> (Table 2) Differences in amino acids between H3 hemagglutinin in dogs and horses<img id="000003" he="129" wi="159" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* Amino acid residues (single letter notation) and position of mature H3 HA. The amino acid codes are A = alanine, D = aspartic acid, G = glycine, I = isoleucine, K = lysine, L = leucine, M = methionine, N = aspartic acid, R = arginine, S = serine, T = threonine, V. = Valin, W = tryptophan. Shows no change from the common horse H3 HA.</p><p num="0150"> (Table 3) Inhibition of hemagglutination of viral isolates by reference antisera for different HA subtypes<img id="000004" he="179" wi="157" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />^a Hemagglutination inhibitory titer against virus isolate derived from canine # 43^{b b} Polyclonal antisera were produced in ferrets, while all other antisera were produced in sheep or goats.</p><p num="0151"> (Table 4) Sequence homology of the A / dog / Florida / 43/2004 (H3N8) gene to equine, avian, porcine and human influenza A strains<img id="000005" he="194" wi="158" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />^a Nucleotide and amino acid (in parentheses) percent sequence identity of the A / canine / Florida / 43/04 (H3N8) gene for most homologous genes of influenza virus isolates from animal species, followed by their Genbank sequence database accession numbers. ..^{b b} Not applicable: N8 neuraminidase has not previously been reported in human or porcine viruses.</p><p num="0152"> (Table 5) Antibody titers of Greyhound sera collected from 1996 to 2003 against A / dog / Florida / 43/04 (H3N8)<img id="000006" he="50" wi="147" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />^a Year of serum sampling from Florida racing greyhounds.^{b b} Microneutralization test antibody titers in serum-positive dogs, including a range of 6 2003 serum-positive dogs.</p><p num="0153"> (Table 6) Antigen-antibody reaction to racing greyhound A / dog / Florida / 43/2004 (H3N8) on four tracks in Florida in June 2004<img id="000007" he="50" wi="156" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />^a Number of clinically diseased dogs tested by HI using A / Dog / Florida / 43/04 (H3N8).^{b b} Percentage of dogs showing a 4-fold increase in antibody titers in acute and convalescent sera.^c Percentage of dogs showing a positive antibody titer (HI antibody titer&gt; 16) in convalescent sera.^d Geometric mean of antibody titers in convalescent sera</p><p num="0154">Materials and Methods of Examples 7-11<u style="single">Dog tissue</u> Six mongrel dogs died in April / May 2005 due to an influenza pandemic in a shelter in northeastern Florida and one pet died in May 2005 in an influenza pandemic in a veterinary clinic in southeastern Florida. The Yorkshire Terrier was tested after death at the Anatomic Pathology Service at the University of Florida College of Veterinary Medicine. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin and 5 μm sections for histopathological diagnosis were stained with hematoxylin and eosin. Unfixed tissue was stored at -80 ° C until virological analysis.</p><p num="0155"><u style="single">Extraction of RNA from canine tissue samples</u> Thaw frozen lung tissue from each of the 7 dogs and use a disposable tissue grinder (Kendall, Lifeline Medical Inc., Danbury, CT) with 0.5% bovine serum albumin (BSA) and antibiotics (gentamicin and). Minimal essential medium (MEM) enriched with ciprofloxacin) was added and homogenized. Total RNA was extracted using a commercially available kit (RNeasy® Mini Kit, QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions and eluted with a final volume of 60 μL buffer. Total RNA was also extracted from lung tissue collected from dogs without respiratory disease.</p><p num="0156"><u style="single">Real-time RT-PCR</u> QuantiTect® probe RT-PCR kit containing ROX as a passive reference dye (QIAGEN Inc., Valencia, Using CA), one-step quantitative real-time RT-PCR was performed on all RNA extracted from canine tissue samples. That is, two primer-probe sets were used to detect influenza A sequences in each sample (Table 7). One primer-probe set was selective for the canine hemagglutinin (H3) sequence. The other primer-probe set targeted the highly conservative region of the influenza A virus matrix (M) gene. In each real-time RT-PCR reaction, 2X QuantiTech® Probe RT-PCR Master Mix 12.5 μL, QuantiTech® RT Mix 0.25 μL, forward and reverse primers (0.4 μM each) in a final volume of 25 μL. Concentration), probe (final concentration of 0.1 μM), and 5 μL of extracted total RNA were added to the reaction mixture containing RNase-free water. TaqMan® Ribosomal RNA Control Reagent (Applied) for the detection of 18S rRNA, an endogenous internal control for the presence of RNA extracted from canine tissue samples. Biosystems, Foster City, CA) was used according to the manufacturer's instructions.</p><p num="0157"> Quantitative one-step real-time RT-PCR was performed on the reaction mixture using the Mx3000P® QPCR System (Stratagene, La Jolla, CA). Cycling conditions included a 30 minute reverse transcription step at 50 ° C, a 15 minute initial denaturation step at 95 ° C to activate HotStarTaq® DNA polymerase, and 40 cycles of amplification. Each amplification cycle included 15 seconds of denaturation at 94 ° C followed by 1 minute of annealing / elongation at 60 ° C. FAM (emission wavelength 518 nm) and VIC (emission wavelength 554 nm) fluorescence signals were recorded at the end of each cycle. The threshold cycle (Ct) was determined by setting the threshold fluorescence (dR) to 1000 in each experiment. For data collection and analysis, Mx3000P® Version 2.0 Software Program (Stratagene, La Jolla, CA) was used. A sample was considered positive for influenza A virus if the threshold cycle (Ct) of the H3 or M gene was 3 units less than the Ct of lung tissue obtained from dogs without respiratory disease. The positive control consisted of an amplification of RNA extracted from the A / dog / FL / 242/03 (H3N8) virus.</p><p num="0158"><u style="single">Isolation of virus in MDCK cells</u> Thaw frozen lung tissue from each of the 7 dogs and homogenize with 0.5% (BSA) and 10 times Dulbecco's modified Eagle's medium (DMEM) fortified with antibiotics (gentamicin and ciprofloxacin). did. DMEM with 1 μg / mL TPCK-treated trypsin (Sigma-Aldrich Corp., St. Louis, MO) and antibiotics (gentamicin and ciprofloxacin) removed by centrifugation to remove solid debris. Madin-Darby canine kidney (MDCK) cells cultured in 1 were inoculated. The cells are 25 cm²5% CO using a flask₂The cells were cultured at 37 ° C in a humidified atmosphere. The culture was observed for morphological changes once daily and collected 5 days after inoculation. The collected cultures were clarified by centrifugation and the supernatant was inoculated into fresh MDCK cells as described in the initial inoculation; two more times for samples that did not show evidence of influenza virus by hemagglutination or RT-PCR. Was succeeded. The hemagglutination activity of the clarified supernatant was measured using 0.5% turkey erythrocytes as previously reported (Burleson, F. et al., 1992; Kendal, P. et al., 1982). RT-PCR was performed as follows.</p><p num="0159"><u style="single">Virus isolation in hatched eggs</u> Homogenates were prepared from frozen lung tissue as described above for inoculation of MDCK cells. Homogenate (0.2 mL) was inoculated into the urinary sac of 10-day-old hatched chicken eggs. After incubation at 35 ° C for 48 hours, eggs were cooled at 4 ° C overnight before recovery of allantois cavity fluid. The hemagglutination activity of the clarified supernatant was measured using 0.5% turkey erythrocytes as previously reported (Burleson, F. et al., 1992; Kendal, P. et al., 1982). RT-PCR was performed as follows. Samples that showed no evidence of influenza virus after the first inoculation were passaged two more times with hatched chicken eggs.</p><p num="0160"><u style="single">RT-PCR, nucleotide sequencing, and phylogenetic analysis</u> Viral RNA was extracted from MDCK supernatant or allantois cavities using the QIAamp® Viral RNA Mini Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. Viral RNA was reverse transcribed into cDNA using the QIAGEN® one-step RT-PCR kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. PCR amplification of the coding regions of the eight influenza virus genes in the cDNA was performed as previously reported (Klimov, A. et al., 1992b) using a generic gene-specific primer set (primer sequences given upon request). .. The resulting DNA unit replication sequence was used as a template for automatic sequencing on the ABI PRISM® 3100 automatic DNA sequencer using the cycle sequencing diterminator chemistry (Applied Biosystems, Foster City, CA). .. The nucleotide sequence is Lasergene 6 Package® (DNASTAR, Inc., Madison, It was analyzed using WI). A PHYLIP version 3.5 (copyright) software program was used to estimate phylogeny from nucleotide sequences and to calculate bootstraps (Felsenstein, J., 1989). The phylogenetic tree is PAUP (copyright) compared to the phylogenetic tree obtained by the neighbor-joining method using the Tamura-Nei model implemented in the MEGA (copyright) program (Kumar, S. et al., 2004). Confirmed by 4.0 beta program (Sinauer Associates, Inc., Sunderland, MA).</p><p num="0161"><u style="single">Hemagglutination inhibition (HI) test</u> Serum samples were incubated with receptor-destroying enzymes (RDE, Denka Biopsy Co., Ltd., Tokyo, Japan) (1 volume of serum: 3 volumes of RDE) prior to heat inactivation at 56 ° C for 30 minutes. Influenza A / dog / Jacksonville / 05 (H3N8) virus uses MDCK cells to 5% CO₂It was grown at 37 ° C for 72 hours. The supernatant of the virus culture was collected, clarified by centrifugation and stored at -80 ° C. All other viruses used in the HI assay were cultured in 10-day-old hatched eggs and the allantois cavity fluid was collected and stored at -80 ° C. The HI assay was performed as previously reported (Kendal, P. et al., 1982). That is, 4 hemagglutination units of virus in 25 μl were added to an equal volume of serum serially diluted on a 96-well plastic plate and incubated for 30 minutes at room temperature. Equal amounts of 0.5% turkey erythrocytes were added and the hemagglutination titer was visually determined 30 minutes later. The endpoint HI titer was defined as the final dilution of serum that completely inhibits hemagglutination.</p><p num="0162"><u style="single">Example 7-Clinical case</u> In April and May 2005, a previously reported (Crawford, PC et al., 2005) respiratory disease outbreak occurred in dogs housed in shelters in northeastern Florida. The pandemic involved at least 58 dogs in the age range from 3 months to 9 years, including pure and mongrel dogs. The most common clinical signs were purulent nasal discharge and cough over 7-21 days. Of the 43 dogs with clinical disease over 7 days, 41 showed HI antibody titers of 32 to&gt; 1024 against dogs / FL / 04 (H3N8). At least 10 dogs progressed to pneumonia, 6 of which were euthanized. These 6 mongrel dogs were 3 male and 3 female from 4 months to 3 years of age. The duration of clinical signs was 2-10 days at the time of euthanasia. On postmortem examination, these dogs showed pulmonary congestion and edema. Histological examination of the respiratory tract revealed rhinitis, tracheitis, bronchitis, bronchiolitis and suppurative bronchopneumonia. Necrosis and erosion of epithelial cells were found in the trachea, bronchi, bronchioles and bronchial glands. Infiltration of neutrophils and macrophages was seen in the respiratory tissue.</p><p num="0163"> In May 2005, a respiratory disease outbreak occurred in 40 pet dogs at a veterinary clinic in southeastern Florida. The most common clinical signs were purulent nasal discharge and cough over a 10-30 day period. Of the 40 dogs, 17 were serum positive for dogs / FL / 04 (H3N8), with HI antibody titers ranging from 32 to&gt; 1024. Serum conversion occurred in 10 dogs for which a pair of acute and convalescent sera were obtained. Three dogs progressed to pneumonia. One of these, a 9-year-old male Yorkshire terrier, died 3 days after the onset of clinical signs. The dog had bronchopneumonia, pulmonary edema and congestion, and severe bronchopneumonia. Necrosis and erosion of epithelial cells of the airways and neutrophil infiltration in the tissues were observed, as in the dogs in the 6 shelters.</p><p num="0164"><u style="single">Example 8-Real-time RT-PCR and virus isolation</u> Lung tissue obtained from 7 dogs was analyzed by a quantitative real-time RT-PCR assay to detect the M gene for influenza A and the H3 gene for canine H3N8 influenza A virus. The lungs of all 7 dogs were positive for both the influenza A M gene and the canine influenza H3 gene (Table 8). Influenza A H3N8 subtype virus was isolated from the lungs of dogs in shelters that died after 3 days of pneumonia after 3 passages with MDCK cells. The virus was named A / Dog / Jacksonville / 05 (H3N8) (Dog / Jax / 05). After two passages in hatched eggs, the H3N8 subtype virus of influenza A was isolated from the lungs of pet dogs that also died after 3 days of pneumonia. The virus was named A / Dog / Miami / 05 (H3N8) (Dog / Miami / 05).</p><p num="0165"><u style="single">Example 9-Gene analysis of canine influenza A H3N8 isolate</u> Sequence analysis of dog / Jax / 05 and dog / Miami / 05 revealed that their hemagglutinin (HA) genes were recovered from the lungs of racing greyhounds that died of pneumonia during the 2004 and 2005 influenza pandemic. It was found to be 98% consistent with the dog / FL / 04, dog / TX / 04 and dog / Iowa / 05 isolates (Crawford, PC, , 2005; Yoon KY et al., 2005). In addition, the canine / Jax / 05 and canine / Miami / 05 HA genes were 98% consistent with modern equine influenza viruses isolated since 2000. In a phylogenetic comparison of HA genes, canine / Jax / 05 and canine / Miami / 05 viruses are greyhound isolates of canine / FL / 04, canine / TX / 04, and canine / Iowa / 05, as well as modern horse isolates. It has been shown to form clusters with objects and to form a different group than the old horse virus isolated in the early 1990s (Fig. 4). In addition, dog / Jax / 05, dog / Miami / 05 and dog / Iowa / 05 isolates were more closely associated with dog / Tx / 04 than dog / FL / 04 or dog / FL / 03. The 2005 isolate forms a subtype that appears to have branched from the previous 2003 and 2004 canine viruses, with approximately 10 destructive and informative sites differing. These differences support the hypothesis of horizontal gene transfer from dog to dog, as opposed to regular reintroduction from external sources of infection. The accumulation of mutations from 2003 to 2005 illustrates the ongoing process of adaptation that must be received after the virus has been transmitted to a new host, as expected to occur in the canine influenza virus. ..</p><p num="0166"><u style="single">Example 10-Amino acid analysis of canine influenza A H3N8 isolate</u> All canine isolates from the 6 specimens had conservative amino acid substitutions that distinguish them from modern equine influenza (Table 9). These conservative substitutions were I15M, N83S, W222L, I328T and N483T. Phylogenetic comparisons of mature HA proteins have shown that canine / Jax / 05, canine / Miami / 05 and canine / Iowa / 05 viruses form subtypes with canine / TX / 04 isolates ( Figure 4). There were three amino acid changes (L118V, K261N and G479E) that distinguish this subtype from other canine viruses (Table 9). There were two amino acid changes (F79L and G218E) that distinguished the 2005 isolates from their roots, canine / TX / 04. In addition, the 2005 isolates from dogs other than Greyhound, Dog / Jax / 05 and Dog / Miami / 05, differed from the Dog / Iowa / 05 Greyhound isolate in R492K, a single amino acid change. Finally, dog / Jax / 05 differed from dog / Miami / 05 in one amino acid S107P. In all other H3N8 horse and canine viruses, S is conserved at position 107, with the exception of A / horse / chirin / 1/89 (Guo Y. et al., 1992) with T.</p><p num="0167"><u style="single">Example 11-Antigen analysis of canine influenza A H3N8 isolate</u> Hemagglutination inhibition (HI) studies were performed using serum from previous and modern equine influenza and canine influenza viruses, as well as from horses and dogs infected with the influenza virus in 2005 (Table 10). The sera of ferrets immunized against dog / FL / 04 were also included in the analysis. HI antibody titers in horse serum were 8 to 16-fold higher when tested with modern horse virus compared to previous isolates, but decreased by at least 1/4 when tested with canine virus. Canine serum was non-reactive with previous horse viruses, but antibody titers increased 4-fold when examined with modern horse and canine isolates. This was also observed for ferrets immunized against canine influenza virus. These serum response patterns demonstrated antigen similarity between canine influenza virus and modern equine influenza virus and were consistent with phylogenetic analysis. The antibody titers of horse, canine and ferret sera to the canine / Miami / 05 isolate were similar to those of the 2003 and 2004 canine isolates. However, the antibody titer of the dog / Jax / 05 isolate was 1/2 to 1/4. This suggests that canine / Jax / 05 may be antigenically different from other canine isolates and may be partially associated with a single amino acid change at position 107 of mature HA.</p><p num="0168"> (Table 7) Influenza A virus matrix genes and canine influenza A (H3N8) H3 genes primers and probes for quantitative real-time RT-PCR analysis<img id="000008" he="194" wi="97" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />^a The underlined letter r indicates the nucleotide of a or g, and the underlined letter k indicates the nucleotide of g or t.^{b b} Uppercase letters indicate fixed nucleic acid residues.</p><p num="0169"> (Table 8) Quantitative real-time RT-PCR and virus isolation performed on lung tissue of dogs that died of pneumonia during a respiratory disease outbreak at a Florida shelter and animal hospital.<img id="000009" he="130" wi="159" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0170"> (Table 9) Comparison of mature HA amino acids in canine influenza virus and modern equine influenza virus<img id="000010" he="217" wi="109" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0171"> (Table 10) Equine, canine and ferret serum antibody titers against pre- and modern equine and canine influenza viruses<img id="000011" he="136" wi="128" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />^a Antibody titers were determined by serial dilution of horse, dog or ferret sera, and hemagglutination inhibition studies performed with the viruses listed on the antigen column.^{b b} Serum of ferrets immunized with canine / FL / 04 virus</p><p num="0172">Examples of materials and methods of Examples 12 to 15<u style="single">Canine influenza virus inoculation</u> The virus inoculum was used to stock Madin-Darby canine kidney (MDCK) epithelial cells with A / Canine / FL / 43/04 (H3N8), a triple-passage of the original isolate from the previous report (Crawford et al., 2005). Prepared by inoculation. Inoculated MDCK cells in Dulbecco's Minimal Essential Medium (DMEM) enriched with 1 μg / mL TPCK-treated trypsin (Sigma-Aldrich Corp., St. Louis, MO) and antibiotics (gentamicin and ciprofloxacin) were 250 cm.²5% CO in a flask₂It was grown at 37 ° C in a humidified atmosphere containing. The culture was observed for morphological changes once daily and collected 5 days after inoculation. The collected cultures were clarified by centrifugation and the supernatant was stored at -80 ° C until inoculation of dogs. A fraction of the supernatant was used to measure virus titers by the Reed and Muench methods. Titer is 10 per mL⁷Half Tissue Culture Infection Dose (TCID)₅₀) A / dog / Florida / 43/04 (dog / FL / 2004).</p><p num="0173"><u style="single">Experimental inoculation</u> Eight 4-month-old colony-breeding four-month-old mongrel dogs (Marshall BioResources, North Rose, NY) (4 males and 4 females) were used in an experimental inoculation study approved by the University of Florida Institutional Animal Care and Use Committee. The dog weighed 13-17 kg. Dogs were healthy based on physical examination, baseline blood tests, and temperature records 2 weeks prior to inoculation. All dogs had no history of exposure to canine influenza virus based on serological tests performed on a pair of serum samples taken upon arrival at the facility and two weeks later. Dogs were anesthetized by intravenous injection of propofol (Diprivan®, Zeneca Pharmaceuticals, 0.4 mg / kg body weight to perform) in preparation for endotracheal tube intubation. 6 dogs (3 males and 3 females) in 5 mL sterile saline via a small diameter rubber catheter inserted into an endotracheal tube 10⁷ TCID₅₀Canine / FL / 04 virus was administered to the distal trachea and inoculated respectively. Two dogs (one male and one female) were sham-inoculated with equal doses of sterile saline. The sham-inoculated control dogs were housed in a separate room from the virus-inoculated dogs and cared for by another person. Physical examination and rectal temperature recording were performed twice daily for 6 days after inoculation (pi).</p><p num="0174"><u style="single">Collection of pharyngeal and rectal swabs</u> To monitor viral shedding, oropharyngeal specimens were taken from each dog twice daily using a polyester swab (Fisher Scientific International Inc., Pittsburgh, PA) on days 0-6. Swabs were placed in 1 mL of sterile phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA). Rectal swabs were collected from each dog once daily from day 0 to day 6. The swab extract was prepared by clarifying the swab transport medium by centrifugation. Swab extract fractions were immediately tested for influenza A virus nucleoprotein using Directigen commercially available immunoassay kits (BD, Franklin Lakes, NJ) according to the manufacturer's instructions. The remaining extract was stored at -80 ° C until other virological assays.</p><p num="0175"><u style="single">Post-mortem inspection</u> On day pi, one sham-inoculated dog and one virus-inoculated dog were euthanized by intravenous infusion of Beuthanasia-D® solution (1 mL / 5 kg body weight; Schering-Plough Animal Health Corp). pi One virus-inoculated dog was similarly euthanized daily from day 2 to day 5. pi The remaining dogs were euthanized on the 6th after fake and virus inoculation. A detailed post-mortem examination was performed by one investigator (WLC). Tissues are fixed in 10% neutral buffered formalin, embedded in paraffin and 5 μm sections stained with hematoxylin eosin for histopathological diagnosis or immunohistochemical as described below. Processed for inspection. Unfixed lung tissue was sent to the Diagnostic Clinical Microbiology / Parasitology / Serology Service of the University of Florida College of Veterinary Medicine for bacterial isolation and identification. Samples were cultured in non-selective medium and selective medium of the genus Bordetella (Regan-Lowe; Remel, Lenexa, KS) and the genus Mycoplasma (Remel). All cultures were maintained for 21 days before reporting no growth. Unfixed tissue was also stored at -80 ° C until virological analysis.</p><p num="0176"><u style="single">Immunohistochemistry</u> Deparaffinized and rehydrated 5 μm tracheal and lung tissue sections were set on Bond-Rite slide glass (Richard-Allan Scientific, Kalamazoo, MI) and proteinase K (DAKO Cytomation, Carpenteria, CA) peroxidase blocking reagent. (DAKO® EnVision® Peroxidase Kit, DAKO Corp., Carpenteria, CA) were treated sequentially. Sections were incubated with 1: 500 dilutions of monoclonal antibody against influenza A H3 (Chemicon International, Inc., Ternecula, CA) for 2 hours at room temperature. Controls include identical sections and mouse IgG (1 mg / mL, Serotec, Inc.). Incubation of Raleigh, NC) and incubation of monoclonal antibodies and normal canine lung sections were included. After treatment with the primary antibody, sections were incubated with secondary immunoperoxidase and peroxidase substrate reagents (Dako® EnVision® Peroxidase Kit, Dako Corp.) according to the manufacturer's instructions. Sections were counterstained with hematoxylin, treated with clarifying agent # 2 and Bluning reagent (Richard-Allan Scientific, Kalamazoo, MI), dehydrated, and covered with Permount (ProSciTech, Queensland, Australia). ..</p><p num="0177"><u style="single">Extraction of RNA from swabs and tissues</u> Thaw lung and tracheal tissue from each dog and use a disposable tissue grinder (Kendall, Lifeline Medical Inc., Danbury, CT) to 0.5% bovine serum albumin (BSA) and antibiotics (gentamicin and ciprofloxacin). Minimal essential medium (MEM) enriched with ciprofloxacin) was added and homogenized. Total RNA was extracted from tissue homogenate and oropharyngeal and rectal swab extracts using a commercially available kit (RNeasy® minikit, QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions to finalize. It was eluted with 60 μL of buffer.</p><p num="0178"><u style="single">Real-time RT-PCR</u> QuantiTect® probe RT-PCR kit containing ROX as a passive reference dye (QIAGEN Inc., Valencia, Total RNA using primer-probe sets (Payungporn S. et al., 2006 a; Payungporn S. et al., 2006b) targeting highly conserved regions of the matrix (M) gene of influenza A virus and influenza A virus. One-step quantitative real-time RT-PCR was performed on the cells. In each real-time RT-PCR reaction, 2X QuantiTech® Probe RT-PCR Master Mix 12.5 μL, QuantiTech® RT Mix 0.25 μL, forward and reverse primers (0.4 μM each) in a final volume of 25 μL. Concentration), probe (final concentration of 0.1 μM), and 5 μL of extracted total RNA were added to the reaction mixture containing RNase-free water. Manufacturers of TaqMan® GAPDH Control Reagents (Applied Biosystems, Foster City, CA) for the detection of GAPDH as an endogenous internal control for the presence of RNA extracted from swabs and tissue samples and as a standardized control. Used according to the instructions in.</p><p num="0179"> Quantitative one-step real-time RT-PCR was performed on the reaction mixture using the Mx3000P® QPCR System (Stratagene, La Jolla, CA). Cycling conditions included a 30 minute reverse transcription step at 50 ° C, a 15 minute initial denaturation step at 95 ° C to activate HotStarTaq® DNA polymerase, and 40 cycles of amplification. Each amplification cycle included 15 seconds of denaturation at 94 ° C followed by 1 minute of annealing / elongation at 60 ° C. FAM (emission wavelength 518 nm) and VIC (emission wavelength 554 nm) fluorescence signals were recorded at the end of each cycle. The threshold cycle (Ct) was determined by setting the threshold fluorescence (dR) to 1000 in each experiment. For data collection and analysis, Mx3000P® Version 2.0 Software Program (Stratagene, La Jolla, CA) was used. The positive control consisted of an amplification of RNA extracted from the A / dog / FL / 242/03 (H3N8) virus. The results were standardized by dividing the M Ct value for each sample by the corresponding GAPDH Ct value.</p><p num="0180"><u style="single">Re-isolation of virus from tissue</u> Frozen lung and tracheal tissues from virus-inoculated dogs were thawed and homogenized in 10-fold DMEM fortified with 0.5% BSA and antibiotics. Solid debris was removed by centrifugation and the supernatant was cultured in 1 μg / mL TPCK-treated trypsin (Sigma-Aldrich Corp., St. Louis, MO) and antibiotic-enriched DMEM as described above. The cells were inoculated. The cells are 25 cm²5% CO using a flask₂The cells were cultured at 37 ° C in a humidified atmosphere. The culture was observed for morphological changes once daily and collected 5 days after inoculation. The collected cultures were clarified by centrifugation and the supernatant was inoculated into fresh MDCK cells as described in the initial inoculation; two more times for samples that did not show evidence of influenza virus by hemagglutination or RT-PCR. Was succeeded. The hemagglutination activity of the clarified supernatant was measured using 0.5% turkey erythrocytes (Crawford et al., 2005) as described above. RT-PCR was performed as follows.</p><p num="0181"><u style="single">RT-PCR, nucleotide sequencing, and phylogenetic analysis</u> Viral RNA was extracted from the MDCK supernatant using the QIAamp® Viral RNA Mini Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. Viral RNA was reverse transcribed into cDNA using the QIAGEN® one-step RT-PCR kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. PCR amplification of the coding regions of the eight influenza virus genes in the cDNA was performed as previously reported (Crawford et al., 2005) using a generic gene-specific primer set (primer sequences given upon request). The resulting DNA unit replication sequence was used as a template for automatic sequencing on the ABI PRISM® 3100 automatic DNA sequencer using the cycle sequencing diterminator chemistry (Applied Biosystems, Foster City, CA). .. The nucleotide sequence is Lasergene 6 Package® (DNASTAR, Inc., Madison, It was analyzed using WI). The nucleotide sequence of the virus recovered from the infected dog was compared to the sequence of the virus in the inoculum to see if any changes occurred during respiratory replication.</p><p num="0182"><u style="single">Example 12-Clinical disease</u> All 6 virus-inoculated dogs developed fever (rectal temperature 39 ° C) during the first 2 days of pi, but showed respiratory symptoms such as cough or nasal discharge throughout the 6-day observation period. There was nothing. The sham-inoculated dogs remained clinically healthy.</p><p num="0183"><u style="single">Example 13-Viral shedding</u> Influenza A nucleoprotein was detected in a pharyngeal swab taken from one dog virus inoculated at pi 24 hours. Oropharyngeal swabs from one dog at pi 72, 84 and 120 hours and from another dog at pi 108, 120 and 132 hours were positive for virus by quantitative real-time RT-PCR. (Table 11). The absolute number of influenza M gene copies per μL of swab extract increased over time from pi 3 to 6 days. No virus was detected in the rectal swab.</p><p num="0184"><u style="single">Example 14-Postmortem examination</u> In contrast to past experimental infections with specific-pathogen-free beagles (Crawford et al., 2005), virus-inoculated mongrel dogs were shown by gross and histological analysis of the lungs from pi 1 to 6 days. As you can see, he developed pneumonia. In addition to pneumonia, dogs presented with rhinitis, tracheitis, bronchitis and bronchiolitis, as reported in naturally infected dogs (Crawford et al., 2005). Epithelial necrosis and erosion were observed in the cells lining the airways and bronchial glands, and infiltration of neutrophils and macrophages was observed in the submucosa (Fig. 5, upper row). Immunohistochemical examination detected viral H3 antigens in epithelial cells of the bronchi, bronchioles and bronchial glands (Fig. 5, bottom). There was no bacterial coinfection. The respiratory tissue of the two pseudo-inoculated dogs was normal.</p><p num="0185"><u style="single">Example 15-Virus replication in the trachea and lungs</u> Trachea and lungs were positive for virus by quantitative real-time RT-PCR in all dogs from pi 1 to 6 days (Table 12). The absolute number of influenza M gene copies per μL of tracheal homogenate increased from 1 to 5 days pi and then decreased at 6 days. The absolute number of M gene copies per μL of lung homogenate decreased from pi 1 to 6 days. Generally, the trachea has more 1 log than the lungs on each pi day for 6 days₁₀Many viruses were included.</p><p num="0186"> (Table 11) Detection of viral shedding in the oropharynx of mongrel dogs inoculated with canine influenza virus by quantitative real-time RT-PCR<img id="000012" he="81" wi="150" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />^a A / dog / FL / 43/04 (H3N8) The time when the oropharyngeal swab was collected from the dog after inoculation with the virus.^{b b} The standardized ratio was calculated by dividing M (Ct) by GAPDH (Ct) for each swab extract.^c Absolute number of matrix gene copies per μL of swab extract.</p><p num="0187"> (Table 12) Detection of viral replication in the trachea and lungs of mongrel dogs inoculated with canine influenza virus by quantitative real-time RT-PCR<img id="000013" he="81" wi="142" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />^a A / dog / FL / 43/04 (H3N8) The time when tissue was collected from dogs after inoculation with the virus.^{b b} The standardized ratio was calculated by dividing M (Ct) by GAPDH (Ct) for each tissue homogenate.^c Absolute number of matrix gene copies per μL of tissue homogenate.</p><p num="0188">Examples of Materials and Methods of Example 16<u style="single">Virus strain</u> Influenza virus strains and virus strains from birds, horses and humans (listed in Table 15) were grown in hatched chicken eggs or MDCK cells and their infectivity was quantified by endpoint dilution or plaque method in chicken embryos. Rapid virus quantification was performed by a hemagglutination test using erythrocytes of turkey red blood cells.</p><p num="0189"><u style="single">Specimen for diagnosis</u> The presence of canine influenza virus was investigated in the lung tissue of a total of 60 dogs collected from cases suspected of having viral respiratory disease in 2005.</p><p num="0190"><u style="single">Extraction of RNA from canine tissue samples</u> A block of lung tissue weighing 20-30 mg was homogenized in a disposable tissue grinder (Kendal). Total RNA was extracted using a commercially available kit (RNeasy mini kit, Qiagen, Valencia, CA) according to the manufacturer's instructions and eluted with a final volume of 60 μL.</p><p num="0191"><u style="single">Primer and probe design</u> The CLUSTAL X program (version 1.8) was used to perform multiple sequence alignments of the H3 and M genes from different subtypes and different animal species. Matrix (M) primers and probes were selected from conserved regions through known sequences corresponding to different subtypes of influenza A virus, and H3 hemagglutinin gene-specific primers and probes were specific for the horse and canine influenza A virus genes. They were selected to match and mismatch the homologous avian and human genes (Table 13). Analysis tools on the web provided by primer design software (OLIGOS version 9.1) and EXIQON (http://lnatools.com) were used to calculate Tm and predict secondary structure and self-hybridization. The conserved region of 18S rRNA was used as an endogenous internal control for the presence of RNA extracted from canine tissue samples. Pre-Developed TaqMan® Assay Reagents for Eukaryotic 18S rRNA (VIC / TAMRA) (Applied Biosystems) was used for real-time detection of 18S rRNA in tissue samples.</p><p num="0192"><u style="single">Real-time RT-PCR conditions</u> One-step quantitative real-time RT-PCR is a Quantitect probe RT-PCR kit containing ROX as a passive reference dye (Qiagen, Valencia, In each real-time RT-PCR reaction performed using CA), 2X QuantiTech probe RT-PCR master mix 10 μL, QuantiTech RT Mix 0.2 μL, primer (final concentration 0.4 μM for H3 gene, M gene) in a final liquid volume of 20 μL. The final concentration is 0.6 μM), the probe (final concentration 0.1 μM for the H3 gene, the final concentration 0.2 μM for the M gene), and 5 μL of RNA sample as a template for combining with the reaction mixture containing RNase-free water. used. One-step real-time RT-PCR was performed using the Mx3005P real-time QPCR system (Stratagene). Cycling conditions included a reverse transcription process at 50 ° C for 30 minutes. After the first denaturation step at 95 ° C for 15 minutes to activate HotStarTaq DNA polymerase, amplification is 40 cycles including denaturation (94 ° C, 15 seconds) and annealing / elongation (60 ° C, 30 seconds). It was carried out during the period of. FAM (emission wavelength 516 nm for H3 and M detection) and VIC (18S) A fluorescence signal with an emission wavelength of 555 nm) was obtained once per cycle for rRNA detection at the end of the extension phase. Data acquisition and analysis of the real-time PCR assay was performed using Mx3005P software version 2.02 (Stratagene).</p><p num="0193"><u style="single">Specificity of H3 primers / probes for canine influenza (H3N8) and versatility of M primer / probe sets for influenza A virus</u> RNA extracted from multiple known subtypes of influenza A virus was used as a template in the real-time RT-PCR assay to test the specificity of each primer / probe set (Table 15).</p><p num="0194"><u style="single">RNA standard for measuring the performance of real-time RT-PCR</u> The gene for canine influenza A virus (A / canine / Florida / 242/2003 (H3N8)) is a PCR unit of H3 (nt 1 to 487) and M (nt 1 to 276) by using a primer bound to the T7 promoter. Used to create duplicate sequences (Table 13). Subsequently, purified PCR unit replication sequences of the H3 and M genes were used as templates for in vitro transcription by using the Riboprobe In vitro Transcription System-T7 (Promega) according to the manufacturer's instructions. The concentration of transcribed RNA was calculated by measuring the absorbance at 260 nm. Then 10 RNAs to perform a susceptibility test⁸Diluted 10-fold in the range of ~ 10 copies / μL. In addition, to examine the overall performance of real-time RT-PCR, the logarithm of the initial RNA template concentration (copy / μL) was plotted against the threshold cycle (Ct) obtained from each dilution to create a standard curve.</p><p num="0195"><u style="single">Comparative susceptibility testing of real-time RT-PCR and Directigen Flu A test kits</u> 10^6.67EID₅₀/ mL (HA = 64) A / Wyoming / 3/2003 (H3N2) and 10^7.17EID₅₀Stock viruses from two strains containing / mL (HA = 16) A / dog / Florida / 242/2003 (H3N8) were used for the detection threshold assay. A fast influenza A antigen detection kit, Directigen Flu A (Becton, Dickinson and Company), was used according to the manufacturer's instructions and a logarithmic dilution of the specimen phosphate buffered saline (PBS) (125 μL) was used. Each Directigen Flu A tester has an H1N1 influenza antigen spot that appears as a purple dot in the center of the membrane, demonstrating test completeness based on a monoclonal antibody against nucleoprotein (NP). The appearance of purple triangles surrounding the dots indicates the presence of influenza NP in the test specimen. The intensity of the purple signal from the triangle is scored as + (triangle outline), ++ (lightly colored triangle), +++ (dark purple triangle) and ++++ (extremely dark purple triangle). .. Viral RNA is the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, Virus RNA was extracted from the 125 μL fraction of each virus diluent using CA) and eluted to a final volume of 50 μL. A 5 μL volume of extracted viral RNA was tested by real-time RT-PCR in a comparative susceptibility test using the Directigen Flu A kit.</p><p num="0196"><u style="single">Example 16</u> Real-time RT-PCR assays for canine influenza rely on information from three molecular probes targeting 18S rRNA from host cells and M and H3 from the influenza A virus genome (Table 14). Host gene amplification is a reporter on sample quality and integrity. Clinical, autopsy or experimental samples containing canine influenza (H3N8) virus were expected to give amplified signals using three probes. Specimens that give an amplified signal when using the M and 18S rRNA probes but are negative for H3 are indicators of influenza virus H3 subtypes derived from humans, pigs or birds, or from subtypes other than H3. These rare cases can be resolved by RT-PCR with HA generic primers to produce unit replication sequence cDNAs that can be analyzed by sequencing. Properly collected and handled influenza A virus-free specimens give only 18S rRNA unit replication sequence signals. 18S The situation where only the rRNA probe and the H3 probe give an amplified signal is an indicator of technical incompleteness unless proven otherwise; either a false negative with the M probe or a false positive with the H3. Must be substantiated. Finally, specimens that are not given an amplification signal using the three probes suggest incomplete sampling, denaturation, inadequate RNA extraction, or the presence of polymerase inhibitors used in PCR.</p><p num="0197"> To investigate the specificity of the H3 primer / probe set for canine influenza A virus (H3N8) and the versatility of the M primer / probe set for influenza A, multiple subtypes of influenza A virus were tested by real-time RT-PCR. .. The results show that the H3 primer / probe set gives a positive amplification signal only with canine influenza (H3N8). No significant false positive or non-specific amplification signals were observed in other subtypes or human H3 strains. The M primer / probe set gave a clear amplification signal in all strains tested (Table 15). These results indicate that the H3 primer / probe specifically detects canine influenza A virus (H3N8) and the M primer / probe detects many subtypes of influenza A virus.</p><p num="0198"> The performance of the real-time RT-PCR assay was evaluated by endpoint dilution of transcribed RNA in vitro for M and H3. As expected, the threshold cycle (Ct) increased in direct correlation with the dilution of the RNA standard. Fluorescent signals were 10 in the dilution of the RNA standard for M and H3, respectively.³And 10²Detectable at low copy / μL values (Figures 6A and 6B). Standard curves for the M and H3 genes were created by plotting the logarithm of RNA concentration at the start against the threshold cycle (Ct) obtained from each dilution (Figures 6C and 6D). The gradient of the standard curve is used to determine the theoretically exponential PCR reaction efficiency; 100% amplification efficiency means doubling the unit replication sequence concentration in each cycle. Standard curves with gradients of about -3.1 to -3.6 are typically acceptable for most applications that require accurate quantification (90 to 110% reaction efficiency). The Rsq value is the fit of all data to the standard curve plot. If all the data are perfectly on the curve, then Rsq is 1.00. As the data move further away from the curve, Rsq decreases. An Rsq value of 0.985 is acceptable for most assays. The gradient of the M standard curve is -3.576 (efficiency = 90.4%), Rsq = 1.00, and the gradient of the H3 standard curve. It was -3.423 (efficiency = 95.9%) and Rsq = 0.999. These values indicate that the amplification efficiency and overall performance of the real-time RT-PCR assay are satisfactory. We found that the low efficiency and sensitivity of the M primer / probe set compared to the H3 primer / probe set broadly covers the variability of the M gene sequence through viruses of many subtypes, hosts and strains. The cause is the N-fold degeneracy of the M primer sequence required to secure it.</p><p num="0199"> The sensitivity of the real-time RT-PCR assay was also compared with the commercially available fast antigen detection assay (Directigen Flu A). Logarithmic dilutions of A / Wyoming / 3/2003 (H3N2) and A / Canine / Florida / 242/2003 (H3N8) were analyzed by Directigen Flu A and real-time RT-PCR. The results of Directigen Flu A showed that the susceptibility to both virus strains was about 100-fold dilution from the virus stock strains used in these experiments (Fig. 7). Canine virus (A / dog / Florida / 242/2003:10^6.xThe signal (purple) generated from the PFU / ml) samples is consistent with the low viral concentrations in these samples, and is consistent with the human virus (A / Wyoming / 3/2003: 10).^7.xIt was much weaker than the signal seen in PFU / ml). Alternatively, the low signal of canine influenza may be due to the molecular specificity of the monoclonal antibody against NP, i.e., the low storage of amino acids within the NP epitope of canine influenza A virus.</p><p num="0200"> Real-time RT-PCR of the M gene gave above-threshold Ct values for 10 and 30 PFU equivalents of virus per reaction of A / Canine / Florida / 242/2003 and A / Wyoming / 3/2003, respectively (Table 16). ). The difference in susceptibility values between the two virus strains is the difference in the original virus titer. Since the H3 primers / probes in our real-time RT-PCR assay method exclusively amplify the canine influenza A virus, no comparison of H3 gene detection between canine and human influenza viruses was performed. RT-PCR susceptibility is 10 more than the fast antigen detection kit⁵It was twice as high.</p><p num="0201"> In order to evaluate the performance of RT-PCR testing in autopsy specimens obtained from dogs with acute respiratory disease, the presence of canine influenza A virus was detected by real-time RT-PCR on 60 canine lung tissue samples submitted in 2005. Examined. A total of 12 of the 60 samples (20%) were positive for both the M and H3 genes, and the remaining 48 samples were negative for both the M and H3 genes. To investigate the specificity of the real-time assay, an attempt was made to isolate the virus by inoculating chicken eggs and MDCK cells; RT-PCR obtained canine influenza virus from 2 of 12 samples that were positive for canine influenza. (Data not shown, manuscript is being prepared). All tissues were taken from dogs with a history of severe respiratory disease, but most samples did not yield canine influenza virus by either real-time RT-PCR or conventional isolation, and Bordetella bronchiseptica, canine distemper or A high incidence of other respiratory pathogens such as parainfluenza virus was suggested. The one-step real-time RT-PCR assay described herein provides a fast and sensitive economical approach for the detection of canine influenza A virus (H3N8). Rapid laboratory diagnosis of canine influenza A virus (H3N8) infection in the early stages of the disease may provide information relevant to clinical patient and institutional operations.</p><p num="0202"> (Table 13) Primers and probes used in real-time RT-PCR detection and in vitro transcription<img id="000014" he="233" wi="123" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* Note: Uppercase = LNA (fixed nucleic acid) residue, r = a or g, k = g or t, underline = T7 promoter sequence</p><p num="0203"> (Table 14) Evaluation of real-time RT-PCR assay<img id="000015" he="58" wi="158" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0204"> (Table 15) Dog H3 primer / probe set specificity test and M primer / probe set versatility test using multiple subtypes of influenza A virus<img id="000016" he="175" wi="149" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* Note that clinical sample subtypes were confirmed by nucleotide sequencing.</p><p num="0205"> (Table 16) Comparative susceptibility study for influenza A virus detection between real-time RT-PCR and Directigen Flu A<img id="000017" he="96" wi="159" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0206"> (Table 17)<img id="000018" he="53" wi="159" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0207"> (Table 18)<img id="000019" he="75" wi="140" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0208"> (Table 19) Differences in amino acids between PB2 proteins of H3N8 horse and canine influenza viruses<img id="000020" he="43" wi="155" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0209"> (Table 20) Differences in amino acids between PB1 proteins of H3N8 horse and canine influenza viruses<img id="000021" he="37" wi="155" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0210"> (Table 21) Differences in amino acids between PA proteins of H3N8 horse and canine influenza viruses<img id="000022" he="66" wi="158" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* Based on the effective genes of the virus isolated from 1963 to 1998.</p><p num="0211"> (Table 22) Differences in amino acids between H3N8 horse and canine influenza virus NP proteins<img id="000023" he="53" wi="157" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0212"> (Table 23) Differences in amino acids between NA proteins of H3N8 horse and canine influenza viruses<img id="000024" he="83" wi="157" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0213"> (Table 24) Differences in amino acids between M1 proteins of H3N8 horse and canine influenza viruses<img id="000025" he="17" wi="157" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* Based on the effective genes of the virus isolated from 1963 to 1998.</p><p num="0214"> (Table 25) Differences in amino acids between NS1 proteins of H3N8 horse and canine influenza viruses<img id="000026" he="42" wi="156" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* Based on the effective genes of the virus isolated from 1963 to 1998.</p><p num="0215"><u style="single">Example 17-Development of a canine influenza stimulation model</u> The canine influenza (canine flu) virus isolated from the outbreak of influenza in Florida has been observed to be the H3N8 influenza virus, an equine influenza virus strain A / horse / ohio. Closely related to / 03 (Crawford et al., SCIENCE Vol. 309, September 2005, incorporated by reference in its entirety into this patent). This study examined the possibility of using the equine influenza virus strain A / horse / ohio / 03 to induce influenza-like illness in dogs.</p><p num="0216"><u style="single">procedure</u>:: Ten male and female 13-week-old beagle dogs were obtained from suppliers and bred in separate cages within the BSL-2 facility. Dogs were randomly assigned to 2 groups of 5 dogs in each group. As shown in Table 26, one group was subjected to intratracheal stimulation and the other group was subjected to intranasal stimulation. The dog was stimulated at 14 weeks of age.</p><p num="0217"> (Table 26) Experimental design<img id="000027" he="24" wi="159" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0218"> Equine influenza virus A / horse / Ohio / 03 grown in cell culture was used as a stimulating virus. In intratracheal stimulation, the irritating virus is transmitted via a delivery tube consisting of a cuffed tracheal tube (size 4.0 / 4.5, Sheridan, USA) and a liquid supply tube (size 5Fr, 1.7 mm, length / 16 inches, Kendall, USA). Then, it was administered in a liquid volume of 0.5 to 1.0 ml. In oral and nasal irritation, irritation virus (10⁷~10⁸ TCID50 / dog) was administered as a mist in a liquid volume of 2-3 ml using a nebulizer (DeVilbiss Ultra-Neb® 99 Ultrasonic Atomizer, Sunrise Medical, USA).</p><p num="0219"> Dogs were observed for influenza-related clinical signs for 14 days after stimulation. Serum from each dog at day 0 (before stimulation) and 7 and 14 days after stimulation to measure HI titers with H3N8 equine influenza virus according to standard protocols (SAM 124, CVB, USDA, Ames, IA). A sample was taken. All dogs were humanely euthanized and lung tissue was harvested in 10% buffered formalin for histopathological evaluation.</p><p num="0220"><u style="single">result</u>:: The results of this experiment are summarized in Table 27. Influenza-related clinical signs were observed in several dogs after stimulation. These signs include fever (&gt; 103)^○F;&gt; 39.4 ° C) and cough were included. Two out of five (ie, 40%) fever (&gt; 103) in one group compared to one in five (ie, 20%) in the two groups.^○F;&gt; 39.4 ° C) was shown. After stimulation, one of the oral and nasal stimulation groups showed sneezing and the other showed cough. In group 1, a HI titer range of 10-80 and a geometric mean titer (GMT) of 20 were observed. In the second group, a titer range of 40 to 160, GMT 86, was observed. One dog in each group showed influenza-compatible or influenza-specific histopathological lesions.</p><p num="0221"> (Table 27) Canine Influenza Stimulation-Clinical Signs, Virus Isolation, Histopathological Results, and Serological Results<img id="000028" he="230" wi="103" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* Animals were stimulated with the equine influenza isolate Ohio 03. ** Rectal temperature? 103^○F; 39.4 ° C</p><p num="0222"><u style="single">Example 18-Equine Influenza Virus Vaccine Efficacy in Dogs</u> The canine influenza (canine influenza) virus isolated from the outbreak of influenza in Florida has been observed to be the H3N8 influenza virus and is closely associated with the equine influenza virus A / horse / ohio / 03 based on sequence similarity. Related to. The following trials were conducted to investigate the efficacy of the experimentally inactivated equine influenza virus vaccine.</p><p num="0223"><u style="single">procedure</u>:: Nine male and female 7-week-old beagle dogs were obtained from suppliers and bred in separate cages within the BSL-2 facility. These dogs were randomly assigned to two groups, as shown in Table 28.</p><p num="0224"> (Table 28) Experimental design<img id="000029" he="23" wi="159" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0225"> The first group consisted of 5 dogs vaccinated with the inactivated and CARBIGEN adjuvanted equine influenza virus A / horse / ohio / 03 vaccine by the subcutaneous (SQ) route at 8-12 weeks of age. It consisted of. A / Horse / Ohio / 03 were inactivated by binary ethyleneimine (BEI) using standard methods. For each dose of vaccine, 5% CARBIGEN based on mass, 4096 HA units of inactivated virus, PBS sufficient for a total dose of 1 ml, and pH adjusted to 7.2-7.4. Sufficient NaOH was included for this. H3N8 Equine Influenza Virus Standard Protocol (SAM 124, CVB, USDA, Ames, Serum samples were taken from all dogs on the first and second vaccination days, and 7 and 14 days after the first and second vaccinations, and prior to stimulation to measure HI titers using IA). did. Equine influenza virus A / horse / ohio in which a second group (ie, control group) consisting of 5 vaccinated dogs and 4 corresponding age dogs was grown in cell culture 3 weeks after the second vaccination. / 03 (10)^7.0~10^8.0 TCID50 / dog) was stimulated by intraoral administration at a liquid volume of 1 to 2 ml per animal. The irritating virus was administered as a mist using a nebulizer (DeVilbiss Ultra-Neb® 99 Ultrasonic Atomizer, Sunrise Medical, USA). Dogs were observed for influenza-related clinical signs for 14 days after stimulation. 5 animals (3 animals in the vaccinated group, 2 animals in the control group) and 4 animals 14 days after stimulation (controls) to collect lung tissue in 10% buffered formalin for histopathological evaluation. Two dogs in the group and two in the vaccinated group) were humanely euthanized.</p><p num="0226"><u style="single">result</u>:: The results of this experiment are summarized in Tables 29 and 30. All vaccinated dogs showed serum conversion after vaccination. A HI titer range of 40-640 and 129 GMTs were observed during the post-vaccination period with equine influenza virus A / horse / ohio / 03, and in the canine influenza isolate A / can / Florida / 242/03. HI titers of 160-320 and geometric mean titers of 211 were observed. 2 out of 6 vaccinated dogs&gt; 103^○Fever of F (&gt; 39.4 ° C) was shown for 1 day, and no other clinical signs were observed in any of the dogs after stimulation.</p><p num="0227"><u style="single">Conclusion</u>:: All vaccinated dogs responded to the CARBIGEN adjuvanted equine influenza virus inactivated. HI titer results using canine influenza virus isolates suggest that the inactivated equine influenza vaccine induced a detectable amount of cross-reactive antibody against canine influenza virus. An inactivated horse vaccine based on HI titer with canine influenza virus isolate, even though the stimulatory virus used in this experiment did not induce any notable clinical disease in beagle dogs. Was concluded to be able to be used to induce cross-reactive antibodies in dogs that could potentially protect dogs from the "canine influenza" disease caused by the H3N8 canine influenza virus.</p><p num="0228"> (Table 29) Serology-HI titers before, after and after vaccination<img id="000030" he="198" wi="99" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* Animals were stimulated with the equine influenza isolate Ohio 03. ** Inactivated equine influenza virus Ohio 03 vaccine adjuvanted with CARBIGEN was used for vaccination. *** Euthanized 7 days after stimulation.</p><p num="0229"> (Table 30) Canine influenza stimulation *-Results of clinical signs, virus isolation, and histopathology<img id="000031" he="64" wi="158" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* Animals were stimulated with the equine influenza isolate Ohio 03. ** Inactivated equine influenza virus Ohio 03 vaccine adjuvanted with CARBIGEN was used for vaccination.</p><p num="0230">Example 19-Equine Influenza Virus Vaccine Efficacy in Dogs The canine influenza virus isolated from the influenza pandemic in Florida was characterized to be closely associated with many H3N8 equine influenza virus isolates. Analysis of DNA and amino acid sequence similarity showed that canine influenza virus was very similar to equine influenza virus A / horse / ohio / 03. The following tests were conducted in dogs to investigate the efficacy of the over-the-counter equine influenza vaccine in dogs.</p><p num="0231"><u style="single">procedure</u>:: Twenty male and female hybrids approximately 16 months old and 20 beagle dogs were used in the study. Dogs were randomly assigned to 6 groups (Table 31) with 6 to 7 dogs in each group. Dogs in groups 1 and 4 were given a commercially available inactivated and adjuvanted equine influenza vaccine (EQUICINE , Intervet Inc., Millsboro, DE) subcutaneously (SQ) at 16 and 17 months of age. Vaccinated by. Dogs in groups 2 and 5 were vaccinated with the modified horse / Kentucky / 91 live influenza vaccine by intranasal administration (single nostril) in 1 ml volume. Vaccination date, after first vaccination (1,, in preparation for HI titer measurement with H3N8 equine and canine influenza viruses using standard protocols (SAM 124, CVB, USDA, Ames, IA) Blood samples were taken 7 and 14 days after (groups 2, 4 and 5) and 2nd vaccination (groups 1 and 4).</p><p num="0232"> A / horse / ohio / 03 (10 per animal) of equine influenza virus strains grown in cell culture in the vaccinated group (72 hours after the last vaccination) and in the control group^7.0~10^8.0 TCID50) was stimulated by intraoral administration in a liquid volume of 1 to 2 ml. The irritating virus was administered to dogs as a mist using a nebulizer (DeVilbiss Ultra-Neb® 99 Ultrasonic Atomizer, Sunrise Medical, USA). Dogs were observed for influenza-related clinical signs for 12 days after stimulation. Nasal and pharyngeal swabs were collected in Earl MEM medium supplemented with antibiotics (neomycin and polymyxin B) daily from 1 to 12 days after stimulation for virus isolation. The presence of the virus in the swab indicates that the animal is shedding the virus in the nasal / oral secretions. In preparation for histopathological evaluation, all dogs were humanely sacrificed 12 days after stimulation and lung tissue was harvested in 10% buffered formalin.</p><p num="0233"> (Table 31) Experimental design<img id="000032" he="42" wi="156" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* Not applicable ** EQUICINE II is marketed as a liquid vaccine by Intervet Inc. EQUICINE II is an inactivated A / Pennsylvania / 63 influenza (ie, "A / Pa / 63") virus and an A / horse / Kentucky / 93 influenza (ie, "A / KY / 93") virus. Is included with Carbopol (ie, HAVLOGEN® (Intervet Inc.)). More specifically, a single dose of EQUICINE II is an inactivated A / Pa / 63-10.^6.0 EID₅₀, Inactivated A / KY / 93-10^6.7 EID₅₀Includes 0.25% carbopol based on volume, and sufficient PBS to create a total volume of 1 ml. *** A / KY / 91 is a lyophilized vaccine redissolved in water. Such redissolution was performed with sufficient vaccine grade water to bring the vaccine dose to a total fluid volume of 1 ml. The vaccine contained the horse / Kentucky / 91 influenza (ie "A / KY / 91") virus. The vaccines are discussed, for example, in US Pat. Nos. 6,436,408, 6,398,774 and 6,177,082, all of which are incorporated herein by reference. When redissolved, the single dose of vaccine is 10 per 1 ml of A / KY / 91^7.2 TCID₅₀, NZ AMINE AS 0.015 g / ml, 0.0025 g / ml gelatin, and 0.04 g / ml D lactose. NZ AMINE AS is a purified source of amino acids and peptides produced by enzymatic hydrolysis of casein. NZ AMINE AS is sold by Kerry Bio-Science (Norwich, NY, USA).</p><p num="0234"><u style="single">result</u>:: All vaccinated dogs showed serum conversion after vaccination, and the HI titer was EQUICINE II compared to 10-40 in dogs in the A / KY / 91 vaccine group using equine influenza virus (H3N8 type). It was 10 to 80 in the vaccine group.</p><p num="0235"> Samples collected 2 weeks after vaccination (after the second vaccination for the EQUICINE II vaccine) were analyzed for HI titer measurement using canine and equine influenza viruses (H3N8). .. The results of HI are shown in Table 32. Clinical signs include fever observed after stimulation (&gt; 103)^○F;&gt; 39.4 ° C), sporadic cough, and mild nasal discharge.</p><p num="0236"> (Table 32) Serology-HI titer 2 weeks after vaccination<img id="000033" he="57" wi="160" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* Not applicable</p><p num="0237"> For beagle dogs, 2 out of 6 dogs in the EQUICINE II vaccine group (1 group), 1 out of 7 dogs in the A / KY / 91 vaccine group (2 groups) and 6 out of 6 dogs in the control group (3 groups). Two dogs showed fever. One of six dogs in the three group (control group) was positive for the virus in the cell culture supernatant of the nasal swab material in a hemagglutination test using 0.25% chicken erythrocytes (CRBC). One in six dogs in the control group (3 groups) and 1 in 7 dogs in the A / KY / 91 vaccine group (2 groups) showed mild nasal secretions during the post-stimulation observation period. In Beagle dogs, there was no statistically significant difference between the control and vaccine groups (P&gt; 0.05).</p><p num="0238"> Among mongrel dogs, 5 out of 7 dogs in the EQUICINE II vaccine group (4 groups), 1 out of 7 dogs in the A / KY / 91 vaccine group (5 groups) and 6 out of 6 dogs in the control group (6 groups). Five dogs showed fever. One dog in each of groups 4 and 6 showed mild nasal secretions, and one dog in groups 5 showed sporadic cough. Two of the seven dogs in the EQUICINE II vaccine group (4 groups) and 3 of 6 dogs in the control group (6 groups) were positive for influenza virus in nasal swabs by HA assay. None of the dogs in the A / KY / 91 group (5 groups) were positive for influenza virus in the nasal swab material.</p><p num="0239"><u style="single">Conclusion</u>:: Serology has shown that vaccination of dogs with a commercially available equine influenza vaccine stimulates a moderate influenza antibody response. There may be slight breed differences in the development of influenza-related clinical signs in dogs after stimulation with H3N8 influenza virus. The attenuated live equine influenza vaccine (A / KY / 91) showed significant (P &lt;0.05) protection from the development of clinical disease in terms of rectal temperature in mongrel dogs. Similarly, live attenuated virus vaccines prevented the elimination of influenza virus in nasal secretions.</p><p num="0240">Example 20-Development of a canine influenza stimulation model Considering the report that the disease was not successfully induced in dogs for the study, the possibility of using the canine influenza virus H3N8 to develop a model of canine influenza stimulation in dogs was examined in the following studies. I examined it in.</p><p num="0241"><u style="single">procedure</u>:: Ten male and female mongrel dogs were obtained from a commercial vendor and kept in cages at the BSL-2 facility. Dogs were randomly assigned to 2 groups of 5 dogs in each group. As shown in Table 33, one group was subjected to intratracheal / intranasal stimulation and the other group was served.</p><p num="0242"> (Table 33) Experimental design<img id="000034" he="17" wi="157" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0243"> The dog was stimulated at about 12 weeks of age. The canine influenza virus (A / dog / Florida / 242/03) virus propagated in hatched chicken eggs was used as a stimulating virus. About 10 in total for each dog^7.2 The TCID50 virus was administered in either 2 ml (intraoral) or 4 ml (intratracheal / intranasal) volumes.</p><p num="0244"> For intratracheal / intranasal stimulation, use a delivery tube consisting of a cuffed tracheal tube (size 4.5 / 5.0, Sheridan, USA) and a liquid supply tube (size 5 Fr, 1.7 mm; 16 inches (41 cm) long, Kendall, USA). First, 3 ml of stimulating virus was administered to the trachea, then 5 ml of PBS was administered to the trachea, and then 1 ml of stimulating virus and 3 ml of air were sequentially administered to the nostrils using a syringe.</p><p num="0245"> For intranasal stimulation, a nebulizer (Nebulair , DVM Pharmaceuticals, Inc., Miami, FL) was used to administer the stimulating virus as a mist in a liquid volume of about 2 ml. Dogs were observed for influenza-related clinical signs for 14 days after stimulation. In preparation for histopathological examination, dogs were euthanized 14 days after stimulation and samples of tissue (lung and trachea) were collected in 10% buffered formalin.</p><p num="0246"><u style="single">result</u>:: All dogs in groups 1 and 2 developed clinical signs of canine influenza within 24-48 hours. Each dog showed two or more of the following clinical signs: fever (&gt; 103.0)^○F;&gt; 39.4 ° C), cough, serous or mucous nasal secretions, serous or mucous nasal discharge, vomiting, diarrhea, depression, weight loss, dry vomiting, hemoptysis and audible la sound. Pulmonary tissue in 5 of 5 dogs in 1 group and 4 of 5 dogs in 2 groups showed histopathological lesions containing one or more of the following: diffuse purulent bronchopneumonia, intracavitary Bronchopneumonia / bronchopneumonia with marked mononuclear cell aggregation in neutrophil exudates plugs and mucosal and peribronchiolitis tissues, mixed exudates in alveoli with numerous foamy macrophages, lymphogenic and traits Cellular and granulocytic cell infiltration, as well as thickening of the alveolar septum with proliferation of type II pulmonary cells that are compatible with influenza virus infection or disease-specific to influenza virus infection. Tracheal tissue samples were normal.</p><p num="0247"><u style="single">Conclusion</u>:: H3N8 canine influenza isolates, such as those used in this study, can be used to induce canine influenza disease in dogs using one of the methods described or similar in this study.</p><p num="0248">Example 21-Development of a canine influenza stimulation model The possibility of using the canine influenza virus H3N8 to develop a canine influenza stimulation model in dogs was further investigated in the following studies.</p><p num="0249"><u style="single">procedure</u>:: Fifteen 17-18 week old mongrel dogs and five 15 week old beagle dogs were obtained from a marketer and housed in cages at a BSL-2 facility. Mongrel dogs were randomly assigned to 3 groups (1 to 3 groups) with 5 dogs in each group. All beagle dogs were divided into one group (4 groups) as shown in Table 34.</p><p num="0250"> (Table 34) Experimental design<img id="000035" he="27" wi="159" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0251"> Intraoral administration of dogs with the pathogenic canine influenza virus A / Canine / Florida / 242/2003 (isolated from the lungs of Greyhound dogs with canine influenza disease) (provided by Dr. Cynda Crawford of the University of Florida) Stimulated by. The irritating virus was administered as a mist in a liquid volume of about 2 ml using a nebulizer (Nebulair ). Dogs were observed for influenza-related clinical signs for 14 days after stimulation.</p><p num="0252"><u style="single">result</u>:: Eighty percent of dogs in groups 1 and 4 (4 of 5) and 100% of dogs in groups 2 and 3 developed clinical signs of canine influenza within 48 hours. Each dog showed one or more of the following clinical signs: fever (&gt; 103.0)^○F;&gt; 39.4 ° C), cough, serous or mucous nasal secretions, serous or mucous nasal discharge, vomiting, diarrhea, depression, weight loss, dry vomiting and rales. The clinical signs observed in beagle dogs were generally milder and shorter than in mongrel dogs.</p><p num="0253"><u style="single">Conclusion</u>:: H3N8 canine influenza isolates, such as those used in this study, were 10^4.8~10^6.8 Within the stimulating dose range of TCID50, it can be used to induce canine influenza-like or Kennel cough-like disease in dogs using the methods described in this study or similar methods. There were some differences in the clinical signs seen in mongrel and beagle dogs. In general, beagle dogs tend to show milder influenza-related clinical signs than mongrel dogs.</p><p num="0254">Example 22-Canine Influenza Vaccine Efficacy Test The following studies were conducted to investigate the efficacy of the H3N8 equine influenza vaccine against canine influenza virus in dogs.</p><p num="0255"><u style="single">procedure</u>:: 17 14-week-old mongrel dogs and 10 8-week-old beagle dogs were obtained from a commercial vendor. Dogs were randomly assigned to 5 study groups and bred in the laboratory as shown in Table 35.</p><p num="0256"> (Table 35) Experimental design<img id="000036" he="42" wi="159" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0257"> The vaccine used in this study was an inactivated equine influenza virus (A / horse / KY / 02) vaccine adjuvanted with HAVLOGEN®. To prepare this vaccine, the virus was inactivated with binary ethyleneimine (BEI) using standard methods. Each vaccine dose is HAVLOGEN® (10% v / v), inactivated virus 6144 HA units, 10% thimerosal 0.1% (v / v), phenol red 0.1% (v / v), pH 6.8 ~ It contained sufficient NaOH to adjust to 7.2 and sufficient PBS to bring the total dose to 1 ml.</p><p num="0258"> Dogs in groups 1 and 4 were vaccinated with two doses of the vaccine. The second dose (ie, booster) was administered 4 weeks after the first dose. Two groups of dogs were vaccinated with a single dose at 18 weeks of age. HI power with standard protocols (eg, SAM 124, CVB, USDA, Ames, LA) using H3N8 canine influenza isolates 0 days (before vaccination), 7 and 14 days after the first and second vaccinations. Blood samples were taken to assess the valence. Approximately 5 days before stimulation, dogs were transferred to a BSL-2 facility and reared in individual cages.</p><p num="0259"> All dogs in the vaccinated group and the corresponding control group of age were subjected to pathogenic influenza virus (A / per animal) 2 weeks after the second vaccination in groups 1 and 4 and 2 weeks after the first vaccination in groups 2. Dog / Florida / 242/2003 10^7.7 It was stimulated by intraoral administration of TCID50). The stimulating virus was administered as a mist in 2 ml per animal using a nebulizer (Nebulair ). Dogs were observed for influenza-related clinical signs for 17 days after stimulation. Post-stimulation-1 day (ie, the day before stimulation) to 17 days, nasal and oropharyngeal swabs were collected in test tubes containing 2 ml of virus transport medium for virus isolation. In preparation for histopathology, all dogs were euthanized 17 days after stimulation and lung and tracheal samples were taken in 10% buffered formalin. Blood samples were taken 7 and 14 days after stimulation to measure HI titers. Table 36 shows the clinical sign score assignments used in post-stimulation observations.</p><p num="0260"><u style="single">result</u>:: All dogs in the double vaccination group (Groups 1 and 4) expressed an HI antibody titer response to the canine influenza virus isolate (Table 37). After stimulation, the titer 14 days after stimulation increased approximately 4-fold in all groups, indirectly indicating that all dogs were exposed to the stimulating virus. All dogs developed one or more of the following signs of canine flu: fever (&gt; 103.0)^○F;&gt; 39.4 ° C), cough, serous or mucous nasal secretions, serous or mucous nasal discharge, vomiting, diarrhea, depression, weight loss, and dyspnea. The vaccinated group showed milder clinical signs compared to the corresponding control group of age (Table 38). Significant reduction in clinical signs was seen due to double dose vaccination in both 8-week-old (P = 0.040) and 14-week-old (P = 0.003) dogs (groups 4 and 1, respectively). Was done. In this experiment, single dose vaccination did not show a significant reduction in clinical signs (P = 0.294) (Group 2).</p><p num="0261"> The virus isolation results are shown in Table 39. In 14-week-old dogs, 2 of 7 (29%) in the 2-dose vaccine group (1 group), 3 of 5 (60%) in the 1-dose vaccine group (2 groups), and the control group. Influenza virus was isolated from swab samples collected from 5 of 5 (100%) dogs in (Group 3). In 8-week-old dogs, the virus was isolated from 1 in 5 dogs (20%) in the double-dose vaccine group (4 groups) and 4 out of 5 dogs (80%) in the control group (5 groups). It was. Significant reduction in the number of dogs positive for canine influenza virus in swab samples due to double dose vaccination (groups 1 and 4) compared to unvaccinated controls (groups 3 and 5) (P = 0.003) was shown. There was a decrease in the number of canine influenza virus-positive dogs in swab samples between the single-dose vaccine group (2 groups) and the control group (3 groups) (60% vs. 100%), but the difference was statistically significant. It was not significant (P = 0.222).</p><p num="0262"> Histopathological evaluation of lung and tracheal tissue samples was performed on the lesions to identify lesions compatible or disease-specific with canine influenza disease. This includes, for example, examining the presence of one or more of the following: regions with purulent bronchiolitis, peribronchiolitis / bronchioles with mononuclear cell aggregation (lymphocytes, plasma cells): Peripheritis; presence of plugs of granulocyte cell debris in the cavity; hyperformation of respiratory epithelium; mixed exudates in alveoli with large amounts of granulocyte cells and cell debris; Aggregation; as well as thickening of the alveolar septum with proliferation of type II lymphocytes.</p><p num="0263"> Table 40 summarizes the extent of lesions in this experiment in dogs. In 14-week-old dogs, lung lesions were smaller and smaller in 5 of 7 dogs in the 2-dose vaccination group (2 groups) and 4 of 5 dogs in the 1-dose vaccination group (1 group). The degree was light. All dogs in the control group (3 groups) showed severe and large lesions, suggesting no protective effect. In 14-week-old dogs, there was no difference in tracheal lesions due to single or double dose vaccination. In 8-week-old dogs, there was no difference in lung lesions between the double-dose and control dogs. None of the dogs showed any tracheal lesions.</p><p num="0264"><u style="single">Conclusion</u>:: The results of this test demonstrate the following: (1) Inactivated H3N8 equine influenza virus can induce canine influenza virus cross-reactive HI antibody response in vaccinated dogs, (2) Use of H3N8 equine influenza virus vaccine can induce canine influenza virus disease in dogs And (3) the use of the H3N8 equine influenza virus vaccine can suppress viral excretion in the nasal and / or oral secretions.</p><p num="0265"> (Table 36) Clinical sign and scoring system<img id="000037" he="107" wi="128" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000038" he="153" wi="128" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0266"> (Table 37) Serology-Hemagglutination inhibitory titer<img id="000039" he="229" wi="147" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* 1st vaccination-Group 1 and Group 4 ** Second vaccination-1 and 4 groups; 1st vaccination-2 groups *** Stimulation day</p><p num="0267"> (Table 38) Analysis of total canine influenza virus disease clinical scores<img id="000040" he="227" wi="81" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* Analyzed using the SAS® version 8.2 NPARIWAY method (vaccine groups were compared using the Wilcoxon rank sum test).</p><p num="0268"> (Table 39) Viral shedding<img id="000041" he="233" wi="145" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0269"> (Table 40) Histopathological evaluation of tissue samples<img id="000042" he="154" wi="152" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />"+" Severe lesions consistent with influenza infection or disease-specific "+/-" Mild lesion (unclear) "-" Normal</p><p num="0270">Example 23-Canine Influenza Vaccine Efficacy Test The following studies were conducted to investigate the efficacy of the polyvalent H3N8 equine influenza vaccine against canine influenza virus in dogs.</p><p num="0271"><u style="single">procedure</u>:: Seventeen 15-week-old beagle dogs were obtained from a commercial vendor. Dogs were randomly assigned to 3 groups and bred in a research facility as shown in Table 41.</p><p num="0272"> (Table 41) Experimental design<img id="000043" he="32" wi="135" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0273"> The vaccine used in this study was the HAVLOGEN® adjuvant inactivated equine influenza (A / horse / KY / 02, A / horse / KY / 93 and A / horse / NM / 2/93) vaccines. there were. To prepare this vaccine, the virus was inactivated with binary ethyleneimine (BEI) using standard methods. Each vaccine dose is HAVLOGEN® (10% v / v), each inactivated virus 2048 HA units, 10% thimerosal 0.1% (v / v), phenol red 0.1% (v / v), pH 6.8 It contained sufficient NaOH to adjust to ~ 7.2 and sufficient PBS to bring the total dose to 1 ml.</p><p num="0274"> One group of dogs was vaccinated with two doses of the vaccine. The second (ie, booster) dose was administered 4 weeks after the first dose. Two groups of dogs were vaccinated with a single dose of vaccine at 19 weeks of age. Blood samples were taken 0 days (before vaccination), 7 and 14 days after the first and second vaccinations to assess HI titers by standard protocol using H3N8 canine influenza isolates. Dogs were transferred to a BSL-2 facility 7 days before stimulation and reared in individual cages.</p><p num="0275"> All dogs in the vaccinated group and the corresponding control group of age were vaccinated with the pathogenic influenza virus (A / dog / per animal) 2 weeks after the second vaccination in group 1 and 2 weeks after the first vaccination in group 2. Florida / 242/2003 10^7.3 It was stimulated by intraoral administration of TCID50). The stimulating virus was administered as a mist in 2 ml per animal using a nebulizer (Nebulair ). Dogs were observed for influenza-related clinical signs for 14 days after stimulation. In preparation for histopathology, all dogs were euthanized 14 days after stimulation and lung and tracheal samples were taken in 10% buffered formalin. Blood samples were taken 7 and 14 days after stimulation to measure HI titers. Table 42 shows the clinical sign score assignments used in post-stimulation observations.</p><p num="0276"><u style="single">result</u>:: All vaccinated dogs expressed an HI antibody titer response to canine influenza virus isolates (Table 43). After stimulation, the HI titer 14 days after stimulation increased approximately 4-fold in all groups compared to the HI titer before stimulation, indirectly indicating that all dogs were exposed to the stimulating virus. .. All dogs developed signs of canine influenza disease, and each dog showed one or more of the following clinical signs: fever (&gt; 103.0)^○F;&gt; 39.4 ° C), cough, serous or mucous nasal secretions, serous or mucous nasal discharge, vomiting, diarrhea, depression, weight loss and dyspnea. The vaccinated group showed milder clinical signs compared to the corresponding control group of age (Table 44). There was a significant (P = 0.028) reduction in clinical signs due to the double dose vaccination in dogs (Group 1). Single dose vaccination did not show a significant reduction in clinical signs (P = 0.068) (Group 2).</p><p num="0277"> Similar to Example 22, a histopathological evaluation of lung and tracheal tissue samples was performed on the lesions to identify lesions compatible or disease-specific with canine influenza disease. Table 45 summarizes the extent of lesions in this experiment in dogs. In 15-week-old dogs, vaccination of single or double dose dogs prevented lung lesions in all dogs. Four of the five (80%) dogs in the control group had severe suppurative bronchopneumonia consistent with influenza disease. Mild bronchitis suggestive of bronchitis that can be attributed to influenza disease in 1 of 7 dogs in the double dose vaccine group (1 group) and 1 in 5 dogs in the control group (3 groups) Had.</p><p num="0278"><u style="single">Conclusion</u>:: The results of this study show that 1) inactivated H3N8 equine influenza virus can elicit a canine influenza virus cross-reactive HI antibody response in vaccinated dogs, and 2) use of the H3N8 equine influenza virus vaccine in dogs. Demonstrate that the degree of canine influenza virus disease can be reduced.</p><p num="0279"> (Table 42) Clinical sign and scoring system<img id="000044" he="249" wi="101" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0280"> (Table 43) Serology-Hemagglutination inhibitory titer<img id="000045" he="209" wi="109" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* 1st vaccination-1 group ** Second vaccination-1 group; 1st vaccination-2 groups *** Stimulation day</p><p num="0281"> (Table 44) Analysis of total canine influenza disease clinical scores<img id="000046" he="208" wi="57" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* Analyzed using the SAS® version 8.2 NPARIWAY method (vaccine groups were compared using the Wilcoxon rank sum test).</p><p num="0282"> (Table 45) Histopathological evaluation of tissue samples<img id="000047" he="104" wi="130" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />"+" Severe lesions consistent with influenza infection or disease-specific "+/-" Mild lesion (unclear) "-" Normal</p><p num="0283">Example 24-Canine Influenza Vaccine Efficacy Test The following studies were conducted to investigate (1) the efficacy of the monovalent vs. polyvalent H3N8 equine influenza vaccine against canine influenza virus in dogs, and (2) the effect of the administration route on vaccine efficacy.</p><p num="0284"><u style="single">procedure</u>:: Thirty 10-week-old beagle dogs were obtained from a commercial vendor. Dogs were randomly assigned to 6 groups as shown in Table 46 and bred in the research facility.</p><p num="0285"> (Table 46) Experimental design<img id="000048" he="47" wi="154" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0286"> Three vaccines (VAX-1, VAX-2 and VAX-3) were used. VAX-1 is a monovalent vaccine of inactivated equine influenza virus (A / horse / KY / 02) adjuvanted with HAVLOGEN®, and each dose is HAVLOGEN® (10% v / v), Inactivated virus 6144 HA units, 10% thimerosal 0.1% (v / v), phenol red 0.1% (v / v), sufficient NaOH to adjust the pH to 6.8-7.2, and a total dose of 1 ml Included enough PBS to do. VAX-2 is a monovalent vaccine of inactivated equine influenza virus (A / horse / KY / 02) adjuvanted with HAVLOGEN®, and each dose of the vaccine is HAVLOGEN® (10% v / v). ), Inactivated virus 4096 HA units, 10% thimerosal 0.1% (v / v), phenol red It contained 0.1% (v / v), sufficient NaOH to adjust the pH to 6.8-7.2, and sufficient PBS to bring the total dose to 1 ml. VAX-3 is a multivalent vaccine of inactivated equine influenza (A / horse / KY / 02, A / horse / KY / 93 and A / horse / NM / 2/93) adjuvanted with HAVLOGEN®. , HAVLOGEN® (10% v / v), inactivated virus per strain 2048 HA units, 10% thimerosal 0.1% (v / v), phenol red 0.1% (v / v), pH 6.8-7.2 It contained sufficient NaOH to regulate and sufficient PBS to bring the total dose to 1 ml. All influenza viruses used for vaccine formulation were inactivated with binary ethyleneimine (BEI) using standard methods.</p><p num="0287"> The vaccines and routes of administration for each group are shown in Table 46. All dogs in the vaccinated group were vaccinated via the intranasal (IN) or subcutaneous (SQ) route, and each dog received two doses. The second (ie, booster) dose was administered 4 weeks after the first dose. Blood samples were taken 0 days (before vaccination), 7 and 14 days after the first and second vaccinations to assess HI titers by standard protocol using H3N8 canine influenza isolates. Dogs were transferred to a BSL-2 facility 7 days before stimulation and reared in individual cages.</p><p num="0288"> All dogs in the vaccinated group and the corresponding age control group were vaccinated with the pathogenic canine influenza virus (A / dog / Florida / 242/2003 10 per dog 2 weeks after the second vaccination.^7.4 It was stimulated by intraoral administration of TCID50). The stimulating virus was administered as a mist in 2 ml per day using a nebulizer (Nebulair ). Dogs were observed for influenza-related clinical signs for 14 days after stimulation. Blood samples were taken 7 and 14 days after stimulation to measure HI titers. In preparation for histopathology, all dogs were euthanized 14 days after stimulation and lung and tracheal samples were taken in 10% buffered formalin. Table 47 shows the clinical sign score assignments used in post-stimulation observations.</p><p num="0289"><u style="single">result</u>:: All dogs vaccinated via the SQ pathway expressed an HI antibody titer response to canine influenza virus isolates, regardless of vaccine type (Table 48). Regardless of the type of vaccine, none of the dogs in the IN vaccination group (ie, groups 1, 3 and 5) developed an HI antibody titer response to the canine influenza virus isolate during the post-vaccination period. It was. However, antibody titers increased 4-fold in all dogs by 14 days after stimulation, indicating indirectly that all dogs were exposed to the stimulating virus (Table 47).</p><p num="0290"> All dogs developed one or more of the following clinical signs of canine flu: fever (&gt; 103.0)^○F;&gt; 39.4 ° C), cough, serous or mucous nasal secretions, serous or mucous nasal discharge, vomiting, diarrhea, depression, weight loss, and dyspnea. The vaccinated group showed milder clinical signs compared to the corresponding control group of age (Table 49). Dogs (group 4) vaccinated with VAX-3 by the SQ route showed marked reduction of clinical signs. In this experiment, IN administration of VAX-1, VAX-2 or VAX-3 showed no significant reduction in clinical signs of canine influenza virus.</p><p num="0291"> Similar to Examples 22 and 23, histopathological evaluation of lung and tracheal tissue samples was performed on the lesions to identify lesions compatible or disease-specific with canine influenza disease. Table 50 summarizes the extent of lesions in this experiment in dogs. Five of the five dogs in the control group (6 groups) showed lung lesions consistent with influenza infection. Any influenza-related dogs in 2 of 5 dogs (2 groups) vaccinated with VAX-2 via the SC route and 3 of 5 dogs (4 groups) vaccinated with VAX-3 via the SC route There were no lung lesions. All dogs that received the vaccine by the intranasal route showed severe lung lesions consistent with influenza infection, regardless of vaccine type. The tracheal lesions observed in this study were extremely mild.</p><p num="0292"><u style="single">Conclusion</u>:: The results of this test demonstrate the following: (1) Inactivated H3N8 equine influenza virus can induce canine influenza virus cross-reactive HI antibody response in dogs vaccinated via the SQ pathway, (2) monovalent (VAX-1 and VAX-2) ) Or intranasal administration of the polyvalent vaccine (VAX-3) was not effective in dogs, and (3) subcutaneous administration of the polyvalent vaccine (VAX-3) significantly marked the extent of canine influenza virus disease in dogs. It was reduced to (P = 0.016).</p><p num="0293"> (Table 47) Clinical sign and scoring system<img id="000049" he="234" wi="134" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000050" he="41" wi="133" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0294"> (Table 48) Serology-Hemagglutination inhibitory titer<img id="000051" he="230" wi="140" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000052" he="229" wi="58" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* First vaccination ** Second vaccination *** Stimulation day</p><p num="0295"> (Table 49) Analysis of total canine influenza disease clinical scores<img id="000053" he="227" wi="72" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* Analyzed using the SAS® version 8.2 NPARIWAY method (vaccine groups were compared using the Wilcoxon rank sum test).</p><p num="0296"> (Table 50) Histopathological evaluation of tissue samples<img id="000054" he="168" wi="151" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />"+" Severe lesions consistent with influenza infection or disease-specific "+/-" Mild lesion (unclear) "-" Normal</p><p num="0297">Example 25-Canine Influenza Vaccine Efficacy Test Canine influenza disease is caused by the H3N8 influenza virus (CIV). CIV is very closely associated with the equine H3N8 virus (Crawford et al., 2005) and infects all exposed dogs. Approximately 80% of exposed dogs developed clinical signs. The following studies examined the efficacy of inactivated H3N8 equine influenza virus vaccine and canine influenza virus vaccine.</p><p num="0298"><u style="single">procedure</u>:: Thirty-five beagle dogs and five mongrel dogs were used in this study. Beagle dogs were randomly assigned to 3 groups (Table 51). All mongrel dogs were assigned to the control group (3 groups). All dogs were bred on a standard growth diet and were given free drinking water.</p><p num="0299"> (Table 51) Experimental design<img id="000055" he="32" wi="139" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0300"> Dogs in groups 1 and 2 were vaccinated with either VAX-1 or VAX-2 (Table 51). VAX-1 was an inactivated equine influenza virus (A / horse / KY / 02) vaccine adjuvanted with HAVLOGEN®. For vaccine preparation, the vaccine virus was inactivated with binary ethyleneimine (BEI) using standard methods. Each dose of vaccine is HAVLOGEN® (10% v / v), inactivated virus 6144 HA units, 10% thimerosal 0.1% (v / v), phenol red 0.1% (v / v), total dose. It contained sufficient PBS to bring the volume to 1 ml and sufficient NaOH to adjust the pH to 6.8-7.2.</p><p num="0301"> VAX-2 was an inactivated canine influenza antigen vaccine (A / canine / Fl / 43/2004) adjuvanted with CARBIGEN . A / dog / Fl / 43/2004 were inactivated by binary ethyleneimine (BEI) using standard methods. For each dose of vaccine, 5% CARBIGEN based on mass, about 1280 HA units of inactivated virus, PBS sufficient for a total dose of 1 ml, and pH adjusted to 7.2-7.4. It contained enough NaOH to do so. First and second vaccination days, first and second vaccines from all dogs to measure HI titers using the H3N8 equine influenza virus standard protocol (SAM 124, CVB, USDA, Ames, IA) Serum samples were taken 7 and 14 days after inoculation and before stimulation. Dogs were transferred to the ABSL-2 facility 7 days before stimulation and reared in individual cages.</p><p num="0302"> All dogs in the vaccinated group and the corresponding age control group were vaccinated with the pathogenic canine influenza virus (A / dog / Florida / 242/2003 10 per dog 2 weeks after the second vaccination.^7.2 It was stimulated by intraoral administration of TCID50). The irritating virus was administered as a mist (2 ml / dog) using a nebulizer (Nebulair ). Dogs were observed for influenza-related clinical signs for 14 days after stimulation. After stimulation-1 day (ie, the day before stimulation) to 14 days, nasal and oropharyngeal swabs were collected daily in tubes containing 2 ml of virus transport medium for virus isolation. Blood samples were taken 7 and 14 days after stimulation to measure HI titers. Table 52 shows the clinical sign score assignments used in post-stimulation observations.</p><p num="0303"><u style="single">result</u>:: All vaccinated dogs (Groups 1 and 2) expressed an HI antibody titer response to canine influenza virus isolates (Table 53). All dogs developed one or more of the following signs of canine flu: fever (&gt; 103.0)^○F;&gt; 39.4 ° C), cough, serous or mucous nasal secretions, serous or mucous nasal discharge, vomiting, diarrhea, depression, and loss of appetite. The vaccinated group showed milder clinical signs compared to the corresponding control group of age (Table 54). Significant (P &lt;0.001) reduction of clinical signs was seen in dogs vaccinated with either VAX-1 (Group 1) or VAX-2 (Group 2).</p><p num="0304"> The virus isolation results are shown in Tables 55 and 56. After stimulation with pathogenic canine influenza virus, 5 out of 15 heads (33%) in group 1 (VAX-1), 0 out of 5 heads (0%) in group 2 (VAX-2), and control group (3 groups) The canine influenza virus was isolated from 17 (85%) of the 20 dogs (). Both the inactivated equine influenza vaccine (VAX-1) and canine influenza virus (VAX-2) vaccinated groups had significant viral shedding in nasal and / or oral secretions (P =) compared to the control group. It showed a decrease of 0.004) (Table 55).</p><p num="0305"><u style="single">Conclusion</u>:: The results of this test demonstrate the following: (1) Inactivated H3N8 horse influenza virus and canine influenza virus vaccines can induce canine influenza virus-reactive HI antibody response in vaccinated dogs, (2) H3N8 horse influenza virus or canine influenza virus vaccines Use can reduce the extent of canine influenza virus disease in dogs, and (3) use of H3N8 horse influenza virus or canine influenza virus vaccine can suppress viral excretion in nasal and / or oral secretions. ..</p><p num="0306"> (Table 52) Clinical sign and scoring system<img id="000056" he="250" wi="109" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0307"> (Table 53) Serology-Hemagglutination inhibitory titer<img id="000057" he="192" wi="145" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000058" he="192" wi="94" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* First vaccination ** Second vaccination *** Stimulation day</p><p num="0308"> (Table 54) Analysis of total canine influenza disease clinical scores<img id="000059" he="41" wi="136" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* Analyzed using the SAS (registered trademark) version 9.1 NPARIWAY method (vaccine groups were compared using the GLM method).</p><p num="0309"> (Table 55) Viral shedding after stimulation<img id="000060" he="46" wi="135" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />* Analyzed using SAS® (version 9.1) FREQ method and P-values associated with Fisher's exact test.</p><p num="0310"> (Table 56) Serology-Hemagglutination inhibitory titer<img id="000061" he="207" wi="141" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000062" he="207" wi="89" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" />No virus was isolated from the N-mouth or nasal swab. P-The virus was isolated from the nose or mouth or nasal and oral swabs.</p><p num="0311"> (Table 57) Similarities in hemagglutinin, neuraminidase and nucleoprotein gene amino acid sequences between influenza viruses<img id="000063" he="163" wi="158" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0312"> The terms "comprise," "comprises," and "comprising" in this patent (including the claims) should be construed in a comprehensive manner rather than exclusively. This interpretation is intended to be the same as the interpretation given under 35 US patent law.</p><p num="0313"> The above detailed description of a preferred embodiment is intended to inform those skilled in the art of the invention, its principles and its practical application, and thus those skilled in the art may be optimal for their particular use requirements. As such, the invention can be adopted and applied in many forms. Therefore, the present invention is not limited to the above-described embodiment, and can be modified in various ways.</p><p num="0314"> It is understood that the examples and embodiments described herein are for illustrative purposes only, and in that regard various modifications or modifications should be suggested to those skilled in the art and included within the spirit and scope of the present application. There must be.</p><p num="0315">reference U.S. Pat. No. 5,106,739 U.S. Pat. No. 5,034,322 U.S. Pat. No. 6,455,760 U.S. Pat. No. 6,696,623 U.S. Pat. No. 4,683,202 U.S. Pat. No. 4,683,195 U.S. Pat. No. 4,800,159 U.S. Pat. No. 4,965,188 U.S. Pat. No. 5,994,056 U.S. Pat. No. 6,814,934 U.S. Pat. No. 6,436,408 U.S. Pat. No. 6,398,774 U.S. Pat. No. 6,177,082 Published U.S. Patent Application No. 20040078841 Published U.S. Patent Application No. 20040067506 Published U.S. Patent Application No. 20040019934 Published U.S. Patent Application No. 20030177536 Published U.S. Patent Application No. 20030084486 Published U.S. Patent Application No. 20040123349<img id="000064" he="34" wi="158" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000065" he="233" wi="159" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000066" he="229" wi="159" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000067" he="221" wi="159" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000068" he="243" wi="159" file="JP5974397B2_D0001.tif" img-format="tif" img-content="drawing" /></p>

0316配列の簡単な説明 配列番号:1は、本発明に従って使用することができるPB2タンパク質をコードするイヌインフルエンザウイルス(フロリダ/43/04)のヌクレオチド配列である。 配列番号:2は、配列番号:1によってコードされるアミノ酸配列である。 配列番号:3は、本発明に従って使用することができるPB1タンパク質をコードするイヌインフルエンザウイルス(フロリダ/43/04)のヌクレオチド配列である。 配列番号:4は、配列番号:3によってコードされるアミノ酸配列である。 配列番号:5は、本発明に従って使用することができるPAタンパク質をコードするイヌインフルエンザウイルス(フロリダ/43/04)のヌクレオチド配列である。 配列番号:6は、配列番号:5によってコードされるアミノ酸配列である。 配列番号:7は、本発明に従って使用することができるNSタンパク質をコードするイヌインフルエンザウイルス(フロリダ/43/04)のヌクレオチド配列である。 配列番号:8は、配列番号:7によってコードされるアミノ酸配列である。 配列番号:9は、本発明に従って使用することができるNPタンパク質をコードするイヌインフルエンザウイルス(フロリダ/43/04)のヌクレオチド配列である。 配列番号:10は、配列番号:9によってコードされるアミノ酸配列である。 配列番号:11は、本発明に従って使用することができるNAタンパク質をコードするイヌインフルエンザウイルス(フロリダ/43/04)のヌクレオチド配列である。 配列番号:12は、配列番号:11によってコードされるアミノ酸配列である。 配列番号:13は、本発明に従って使用することができるMAタンパク質をコードするイヌインフルエンザウイルス(フロリダ/43/04)のヌクレオチド配列である。 配列番号:14は、配列番号:13によってコードされるアミノ酸配列である。 配列番号:15は、本発明に従って使用することができるHAタンパク質をコードするイヌインフルエンザウイルス(フロリダ/43/04)のヌクレオチド配列である。 配列番号:16は、配列番号:15によってコードされるアミノ酸配列である。 配列番号:17は、本発明に従って使用することができるPB2タンパク質をコードするイヌインフルエンザウイルス(FL/242/03)のヌクレオチド配列である。 配列番号:18は、配列番号:17によってコードされるアミノ酸配列である。 配列番号:19は、本発明に従って使用することができるPB1タンパク質をコードするイヌインフルエンザウイルス(FL/242/03)のヌクレオチド配列である。 配列番号:20は、配列番号:19によってコードされるアミノ酸配列である。 配列番号:21は、本発明に従って使用することができるPAタンパク質をコードするイヌインフルエンザウイルス(FL/242/03)のヌクレオチド配列である。 配列番号:22は、配列番号:21によってコードされるアミノ酸配列である。 配列番号:23は、本発明に従って使用することができるNSタンパク質をコードするイヌインフルエンザウイルス(FL/242/03)のヌクレオチド配列である。 配列番号:24は、配列番号:23によってコードされるアミノ酸配列である。 配列番号:25は、本発明に従って使用することができるNPタンパク質をコードするイヌインフルエンザウイルス(FL/242/03)のヌクレオチド配列である。 配列番号:26は、配列番号:25によってコードされるアミノ酸配列である。 配列番号:27は、本発明に従って使用することができるNAタンパク質をコードするイヌインフルエンザウイルス(FL/242/03)のヌクレオチド配列である。 配列番号:28は、配列番号:27によってコードされるアミノ酸配列である。 配列番号:29は、本発明に従って使用することができるMAタンパク質をコードするイヌインフルエンザウイルス(FL/242/03)のヌクレオチド配列である。 配列番号:30は、配列番号:29によってコードされるアミノ酸配列である。 配列番号:31は、本発明に従って使用することができるHAタンパク質をコードするイヌインフルエンザウイルス(FL/242/03)のヌクレオチド配列である。 配列番号:32は、配列番号:31によってコードされるアミノ酸配列である。 配列番号:33は、N末端の16のアミノ酸シグナル配列が除去されている配列番号:16に示すHAタンパク質の成熟型である。 配列番号:34は、N末端の16のアミノ酸シグナル配列が除去されている配列番号:32に示すHAタンパク質の成熟型である。 配列番号:35は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:36は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:37は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:38は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:39は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:41は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:42は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:43は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:44は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:45は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:46は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:47は、本発明に従って使用することができるPB2タンパク質をコードするイヌインフルエンザウイルス(マイアミ/2005)のヌクレオチド配列である。 配列番号:48は、配列番号:47によってコードされるアミノ酸配列である。 配列番号:49は、本発明に従って使用することができるPB1タンパク質をコードするイヌインフルエンザウイルス(マイアミ/2005)のヌクレオチド配列である。 配列番号:50は、配列番号:49によってコードされるアミノ酸配列である。 配列番号:51は、本発明に従って使用することができるPAタンパク質をコードするイヌインフルエンザウイルス(マイアミ/2005)のヌクレオチド配列である。 配列番号:52は、配列番号:51によってコードされるアミノ酸配列である。 配列番号:53は、本発明に従って使用することができるNSタンパク質をコードするイヌインフルエンザウイルス(マイアミ/2005)のヌクレオチド配列である。 配列番号:54は、配列番号:53によってコードされるアミノ酸配列である。 配列番号:55は、本発明に従って使用することができるNPタンパク質をコードするイヌインフルエンザウイルス(マイアミ/2005)のヌクレオチド配列である。 配列番号:56は、配列番号:55によってコードされるアミノ酸配列である。 配列番号:57は、本発明に従って使用することができるNAタンパク質をコードするイヌインフルエンザウイルス(マイアミ/2005)のヌクレオチド配列である。 配列番号:58は、配列番号:57によってコードされるアミノ酸配列である。 配列番号:59は、本発明に従って使用することができるMAタンパク質をコードするイヌインフルエンザウイルス(マイアミ/2005)のヌクレオチド配列である。 配列番号:60は、配列番号:59によってコードされるアミノ酸配列である。 配列番号:61は、本発明に従って使用することができるHAタンパク質をコードするイヌインフルエンザウイルス(マイアミ/2005)のヌクレオチド配列である。 配列番号:62は、配列番号:61によってコードされるアミノ酸配列である。 配列番号:63は、本発明に従って使用することができるPB2タンパク質をコードするイヌインフルエンザウイルス(ジャクソンビル/2005)のヌクレオチド配列である。 配列番号:64は、配列番号:63によってコードされるアミノ酸配列である。 配列番号:65は、本発明に従って使用することができるPB1タンパク質をコードするイヌインフルエンザウイルス(ジャクソンビル/2005)のヌクレオチド配列である。 配列番号:66は、配列番号:65によってコードされるアミノ酸配列である。 配列番号:67は、本発明に従って使用することができるPAタンパク質をコードするイヌインフルエンザウイルス(ジャクソンビル/2005)のヌクレオチド配列である。 配列番号:68は、配列番号:67によってコードされるアミノ酸配列である。 配列番号:69は、本発明に従って使用することができるNSタンパク質をコードするイヌインフルエンザウイルス(ジャクソンビル/2005)のヌクレオチド配列である。 配列番号:70は、配列番号:69によってコードされるアミノ酸配列である。 配列番号:71は、本発明に従って使用することができるNPタンパク質をコードするイヌインフルエンザウイルス(ジャクソンビル/2005)のヌクレオチド配列である。 配列番号:72は、配列番号:71によってコードされるアミノ酸配列である。 配列番号:73は、本発明に従って使用することができるNAタンパク質をコードするイヌインフルエンザウイルス(ジャクソンビル/2005)のヌクレオチド配列である。 配列番号:74は、配列番号:73によってコードされるアミノ酸配列である。 配列番号:75は、本発明に従って使用することができるMAタンパク質をコードするイヌインフルエンザウイルス(ジャクソンビル/2005)のヌクレオチド配列である。 配列番号:76は、配列番号:75によってコードされるアミノ酸配列である。 配列番号:77は、本発明に従って使用することができるHAタンパク質をコードするイヌインフルエンザウイルス(ジャクソンビル/2005)のヌクレオチド配列である。 配列番号:78は、配列番号:77によってコードされるアミノ酸配列である。 配列番号:79は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:80は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:81は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:82は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:83は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:84は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:85は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:86は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:87は、本発明に従って用いることができるオリゴヌクレオチドである。 配列番号:88は、本発明に従って用いることができるオリゴヌクレオチドである。