RU2802222C2 - Influenza virus capable of infecting canines and its use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: canine influenza virus is proposed, containing a polynucleotide encoding a hemagglutinin (HA) protein containing the amino acid sequence under the following SEQ ID NO: 62 and the following SEQ ID NO: 78, or its mature form, in which the N-terminal 16 amino acids are removed from the above, or a sequence that is more than 95% identical to the sequence of the mature form, where the indicated mature HA protein contains Ser at amino acid sequence position 82, Leu at 221, Thr at 327 and Thr at 482. Also a vaccine containing an effective amount of canine influenza virus is provided. Besides, a method of inducing an immune response in animals against canine influenza virus by administering an effective amount of the said virus or vaccine is provided.

EFFECT: inventions allow to provide effective treatment of influenza virus infection in canines.

16 cl, 7 dwg, 57 tbl, 25 ex

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application is a partial continuation of US application 11/409416, filed April 21, 2006; and this application claims the priority of U.S. Applications 60/728449, filed October 19, 3005; 60/754991, filed December 29, 2005; 60/759162, filed January 14, 2006; 60/761451, filed January 23, 2006 and 60/779080, filed March 3, 2006, the description of each of which is incorporated here by reference in its entirety, including a brief summary, detailed descriptions of this invention, examples, claims, abstract, figures, tables, nucleic acid sequences, amino acid sequences and figures.

BACKGROUND OF THE INVENTION

"Kennel cough" or infectious canine tracheobronchitis (ITB) is an acute, contagious respiratory infection in dogs characterized primarily by coughing (Ford et al., 1998). Canine ITB is considered one of the most prevalent infectious diseases in dogs worldwide and outbreaks of this infectious disease can reach epidemic proportions when dogs are kept in high population density settings such as kennels. Most outbreaks are due to direct dog-to-dog contact or aerosolization of respiratory secretions (Ford et al., 2998). Clinical symptoms are due to infection with a single bacterial or viral agent, or a combination of bacterial and viral agents that colonize the epithelium of the upper and lower respiratory tract. Canine parainfluenza virus (CPiV) and Bordetella bronchiseptica bacteria are the most common organisms isolated from affected dogs, but several other viruses, such as canine distemper virus (CDV) and adenovirus-1 and -2 (CAV-1, CAV-2), together with bacteria such as Streptococcus sp., Pasteurella multicoda and Escherichia coli, may influence the clinical course and outcome of the disease (Ford et al, 1998). Although outbreaks occur most effectively and rapidly in high-density populations with high prevalence, complicated respiratory infections and death are rare. Although life-threatening secondary bacterial pneumonia may develop, most cases of ITB are self-limiting and resolve without any treatment (Ford et al., 1998).

In July 1992, a respiratory infection believed to be "kennel cough" became epidemic in several greyhound tracks in New England, Florida, West Virginia, Wisconsin, Kansas, Colorado, Oklahoma, and Arizona. According to veterinary reports, most of the affected dogs had a mild cough that disappeared, but more than a dozen greyhounds developed acute hemorrhagic pneumonia followed by rapid death (Greyhound Daily News, 1999).

In the late 1998s and early 1999s, several outbreaks of "kennel cough" occurred in kennels (kennels) of beagle dogs (greyhounds) throughout the country, which led to the forced closure of tracks (tracks) and the quarantine of all beagle greyhounds in the United States for a few weeks (Greyhound Daily News, 1999). At one track in Florida (Palm Beach Kennel Club), coughing was reported in nearly 40% of the dog population in one day (Personal communication from Dr. William Duggar). Similar to the 1992 outbreak, this cough cleared up on its own in most greyhounds, but 10 dogs in Florida died of a hemorrhagic pneumonia syndrome uncharacteristic of "kennel cough" (Putnam, 1999).

In March-April 2003, another outbreak of "kennel cough" occurred at greyhound tracks in the eastern US. This outbreak is believed to have originated from dog kennels at four tracks in Florida and caused the suspension of dog racing and the quarantine of dogs for nearly three weeks. Approximately 25% of the dogs on the track at West Palm Beach were affected, while almost 50% of the 1,400 dogs at Derby Lane in St. Petersburg developed a cough. Again, most of the dogs recovered, but a few dogs died from this respiratory infection. The estimated economic impact of the Derby Lane respiratory outbreak was $2 million.

There are no published reports documenting the etiology or clinical pathology of the "kennel cough" epidemic in 1992, 1998-1999 or 2003 greyhound kennels. These infections were thought to be due to CPiV and/or B. bronchiseptica, the two most common causes of kennel cough. Anecdotal reports such as websites attributed the lethal hemorrhagic pneumonias reported in some coughing dogs to infections with β-hemolytic subspecies of Streptococcus equi zooepidemicus, and this syndrome was referred to as "canine streptococcal toxic shock."

Transmission of a virus from one host species to another is a critical feature of influenza virus ecology and epidemiology (Webster, 1998). Two main mechanisms of interspecies transmission of influenza virus infection are possible (Webster et al, 1992; Lipatov et al, 2004). One is the direct transfer of an essentially unmodified virus from one species to another. Examples of this mechanism include recent human infections with the H5N1 subtype of the avian influenza virus (Subbarao et al, 1998; Peiris et al, 2004; Guan et al, 2004) and possibly the 1918 pandemic influenza known as Spanish influenza (Reid et al , 2004). The second mechanism is a result of the segmented nature of the influenza virus genome. Co-infection of a host with viruses from different species can lead to genetic recombination of segmented viral genes and the generation of a recombinant with the ability to infect other species. For example, new viruses generated by gene recombination between avian and human influenza viruses led to the human influenza pandemics of 1957 and 1968 (Webster et al, 1992; Lipatov et al, 2004; Kawaoka et al, 1989).

The most direct transmissions of influenza viruses from natural host species to other species are critical events, as prolonged transmission between individuals of these new species cannot occur. Multiple virus-host interactions are required for replication and "horizontal" human-to-human transmission and provide a significant barrier to the persistence of influenza viruses in a new host (Webby et al, 2004). Thus, the establishment of new influenza virus host-specific lineages is rare and occurs only in poultry, pigs, horses, and humans (Webster et al, 1992; Lipatov et al, 2004).

Due to the serious nature of influenza virus infection, there remains a need for methods for diagnosing, preventing, and treating influenza virus infection.

SUMMARY OF THE INVENTION

The present invention relates to an isolated influenza virus that is capable of infecting canines and causing respiratory disease in canines. This invention also relates to compositions and methods for inducing an immune response against influenza virus according to this invention. This invention also relates to compositions and methods for identifying a virus of this invention and diagnosing infection of an animal with a virus of this invention.

One aspect of this invention relates to vaccines and methods for protecting canines from canine virus, kits containing such vaccines, and methods of using such vaccines. This protection includes reducing the risk, delaying the onset, reducing the spread, attenuating, suppressing and/or killing the influenza virus and/or one or more (usually two or more) of its symptoms. It is believed that the vaccines, kits and methods of this invention are generally suitable for use in canines. Canines include wild canines, zoo canines, and domestic canines such as wolves, coyotes, and foxes. Canine also includes dogs, in particular domestic dogs, such as, for example, purebred and/or mongrel companion dogs, show dogs, working dogs, herding dogs, hunting dogs, guard dogs, police dogs, hounds. dogs and/or laboratory dogs.

This invention also relates, in part, to a method of protecting canines from influenza virus infection {ie, preventing, reducing the risk of, or delaying the onset, suppression, attenuation or elimination of influenza virus infection). The method comprises administering a therapeutically effective amount of a vaccine that comprises at least one equine influenza virus antigen, at least one H3 influenza virus antigen, and/or at least one H7 influenza virus antigen.

This invention also relates, in part, to a method for protecting canines from respiratory injury (ie, preventing, reducing the risk or delaying the onset, suppressing, attenuating or eliminating respiratory injury) caused by canine influenza virus. The method comprises administering to the dog a therapeutically effective amount of a vaccine that comprises at least one equine influenza virus antigen, at least one H3 influenza virus antigen, and/or at least one H7 influenza virus antigen.

This invention also relates, in part, to a method for protecting canines from the presence of canine influenza virus in a nasal or oral discharge (i.e., preventing, reducing the risk of or delaying the appearance, suppression, attenuation, or elimination of canine influenza virus in a nasal discharge). or mouth) caused by canine influenza virus. The method comprises administering to the dog a therapeutically effective amount of a vaccine that comprises at least one equine influenza virus antigen, at least one H3 influenza virus antigen, and/or at least one H7 influenza virus antigen.

This invention relates, in part, to 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.

This invention relates, in part, to a kit for protecting canines from influenza virus infection. This kit contains a therapeutically effective amount of a vaccine that contains 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, this set contains at least one of the following:

device for administering a vaccine to dogs,

a pharmaceutically acceptable excipient that facilitates administration of this vaccine to dogs,

a pharmaceutically acceptable excipient that enhances the canine's immune response to this vaccine,

food for consumption by dogs at the same time as this vaccine and/or

treat for canine consumption at the same time as this vaccine.

Additional advantages of the applicants' invention will be apparent to a person skilled in the art upon reading this application.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1A-1B show phylogenetic relationships among hemagglutinin genes. 1A shows the HA gene tree from representative canine, human, avian, porcine, and equine isolates, including A/Budgerigar/Hokkaido/1/77 (H4) as an outgroup. 1B shows a canine influenza virus gene tree with modern and older equine HA genes using A/Duck/Ukraine/63 (H3) as an outgroup. Phylogenetic trees are derived from nucleotide sequences by neighbor joining and bootstrap values >90% are shown. The stem denotes the number of nucleotide changes per unit length of the horizontal branches of the tree.

Fig. 2A-2B show immunohistochemical detection of influenza H3 antigen in the lungs. Lung tissue sections were probed with mouse anti-hemagglutinin H3 monoclonal antibody and binding was detected by immunoperoxidase reaction (brown pellet). Figure 2A shows bronchial epithelium from a spontaneously diseased greyhound. The H3 virus antigen was detected in the cytoplasm of bronchial epithelial cells and in macrophages in the airway lumen and in the alveolar spaces. 2B shows bronchial epithelium from a dog 5 days after A/canine/Florida/43/2004 (H3N8) inoculation. The H3 virus antigen was detected in the cytoplasm of bronchial epithelial cells. Scale rod, 66 µm.

3 shows characteristic histological changes in the bronchi of greyhounds that died of hemorrhagic pneumonia associated with influenza virus infection. The tissues were stained with H&E. Upper panel: Healthy bronchus with ciliated epithelial cells, mucosal cells, and basal cells. Bottom panel: Borzoi bronchus with spontaneous influenza. There is necrosis and erosion of bronchial ciliated epithelial cells. Scale rod, 100 µm.

Fig. 4A-4B show the phylogenetic relationships of the H3 hemagglutinin genes. 4A shows the phylogenetic tree of canine influenza HA virus genes with modern and older equine HA virus genes. 4B shows the phylogenetic tree of the HA virus protein with modern and older equine HA. Phylogenetic trees were derived from genetic or amino acid sequences by neighbor joining method (method of deriving a phylogenetic tree based on the principle of minimal evolution (ME) and bootstrap analysis values >80% are shown. The bar denotes the number of amino acid residues per unit length of the horizontal branches of the tree.

5 shows the H3 influenza virus protein in bronchial and bronchial gland epithelial cells in the lungs of dogs that died of pneumonia associated with influenza virus infection. Upper panels: Erosion of ciliated bronchial epithelial cells in the bronchi. The tissues were stained with H&E. Lower panels: H3 influenza virus protein in the cytoplasm of bronchial cells (left) and epithelial cells of the bronchial gland (right). Tissues were stained with monoclonal antibody to hemagglutinin H3 and binding was detected by immunoperoxidase reaction (brown precipitate) and counterstained with hematoxylin.

Fig. 6A-6D show amplification plots of the H3 gene and matrix gene (FIG. 6A and FIG. 6B) obtained from the amplification of 10-fold serially diluted in vitro transcribed RNA standards. H3 gene and matrix gene calibration curves (FIG. 6C and FIG. 6D) were obtained by plotting log of initial RNA concentrations as a function of the cycle threshold (Ct) obtained from each dilution.

7 shows the sensitivity of Directigen Flu A, which was tested with 10-fold serially diluted virus stock solutions, including A/Wyoming/3/2003 and A/canine/FL/242/2003. The purple triangle indicates a positive result.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1 is the nucleotide sequence of canine influenza virus (Florida/43/04) encoding a PB2 protein that can be used in accordance with the present invention.

SEQ ID NO: 2 is the amino acid sequence encoded by SEQ ID NO: 1.

SEQ ID NO: 3 is the nucleotide sequence of canine influenza virus (Florida/43/04) encoding a PBl protein that can be used in accordance with the present invention.

SEQ ID NO: 4 is the amino acid sequence encoded by SEQ ID NO: 3.

SEQ ID NO: 5 is the nucleotide sequence of canine influenza virus (Florida/43/04) encoding a PA protein that can be used in accordance with the present invention.

SEQ ID NO: 6 is the amino acid sequence encoded by SEQ ID NO: 5.

SEQ ID NO: 7 is the nucleotide sequence of canine influenza virus (Florida/43/04) encoding an NS protein that can be used in accordance with the present invention.

SEQ ID NO: 8 is the amino acid sequence encoded by SEQ ID NO: 7.

SEQ ID NO: 9 is the nucleotide sequence of canine influenza virus (Florida/43/04) encoding an NP protein that can be used in accordance with the present invention.

SEQ ID NO: 10 is the amino acid sequence encoded by SEQ ID NO: 9.

SEQ ID NO: 11 is the nucleotide sequence of canine influenza virus (Florida/43/04) encoding an NA protein that can be used in accordance with the present invention.

SEQ ID NO: 12 is the amino acid sequence encoded by SEQ ID NO: 11.

SEQ ID NO: 13 is the nucleotide sequence of canine influenza virus (Florida/43/04) encoding the MA protein, which can be used in accordance with the present invention.

SEQ ID NO: 14 is the amino acid sequence encoded by SEQ ID NO: 13.

SEQ ID NO: 15 is the nucleotide sequence of canine influenza virus (Florida/43/04) encoding an HA protein that can be used in accordance with the present invention.

SEQ ID NO: 16 is the amino acid sequence encoded by SEQ ID NO: 15.

SEQ ID NO: 17 is the nucleotide sequence of canine influenza virus (FL/242/03) encoding the PB2 protein, which can be used in accordance with the present invention.

SEQ ID NO: 18 is the amino acid sequence encoded by SEQ ID NO: 17.

SEQ ID NO: 19 is the nucleotide sequence of canine influenza virus (FL/242/03) encoding a PB1 protein that can be used in accordance with the present invention.

SEQ ID NO: 20 is the amino acid sequence encoded by SEQ ID NO: 19.

SEQ ID NO: 21 is the nucleotide sequence of canine influenza virus (FL/242/03) encoding a PA protein that can be used in accordance with the present invention.

SEQ ID NO: 22 is the amino acid sequence encoded by SEQ ID NO: 21.

SEQ ID NO: 23 is the nucleotide sequence of canine influenza virus (FL/242/03) encoding an NS protein that can be used in accordance with the present invention.

SEQ ID NO: 24 is the amino acid sequence encoded by SEQ ID NO: 23.

SEQ ID NO: 25 is the nucleotide sequence of canine influenza virus (FL/242/03) encoding an NP protein that can be used in accordance with the present invention.

SEQ ID NO: 26 is the amino acid sequence encoded by SEQ ID NO: 25.

SEQ ID NO: 27 is the nucleotide sequence of canine influenza virus (FL/242/03) encoding an NA protein that can be used in accordance with the present invention.

SEQ ID NO: 28 is the amino acid sequence encoded by SEQ ID NO: 27.

SEQ ID NO: 29 is the nucleotide sequence of canine influenza virus (FL/242/03) encoding the MA protein, which can be used in accordance with the present invention.

SEQ ID NO: 30 is the amino acid sequence encoded by SEQ ID NO: 29.

SEQ ID NO: 31 is the nucleotide sequence of canine influenza virus (FL/242/03) encoding an HA protein that can be used in accordance with the present invention.

SEQ ID NO: 32 is the amino acid sequence encoded by SEQ ID NO: 31.

SEQ ID NO: 33 is the mature form of the HA protein shown in SEQ ID NO: 16 where the N-terminal 16 amino acid signal sequence has been deleted.

SEQ ID NO: 34 is the mature form of the HA protein shown in SEQ ID NO: 32 where the N-terminal 16 amino acid signal sequence has been deleted.

SEQ ID NO: 35 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 36 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 37 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 38 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 39 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 41 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 42 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 43 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 44 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 45 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 46 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 47 is the nucleotide sequence of Canine Influenza Virus (Miami/2005) encoding a PB2 protein that can be used in accordance with the present invention.

SEQ ID NO: 48 is the amino acid sequence encoded by SEQ ID NO: 47.

SEQ ID NO: 49 is the nucleotide sequence of Canine Influenza Virus (Miami/2005) encoding a PB1 protein that can be used in accordance with the present invention.

SEQ ID NO: 50 is the amino acid sequence encoded by SEQ ID NO: 49.

SEQ ID NO: 51 is the nucleotide sequence of canine influenza virus (Miami/2005) encoding a PA protein that can be used in accordance with the present invention.

SEQ ID NO: 52 is the amino acid sequence encoded by SEQ ID NO: 51.

SEQ ID NO: 53 is the nucleotide sequence of canine influenza virus (Miami/2005) encoding an NS protein that can be used in accordance with the present invention.

SEQ ID NO: 54 is the amino acid sequence encoded by SEQ ID NO: 53.

SEQ ID NO: 55 is the nucleotide sequence of canine influenza virus (Miami/2005) encoding an NP protein that can be used in accordance with the present invention.

SEQ ID NO: 56 is the amino acid sequence encoded by SEQ ID NO: 55.

SEQ ID NO: 57 is the nucleotide sequence of canine influenza virus (Miami/2005) encoding an NA protein that can be used in accordance with the present invention.

SEQ ID NO: 58 is the amino acid sequence encoded by SEQ ID NO: 57.

SEQ ID NO: 59 is the nucleotide sequence of canine influenza virus (Miami/2005) encoding the MA protein, which can be used in accordance with the present invention.

SEQ ID NO: 60 is the amino acid sequence encoded by SEQ ID NO: 59.

SEQ ID NO: 61 is the nucleotide sequence of canine influenza virus (Miami/2005) encoding an HA protein that can be used in accordance with the present invention.

SEQ ID NO: 62 is the amino acid sequence encoded by SEQ ID NO: 61.

SEQ ID NO: 63 is the nucleotide sequence of Canine Influenza Virus (Jacksonville/2005) encoding a PB2 protein which can be used in accordance with the present invention.

SEQ ID NO: 64 is the amino acid sequence encoded by SEQ ID NO: 63.

SEQ ID NO: 65 is the nucleotide sequence of Canine Influenza Virus (Jacksonville/2005) encoding a PB1 protein that can be used in accordance with the present invention.

SEQ ID NO: 66 is the amino acid sequence encoded by SEQ ID NO: 65.

SEQ ID NO: 67 is the nucleotide sequence of canine influenza virus (Jacksonville/2005) encoding a PA protein which can be used in accordance with the present invention.

SEQ ID NO: 68 is the amino acid sequence encoded by SEQ ID NO: 67.

SEQ ID NO: 69 is the nucleotide sequence of canine influenza virus (Jacksonville/2005) encoding an NS protein that can be used in accordance with the present invention.

SEQ ID NO: 70 is the amino acid sequence encoded by SEQ ID NO: 69.

SEQ ID NO: 71 is the nucleotide sequence of canine influenza virus (Jacksonville/2005) encoding an NP protein that can be used in accordance with the present invention.

SEQ ID NO: 72 is the amino acid sequence encoded by SEQ ID NO: 71.

SEQ ID NO: 73 is the nucleotide sequence of canine influenza virus (Jacksonville/2005) encoding an NA protein that can be used in accordance with the present invention.

SEQ ID NO: 74 is the amino acid sequence encoded by SEQ ID NO: 73.

SEQ ID NO: 75 is the nucleotide sequence of canine influenza virus (Jacksonville/2005) encoding the MA protein, which can be used in accordance with the present invention.

SEQ ID NO: 76 is the amino acid sequence encoded by SEQ ID NO: 75.

SEQ ID NO:77 is the nucleotide sequence of canine influenza virus (Jacksonville/2005) encoding an HA protein that can be used in accordance with the present invention.

SEQ ID NO: 78 is the amino acid sequence encoded by SEQ ID NO: 77.

SEQ ID NO: 79 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 80 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 81 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 82 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 83 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 84 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 85 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 86 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 87 is an oligonucleotide that can be used in accordance with this invention.

SEQ ID NO: 88 is an oligonucleotide that can be used in accordance with this invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to an isolated influenza virus that is capable of infecting canines and causing respiratory disease. In one embodiment, the influenza virus of this invention comprises a polynucleotide that encodes a protein having the amino acid sequence shown in any of 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 its functional and /or immunogenic fragment or variant. In a specific embodiment, this polynucleotide contains the nucleotide sequence shown in any of 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, or a fragment or variant thereof. The influenza virus of the present invention may have the HA subtype Hl, H2, H3, H4, H5, H6, H7, H8 and H9, H10, H1l, H12, H13, H14, H15 or H16, or the NA subtype Nl, N2, N3, N4, N5, N6, N7, N8 or N9. In a specific embodiment, the influenza virus of this invention is the H3 subtype. The virus can be isolated from infected dogs and cultured in cells or eggs in accordance with the experiments described here. In an exemplary embodiment, the influenza virus is influenza A.

The present invention also relates to polynucleotides that contain all or part of a gene or genes or a genomic segment of an influenza virus of the present invention. In one embodiment, a polynucleotide of this invention comprises an influenza hemagglutinin (HA) gene, a neuraminidase (NA) gene, a nucleoprotein (NP) gene, a matrix protein (MA or M) gene, a polymerase basic protein (PB) gene, an acidic protein (PA) gene ) polymerase, a non-structural protein (NS) gene, or a functional fragment or variant of any of these genes. In a specific embodiment, the polynucleotide of this invention contains a hemagglutinin (HA) gene or a functional fragment or variant thereof. In a further embodiment, the HA gene encodes a hemagglutinin protein having one of the following: serine at position 83; leucine at position 222; threonine at position 328 and/or threonine at position 483 compared to the amino acid sequence of the equine H3 consensus sequence. In one embodiment, the HA gene encodes a polypeptide having the amino acid sequence shown 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 contains the nucleotide sequence shown in SEQ ID NO: 15, 31, 61, or 77.

In one embodiment, a polynucleotide of this invention encodes a polypeptide having the amino acid sequence shown in any of 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 functional and/or immunogenic fragment thereof, or option. In a specific embodiment, a polynucleotide encoding the amino acid sequence shown in 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, contains the nucleotide sequence shown in 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, respectively, or a sequence encoding a functional and/or immunogenic fragment or variant of any of 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. Thus, the present invention relates to polynucleotide sequences containing the nucleotide sequence shown in any of 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, or a fragment or variant, including a degenerate variant, any of 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, 65, 67, 69, 71, 73, 75 or 77. In the following specific embodiment, the polynucleotide of this invention may contain: nucleotides 1-2271 of SEQ ID NO: 3; nucleotides 1-2148 SEQ ID NO: 5; nucleotides 1-657 SEQ ID NO: 7; nucleotides 1-1494 SEQ ID NO: 9; nucleotides 1-1410 SEQ ID NO: 11; nucleotides 1-756 SEQ ID NO: 13; nucleotides 1-1695 SEQ ID NO: 15; nucleotides 1-2271 SEQ ID NO: 19; nucleotides 1-2148 SEQ ID NO: 21; nucleotides 1-657 SEQ ID NO: 23; nucleotides 14494 SEQ ID NO: 25; nucleotides 1-756 SEQ ID NO: 29; nucleotides 1-1695 SEQ ID NO: 31; nucleotides 1-2277 SEQ ID NO: 47; nucleotides 1-2271 SEQ ID NO: 49; nucleotides 1-2148 SEQ ID NO: 51; nucleotides 1-690 SEQ ID NO: 53; nucleotides 1-1494 SEQ ID NO: 55; nucleotides 1-1410 SEQ ID NO: 57; nucleotides 1-756 SEQ ID NO: 59; nucleotides 1-1695 SEQ ID NO: 61; nucleotides 1-2277 SEQ ID NO: 63; nucleotides 1-2271 SEQ ID NO: 65; nucleotides 1-2148 SEQ ID NO: 67; nucleotides 1-690 SEQ ID NO: 69; nucleotides 1-1494 SEQ ID NO: 71; nucleotides 1-1410 SEQ ID NO: 73; nucleotides 1-756 of SEQ ID NO: 75; and nucleotides 1-1695 of SEQ ID NO: 77. DQ124147 - DQ124161 and DQ124190, the description of which is included here by reference.

The present invention also relates to polypeptides encoded by the influenza virus polynucleotides of the present invention. The present invention also relates to functional and/or immunogenic fragments and variants of the subject polypeptides. Contemplated polypeptides include the HA protein, the NA protein, the NS protein, the nucleoprotein, the basic polymerase protein, the polymerase acidic protein, and the influenza virus matrix protein of the invention. In an exemplary embodiment, a polypeptide of this invention has the amino acid sequence shown in any of 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 functional and/or immunogenic fragment or variant thereof.

The present invention also relates to constructs containing the polynucleotide sequence of the present invention. In one embodiment, the expression construct of this invention comprises a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in any of 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 its functional and /or immunogenic fragment or variant. In a particular embodiment, a polynucleotide encoding the amino acid sequence shown in 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, contains the nucleotide sequence shown in 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, respectively, or a sequence encoding a functional and/or immunogenic fragment or variant of any of 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. Thus, the present invention relates to expression constructs containing a polynucleotide sequence comprising the nucleotide sequence shown in any of 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, or a fragment or variant, including a degenerate variant, any of 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, 65, 67, 69, 71, 73, 75, or 77. In a preferred embodiment, the expression construct of the invention allows for overexpression of an operably linked polynucleotide of the invention.

The expression constructs of this invention typically include regulatory elements that are functional in the intended host cells in which the expression construct is to be expressed. Thus, one of ordinary skill 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, these regulatory elements are regulatory elements functional in canine 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 allows transcription of an operably linked nucleic acid sequence. In this context, the term "operably linked" refers to the direct contact of the described components, where these components are in such a relationship that allows them to function in their intended way. Typically, functionally related components are adjacent to each other.

The expression construct of this invention may contain a promoter sequence operably linked to a polynucleotide sequence encoding a polypeptide of this invention. Promoters can be incorporated into a polynucleotide using standard methods known in the art. Multiple copies of promoters or multiple promoters can be used in the expression construct of this invention. In a preferred embodiment, the promoter may be located at about the same distance from the transcription start site in the expression construct as it is from the transcription start site in its natural environment. Some variation in this distance is acceptable without a significant reduction in promoter activity. The site of transcription initiation is usually included in the expression construct. Preferably, the promoter associated with the expression construct of this invention allows overexpression of the operably linked polynucleotide of this invention.

Promoters for use with the expression construct of this invention in eukaryotic cells may be promoters of viral or cellular origin. Viral promoters include, but are not limited to, cytomegalovirus (CMV) gene promoters, SV40 early or late promoters, or Rous sarcoma virus (RSV) gene promoters. Promoters of cellular origin include, but are not limited to, the desmin gene promoter and the actin gene promoter. Promoters suitable for use with the expression construct of this invention in yeast cells include, but are not limited to, the 3-phosphoglycerate kinase promoter, the glyceraldehyde-3-phosphate dehydrogenase promoter, the metallothionein promoter, the alcohol dehydrogenase promoter, and the hexokinase promoter.

If the expression construct is to be provided in a plant cell or to be introduced into a plant cell, plant virus promoters can be used, such as, for example, cauliflower mosaic virus (CaMV) 35S (including the boosted CaMV 35S promoter (see, for example, US Pat. No. 5,106,739 and An, 1997)) or the CaMV 19S promoter. Other promoters that can be used for expression constructs in plants include, for example, prolifera promoter, Ap3 promoter, heat shock protein promoters, A. tumefaciens 1' or 2' T-DNA promoter, polygalacturonase promoter, chalcone synthase promoter A (CHS-A) from petunia, tobacco PR-1a promoter, ubiquitin promoter, actin promoter, alcA gene promoter, pin2 promoter (Xu et al., 1993), maize WipI promoter, maize trpA gene promoter (U.S. Patent No. 5,625,136) , the maize CDPK gene promoter, and the RUBISCO SSU promoter (US Pat. No. 5,034,322). Root-specific promoters, such as any of the promoter sequences described in US Patent Nos. 6,455,760 or 6,696,623 or US Published Application Nos. 20040078841; 20040067506; 20040019934; 20030177536; 20030084486 or 20040123349 may be used with the expression construct of this invention. Constitutive promoters (such as the CaMV, ubiquitin, actin, or NOS promoter), developmentally regulated promoters, and inducible promoters (such as promoters that can be induced by heat, light, hormones, or chemicals) are also discussed for use with the polynucleotide expression constructs of this invention. . Tissue-specific promoters may also be used, for example, fruit-specific promoters such as the tomato E8 promoter (accession number: AF515784; Good et al. (1994)). Seed-specific promoters may also be used, such as the promoter from the β-phaseolin gene (eg, common bean) or glycinin gene (eg, soybean) and others.

For expression in prokaryotic systems, the expression construct of this invention may contain promoters such as, for example, alkaline phosphatase promoter, tryptophan (trp) promoter, lambda _PL promoter, β-lactamase promoter, lactose promoter, phoA promoter, T3 promoter, T7 promoter or the tac promoter (de Boer et al., 1983).

The expression constructs of this invention may optionally contain a transcription termination sequence, a translation termination sequence, a sequence encoding a signal peptide, and/or enhancing elements. Transcription termination regions can usually be derived from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences may be located to the right of the coding sequence to ensure efficient termination. A signal peptide sequence is a short amino acid sequence, usually present at the amino terminus of a protein, that is responsible for the movement of an operably linked mature polypeptide to a wide range of post-translational cellular destinations, from a specific organelle compartment to protein action sites and the extracellular environment. For use with the polypeptides of this invention, targeting gene products to their intended cellular and/or extracellular destination through the use of an operably linked signal peptide sequence is discussed. Classic enhancers are cis-acting elements that increase gene transcription and can also be included in this expression construct. Classic enhancers are known in the art and include, but are not limited to, the CaMV 35S enhancer, the cytomegalovirus (CMV) early promoter enhancer, and the SV40 enhancer. Also known in the art are intron-mediated enhancer elements that enhance gene expression. These elements must be present in the transcribed region and are orientation dependent.

DNA sequences that direct polyadenylation of mRNA transcribed from this expression construct can also be included in this expression construct and include, but are not limited to, an octopine synthase or nopaline synthase signal.

Expression constructs may also include one or more dominant selectable marker genes, including, for example, genes encoding antibiotic resistance and/or herbicide resistance, for selection of transformed cells. Antibiotic resistance genes can confer resistance to one or more of the following antibiotics: hygromycin, kanamycin, bleomycin, G41S, streptomycin, paromomycin, neomycin, and spectinomycin. Kanamycin resistance can be conferred by neomycin phosphotransferase (NPT II). Herbicide resistance genes can confer resistance to phosphinothricin acetyltransferase or glyphosate. Other markers used to screen for cell transformation include, but are not limited to, genes encoding β-glucuronidase (GUS), β-galactosidase, luciferase, nopaline synthase, chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), or enhanced GFP (Yang et al, 1996).

The present invention also relates to polynucleotide vectors containing the polynucleotide sequence of this invention which encodes a polypeptide of this invention. Unique restriction enzyme sites can be included at the 5' and 3' ends of an expression construct or polynucleotide of this invention to allow insertion into a polynucleotide vector. As used herein, the term "vector" refers to any genetic element, including, for example, plasmids, cosmids, chromosomes, phage, virus, etc., that is capable of replication when bound to the correct regulatory elements and that can carry polynucleotide sequences between cells. The vectors contain a nucleotide sequence that allows the vector to replicate in the selected host cell. A number of vectors are available for expression and/or cloning and include, but are not limited to, pBR322, the pUC series, the M13 series, the pGEM series, and the pBLUESCREPT vectors (Stratagene, La Jolla, CA and Promega, Madison, WI). The invention also relates to oligonucleotide probes and primers, such as polymerase chain reaction (PCR) primers, which can hybridize to the coding or non-coding sequence of a polynucleotide of the invention. The oligonucleotide probes of this invention can be used in methods for detecting influenza virus nucleic acids. The oligonucleotide primers of this invention can be used in PCR methods and other methods involving nucleic acid amplification. In a preferred embodiment, the probe or primer of this invention can hybridize to a polynucleotide of this invention under stringent conditions. The probes and primers of this invention may optionally contain a detectable label or reporter molecule, such as fluorescent molecules, enzymes, a radioactive moiety, and the like. The probes and primers of this invention may be of any suitable length for the method or assay in which they are used. Typically, the probes and primers of this invention will be 10-500 or more nucleotides in length. Probes and primers that are 10-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, or 101 or more nucleotides in length are discussed in within the scope of this invention. In one embodiment, the probes and primers are any of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. The probes and primers of this invention may have complete (100%) nucleotide sequence identity with this polynucleotide sequence, or sequence identity may be less than 100%. For example, the sequence identity between the probe and the primer and that sequence may be 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, or any other percent sequence identity, as long as this probe or primer can hybridize under stringent conditions to the nucleotide sequence of this invention. Examples of probes and primers of this invention include probes and primers having the nucleotide sequence shown in any of 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, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, or a functional fragment or variant of any from SEQ ID NO: 35-46.

As used herein, the terms "nucleic acid", "polynucleotide", and "oligonucleotide" refer to a deoxyribonucleotide, ribonucleotide, or mixed polymer of deoxyribonucleotide and ribonucleotide in single-stranded or double-stranded form and, unless otherwise indicated, may include known natural nucleotide analogs that can function similarly to the functioning of naturally occurring nucleotides. Polynucleotide sequences include a DNA chain sequence that can be transcribed into RNA and an RNA chain that can be translated into protein. The complementary sequence of any nucleic acid, any polynucleotide or oligonucleotide of this invention is also discussed within the scope of this invention. Polynucleotide sequences also include both full length sequences and shorter sequences derived from these full length sequences. The invention also includes polynucleotides that are complementary in their sequence to the polynucleotides described herein. The polynucleotides and polypeptides of this invention may be provided in purified or isolated form.

Due to the degeneracy of the genetic code, different polynucleotide sequences may encode a polypeptide of the present invention. A table showing all possible triplet codons (where U also stands for T) and the amino acid encoded by each codon is described in Lewin (1985). In addition, it is within the skill of one skilled in the art to generate alternative polynucleotide sequences encoding the same, or substantially the same, polypeptides of the present invention. These degenerate variant and alternative polynucleotide sequences are within the scope of this invention. In this context, references to "substantially the same" sequence refer to sequences that encode amino acid substitutions, deletions, additions, or insertions that do not actually alter the functional and/or immunogenic activity of the polypeptide encoded by the polynucleotides of this invention.

The present invention also relates to variants of the polynucleotides of the present invention which encode the polypeptides of the present invention. Variant sequences include sequences in which one or more nucleotides of that sequence have been changed, deleted and/or inserted. Nucleotides that can replace natural DNA nucleotides have a base, which may include, but is not limited to, 5-fluorouracil, 5-bromouracil, hypoxanthine, 1-methylguanine, 5-methylcytosine, and tritylated bases. The sugar portion of a nucleotide in the sequence may also be modified and includes, but is not limited to, arabinose, xylulose, and hexose. In addition, the bases adenine, cytosine, guanine, thymine and uracil of these nucleotides can be modified with acetyl, methyl and/or thio groups. Sequences containing nucleotide substitutions, deletions and/or insertions can be generated and analyzed using standard methods known in the art.

Amino acid substitutions other than the amino acids specifically exemplified or naturally present in the polypeptide of this invention are also discussed within the scope of this invention. For example, non-natural amino acids can replace the amino acids of a polypeptide, as long as the polypeptide having the amino acid substitutions retains substantially the same functional activity as the polypeptide in which the amino acids have not been replaced. Examples of non-natural amino acids include, but are not limited to, ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, γ- aminobutyric acid, e-aminohexanoic acid, 6-aminohexanoic acid, 2-aminoisobutyric acid, 3-aminopropionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, f-butylglycine, f-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, engineered amino acids such as β-methylamino acids, C-methylamino acids, N-methylamino acids, and amino acid analogs in general. Non-natural amino acids also include amino acids having derivatized side groups. In addition, any of the amino acids in a protein can be in the D (dextrorotatory) form or L (left-handed) form. Allelic variants of the protein sequence of the polypeptide of the present invention are also discussed within the scope of this invention.

Amino acids can be generally divided into the following classes: non-polar, uncharged polar, basic and acidic. Conservative substitutions whereby a polypeptide of the present invention having an amino acid of one class is replaced with a polypeptide having another amino acid of the same class are included within the scope of this invention as long as the polypeptide having the substitution still retains substantially the same functional activity as a polypeptide that does not have this substitution. Polynucleotides encoding a polypeptide having one or more amino acid substitutions in sequence are contemplated within the scope of this invention. Table 11 below provides a list of examples of amino acids belonging to each class. Single letter amino acid abbreviations are defined in Table 12.

Fragments and polypeptide variants of the influenza virus of this invention can be obtained using standard methods known in this field, and examined for the presence of function or immunogenicity using standard methods known in this field. For example, to study fragments and/or variants of the neuraminidase polypeptide of this invention, neuraminidase activity can be analyzed. Thus, one of ordinary skill in the art can easily prepare and examine fragments and variants of a polypeptide of this invention and determine whether the fragment or variant retains activity relative to the full-length or non-variant polypeptide.

The polynucleotides and polypeptides discussed within the scope of this invention may also be defined with respect to more specific ranges of identity and/or similarity to the sequences of this invention specifically exemplified herein. The sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and may be greater than 95%. Sequence identity and/or similarity can be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 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, 87, 88, 89, 90, 91, 92, 93, 94, 95%, 96%, 97%, 98% or 99% compared to the sequence shown here as an example. Unless otherwise indicated, sequence identity and/or similarity reported herein can be determined using the algorithm of Karlin and Altschul (1990) modified as described in Karlin and Altschul (1993). Such an algorithm is included in the NBLAST and XBLAST programs in Altschul et al (1990). BLAST searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain the desired percent sequence identity. Gapped BLAST programs can be used to generate gap matches for comparison purposes, as described in Altschul et al. (1997). When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used. See the NCBI/NIH website.

The invention also contemplates polynucleotide molecules having sequences that are sufficiently homologous to the polynucleotide sequences exemplified herein such that hybridization to that sequence is possible under standard stringent conditions and using standard methods (Maniatis et al., 1982). In this context, "stringent" hybridization conditions are those under which hybridization is carried out typically at 20-25° C. below the melting point of the Tm DNA hybrid in 6X SSPE, 5X Denhardt's solution, 0.1% SDS, 0.1 mg/mL denatured DNA. The melting point, Tm, is described by the following formula (Beltz et al, 1983):

Tm=81.5°C+16.6 Log[Na+]+0.41(%G+C)-0.61(% formamide)-600/duplex length in b.p.

Washes are usually carried out as follows:

(1) Twice at room temperature for 15 minutes in 1H SSPE, 0.1% SDS (low stringency wash).

(2) Once at Tm-20 ^° C for 15 minutes in 0.2H SSPE, 0.1% SDS (medium stringency wash).

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

This invention also relates to compositions and methods for inducing an immune response against an influenza virus that is capable of infecting a susceptible animal host and causing respiratory illness. This invention can be used to induce an immune response against influenza virus of any subtype in a susceptible host animal. For example, an influenza virus may be the HA subtype Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16, and the NA subtype N1, N2, N3, N4, N5, N6, N7, N8 or N9. 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, an immune response is induced against influenza virus subtype H3N8. In one embodiment, the host animal is a canine. Canines include wild animals, zoo animals, and pets such as wolves, coyotes, and foxes. Canine also includes dogs, in particular domestic dogs, such as, for example, purebred and/or mongrel companion dogs, show dogs, working dogs, herding dogs, hunting dogs, guard dogs, police dogs, hounds. dogs and/or laboratory dogs. In a specific embodiment, the host animal is a domestic dog such as a greyhound. In one embodiment, an effective amount of an immunogenic composition of this invention is administered to an animal sufficient to induce an immune response against an influenza virus of this invention. Such an immune response may be a humoral and/or cellular immune response. In a specific embodiment, such an immune response is a protective immune response that is capable of preventing or minimizing viral infection in an immunized host animal for a period of time following that immunization. Thus, this invention also relates to vaccine compositions and methods that can provide a vaccinated animal with a protective immune response to the virus of this invention.

As described herein, the vaccine or immunogenic compositions of this invention may contain a cell-free whole virus, including an attenuated or inactivated virus, or parts of a virus, including subvirion particles (including a "subunit vaccine", where the virion is processed to remove some or all viral lipids), viral proteins (including individual proteins and macromolecular complexes of multiple proteins), polypeptides and peptides, as well as virus-infected cell lines, or combinations of any of them. Vaccine or immunogenic compositions containing virus-infected cell lines may contain multiple cell lines, each infected with a different viral strain.

In one embodiment, the canine may be immunized with one or more inactivated (i.e., killed) and/or live attenuated influenza virus vaccines or vaccines containing one or more influenza virus antigens from one or more virus isolates. In one embodiment, the influenza virus is a canine influenza virus. In another embodiment, the influenza virus is an equine influenza virus that encodes or expresses a polypeptide that has at least about 90%, or at least about 95%, or at least about 96%, or 97%, or 98%, or 99% or greater amino acid sequence identity with a canine influenza virus polypeptide. In one embodiment, the influenza antigen used in the vaccine of this invention has at least about 96% sequence identity with the canine influenza virus HA antigen and/or NA antigen.

An example of an inactivated vaccine is EQUICINE II™, which was marketed by Intervet Inc. (Millsboro, DE, USA) as a liquid vaccine. EQUICINE II™ contains inactivated influenza A/Pennsylvania/63 ("A/Pa/63") and influenza A/equine/Kentucky/93 ("A/KY/93") with carbopol (ie, HAVLOGEN® (Intervet Inc. .)). More specifically, one dose of EQUICINE II™ contains: inactivated A/Pa/63 at 10 ^6.0 EID ₅₀ , inactivated A/KY/93 at 10 ^6.7 EID ₅₀ , 0.25% v/v carbopol, and sufficient PBS to create a total volume of 1 ml.

Another example of a live attenuated vaccine is equine influenza virus A/equine/Ohio/03 ("Ohio 03"). In some embodiments, such a vaccine contains CARBIGEN™, which is a polymer-based emulsifiable adjuvant commercially available from MVP Laboratories, Inc. (Ralston, N.E.). In such vaccines, the dosage unit typically contains at least about 250 HA units of the virus, from about 250 to about 12500 HA units of the virus, or from about 1000 to about 6200 HA units of the virus. The recommended concentration of CARBIGEN™ is from about 5 to about 30% by weight.

An example of a live attenuated vaccine is modified live equine/Kentucky/91 ("A/KY/91") influenza virus in the form of a lyophilized vaccine that can be reconstituted with water. In some embodiments, such reconstitution is carried out using sufficient vaccine water to bring the vaccine dose to a total volume of 1 ml. Options for such vaccines are discussed, for example, in US Patent Nos. 6,436,408; 6398774 and 6177082, which are incorporated by reference in their entirety in this patent. Once reconstituted, a dose of such a vaccine may, for example, contain A/KY/91 at 10 ^7.2 TCID _{5 O} per ml, 0.015 grams of NZ AMINE AS™ per ml, 0.0025 grams of gelatin per ml, and 0.04 grams of D-lactose per ml. ml. NZ AMINE AS™ is a purified source of amino acids and peptides derived from the enzymatic hydrolysis of casein. NZ AMINE AS™ is sold by Kerry Bio-Science (Norwich, NY, USA).

In a preferred embodiment, the vaccine contains an H3 influenza virus antigen having at least about 93% homology to Florida/43/2004 in HA coding sequences, such as, for example, the equine/New Market/79 strain. Preferred homology is at least about 96%, such as that found in strains equine/Alaska/1/91 and equine/Santiago/85. In the examples that follow, equine/Kentucky/91, equine-2/Kentucky/93, equine-1/Pennsylvania/63 and equine/Ohio/03 influenza antigens are included in the vaccines. Preferred vaccines also include those containing equine/Wisconsin/03, equine/Kentucky/02, equine/Kentucky/93 and equine/New Market 2/93. The examples that follow use H3N8 viruses. However, the authors believe that other H3 influenza viruses can be used in accordance with this invention.

Live attenuated vaccines can be prepared by conventional methods. Such methods typically include, for example, modification of pathogenic strains by in vitro passaging, cold adaptation, modification of the organism's pathogenicity by genetic manipulation, generation of chimeras, incorporation of antigens into viral vectors, selection of non-virulent wild-type strains, and the like.

In some embodiments, a live attenuated virus strain is obtained by serial passage of wild-type virus through cell culture, laboratory animals, non-host animals, or eggs. The accumulation of a genetic mutation during such passage(s) usually results in a progressive loss of the organism's virulence relative to the original host.

In some embodiments, a live attenuated virus strain is produced by coinfection of permissive cells with an attenuated mutant virus and a pathogenic virus. The desired resulting recombinant virus has the safety of an attenuated virus with genes encoding protective antigens from the pathogenic virus.

In some embodiments, a live attenuated virus strain is produced by cold adaptation. A cold-adapted virus has the advantage of replicating only at the temperature found in the upper respiratory tract. A method for producing a cold-adapted equine influenza virus has been described in US Patent No. 6,177,082. The desired cold-adapted virus produced confers one or more of the following phenotypes: cold adaptation, temperature sensitivity, dominant interference, and/or attenuation.

In some embodiments, a live attenuated virus strain is produced by molecular means, such as point mutation, deletion, or insertion, to convert the pathogenic virus to a non-pathogenic or less pathogenic virus relative to the parent virus, while retaining the protective properties of the parent virus.

In some embodiments, a live attenuated virus is obtained by cloning a candidate defense antigen gene into the genome of a non-pathogenic or less pathogenic virus or other organism.

Inactivated (ie, "killed") viral vaccines can be obtained by inactivating the virus using conventional methods. Typically, such vaccines include excipients that can enhance the immune response, as well as other excipients that are commonly used in vaccines. For example, in the examples that follow, EQUICINE II™ contains HAVLOGEN®. Inactivation of this virus can be accomplished by treating this virus with inactivating chemicals (eg, formalin, beta-propiolactone (“BPL”), bromoethylamine (“BEA”), and binary ethyleneimine (“BEI”)), or by non-chemical methods (eg, heating, freezing/ thawing or sonication) to suppress the replication ability of this virus.

In the examples that follow, the equine/Ohio/03 virus was used as the infection virus. It is known to have approximately 99% homology with Florida/43/04 isolates and has been shown to induce symptoms of infection and seroconversion in dogs. Example 18 illustrates the efficacy of an equine influenza vaccine in dogs, as can be seen from the inhibition of haemagglutination (or "HI" or "HAI") titers in dogs vaccinated with the inactivated Ohio 03 antigen in a vaccine composition containing a CARBIGEN™ adjuvant. Table 29 shows the titers before vaccination, after vaccination and after the second vaccination, as well as after challenge. These results show HI titers at each stage post-vaccination for vaccinated dogs, with little or no increase for controls. Table 30 shows clinical symptoms, host virus isolation, and histopathology results from the same study. Although immunized animals showed no clinical symptoms, virus isolation, or positive histopathology, positive HI titers (Table 29) indicate significant antibody titers in immunized animals.

It should be noted that other H3 influenza virus antigen vaccines are also included in this invention. The vaccines described in this specification and the following examples are provided to illustrate this invention and its preferred embodiments, but not to limit the scope of the claimed invention.

In addition, it should be noted that influenza antigens other than influenza H3 virus antigens can be used in accordance with this invention. Such antigens include, for example, those from equine/PA/63, which is an equine subtype of A1 (H7N7). It is contemplated that one or more of these antigens may be used with or without one or more influenza H3 antigens.

Typically, this vaccine is administered in a therapeutically effective amount. A "therapeutically effective amount" is an amount sufficient to induce a protective response in a canine patient against the target virus. Generally, a dose is "therapeutically effective" if it prevents, reduces risk, delays the onset, reduces the spread, relieves, suppresses, or eliminates influenza or one or more (usually two or more) of its symptoms. Typical flu symptoms include, for example, fever (usually ≥103.0°F; ≥39.4°C in dogs), coughing, sneezing, histopathological lesions, ocular discharge, nasal discharge, vomiting, diarrhoea, depression, weight loss, gagging, hemoptysis and/or audible wheezing. Other often more severe symptoms may include, for example, bleeding in the lungs, mediastinum, or pleural space; tracheitis; bronchitis; bronchiolitis; replacement bronchopneumonia and/or infiltration of the epithelial lining and respiratory lumen of the lungs by neutrophils and/or macrophages.

This vaccine may be administered as part of a combination therapy, ie. therapy, which includes, in addition to the vaccine itself, the administration of one or more additional active agents, adjuvants, therapeutic agents, etc. In this case, it should be understood that the amount of vaccine that constitutes a "therapeutically effective" amount may be less than the amount of vaccine that would constitute a "therapeutically effective" amount if that vaccine alone were administered. Other therapies may include therapies known in the art such as, for example, antiviral drugs, analgesics, fever-reducing drugs, expectorants, anti-inflammatory drugs, antihistamines, antibiotics to treat a bacterial infection that originates from the infection. influenza virus, rest and/or fluids. In some embodiments, the vaccine of this invention is administered in combination with a Bordetella vaccine, an adenovirus vaccine, and/or a parainfluenza virus vaccine.

In some embodiments, a typical dose for a live attenuated vaccine is at least about 10 ³ pfu/animal (Canidae); and more typically about 10 ³ to about 10 ⁹ fu/animal (Canidae). In this patent, "pf" means "plaque forming units". In some embodiments, a typical dose for a live attenuated vaccine is at least about 10 ³ TCDD ₅₀ /animal (Canidae) and more typically about 10 ³ - about 10 ⁹ TCID ₅₀ /animal (Canidae) In some embodiments, a typical dose for a live attenuated vaccine is at least about 10 ³ EID ₅₀ /animal (Canidae) and more typically about 10 ³ to about 10 ⁹ EID ₅₀ /animal (Canidae). in some embodiments, a typical dose for a killed vaccine is at least about 40 HA units, typically about 40 to about 10,000 HA units, and more often about 500 to about 6200 HA units.In some embodiments, this dose is about 6100 to about 6200 NA units.

In some embodiments, the vaccine contains the live attenuated vaccine at a concentration that is at least about 10 ^0.5 pfu/animal (Canidae) greater than the level of immunogenicity. In some embodiments, the vaccine contains the live attenuated vaccine at a concentration that is at least about ^100.5 TCID ₅₀ /animal (Canidae) greater than the level of immunogenicity. In some embodiments, the vaccine contains the live attenuated vaccine at a concentration that is at least about ^100.5 EID ₅₀ /animal (Canidae) greater than the level of immunogenicity.

The level of immunogenicity can be determined experimentally by immunization dose titration testing methods commonly known in the art. Such methods typically involve vaccinating a number of canines with the vaccine at various doses and then infecting these animals with a virulent virus to detect the minimum protective dose.

Factors influencing the preferred dosage regimen may include, for example, type (eg, species and breed), age, weight, sex, diet, activity, lung size, and condition of the subject; method of administration; the efficacy, safety and duration profiles of the specific vaccine used; use of the delivery system; administering the vaccine as part of a drug and/or vaccine combination. Thus, the actual dose used may vary for individual animals and therefore may deviate from the above typical doses. Determination of such dose adjustments using conventional means is usually within the skill of those skilled in the art. In addition, it should be noted that live attenuated viruses are usually self-replicating; thus, the specific amount of such virus administered is not necessarily critical.

It is anticipated that this vaccine may be administered to canine patients once; or alternatively two or more times over days, weeks, months or years. In some embodiments, the vaccine is administered at least twice. In some such embodiments, the vaccine is administered, for example, twice, with the second dose (eg, booster) administered at least 2 weeks after the first dose. In some embodiments, the vaccine is administered, for example, twice, with the second dose (eg, booster) no more than 8 weeks apart after the first dose. In some embodiments, the second dose is administered at about 2 weeks 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. 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 in form. However, often these doses are the same in number and form. When only a single dose is administered, the amount of vaccine in that single dose will typically contain a therapeutically effective amount of that vaccine. However, when more than one dose is administered, the amounts of the vaccine at those doses together may constitute a therapeutically effective amount.

In some embodiments, the vaccine is administered prior to infection of the canine influenza recipient. In such embodiments, the vaccine may be administered to prevent, reduce the risk of, or delay the onset of influenza or one or more (usually two or more) influenza symptoms.

In some embodiments, the vaccine is administered following influenza infection of a canine recipient. In such embodiments, the vaccine may, for example, relieve, suppress, or eliminate influenza or one or more (usually two or more) influenza symptoms.

The preferred composition of a vaccine depends, for example, on whether the vaccine is an inactivated vaccine, a live attenuated vaccine, or both. This composition also depends on the route of administration. This vaccine is expected to contain one or more conventional pharmaceutically acceptable carriers, adjuvants, other immune response enhancers and/or carriers (collectively referred to as "excipients"). Such excipients are typically chosen to be compatible with the active ingredient(s) in the vaccine. The use of fillers is generally known to those skilled in the art.

The term "pharmaceutically acceptable" is used as a definition and means that the modified noun is suitable for use in a pharmaceutical product. For example, when used to describe an excipient in a pharmaceutical vaccine, the term characterizes the excipient as being compatible with the other ingredients of the composition and not disadvantageously harmful to the intended canine recipient.

These vaccines may be administered by conventional means, including, for example, mucosal administration (eg, intranasal, oral, intratracheal, and intraocular) and parenteral administration. Mucosal administration is often particularly preferred for live attenuated vaccines. Parenteral administration is often particularly preferred for inactivated vaccines.

Mucosal vaccines may be, for example, liquid dosage forms such as pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. Suitable excipients for such vaccines include, for example, inert solvents commonly used in the art such as water, saline, dextrose, glycerol, lactose, sucrose, starch powder, cellulose alkanoic acid esters, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, gum arabic, sodium alginate, polyvinylpyrrolidone and/or polyvinyl alcohol. The excipients may also contain various wetting, emulsifying, suspending, flavoring (eg sweetening) and/or flavoring agents.

Oral mucosal vaccines may also be, for example, tableted or encapsulated for convenient administration. Such capsules or tablets may contain a controlled release form. In the case of capsules, tablets and pills, these dosage forms may also contain buffering agents such as sodium citrate or magnesium or calcium carbonate or bicarbonate. Tablets and pills may be prepared with an enteric coating.

It is contemplated that this vaccine may be administered through the drinking water and/or food of a canine patient. In addition, it is contemplated that this vaccine may be administered in the form of a treat or toy.

"Parenteral administration" includes subcutaneous injections, submucosal injections, intravenous injections, intramuscular injections, intrasternal injections, transdermal injections and infusion. Injectable preparations (eg, sterile injectable aqueous or oily suspensions) may be prepared according to known methods using suitable excipients such as carriers, diluents, dispersing, wetting agents, emulsifying agents and/or suspending agents. They usually include, for example, water, saline, dextrose, glycerin, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, 1,3-butanediol, Ringer's solution, isotonic sodium chloride solution, tasteless non-volatile oils (eg synthetic mono- or diglycerides), fatty acids (eg oleic acid), dimethylacetamide, surfactants (eg ionic and non-ionic detergents), propylene glycol and/or polyethylene glycols. Fillers may also include small amounts of other excipients such as pH buffering agents.

This vaccine may include one or more excipients that enhance the canine patient's immune response (which may be an antibody response, a cellular response, or both), thereby increasing the efficacy of the vaccine. The use of such excipients (or "adjuvants") can be particularly useful when using an inactivated vaccine. The adjuvant(s) can be a substance that has a direct (eg, cytokine or Bacillus Calmette-Guerin (“BCG”) or indirect effect (liposomes) on the 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 liposaccharides, Freund's adjuvants and/or MDP), plant components (eg Quil A ) and/or one or more substances which may be carriers (eg bentonite, latex particles, liposomes and/or Quil A, ISCOM). As noted above, adjuvants also include, for example, CARBIGEN™ and carbopol. It should be understood that this invention includes both vaccines that contain an adjuvant(s) and vaccines that do not contain any adjuvants.

It is contemplated that the vaccine may be lyophilized (or otherwise reduced in liquid volume) for storage and then reconstituted in liquid before or during administration. Such reconstitution can be achieved using, for example, vaccine grade water.

In addition, the present invention provides kits that are suitable for use in performing the methods described above. The kit contains a dosage form containing the vaccine described above. This kit also contains at least one additional component and usually instructions for using this vaccine with the additional component (components). This additional component (s) may be, for example, one or more additional ingredients (such as, for example, one or more of the excipients discussed above, food and/or treat) that can be mixed with this vaccine before or during administration. This additional component(s) may alternatively (or additionally) comprise one or more devices for administering the vaccine to the canine patient. Such a device may be, for example, a syringe, inhaler, nebulizer, pipette, tweezers, or any medically applicable delivery device. In some embodiments, this device is suitable for intranasal administration of a vaccine.

Other excipients and routes of administration known in the pharmaceutical and biological fields may also be used.

The vaccine or immunogenic compositions of the present invention also include recombinant viral vector constructs which may contain, for example, genes encoding the HA protein, NA protein, nucleoprotein, polymerase basic protein, polymerase acidic protein and/or influenza virus matrix protein of the present invention. . Any suitable viral vector that can be used to produce a recombinant vector/virus construct is contemplated for use with the present invention. For example, viral vectors derived from adenovirus, avipoxvirus, herpesvirus, vaccinia virus, canaripoxvirus, entomopoxvirus, swine pox virus, West Nile virus, and others known in the art may be used with the compositions and methods of this invention. Recombinant polynucleotide vectors that encode and express these components can be constructed using standard genetic engineering techniques known in the art. In addition, the various vaccine compositions described herein may be used alone and in combination with each other. For example, primary immunizations of an animal may use recombinant vector-based constructs having single-strain or multiple-strain components, followed by booster immunizations with vaccine compositions containing inactivated virus or inactivated virus-infected cell lines. Other protocols for immunization with the vaccine compositions of this invention will be apparent to those skilled in the art and are intended to be included within the scope of this invention.

The present invention also relates to a chimeric virus comprising at least one gene or genomic segment of an influenza virus of the present invention and the remaining viral genes or genomic segments from a different influenza virus of the present invention or from an influenza virus other than a virus of the present invention. A chimeric virus can be obtained by genetically reassorting a nucleic acid of a donor influenza virus of the present invention with a nucleic acid of a recipient influenza virus and then selecting a chimeric virus that includes the nucleic acid of the donor virus. Methods for obtaining and isolating a chimeric virus are well known in the art (Fields et al., 1996). In one embodiment, the chimeric virus of this invention contains genes or genomic segments of a human, avian, porcine, or equine influenza virus. The chimeric virus of this invention may include any combination of nucleic acid from the donor and recipient influenza virus, as long as the chimeric virus contains at least one gene or genomic segment from the donor influenza virus of the invention. In one embodiment, the recipient influenza virus may be an equine influenza virus.

Natural, recombinant or synthetic viral protein polypeptides and peptide fragments thereof may also be used as vaccine compositions according to the methods in question. In one embodiment, the vaccine composition comprises a canine influenza virus polynucleotide or polypeptide. In one embodiment, the vaccine composition contains a polynucleotide encoding a polypeptide having the amino acid sequence shown in any of 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 its functional and/or immunogenic fragment or variant. In a specific embodiment, a polynucleotide encoding the amino acid sequence shown in 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, contains the nucleotide sequence shown in 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, respectively, or a sequence encoding a functional and/or immunogenic variant of any of 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. In the following specific embodiment, , the polynucleotide of this invention may contain: nucleotides 1-2271 of SEQ ID NO: 3; nucleotides 1-2148 SEQ ID NO: 5; nucleotides 1-657 SEQ ID NO: 7; nucleotides 1-1494 SEQ ID NO: 9; nucleotides 1-1410 SEQ ID NO: 11; nucleotides 1-756 SEQ ID NO: 13; nucleotides 1-1695 SEQ ID NO: 15; nucleotides 1-2271 SEQ ID NO: 19; nucleotides 1-2148 SEQ ID NO: 21; nucleotides 1-657 SEQ ID NO: 23; nucleotides 14494 SEQ ID NO: 25; nucleotides 1-756 SEQ ID NO: 29; nucleotides 1-1695 SEQ ID NO: 31; nucleotides 1-2277 SEQ ID NO: 47; nucleotides 1-2271 SEQ ID NO: 49; nucleotides 1-2148 SEQ ID NO: 51; nucleotides 1-690 SEQ ID NO: 53; nucleotides 1-1494 SEQ ID NO: 55; nucleotides 1-1410 SEQ ID NO: 57; nucleotides 1-756 SEQ ID NO: 59; nucleotides 1-1695 SEQ ID NO: 61; nucleotides 1-2277 SEQ ID NO: 63; nucleotides 1-2271 SEQ ID NO: 65; nucleotides 1-2148 SEQ ID NO: 67; nucleotides 1-690 SEQ ID NO: 69; nucleotides 1-1494 SEQ ID NO: 71; nucleotides 1-1410 SEQ ID NO: 73; nucleotides 1-756 of SEQ ID NO: 75; and nucleotides 1-1695 of SEQ ID NO: 77. In another embodiment, the vaccine composition comprises a polypeptide having the amino acid sequence shown in any of 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, or a functional and/or immunogenic fragment or variant thereof. In a further embodiment, the vaccine composition comprises an equine influenza virus polynucleotide or polypeptide, wherein the polynucleotide or polypeptide is at least about 90%, or at least about 95%, or at least about 96%, or 97%, or 98 %, or 99% or greater sequence identity with a canine influenza virus polynucleotide or polypeptide. In one embodiment, viral polypeptides derived from multiple strains can be combined in a vaccine composition and used to vaccinate a host animal. For example, polypeptides based on the viral HA protein from at least two different strains of the influenza virus of the present invention can be combined in this vaccine. These polypeptides may be homologous to one strain, or may contain "hybrid" or "chimeric" polypeptides whose amino acid sequence is produced by combining or linking polypeptides from at least two different strains. Procedures for obtaining viral polypeptides are well known in the art. For example, viral polypeptides and peptides can be synthesized using solid phase synthesis methods (Merrifield, 1963). Viral polypeptides and peptides can also be produced using recombinant DNA methods, in which a polynucleotide molecule encoding a viral protein or peptide is expressed in a host cell such as bacteria, yeast, or mammalian cell lines, and the expressed protein is purified using standard methods of this areas.

The vaccine compositions of this invention also include naked DNA compositions. In one embodiment, the nucleic acid may comprise a nucleotide sequence encoding the HA protein and/or NA protein of the influenza virus of the invention. Methods for vaccinating with nucleic acids are known in the art and are described, for example, in US Patent Nos. 6,063,385 and 6,472,375. The nucleic acid may be in the form of a plasmid or a gene expression cassette. In one embodiment, the nucleic acid is provided encapsulated in a liposome, which is administered to an animal.

Vaccine compositions and immunogens, such as polypeptides and nucleic acids, which can be used in accordance with this invention, can be obtained with a pharmaceutically acceptable carrier or diluent. Compounds and compositions useful in this invention can be prepared in accordance with known methods for the preparation of pharmaceutically acceptable compositions. The formulations are described in detail in a number of literatures that are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science by EW Martin, Easton Pennsylvania, Mack Publishing Company, ^19th ed., 1995, describes formulations that may be used in connection with the present invention. Generally, the compositions of this invention will be formulated such that an effective amount of the immunogen is combined with a suitable carrier to facilitate effective administration of the composition, and the compositions used in these methods may also be in a variety of forms. They include, for example, solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable or infusible solutions and sprays. The preferred form depends on the intended route of administration and therapeutic application. These compositions preferably also include conventional pharmaceutically acceptable carriers and diluents which are known to those skilled in the art. Examples of carriers and diluents for use with the present peptidomimetics include, but are not limited to, water, saline, oils, including mineral oil, ethanol, dimethyl sulfoxide, gelatin, cyclodextrans, magnesium stearate, dextrose, cellulose, sugars, carbonate calcium, glycerin, alumina, starch and equivalent carriers and diluents or mixtures of any of them. The immunogen formulations of this invention may also contain suspending agents, protective agents, buffers, preservatives and stabilizers. To ensure the introduction of such doses for the desired therapeutic treatment, the pharmaceutical compositions of this invention will preferably contain approximately 0.1% of the mass. - 45% of the mass. and, in particular, 1% of the mass. - 15% of the mass. an immunogen or immunogens of a general composition comprising a carrier or diluent.

Vaccine and immunogenic compositions of the present invention may be prepared by procedures well known in the art. For example, the vaccine or immunogens are usually prepared in injectable forms, such as liquid solutions or suspensions. These vaccine or immunogens are administered in a manner that is compatible with the dosage form and in such amount as will be therapeutically effective and immunogenic in the recipient. Optimal dosages and administration schedules for a particular vaccine or immunogen preparation can be readily determined by one of skill in the art.

The peptides and/or polypeptides of the present invention may also be provided in the form of a multiple antigenic peptide (MAP) construct. The preparation of MAP constructs has been described in Tam (1988). MAP constructs use a core matrix of lysine residues from which multiple copies of the immunogen are synthesized (Posnett et al, 1988). Multiple MAP constructs, each containing the same or different immunogens, can be prepared and incorporated into a vaccine composition in accordance with the methods of this invention. In one embodiment, a MAP construct is provided with one and/or more adjuvants and/or administered with one or more adjuvants. The influenza virus polypeptides of this invention may also be prepared and administered as macromolecular protein structures containing one or more polypeptides. US Published Application US 2005/0009008 describes methods for producing virus-like particles as an influenza vaccine.

According to the methods of the present invention, the vaccine and immunogenic compositions described herein are administered to susceptible hosts, typically canines, and more commonly domestic dogs, in an effective amount and in an effective manner to induce protective immunity against subsequent challenge or infection of that host with a virus. In one embodiment, the host animal is a member of the canine family. Canines include wild canines, zoo canines, and domestic canines such as wolves, coyotes, and foxes. Canine also includes dogs, in particular domestic dogs, such as, for example, purebred and/or mongrel companion dogs, show dogs, working dogs, herding dogs, hunting dogs, guard dogs, police dogs, hounds. dogs and/or laboratory dogs. In a specific embodiment, the host animal is a domestic dog such as a greyhound. These vaccines or immunogens are usually administered parenterally, by injection, for example, subcutaneously, intraperitoneally or intramuscularly. Other suitable routes of administration include oral or nasal administration. Typically, these vaccines or immunogens are administered to the animal at least twice, with an interval of one or more weeks between administrations. However, other schedules for initial and booster administrations of the vaccine or immunogens are contemplated and may depend on the judgment of the practitioner and the particular host animal being treated.

Viral and virus-infected cells in a vaccine preparation can be inactivated or attenuated using methods known in the art. For example, whole virus and infected cells can be inactivated or attenuated by exposure to paraformaldehyde, formalin, beta-propiolactone (BPL), bromoethylamine (BEA), binary ethyleneimine (BEI), phenol, ultraviolet light (UV), heat, freezing and thawing, sound (including sonication), etc. The amount of cell-free whole virus in a vaccine dose can be in the range of about 0.1 mg to about 5 mg, and more often is in the range of about 0.2 mg to about 2 mg. A dose for vaccine preparations containing virus-infected cell lines will typically contain about 10 ⁶ to about 10 ⁸ cells per dose, and more typically about 5×10 ⁶ to about 7.5×10 ⁷ cells per dose. The amount of protein or peptide immunogen per animal dose may vary from about 0.1 µg to about 10,000 µg, or about 1 µg to about 5000 µg, or about 10 µg to about 1000 µg, or about 25 µg to about 750 µg, or approximately 50 micrograms - approximately 500 micrograms, or 100 micrograms - approximately 250 micrograms, depending on size, age, etc. animal receiving this dose.

An immunogenic or vaccine composition of this invention, such as a virus or virus-infected cells or viral proteins or peptides, can be combined with an adjuvant, usually just prior to administration. Adjuvants contemplated for use in these vaccine forms typically include threonylmuramyl dipeptide (MDP) (Byars et al, 1987), saponin, Cornebacterium parvum, Freund's complete adjuvant and Freund's incomplete adjuvant, aluminum, or a mixture of any of these. Various other adjuvants suitable for use with the methods and vaccines of this invention, such as alum, are well known in the art and are contemplated for use with this invention.

This invention also relates to antibodies that bind specifically to the protein or peptide of this invention. The antibodies of this invention include compositions of monoclonal and polyclonal antibodies. Preferably, the antibodies of this invention are monoclonal antibodies. The present invention contemplates whole antibodies and antigen-binding fragments. For example, suitable antigen-binding fragments include Fab ₂ , Fab and Fv antibody fragments. The antibodies of this invention may be labeled with a detectable moiety, such as a fluorescent molecule (eg, fluorescein or an enzyme).

The present invention also relates to methods and compositions for detecting and identifying an influenza virus of this invention and for diagnosing infection of an animal with an influenza virus of this invention. The methods of this invention include detecting the presence of canine influenza in a biological sample from an animal. The detection of canine influenza in a sample is useful for diagnosing canine influenza in an animal. In turn, this information may allow the prediction of an animal to be determined based on varying levels of canine influenza virus over time, and may assist in the selection of therapeutic agents and treatments for that animal and facilitate monitoring of therapy. This method also makes it possible to establish the absence of canine influenza in the test animal.

The ability to detect canine influenza in an animal allows evaluation of canine influenza outbreaks in different geographic locations. This information also enables early detection, so that infected animals can be isolated to limit the spread of the disease, and enables early intervention on treatment options. In addition, the availability of this information may enable the mobilization of medical personnel to treat large numbers of sick animals, including the collection of medical supplies and vaccines, if available.

In one embodiment, the method of this invention includes taking a biological sample from a test animal, such as a canine. This biological sample can be any biological material, including cells, tissue, hair, whole blood, serum, plasma, breast aspirate, pulmonary lavage, cerebrospinal fluid, saliva, sweat and tears.

An animal test sample may be from an animal suspected of having influenza virus, whether or not the animal exhibits symptoms of the disease. Control samples may also be provided or collected from animals known not to have canine influenza. Additional controls may also be provided, for example, to reduce false positives and false negatives and confirm that the reagents in this assay are actively detecting canine influenza A virus.

In addition to detecting the presence or absence of canine influenza in a biological sample, the detection methods used in this invention can detect mutations in the canine influenza virus, such as changes in the nucleic acid sequence that may occur due to environmental, drug treatment, genetic manipulation or mutations. , injury, change in diet, age or any other characteristic(s) of the animal. Mutations can also make the canine influenza A virus resistant to a previously effective drug, or make the virus able to infect and replicate in different animal and human species. For example, the avian influenza A virus has been shown to infect other animals and humans.

In one embodiment of the detection of influenza virus in an animal, diagnosis is facilitated by the collection of high quality samples, their rapid transport to testing equipment, and proper storage prior to laboratory examination. The virus is best detected in samples containing infected cells and secretions. In one embodiment, samples for direct detection of viral antigens and/or for isolation of nucleic acids and/or virus in cell culture are taken during the first 3 days after the onset of clinical symptoms. A number of sample types are useful for diagnosing viral infections of the upper respiratory tract, including, but not limited to, nasal swab, nasopharyngeal swab, nasopharyngeal aspirate, nasal wash, and throat swab. In addition to swabs, tissue or serum samples may be taken and invasive procedures may also be performed.

In one embodiment, respiratory samples are collected and transported in 1-5 ml of virus transport medium. A number of media that are satisfactory for the extraction of a wide variety of viruses are commercially available. Clinical specimens are added to the transport medium. Nasal swabs or nasopharyngeal swabs may also be transported in a virus transport medium. One example of a transport medium is 10 g of meat-peptone culture broth and 2 g of bovine albumin fraction V supplemented with sterile distilled water to 400 ml. Antibiotics such as 0.8 ml gentamicin sulfate solution (50 mg/ml) and 3.2 ml amphotericin B (250 µg/ml) may also be added. This medium is preferably sterilized by filtration. Nasal swabs, such as sterile saline (0.85% NaCl), can also be used to collect respiratory virus samples.

In one embodiment, serum is collected in an amount of 1-5 ml of whole blood from an acute phase animal, shortly after the onset of clinical symptoms and preferably no later than 7 days. A convalescent phase sample may also be taken, for example at about 14 days after the onset of symptoms. Serum samples may be useful for detecting antibodies against respiratory viruses in a neutralization test.

In some cases, samples may be collected from individual animals over a period of time (eg, once a day, once a week, once a month, twice a year, or once a year). Obtaining multiple samples from an individual animal over time can be used to confirm the results of earlier detections and/or to identify response or resistance to a particular treatment, such as a selected therapeutic drug.

The methods of the present invention can be used to detect the presence of one or more pathological agents in a test sample from an animal and the level of each pathological agent. Any method for detecting a pathological agent can be used, including, but not limited to, antibody assays, including enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody (IFA) tests, hemagglutination and hemagglutination inhibition (HI) and Western- blotting. Known methods using cell cultures can also be used. Positive cultures can be further identified using cell culture immunofluorescence or HI analysis of the cell culture medium (supernatant).

In addition, nucleic acid (DNA or RNA) or protein detection methods can be used. Such methods include, but are not limited to, polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR) assays and real-time PCR assays and assays using nuclease protection. There are commercially available test kits to perform these assays. For example, QIAGEN (Valencia, CA) sells a one-step RT-PCR kit and a viral RNA extraction kit.

In one embodiment, this method uses an antibody specific for the virus or viral protein of the invention. In a particular 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, for example from the nasal or nasopharyngeal region, is obtained from an animal and the virus or viral protein is isolated from it. These viral components are then screened for binding of an antibody specific for a protein, such as HA or NP, of the virus of this invention. In another embodiment, a serum sample (or sample containing another antibody) is obtained from the animal and the serum is screened for the presence of an antibody that binds to a virus protein of the invention. For example, an ELISA assay may be performed in which the walls of the plate have the HA and/or NP protein, or peptide fragment thereof, associated with the wall. The wall of the plate is then contacted with serum or antibody from the test animal. The presence in an animal of an antibody that binds specifically to the HA and/or NP protein indicates that the test animal is or has been infected with the influenza virus of the present invention.

In one embodiment, the presence of a pathological agent is detected by determining the presence or absence of antibodies against this agent in a biological sample. It may take some time (eg, months) after an animal is infected before antibodies can be detected in a blood test. Once formed, antibodies usually persist for many years, even after successful treatment of the disease. The detection of antibodies to the canine influenza virus cannot indicate whether the infection was recent or sometime in the past.

Antibody testing can also be performed on liquid(s). Antibody assays include enzyme-linked immunosorbent assays (ELISA), indirect fluorescent antibody (IFA) tests, and Western blotting. Preferably, antibody testing is performed using multiple assays, such as ELISA or IFA followed by Western blotting. ELISA is considered more reliable and accurate than IFA, but IFA can be used if ELISA is not available. Western blot analysis (which is a more specific test) can be performed in all animals, especially in animals that give positive or ambiguous (indeterminate) results in the ELISA or IFA analysis.

Other antibody-based tests that can be used to detect influenza virus include hemagglutination inhibition assays. Haemagglutination activity can be detected in a biological sample from an animal using chicken or turkey red blood cells, as described (Burleson et al., 1992 and Kendal et al., 1982). In one embodiment, an influenza virus or a HA protein or peptide of the invention is contacted with a test sample containing serum or antibody. Then red blood cells (RBC) from an animal such as a bird are added. If an anti-HA antibody is present, then these RBCs will agglutinate in the presence of HA. Variations and modifications to standard hemagglutination inhibition assays are known in the art and are contemplated within the scope of this invention.

An animal's infection can also be determined by virus isolation from a sample, such as a nasal swab or nasopharyngeal swab. Virus isolation can be performed using standard techniques, including cell culture and egg inoculation.

In a further embodiment, a nucleic acid-based assay may be used to detect the virus of the present invention. In one embodiment, a nucleic acid probe is obtained from an animal and the nucleic acid is subjected to PCR using primers that will generate an amplification product if the nucleic acid contains an influenza virus specific sequence of the invention. In a specific embodiment, RT-PCR is used in the assay for a given virus. In an exemplary embodiment, real-time RT-PCR is used to analyze the influenza virus of the present invention. PCR, RT-PCR and real-time PCR methods are known in the art and have been described in US Patent Nos. 4,683,202; 4683195; 4800159; 4965188; 5994056; 6814934 and in Saiki et al. (1985); Sambrook et al. (1989); Lee et al (1993) and Livak et al. (1995). In one embodiment, the PCR assay uses oligonucleotides specific for the influenza matrix (MA) gene and/or the HA gene. The amplification product can also be sequenced to determine if the product has the sequence of the influenza virus of the present invention. Other nucleic acid-based assays may be used to detect and diagnose viral infection with the virus of this invention, and such assays are contemplated within the scope of this invention. In one embodiment, a probe containing a nucleic acid is subjected to PCR-based amplification using forward and reverse primers, where these primers are specific for a viral polynucleotide or gene sequence. If the nucleic acid in this sample is RNA, then RT-PCR can be performed. For real-time PCR with these primers, a detectable probe is used.

Primer sets specific for the hemagglutinin (HA) gene of many circulating influenza viruses are known and are being continuously developed. The influenza virus genome is a single-stranded RNA and a DNA copy (cDNA) must be made using reverse transcriptase (RT) polymerase. Amplification of the RNA genome, for example using RT-PCR, requires a pair of oligonucleotide primers, usually designed based on the known sequence of the HA subtypes of influenza A and neuraminidase (NM)-I. These primers can be chosen such that they will specifically amplify the RNA of only one virus subtype. DNA generated using subtype specific primers can be further analyzed by molecular genetic methods such as sequencing. This test is preferably performed with a positive control or the products are confirmed by sequencing and comparison to known sequences. The absence of PCR target products (i.e. "negative (negative) result")) may not exclude the presence of this virus. Results can then be available within hours, either from clinical swabs or from infected cell cultures. PCR and RT-PCR tests for influenza A virus are described by Fouchier et at, 2000 and Maertzdorf et al., 2004.

The present invention also relates to methods for screening for compounds or drugs that have antiviral activity against the virus of the present invention. In one embodiment, cells infected with a virus of this invention are contacted with a test compound or drug. Then determine the amount of virus or viral activity after contact. Compounds or drugs that exhibit antiviral activity may be selected for further evaluation.

The present invention also relates to isolated cells infected with the influenza virus of the present invention. In one embodiment, these cells are cells from a canine animal, for example, canine kidney epithelial cells.

The present invention also relates to cells transformed with a polynucleotide of the present invention encoding a polypeptide of the present invention. Preferably, this polynucleotide sequence is provided in the expression construct of this invention. More preferably, this expression construct allows for overexpression in the cell of an operably linked polynucleotide of this invention. In one embodiment, the cell is transformed with a polynucleotide sequence containing a sequence encoding the amino acid sequence shown in any of 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 functional fragment or variant thereof . In a specific embodiment, this cell is transformed with a polynucleotide encoding the amino acid sequence shown in 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 and contains the nucleotide sequence shown in 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, 65 , 67, 69, 71, 73, 75, or 77, respectively, or a sequence encoding a functional fragment or variant of any of 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. Thus, the present invention relates to cells transformed with a polynucleotide sequence containing the nucleotide sequence shown in any of 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, or a fragment or variant, including a degenerate variant, any of 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, 65, 67, 69, 71, 73, 75 or 77.

The transformed cell may be a eukaryotic cell, eg a plant cell, including a protoplast, or the transformed cell may be a prokaryotic cell, eg a bacterial cell, such as E. coli or B. subtilis. Animal cells include human cells, mammalian cells, in particular canine cells, avian cells and insect cells. Plant cells include, but are not limited to, dicotyledonous, monocotyledonous, and coniferous plant cells.

The present invention also relates to plants, including transgenic plants, which express and produce the viral protein or polypeptide of the present invention. This invention contemplates plants, plant tissues and plant cells transformed or bred to contain a polynucleotide of this invention. Preferably, the polynucleotide of this invention is overexpressed in the plant, plant tissue or plant cell. Plants can be used to make vaccine compositions of this invention and these vaccines can be administered through consumption of this plant (see, for example, US Patent Nos. 5,484,719 and 6,136,320).

The present invention also relates to kits for detecting a virus or diagnosing infection with a virus of the present invention. In one embodiment, the kit contains an antibody of this invention that specifically binds to an influenza virus of this invention, or an antigenic portion thereof. In another embodiment, the kit contains one or more polypeptides or peptides of the present invention. In a specific embodiment, these polypeptides have the amino acid sequence shown in any of 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, or a functional and/or immunogenic fragment or variant thereof. In an additional embodiment, the kit contains one or more polynucleotides or oligonucleotides of the present invention. In a specific embodiment, these polynucleotides have the nucleotide sequence shown in any of 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, or a fragment or variant thereof. The kit may optionally contain one or more control antibodies, control polypeptides or peptides and/or control polynucleotides or oligonucleotides. The antibody, polypeptides, peptides, polynucleotides and/or oligonucleotides of this kit may be provided in a suitable container or suitable package.

The present application also relates to the use of non-purebred dogs (mutts) as a model for the infection and pathogenesis of the influenza virus. In one embodiment, the off-bred dog is inoculated with an influenza virus, such as the canine influenza virus of the present invention. Optionally, the dog may be administered therapeutic agents after inoculation. The dog may also be administered a composition to generate an immune response against the influenza virus prior to inoculation with the virus. Tissue, blood, serum, and other samples may be obtained before and/or after inoculation and tested for virus presence and tissue pathogenesis using methods known in the art, including, but not limited to, PCR, RT-PCR, sequencing. nucleic acids and immunohistochemistry.

Canine influenza virus strains (designated “A/canine/Florida/43/2004” and “A/canine/Florida/242/2003”) have been deposited with the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, October 9, 2006. Canine influenza virus strains (designated “canine/Jax/05” and “canine/Miami/05”) were deposited with the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, October 17, 2006. The submitted viral strains have been deposited under conditions that ensure that access to these cultures will be granted during the pending period of this patent application to a person designated by the Commissioner of Patents and Trademarks, who is entitled to do so under 37 CFR 1.14 and 35 U.S.C. 122. This deposit will be made available as required by foreign patent laws in the countries in which copies of the submitted application or continuation thereof are registered. However, it should be understood that the availability of a deposit does not confer a license to practice a submitted invention that derogates from patent rights granted by a government decision.

In addition, the virus deposits provided will be stored and made available to the public in accordance with the terms of the Budapest Microorganism Deposit Treaty, i.e. they will be kept with all precautions necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for a deposit sample, and in any event for a period of at least thirty (30) years after the date of deposit, or during the enforceable term of any patent that may challenge the disclosure of that crop. The depositor acknowledges the obligation to replace the deposit if the depositary is unable to provide the requested sample due to the condition of the deposit. All restrictions regarding the availability to the public of the provided culture deposit will be unconditionally removed upon the issuance of a patent disclosing it.

Table 57 illustrates the similarities among the amino acid sequences encoded by the hemagglutinin (or “HA”), neuraminidase (or “NA”), and nucleoprotein (NP) genes of canine influenza virus identified as A/canine/Florida/43/2004 (Ca/Fla/ 43/04), with equine H3N8 isolates, as well as canine/Florida/242/2003 isolate.

Any element of any embodiment disclosed herein may be combined with any other element or embodiment disclosed herein, and such combinations are specifically intended to be within the scope of this invention.

All patents, patent applications, and publications referenced and cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent that they are not inconsistent with the exact statements of this application.

MATERIALS AND METHODS FOR EXAMPLES 1-6

Collection of blood and nasal swabs from greyhounds

Acute and convalescent blood samples were collected by jugular vein puncture from clinically ill or healthy greyhounds in dog racing kennels where outbreaks of respiratory disease occurred. Samples in the convalescent phase were collected 4–12 weeks after the collection of acute phase samples. Serum was collected and stored at -80°C. Nasal swabs were collected and placed on Amies charcoal transport medium (Becton Dickinson Biosciences) pending submission for bacterial isolation.

Post-mortem study of greyhounds (autopsy)

Complete post-mortem examinations were performed by the Anatomic Pathology Service at the University of Florida College of Veterinary Medicine (UF CVM) on 5 of 8 greyhounds that died during an outbreak in January 2004 at a track in Florida. Post-mortem examination of another dog was performed at a private veterinary clinic with tissues provided in the UF CVM for histopathological diagnosis. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and 5 μm sections were either stained with hematoxylin and eosin for histopathological diagnosis or processed for immunohistochemistry as described below. Unfixed tissues were provided for bacterial culture and also stored at -80°C.

Serological tests for canine viral respiratory pathogens

Paired acute and convalescent samples were provided to the Animal Health Diagnostic Laboratory (AHDL) at the Cornell University College of Veterinary Medicine for serum neutralization assays against canine distemper virus, adenovirus type 2, and parainfluenza virus. Antibody titers are expressed as the last serum dilution that inhibited viral infection of cell cultures. Seroconversion, defined as a ≥4-fold increase in antibody titer between acute and convalescent samples, was indicative of viral infection. No seroconversions were detected against these viral pathogens.

Microbial tests for canine bacterial respiratory pathogens

Paired swab and mortem samples were provided to the Diagnostic Clinical Microbiology/Parasitology/Serology Service at the UF CVM for bacterial isolation and identification. These samples were cultured on non-selective media as well as selective media for Bordetella species (Regan-Lowe; Remel) and Mycoplasma species (Remel). All cultures were held for 21 days before reporting no growth. Nose swabs from some of these greyhounds were also provided to the Department of Diagnostic Medicine/Pathobiology at the Kansas State University College of Veterinary Medicine for bacterial culture. Of 70 clinically ill dogs tested, Bordetella bronchiseptica was isolated from the nasal cavity of 1 dog, while Mycoplasma spp. isolated from the nasal cavity of 33 dogs. Pasteurella multocida has been commonly isolated from the nasal cavity of dogs with purulent nasal discharge. Two of these dogs, which died during the January 2004 outbreak, had limited growth of Escherichia coli in the lungs after death, one dog had limited growth of E. coli and Streptococcus canis, and the other had limited growth of Pseudomonas aeruginosa and yeast. Neither Bordetella bronchiseptica nor Mycoplasma have been isolated from the trachea or lungs of dogs that have died.

Isolation of the virus from post-mortem tissues

Frozen tissues were thawed and homogenized in 10 volumes of minimal maintenance medium (MEM) supplemented with 0.5% bovine serum albumin (BSA) and antibiotics. Cell solids were removed by centrifugation and supernatants were inoculated onto cultured cells or into 10 day old embryonated hen eggs. Tissue homogenates from greyhounds that had died were inoculated into various cell cultures that supported the replication of a wide range of viral pathogens. These cell cultures included Vero cells (African green monkey kidney epithelial cells, ATCC No. CCL-81), A-72 (canine tumor fibroblasts, CRL-1542), HRT-18 (human rectal epithelial cells, CRL-11663) , MDCK (canine kidney epithelial cells, CCL-34), primary canine kidney epithelial cells (AHDL, Cornell University), primary canine lung epithelial cells (AHDL), and primary testicular bovine cells (AHDL). MDCK and HRT cells were cultured in MEM supplemented with 2.5 μg/ml TPCK-treated trypsin (Sigma); the remaining cell lines were cultured in MEM supplemented with 10% fetal calf serum and antibiotics. Cells were grown in flasks 25 cm ² at 37°C in a humidified atmosphere containing 5% CO ₂ . A control culture was inoculated with supplemented MEM. These cultures were observed every day for morphological changes and harvested at 5 days post-inoculation. Collected fluids and cells were clarified by centrifugation and inoculated onto fresh cells as described for initial inoculation; performed two blind passages. Haemagglutination activity in clarified supernatants was determined using chicken or turkey erythrocytes as described (Burleson et al, 1992; Kendal et al, 1982). For virus isolation in chick embryos, 0.1 ml of tissue homogenate was inoculated into allantois and incubated for 48 hours at 35°C. After two blind passages, hemagglutination activity in allantoic fluids was determined as described (Burleson et al, 1992; Kendal et al, 1982).

RT-PCR, nucleotide sequencing and phylogenetic analyzes

Total RNA was extracted from tissue culture supernatant or allantoic 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 region of 8 genes of influenza viruses in cDNA was performed as previously described (Klimov et al, 1992a), using universal sets of gene-specific primers. These resulting DNA amplicons were used as templates for automated sequencing on an Applied Biosystems 3100 automated synthesizer using dye-terminated cycle sequencing (ABI) chemistry. Nucleotide sequences were analyzed using GCG Package ^® , Version 10.0 (Accelyrs) (Womble, 2000). Phylogeny Inference Package Version 3.5 was used to evaluate phylogenies and calculate bootstrap values from these nucleotide sequences (Felsenstein, 1989). Phylogenetic trees were compared with phylogenetic trees obtained by neighbor joining analysis (method of constructing a phylogenetic tree based on the principle of minimal evolution) using the Tamura-Nei gamma model implemented in the MEGA ^® software (Kumar et al, 2004) and validated using the PAUP ^® software 4.0 Beta (Sinauer Associates).

Experimental inoculation of dogs

Four 6-month-old specific pathogen-free beagles (short-legged beagle dogs) [(2 males and 2 females (Liberty Research)] were used. Physical examination and background blood tests, including CBC/leukocyte count, serum chemistry panel, and urinalysis determined that these animals were healthy.They were housed together in BSL2-enhanced equipment provided by the Association for Assessment and Accreditation of Laboratory Animal Care.Background rectal temperatures were recorded twice daily for 7 days.Dogs were anesthetized with an intravenous injection of propofol ( Diprivan®, Zeneca Pharmaceuticals, 0.4 mg/kg bw for effect) for endotracheal tube intubation Each dog was inoculated with a total dose of ^106.6 medium tissue culture infectious doses (TCID ₅₀ ) of virus A/Canine/Florida/43/ 2004 (Canine/FL/04) (H3N8), with half of this dose administered into the distal trachea via an endotracheal tube and the other half administered into the deep nasal passage via a catheter. Physical examinations and rectal temperature recordings were performed twice daily for 14 days post-inoculation (pi). Blood samples (4 ml) were collected by jugular vein venipuncture on days 0, 3, 5, 7, 10, and 14 pi Nasal and nasopharyngeal samples were collected with a polyester fiber swab (Fisher Scientific) from each dog on days 0 to 5, 7, 10 and 14 pi These swabs were placed in medium for virus transport (Remel) and stored at –80°C. Two dogs (1 male and 1 female) were euthanized by intravenous inoculation with Beuthanasia-D® solution (1 ml/5 kg body weight; Schering-Plough Animal Health Corp) on day 5 pi and the remaining 2 dogs on day 14 for post-mortem examination. Tissues for histological analysis were processed as described. Virus culture tissues were stored at -80°C. This study was approved by the University of Florida Institutional Animal Care and Use Committee.

Isolation of the virus by the host from experimentally inoculated animals

Serial dilutions of lung homogenates and swab extracts obtained by clarification of media for transport of smears by centrifugation were placed in MEM supplemented with 0.5% BSA and antibiotics. Plaque assays were performed as described (Burleson et al, 1992) using monolayers of MDCK cells in 6-well tissue culture dishes. Monolayers of inoculated cells were overlaid with supplemented MEM containing 0.8% agarose and 1.5 μg/ml TPCK-trypsin. Cells were cultured for 72 hours at 37° C. in a humidified atmosphere containing 5% CO ₂ before being fixed and stained with crystal violet. Virus concentration was expressed as plaque forming units (PFU) per gram of tissue or per swab.

Immunohistochemistry

Deparaffinized and rehydrated 5 µm sections of lung tissue from greyhounds and beagles were mounted as preparations on Bond-Rite™ slides (Richard-Allan Scientific, Kalamazoo, MI) and then treated with proteinase K (DakoCytomation, Carpenteria, CA) followed by peroxidase blocking reagent (Dako® EnVision™ Peroxidase Kit, Dako Corp.). These sections were incubated with 1:500 dilutions of monoclonal antibodies to canine distemper virus (VMRD, Inc.), canine adenovirus type 2 (VMRD, Inc.), canine parainfluenza virus (VMRD, Inc.), or influenza A virus H3 (Chemicon International, Inc.) for 2 hours at room temperature. Controls included incubating the same sections with mouse IgG (1 mg/ml, Serotec, Inc.) and incubating these monoclonal antibodies with healthy canine lung sections. After treatment with primary antibodies, these sections were incubated with secondary immunoperoxidase reagents and peroxidase substrate (Dako® EnVision™ Peroxidase Kit, Dako Corp.) according to the manufacturer's instructions. These sections were counterstained with hematoxylin, treated with Clarifier #2 and Bluing Reagent (Richard-Allan Scientific, Kalamazoo, MI), dehydrated, and cover slips placed using permount (ProSciTech).

Hemagglutination inhibition (HI) assay

Serum samples were incubated with receptor-degrading enzyme (RDE, Denka) (1 part serum: 3 parts RDE) for 16 hours at 37°C before heat inactivation for 60 minutes at 56°C. Virus A/Canine/FL/04 (H3N8) was grown in MDCK cells for 36-48 hours at 37°C. Virus culture supernatants were collected, clarified by centrifugation and stored at -80°C. HI analysis was performed as previously described (Kendal et al, 1982). Briefly, 4 hemagglutination units of virus in 25 μl were added to an equal volume of serially diluted serum in microtiter wells and incubated at room temperature for 30 minutes. An equal volume of 0.5% v/v was added. turkey erythrocytes and hemagglutination titers were determined approximately visually after 30 minutes. The final HI titer was defined as the last serum dilution that completely inhibited hemagglutination. Seroconversion was defined as a ≥ 4-fold increase in titer between paired acute and convalescent samples. Individual sample seropositive was defined as HI antibody titer ≥1:32.

Microneutralization (MN) analysis

Serum neutralizing antibody responses to A/Canine/FL/04 (H3N8) virus were detected by the MN assay as described previously (Rowe et al, 1999), except that canine sera were treated with RDE as described above prior to this assay. The final titer was defined as the highest serum dilution that gave 50% neutralization of 100 TCID _5O of the virus. Seroconversion was defined as a ≥4-fold increase in MN titer between paired acute and convalescent phase trials. The seropositive reaction of an individual sample was defined as an MN titer ≥1:80.

The following describes examples that illustrate procedures for practicing this invention. These examples should not be considered as limiting this invention. All percentages are wt % and all solvent mixture ratios are by volume unless otherwise indicated.

EXAMPLE 1

In January 2004, there was an outbreak of a respiratory disease in 22 greyhounds kept in 2 kennels at a track (dog racing track) in Florida and a local farm that supplied dogs to these kennels. Approximately 60 dogs were kept in each kennel building and 300 dogs were kept on the farm. This outbreak occurred over a 6-day period after which no new cases were detected. Fourteen of the 22 dogs had fevers of 39.5–41.5°C, mild cough with gagging within 10–14 days, and eventually recovery. Of the remaining 8 dogs, 6 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 mouth and nose bleeding due to rapid deterioration. Both of these dogs had a temperature of 41°C. Four of the 8 deaths occurred in the kennel buildings and 4 occurred on the farm. Fifty percent of deaths occurred on the 3rd day of the outbreak (epidemic). The 22 dogs had an age range of 17 months to 4 years, but 73% were 17 months to 33 months old.

Two clinical syndromes were evident: a milder illness characterized by an initial fever and then a cough within 10-14 days (14 dogs) followed by recovery, or a fulminant death associated with respiratory haemorrhage (8 dogs with a mortality rate of 36%). Post-mortem examinations were performed in 6 of 8 deaths. All dogs had extensive bleeding in the lungs, mediastinum and pleural cavity. Histological examination of the respiratory tract revealed that, along with pulmonary bleeding, all dogs had tracheitis, bronchitis, bronchiolitis and purulent bronchopneumonia (figure 3). The epithelial lining and airways in these tissues were infiltrated with neutrophils and macrophages. Lung homogenates obtained from these dogs were inoculated into various simian, human, bovine and canine cell lines to obtain a virus culture. A single dog lung homogenate produced cytopathic (showing cell damage) effects in trypsin-cultured Madin-Darby canine kidney epithelial cells (MDCK) and agglutinated chicken erythrocyte cell culture supernatant. Preliminary evidence for influenza A virus was obtained by commercial ELISA for the detection of the nucleoprotein of influenza A and B viruses and PCR analysis using primers specific for the influenza A virus matrix gene. antisera specific for H1-H11 and H113 human influenza A subtypes (Table 3). To characterize the molecular properties of this virus, the inventors determined the nucleotide sequences of 8 RNA segments of this viral genome. Sequence comparisons with known influenza virus genes and phylogenetic analyzes indicated that the 8 canine isolate genes were most similar to genes from modern equine influenza A (H3N8) viruses, with which they shared ≥96-97% sequence identity (Figure 1A, Table 4 ). In contrast, representative genes from avian, porcine, and human influenza A isolates had ≤94% identity with this canine isolate (Table 4). These data identified the canine isolate A/Canine/Florida/43/2004 (Canine/FL/04) as H3N8 influenza A virus, closely related to modern lineages of equine influenza viruses. Since all the genes of the canine isolate were derived from the equine influenza virus, the inventors concluded that the entire genome of the equine influenza virus had been transferred to the dog.

EXAMPLE 2

To investigate the role of the Canine/FL/04 virus in clinical and pathological observations in greyhounds, the inventors performed immunohistochemical staining (IHC) on lung tissues using a monoclonal antibody to H3 virus A. The H3 viral antigen was consistently detected in the cytoplasm of bronchial and bronchiolar epithelial cells, cells of the bronchial gland and macrophages in the lumen of the respiratory tract and alveolar spaces (Fig.2A). These data support the diagnosis of pulmonary H3 influenza virus infection in multiple dogs.

EXAMPLE 3

To determine the involvement of Canine/FL/04-like virus in the etiology of a respiratory outbreak, the inventors analyzed paired acute and convalescent sera from 11 sick dogs and 16 asymptomatic dogs exposed to this virus by haemagglutination (HI) and microneutralization (MN) assays. . Seroconversion, defined as a ≥ 4-fold increase in anti-Canine/FL/04 antibody titer from acute to convalescent phase, occurred in 8 of 11 (73%) affected dogs in both analyzes (Table 1). Seroconversion occurred in 6 of 16 (38%) asymptomatic contact dogs in the HI assay, while 8 of 16 (50%) seroconverted in the MN assay (Table 1). These seroconversion results demonstrated infection of the dogs with Canine/FL/04-like virus, which coincided with the onset of respiratory disease in most of the animals.

Single sera samples were collected 3 months after the outbreak from an additional 46 asymptomatic dogs housed with affected dogs. Of these, 43 (93%) were seropositive in both analyses, including 82% (9/11) of affected dogs and 95% (59/62) of healthy contact dogs. This high seroprevalence in dogs with no history of respiratory disease indicates that most canine influenza virus infections are subclinical and suggest effective spread of the virus in dogs. It is known that subclinical infections contribute to the spread of the virus.

EXAMPLE 4

To better understand the ability of the Canine/FL/04 virus to infect dogs, four 6-month-old breeding beagles were each inoculated with ^106.6 tissue culture median infective doses (infectious units) (TCID ₅₀ ) by intratracheal and intranasal routes. All dogs developed fever (rectal temperature ≥39°C) during the first 2 days post-inoculation (pi), but none exhibited respiratory symptoms such as cough or nasal discharge during the 14 day time period. The release of the virus into the environment was investigated by quantitative determination of the virus in swabs from the nose and oropharyngeal swabs. Only 2 out of 4 dogs shed detectable amounts of virus. One dog shed virus on days 1 and 2 pi (1.0-2.5 log ₁₀ PFU per swab), while the other dog shed virus for 4 consecutive days post-inoculation (1.4-4.5 log ₁₀ PFU per swab). smear). Pathological examination of 2 dogs on day 5 pi revealed necrotizing and hyperplastic tracheitis, bronchitis, and bronchiolitis similar to the tracheitis, bronchitis, and bronchiolitis found in spontaneous disease in Greyhounds, but there was no pulmonary hemorrhage or bronchopneumonia. Viral H3 antigen was detected in the cytoplasm of epithelial cells of the bronchi, bronchioles and bronchial glands using IHC (FIG. 2B). The infectious virus was isolated from the lung tissue of one of these dogs. Pathological examination of the remaining 2 dogs on day 14 pi showed minimal histological changes in respiratory tissues, no viral antigen according to IHC, and no virus isolation from lung homogenates. Seroconversion in these last 2 dogs was detected in MN assays on day 7 pi, with an additional 2-3-fold increase in antibody titers on day 14. These results established the dogs' susceptibility to Canine/FL/04 infection as evidenced by a febrile reaction, the presence of viral antigen and infectious virus in the lung parenchyma, histopathological features typical of influenza, and seroconversion. The lack of success in reproducing severe disease and death in experimentally inoculated Beagles is not surprising, as a large proportion of naturally infected greyhounds were asymptomatic.

EXAMPLE 5

To investigate whether a Canine/FL/04-like influenza virus was circulating in Florida greyhound populations prior to the January 2004 outbreak, archival sera from 65 greyhounds were tested for the presence of antibodies to Canine/FL/04 using HI and MN assays. . There were no detectable antibodies in 33 dogs sampled from 1996 to 1999. Of 32 dogs sampled between 2000 and 2003, 9 were seropositive in both assays: 1 in 2000, 2 in 2002, and 6 in 2003 (Table 5). Seropositive dogs have been found in racetracks in Florida during outbreaks of respiratory disease of unknown etiology in 1999-2003, suggesting that Canine/FL/04-like virus may be the etiological factor in these outbreaks. To further explore this possibility, the inventors tested archival tissues from greyhounds that died of hemorrhagic bronchopneumonia in March 2003. Lung homogenates from one dog, inoculated into MDCK cells and chick embryos, gave rise to H3N8 influenza virus named A/Canine/Florida/242/2003 (Canine/FL/03). Whole genome sequencing analysis of Canine/FL/03 revealed >99% identity with Canine/FL/04 (Table 4), indicating that Canine/FL/04-like viruses were still infecting greyhounds prior to 2004.

EXAMPLE 6

From June to August 2004, outbreaks of respiratory disease occurred in thousands of greyhounds at 14 racetracks in Florida, Texas, Alabama, Arkansas, West Virginia and Kansas.

Officials at some of these tracks estimated that at least 80% of their dog population had clinical disease. Most of these dogs had clinical symptoms of fever (≥39°C) and cough similar to those of dogs in the January 2004 epidemic, but many dogs also had mucopurulent nasal discharge. Multiple deaths were reported, but an exact death rate could not be determined.

The present inventors collected paired acute and convalescent sera from 94 dogs on 4 tracks in Florida: 56% of these dogs had ≥4-fold increases in anti-Canine/FL/04 antibody titers and 100% were seropositive (Table 6) . Convalescent sera from 29 dogs in West Virginia and Kansas also had antibodies to Canine/FL/04. The inventors isolated influenza A (H3N8) virus from the lungs of a greyhound that died of hemorrhagic bronchopneumonia on a track in Texas. Full genome sequencing analysis of this isolate, named A/Canine/Texas/1/2004 (Canine/TX/04), revealed ≥ 99% identity to Canine/FL/04 (Table 4). The isolation of three closely related influenza viruses from canine deaths over a 13-month time period and from different geographic locations, together with substantial serological evidence of widespread infection in racing greyhounds, suggested sustained circulation of a Canine/FL/04-like virus in the dog population. .

Phylogenetic analysis of the Canine/FL/03, Canine/FL/04 and Canine/TX/04 HA genes showed that they consist of a monophyletic (monocentric) group with a strong (sustainable) bootstrap support, which is clearly different from the modern H3 genes of equine viruses. isolated in 2002 and 2003 (Figure 1B). Phylogenetic analyzes and pairwise comparisons of the nucleotide sequences of the other 7 genomic segments supported the segregation of canine genes into a distinct sublineage most closely related to the equine virus lineage (data not shown and Table 4). Clustering of canine influenza genes as a monophyletic group separate from equine virus is also confirmed by the presence of 4-signature amino acid changes in HA (Table 2). Together with serological results from 2003 and 2004, these data are consistent with a single event of transmission of the virus from horses to dogs, followed by a "horizontal" spread of this virus in the greyhound population. However, repeated introductions of this unique influenza virus lineage from unidentified reservoir viruses cannot formally be ruled out, improbable as it may seem.

Viral HA is a critical determinant of influenza virus host species specificity (Suzuki et al, 2000). To identify residues in HA that may be associated with adaptation in the canine host, the present inventors compared the deduced amino acid sequence of canine HA with that of modern equine viruses. Four amino acid changes differentiate the mature equine and canine HA consensus amino acid sequences: N83S, W222L, I328T, and N483T (see Table 2). Canine viruses have an amino acid deletion compared to the equine consensus. Thus amino acid position 7 in the equine HA sequence is position 6 in the canine HA sequence, amino acid position 29 in the equine HA sequence is position 28 in the canine HA sequence, position 83 is position in the equine HA sequence is position 82 in the canine HA sequence, and etc. Thus, these four amino acid substitutions are at positions 82, 221, 327, and 482 of the amino acid sequence shown in SEQ ID NO: 33 and SEQ ID NO: 34. The substitution of serine for asparagine at position 83 of the consensus sequence is a change of unknown functional significance, since different polar residues have been found in H3 molecules from other species. The strongly conserved isoleucine at position 328 of the consensus sequence near the HA H3 cleavage site was replaced by threonine. The central role of HA cleavage by host proteases in pathogenesis suggests that this change deserves further investigation. The tryptophan leucine substitution at position 222 of the consensus sequence is important as it is a non-conservative change adjacent to the sialic acid binding pocket that could modulate receptor function (Weis et al, 1988). Interestingly, the leucine at position 222 is not unique to canine H3 HA, as it is commonly found in the H4, H8, H9, and H12 subtypes of HA (Nobusawa et al, 1991; Kovacova et al, 2002). This leucine substitution may be more consistent with the mammalian host specificity of the virus, as infections of pigs with the H4 subtype (Karasin et al, 2000) and humans and pigs with the H9 subtype (Peiris et al, 1999) of the viruses have been reported. Replacement of asparagine with threonine at position 483 of the consensus sequence resulted in the loss of a glycosylation site in the HA2 subunit, which is conserved across all HA subtypes (Wagner et al, 2002). Although the importance of these amino acid changes in HA for adaptation of the equine virus to dogs has yet to be determined, similar amino acid changes have been observed previously in association with interspecies transfer (Vines et al, 1998; Matrosovich et al, 2000). Amino acid differences between other influenza viral proteins of this invention and the equine consensus sequence are shown in Tables 19-25.

The source of the equine influenza virus that originally infected greyhounds remains speculative. Track kennels are not located near racetracks (racing tracks), suggesting that contact between greyhounds and virus-releasing horses is not a sufficient explanation for the multiple outbreaks in various states in 2004. A potential source of exposure to the equine virus is the feeding of horsemeat to greyhounds whose diet is supplemented with raw meat supplied by meat processing plants that process carcasses, including horses that may carry the influenza virus. Precedents for this mode of infection include reports of interspecies transmission of the H5N1 avian influenza virus to zoo pigs and felines fed chicken carcasses (Webster, 1998; Keawcharoen et al, 2004; Kuiken et al, 2004). Although this route is a likely route for the initial introduction of equine influenza virus into dogs, it does not explain the recent numerous outbreaks of influenza in thousands of dogs in various states. An experimental study of the inoculation of this invention showed the presence of the virus in the nasal passages and oropharynx of dogs, albeit in moderate titers. However, these results indicate that environmental isolation of the virus is possible and that dog-to-dog transmission of the virus by large droplet aerosols, vomit, or direct mucosal contact could play a role in the epidemiology of this disease.

Cross-species transfer of an entire mammalian influenza virus into unrelated mammalian species is a rare event. Earlier studies provided limited serological or virological evidence, but not both, of transient infection of dogs with human influenza A (H3N2) viruses (Nikitin et al., 1972, Kilbourne, et al, 1975; Chang et al, 1976; Houser et al, 1980). However, there is no evidence of sustained circulation in the canine host. Although the direct transfer of swine influenza viruses from pigs to humans is well documented (Dacso et al, 1984; Kimura et al, 1998; Patriarca et al, 1984; Top et al, 1977), there is no evidence for adaptation of swine viruses in the human host. In this communication, the present inventors provide virological, serological and molecular evidence for cross-species transmission of complete influenza A (H3N8) virus into another mammalian species, the dog. The unique amino acid substitutions in canine virus HA, coupled with serological confirmation of canine infection in numerous states in the US, suggest adaptation of this virus to the canine host. Because dogs are primarily a companion animal for humans, these discoveries have implications for public health; dogs may create a new source for the transfer of new influenza A viruses to humans.

Table 1 Antibody reaction to A/Canine/Florida/43/2004 (H3N8) Sick dogs (11) ^a Healthy, in contact with diseased dogs (16) ^b Reaction HI ^{c SNd} HI SN Seroconversion (%) ^e 73 73 38 50 Seropositive (%) ^f 82 82 100 100 Geometric mean titer ^g 329 424 268 431 ^a Number of dogs with clinical signs of disease.
^b Number of asymptomatic dogs kept in contact with clinically ill dogs.
^c Agglutination inhibition (HI) assay using virus A/Canine/Florida/43/2004.
^d Microneutralization (MN) assay using virus A/Canine/Florida/43/2004.
^e Percentage of dogs with at least a 4-fold increase in antibody titer in paired acute and convalescent sera.
^f Percentage of dogs with a positive antibody titer (HI titer ≥32: MN titer ≥80) in convalescent sera.
^g Geometric mean antibody titer for convalescent sera.

table 2 Amino acid differences between canine and equine H3 hemagglutinins Consensus H3 equine Can/FL/03 Can/FL/04 Can/TX/04 Potential functional sequence G7* D _† - D is also found in duck and human H3 HA 129 - M M I is conserved in H3 HA of all species N83 S S S Various polar amino acids are present at this position in the H3 HA of other species. S92 - N - N is present in some H3 HA L118 - - V L retained in all H3 ON W222 L L L W is retained in most H3 HAs of all species; localized near the receptor binding site A272 V A V V is present in some recent equine isolates I328 T T T T is strictly conservative in all HA in birds, pigs, or humans. N483 T T T N occurs in all RA and other HA subtypes. The substitution results in the loss of a glycosylation site K541 - R - Conservative replacement of a basic amino acid * Amino acid residue (single letter code) and position in mature HA H3. Amino acid code: A=alanine, D=aspartic acid, G=glycine, I=isoleucine, K=lysine, L=leucine, M=methionine, N=asparagine, R=arginine, S=serine, T=threonine, V= valine, W=tryptophan. † Denotes no change from H3 equine consensus.

Table 3 Inhibition of haemagglutination of virus isolate by reference antisera to various HA serotypes Reference antisera Specificity for NA HI ^a titer Puerto Rico/8/34 H1 5 Pig /Iowal 5/30 H1 5 Singapore/01/57 H2 5 Shanghai/11/87 ^H3b 5 Equine/Miami/1/63 H3 160 Duck/Czechoslovakia/5 6 H4 5 Triad/South Africa/61 H5 5 Turkey/Massachussetts/65 H6 5 Fowl Plague/Dutch/27 H7 5 Fowl Plague/Rostock/34 H7 5 Equine/Prague/1/56 H7 5 Turkey/Ontario/6118/68 H8 5 Quail/Hong Kong/Gl/97 ^H9b 5 Chicken/Hong Kong/G9/97 ^H9b 5 Chicken/Germany/49 H10 5 Duck/England/56 H11 5 Seagull/Maryland/704/77 H13 5 Healthy Sheep Serum - 5 Healthy ferret serum - 5 ^a Haemagglutination inhibition titer against virus isolate from dog no. 43.
^b Polyclonal antisera were obtained in ferrets, while all other antisera were obtained in sheep or goats.

Table 4

Homology of the gene sequence A/Canine/Florida/43/2004 (H3N8) with influenza A strains of horses, birds, pigs and humans

^a Percent nucleotide and amino acid sequence identity (in parentheses) of the Canine/Florida/43/2004 (H3N8) genes with the most homologous gene of influenza virus isolates of the species, followed by GenBank sequence database access numbers.

^b Not applicable: N8 neuraminidase has never been reported in human or porcine viruses.

Table 5 Antibody titers to A/Canine/Florida/43/2004 (H3N8) in greyhound sera collected in 1996-2003 Year ^a 1996 1997 1998 2000 2002 2003 Number of dogs tested 8 6 19 4 6 22 Number of seropositive dogs 0 0 0 1 2 6 Antibody titers 512 232.524 280-2242 ^a Year of serum sampling from beagle hounds in Florida.
^b Antibody titers using microneutralization assay for seropositive dogs, including the 2003 range for six seropositive dogs.

Table 6 Antibody reaction to A/Canine/Florida/43/2004 (H3N8) in 4-track Florida Greyhounds in June 2004 Reaction Track A Track B Track C Track D Number of dogs tested ^a 37 10 22 25 Seroconversion (%) ^b 46 90 100 64 Seropositive (%) ^with 100 100 100 100 Geometric mean titer ^d 401 512 290 446 ^a Number of clinically ill dogs tested with HI using A/canine/Florida/43/2004 (H3N8).
^b Percentage of dogs with a ≥4-fold increase in antibody titer between acute and convalescent sera.
^With. Percentage of dogs with positive antibody titer (HI titer >16) in convalescent sera.
^d Geometric mean antibody titer for convalescent sera.

MATERIALS AND METHODS FOR EXAMPLES 7-11

Canine fabrics

Post mortem examinations were performed by the Anatomic Pathology Service at the University of Florida College of Veterinary Medicine on 6 mixed breed dogs that died in April/May 2005 during an influenza outbreak in an enclosed facility in northeast Florida, and on a domestic dog, a Yorkshire Terrier who died in May 2005 during an influenza outbreak at a veterinary clinic in southeast Florida. Tissues were fixed with 10% neutral buffered formalin, embedded in paraffin, and 5 µm sections were stained with hematoxylin and eosin for histopathological diagnosis. Unfixed tissues were stored at -80° C. pending virological analyses.

Extraction of RNA from canine tissue samples

Frozen lung tissues from each of 7 dogs were thawed and homogenized in minimal maintenance medium (MEM) supplemented with 0.5% bovine serum albumin (BSA) and antibiotics (gentamicin and ciprofloxacin) using a commercially available tissue homogenizer (Kendall, Lifeline Medical Inc. , Danbury, CT). Total RNA was extracted using a commercial kit ( ^RNeasy® Mini Kit, QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions and eluted in a final volume of 60 μl of buffer. Total RNA was also extracted from lung tissue collected from dogs without respiratory disease.

real-time RT-PCR

One-step quantitative real-time RT-PCR was performed on total RNA extracted from canine tissue samples using a ^QuantiTect® Probe RT-PCR kit containing ROX as a passive reference dye (QIAGEN Inc., Valencia, CA). Briefly, 2 sets of primer probes were used to detect influenza A sequences in each sample (Table 7). One set of primer-probes was selective for canine hemagglutinin (H3) gene sequences. Another set of primer-probes targeted the highly conserved ^region of the matrix gene (M) of influenza type A virus. PCR Master Mix, 0.25 µl ^QuantiTech® RT Mix, forward and reverse primers (0.4 µM final concentration each), probe (0.1 µM final concentration) and RNase-free water in a final volume of 25 µl. ^TaqMan® ribosomal RNA control reagents (Applied Biosystems, Foster City, CA) were used according to the manufacturer's instructions to detect 18S rRNA as an endogenous internal control for the presence of RNA extracted from canine tissue samples.

Quantitative one-step real-time RT-PCR was performed on reaction mixtures in the ^Mx3000P® QPCR System (Stratagene, La Jolla, CA). Cycling conditions included a reverse transcription step at 50°C for 30 minutes, an initial denaturation step at 95°C for 15 minutes to activate ^HotStarTaq® DNA polymerase, and amplification for 40 cycles. Each amplification cycle included denaturation at 94°C for 15 seconds followed by annealing/extension at 60°C for 1 minute. Fluorescent signals FAM (emission wavelength 518 nm) and VIC (emission wavelength 554 nm) were recorded at the end of each cycle. The threshold cycle (Ct) was determined by setting the threshold fluorescence (dR) at 1000 in each individual experiment. The Mx3000P ^® version 2.0 software (Stratagene, La Jolla, CA) was used for data acquisition and analysis. Samples were considered positive for influenza A virus when the threshold cycle (Ct) for the H3 or M gene was 3 units less than lung tissue from dogs without respiratory disease. This positive control consisted of an amplification of RNA extracted from A/canine/FL/242/03 (H3N8) virus.

Virus isolation in MDCK cells

Frozen lung tissues from each of 7 dogs were thawed and homogenized in 10 volumes of Dulbecco's modified Eagle's medium supplemented with 0.5% bovine serum albumin (BSA) and antibiotics (gentamicin and ciprofloxacin). Cell debris was removed by centrifugation and supernatants were inoculated into Madin-Darby canine kidney (MDCK) cells cultured in DMEM supplemented with 1 μg/ml TPCK-treated trypsin (Sigma-Aldrich Corp., St. Louis, MO) and antibiotics (gentamicin and ciprofloxacin). Cells were grown in 25 cm ² flasks at 37° C. in a humidified atmosphere containing 5% CO ₂ . Cultures were observed daily for morphological changes and harvested at 5 days post-inoculation. Harvested cultures were clarified by centrifugation and supernatants were inoculated onto fresh MDCK cells as described for initial inoculation; performed two additional passages for samples that did not detect the presence of influenza virus by hemagglutination or RT-PCR. Haemagglutination activity in clarified supernatants was determined using 0.5% turkey erythrocytes as previously described (Burleson et al, 1992; Kendal et al, 1982). RT-PCR was performed as described below.

Isolation of the virus in eggs with developing embryos

Homogenates were prepared from frozen lung tissues as described above for inoculation of MDCK cells. These homogenates (0.2 ml) were inoculated into the allantois of 10 day old embryonated hen eggs. After 48 hours of incubation at 35°C, these eggs were cooled at 4°C overnight before collecting allantoic fluid. Haemagglutination activity in clarified supernatants was determined using 0.5% turkey erythrocytes as previously described (Burleson, F. et al., 1992; Kendal, P. et al, 1982). RT-RCR was performed as described below. Two additional passages were performed for samples that did not detect the presence of influenza virus after the initial inoculation.

RT-PCR, nucleotide sequencing and phylogenetic analyzes

Viral RNA was extracted from the MDCK supernatant or allantoic fluid using the ^QIAamp® Viral RNA mini kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. This viral RNA was reverse-transcribed into cDNA using the ^QIAGEN® OneStep RT-PCR kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. PCR amplification of the coding region of 8 influenza virus genes in this cDNA was performed as previously described (Klimov, A. et al, 1992b) using universal gene-specific primer sets (primer sequences available on request). The resulting DNA amplicons were used as templates for automated sequencing in an ABI ^PRISM® 3100 automated DNA sequencer using a dye terminator cycle sequencing chemistry (Applied Biosystems, Foster City, CA). Nucleotide sequences were analyzed using Lasergene 6 ^Package® software (DNASTAR, Inc., Madison, WI). PHYLIP Version ^3.5® was used to approximate phylogenies and calculate bootstrap values from these nucleotide sequences (Felsenstein, J., 1989). Phylogenetic trees were compared with phylogenetic trees obtained by neighbor joining analysis (method of constructing a phylogenetic tree based on the principle of minimal evolution) using the Tamura-Nei model implemented in the MEGA ^® software (Kumar et al, 2004) and confirmed using the PAUP ^® 4.0 software Beta (Sinauer Associates, Inc., Sunderland, MA).

Hemagglutination inhibition (HI) assay

Serum samples were incubated with receptor-degrading enzyme (RDE, DENKA SEIKEN Co., Ltd., Tokyo, Japan) (1 part serum:3 parts RDE) for 16 hours at 37°C before heat inactivation for 30 minutes at 56°C . Influenza virus A/Canine/Jacksonville/05 (H3N8) was grown in MDCK cells for 72 hours at 37°C in 5% CO ₂ . Virus culture supernatants were collected, clarified by centrifugation and stored at -80°C. All other viruses used in the HI assay were grown in 10-day-old embryonated hen eggs, from which the allantoic fluid was collected and stored at -80°C. HI analysis was performed as previously described (Kendal et al, 1982). Briefly, 4 hemagglutination units of virus in 25 μl were added to an equal volume of serially diluted serum in microtiter 96-well plastic plates and incubated at room temperature for 30 minutes. An equal volume of 0.5% turkey erythrocytes was added and hemagglutination titers were determined approximately visually after 30 minutes. The final HI titer was defined as the last serum dilution that completely inhibited hemagglutination.

EXAMPLE 7 - CLINICAL CASES

In April and May 2005, there was a previously described (Crawford, P.C. et al, 2005) outbreak of a respiratory disease in dogs kept in an enclosed facility in northeast Florida. This outbreak included at least 58 dogs aged 3 months to 9 years and included both purebreds and mixed breeds. The most common clinical signs were purulent nasal discharge and cough for 7 days to 21 days. Of the 43 dogs that had been clinically ill for ≥7 days, 41 dogs had anti-canine/FL/04 (H3N8) HI antibody titers ranging from 32->1024. At least 10 dogs progressed to pneumonia, of which 6 dogs were euthanized. These 6 mixed breed dogs included 3 males and 3 females, ranging in age from 2 days to 10 days at the time of euthanasia. At post-mortem examination, these dogs had pulmonary congestion and edema. Histological examination of the respiratory tract revealed rhinitis, tracheitis, bronchitis, bronchiolitis and purulent bronchopneumonia. Observed necrosis of epithelial cells and erosion in the trachea, bronchi, bronchioles and bronchial glands. Respiratory tissues were infiltrated with neutrophils and macrophages.

In May 2005, a respiratory disease outbreak occurred in 40 pet dogs at a veterinary clinic in southeast Florida. The most common clinical signs were purulent nasal discharge and coughing for 10–30 days. Of the 40 dogs, 17 were canine/FL/04 (H3N8) seropositive with HI antibody titers ranging from 32->1024. Seroconversion occurred in 10 dogs for which paired acute and convalescent phase sera were available. Three dogs progressed to pneumonia. One of these dogs, a 9-year-old male Yorkshire Terrier, died 3 days after the onset of clinical signs. This dog had tracheobronchitis, pulmonary edema and congestion, and severe bronchopneumonia. Like 6 dogs from the indoor facility, there was epithelial cell necrosis and airway erosion and neutrophilic infiltrates in these tissues.

EXAMPLE 8 - REAL-TIME RT-PCR AND VIRUS ISOLATION

Lung tissues from these 7 dogs were analyzed by quantitative real-time RT-PCR assays that detect the M gene of influenza A virus and the H3 gene of H3N8 canine influenza A virus. The lungs from all 7 dogs were positive for both influenza A M gene and canine A virus H3 gene (Table 8). After 3 passages in MDCK cells, influenza A subtype H3N8 virus was isolated from the lungs of a sheltered dog that died after 3 days of pneumonia. This virus was named A/canine/Jacksonville/05 (H3N8) (canine/Jax/05). After 2 passages in embryonated hen eggs, influenza A subtype H3N8 virus was recovered from the lungs of a domestic dog that also died after 3 days of pneumonia. This virus has been named A/canine/Miami//05 (H3N8) (canine/Miami/05).

EXAMPLE 9 - GENETIC ANALYSIS OF CANINE INFLUENZA A VIRUS H3N8

Sequence analyzes of canine/Jax/05 and canine/Miami/05 revealed that their hemagglutinin (HA) genes were 98% identical to canine/FL/04, canine/TX/04 and canine/Iowa/05 isolates isolated from lung beagles greyhounds that died of pneumonia during the 2004 and 2005 track flu outbreaks (Crawford, P. C. et al, 2005; Yoon K-Y. et al, 2005). In addition, the canine/Jax/05 and canine/Miami/05 HA genes were 98% identical to equine influenza viruses of the same time period isolated after 2000. Phylogenetic comparisons of HA genes showed that canine/Jax/05 and canine/Miami/05 viruses clustered with canine/FL/04, canine/TX/04 and canine/Iowa/05 greyhound isolates and equine isolates of the same time, forming a different group compared to older equine viruses isolated in the early 1990s (FIG. 4). In addition, canine/Jax/05, canine/Miami/05 and canine/Iowa/05 isolates were more closely related to canine/Tx/04 than canine/FL/04 or canine/FL/03. These 2005 isolates form a subset that appears to branch off from the earlier 2003 and 2004 canine isolates with differences in approximately 10 sites of economic information. These differences support the hypothesis that canine influenza virus is horizontally transmitted from dog to dog, as opposed to repeated periodic introduction from an external source. The accumulation of mutations from 2003 to 2005 illustrates the ongoing process of adaptation that this virus must undergo after transfer to a new host, as has happened, as expected, for canine influenza viruses.

EXAMPLE 10 - AMINO ACID TESTS OF CANINE INFLUENZA A VIRUS H3N8 ISOLATES

All 6 canine isolates had conservative amino acid substitutions that distinguished them from equine influenza viruses of the same time (Table 9). These conservative substitutions were I15M, N83S, W222L, I328T and N483T. Phylogenetic comparisons of the mature HA protein showed that the canine/Jax/05, canine/Miami/05 and canine/Iowa/05 viruses formed a subgroup with the canine/TX/04 isolate (FIG. 4). There were 3 amino acid changes (Ll18V, K261N and G479E) that distinguished this subgroup from other canine viruses (Table 9). There were 2 amino acid changes (F79L and G218E) that distinguished the 2005 isolates from their progenitor canine/TX/04. In addition, the 2005 non-greyhound isolates canine/Jax/05 and canine/Miami/05 differed from the greyhound isolate canine/Iowa/05 in one amino acid change, R492K. Finally, canine/Jax/05 differed from canine/Miami/05 in a single amino acid, S107P. In all other equine and canine H3N8 viruses, the S was retained at position 107, except for A/Equine/Jilin/1/89, which had a T (Guo Y. et al, 1992).

EXAMPLE 11 - ANTIGEN ASSAYS OF CANINE H3N8 VIRUS ISOLATES

Hemagglutination inhibition (HI) tests were performed using a panel of older and more recent equine and canine influenza viruses and sera were collected in 2005 from horses and dogs that were infected with influenza virus (Table 10). Serum from ferrets immunized against canine/FL/04 was also included in these assays. Horse serum HI antibody titers were 8-16-fold higher when tested with modern equine viruses compared to older isolates, but decreased by at least 4-fold when tested with canine viruses. Canine sera were non-reactive with older equine viruses, but these antibody titers increased 4-fold when tested with modern equine and canine isolates. This was also observed for sera from ferrets immunized against canine influenza virus. These seroreactivity distributions demonstrated antigenic similarity between canine influenza viruses and modern equine influenza viruses and were consistent with phylogenetic analyses. Antibody titers in equine, canine, and ferret sera against the canine/Miami/05 isolate were similar to those against the 2003 and 2004 canine isolates. However, these titers were 2-4 times lower for the canine/Jax/05 isolate. This suggests that canine/Jax/05 is antigenically distinct from other canine isolates, which may be due in part to a single amino acid change at position 107 in mature HA.

Table 7

Primers and probes for quantitative analysis of real-time RT-PCR for the influenza A virus matrix gene and the H3 gene of canine influenza A (H3N8) virus

^a The underlined letter r stands for nucleotide a or g, and the underlined letter k stands for nucleotide g or t.

^b Capital letters denote "locked" nucleic acid residues.

Table 8 Real-time quantitative RT-PCR and virus isolation performed on lung tissue from dogs that died of pneumonia during outbreaks of respiratory disease in a sheltered facility (kennel) and veterinary clinic in Florida Dog ID Location Duration of clinical illness real-time RT-PCR Virus isolation M(Ct) HA
(ct) A/canine/FL/242/03
positive control 28.15 27.36 1079 Indoor facility (NE FL) 2 days 29.81 28.84 No 1078 Indoor facility (NE FL) 3 days 30.37 29.71 3rd passage in MDCK 318 Indoor facility (NE FL) 9 days 33.89 32.97 No 320 Indoor facility (NE FL) 10 days 39.44 37.09 No 319 Indoor facility (NE FL) 6 days 33.87 32.23 No 1080 Indoor facility (NE FL) 6 days 38.87 38.23 No 374 Veterinary Clinic (SEFL) 3 days 24.05 22.65 2nd passage in eggs

Table 9

Comparison of Mature HA Amino Acids for Canine and Modern Equine Influenza Viruses

Table 10

Serum antibody titers in equine, canine and ferrets against older and more recent equine and canine influenza viruses

^a Antibody titers were determined in a hemagglutination inhibition assay performed with serial dilutions of equine, canine, and ferret sera, and these viruses are listed in the antigen column.

^b Serum from ferrets immunized with canine/FL/04 virus.

MATERIALS AND EXAMPLES OF METHODS FOR EXAMPLES 12-15

Canine influenza virus inoculum

An inoculum of this virus was obtained by inoculation of Madin-Darby canine kidney epithelial cells (MDCK) with A/canine/FL/43/04 (H3N8) stock solution, passage 3 of the stock isolate previously described (Crawford et al, 2005). Inoculated MDCK cells in Dulbecco's Minimal Maintenance Medium (DMEM) supplemented with 1 μg/ml TPCK-treated trypsin (Sigma-Aldrich Corp., St. Louis, MO) and antibiotics (gentamicin and ciprofloxacin) were grown in 25 cm ² flasks at 37°C. C in a humidified atmosphere containing 5% CO ₂ . These cultures were observed every day for morphological changes and harvested at 5 days post-inoculation. The harvested cultures were clarified by centrifugation and the supernatants were stored at -80° C. pending inoculation of the dogs. An aliquot of the supernatant was used to determine the virus titer according to the method of Reed and Muench. This titer was equal to 10 ⁷ tissue culture median infectious doses (TCID ₅₀ ) A/canine/Florida/43/2004 (canine/FL/04) per ml.

Experimental inoculation

Eight 4-month-old colony-bred, non-purebred 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 body weights of these dogs were in the range of 13–17 kg. These dogs were healthy based on physical examination, background blood tests, and body temperature records for 2 weeks prior to inoculation. All dogs were previously free of canine influenza virus based on serological tests performed on paired sera samples collected at the time of arrival at this facility and 2 weeks later. Dogs were anesthetized with an intravenous injection of propofol (Diprivan®, Zeneca Pharmaceuticals, 0.4 mg/kg body weight to effect) for endotracheal tube intubation. Six dogs (3 males and 3 females) each were inoculated with 10 ⁷ TCID ₅₀ of canine/FL/04 virus in 5 ml of sterile saline injected into the distal trachea through a small diameter rubber catheter inserted into the endotracheal tube. Two dogs (1 male and 1 female) were sham-inoculated with an equal volume of sterile saline. The mock-inoculated control dogs were kept in a separate room from the virus-inoculated dogs and were cared for by different staff. Physical examinations and rectal temperature recordings were performed twice daily for 6 days post-inoculation (pi).

Collection of pharyngeal and rectal swabs

To monitor virus shedding, oropharyngeal samples were collected twice daily from each dog on days 0-6 p.i. using polyester fiber swabs (Fisher Scientific International Inc., Pittsburgh, PA). These swabs were placed in 1 ml of sterile phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA). Rectal swabs were taken from each dog once a day from day 0 to day 6. Swab extracts were prepared by clarification of media for transport of swabs by centrifugation. An aliquot of the swab extract was tested immediately for influenza A nucleoprotein using a commercial Directigen™ immunoassay kit (BD, Franklin Lakes, NJ) according to the manufacturer's instructions. The remaining extract was stored at -80°C pending other virological analyses.

Pathological anatomical studies

On day 1 pi, one sham-inoculated dog and one virus-inoculated dog were euthanized by intravenous inoculation with Beuthanasia- ^D® solution (1 ml/5 kg body weight; Schering-Plough Animal Health Corp). One virus-inoculated dog was similarly euthanized each day from days 2-5 pi On day 6 pi, the remaining mock-inoculated and virus-inoculated dog was euthanized. Full post-mortem examinations were performed by one of the investigators (WLC). Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and 5 μm sections were either stained with hematoxylin and eosin for histopathological diagnosis or processed for immunohistochemistry as described below. Unfixed lung tissue was provided to the Diagnostic Clinical Microbiology/Parasitology/Serology Service at the University of Florida College of Veterinary Medicine for isolation and identification of the bacteria. Samples were cultured on non-selective media, as well as selective media for Bordetella species (Regan-Lowe; Remel, Lenexa, KS) and Mycoplasma species (Remel). All cultures were held for 21 days before reporting no growth. Unfixed tissues were also stored at -80° C. pending virological analyses.

Immunochemistry

Deparaffinized and rehydrated 5 µm sections of tracheal and lung tissue were mounted as preparations on Bond-Rite™ slides (Richard-Allan Scientific, Kalamazoo, MI) and then treated with Proteinase K (DakoCytomation, Carpenteria, CA) followed by peroxidase blocking reagent ( Dako® EnVision™ Peroxidase Kit, Dako Corp., Carpenteria, CA). The sections were incubated with a 1:500 dilution of anti-influenza A virus monoclonal antibody H3 (Chemicon International, Inc.) for 2 hours at room temperature. Controls included incubating the same sections with mouse IgG (1 mg/mL, Serotec, Inc. Releigh, NC) and incubating this monoclonal antibody with healthy canine lung sections. After primary antibody treatment, these sections were incubated with secondary immunoperoxidase reagents and peroxidase substrate (Dako® EnVision™ Peroxidase Kit, Dako Corp.) according to the manufacturer's instructions. These sections were counterstained with hematoxylin, treated with Clarifier #2 and Bluing Reagent (Richard-Allan Scientific, Kalamazoo, MI), dehydrated, and cover slips placed using permount (ProSciTech, Queensland, Australia).

Extraction of RNA from swabs and tissues

Lung and tracheal tissues from each dog were thawed and homogenized in minimal maintenance medium (MEM) supplemented with 0.5% bovine serum albumin (BSA) and antibiotics (gentamicin and ciprofloxacin) using a commercially available tissue homogenizer (Kendall, Lifeline Medical Inc., Danbury , CT). Total RNA was extracted from tissue homogenates and oropharyngeal and rectal swab extracts using a commercial kit ( ^RNeasy® Mini Kit, QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions and eluted in a final volume of 60 µl of buffer.

real-time RT-PCR

One-step quantitative real-time RT-PCR was performed on total RNA using a ^QuantiTect® Probe RT-PCR kit containing ROX as a passive reference dye (QIAGEN Inc., Valencia, CA) and a set of primer-probes that target the highly conserved region of the gene matrix (M) of type A influenza virus (Payungporn S. et al, 2006a; Payungporn S. et al, 2006b). For each real-time RT-RCR reaction, 5 µl of extracted total RNA was added to a reaction mixture containing 12.5 µl of 2X ^QuantiTech® Probe RT-PCR Master Mix, 0.25 µl of ^QuantiTech® RT Mix, forward and reverse primers (0 .4 μM final concentration for each), probe (0.1 μM final concentration) and RNase-free water in a final volume of 25 μl. ^TaqMan® ribosomal RNA control reagents (Applied Biosystems, Foster City, CA) were used according to the manufacturer's instructions for GAPDH detection as an endogenous internal control for the presence of RNA extracted from swab and tissue samples and as a normalization control.

Quantitative one-step real-time RT-PCR was performed on reaction mixtures in the ^Mx3000P® QPCR system (Stratagene, La Jolla, CA). Cycling conditions included a reverse transcription step at 50 ^° C. for 30 minutes, a denaturation step at 95° C. for 15 minutes to activate ^HotStarTaq® DNA polymerase, and amplification for 40 cycles. Each amplification cycle included denaturation at 94°C for 15 seconds followed by annealing/extension at 60°C for 1 minute. Fluorescent signals FAM (emission wavelength 518 nm) and VIC (emission wavelength 554 nm) were recorded at the end of each cycle. The threshold cycle (Ct) was determined by setting the threshold fluorescence (dR) at 1000 in each individual experiment. The Mx3000P ^® version 2.0 software (Stratagene, La Jolla, CA) was used for data acquisition and analysis. The positive control consisted of amplification of RNA extracted from A/canine/FL/242/03 (H3N8) virus. The results were normalized by dividing the Ct M value by the corresponding GAPDH Ct value for each sample.

Reisolation of the virus from tissues

Frozen lung and trachea tissues from virus-inoculated dogs were thawed and homogenized in 10 volumes of DMEM supplemented with 0.5% BSA and antibiotics. Solid cell debris was removed by centrifugation and supernatants were inoculated onto MDCK cells cultured in DMEM supplemented with 1 μg/ml TPCK-treated trypsin (Sigma-Aldrich Corp., St. Louis, MO) and antibiotics as described above. Cultures were observed daily for morphological changes and harvested at 5 days post-inoculation. Harvested cultures were clarified by centrifugation and supernatants were inoculated onto fresh MDCK cells as described for initial inoculation; performed two additional passages for samples that did not detect the presence of influenza virus by hemagglutination or RT-PCR. Hemagglutination activity in clarified supernatants was determined using 0.5% turkey erythrocytes as described previously (Crawford et al., 2005). RT-PCR was performed as described below.

RT-PCR, nucleotide sequencing and phylogenetic analyzes

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. This viral RNA was reverse-transcribed into cDNA using the ^QIAGEN® OneStep RT-PCR kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. PCR amplification of the coding region of 8 influenza virus genes in this cDNA was performed as previously described (Crawford e 2005) using universal gene-specific primer sets (primer sequences available on request). The resulting DNA amplicons were used as templates for automated sequencing in an ABI ^PRISM® 3100 automated DNA sequencer using a dye terminator cycle sequencing chemistry (Applied Biosystems, Foster City, CA). Nucleotide sequences were analyzed using Lasergene 6 ^Package® software (DNASTAR, Inc., Madison, WI). Nucleotide sequences for viruses derived from infected dogs were compared with those of the virus in this inoculum to determine if any changes occurred during replication in the respiratory tract.

EXAMPLE 12 - CLINICAL DISEASE

All 6 virus-inoculated dogs developed transient fever (rectal temperature ≥39°C) during the first 2 days p.i., but none showed respiratory signs such as cough or nasal discharge during the 6 day period observations. The sham-inoculated dogs remained clinically healthy.

EXAMPLE 13 VIRUS ISOLATION

Influenza A virus nucleoprotein was detected in an oropharyngeal swab collected from one of the virus-inoculated dogs at 24 hours p.i. Oropharyngeal swabs collected from one dog at 72, 84, and 120 p.i. and another dog at 108, 120, and 132 p.i. were positive for virus by real-time quantitative RT-PCR (Table 11). The absolute number of influenza A virus M gene copies per µl of swab extract increased over time from 3 days to 6 days p.i. The virus was not detected in rectal swabs.

EXAMPLE 14 - PATHOLOGICAL STUDIES

In contrast to previous experimental infection using specific pathogen-free beagles (short-legged beagle dogs) (Crawford et al, 2005), virus-inoculated mongrel dogs (mutts) had pneumonia, as shown by macroscopic and histological analyzes of the lungs, from 1 to 6 days p.i. In addition to pneumonia, these dogs had rhinitis, tracheitis, bronchitis, and bronchiolitis, similar to those described for naturally infected dogs (Crawford et al, 2005). There was epithelial necrosis and erosion of the lining of the airways and bronchial glands with neutrophil and macrophage infiltration of the submucosal tissues (FIG. 5, upper panels). Immunochemistry detected the H3 viral antigen in epithelial cells of the bronchi, bronchioles, and bronchial glands (FIG. 5, bottom panels). There was no bacterial overinfection. Respiratory tissues from 2 sham-inoculated dogs were normal.

EXAMPLE 15 - VIRUS REPLICATION IN THE TRACHEA AND LUNGS

The trachea and lungs were positive for virus by real-time quantitative RT-PCR in all dogs from 1 to 6 days pi (Table 12). The absolute number of copies of the M gene of influenza A virus per μl of tracheal homogenate increased from 1 to 5 days pi, then decreased on day 6. The absolute number of copies of the M gene per μl of lung homogenate decreased from 1 to 6 days pi Typically, the trachea contained more than ≥ one log _1O , more virus than lung, on each of 6 days pi

Table 11

Detection of Virus Shedding in the Oropharynx of Non-Purebred Dogs Inoculated with Canine Influenza Virus by Real Time Quantitative RT-PCR

^a Time when oropharyngeal swabs were collected from dogs after inoculation with A/canine/FL/43/04 (H3N8) virus.

^b Normalization ratios were calculated by dividing M (Ct) by GAPDH (Ct) for each smear extract.

^c Absolute number of matrix gene copies per µL of swab extract.

Table 12

Detection of virus replication in the trachea and lung of non-purebred dogs inoculated with canine influenza virus by real-time quantitative RT-PCR

^a Time when oropharyngeal swabs were collected from dogs after inoculation with A/canine/FL/43/04 (H3N8) virus.

^b Normalization ratios were calculated by dividing M (Ct) by GAPDH (Ct) for each smear extract.

^c Absolute number of matrix gene copies per µL of swab extract.

MATERIALS AND EXAMPLES OF METHODS FOR EXAMPLE 16

Viral strains

Canine influenza virus strains as well as avian, equine and human influenza virus strains (listed in Table 15) were propagated in embryonated eggs or MDCK cells and their infectivity was titrated by final dilution in chick embryos or plaque assay. Rapid virus quantification was performed by haemagglutination analysis using turkey erythrocytes.

Diagnostic Samples

Altogether, lung tissues from 60 canines collected from suspected cases of viral respiratory disease in 2005 were examined for the presence of canine influenza virus.

Extraction of RNA from canine tissue samples

Blocks of lung tissue weighing 20–30 mg were homogenized in an available tissue homogenizer (Kendal). Total RNA was extracted using a commercial kit (RNeasy Mini Kit, Qiagen, Valencia, CA) and eluted in a final volume of 60 µl according to the manufacturer's recommendations.

Design of primers and probes

Multiple alignments of the H3 and M gene sequences from different subtypes and from different species were performed using the CLUSTAL X program (Version 1.8). The primers and probe for the matrix (M) gene were selected from conserved regions along the known sequences corresponding to different subtypes of influenza A virus, while the set of gene-specific primers and probes for the H3 hemagglutinin gene were selected such that they corresponded to the genes of the equine influenza A virus. and canine and did not correspond to the homologous genes of birds and humans (Table 13). A primer design program (OLIGOS Version 9.1) and web-based analysis tools provided by EXIQON ( http://lnatools.com ) were used for Tm calculation and secondary structure prediction as well as self-hybridization. The conserved region of the 18S rRNA gene was used as an endogenous internal control for the presence of RNA extracted from the canine tissue sample. Previously developed TaqMan® 18S eukaryotic rRNA assay reagents (VIC/TAMRA) (Applied Biosystems) were used for real-time detection of 18S rRNA in tissue samples.

Real-time RT-PCR conditions

One-step real-time RT-PCR was performed using a ^QuantiTect® Probe RT-PCR kit containing ROX as a passive reference dye (QIAGEN Inc., Valencia, CA). In each real-time RT-RCR reaction, 5 µl of RNA probe was used as template to combine with a reaction mixture containing 10 µl of 2X ^QuantiTech® Probe RT-PCR Master Mix, 0.25 µl of ^QuantiTech® RT Mix, primers (0, 4 μM final concentration for H3 gene or 0.6 μM final concentration for M gene), probe (0.1 μM final concentration for H3 gene or 0.2 μM final concentration for M gene) and RNase-free water in final volume 20 µl. One-step real-time RT-PCR was performed on an ^Mx3000P® QPCR system (Stratagene). Cycling conditions included a reverse transcription step at 50°C for 30 minutes. After an initial denaturation step at 95°C for 15 minutes to activate the ^HotStarTaq® DNA polymerase, 40 cycles of amplification was performed, including denaturation (94°C for 15 seconds) and annealing/extension (60°C for 30 seconds). Fluorescent signals FAM (516 nm emission wavelength for H3 and M detection) and VIC (555 nm emission wavelength for 18S rRNA detection) were obtained once per cycle at the end of the extension step. Real-time PCR data acquisition and analysis was performed using the Mx3000P version 2.0 program (Stratagene).

H3 Primer/Probe Set Specificity for Canine Influenza (H3N8) Virus and Versatility of the M Primer/Probe Set for Influenza A Virus

To examine the specificity of each primer/probe set, RNA extracted from several known influenza A virus subtypes was used as a template in a real-time RT-PCR assay (Table 15).

RNA standard for determining the performance of real-time RT-PCR

Canine influenza A virus genes (A/canine/Florida/242/2003(H3N8)) were used to generate PCR amplicons for H3 (nucleotides 1-487) and M (nucleotides 1-276) using primers linked to the T7 promoter ( table 13). The purified PCR amplicons of the H3 and M genes were then used as templates for in vitro transcription using the Riboprobe in vitro Transcription System-T7 (Promega) according to the manufacturer's recommendations. The concentration of transcribed RNAs was calculated by measuring the optical density at 260 nm. These RNAs were then serially diluted 10-fold, in the range of 10 ⁸ -10 copies/µl, to perform sensitivity tests. In addition, a calibration curve was generated by plotting log starting template RNA concentrations (copies/µl) versus cycle threshold (Ct) obtained from each dilution to determine the overall efficiency of real-time RT-PCR.

Comparative sensitivity tests between real-time RT-PCR and Directigen Flu A test kit

The original viruses of two viral strains, including A/Wyoming/3/2003 (H3N2) at 10 ^6.67 EID ₅₀ /ml (HA=64) and A/canine/Florida/242/2003(H3N8) at 10 ^{7, 17} EID ₅₀ /ml (HA=16) was used for threshold detection assay. Logarithmic dilutions of samples in phosphate buffered saline (PBS) (125 μl) were used in the Rapid Influenza A Antigen Detection Kit, Directigen Flu A, (Becton, Dickinson and Company), according to the manufacturer's instructions. Each Directigen Flu A test device has an influenza virus H1N1 antigen spot in the center of the membrane that develops as a purple dot and indicates the integrity (purity) of this test, which is based on an anti-nucleoprotein (NP) monoclonal antibody. The development of a purple triangle surrounding this point indicates the presence of influenza NP in the test sample. The intensity of the development of the magenta signal from this triangle is rated as + (outline of the triangle in general), ++ (slightly colored triangle), +++ (dark magenta triangle) and ++++ (very dark magenta triangle). Viral RNA was extracted with 125 µl aliquots of each dilution using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA) and eluting in a final volume of 50 µl. A volume of 5 µl of the extracted viral RNAs was examined by real-time RT-PCR for a comparative sensitivity test with the Directigen Flu A kit.

EXAMPLE 16

Canine influenza real-time RT-PCR analysis is based on information from three molecular probes that target 18S rRNA from the host cell and M and H3 from the influenza A virus genome (Table 14). Host gene amplification is a reporter of sample quality and integrity. Clinical specimens, autopsy specimens, or laboratory specimens containing canine influenza virus (H3N8) are expected to give an amplification signal with these three probes. Specimens that give amplification signal with M probes and 18S rRNA but are negative for H3 will indicate the H3 subtype of human, porcine or avian influenza virus or non-H3 subtypes. These rare cases could be resolved by RT-PCR using universal HA primers to generate a cDNA amplicon that can be analyzed by sequencing. Properly collected and marketed samples devoid of Influenza A virus provide only the 18S rRNA amplification signal. Situations in which only the 18S rRNA probe and H3 probes produce an amplification signal are indicative of a flawed method, unless proven otherwise; or either a false negative for M or a false positive for H3 must be demonstrated. Finally, samples giving no amplification signals with these three probes are indicative of defective sampling, degradation, erroneous RNA extraction, or the presence of polymerase inhibitors used in PCR.

To investigate the specificity of the H3 primer/probe set for canine influenza A (H3N8) and the versatility of the M primer/probe set for influenza type A, several subtypes of influenza A viruses were examined by real-time RT-PCR. These results show that the H3 primer/probe set gave a positive amplification signal with canine influenza (H3N8) only. No significant positive or non-specific amplification signals were observed in other human H3 subtypes or strains. The primer/probe set M gave a positive amplification signal with all strains tested (Table 15). These results show that H3 primers/probe specifically detects canine influenza A (H3N8) virus, while M primers/probe detects numerous influenza A viruses.

The performance of real-time RT-PCR assays was assessed by final dilutions of in vitro transcribed M and H3 RNAs. As expected, the cycle threshold (Ct) increased in direct correlation with the dilution of the RNA standards. Fluorescence signals can be detected at dilutions of the M and H3 RNA standards as low as 10 ³ and 10 ² copies/µl, respectively (FIGS. 6A and 6B). Calibration curves for the M and H3 genes were generated by plotting log baseline RNA concentrations versus cycle threshold (Ct) obtained from each dilution (FIGS. 6C and 6D). The slope of the calibration curve is used to determine the efficiency of the PCR reaction, which is theoretically exponential; 100% amplification efficiency assumes doubling the concentration of amplicons each cycle. Calibration curves with a slope of approximately -3.1 - -3.6 are generally acceptable for most applications requiring accurate quantitation (90-110% reaction efficiency). The Rsq value is the fit of all data to this calibration curve. If all data lie exactly on this line, Rsq should be equal to 1.00. When this data falls further from this line, Rsq decreases. An Rsq value of ≥0.985 is acceptable for most analyses. The M calibration curve showed a slope of -3.576 (efficiency=90.4%) and Rsq=1.00, while the H3 calibration curve showed a slope of -3.423 (efficiency=95.9%) and Rsq=0.999. These values indicate satisfactory performance and overall performance of real-time RT-PCR assays. The inventors attribute the lower potency and sensitivity of the M primer/probe set compared to the H3 primer/probe set to the N-fold degeneracy of the M primer sequences required to ensure wide coverage of M gene sequence variability in all viruses of multiple subtypes, hosts and lineages.

The sensitivity of the real-time RT-PCR assay was also compared to a commercial rapid antigen detection assay (Directigen Flu A). Log dilutions of A/Wyoming/3/2003 (H3N2) and A/canine/Florida/242/2003(H3N8) were analyzed using Directigen Flu A and real time RT-PCR. The results of Directigen Flu A showed that the susceptibility against both viral strains equal approximately 100-fold dilution of the original viruses used in these experiments (figure 7). The signals (magenta color) generated by the canine virus samples (A/canine/Florida/242/2003: ^106.x PFU/ml) were much weaker than the signals found in the human virus (A/Wyoming/3/2003 : 10 ^7.x PFU/ml), corresponding to the lower concentration in these samples. Alternatively, the lower canine virus signal could be attributed to the molecular specificity of the anti-NP monoclonal antibodies; those. poor conservation of amino acids in the NP epitope of canine influenza A viruses.

Real-time RT-PCR of the M gene gave Ct values above the thresholds with 10 and 30 PFU equivalents for reaction with A/canine/Florida/242/2003 and A/Wyoming/3/2003 viruses, respectively (Table 16). Differences between the values of sensitivity 2 viral strains associated with differences in the initial titers of the viruses. Comparison of H3 gene detection between canine and human influenza viruses was not performed because the H3 primers/probe in our RT-PCR assay amplifies exclusively canine influenza virus. RT-PCR was 10 ⁵ times more sensitive than the rapid antigen detection kit.

To evaluate the performance of the RT-PCR test in autopsy specimens from dogs with acute respiratory disease, sixty lung tissue samples from dogs provided during 2005 were tested for the presence of canine influenza A virus by real-time RT-PCR. A total of 12 out of 60 samples (20%) were positive for both the M gene and the H3 gene, while the remaining 48 samples were negative for both the M gene and the H3 gene. Virus isolation attempts were made by inoculating eggs and MDCK cells to assess the specificity of this real-time assay; 2 out of 12 samples that were positive for canine influenza by RT-PCR detected canine influenza virus (data not shown, manuscript under preparation). Although all of these tissues were collected from dogs with a history of severe respiratory disease, most of these samples did not detect canine influenza virus using either real-time PCR or conventional isolation, suggesting a high incidence of other respiratory pathogens such as Bordetella bronchiseptica, distemper virus canine or parainfluenza virus. One-step real-time RT-PCR analysis provides the present invention with a fast, sensitive, and economical approach for detecting canine influenza A (H3N8) virus. Rapid laboratory diagnosis of canine influenza A (H3N8) virus infections at an early stage of the disease may provide information relevant to dogs kept in veterinary clinics and kennels.

Table 13

Primers and probes used for in vitro detection of real-time RT-RCR and transcription

*Note: Capital letters = LNA (Locked Nucleic Acid) residues, r = a or g, k = g or t, underlined = T7 promoter sequence

Table 14 Real-time RT-PCR analysis interpretation Interpretation results M H3 rRNA 18S Canine influenza A virus (H3N8) positive + + + Positive for influenza A virus (subtype unknown) + - + Negative for influenza A virus - - + An error in RNA extraction or the presence of an RCR inhibitor - - -

Table 15 H3 primer set/probe specificity test and M primer set/probe versatility test with multiple subtypes of influenza A viruses Subtypes Strain name Master Real-time RT-PCR detection Gene H3 (Ct) Gene M (Ct) H1 A/Ohio/1983 Human No Ct 15.40 A/WSN/1933 Human No Ct 20.09 H3 A/Wyoming/3/2003 Human No Ct 28.85 A/Victoria/3/1975 Human No Ct 16.62 A/canine/FL/242/2003 canine 28.43 29.25 H4 Turkey/MN/1066/1980 Birds No Ct 17.49 Clinical trial* Birds No Ct 20.87 H5 AChicken/Thailand/CUK2/2004 Birds No Ct 20.13 A/Pheasant/NJ/1335/1998 Birds No Ct 16.64 H6 Clinical trial* Birds No Ct 19.52 H10 Clinical trial* Birds No Ct 25.64 Clinical trial* Birds No Ct 19.59 H11 Clinical trial* Birds No Ct 15.72 Clinical trial* Birds No Ct 25.55 *Please note that subtypes of clinical samples have been confirmed by nucleotide sequencing

Table 16

Comparative sensitivity tests for the detection of influenza A virus between real-time RT-PCR and Directigen Flu A

Table 17

Table 18 Letter symbol Amino acid Letter symbol Amino acid A Alanine M Methionine B Asparagine or aspartic acid N Asparagine WITH Cysteine P Proline D Aspartic acid Q Glutamine E Glutamic acid R Arginine F Phenylalanine S Serene G Glycine T Threonine H Histidine V Valine I Isoleucine W tryptophan K Lysine Z Tyrosine L Leucine Glutamine or glutamic acid

Table 19

Amino acid differences between PB2 proteins of H3N8 influenza viruses of horse and canine

Table 20

Amino acid differences between PB1 proteins of H3N8 influenza viruses of horse and canine

Table 21

Amino Acid Differences Between Equine and Canine Influenza H3N8 RA Proteins

*based on available virus genes isolated between 1963 and 1998.

Table 22

Amino acid differences between NP proteins of equine and canine H3N8 influenza viruses

Table 23

Amino Acid Differences Between Equine and Canine H3N8 Influenza NA Proteins

Table 24

Amino acid differences between M1 proteins of equine and canine H3N8 influenza viruses

*based on available virus genes isolated between 1963 and 1998.

Table 25

Amino acid differences between NS1 proteins of equine and canine H3N8 influenza viruses

*based on available virus genes isolated between 1963 and 1998.

EXAMPLE 17 - DEVELOPMENT OF THE CANINE INFLUENZA MODEL.

Canine flu virus, which was isolated from Florida influenza outbreaks, was observed to be an H3N8 influenza virus and closely related to the equine influenza virus strain, A/equine/Ohio/03 (Crawford et al, SCIENCE Vol. 309). , September 2005, incorporated by reference in its entirety in this patent). This study investigated the potential for using the A/equine/Ohio/03 strain of equine influenza virus to induce influenza-like disease in dogs.

Procedure: Ten 13-week-old Beagles (short-legged beagles) were obtained from a commercial supplier and housed in individual cages in a BSL-2 facility. These dogs were randomly assigned to two groups of 5 dogs each. As shown in Table 26, one group was subjected to intratracheal infection and the other group was subjected to oronasal infection. Dogs were challenged at 14 weeks of age.

Table 26 Experiment plan Group Number of dogs Method of infection 1 5 intratracheal 2 5 rotonosova

Cell cultured equine influenza virus A/equine/Ohio/03 was used as the challenge virus. For intratracheal challenge, the challenge virus was administered through a delivery tube, which consisted of a tracheal tube with an inflatable cuff (Size 4.0/4.5, Sheridan, USA) and a feeding tube (for tube feeding) (size 5Fr, 1.7 mm ,/16 inches long, Kendall, USA) in a volume of 0.5-1.0 ml. For oronasal challenge, challenge virus (10 x 7.0 - 10 x 8.0 TCID ₅₀ per dog) was administered as an aerosol ("fog") using a nebulizer (DeVilbiss Ultra-Neb®99 ultrasonic nebulizer, Sunrise Medical, USA) in volume 2–3 ml.

These dogs were observed for influenza-related signs within 14 days of infection. Serum samples were collected from each dog on day 0 (pre-challenge) and days 7 and 14 post-challenge for determination of HI titer using H3N8 equine influenza virus standard protocol (SAM 124, CVB, USDA, Ames, IA). All dogs were humanely euthanized and lung tissues were collected in 10% buffered formalin for histopathological evaluation.

results

The results of this experiment are summarized in Table 27. Influenza-related symptoms were observed in a small number of dogs after infection. These symptoms included fever (>103°F; >39.4°C) and cough. Two of 5 dogs (i.e., 40%) had a fever (>103°F; >39.4°C) in group 1, compared to 1 of 5 (i.e., 20%) in group 2 One dog in the oronasal infection group had a sneeze and the other had a cough after the infection. For group 1, an HI titer range of 10–80 was observed, with a geometric mean titer (GMT) of 20. For group 2, an HI titer range of 40–160, with a geometric mean titer (GMT) of 86, was observed. One dog from each group had histopathological lesions consistent with with the flu or characteristic of the flu.

Table 27 Canine Influenza Infection - Clinical Signs, Virus Isolation, Histopathological and Serological Findings Dog ID Method of infection Clinical symptoms Virus isolation Microscopic damage (histopathology) Serology (HI titer) Nasal/oral swab Tracheal scraping lung tissue Before infection 7 days after infection 14 days after infection AAN intratracheal No Negative Negative Negative Negative 10 10 20 adb intratracheal No Negative Negative Negative Negative 10 80 20 ADC intratracheal Fever* Negative Negative Negative Negative 10 20 20 AEB intratracheal Fever Negative Negative Negative Negative 10 40 20 AEE intratracheal No Negative Negative Negative Inclusive 10 20 10 AAE rotonosova No Negative Negative Negative Negative 10 80 80 AAG rotonosova No Negative Negative Negative Negative 10 40 80 ABY rotonosova episodic sneezing, episodic cough Negative Negative Negative Negative 10 80 160 ADY rotonosova Fever, episodic sneezing Negative Negative Negative Negative 10 80 80 ADZ rotonosova No Negative Negative Negative Negative 10 80 160 * These animals were challenged with Ohio 03 equine influenza isolate.
** Rectal temperature >103°F;>39.4°C

EXAMPLE 18 - EFFECTIVENESS OF EQUINE INFLUENZA VIRUS VACCINE ON DOGS

The canine flu virus isolated from the Florida influenza outbreaks was observed to be an H3N8 influenza virus and closely related to the equine influenza virus strain, A/equine/Ohio/03 based on sequence similarity. The following study was conducted to determine the efficacy of an experimentally inactivated equine influenza virus vaccine.

Procedure: Nine 7-week-old beagles (short-legged beagles) were obtained from a commercial supplier and kept in individual cages in a BSL-2 facility. These dogs were randomly assigned to two groups, summarized in Table 28:

Table 28 Experiment plan Group Number of dogs Treatment 1 5 Vaccine 2 4 Control

The first group consisted of 5 dogs that were vaccinated with an inactivated, CARBIGEN™ adjuvanted equine influenza A/equine/Ohio/03 vaccine at 8 and 12 weeks of age by the subcutaneous (SQ) route. A/equine/Ohio/03 was inactivated with binary ethyleneimine (“BEI”) using the standard method. Each dose of this vaccine contained 5% of the mass. CARBIGEN™, 4096 HA units of inactivated virus, enough PBS to bring the total volume of this dose to 1 ml, and enough NaOH to adjust the pH to 7.2-7.4. Serum samples were collected from all dogs on the day of the first and second vaccinations and before challenge for determination of HI titer using the H3N8 equine influenza virus standard protocol (SAM 124, CVB, USDA, Ames, IA). At 3 weeks after the second vaccination, 5 vaccinated dogs and a second group (i.e. control group) consisting of 4 dogs of the same age were oronasally challenged with cell cultured equine influenza virus A/equine/Ohio/03 (107.0 -108.0 TCID ₅₀ per dog) in a volume of 1-2 ml per dose. This challenge virus was administered to dogs as an aerosol ("fog") using a nebulizer (DeVilbiss Ultra-Neb®99 ultrasonic nebulizer, Sunrise Medical, USA). Dogs were observed for influenza-related clinical symptoms for 14 days post infection. Five dogs (3 vaccinates and 2 controls) 7 days post challenge and 4 dogs (2 controls and 2 vaccinates) 14 days post challenge were humanely euthanized to collect lung tissue in 10% buffered formalin for histopathological evaluation.

results

The results of this experiment are summarized in Tables 29 and 30. All vaccinated dogs seroconverted (ie changed serological specificity) after vaccination. An HI titer range of 40-640 with a GMT of 129 was observed during the post-vaccination period with equine influenza virus A/equine/Ohio/03 and a HI titer range of 160-320, with a geometric mean titer of 211, with a canine virus isolate, A/canine/Florida/ 242/03. Two of the 6 vaccinates had a fever >103°F (>39.4°C) on one day and no other symptoms were observed in any dog after challenge.

Conclusion

All vaccinated dogs responded to the inactivated CARBIGEN™ adjuvanted equine influenza virus vaccine. The HI titer results with canine influenza virus isolate suggest that this inactivated equine influenza vaccine did not induce a detectable level of cross-reactive antibody to canine influenza virus. Even though the challenge virus used in this experiment did not induce any noteworthy clinical disease in beagle dogs, based on the HI titer with the canine influenza virus isolate, it was concluded that an inactivated equine vaccine could be used in dogs to cross-induce reactive antibodies that could potentially protect dogs against "canine flu" disease caused by H3N8 canine influenza viruses.

Table 29 Serology - HI titers before and after vaccination and after infection Dog* Group Titles HI Before vaccination After 1st vaccination After the 2nd vaccination After infection * 7d 14-d 7d 14-d 21-d 7d 14-d AKT Vaccinate (vaccinated dog)** <10 40 80 640 640 640 320 320 ALH Vaccinate (vaccinated dog)** <10 40 80 320 160 160 80 *** ALU Vaccinate (vaccinated dog)** <10 40 80 320 160 160 80 80 ANJ Vaccinate (vaccinated dog)** <10 40 80 320 160 80 320 *** ANU Vaccinate (vaccinated dog)** <10 40 80 320 160 80 160 *** AJW Control <10 <10 <10 <10 <10 <10 10 *** AKR Control <10 <10 <10 <10 <10 <10 10 *** ALZ Control <10 <10 <10 <10 <10 <10 20 20 ARC Control <10 <10 <10 <10 <10 <10 10 10 * These animals were challenged with Ohio 03 equine influenza isolate
** Vaccination used CARBIGEN™ adjuvanted inactivated Ohio 03 Equine Influenza Virus Vaccine
*** Euthanized 7 days after infection

Table 30 Infection with Canine Influenza Virus* - Clinical Symptoms, Virus Isolation, Histopathological Findings Dog ID Processing group Clinical symptoms Virus isolation Microscopic damage (histopathology), Nose swab Tracheal scraping lung tissue AKT Vaccinate** No Negative Negative Negative Negative ALH Vaccinate** No Negative Negative Negative Negative ALU Vaccinate** No Negative Negative Negative Negative ANJ Vaccinate** No Negative Negative Negative Negative ANU Vaccinate** No Negative Negative Negative Negative AJW Control No Negative Negative Negative Negative AKR Control No Negative Negative Negative Negative ALZ Control No Negative Negative Negative Negative ARC Control No Negative Negative Negative Negative * These animals were challenged with Ohio 03 equine influenza isolate
** Vaccination used CARBIGEN™ adjuvanted inactivated Ohio 03 Equine Influenza Virus Vaccine

EXAMPLE 19 - EFFECTIVENESS OF EQUINE INFLUENZA VIRUS VACCINE FOR DOGS

Canine influenza virus isolated from Florida influenza outbreaks has been characterized and is closely related to a number of H3N8 equine influenza virus isolates. By analyzing the similarity of the DNA sequences and amino acid sequences, it was demonstrated that the canine influenza virus is very similar to the equine influenza virus, A/equine/Ohio/03. The following study was conducted in dogs to determine the efficacy of commercially available equine influenza virus vaccines in dogs.

Procedure

Approximately 16 months old, 20 mixed breed dogs and 20 mixed sex Beagles were used in this study. Dogs were randomly assigned to 6 groups (Table 31) of 6-7 dogs each. Dogs in groups 1 and 4 were vaccinated with a commercially available inactivated, adjuvanted equine influenza vaccine (EQUICINE II™ Intervet Inc., Millsboro, DE) at 16 and 17 months of age by subcutaneous (SQ) route. Dogs in groups 2 and 5 were vaccinated with modified live influenza equine/Kentucky/91 vaccine in a volume of 1 ml intranasally (in one nostril). Blood samples were collected on the day of vaccination, on day 7 and day 14 after the first vaccination (groups 1, 2, 4 and 5) and after the second vaccination (groups 1 and 4) to determine the HI titer using equine influenza virus and canine influenza virus with using a standard protocol (SAM 124, CVB, USDA, Ames, IA).

Vaccinated dogs (vaccinates) (at 72 days after final vaccination) and controls were oronasally challenged with cell cultured equine influenza virus strain A/equine/Ohio/03 (10 x 7.0 - 10 x 8.0 TCID ₅₀ per dog) in a volume of 1-2 ml. The challenge virus was administered to dogs as an aerosol ("fog") using a nebulizer (DeVilbiss Ultra-Neb®99 ultrasonic nebulizer, Sunrise Medical, USA). Dogs were observed for influenza-related clinical symptoms for 12 days post-challenge. Nasal and oropharyngeal swabs were collected in Earl's MEM medium with antibiotics (neomycin and polymyxin B) once daily from day -1 to day 12 post challenge for virus isolation. The presence of virus in these swabs indicates that the animal is excreting the virus in nasal/oral secretions. All dogs were humanely euthanized on day 12 post challenge and lung tissue was collected in 10% buffered formalin for histological evaluation.

Table 31 Experiment plan Group Number of dogs Breed Treatment Number of doses Method of vaccination 1 7 Beagle EQUICINEII™** 2 Subcutaneous 2 7 Beagle A/KY/91*** 1 intranasal 3 6 Beagle Control N/A* N/A* 4 7 Mongrel dog EQUICINE II™ 2 Subcutaneous 5 7 Mongrel dog A/KY/91 1 intranasal 6 6 Mongrel dog Control N/A* N/A* * Not used
** EQUICINE II™ is sold by Intervet Inc. as a liquid vaccine. EQUICINE II™ contains inactivated influenza A/Pennsylvania/63 (or "A/Pa/63") and influenza A/equine/Kentucky/93 (or "A/KY/93") with carbopol (ie, HAVLOGEN® ( Intervet Inc. More specifically, a dose of EQUICINE II™ contains: inactivated A/Pa/63 at 10 ^6.0 EID ₅₀ , inactivated A/KY/93 at 10 ^6.7 EID _{5 O} , 0.25% v/v carbopol and sufficient PBS to bring total volume up to 1 ml.
*** A/KY/91 is a dried vaccine that has been reconstituted by adding water. This reconstitution was performed using vaccine grade water in an amount sufficient to bring the dose of vaccine to a total volume of 1 ml. This vaccine contained equine/Kentucky/91 (or "A/KY/91") influenza virus and is discussed in, for example, US Patent Nos. 6,436,408; 6398774 and 6177082, which are incorporated by reference in their entirety in this patent. When reconstituted, the vaccine dose contained A/KY/91 at 10 ^7.2 TCID _{5 O} per ml, 0.015 grams of NZ AMINE AS™ per ml, 0.0025 grams of gelatin per ml, and 0.04 grams of D-lactose per ml. NZ AMINE AS™ is a purified source of amino acids and peptides derived from the enzymatic hydrolysis of casein. NZ AMINE AS™ is sold by Kerry Bio-Science (Norwich, NY, USA).

results

All vaccinated dogs seroconverted (i.e. mutated serological specificity) after vaccination and HI titers ranged from 10-80 for dogs in the EQUICINE II™ vaccine group compared to 10-40 for dogs in the A/KY/91 vaccine group when used equine influenza virus (H3N8 type).

Samples collected at 2 weeks post-vaccination (following the second vaccination for the EQUICINE II™ vaccine) were analyzed to determine the HI titer with canine influenza virus as well as with equine influenza virus (type H3N8). These HI results are shown in Table 32. Clinical symptoms include fever (>103°F; >39.4°C), occasional cough, and mild nasal discharge observed after infection.

Table 32 Serology - HI titers at 2 weeks post vaccination Group Number of dogs Breed Treatment Titer HI with equine influenza virus canine influenza virus Range GMT Range GMT 1 7 Beagle Equicine II ^™ 10-80 36 10-80 33 2 7 Beagle A/KY/91 10-20 12 20-160 54 3 6 Beagle Control N/A* N/A* N/A* N/A* 4 7 Cur Equicine II ^™ 40-80 54 40-80 50 5 7 Cur A/KY/91 10-40 24 40-80 49 6 6 Cur Control N/A* N/A* N/A* N/A* * Not used

Among Beagles, 2 of 6 dogs in the EQUICINE II™ vaccine group (Group 1), 1 of 7 dogs in the A/KY/91 vaccine group (Group 2) and 2 of 6 dogs in the control group (Group 3) had a fever. One of the 6 dogs in Group 3 (control) was positive for virus in the cell culture supernatant of nasal swab material in a 0.25% chicken erythrocyte (CRBC) haemagglutination assay. One of 6 dogs in this control group (Group 3) and 1 of 7 dogs in the A/KY/91 vaccine group (Group 2) had a mild nasal discharge during the post-challenge observation period. There was no statistically significant difference (P>0.05) between the control and beagle dog vaccine groups.

Among non-purebred dogs, 5 of 7 dogs in the EQUICINE II™ vaccine group (Group 4), 1 of 7 dogs in the A/KY/91 vaccine group (Group 5) and 5 of 6 dogs in the control group (Group 6) had a fever. One dog from each of groups 4 and 6 had mild nasal discharge and one dog from group 5 had an occasional cough. Two of 7 dogs in the EQUICINE II™ vaccine group (Group 4) and 3 of 6 dogs in the control group (Group 6) were positive for influenza virus in a nasal swab on HA analysis. None of the dogs in group A/KY/91 (Group 5) were positive for influenza virus in nasal swabs.

Conclusion

It has been demonstrated through serology that vaccination of dogs with commercially available equine influenza vaccines stimulated a moderate level of anti-influenza antibody response. There may be some breed-to-breed variation in the development of influenza-associated clinical symptoms in dogs following infection with the H3N8 type influenza virus. Live attenuated equine influenza vaccine (A/KY/91) provided significant (P<0.05) protection against clinical disease progression on rectal temperature in non-purebred dogs. This live attenuated virus vaccine also prevented the isolation of influenza virus in nasal secretions.

EXAMPLE 20 - DEVELOPMENT OF THE CANINE INFLUENZA MODEL

In view of reports that disease induction in canines was not successful for research purposes, the potential use of canine influenza virus, H3N8, to develop a canine influenza virus immunization model in dogs was explored in the following study.

Procedure

Ten non-purebred mixed sex dogs were obtained from a commercial supplier and kept in cages in the BSL-2 facility. These dogs were randomly assigned to two groups of 5 dogs each. As shown in Table 33, one group was subjected to intratracheal/nasal infection and the other group was subjected to oronasal infection.

Table 33 Invention Plan Group Number of dogs Method of infection 1 5 Intratracheal/Intranasal 2 5 rotonosova

These dogs were challenged at approximately 12 weeks of age. Canine influenza virus grown in embryonated hen eggs (A/canine/Florida/242/03) was used as the challenge virus. Each dog received a total of approximately 107.2 TCID ₅₀ virus in a volume of either 2 ml (for the oronasal route) or 4 ml (for the intratracheal/nasal route).

For intratracheal/nasal challenge, 3 ml of challenge virus was injected first into the trachea followed by 5 ml of PBS using a delivery tube that consisted of a tracheal tube with an inflatable cuff (size 4.5/5.0, Sheridan, USA) and nutritional tubing (5Fr size, 1.7 mm; 16 inches (41 cm) long, Kendall, USA), and 1 ml challenge virus followed by 3 ml atmospheric air were injected into the nostrils using a syringe. For oronasal challenge, challenge virus was administered as an aerosol ("fog") using a nebulizer (Nebulair™, DVM Pharmaceuticals, Inc., Miami, FL) in a volume of approximately 2 ml. These dogs were observed for influenza-related symptoms within 14 days of infection. Dogs were euthanized on day 14 post challenge and tissue samples (lung and trachea) were collected in 10% buffered formalin for histopathological testing.

results

All dogs in groups 1 and 2 developed clinical symptoms of canine influenza within 24-48 hours. Each dog had 2 or more of the following clinical signs: fever (>103.0°F; >39.4°C), cough, serous or mucopurulent ocular discharge, serous or mucopurulent nasal discharge, vomiting, diarrhea, depression, weight loss, vomiting, hemoptysis and audible wheezing. Lung tissues from 5 of 5 dogs from group 1 and 4 and 4 of 5 from group 2 had histopathological lesions that included one or more of the following: diffuse purulent bronchopneumonia, bronchitis/bronchiolitis with plugs of neutrophilic exudate in the cavity, and marked aggregation mononuclear cells in the mucosa and peribronchiolar tissue, mixed exudate in the alveoli with large numbers of foamy macrophages, infiltration of lymphocytes and plasma cells, as well as granulocytes and thickening of the interalveolar septa with proliferation of type II pneumocytes, consistent with influenza infection. Tracheal tissue samples were normal.

Conclusion

An H3N8 canine influenza isolate, such as the isolate used in this study, can be used to induce canine influenza disease in dogs using one of the methods described in this study or a similar method.

EXAMPLE 21 - DEVELOPMENT OF THE CANINE INFLUENZA MODEL

The potential use of canine influenza virus, H3N8, to develop a canine influenza infection model in dogs was explored further in the following study.

Procedure

Fifteen non-purebred dogs aged 17-18 weeks and five beagles aged 15 weeks were obtained from commercial suppliers and kept in BSL-2 room cages. Non-purebred dogs were randomly assigned to 3 groups (Groups 1-3) of 5 dogs each. All Beagles were assigned to one group (Group 4) as shown in Table 34.

Table 34 Experiment plan Group Breed Number of dogs Dose of virus infection 1 Mongrels 5 10 ^6.8 TCID ₅₀ 2 Mongrels 5 10 ^5.8 TCID ₅₀ 3 Mongrels 5 10 ^4.8 TCID ₅₀ 4 Beagles 5 10 ^6.8 TCID ₅₀

These dogs were challenged oronasally with virulent canine influenza virus, A/Canine/Florida/242/2003 (isolated from the lung of a dog (greyhound) with canine influenza disease (provided by Dr. Cynda Crawford at the University of Florida)). This challenge virus was administered as an aerosol ("fog") using a nebulizer (Nebulair™) in a volume of approximately 2 ml. These dogs were observed for influenza-related symptoms within 14 hours of infection.

results

Eighty percent (4 out of 5) of dogs in groups 1 and 4, 100% of dogs in groups 2 and 3 developed clinical symptoms of canine influenza virus within 48 hours. Each dog had one or more of the following clinical signs: fever (>103.0°F; >39.4°C), cough, serous or mucopurulent discharge from the eyes, serous or mucopurulent nasal discharge, vomiting, diarrhea, depression, weight loss, gagging and wheezing. These clinical symptoms seen in Beagles were usually milder in short duration compared to non-purebred dogs.

Conclusion

An H3N8 canine influenza isolate, such as the isolate used in this study, can be used to induce a canine influenza-like or kennel cough-like disease in dogs using the method described in this study or a similar method with an infection dose range of 104.8– _106.8TCID50 . There were some differences in the clinical symptoms seen in non-purebred dogs and Beagles. Beagles generally tend to exhibit milder influenza-related clinical symptoms than non-purebred dogs.

EXAMPLE 22 - STUDY OF THE EFFICACY OF THE VACCINE AGAINST CANINE INFLUENZA.

The following study was conducted to evaluate the effectiveness of the H3N8 equine influenza vaccine in dogs against canine influenza virus.

Procedure

Seventeen off-bred dogs at 14 weeks of age and ten Beagles at 8 weeks of age were obtained from commercial suppliers. These dogs were randomly assigned to 5 groups as shown in Table 35 and kept in the study room.

Table 35 Experiment plan Group Age Number of dogs Treatment Number of doses Age at vaccination (weeks) 1 14 weeks 7 Vaccinate 2 14&18 2 14 weeks 5 Vaccinate 1 18 3 14 weeks 5 Control — — 4 8 weeks 5 Vaccinate 2 8&12 5 8 weeks 5 Control — —

The vaccine used in this study was adjuvanted HAVLOGEN®, inactivated equine influenza virus vaccine (A/equine/KY/02). To obtain this vaccine, the virus was inactivated with binary ethyleneimine (BEI) using a standard method. Each vaccine dose contained HAVLOGEN® (10% v/v), 6144 HA units of inactivated virus, 0.1% (v/v) 10% thimerosal, 0.1% (v/v) phenol red , enough NaOH to adjust the pH to 6.8-7.2, and enough PBS to bring the total dose volume to 1 ml.

Dogs in groups 1 and 4 were vaccinated with 2 doses of this vaccine. The second dose (ie booster) was administered 4 weeks after the first. Dogs in group 2 were vaccinated with 1 dose at 18 weeks of age. Blood samples were collected for YI titer assessment using a standard protocol (eg, SAM 124, CVB, USDA, Ames, IA) with H3N8 influenza isolate on days 0 (pre-vaccination), 7 and 14 after the first and second vaccinations. Approximately 5 days prior to challenge, these dogs were moved to the BSL-2 facility and housed in individual cages.

All vaccinated dogs (vaccinations) and control dogs of the same age were challenged oronasally with virulent canine influenza virus (107.7 TCID ₅₀ A/Canine/Florida/242/2003 per dog) at 2 weeks after the second vaccination of groups 1 and 4 and the first group 2 vaccinations. The challenge virus was administered as an aerosol ("fog") using a nebulizer (Nebulair™) at 2 ml per dog. These dogs were observed for influenza-related clinical symptoms for 17 days post infection. Nasal swabs and oropharyngeal swabs were collected in tubes containing 2 ml of virus transport medium for virus isolation from day -1 (ie one day before challenge) to day 17 post challenge. All dogs were euthanized on day 17 and lung and tracheal samples were collected in 10% buffered formalin for histopathology. Blood samples were collected on days 7 and 14 post challenge to determine the HI titer. The score assignments used for clinical symptoms during post-challenge observation are shown in Table 36.

results

All dogs in the 2-dose vaccination groups (groups 1 and 4) developed HI antibody titer responses against canine influenza virus isolate (Table 37). Post-challenge, an approximately 4-fold increase in titer on day 14 post-challenge in all groups indirectly indicated that all dogs had been exposed to the challenge virus. All dogs exhibited one or more of the following canine influenza symptoms: fever (>103.0°F; >39.4°C), cough, serous or mucopurulent eye discharge, serous or mucopurulent nasal discharge, vomiting , diarrhea, depression, weight loss and shortness of breath. The vaccines had less severe clinical symptoms compared to age-matched controls (Table 38). There was a significant reduction in clinical symptoms due to 2-dose vaccination in both 8-week-old (P=0.040) and 14-week-old (P=0.003) dogs (groups 4 and 1, respectively). In this experiment, single-dose vaccination did not result in a significant (P=0.294) reduction in clinical symptoms (Group 2).

The results of virus isolation are shown in Table 39. Among 14-week-old dogs, virus was isolated from swab samples collected from 2 of 7 dogs (29%) from the 2-dose vaccine group (Group 1), 3 of 5 dogs (60% ) from the single dose vaccine group (Group 2) and 5 of 5 dogs (100%) from the control group (Group 3). Among 8-week-old dogs, virus was isolated from 1 of 5 dogs (20%) from the 2-dose vaccine group (Group 4), and 4 of 5 dogs (80%) from the control group (Group 5). There was a significant decrease (P = 0.003) in the number of dogs positive for canine influenza virus in swab samples due to 2-dose vaccination (groups 1 and 4) compared to unvaccinated controls (groups 3 and 5). Although there was a decrease (60% vs 100%) in the number of dogs positive for canine influenza virus in swab samples between the single dose vaccine group (Group 2) and the control group (Group 3), this difference was not statistically significant (P = 0.222 ).

Histopathological evaluation of lung and tracheal tissue samples for lesions was performed to identify lesions consistent with canine influenza disease or pathognomic (characteristic) of canine influenza disease. It includes, for example, determining whether one or more of the following are present: areas of pyogenic bronchopneumonia; peribronchitis/peribronchiolitis with aggregation of mononuclear cells (lymphocytes, plasma cells); the presence of plugs of granulocytic cell debris in the lumen; hyperplasia of the respiratory epithelium; mixed exudate in the alveoli with a large number of granulocytic cells and cell debris; aggregates of (foamy) macrophages, plasma cells and lymphocytes and thickening of the interalveolar septa with proliferation of type II pneumocytes.

Table 40 provides a summary of the extent of damage in this experiment for the dogs tested. Among the 14-week-old dogs, lung lesions were less extensive and less severe in 5 of 7 dogs in the 2-dose vaccination group (Group 2) and 4 of 5 dogs in the 1-dose vaccination group (Group 1). All control dogs (Group 3) had severe and extensive lesions suggesting a lack of protection. There was no difference in tracheal injury due to 1- or 2-dose vaccination among 14 week old dogs. Among 8-week-old dogs, there was no difference in lung injury between 2-dose vaccinates and control dogs. None of these dogs had any tracheal lesions.

Conclusion

The results from this study demonstrate that: (1) an inactivated H3N8 equine influenza virus can induce HI antibody cross-reactivity with canine influenza virus in vaccinated dogs, (2) administration of an equine H3N8 vaccine can reduce the severity of canine influenza virus disease in dogs, and (3) the use of H3N8 equine influenza virus vaccine may reduce viral excretion in nasal and/or oral secretions.

Table 36 Clinical symptoms and scoring system Clinical symptoms Rating for the day Temperature <103.0°F (<39.4°C) 0 103.0-103.9°F (39.4- 2 104.0-104.9°F (40.0-40.5°C) 3 >105.0°F (>40.6°C) 4 Cough No cough 0 Episodic 2 Paroxysmal 4 sneezing No sneeze 0 episodic 1 Paroxysmal 2 Discharge from the nose No selection 0 Serous - minor 1 Serous - copious 1 Mucopurulent - weak 2 Mucopurulent - copious 3 Ocular discharge No selection 0 Serous - weak 1 Serous - copious 1 Mucopurulent - weak 2 Mucopurulent - copious 3 Hemoptysis No 0 Yes 5 Depression No 0 Yes 1 Anorexia No 0 Yes 1 Respiratory symptoms No 0 Wheezing 3 Dyspnea 4 Suffocation 5 Mucous sputum No 0 Yes 2 Vomit No 0 Yes 1 Fecal pathologies No 0 Yes 1

Table 37

Serology - Haemagglutination Inhibition Titers

*First vaccination - groups 1 and 4

**Second vaccination - groups 1 and 4; First vaccination - group 2

***Day of infection

Table 38 Analysis of common clinical assessments of canine influenza disease Group Treatment Number of vaccine doses Age at first vaccination groups 1 and 4 Average overall rating per dog P-value* 1 Vaccinate 2 14 weeks 8.7 0.003 (Group 1 vs. 3) 2 Vaccinate 1 14 weeks (these dogs were vaccinated once when they were 18 weeks old)) 21.8 0.294 (Group 2 vs. 3) 3 Control - 14 weeks (these dogs were not vaccinated) 25.4 - 4 Vaccinate 2 8 weeks 2.0 0.040 (Group 4 vs. 5) 5 Control - 8 weeks (these dogs were not vaccinated) 5.4 - * Analyzed using the NPARIWAY SAS® Version 8.2 procedure (vaccine groups compared using the Wilcoxon rank sum test)

Table 39

Isolation of the virus by the host

Table 40

Histopathological evaluation of tissue samples

"+" Severe lesion consistent with influenza infection or pathognomic for influenza infection

"+/-" Light damage (inconclusive)

"-" Normal condition

EXAMPLE 23 - CANINE INFLUENZA VACCINE EFFECTIVE STUDY

The following study was conducted to determine the effectiveness of the H3N8 multivalent equine influenza vaccine against canine influenza virus in dogs.

Procedure

Seventeen 15 week old beagles were obtained from a commercial supplier. These dogs were randomly assigned to 3 groups as shown in Table 41 and kept in the study room.

Table 41 Experiment plan Group Number of dogs Treatment Number of doses Age at vaccination (weeks) 1 7 Vaccinate 2 15 and 19 2 5 Vaccinate 1 19 3 5 Control - -

The vaccine used in this study was HAVLOGEN® adjuvanted inactivated equine influenza vaccine (A/equine/KY/02, A/equine/KY/93 and A/equine/NM/2/93). To obtain this vaccine, the viruses were inactivated with binary ethyleneimine (BEI) using a standard method. Each vaccine dose contained HAVLOGEN® (10% v/v), 2048 HA units of each inactivated virus, 0.1% (v/v) 10% thimerosal, 0.1% (v/v) phenol red, enough NaOH to adjust the pH to 6.8-7.2, and enough PBS to bring the total dose volume to 1 ml.

Dogs in group 1 were vaccinated with 2 doses of this vaccine. The second dose (ie booster) was administered 4 weeks after the first dose. Dogs in group 2 were vaccinated with 1 dose of vaccine at 9 weeks of age. Blood samples were collected for HI titer assessment using a standard canine influenza H3N8 isolate protocol on days 0 (pre-vaccination), 7 and 14 after the first and second vaccinations. 7 days prior to challenge, these dogs were moved to the BSL-2 facility and housed in individual cages.

All vaccinated dogs (vaccinates) and control dogs of the same age were challenged oronasally with virulent canine influenza virus (107.3 TCID ₅₀ A/Canine/Florida/242/2003 per dog) at 2 weeks after the second vaccination of group 1 and the first vaccination of group 2. The challenge virus was administered as an aerosol ("fog") using a nebulizer (Nebulair™) at 2 ml per dog. These dogs were observed for influenza-associated clinical symptoms within 14 days of infection. All dogs were euthanized on day 14 and lung and tracheal samples were collected in 10% buffered formalin for histopathology. Blood samples were collected on days 7 and 14 post challenge to determine the HI titer. The score assignments used for clinical symptoms during post-challenge observation are shown in Table 42.

results

All vaccinated dogs developed HI antibody titer responses against canine influenza virus isolate (Table 43). Post-challenge, an approximately 4-fold increase in titer on day 14 post-challenge in all groups indirectly indicated that all dogs had been exposed to the challenge virus. All dogs exhibited one or more of the following canine influenza symptoms: fever (>103.0°F; >39.4°C), cough, serous or mucopurulent eye discharge, serous or mucopurulent nasal discharge, vomiting , diarrhea, depression, weight loss and shortness of breath. The vaccines had less severe clinical symptoms compared to age-matched controls (Table 44). There was a significant reduction in clinical symptoms due to 2-dose vaccination (P=0.028) (group 1). Single-dose vaccination did not result in a significant (P=0.068) reduction in clinical symptoms (Group 2).

As in Example 22, histopathological evaluation of lung and tracheal samples for lesions was performed to identify lesions consistent with canine influenza disease or pathognomic for canine influenza disease. Table 45 provides a summary of the extent of lesions in this experiment for these dogs. Among 15 week old dogs, vaccination of dogs with either 1 dose or 2 doses prevented lung injury in all dogs. Four of the 5 control dogs (80%) had severe pyogenic bronchopneumonia consistent with influenza. One of 7 dogs in the 2-dose vaccination group (Group 1) and 1 of 5 dogs in the control group (Group 3) had mild tracheal lesions suggestive of tracheitis, which could be attributed to influenza.

Conclusion

The results from this study demonstrate that: (1) an inactivated H3N8 equine influenza virus can induce HI antibody cross-reactivity with canine influenza virus in vaccinated dogs, (2) administration of an equine H3N8 vaccine can reduce the severity of canine influenza virus disease in dogs, and (3) the use of H3N8 equine influenza virus vaccine may reduce viral excretion in nasal and/or oral secretions.

Table 42. Clinical symptoms and scoring system

Table 42 Clinical symptoms and scoring system Clinical symptoms Rating for the day Temperature <103.0°F (<39.4°C) 0 103.0-103.9°F (39.4- 2 104.0-104.9°F (40.0- 3 >105.0°F (>40.6°C) 4 Cough No cough 0 Episodic 2 Paroxysmal 4 sneezing No sneeze 0 episodic 1 Paroxysmal 2 Discharge from the nose No selection 0 Serous - minor 1 Serous - copious 1 Mucopurulent - weak 2 Mucopurulent - copious 3 Ocular discharge No selection 0 Serous - weak 1 Serous - copious 1 Mucopurulent - weak 2 Mucopurulent - copious 3 Hemoptysis No 0 Yes 5 Depression No 0 Yes 1 Anorexia No 0 Yes 1 Respiratory symptoms No 0 Wheezing 3 Dyspnea 4 Suffocation 5 Mucous sputum No 0 Yes 2 Vomit No 0 Yes 1 Fecal pathologies No 0 Yes 1

Table 43

Serology - Haemagglutination Inhibition Titers

*First vaccination - group 1

**Second vaccination - group 1; First vaccination - group 2

***Day of infection

Table 44 Analysis of common clinical assessments of canine influenza disease Group Treatment Number of doses Age at first vaccination group 1 Average overall rating per dog P-value 1 Vaccinate 2 15 weeks 6.3 0.028
(Group 1 vs 3) 2 Vaccinate 1 15 weeks (these dogs were vaccinated once when they were 19 weeks old) 14.2 0.068
(Group 2 vs 3) 3 Control - 15 weeks (these dogs were not vaccinated) 22.4 - * Analyzed using the NPARIWAY SAS® Version 8.2 procedure (vaccine groups compared using the Wilcoxon rank sum test)

Table 45

Histopathological evaluation of tissue samples

"+" Severe lesion consistent with influenza infection or pathognomic for influenza infection

"+/-" Light damage (inconclusive)

"-" Normal condition

EXAMPLE 24 - CANINE INFLUENZA VACCINE EFFECTIVE STUDY

The following study was conducted to determine: (1) the efficacy of the monovalent vaccine versus multivalent H3N8 equine influenza vaccines against canine influenza virus in dogs and (2) the effect of the route of administration on the efficacy of the vaccine.

Procedure

Thirty 1-week-old off-bred Beagle dogs were obtained from a commercial supplier. These dogs were randomly assigned to 6 groups as shown in Table 46 and kept in the study room.

Table 46 Experiment plan Group Number of dogs Treatment Method of vaccination Number of doses Age at vaccination (weeks) 1 5 VAX-1 IN 2 10&14 2 5 VAX-2 SQ 2 10&14 3 5 VAX-2 IN 2 10&14 4 5 VAX-3 SQ 2 10&14 5 5 VAX-3 IN 2 10&14 6 5 Control — — —

Three types of vaccines were used (VAX-1, VAX-2 and VAX-3). VAX-1 was adjuvanted with HAVLOGEN®, a non-inactivated monovalent equine influenza virus vaccine (A/equine/KY/02), and each dose contained HAVLOGEN® (10% v/v), 6144 HA units of inactivated virus, 0.1 % (v/v) 10% thimerosal, 0.1% (v/v) phenol red, enough NaOH to adjust the pH to 6.8-7.2, and enough PBS to bring the total dose volume to 1 ml VAX-2 was adjuvanted with HAVLOGEN®, a non-inactivated monovalent equine influenza virus vaccine (A/equine/KY/02), and each dose contained HAVLOGEN® (10% v/v), 4096 HA units of inactivated virus, 0.1 % (v/v) 10% thimerosal, 0.1% (v/v) phenol red, enough NaOH to adjust the pH to 6.8-7.2, and enough PBS to bring the total dose volume to 1 ml VAX-3 was adjuvanted with HAVLOGEN®, a non-inactivated multivalent equine influenza virus vaccine (A/equine/KY/02, A/equine/KY/93 and A/equine/NM/2/93) and contained HAVLOGEN® (10% v/v ./v), 2048 HA units of inactivated virus per strain, 0.1% (v/v) 10% thimerosal, 0.1% (v/v) phenol red, enough NaOH to adjust pH to 6.8-7.2 and enough PBS to bring the total dose volume to 1 ml. All influenza viruses used to prepare the vaccine composition were inactivated with binary ethyleneimine (BEI) using a standard method.

These vaccines and routes of administration for each group are described in Table 46. All dogs in the vaccinated groups were vaccinated either intranasally (IN) or subcutaneously (SQ) and each dog received 2 doses. The second (ie booster) dose was administered 4 weeks after the first dose. Blood samples were collected for HI titer assessment using a standard canine influenza H3N8 isolate protocol on days 0 (pre-vaccination), 7 and 14 after the first and second vaccinations. 7 days prior to challenge, these dogs were moved to the BSL-2 facility and housed in individual cages.

All vaccinated dogs (vaccinates) and control dogs of the same age were challenged oronasally with virulent canine influenza virus (107.4 TCID ₅₀ A/Canine/Florida/242/2003 per dog) at 2 weeks after the second vaccination. The challenge virus was administered as an aerosol ("fog") using a nebulizer (Nebulair™) at a volume of 2 ml per day. These dogs were observed for influenza-associated clinical symptoms within 14 days of infection. Blood samples were collected on days 7 and 14 post challenge to determine the HI titer. All dogs were euthanized on day 14 and lung and tracheal samples were collected in 10% buffered formalin for histopathology. The score assignments used for clinical symptoms during post-challenge observation are shown in Table 47.

results

All SQ-vaccinated dogs developed HI antibody titer responses against canine influenza virus isolate, regardless of vaccine type (Table 48). None of the dogs in the IN vaccination groups (ie, groups 1, 3, and 5) developed HI antibody titer responses against canine influenza virus isolate, regardless of vaccine type, during the post-vaccination period. However, there was a 4-fold increase in titer on day 14 post-challenge in all dogs by indirect determination, indicating that all dogs were exposed to the challenge virus (Table 47).

All dogs exhibited one or more of the following clinical signs of canine influenza: fever (>103.0°F; >39.4°C), cough, serous or mucopurulent discharge from the eyes, serous or mucopurulent discharge from the nose, vomiting, diarrhea, depression, weight loss and shortness of breath. The vaccines had less severe clinical symptoms compared to age-matched controls (Table 49). There was a significant reduction in clinical symptoms in dogs vaccinated with VAX-3 using the SQ method (Group 4). In this experiment, administration of IN VAX-1, VAX-2, or VAX-3 showed no significant reduction in clinical symptoms of canine influenza virus.

As in Examples 22 and 23, histopathological evaluation of lung and tracheal lesions for lesions was performed to identify lesions consistent with canine influenza disease or pathognomic for canine influenza disease. Table 50 provides a summary of the extent of damage in this experiment for these dogs. Five of the 5 control dogs (Group 6) had lung injury consistent with influenza infection. Two of 5 dogs vaccinated with VAX-2 method SQ (group 2) and 3 of 5 dogs vaccinated with VAX-3 method SQ (group 4) did not have any influenza-associated lung injury. All dogs that received intranasal vaccine, regardless of vaccine type, had severe lung injury consistent with influenza infection. Tracheal lesions observed in this study were very mild.

Conclusion

The results from this study demonstrate that: (1) inactivated H3N8 equine influenza virus can induce HI antibody cross-reactivity in SQ-vaccinated dogs, (2) intranasal administration of a monovalent (VAX-1 and VAX-2) or multivalent vaccine ( VAX-3) was not effective in dogs, and (3) subcutaneous administration of the multivalent vaccine (VAX-3) provided a significant (P=0.016) reduction in the severity of canine influenza virus disease in dogs.

Table 47 Clinical symptoms and scoring system Clinical symptoms Rating for the day Temperature <103.0°F (<39.4°C) 0 103.0-103.9°F (39.4- 2 104.0-104.9°F (40.0- 3 >105.0°F (>40.6°C) 4 Cough No cough 0 Episodic 2 Paroxysmal 4 sneezing No sneeze 0 episodic 1 Paroxysmal 2 Discharge from the nose No selection 0 Serous - minor 1 Serous - copious 1 Mucopurulent - weak 2 Mucopurulent - copious 3 Ocular discharge No selection 0 Serous - weak 1 Serous - copious 1 Mucopurulent - weak 2 Mucopurulent - copious 3 Hemoptysis No 0 Yes 5 Depression No 0 Yes 1 Anorexia No 0 Yes 1 Respiratory symptoms No 0 Wheezing 3 Dyspnea 4 Suffocation 5 Mucous sputum No 0 Yes 2 Vomit No 0 Yes 1 Fecal pathologies No 0 Yes 1

Table 48

Serology - Haemagglutination Inhibition Titers

*First vaccination

**Second vaccination

***Day of infection

Table 49

Analysis of common clinical assessments of canine influenza disease

Group Treatment Method of vaccination Average overall rating per dog P-value* 1 VAX-1 IN 35.2 .500 (Group 1 vs. 6) 2 VAX-2 SQ 31.0 .345 (Group 2 vs. 6) 3 VAX-2 IN 39.4 0.631 (Group 3 vs. 6) 4 VAX-3 SQ 13.0 0.016 (Group 4 vs. 6) 5 VAX-3 IN 42.6 .790 (Group 4 vs. 6) 6 Control — 36.8 — * Analyzed using the NPARIWAY SAS® Version 8.2 procedure (vaccine groups compared using the Wilcoxon rank sum test)

Table 50

Histopathological evaluation of tissue samples

"+" Severe lesion consistent with influenza infection or pathognomic for influenza infection

"+/-" Light damage (inconclusive)

"-" Normal condition

EXAMPLE 25 - CANINE INFLUENZA VACCINE EFFECTIVE STUDY

Canine influenza is caused by the H3N8 influenza virus (CIV). CIV is very closely related to the equine H3N8 viruses (Crawford et al, 2005) and infects all exposed dogs.

Approximately 80% of exposed dogs develop clinical symptoms. In the following study, the effectiveness of an inactivated H3N8 equine influenza virus vaccine and a canine influenza virus vaccine was determined.

Procedure

Thirty-five Beagles and five non-purebred dogs were used in this study. Beagles were randomly assigned to three groups (Table 51). All non-purebred dogs were assigned to the control group (Group 3). All dogs were fed a standard growth diet and water was available ad libitum.

Table 51 Experiment plan Group Treatment Method of vaccination Number of dogs Age at vaccination (weeks) Infection 1 VAX-1 IM 15 8&12 Yes 2 VAX-2 SC 5 8&12 Yes 3 Control N/A 20 N/A Yes

Dogs in groups 1 and 2 were vaccinated with either VAX-1 or VAX-2 (Table 51). VAX-1 was adjuvanted with HAVLOGEN®, an inactivated equine influenza virus vaccine (A/equine/KY/02). To obtain a vaccine, the vaccine virus was inactivated with binary ethyleneimine (BEI) using a standard method. Each vaccine dose contained HAVLOGEN® (10% v/v), 6144 HA units of inactivated virus, 0.1% (v/v) 10% thimerosal, 0.1% (v/v) phenol red , enough PBS to bring the total dose volume to 1 ml and enough NaOH to bring the pH to 6.8-7.2.

VAX-2 was an inactivated, CARBIGEN™ adjuvanted canine influenza virus vaccine (A/canine/F1/43/2004). (A/canine/F1/43/2004) was inactivated with binary ethyleneimine (BEI) using the standard method. Each dose of this vaccine contained 5% wt. CARBIGEN™, approximately 1280 HA units of inactivated virus, enough PBS to bring the total dose volume to 1 ml, and enough NaOH to adjust the pH to 7.2-7.4. Serum samples were collected from all dogs on the first and second vaccination days, days 7 and 14 after the first and second vaccinations, and before challenge to determine HI titers using the standard protocol for H3N8 equine influenza virus (SAM 124, CVB, USDA, Ames, IA) . Seven days prior to challenge, these dogs were moved to the ABSL-2 facility and housed in individual cages.

All vaccinated dogs and control dogs of the same age were challenged oronasally with virulent canine influenza virus (107.2 TCID ₅₀ A/Canine/Florida/242/2003 per dog) at 2 weeks after the second vaccination. The challenge virus was administered as an aerosol ("fog") (2 ml/dog) using a nebulizer (Nebulair™). These dogs were observed for influenza-associated clinical symptoms within 14 days of infection. Nasal swabs and oropharyngeal swabs were collected once daily in tubes containing 2 ml of virus transport medium for virus isolation from day -1 (ie, one day before challenge) until day 14 post challenge. Blood samples were collected on days 7 and 14 post challenge to determine the HI titer. The score assignments used for clinical symptoms during post-challenge observation are shown in Table 52.

results

All vaccinated dogs (Groups 1 and 2) developed HI antibody titer responses against canine influenza virus isolate, regardless of vaccine type (Table 53). All dogs exhibited one or more of the following canine influenza symptoms: fever (>103.0°F; >39.4°C), cough, serous or mucopurulent eye discharge, serous or mucopurulent nasal discharge, vomiting , diarrhea, depression, weight loss and anorexia. The vaccines had less severe clinical symptoms compared to age-matched controls (Table 54). There was a significant (P<0.001) reduction in clinical symptoms in dogs vaccinated with either VAX-1 (Group 1) or VAX-2 (Group 2).

The results of virus isolation are shown in Tables 55 and 56. After infection with virulent canine influenza virus, canine influenza virus was isolated from 5 of 15 (33%) dogs from group 1 (VAX-1), 0 of 5 (0%) dogs from group 2 (VAX-2) and 17 of 20 (85%) controls (Group 3). Both vaccinated with inactivated equine influenza virus vaccine (VAX-1) and vaccinated with canine influenza virus vaccine (VAX-2) showed a significant (P = 0.004) reduction in host shedding of virus in nasal and oral secretions or both ( table 55) in comparison with controls.

Conclusion

The results from this study demonstrate that: (1) inactivated equine and canine H3N8 influenza vaccines can induce HI antibody cross-reactivity in vaccinated dogs, (2) administration of H3N8 equine influenza or canine influenza virus vaccine can reduce disease severity, caused by canine influenza virus in dogs and (3) the use of H3N8 equine influenza virus vaccine or canine influenza virus vaccine may reduce virus excretion in nasal and/or oral secretions.

Table 52 Clinical symptoms and scoring system Clinical symptoms Rating for the day Temperature <103.0°F (<39.4°C) 0 103.0-103.9°F (39.4- 2 104.0-104.9°F (40.0-40.5°C) 3 >105.0°F (>40.6°C) 4 Cough No cough 0 Episodic 2 Paroxysmal 4 sneezing No sneeze 0 episodic 1 Paroxysmal 2 Discharge from the nose No selection 0 Serous - minor 1 Serous - copious 1 Mucopurulent - weak 2 Mucopurulent - copious 3 Ocular discharge No selection 0 Serous - weak 1 Serous - copious 1 Mucopurulent - weak 2 Mucopurulent - copious 3 Hemoptysis No 0 Yes 5 Depression No 0 Yes 1 Anorexia No 0 Yes 1 Respiratory symptoms No 0 Wheezing 3 Dyspnea 4 Suffocation 5 Mucous sputum No 0 Yes 2 Vomit No 0 Yes 1 Fecal pathologies No 0 Yes 1

Table 53

Serology - Haemagglutination Inhibition Titers

*First vaccination

**Second vaccination

***Day of infection

Table 54 Analysis of common clinical assessments of canine influenza disease Group Treatment Average overall rating per dog P-value 1 VAX-1 9.1 <0.001 (Group 1 vs. 3) 2 VAX-2 5.4 <0.001 (Group 2 vs. 3) 3 Control 24.1 — * Analyzed using the NPARIWAY SAS® Version 9.1 procedure (vaccine groups compared using the GLM procedure)

Table 55 Isolation of the virus after infection Group Treatment Percentage of dogs excreting the virus P value 1 VAX-1 33% (5/15) 0.004 (Group 1 vs. 3) 2 VAX-2 0% (0/5) 0.004 (Group 2 vs. 3) 3 Control 85% (17/20) — * Analyzed using the FREQ SAS® procedure (Version 9.1), and the P value is associated with Fisher's exact test

Table 56

Serology - Haemagglutination Inhibition Titers

N - the virus was not isolated from swabs from the oral cavity of the nose or from the nose

P - virus isolated from nasal or oral swabs or from nasal and oral swabs

Table 57

Similarities of amino acid sequences of hemagglutinin, neuraminidase and nucleoprotein genes among influenza viruses

The words "comprise", "comprises" and "comprising" in this patent (including in the claims) are to be interpreted as "inclusive" and not as "exclusively". This interpretation is meant to be the same as the interpretation given to these words under US Patent Law.

The foregoing detailed description of the preferred embodiments is only intended to familiarize other skilled artisans in the art with this invention, its principles, and its practice, so that other skilled artisans in the art may adapt and use this invention in its many forms, so that they will best suit the requirements of the particular application. Thus, this invention is not limited to the above embodiments, and can be modified in a variety of ways.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light of this description will be prompted by those skilled in the art and should be within the spirit and scope of this application.

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SEQUENCE LIST

<110> Crawford, Patti Cynthia

Gibbs, E. Paul J.

Dubovi, Edward J.

Donis, Ruben Omar

Katz, Jacqueline M.

Klimov, Alexander I.

Lakshmanan, Nallakannu P.

Lum, Melissa Anne

Goovaerts, Daniel Ghislena Emiel

Mellencamp, Mark William

<120> Influenza virus capable of infecting canines and its uses

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<150> US 11/409,416

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<150> US 60/728,449

<151> 2005-10-19

<150> US 60/754,881

<151> 2005-12-29

<150> US 60/759,162

<151> 2006-01-14

<150> US 60/761,451

<151> 2006-01-23

<150> US 60/779,080

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690 695 700

ggc cca gca cta agc atc aat gaa cta agc aaa ctt gca aaa ggg gag 2160

Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu

705 710 715 720

aaa gcc aat gta cta att ggg caa ggg gac gta gtg ttg gta atg aaa 2208

Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys

725 730 735

cgg aaa cgt gac tct agc ata ctt act gac agc cag aca gcg acc aaa 2256

Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys

740 745 750

agg att cgg atg gcc atc aat 2277

Arg Ile Arg Met Ala Ile Asn

755

<210> 2

<211> 759

<212> PRT

<213> Influenza virus

<400> 2

Met Glu Arg Ile Glu Glu Leu Arg Asp Leu Met Leu Gln Ser Arg Thr

1 5 10 15

Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys

20 25 30

Lys Tyr Thr Ser Glu Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys

35 40 45

Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met

50 55 60

Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys

65 70 75 80

Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val

85 90 95

Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Ile His Tyr Pro

100 105 110

Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly

115 120 125

Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg

130 135 140

Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln

145 150 155 160

Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Ile Ile

165 170 175

Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu

180 185 190

Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu

195 200 205

Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr

210 215 220

Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp

225 230 235 240

Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Ile Asp

245 250 255

Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val

260 265 270

Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln

275 280 285

Ile Gly Gly Ile Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu

290 295 300

Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser

305 310 315 320

Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser

325 330 335

Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu

340 345 350

Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg

355 360 365

Arg Ala Thr Ala Ile Ile Lys Lys Ala Thr Arg Arg Leu Ile Gln Leu

370 375 380

Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val

385 390 395 400

Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly

405 410 415

Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His

420 425 430

Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe His Asn

435 440 445

Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu

450 455 460

Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val

465 470 475 480

Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val

485 490 495

Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Ile Leu

500 505 510

Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr

515 520 525

Ile Ile Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser

530 535 540

Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Ile Val

545 550 555 560

Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Ile Glu

565 570 575

Phe Glu Pro Phe Gln Ser Leu Val Pro Arg Ala Thr Arg Ser Gln Tyr

580 585 590

Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly

595 600 605

Thr Phe Asp Thr Ala Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala

610 615 620

Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val

625 630 635 640

Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe

645 650 655

Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala

660 665 670

Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser

675 680 685

Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asn Lys Arg Tyr

690 695 700

Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu

705 710 715 720

Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys

725 730 735

Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys

740 745 750

Arg Ile Arg Met Ala Ile Asn

755

<210> 3

<211> 2274

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(2274)

<400> 3

atg gat gtc aat ccg act cta ctc ttc tta aag gtg cca gcg cag aat 48

Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn

1 5 10 15

gct ata agc aca aca ttc cct tat act gga gat cct ccc tac agt cat 96

Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His

20 25 30

gga aca ggg aca gga tac acc atg gat act gtc aac aga aca cac caa 144

Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln

35 40 45

tat tca gaa aaa ggg aaa tgg aca aca aac act gag att gga gca cca 192

Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro

50 55 60

caa ctt aat cca atc gat gga cca ctt cct gaa gac aat gaa cca agc 240

Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser

65 70 75 80

ggg tac gcc caa aca gat tgt gta ttg gaa gca atg gct ttc ctt gaa 288

Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu

85 90 95

gaa tcc cat cca gga atc ttt gaa aat tcg tgt ctt gaa acg atg gag 336

Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu

100 105 110

gtg att cag cag aca aga gtg gac aaa cta aca caa ggc cga caa act 384

Val Ile Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr

115 120 125

tat gat tgg acc ttg aat agg aat caa cct gcc gca aca gca ctt gct 432

Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala

130 135 140

aat acg att gaa gta ttc aga tca aat ggt ctg act tcc aat gaa tcg 480

Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr Ser Asn Glu Ser

145 150 155 160

ggg aga ttg atg gac ttc ctc aaa gat gtc atg gag tcc atg aac aag 528

Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys

165 170 175

gaa gaa atg gaa ata aca aca cac ttc caa cgg aag aga aga gta aga 576

Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg

180 185 190

gac aac atg aca aag aga atg gta aca cag aga acc ata ggg aag aaa 624

Asp Asn Met Thr Lys Arg Met Val Thr Gln Arg Thr Ile Gly Lys Lys

195 200 205

aaa caa cga tta aac aga aag agc tat cta atc aga aca tta acc cta 672

Lys Gln Arg Leu Asn Arg Lys Ser Tyr Leu Ile Arg Thr Leu Thr Leu

210 215 220

aac aca atg acc aag gac gct gag aga ggg aaa ttg aaa cga cga gca 720

Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala

225 230 235 240

atc gct acc cca ggg atg cag ata aga ggg ttt gta tat ttt gtt gaa 768

Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu

245 250 255

aca cta gct cga aga ata tgt gaa aag ctt gaa caa tca gga ttg cca 816

Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro

260 265 270

gtt ggc ggt aat gag aaa aag gcc aaa ctg gct aat gtc gtc aga aaa 864

Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys

275 280 285

atg atg act aat tcc caa gac act gaa ctc tcc ttc acc atc act ggg 912

Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly

290 295 300

gac aat acc aaa tgg aat gaa aat cag aac cca cgc ata ttc ctg gca 960

Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Ile Phe Leu Ala

305 310 315 320

atg atc aca tac ata act aga aac cag cca gaa tgg ttc aga aat gtt 1008

Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val

325 330 335

cta agc att gca ccg att atg ttc tca aat aaa atg gca aga ctg ggg 1056

Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly

340 345 350

aaa gga tat atg ttt gaa agc aaa agt atg aaa ttg aga act caa ata 1104

Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile

355 360 365

cca gca gaa atg cta gca agc att gac cta aaa tat ttc aat gat tca 1152

Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser

370 375 380

aca aaa aag aaa att gag aag ata cga cca ctt ctg gtt gac ggg act 1200

Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Val Asp Gly Thr

385 390 395 400

gct tca ctg agt cct ggc atg atg atg gga atg ttc aac atg ttg agc 1248

Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser

405 410 415

act gtg ctg ggt gta tcc ata tta aac ctg ggc cag agg aaa tac aca 1296

Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Arg Lys Tyr Thr

420 425 430

aag acc aca tac tgg tgg gat ggt ctg caa tca tcc gat gac ttt gct 1344

Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala

435 440 445

ttg ata gtg aat gcg cct aat cat gaa gga gta caa gct gga gta gac 1392

Leu Ile Val Asn Ala Pro Asn His Glu Gly Val Gln Ala Gly Val Asp

450 455 460

aga ttc tat aga act tgc aaa ctg gtc ggg atc aac atg agc aaa aag 1440

Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys

465 470 475 480

aag tcc tac ata aat aga act gga aca ttc gaa ttc aca agc ttt ttc 1488

Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe

485 490 495

tac cgg tat ggt ttt gta gcc aat ttc agc atg gaa cta ccc agt ttt 1536

Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe

500 505 510

ggg gtt tcc gga ata aat gaa tct gca gac atg agc att gga gtg aca 1584

Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr

515 520 525

gtc atc aaa aac aac atg ata aat aat gat ctc ggt cct gcc acg gca 1632

Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala

530 535 540

caa atg gca ctc caa ctc ttc att aag gat tat cgg tac aca tac cgg 1680

Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg

545 550 555 560

tgc cat aga ggt gat acc cag ata caa acc aga aga tct ttt gag ttg 1728

Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu

565 570 575

aag aaa ctg tgg gaa cag act cga tca aag act ggt cta ctg gta tca 1776

Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Thr Gly Leu Leu Val Ser

580 585 590

gat ggg ggt cca aac cta tat aac atc aga aac cta cac atc ccg gaa 1824

Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu

595 600 605

gtc tgt tta aaa tgg gag cta atg gat gaa gat tat aag ggg agg cta 1872

Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Lys Gly Arg Leu

610 615 620

tgc aat cca ttg aat cct ttc gtt agt cac aaa gaa att gaa tca gtc 1920

Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val

625 630 635 640

aac agt gca gta gta atg cct gcg cat ggc cct gcc aaa agc atg gag 1968

Asn Ser Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu

645 650 655

tat gat gct gtt gca aca aca cat tct tgg atc ccc aag agg aac cgg 2016

Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg

660 665 670

tcc ata ttg aac aca agc caa agg gga gta ctc gaa gat gag cag atg 2064

Ser Ile Leu Asn Thr Ser Gln Arg Gly Val Leu Glu Asp Glu Gln Met

675 680 685

tat cag aaa tgc tgc aac ctg ttt gaa aaa ttc ttc ccc agc agc tca 2112

Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser

690 695 700

tac aga aga cca gtc gga att tct agt atg gtt gag gcc atg gtg tcc 2160

Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser

705 710 715 720

agg gcc cgc att gat gca cga att gac ttc gaa tct gga cgg ata aag 2208

Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys

725 730 735

aag gat gag ttc gct gag atc atg aag atc tgt tcc acc att gaa gag 2256

Lys Asp Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu

740 745 750

ctc aga cgg caa aaa tag 2274

Leu Arg Arg Gln Lys

755

<210> 4

<211> 757

<212> PRT

<213> Influenza virus

<400> 4

Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn

1 5 10 15

Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His

20 25 30

Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln

35 40 45

Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro

50 55 60

Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser

65 70 75 80

Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu

85 90 95

Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu

100 105 110

Val Ile Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr

115 120 125

Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala

130 135 140

Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr Ser Asn Glu Ser

145 150 155 160

Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys

165 170 175

Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg

180 185 190

Asp Asn Met Thr Lys Arg Met Val Thr Gln Arg Thr Ile Gly Lys Lys

195 200 205

Lys Gln Arg Leu Asn Arg Lys Ser Tyr Leu Ile Arg Thr Leu Thr Leu

210 215 220

Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala

225 230 235 240

Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu

245 250 255

Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro

260 265 270

Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys

275 280 285

Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly

290 295 300

Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Ile Phe Leu Ala

305 310 315 320

Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val

325 330 335

Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly

340 345 350

Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile

355 360 365

Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser

370 375 380

Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Val Asp Gly Thr

385 390 395 400

Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser

405 410 415

Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Arg Lys Tyr Thr

420 425 430

Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala

435 440 445

Leu Ile Val Asn Ala Pro Asn His Glu Gly Val Gln Ala Gly Val Asp

450 455 460

Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys

465 470 475 480

Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe

485 490 495

Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe

500 505 510

Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr

515 520 525

Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala

530 535 540

Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg

545 550 555 560

Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu

565 570 575

Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Thr Gly Leu Leu Val Ser

580 585 590

Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu

595 600 605

Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Lys Gly Arg Leu

610 615 620

Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val

625 630 635 640

Asn Ser Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu

645 650 655

Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg

660 665 670

Ser Ile Leu Asn Thr Ser Gln Arg Gly Val Leu Glu Asp Glu Gln Met

675 680 685

Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser

690 695 700

Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser

705 710 715 720

Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys

725 730 735

Lys Asp Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu

740 745 750

Leu Arg Arg Gln Lys

755

<210> 5

<211> 2151

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(2151)

<400> 5

atg gaa gac ttt gtg cga cag tgc ttc aat cca atg atc gtc gag ctt 48

Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu

1 5 10 15

gcg gaa aag gca atg aaa gaa tat gga gag aac ccg aaa atc gaa aca 96

Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asn Pro Lys Ile Glu Thr

20 25 30

aac aaa ttt gca gca ata tgc act cac ttg gaa gtc tgc ttc atg tac 144

Asn Lys Phe Ala Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr

35 40 45

tcg gat ttt cac ttt att aat gaa ctg ggt gag tca gtg gtc ata gag 192

Ser Asp Phe His Phe Ile Asn Glu Leu Gly Glu Ser Val Val Ile Glu

50 55 60

tct ggt gac cca aat gct ctt ttg aaa cac aga ttt gaa atc att gag 240

Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu

65 70 75 80

ggg aga gat cga aca atg gca tgg aca gta gta aac agc atc tgc aac 288

Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn

85 90 95

acc aca aga gct gaa aaa cct aaa ttt ctt cca gat tta tac gac tat 336

Thr Thr Arg Ala Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr

100 105 110

aag gag aac aga ttt gtt gaa att ggt gtg aca agg aga gaa gtt cac 384

Lys Glu Asn Arg Phe Val Glu Ile Gly Val Thr Arg Arg Glu Val His

115 120 125

ata tac tac ctg gag aag gcc aac aaa ata aag tct gag aaa aca cat 432

Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Lys Thr His

130 135 140

atc cac att ttc tca ttt aca gga gag gaa atg gct aca aaa gcg gac 480

Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys Ala Asp

145 150 155 160

tat act ctt gat gaa gag agt aga gcc agg atc aag acc aga cta ttc 528

Tyr Thr Leu Asp Glu Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe

165 170 175

acc ata aga caa gaa atg gcc agt aga ggc ctc tgg gat tcc ttt cgt 576

Thr Ile Arg Gln Glu Met Ala Ser Arg Gly Leu Trp Asp Ser Phe Arg

180 185 190

cag tcc gag aga ggc gaa gag aca att gaa gaa aga ttt gaa ata aca 624

Gln Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr

195 200 205

ggg acg atg cgc aag ctt gcc aat tac agt ctc cca ccg aac ttc tcc 672

Gly Thr Met Arg Lys Leu Ala Asn Tyr Ser Leu Pro Pro Asn Phe Ser

210 215 220

agc ctt gaa aat ttt aga gtc tat gtg gat gga ttc gaa ccg aac ggc 720

Ser Leu Glu Asn Phe Arg Val Tyr Val Asp Gly Phe Glu Pro Asn Gly

225 230 235 240

tgc att gag agt aag ctt tct caa atg tcc aaa gaa gta aat gcc aga 768

Cys Ile Glu Ser Lys Leu Ser Gln Met Ser Lys Glu Val Asn Ala Arg

245 250 255

atc gaa cca ttt tca aag aca aca ccc cga cca ctc aaa atg cca ggt 816

Ile Glu Pro Phe Ser Lys Thr Thr Pro Arg Pro Leu Lys Met Pro Gly

260 265 270

ggt cca ccc tgc cat cag cga tct aaa ttc ttg cta atg gat gct ctg 864

Gly Pro Pro Cys His Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu

275 280 285

aaa ctg agc att gag gac cca agt cac gag gga gag gga ata cca cta 912

Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu

290 295 300

tat gat gca atc aaa tgc atg aaa act ttc ttt gga tgg aaa gag ccc 960

Tyr Asp Ala Ile Lys Cys Met Lys Thr Phe Phe Gly Trp Lys Glu Pro

305 310 315 320

agt att gtt aaa cca cat gaa aag ggt ata aac ccg aac tat ctc caa 1008

Ser Ile Val Lys Pro His Glu Lys Gly Ile Asn Pro Asn Tyr Leu Gln

325 330 335

act tgg aag caa gta tta gaa gaa ata caa gac ctt gag aac gaa gaa 1056

Thr Trp Lys Gln Val Leu Glu Glu Ile Gln Asp Leu Glu Asn Glu Glu

340 345 350

agg acc ccc aag acc aag aat atg aaa aaa aca agc caa ttg aaa tgg 1104

Arg Thr Pro Lys Thr Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp

355 360 365

gca cta ggt gaa aat atg gca cca gag aaa gtg gat ttt gag gat tgt 1152

Ala Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Glu Asp Cys

370 375 380

aaa gac atc aat gat tta aaa caa tat gac agt gat gag cca gaa gca 1200

Lys Asp Ile Asn Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Ala

385 390 395 400

agg tct ctt gca agt tgg att caa agt gag ttc aac aaa gct tgt gag 1248

Arg Ser Leu Ala Ser Trp Ile Gln Ser Glu Phe Asn Lys Ala Cys Glu

405 410 415

ctg aca gat tca agc tgg ata gag ctc gat gaa att ggg gag gat gtc 1296

Leu Thr Asp Ser Ser Ser Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val

420 425 430

gcc cca ata gaa tac att gcg agc atg agg aga aat tat ttt act gct 1344

Ala Pro Ile Glu Tyr Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala

435 440 445

gag att tcc cat tgt aga gca aca gaa tat ata ata aaa gga gtg tac 1392

Glu Ile Ser His Cys Arg Ala Thr Glu Tyr Ile Ile Lys Gly Val Tyr

450 455 460

atc aac act gct cta ctc aat gca tcc tgt gct gcg atg gat gaa ttt 1440

Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Glu Phe

465 470 475 480

caa tta att ccg atg ata agt aaa tgc agg acc aaa gaa ggg aga agg 1488

Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg

485 490 495

aaa aca aat tta tat gga ttc ata ata aag gga agg tcc cat tta aga 1536

Lys Thr Asn Leu Tyr Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg

500 505 510

aat gat act gac gtg gtg aac ttt gta agt atg gaa ttt tct ctc act 1584

Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr

515 520 525

gat cca aga ttt gag cca cac aaa tgg gaa aaa tac tgc gtt cta gaa 1632

Asp Pro Arg Phe Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu

530 535 540

att gga gac atg ctt cta aga act gct gta ggt caa gtg tca aga ccc 1680

Ile Gly Asp Met Leu Leu Arg Thr Ala Val Gly Gln Val Ser Arg Pro

545 550 555 560

atg ttt ttg tat gta agg aca aat gga acc tct aaa att aaa atg aaa 1728

Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys

565 570 575

tgg gga atg gaa atg agg cgc tgc ctc ctt cag tct ctg caa cag att 1776

Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile

580 585 590

gaa agc atg atc gaa gct gag tcc tca gtc aaa gaa aag gac atg acc 1824

Glu Ser Met Ile Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr

595 600 605

aaa gaa ttt ttt gag aac aaa tca gag aca tgg cct ata gga gag tcc 1872

Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser

610 615 620

ccc aaa gga gtg gaa gag ggc tca atc ggg aag gtt tgc agg acc tta 1920

Pro Lys Gly Val Glu Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu

625 630 635 640

tta gca aaa tct gtg ttt aac agt tta tat gca tct cca caa ctg gaa 1968

Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu

645 650 655

ggg ttt tca gct gaa tct agg aaa tta ctt ctc att gtt cag gct ctt 2016

Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu

660 665 670

aag gat gac ctg gaa cct gga acc ttt gat att ggg ggg tta tat gaa 2064

Lys Asp Asp Leu Glu Pro Gly Thr Phe Asp Ile Gly Gly Leu Tyr Glu

675 680 685

tca att gag gag tgc ctg att aat gat ccc tgg gtt ttg ctt aat gca 2112

Ser Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala

690 695 700

tct tgg ttc aac tcc ttc ctt aca cat gca ctg aag tag 2151

Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Lys

705 710 715

<210> 6

<211> 716

<212> PRT

<213> Influenza virus

<400> 6

Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu

1 5 10 15

Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asn Pro Lys Ile Glu Thr

20 25 30

Asn Lys Phe Ala Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr

35 40 45

Ser Asp Phe His Phe Ile Asn Glu Leu Gly Glu Ser Val Val Ile Glu

50 55 60

Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu

65 70 75 80

Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn

85 90 95

Thr Thr Arg Ala Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr

100 105 110

Lys Glu Asn Arg Phe Val Glu Ile Gly Val Thr Arg Arg Glu Val His

115 120 125

Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Lys Thr His

130 135 140

Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys Ala Asp

145 150 155 160

Tyr Thr Leu Asp Glu Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe

165 170 175

Thr Ile Arg Gln Glu Met Ala Ser Arg Gly Leu Trp Asp Ser Phe Arg

180 185 190

Gln Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr

195 200 205

Gly Thr Met Arg Lys Leu Ala Asn Tyr Ser Leu Pro Pro Asn Phe Ser

210 215 220

Ser Leu Glu Asn Phe Arg Val Tyr Val Asp Gly Phe Glu Pro Asn Gly

225 230 235 240

Cys Ile Glu Ser Lys Leu Ser Gln Met Ser Lys Glu Val Asn Ala Arg

245 250 255

Ile Glu Pro Phe Ser Lys Thr Thr Pro Arg Pro Leu Lys Met Pro Gly

260 265 270

Gly Pro Pro Cys His Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu

275 280 285

Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu

290 295 300

Tyr Asp Ala Ile Lys Cys Met Lys Thr Phe Phe Gly Trp Lys Glu Pro

305 310 315 320

Ser Ile Val Lys Pro His Glu Lys Gly Ile Asn Pro Asn Tyr Leu Gln

325 330 335

Thr Trp Lys Gln Val Leu Glu Glu Ile Gln Asp Leu Glu Asn Glu Glu

340 345 350

Arg Thr Pro Lys Thr Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp

355 360 365

Ala Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Glu Asp Cys

370 375 380

Lys Asp Ile Asn Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Ala

385 390 395 400

Arg Ser Leu Ala Ser Trp Ile Gln Ser Glu Phe Asn Lys Ala Cys Glu

405 410 415

Leu Thr Asp Ser Ser Ser Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val

420 425 430

Ala Pro Ile Glu Tyr Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala

435 440 445

Glu Ile Ser His Cys Arg Ala Thr Glu Tyr Ile Ile Lys Gly Val Tyr

450 455 460

Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Glu Phe

465 470 475 480

Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg

485 490 495

Lys Thr Asn Leu Tyr Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg

500 505 510

Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr

515 520 525

Asp Pro Arg Phe Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu

530 535 540

Ile Gly Asp Met Leu Leu Arg Thr Ala Val Gly Gln Val Ser Arg Pro

545 550 555 560

Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys

565 570 575

Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile

580 585 590

Glu Ser Met Ile Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr

595 600 605

Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser

610 615 620

Pro Lys Gly Val Glu Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu

625 630 635 640

Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu

645 650 655

Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu

660 665 670

Lys Asp Asp Leu Glu Pro Gly Thr Phe Asp Ile Gly Gly Leu Tyr Glu

675 680 685

Ser Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala

690 695 700

Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Lys

705 710 715

<210> 7

<211> 838

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(657)

<400> 7

atg gat tcc aac act gtg tca agc ttt cag gta gac tgt ttt ctt tgg 48

Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

cat gtc cgc aaa cga ttc gca gac caa gaa ctg ggt gat gcc cca ttc 96

His Val Arg Lys Arg Phe Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

ctt gac cgg ctt cgc cga gac cag aag tcc cta agg gga aga ggt agc 144

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

act ctt ggt ctg gac atc gaa aca gcc act cat gca gga aag cag ata 192

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr His Ala Gly Lys Gln Ile

50 55 60

gtg gag cag att ctg gaa aag gaa tca gat gag gca ctt aaa atg acc 240

Val Glu Gln Ile Leu Glu Lys Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

att gcc tct gtt cct act tca ctc tac tta act gac atg act ctt gat 288

Ile Ala Ser Val Pro Thr Ser Leu Tyr Leu Thr Asp Met Thr Leu Asp

85 90 95

gag atg tca aga gac tgg ttc atg ctc atg ccc aag caa aaa gta aca 336

Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Thr

100 105 110

ggc tcc cta tgt ata aga atg gac cag gca atc atg gat aag aac atc 384

Gly Ser Leu Cys Ile Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile

115 120 125

ata ctt aaa gca aac ttt agt gtg att ttc gaa ggg ctg gaa aca cta 432

Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Glu Gly Leu Glu Thr Leu

130 135 140

ata cta ctt aga gcc ttc acc gaa gaa gga gca gtc gtt ggc gaa att 480

Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile

145 150 155 160

tca cca tta cct tct ctt cca gga cat act aat gag gat gtc aaa aat 528

Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn

165 170 175

gca att ggg gtc ctc atc gga gga ctt aaa tgg aat gat aat acg gtt 576

Ala Ile Gly Val Leu Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val

180 185 190

aga atc tct gaa act cta cag aga ttc gct tgg aga agc agt cat gag 624

Arg Ile Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser His Glu

195 200 205

aat ggg aga cct tca ttc cct tca aag cag aaa tgaaaaatgg agagaacaat 677

Asn Gly Arg Pro Ser Phe Pro Ser Lys Gln Lys

210 215

taagccagaa atttgaagaa ataagatggt tgattgaaga agtgcgacat agattgaaaa 737

atacagaaaa tagttttgaa caaataacat ttatgcaagc cttacaacta ttgcttgaag 797

tagaacaaga gataagaact ttctcgtttc agcttattta a 838

<210> 8

<211> 219

<212> PRT

<213> Influenza virus

<400> 8

Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

His Val Arg Lys Arg Phe Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr His Ala Gly Lys Gln Ile

50 55 60

Val Glu Gln Ile Leu Glu Lys Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

Ile Ala Ser Val Pro Thr Ser Leu Tyr Leu Thr Asp Met Thr Leu Asp

85 90 95

Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Thr

100 105 110

Gly Ser Leu Cys Ile Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile

115 120 125

Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Glu Gly Leu Glu Thr Leu

130 135 140

Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile

145 150 155 160

Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn

165 170 175

Ala Ile Gly Val Leu Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val

180 185 190

Arg Ile Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser His Glu

195 200 205

Asn Gly Arg Pro Ser Phe Pro Ser Lys Gln Lys

210 215

<210> 9

<211> 1497

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(1497)

<400> 9

atg gcg tct caa ggc acc aaa cga tcc tat gaa cag atg gaa act gat 48

Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp

1 5 10 15

ggg gaa cgc cag aat gca act gaa atc aga gca tct gtc gga agg atg 96

Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Arg Met

20 25 30

gtg gga gga atc ggc cga ttt tat gtt cag atg tgt act gag ctt aaa 144

Val Gly Gly Ile Gly Arg Phe Tyr Val Gln Met Cys Thr Glu Leu Lys

35 40 45

cta aac gac cat gaa ggg cgg ctg att cag aac agc ata aca ata gaa 192

Leu Asn Asp His Glu Gly Arg Leu Ile Gln Asn Ser Ile Thr Ile Glu

50 55 60

agg atg gta ctt tcg gca ttc gac gaa aga aga aac aag tat ctc gag 240

Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu

65 70 75 80

gag cat ccc agt gct ggg aaa gac cct aag aaa acg gga ggc ccg ata 288

Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile

85 90 95

tac aga agg aaa gat ggg aaa tgg atg agg gaa ctc atc ctc cat gat 336

Tyr Arg Arg Lys Asp Gly Lys Trp Met Arg Glu Leu Ile Leu His Asp

100 105 110

aaa gaa gaa atc atg aga atc tgg cgt cag gcc aac aat ggt gaa gac 384

Lys Glu Glu Ile Met Arg Ile Trp Arg Gln Ala Asn Asn Gly Glu Asp

115 120 125

gct act gct ggt ctt act cat atg atg atc tgg cac tcc aat ctc aat 432

Ala Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn

130 135 140

gac acc aca tac caa aga aca agg gct ctt gtt cgg act ggg atg gat 480

Asp Thr Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp

145 150 155 160

ccc aga atg tgc tct ctg atg caa ggc tca acc ctc cca cgg aga tct 528

Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser

165 170 175

gga gcc gct ggt gct gca gta aaa ggt gtt gga aca atg gta atg gaa 576

Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu

180 185 190

ctc atc aga atg atc aaa cgc gga ata aat gat cgg aat ttc tgg aga 624

Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg

195 200 205

ggt gaa aat ggt cga aaa acc aga att gct tat gaa aga atg tgc aat 672

Gly Glu Asn Gly Arg Lys Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn

210 215 220

atc ctc aaa ggg aaa ttt cag aca gca gca caa cgg gct atg atg gac 720

Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp

225 230 235 240

cag gtg agg gaa ggc cgc aat cct gga aac gct gag att gag gat ctc 768

Gln Val Arg Glu Gly Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu

245 250 255

att ttc ttg gca cga tca gca ctt att ttg aga gga tca gta gcc cat 816

Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His

260 265 270

aaa tca tgc cta cct gcc tgt gtt tat ggc ctt gca ata acc agt ggg 864

Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala Ile Thr Ser Gly

275 280 285

tat gac ttt gag aag gaa gga tac tct ctg gtt gga att gat cct ttc 912

Tyr Asp Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe

290 295 300

aaa cta ctc cag aac agt caa att ttc agt cta atc aga cca aaa gaa 960

Lys Leu Leu Gln Asn Ser Gln Ile Phe Ser Leu Ile Arg Pro Lys Glu

305 310 315 320

aac cca gca cac aag agc cag ttg gtg tgg atg gca tgc cat tct gca 1008

Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala

325 330 335

gca ttt gag gac ctg aga gtt tta aat ttc att aga gga acc aaa gta 1056

Ala Phe Glu Asp Leu Arg Val Leu Asn Phe Ile Arg Gly Thr Lys Val

340 345 350

atc cca aga gga cag tta aca acc aga gga gtt caa att gct tca aat 1104

Ile Pro Arg Gly Gln Leu Thr Thr Arg Gly Val Gln Ile Ala Ser Asn

355 360 365

gaa aac atg gag aca ata aat tct agc aca ctt gaa ctg aga agc aaa 1152

Glu Asn Met Glu Thr Ile Asn Ser Ser Thr Leu Glu Leu Arg Ser Lys

370 375 380

tat tgg gca ata agg acc aga agc gga gga aac acc agt caa cag aga 1200

Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Ser Gln Gln Arg

385 390 395 400

gca tct gca gga cag ata agt gtg caa cct act ttc tca gta cag aga 1248

Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg

405 410 415

aat cta ccc ttt gag aga gcg acc att atg gct gca ttc act ggt aac 1296

Asn Leu Pro Phe Glu Arg Ala Thr Ile Met Ala Ala Phe Thr Gly Asn

420 425 430

act gaa ggg agg act tcc gac atg aga acg gaa atc ata agg atg atg 1344

Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met

435 440 445

gaa aat gcc aaa tca gaa gat gtg tct ttc cag ggg cgg gga gtc ttc 1392

Glu Asn Ala Lys Ser Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe

450 455 460

gag ctc tcg gac gaa aag gca acg aac ccg atc gtg cct tcc ttt gac 1440

Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp

465 470 475 480

atg agc aat gaa ggg tct tat ttc ttc gga gac aat gct gag gag ttt 1488

Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Phe

485 490 495

gac agt taa 1497

Asp Ser

<210> 10

<211> 498

<212> PRT

<213> Influenza virus

<400> 10

Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp

1 5 10 15

Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Arg Met

20 25 30

Val Gly Gly Ile Gly Arg Phe Tyr Val Gln Met Cys Thr Glu Leu Lys

35 40 45

Leu Asn Asp His Glu Gly Arg Leu Ile Gln Asn Ser Ile Thr Ile Glu

50 55 60

Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu

65 70 75 80

Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile

85 90 95

Tyr Arg Arg Lys Asp Gly Lys Trp Met Arg Glu Leu Ile Leu His Asp

100 105 110

Lys Glu Glu Ile Met Arg Ile Trp Arg Gln Ala Asn Asn Gly Glu Asp

115 120 125

Ala Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn

130 135 140

Asp Thr Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp

145 150 155 160

Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser

165 170 175

Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu

180 185 190

Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg

195 200 205

Gly Glu Asn Gly Arg Lys Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn

210 215 220

Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp

225 230 235 240

Gln Val Arg Glu Gly Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu

245 250 255

Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His

260 265 270

Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala Ile Thr Ser Gly

275 280 285

Tyr Asp Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe

290 295 300

Lys Leu Leu Gln Asn Ser Gln Ile Phe Ser Leu Ile Arg Pro Lys Glu

305 310 315 320

Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala

325 330 335

Ala Phe Glu Asp Leu Arg Val Leu Asn Phe Ile Arg Gly Thr Lys Val

340 345 350

Ile Pro Arg Gly Gln Leu Thr Thr Arg Gly Val Gln Ile Ala Ser Asn

355 360 365

Glu Asn Met Glu Thr Ile Asn Ser Ser Thr Leu Glu Leu Arg Ser Lys

370 375 380

Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Ser Gln Gln Arg

385 390 395 400

Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg

405 410 415

Asn Leu Pro Phe Glu Arg Ala Thr Ile Met Ala Ala Phe Thr Gly Asn

420 425 430

Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met

435 440 445

Glu Asn Ala Lys Ser Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe

450 455 460

Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp

465 470 475 480

Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Phe

485 490 495

Asp Ser

<210> 11

<211> 1413

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(1413)

<400> 11

atg aat cca aat caa aag ata ata gca att gga ttt gca tca ttg ggg 48

Met Asn Pro Asn Gln Lys Ile Ile Ala Ile Gly Phe Ala Ser Leu Gly

1 5 10 15

ata tta atc att aat gtc att ctc cat gta gtc agc att ata gta aca 96

Ile Leu Ile Ile Asn Val Ile Leu His Val Val Ser Ile Ile Val Thr

20 25 30

gta ctg gtc ctc aat aac aat aga aca gat ctg aac tgc aaa ggg acg 144

Val Leu Val Leu Asn Asn Asn Arg Thr Asp Leu Asn Cys Lys Gly Thr

35 40 45

atc ata aga aag tac aat gaa aca gta aga gta gaa aaa att act caa 192

Ile Ile Arg Lys Tyr Asn Glu Thr Val Arg Val Glu Lys Ile Thr Gln

50 55 60

tgg tat aat acc agt aca att aag tac ata gag aga cct tca aat gaa 240

Trp Tyr Asn Thr Ser Thr Ile Lys Tyr Ile Glu Arg Pro Ser Asn Glu

65 70 75 80

tac tac atg aac aac act gaa cca ctt tgt gag gcc caa ggc ttt gca 288

Tyr Tyr Met Asn Asn Thr Glu Pro Leu Cys Glu Ala Gln Gly Phe Ala

85 90 95

cca ttt tcc aaa gat aat gga ata cga att ggg tcg aga ggc cat gtt 336

Pro Phe Ser Lys Asp Asn Gly Ile Arg Ile Gly Ser Arg Gly His Val

100 105 110

ttt gtg ata aga gaa cct ttt gta tca tgt tcg ccc tca gaa tgt aga 384

Phe Val Ile Arg Glu Pro Phe Val Ser Cys Ser Pro Ser Glu Cys Arg

115 120 125

acc ttt ttc ctc aca cag ggc tca tta ctc aat gac aaa cat tct aac 432

Thr Phe Phe Leu Thr Gln Gly Ser Leu Leu Asn Asp Lys His Ser Asn

130 135 140

ggc aca gta aag gac cga agt ccg tat agg act ttg atg agt gtc aaa 480

Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser Val Lys

145 150 155 160

ata ggg caa tca cct aat gta tat caa gct aga ttt gaa tcg gta gca 528

Ile Gly Gln Ser Pro Asn Val Tyr Gln Ala Arg Phe Glu Ser Val Ala

165 170 175

tgg tca gca aca gca tgc cat gat gga aaa aaa tgg atg aca gtt gga 576

Trp Ser Ala Thr Ala Cys His Asp Gly Lys Lys Trp Met Thr Val Gly

180 185 190

gtc aca ggg ccc gac aat caa gca att gca gta gtg aac tat gga ggt 624

Val Thr Gly Pro Asp Asn Gln Ala Ile Ala Val Val Asn Tyr Gly Gly

195 200 205

gtt ccg gtt gat att att aat tca tgg gca ggg gat att tta aga acc 672

Val Pro Val Asp Ile Ile Asn Ser Trp Ala Gly Asp Ile Leu Arg Thr

210 215 220

caa gaa tca tca tgc acc tgc att aaa gga gac tgt tat tgg gta atg 720

Gln Glu Ser Ser Cys Thr Cys Ile Lys Gly Asp Cys Tyr Trp Val Met

225 230 235 240

act gat gga ccg gca aat agg caa gct aaa tat agg ata ttc aaa gca 768

Thr Asp Gly Pro Ala Asn Arg Gln Ala Lys Tyr Arg Ile Phe Lys Ala

245 250 255

aaa gat gga aga gta att gga cag act gat ata agt ttc aat ggg gga 816

Lys Asp Gly Arg Val Ile Gly Gln Thr Asp Ile Ser Phe Asn Gly Gly

260 265 270

cac ata gag gag tgt tct tgt tac ccc aat gaa ggg aag gtg gaa tgc 864

His Ile Glu Glu Cys Ser Cys Tyr Pro Asn Glu Gly Lys Val Glu Cys

275 280 285

ata tgc agg gac aat tgg act gga aca aat aga cca gtt ctg gta ata 912

Ile Cys Arg Asp Asn Trp Thr Gly Thr Asn Arg Pro Val Leu Val Ile

290 295 300

tct tct gat cta tcg tac aca gtt gga tat ttg tgt gct ggc att ccc 960

Ser Ser Asp Leu Ser Tyr Thr Val Gly Tyr Leu Cys Ala Gly Ile Pro

305 310 315 320

act gac act cct agg gga gag gat agt caa ttc aca ggc tca tgt aca 1008

Thr Asp Thr Pro Arg Gly Glu Asp Ser Gln Phe Thr Gly Ser Cys Thr

325 330 335

agt cct ttg gga aat aaa gga tac ggt gta aaa ggt ttc ggg ttt cga 1056

Ser Pro Leu Gly Asn Lys Gly Tyr Gly Val Lys Gly Phe Gly Phe Arg

340 345 350

caa gga act gac gta tgg gcc gga agg aca att agt agg act tca aga 1104

Gln Gly Thr Asp Val Trp Ala Gly Arg Thr Ile Ser Arg Thr Ser Arg

355 360 365

tca gga ttc gaa ata ata aaa atc agg aat ggt tgg aca cag aac agt 1152

Ser Gly Phe Glu Ile Ile Lys Ile Arg Asn Gly Trp Thr Gln Asn Ser

370 375 380

aaa gac caa atc agg agg caa gtg att atc gat gac cca aat tgg tca 1200

Lys Asp Gln Ile Arg Arg Gln Val Ile Ile Asp Asp Pro Asn Trp Ser

385 390 395 400

gga tat agc ggt tct ttc aca ttg ccg gtt gaa cta aca aaa aag gga 1248

Gly Tyr Ser Gly Ser Phe Thr Leu Pro Val Glu Leu Thr Lys Lys Gly

405 410 415

tgt ttg gtc ccc tgt ttc tgg gtt gaa atg att aga ggt aaa cct gaa 1296

Cys Leu Val Pro Cys Phe Trp Val Glu Met Ile Arg Gly Lys Pro Glu

420 425 430

gaa aca aca ata tgg acc tct agt agc tcc att gtg atg tgt gga gta 1344

Glu Thr Thr Ile Trp Thr Ser Ser Ser Ser Ile Val Met Cys Gly Val

435 440 445

gat cat aaa att gcc agt tgg tca tgg cac gat gga gct att ctt ccc 1392

Asp His Lys Ile Ala Ser Trp Ser Trp His Asp Gly Ala Ile Leu Pro

450 455 460

ttt gac atc gat aag atg taa 1413

Phe Asp Ile Asp Lys Met

465 470

<210> 12

<211> 470

<212> PRT

<213> Influenza virus

<400> 12

Met Asn Pro Asn Gln Lys Ile Ile Ala Ile Gly Phe Ala Ser Leu Gly

1 5 10 15

Ile Leu Ile Ile Asn Val Ile Leu His Val Val Ser Ile Ile Val Thr

20 25 30

Val Leu Val Leu Asn Asn Asn Arg Thr Asp Leu Asn Cys Lys Gly Thr

35 40 45

Ile Ile Arg Lys Tyr Asn Glu Thr Val Arg Val Glu Lys Ile Thr Gln

50 55 60

Trp Tyr Asn Thr Ser Thr Ile Lys Tyr Ile Glu Arg Pro Ser Asn Glu

65 70 75 80

Tyr Tyr Met Asn Asn Thr Glu Pro Leu Cys Glu Ala Gln Gly Phe Ala

85 90 95

Pro Phe Ser Lys Asp Asn Gly Ile Arg Ile Gly Ser Arg Gly His Val

100 105 110

Phe Val Ile Arg Glu Pro Phe Val Ser Cys Ser Pro Ser Glu Cys Arg

115 120 125

Thr Phe Phe Leu Thr Gln Gly Ser Leu Leu Asn Asp Lys His Ser Asn

130 135 140

Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser Val Lys

145 150 155 160

Ile Gly Gln Ser Pro Asn Val Tyr Gln Ala Arg Phe Glu Ser Val Ala

165 170 175

Trp Ser Ala Thr Ala Cys His Asp Gly Lys Lys Trp Met Thr Val Gly

180 185 190

Val Thr Gly Pro Asp Asn Gln Ala Ile Ala Val Val Asn Tyr Gly Gly

195 200 205

Val Pro Val Asp Ile Ile Asn Ser Trp Ala Gly Asp Ile Leu Arg Thr

210 215 220

Gln Glu Ser Ser Cys Thr Cys Ile Lys Gly Asp Cys Tyr Trp Val Met

225 230 235 240

Thr Asp Gly Pro Ala Asn Arg Gln Ala Lys Tyr Arg Ile Phe Lys Ala

245 250 255

Lys Asp Gly Arg Val Ile Gly Gln Thr Asp Ile Ser Phe Asn Gly Gly

260 265 270

His Ile Glu Glu Cys Ser Cys Tyr Pro Asn Glu Gly Lys Val Glu Cys

275 280 285

Ile Cys Arg Asp Asn Trp Thr Gly Thr Asn Arg Pro Val Leu Val Ile

290 295 300

Ser Ser Asp Leu Ser Tyr Thr Val Gly Tyr Leu Cys Ala Gly Ile Pro

305 310 315 320

Thr Asp Thr Pro Arg Gly Glu Asp Ser Gln Phe Thr Gly Ser Cys Thr

325 330 335

Ser Pro Leu Gly Asn Lys Gly Tyr Gly Val Lys Gly Phe Gly Phe Arg

340 345 350

Gln Gly Thr Asp Val Trp Ala Gly Arg Thr Ile Ser Arg Thr Ser Arg

355 360 365

Ser Gly Phe Glu Ile Ile Lys Ile Arg Asn Gly Trp Thr Gln Asn Ser

370 375 380

Lys Asp Gln Ile Arg Arg Gln Val Ile Ile Asp Asp Pro Asn Trp Ser

385 390 395 400

Gly Tyr Ser Gly Ser Phe Thr Leu Pro Val Glu Leu Thr Lys Lys Gly

405 410 415

Cys Leu Val Pro Cys Phe Trp Val Glu Met Ile Arg Gly Lys Pro Glu

420 425 430

Glu Thr Thr Ile Trp Thr Ser Ser Ser Ser Ile Val Met Cys Gly Val

435 440 445

Asp His Lys Ile Ala Ser Trp Ser Trp His Asp Gly Ala Ile Leu Pro

450 455 460

Phe Asp Ile Asp Lys Met

465 470

<210> 13

<211> 982

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(756)

<400> 13

atg agt ctt cta acc gag gtc gaa acg tac gtt ctc tct atc gta cca 48

Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro

1 5 10 15

tca ggc ccc ctc aaa gcc gag atc gcg cag aga ctt gaa gat gtc ttt 96

Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe

20 25 30

gca gga aag aac acc gat ctt gag gca ctc atg gaa tgg cta aag aca 144

Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr

35 40 45

aga cca atc ctg tca cct ctg act aaa ggg att tta gga ttt gta ttc 192

Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe

50 55 60

acg ctc acc gtg ccc agt gag cga gga ctg cag cgt aga cgc ttt gtc 240

Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val

65 70 75 80

caa aat gcc ctt agt gga aac gga gat cca aac aac atg gac aga gca 288

Gln Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala

85 90 95

gta aaa ctg tac agg aag ctt aaa aga gaa ata aca ttc cat ggg gca 336

Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala

100 105 110

aaa gag gta gca ctc agc tat tcc act ggt gca cta gcc agc tgc atg 384

Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met

115 120 125

gga ctc ata tac aac aga atg gga act gtt aca acc gaa gtg gca ttt 432

Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe

130 135 140

ggc ctg gta tgc gcc aca tgt gaa cag att gct gat tcc cag cat cga 480

Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg

145 150 155 160

gct cac agg cag atg gtg aca aca acc aac cca ttg atc aga cat gaa 528

Ala His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu

165 170 175

aac aga atg gta tta gcc agt acc acg gct aaa gcc atg gaa cag atg 576

Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met

180 185 190

gca gga tcg agt gag cag gca gca gag gcc atg gag gtt gct agt agg 624

Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Arg

195 200 205

gct agg cag atg gta cag gca atg aga acc att ggg acc cac cct agc 672

Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser

210 215 220

tcc agt gcc ggt ttg aaa gat gat ctc ctt gaa aat tta cag gcc tac 720

Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr

225 230 235 240

cag aaa cgg atg gga gtg caa atg cag cga ttc aag tgatcctctc 766

Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys

245 250

gttattgcag caagtatcat tgggatcttg cacttgatat tgtggattct tgatcgtctt 826

ttcttcaaat tcatttatcg tcgccttaaa tacgggttga aaagaggggcc ttctacggaa 886

ggagtacctg agtctatgag ggaagaatat cggcaggaac agcagaatgc tgtggatgtt 946

gacgatggtc attttgtcaa catagagctg gagtaa 982

<210> 14

<211> 252

<212> PRT

<213> Influenza virus

<400> 14

Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro

1 5 10 15

Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe

20 25 30

Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr

35 40 45

Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe

50 55 60

Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val

65 70 75 80

Gln Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala

85 90 95

Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala

100 105 110

Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met

115 120 125

Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe

130 135 140

Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg

145 150 155 160

Ala His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu

165 170 175

Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met

180 185 190

Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Arg

195 200 205

Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser

210 215 220

Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr

225 230 235 240

Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys

245 250

<210> 15

<211> 1698

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(1698)

<400> 15

atg aag aca acc att att ttg ata cta cta acc cat tgg gcc tac agt 48

Met Lys Thr Thr Ile Ile Leu Ile Leu Leu Thr His Trp Ala Tyr Ser

1 5 10 15

caa aac cca atc agt ggc aac aac aca gcc aca ctg tgt ctg gga cac 96

Gln Asn Pro Ile Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His

20 25 30

cat gca gta gca aat gga aca ttg gta aaa aca atg agt gat gat caa 144

His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Met Ser Asp Asp Gln

35 40 45

att gag gtg aca aat gct aca gaa tta gtt cag agc att tca atg ggg 192

Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ile Ser Met Gly

50 55 60

aaa ata tgc aac aaa tca tat aga att cta gat gga aga aat tgc aca 240

Lys Ile Cys Asn Lys Ser Tyr Arg Ile Leu Asp Gly Arg Asn Cys Thr

65 70 75 80

tta ata gat gca atg cta gga gac ccc cac tgt gac gcc ttt cag tat 288

Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gln Tyr

85 90 95

gag agt tgg gac ctc ttc ata gaa aga agc aac gct ttc agc aat tgc 336

Glu Ser Trp Asp Leu Phe Ile Glu Arg Ser Asn Ala Phe Ser Asn Cys

100 105 110

tac cca tat gac atc cct gac tat gca tcg ctc cga tcc att gta gca 384

Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Ser Leu Arg Ser Ile Val Ala

115 120 125

tcc tca gga aca ttg gaa ttc aca gca gag gga ttc aca tgg aca ggt 432

Ser Ser Gly Thr Leu Glu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly

130 135 140

gtc act caa aac gga aga agt gga gcc tgc aaa agg gga tca gcc gat 480

Val Thr Gln Asn Gly Arg Ser Gly Ala Cys Lys Arg Gly Ser Ala Asp

145 150 155 160

agt ttc ttt agc cga ctg aat tgg cta aca aaa tct gga agc tct tac 528

Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Lys Ser Gly Ser Ser Tyr

165 170 175

ccc aca ttg aat gtg aca atg cct aac aat aaa aat ttc gac aag cta 576

Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu

180 185 190

tac atc tgg ggg att cat cac ccg agc tca aat caa gag cag aca aaa 624

Tyr Ile Trp Gly Ile His Pro Ser Ser Asn Gln Glu Gln Thr Lys

195 200 205

ttg tac atc caa gaa tca gga cga gta aca gtc tca aca aaa aga agt 672

Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser

210 215 220

caa caa aca ata atc cct aac atc gga tct aga ccg ttg gtc aga ggt 720

Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Leu Val Arg Gly

225 230 235 240

caa tca ggc agg ata agc ata tac tgg acc att gta aaa cct gga gat 768

Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp

245 250 255

atc cta atg ata aac agt aat ggc aac tta gtt gca ccg cgg gga tat 816

Ile Leu Met Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr

260 265 270

ttt aaa ttg aaa aca ggg aaa agc tct gta atg aga tca gat gca ccc 864

Phe Lys Leu Lys Thr Gly Lys Ser Ser Val Met Arg Ser Asp Ala Pro

275 280 285

ata gac att tgt gtg tct gaa tgt att aca cca aat gga agc atc tcc 912

Ile Asp Ile Cys Val Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Ser

290 295 300

aac gac aag cca ttc caa aat gtg aac aaa gtt aca tat gga aaa tgc 960

Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys

305 310 315 320

cct aag tat atc agg caa aac act tta aag ctg gcc act ggg atg agg 1008

Pro Lys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg

325 330 335

aat gta cca gaa aag caa acc aga gga atc ttt gga gca ata gcg gga 1056

Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala Gly

340 345 350

ttc atc gaa aac ggc tgg gaa gga atg gtt gat ggg tgg tat ggg ttc 1104

Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe

355 360 365

cga tat caa aac tct gaa gga aca ggg caa gct gca gat cta aag agc 1152

Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser

370 375 380

act caa gca gcc atc gac cag att aat gga aag tta aac aga gtg att 1200

Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile

385 390 395 400

gaa aga acc aat gag aaa ttc cat caa ata gag aag gaa ttc tca gaa 1248

Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu

405 410 415

gta gaa gga aga att cag gac ctg gag aaa tat gta gaa gac acc aaa 1296

Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys

420 425 430

ata gac cta tgg tcc tac aat gca gaa ttg ctg gtg gct cta gaa aat 1344

Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn

435 440 445

caa cat aca att gac tta aca gat gca gaa atg aat aaa tta ttt gag 1392

Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu

450 455 460

aag act aga cgc cag tta aga gaa aac gca gaa gac atg gga ggt gga 1440

Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly

465 470 475 480

tgt ttc aag att tac cac aaa tgt gat aat gca tgc att gga tca ata 1488

Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile

485 490 495

aga act ggg aca tat gac cat tac ata tac aga gat gaa gca tta aac 1536

Arg Thr Gly Thr Tyr Asp His Tyr Ile Tyr Arg Asp Glu Ala Leu Asn

500 505 510

aac cga ttt cag atc aaa ggt gta gag ttg aaa tca ggc tac aaa gat 1584

Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp

515 520 525

tgg ata ttg tgg att tca ttc gcc ata tca tgc ttc tta att tgc gtt 1632

Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Ile Cys Val

530 535 540

gtt cta ttg ggt ttc att atg tgg gct tgc caa aga ggc aac atc aga 1680

Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Arg Gly Asn Ile Arg

545 550 555 560

tgc aac att tgc att tga 1698

Cys Asn Ile Cys Ile

565

<210> 16

<211> 565

<212> PRT

<213> Influenza virus

<400> 16

Met Lys Thr Thr Ile Ile Leu Ile Leu Leu Thr His Trp Ala Tyr Ser

1 5 10 15

Gln Asn Pro Ile Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His

20 25 30

His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Met Ser Asp Asp Gln

35 40 45

Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ile Ser Met Gly

50 55 60

Lys Ile Cys Asn Lys Ser Tyr Arg Ile Leu Asp Gly Arg Asn Cys Thr

65 70 75 80

Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gln Tyr

85 90 95

Glu Ser Trp Asp Leu Phe Ile Glu Arg Ser Asn Ala Phe Ser Asn Cys

100 105 110

Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Ser Leu Arg Ser Ile Val Ala

115 120 125

Ser Ser Gly Thr Leu Glu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly

130 135 140

Val Thr Gln Asn Gly Arg Ser Gly Ala Cys Lys Arg Gly Ser Ala Asp

145 150 155 160

Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Lys Ser Gly Ser Ser Tyr

165 170 175

Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu

180 185 190

Tyr Ile Trp Gly Ile His Pro Ser Ser Asn Gln Glu Gln Thr Lys

195 200 205

Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser

210 215 220

Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Leu Val Arg Gly

225 230 235 240

Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp

245 250 255

Ile Leu Met Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr

260 265 270

Phe Lys Leu Lys Thr Gly Lys Ser Ser Val Met Arg Ser Asp Ala Pro

275 280 285

Ile Asp Ile Cys Val Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Ser

290 295 300

Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys

305 310 315 320

Pro Lys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg

325 330 335

Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala Gly

340 345 350

Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe

355 360 365

Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser

370 375 380

Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile

385 390 395 400

Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu

405 410 415

Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys

420 425 430

Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn

435 440 445

Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu

450 455 460

Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly

465 470 475 480

Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile

485 490 495

Arg Thr Gly Thr Tyr Asp His Tyr Ile Tyr Arg Asp Glu Ala Leu Asn

500 505 510

Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp

515 520 525

Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Ile Cys Val

530 535 540

Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Arg Gly Asn Ile Arg

545 550 555 560

Cys Asn Ile Cys Ile

565

<210> 17

<211> 2277

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(2277)

<220>

<221> misc_feature

<222> (731)..(731)

<223> The 'Xaa' at location 731 stands for Val, or Ile

<400> 17

atg gag aga ata aaa gaa ctg aga gat ctg atg tta caa tcc cgc acc 48

Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Leu Gln Ser Arg Thr

1 5 10 15

cgc gag ata cta aca aaa act act gtg gac cac atg gcc ata atc aag 96

Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys

20 25 30

aaa tac aca tca gga aga caa gag aag aac cct gca ctt agg atg aaa 144

Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys

35 40 45

tgg atg atg gca atg aaa tac cca att aca gca gat aag agg ata atg 192

Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met

50 55 60

gag atg att cct gag aga aat gaa cag gga caa acc ctt tgg agc aaa 240

Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys

65 70 75 80

acg aac gat gct ggc tca gac cgc gta atg gta tca cct ctg gca gtg 288

Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val

85 90 95

aca tgg tgg aat agg aat gga cca aca acg agc aca att cat tat cca 336

Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Ile His Tyr Pro

100 105 110

aaa gtc tac aaa act tat ttt gaa aag gtt gaa aga ttg aaa cac gga 384

Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly

115 120 125

acc ttt ggc ccc gtt cat ttt agg aat caa gtc aag ata aga cga aga 432

Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg

130 135 140

gtt gat gta aac cct ggt cac gcg gac ctc agt gcc aaa gaa gca caa 480

Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln

145 150 155 160

gat gtg atc atg gaa gtt gtt ttc cca aat gaa gtg gga gcc aga att 528

Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile

165 170 175

cta aca tcg gaa tca caa cta aca ata acc aaa gag aaa aag gaa gaa 576

Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu

180 185 190

ctt cag gac tgc aaa att gct ccc ttg atg gta gca tac atg cta gaa 624

Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu

195 200 205

aga gag ttg gtc cga aaa aca agg ttc ctc cca gta gca ggc gga aca 672

Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr

210 215 220

agc agt gta tac att gaa gtg ttg cat ctg act cag gga aca tgc tgg 720

Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp

225 230 235 240

gag caa atg tac acc cca gga gga gaa gtt aga aac gat gat att gat 768

Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Ile Asp

245 250 255

caa agt tta att att gca gcc cgg aac ata gtg aga aga gcg aca gta 816

Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val

260 265 270

tca gca gat cca cta gca tcc cta ctg gaa atg tgc cac agt aca cag 864

Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln

275 280 285

att ggt gga ata agg atg gta gac atc ctt aag cag aat cca aca gag 912

Ile Gly Gly Ile Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu

290 295 300

gaa caa gct gtg gat ata tgc aaa gca gca atg gga ttg aga att agc 960

Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser

305 310 315 320

tca tca ttc agc ttt ggt gga ttc acc ttc aaa aga aca agt gga tca 1008

Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser

325 330 335

tca gtc aag aga gaa gaa gaa atg ctt acg ggc aac ctt caa aca ttg 1056

Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu

340 345 350

aaa ata aga gtg cat gag ggc tat gaa gaa ttc aca atg gtc gga aga 1104

Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg

355 360 365

aga gca aca gcc att atc aga aag gca acc aga aga ttg att caa ttg 1152

Arg Ala Thr Ala Ile Ile Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu

370 375 380

ata gta agt ggg aga gat gaa caa tca att gct gaa gca ata att gta 1200

Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val

385 390 395 400

gcc atg gtg ttt tcg caa gaa gat tgc atg ata aaa gca gtt cga ggc 1248

Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly

405 410 415

gat ttg aac ttt gtt aat aga gca aat cag cgt ttg aac ccc atg cat 1296

Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His

420 425 430

caa ctc ttg agg cat ttc caa aaa gat gca aaa gtg ctt ttc caa aat 1344

Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn

435 440 445

tgg gga att gaa ccc atc gac aat gta atg ggg atg att gga ata ttg 1392

Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu

450 455 460

cct gac atg acc cca agc acc gag atg tca ttg aga gga gtg aga gtc 1440

Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val

465 470 475 480

agc aaa atg gga gtg gat gag tac tcc agc act gag aga gtg gtg gtg 1488

Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val

485 490 495

agc att gac cgt ttt tta aga gtt cgg gat caa agg gga aac ata cta 1536

Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Ile Leu

500 505 510

ctg tcc cct gaa gaa gtc agt gaa aca caa gga acg gaa aag ctg aca 1584

Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr

515 520 525

ata att tat tcg tca tca atg atg tgg gag att aat ggt ccc gaa tca 1632

Ile Ile Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser

530 535 540

gtg ttg gtc aat act tat caa tgg atc atc agg aac tgg gaa att gta 1680

Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Ile Val

545 550 555 560

aaa att cag tgg tca cag gac ccc aca atg tta tac aat aag ata gaa 1728

Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Ile Glu

565 570 575

ttt gag cca ttc caa tcc ctg gtc cct agg gcc acc aga agc caa tac 1776

Phe Glu Pro Phe Gln Ser Leu Val Pro Arg Ala Thr Arg Ser Gln Tyr

580 585 590

agc ggt ttc gta aga acc ctg ttt cag caa atg cga gat gta ctt gga 1824

Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly

595 600 605

aca ttt gat act gct caa ata ata aaa ctc ctc cct ttt gcc gct gct 1872

Thr Phe Asp Thr Ala Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala

610 615 620

cct ccg gaa cag agt agg atg cag ttc tct tct ttg act gtt aat gta 1920

Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val

625 630 635 640

aga ggt tcg gga atg agg ata ctt gta aga ggc aat tcc cca gtg ttc 1968

Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe

645 650 655

aac tac aat aaa gcc act aaa agg ctc aca gtc ctc gga aag gat gca 2016

Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala

660 665 670

ggt gcg ctt act gag gac cca gat gaa ggt acg gct gga gta gaa tct 2064

Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser

675 680 685

gct gtt cta aga ggg ttt ctc att tta ggt aaa gaa aac aag aga tat 2112

Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asn Lys Arg Tyr

690 695 700

ggc cca gca cta agc atc aat gaa cta agc aaa ctt gca aaa ggg gag 2160

Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu

705 710 715 720

aaa gcc aat gta cta att ggg caa ggg gac rta gtg ttg gta atg aaa 2208

Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Xaa Val Leu Val Met Lys

725 730 735

cgg aaa cgt gac tct agc ata ctt act gac agc cag aca gcg acc aaa 2256

Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys

740 745 750

agg att cgg atg gcc atc aat 2277

Arg Ile Arg Met Ala Ile Asn

755

<210> 18

<211> 759

<212> PRT

<213> Influenza virus

<220>

<221> misc_feature

<222> (731)..(731)

<223> The 'Xaa' at location 731 stands for Val, or Ile.

<400> 18

Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Leu Gln Ser Arg Thr

1 5 10 15

Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys

20 25 30

Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys

35 40 45

Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met

50 55 60

Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys

65 70 75 80

Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val

85 90 95

Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Ile His Tyr Pro

100 105 110

Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly

115 120 125

Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg

130 135 140

Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln

145 150 155 160

Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile

165 170 175

Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu

180 185 190

Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu

195 200 205

Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr

210 215 220

Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp

225 230 235 240

Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Ile Asp

245 250 255

Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val

260 265 270

Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln

275 280 285

Ile Gly Gly Ile Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu

290 295 300

Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser

305 310 315 320

Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser

325 330 335

Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu

340 345 350

Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg

355 360 365

Arg Ala Thr Ala Ile Ile Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu

370 375 380

Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val

385 390 395 400

Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly

405 410 415

Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His

420 425 430

Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn

435 440 445

Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu

450 455 460

Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val

465 470 475 480

Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val

485 490 495

Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Ile Leu

500 505 510

Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr

515 520 525

Ile Ile Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser

530 535 540

Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Ile Val

545 550 555 560

Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Ile Glu

565 570 575

Phe Glu Pro Phe Gln Ser Leu Val Pro Arg Ala Thr Arg Ser Gln Tyr

580 585 590

Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly

595 600 605

Thr Phe Asp Thr Ala Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala

610 615 620

Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val

625 630 635 640

Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe

645 650 655

Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala

660 665 670

Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser

675 680 685

Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asn Lys Arg Tyr

690 695 700

Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu

705 710 715 720

Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Xaa Val Leu Val Met Lys

725 730 735

Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys

740 745 750

Arg Ile Arg Met Ala Ile Asn

755

<210> 19

<211> 2274

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(2274)

<400> 19

atg gat gtc aat ccg act cta ctt ttc tta aag gtg cca gcg caa aat 48

Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn

1 5 10 15

gct ata agc aca aca ttc cct tat act gga gat cct ccc tac agt cat 96

Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His

20 25 30

gga aca ggg aca gga tac acc atg gat act gtc aac aga aca cac caa 144

Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln

35 40 45

tat tca gaa aaa ggg aaa tgg aca aca aac act gag att gga gca cca 192

Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro

50 55 60

caa ctt aat cca atc gat gga cca ctt cct gaa gac aat gaa cca agt 240

Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser

65 70 75 80

ggg tac gcc caa aca gat tgt gta ttg gaa gca atg gct ttc ctt gaa 288

Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu

85 90 95

gaa tcc cat ccc gga atc ttt gaa aat tcg tgt ctt gaa acg atg gag 336

Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu

100 105 110

gtg att cag cag aca aga gtg gac aaa cta aca caa ggc cga caa act 384

Val Ile Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr

115 120 125

tat gat tgg acc ttg aat agg aat caa cct gcc gca aca gca ctt gct 432

Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala

130 135 140

aat acg att gaa gta ttc aga tca aat ggt ctg act tcc aat gaa tcg 480

Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr Ser Asn Glu Ser

145 150 155 160

ggg aga ttg atg gac ttc ctc aaa gat gtc atg gag tcc atg aac aag 528

Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys

165 170 175

gaa gaa atg gaa ata aca aca cac ttc caa cgg aag aga aga gta aga 576

Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg

180 185 190

gac aac atg aca aag aga atg gta aca cag aga acc ata ggg aag aaa 624

Asp Asn Met Thr Lys Arg Met Val Thr Gln Arg Thr Ile Gly Lys Lys

195 200 205

aaa caa cga tta aac aga aag agc tat cta atc aga aca tta acc cta 672

Lys Gln Arg Leu Asn Arg Lys Ser Tyr Leu Ile Arg Thr Leu Thr Leu

210 215 220

aac aca atg acc aag gac gct gag aga ggg aaa ttg aaa cga cga gca 720

Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala

225 230 235 240

atc gct acc cca ggg atg cag ata aga ggg ttt gta tat ttt gtt gaa 768

Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu

245 250 255

aca cta gcc cga aga ata tgt gaa aag ctt gaa caa tca gga ttg cca 816

Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro

260 265 270

gtt ggc ggt aat gag aaa aag gcc aaa ctg gct aat gtc gtc aga aaa 864

Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys

275 280 285

atg atg act aat tcc caa gac act gaa ctc tcc ttc acc atc act ggg 912

Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly

290 295 300

gac aat acc aaa tgg aat gaa aat cag aac cca cgc ata ttc ctg gca 960

Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Ile Phe Leu Ala

305 310 315 320

atg atc aca tac ata act aga aac cag cca gaa tgg ttc aga aat gtt 1008

Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val

325 330 335

cta agc att gca ccg att atg ttc tca aat aaa atg gca aga ctg ggg 1056

Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly

340 345 350

aaa gga tat atg ttt gaa agc aaa agt atg aaa ttg aga act caa ata 1104

Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile

355 360 365

cca gca gaa atg cta gca agc att gac cta aaa tat ttc aat gat tca 1152

Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser

370 375 380

aca aaa aag aaa att gaa aag ata cga cca ctt ctg gtt gac ggg act 1200

Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Val Asp Gly Thr

385 390 395 400

gct tca ctg agt cct ggc atg atg atg gga atg ttc aac atg ttg agc 1248

Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser

405 410 415

act gtg ctg ggt gta tcc ata tta aac ctg ggc cag agg aaa tac aca 1296

Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Arg Lys Tyr Thr

420 425 430

aag acc aca tac tgg tgg gat ggt ctg caa tca tcc gat gac ttt gct 1344

Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala

435 440 445

ttg ata gtg aat gcg cct aat cat gaa gga ata caa gct gga gta gac 1392

Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp

450 455 460

aga ttc tat aga act tgc aaa ctg gtc ggg atc aac atg agc aaa aag 1440

Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys

465 470 475 480

aaa tcc tac ata aat aga act gga aca ttc gaa ttc aca agc ttt ttc 1488

Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe

485 490 495

tac cgg tat ggt ttt gta gcc aat ttc agc atg gaa cta ccc agt ttt 1536

Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe

500 505 510

ggg gtt tcc gga ata aat gaa tct gca gac atg agc att gga gtg aca 1584

Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr

515 520 525

gtc atc aaa aac aac atg ata aat aat gat ctc ggt cct gcc acg gca 1632

Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala

530 535 540

caa atg gca ctc caa ctc ttc att aag gat tat cgg tac aca tac cgg 1680

Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg

545 550 555 560

tgc cat aga ggt gat acc cag ata caa acc aga aga tct ttt gag ttg 1728

Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu

565 570 575

aag aaa ctg tgg gaa cag act cga tca aag act ggt cta ctg gta tca 1776

Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Thr Gly Leu Leu Val Ser

580 585 590

gat ggg ggt cca aac cta tat aac atc aga aac cta cac atc ccg gaa 1824

Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu

595 600 605

gtc tgt tta aaa tgg gag cta atg gat gaa gat tat aag ggg agg cta 1872

Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Lys Gly Arg Leu

610 615 620

tgc aat cca ttg aat cct ttc gtt agt cac aaa gaa att gaa tca gtc 1920

Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val

625 630 635 640

aac agt gca gta gta atg cct gcg cat ggc cct gcc aaa agc atg gag 1968

Asn Ser Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu

645 650 655

tat gat gct gtt gca aca aca cat tct tgg atc ccc aag agg aac cgg 2016

Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg

660 665 670

tcc ata ttg aac aca agc caa agg gga ata ctc gaa gat gag cag atg 2064

Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met

675 680 685

tat cag aaa tgc tgc aac ctg ttt gaa aaa ttc ttc ccc agc agc tca 2112

Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser

690 695 700

tac aga aga cca gtc gga att tct agt atg gtt gag gcc atg gtg tcc 2160

Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser

705 710 715 720

agg gcc cgc att gat gca cga att gac ttc gaa tct gga cgg ata aag 2208

Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys

725 730 735

aag gat gag ttc gct gag atc atg aag atc tgt tcc acc att gaa gag 2256

Lys Asp Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu

740 745 750

ctc aga cgg caa aaa tag 2274

Leu Arg Arg Gln Lys

755

<210> 20

<211> 757

<212> PRT

<213> Influenza virus

<400> 20

Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn

1 5 10 15

Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His

20 25 30

Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln

35 40 45

Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro

50 55 60

Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser

65 70 75 80

Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu

85 90 95

Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu

100 105 110

Val Ile Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr

115 120 125

Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala

130 135 140

Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr Ser Asn Glu Ser

145 150 155 160

Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys

165 170 175

Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg

180 185 190

Asp Asn Met Thr Lys Arg Met Val Thr Gln Arg Thr Ile Gly Lys Lys

195 200 205

Lys Gln Arg Leu Asn Arg Lys Ser Tyr Leu Ile Arg Thr Leu Thr Leu

210 215 220

Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala

225 230 235 240

Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu

245 250 255

Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro

260 265 270

Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys

275 280 285

Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly

290 295 300

Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Ile Phe Leu Ala

305 310 315 320

Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val

325 330 335

Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly

340 345 350

Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile

355 360 365

Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser

370 375 380

Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Val Asp Gly Thr

385 390 395 400

Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser

405 410 415

Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Arg Lys Tyr Thr

420 425 430

Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala

435 440 445

Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp

450 455 460

Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys

465 470 475 480

Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe

485 490 495

Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe

500 505 510

Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr

515 520 525

Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala

530 535 540

Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg

545 550 555 560

Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu

565 570 575

Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Thr Gly Leu Leu Val Ser

580 585 590

Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu

595 600 605

Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Lys Gly Arg Leu

610 615 620

Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val

625 630 635 640

Asn Ser Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu

645 650 655

Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg

660 665 670

Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met

675 680 685

Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser

690 695 700

Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser

705 710 715 720

Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys

725 730 735

Lys Asp Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu

740 745 750

Leu Arg Arg Gln Lys

755

<210> 21

<211> 2151

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(2151)

<400> 21

atg gaa gac ttt gtg cga cag tgc ttc aat cca atg atc gtc gag ctt 48

Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu

1 5 10 15

gcg gaa aag gca atg aaa gaa tat gga gag aac ccg aaa atc gaa aca 96

Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asn Pro Lys Ile Glu Thr

20 25 30

aac aaa ttt gca gca ata tgc act cac ttg gaa gtc tgc ttc atg tac 144

Asn Lys Phe Ala Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr

35 40 45

tcg gat ttt cac ttt att aat gaa ctg ggt gag tca gtg gtc ata gag 192

Ser Asp Phe His Phe Ile Asn Glu Leu Gly Glu Ser Val Val Ile Glu

50 55 60

tct ggt gac cca aat gct ctt ttg aaa cac aga ttt gaa atc att gag 240

Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu

65 70 75 80

ggg aga gat cga aca atg gca tgg aca gta gta aac agc atc tgc aac 288

Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn

85 90 95

acc aca aga gct gaa aaa cct aaa ttt ctt cca gat tta tac gac tat 336

Thr Thr Arg Ala Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr

100 105 110

aag gag aac aga ttt gtt gaa att ggt gtg aca agg aga gaa gtt cac 384

Lys Glu Asn Arg Phe Val Glu Ile Gly Val Thr Arg Arg Glu Val His

115 120 125

ata tac tac ctg gag aag gcc aac aaa ata aag tct gag aaa aca cat 432

Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Lys Thr His

130 135 140

atc cac att ttc tca ttt aca gga gag gaa atg gct aca aaa gcg gac 480

Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys Ala Asp

145 150 155 160

tat act ctt gat gaa gag agt aga gcc agg atc aag acc aga cta ttc 528

Tyr Thr Leu Asp Glu Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe

165 170 175

act ata aga caa gaa atg gcc agt aga ggc ctc tgg gat tcc ttt cgt 576

Thr Ile Arg Gln Glu Met Ala Ser Arg Gly Leu Trp Asp Ser Phe Arg

180 185 190

cag tcc gag aga ggc gaa gag aca att gaa gaa aga ttt gaa atc aca 624

Gln Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr

195 200 205

ggg acg atg cgc aag ctt gcc aat tac agt ctc cca ccg aac ttc tcc 672

Gly Thr Met Arg Lys Leu Ala Asn Tyr Ser Leu Pro Pro Asn Phe Ser

210 215 220

agc ctt gaa aat ttt aga gtc tat gtg gat gga ttc gaa ccg aac ggc 720

Ser Leu Glu Asn Phe Arg Val Tyr Val Asp Gly Phe Glu Pro Asn Gly

225 230 235 240

tgc att gag agt aag ctt tct caa atg tcc aaa gaa gta aat gcc aga 768

Cys Ile Glu Ser Lys Leu Ser Gln Met Ser Lys Glu Val Asn Ala Arg

245 250 255

atc gaa cca ttt tca aag aca aca ccc cga cca ctc aaa atg cca ggt 816

Ile Glu Pro Phe Ser Lys Thr Thr Pro Arg Pro Leu Lys Met Pro Gly

260 265 270

ggt cca ccc tgc cat cag cga tct aaa ttc ttg cta atg gat gct ctg 864

Gly Pro Pro Cys His Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu

275 280 285

aaa ctg agc att gag gac cca agt cac gag gga gag gga ata cca cta 912

Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu

290 295 300

tat gat gca atc aaa tgc atg aaa act ttc ttt gga tgg aaa gag ccc 960

Tyr Asp Ala Ile Lys Cys Met Lys Thr Phe Phe Gly Trp Lys Glu Pro

305 310 315 320

agt att gtt aaa cca cat gaa aag ggt ata aac ccg aac tat ctc caa 1008

Ser Ile Val Lys Pro His Glu Lys Gly Ile Asn Pro Asn Tyr Leu Gln

325 330 335

act tgg aag caa gta tta gaa gaa ata caa gac ctt gag aac gaa gaa 1056

Thr Trp Lys Gln Val Leu Glu Glu Ile Gln Asp Leu Glu Asn Glu Glu

340 345 350

agg acc ccc aag acc aag aat atg aaa aaa aca agc caa ttg aaa tgg 1104

Arg Thr Pro Lys Thr Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp

355 360 365

gca cta ggt gaa aat atg gca cca gag aaa gtg gat ttt gag gat tgt 1152

Ala Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Glu Asp Cys

370 375 380

aaa gac atc aat gat tta aaa caa tat gac agt gat gag cca gaa aca 1200

Lys Asp Ile Asn Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Thr

385 390 395 400

agg tct ctt gca agt tgg att caa agt gag ttc aac aaa gct tgt gag 1248

Arg Ser Leu Ala Ser Trp Ile Gln Ser Glu Phe Asn Lys Ala Cys Glu

405 410 415

ctg aca gat tca agc tgg ata gag ctc gat gaa att ggg gag gat gtc 1296

Leu Thr Asp Ser Ser Ser Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val

420 425 430

gcc cca ata gaa tac att gcg agc atg agg aga aat tat ttt act gct 1344

Ala Pro Ile Glu Tyr Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala

435 440 445

gag att tcc cat tgt aga gca aca gaa tat ata atg aaa gga gtg tac 1392

Glu Ile Ser His Cys Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr

450 455 460

atc aac act gct cta ctc aat gca tcc tgt gct gcg atg gat gaa ttt 1440

Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Glu Phe

465 470 475 480

caa tta att ccg atg ata agt aaa tgc agg acc aaa gaa ggg aga agg 1488

Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg

485 490 495

aaa aca aat tta tat gga ttc ata ata aag gga agg tcc cat tta aga 1536

Lys Thr Asn Leu Tyr Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg

500 505 510

aat gat act gac gtg gtg aac ttt gta agt atg gaa ttt tct ctc act 1584

Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr

515 520 525

gat cca aga ttt gag cca cac aaa tgg gaa aaa tac tgc gtt cta gaa 1632

Asp Pro Arg Phe Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu

530 535 540

att gga gac atg ctt cta aga act gct gta ggt caa gtg tca aga ccc 1680

Ile Gly Asp Met Leu Leu Arg Thr Ala Val Gly Gln Val Ser Arg Pro

545 550 555 560

atg ttt ttg tat gta agg aca aat gga acc tct aaa att aaa atg aaa 1728

Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys

565 570 575

tgg gga atg gaa atg agg cgc tgc ctc ctt cag tct ctg caa cag att 1776

Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile

580 585 590

gaa agc atg atc gaa gct gag tcc tca gtc aaa gaa aag gac atg acc 1824

Glu Ser Met Ile Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr

595 600 605

aaa gaa ttt ttt gag aac aaa tca gag aca tgg cct ata gga gag tcc 1872

Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser

610 615 620

ccc aaa gga gtg gaa gag ggc tca atc ggg aag gtt tgc agg acc tta 1920

Pro Lys Gly Val Glu Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu

625 630 635 640

tta gca aaa tct gtg ttt aac agt tta tat gca tct cca caa ctg gaa 1968

Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu

645 650 655

ggg ttt tca gct gaa tct agg aaa tta ctt ctc att gtt cag gct ctt 2016

Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu

660 665 670

agg gat gac ctg gaa cct gga acc ttt gat att ggg ggg tta tat gaa 2064

Arg Asp Asp Leu Glu Pro Gly Thr Phe Asp Ile Gly Gly Leu Tyr Glu

675 680 685

tca att gag gag tgc ctg att aat gat ccc tgg gtt ttg ctt aat gca 2112

Ser Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala

690 695 700

tct tgg ttc aac tcc ttc ctt aca cat gca ctg aag tag 2151

Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Lys

705 710 715

<210> 22

<211> 716

<212> PRT

<213> Influenza virus

<400> 22

Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu

1 5 10 15

Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asn Pro Lys Ile Glu Thr

20 25 30

Asn Lys Phe Ala Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr

35 40 45

Ser Asp Phe His Phe Ile Asn Glu Leu Gly Glu Ser Val Val Ile Glu

50 55 60

Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu

65 70 75 80

Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn

85 90 95

Thr Thr Arg Ala Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr

100 105 110

Lys Glu Asn Arg Phe Val Glu Ile Gly Val Thr Arg Arg Glu Val His

115 120 125

Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Lys Thr His

130 135 140

Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys Ala Asp

145 150 155 160

Tyr Thr Leu Asp Glu Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe

165 170 175

Thr Ile Arg Gln Glu Met Ala Ser Arg Gly Leu Trp Asp Ser Phe Arg

180 185 190

Gln Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr

195 200 205

Gly Thr Met Arg Lys Leu Ala Asn Tyr Ser Leu Pro Pro Asn Phe Ser

210 215 220

Ser Leu Glu Asn Phe Arg Val Tyr Val Asp Gly Phe Glu Pro Asn Gly

225 230 235 240

Cys Ile Glu Ser Lys Leu Ser Gln Met Ser Lys Glu Val Asn Ala Arg

245 250 255

Ile Glu Pro Phe Ser Lys Thr Thr Pro Arg Pro Leu Lys Met Pro Gly

260 265 270

Gly Pro Pro Cys His Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu

275 280 285

Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu

290 295 300

Tyr Asp Ala Ile Lys Cys Met Lys Thr Phe Phe Gly Trp Lys Glu Pro

305 310 315 320

Ser Ile Val Lys Pro His Glu Lys Gly Ile Asn Pro Asn Tyr Leu Gln

325 330 335

Thr Trp Lys Gln Val Leu Glu Glu Ile Gln Asp Leu Glu Asn Glu Glu

340 345 350

Arg Thr Pro Lys Thr Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp

355 360 365

Ala Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Glu Asp Cys

370 375 380

Lys Asp Ile Asn Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Thr

385 390 395 400

Arg Ser Leu Ala Ser Trp Ile Gln Ser Glu Phe Asn Lys Ala Cys Glu

405 410 415

Leu Thr Asp Ser Ser Ser Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val

420 425 430

Ala Pro Ile Glu Tyr Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala

435 440 445

Glu Ile Ser His Cys Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr

450 455 460

Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Glu Phe

465 470 475 480

Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg

485 490 495

Lys Thr Asn Leu Tyr Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg

500 505 510

Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr

515 520 525

Asp Pro Arg Phe Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu

530 535 540

Ile Gly Asp Met Leu Leu Arg Thr Ala Val Gly Gln Val Ser Arg Pro

545 550 555 560

Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys

565 570 575

Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile

580 585 590

Glu Ser Met Ile Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr

595 600 605

Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser

610 615 620

Pro Lys Gly Val Glu Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu

625 630 635 640

Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu

645 650 655

Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu

660 665 670

Arg Asp Asp Leu Glu Pro Gly Thr Phe Asp Ile Gly Gly Leu Tyr Glu

675 680 685

Ser Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala

690 695 700

Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Lys

705 710 715

<210> 23

<211> 838

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(657)

<400> 23

atg gat tcc aac act gtg tca agc ttt cag gta gac tgt ttt ctt tgg 48

Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

cat gtc cgc aaa cga ttc gca gac caa gaa ctg ggt gat gcc cca ttc 96

His Val Arg Lys Arg Phe Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

ctt gac cgg ctt cgc cga gac cag aag tcc cta agg gga aga ggt agc 144

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

act ctt ggt ctg gac atc gaa aca gcc act cat gca gga aag cag ata 192

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr His Ala Gly Lys Gln Ile

50 55 60

gtg gag cag att ctg gaa aag gaa tca gat gag gca ctt aaa atg acc 240

Val Glu Gln Ile Leu Glu Lys Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

att gcc tct gtt cct act tca cgc tac tta act gac atg act ctt gat 288

Ile Ala Ser Val Pro Thr Ser Arg Tyr Leu Thr Asp Met Thr Leu Asp

85 90 95

gag atg tca aga gac tgg ttc atg ctc atg ccc aag caa aaa gta aca 336

Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Thr

100 105 110

ggc tcc cta tgt ata aga atg gac cag gca atc atg gat aag aac atc 384

Gly Ser Leu Cys Ile Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile

115 120 125

ata ctt aaa gca aac ttt agt gtg att ttc gaa ggg ctg gaa aca cta 432

Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Glu Gly Leu Glu Thr Leu

130 135 140

ata cta ctt aga gcc ttc acc gaa gaa gga gca gtc gtt ggc gaa att 480

Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile

145 150 155 160

tca cca tta cct tct ctt cca gga cat act aat gag gat gtc aaa aat 528

Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn

165 170 175

gca att ggg gtc ctc atc gga gga ctt aaa tgg aat gat aat acg gtt 576

Ala Ile Gly Val Leu Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val

180 185 190

aga atc tct gaa act cta cag aga ttc gct tgg aga agc agt cat gag 624

Arg Ile Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser His Glu

195 200 205

aat ggg aga cct tca ttc cct tca aag cag aaa tgaaaaatgg agagaacaat 677

Asn Gly Arg Pro Ser Phe Pro Ser Lys Gln Lys

210 215

taagccagaa atttgaagaa ataagatggt tgattgaaga agtgcgacat agattgaaaa 737

atacagaaaa tagttttgaa caaataacat ttatgcaagc cttacaacta ttgcttgaag 797

tagaacaaga gataagaact ttctcgtttc agcttattta a 838

<210> 24

<211> 219

<212> PRT

<213> Influenza virus

<400> 24

Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

His Val Arg Lys Arg Phe Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr His Ala Gly Lys Gln Ile

50 55 60

Val Glu Gln Ile Leu Glu Lys Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

Ile Ala Ser Val Pro Thr Ser Arg Tyr Leu Thr Asp Met Thr Leu Asp

85 90 95

Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Thr

100 105 110

Gly Ser Leu Cys Ile Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile

115 120 125

Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Glu Gly Leu Glu Thr Leu

130 135 140

Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile

145 150 155 160

Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn

165 170 175

Ala Ile Gly Val Leu Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val

180 185 190

Arg Ile Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser His Glu

195 200 205

Asn Gly Arg Pro Ser Phe Pro Ser Lys Gln Lys

210 215

<210> 25

<211> 1497

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(1497)

<400> 25

atg gcg tct caa ggc acc aaa cga tcc tat gaa cag atg gaa act gat 48

Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp

1 5 10 15

ggg gaa cgc cag aat gca act gaa atc aga gca tct gtc gga agg atg 96

Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Arg Met

20 25 30

gtg gga gga atc ggc cgg ttt tat gtt cag atg tgt act gag ctt aaa 144

Val Gly Gly Ile Gly Arg Phe Tyr Val Gln Met Cys Thr Glu Leu Lys

35 40 45

cta aac gac cat gaa ggg cgg ctg att cag aac agc ata aca ata gaa 192

Leu Asn Asp His Glu Gly Arg Leu Ile Gln Asn Ser Ile Thr Ile Glu

50 55 60

agg atg gta ctt tcg gca ttc gac gaa aga aga aac aag tat ctc gag 240

Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu

65 70 75 80

gag cat ccc agt gct ggg aaa gac cct aag aaa acg gga ggc ccg ata 288

Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile

85 90 95

tac aga agg aaa gat ggg aaa tgg atg agg gaa ctc atc ctc cat gat 336

Tyr Arg Arg Lys Asp Gly Lys Trp Met Arg Glu Leu Ile Leu His Asp

100 105 110

aaa gaa gaa atc atg aga atc tgg cgt cag gcc aac aat ggt gaa gac 384

Lys Glu Glu Ile Met Arg Ile Trp Arg Gln Ala Asn Asn Gly Glu Asp

115 120 125

gct act gct ggt ctt act cat atg atg atc tgg cac tcc aat ctc aat 432

Ala Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn

130 135 140

gac acc aca tac caa aga aca agg gct ctt gtt cgg act ggg atg gat 480

Asp Thr Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp

145 150 155 160

ccc aga atg tgc tct ctg atg caa ggc tca acc ctc cca cgg aga tct 528

Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser

165 170 175

gga gcc gct ggt gct gca gta aaa ggt gtt gga aca atg gta atg gaa 576

Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu

180 185 190

ctc atc aga atg atc aaa cgc gga ata aat gat cgg aat ttc tgg aga 624

Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg

195 200 205

ggt gaa aat ggt cga aga acc aga att gct tat gaa aga atg tgc aat 672

Gly Glu Asn Gly Arg Arg Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn

210 215 220

atc ctc aaa ggg aaa ttt cag aca gca gca caa cgg gct atg atg gac 720

Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp

225 230 235 240

cag gtg agg gaa ggc cgc aat cct gga aac gct gag att gag gat ctc 768

Gln Val Arg Glu Gly Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu

245 250 255

att ttc ttg gca cga tca gca ctt att ttg aga gga tca gta gcc cat 816

Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His

260 265 270

aaa tca tgc cta cct gcc tgt gtt tat ggc ctt gca gta acc agt ggg 864

Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala Val Thr Ser Gly

275 280 285

tat gac ttt gag aag gaa gga tac tct ctg gtt gga att gat cct ttc 912

Tyr Asp Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe

290 295 300

aaa cta ctc cag aac agt caa att ttc agt cta atc aga cca aaa gaa 960

Lys Leu Leu Gln Asn Ser Gln Ile Phe Ser Leu Ile Arg Pro Lys Glu

305 310 315 320

aac cca gca cac aag agc cag ttg gtg tgg atg gca tgc cat tct gca 1008

Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala

325 330 335

gca ttt gag gac ctg aga gtt tta aat ttc att aga gga acc aaa gta 1056

Ala Phe Glu Asp Leu Arg Val Leu Asn Phe Ile Arg Gly Thr Lys Val

340 345 350

atc cca aga gga cag tta aca acc aga gga gtt caa att gct tca aat 1104

Ile Pro Arg Gly Gln Leu Thr Thr Arg Gly Val Gln Ile Ala Ser Asn

355 360 365

gaa aac atg gag aca ata gat tct agc aca ctt gaa ctg aga agc aaa 1152

Glu Asn Met Glu Thr Ile Asp Ser Ser Thr Leu Glu Leu Arg Ser Lys

370 375 380

tat tgg gca ata agg acc aga agc gga gga aac acc agt caa cag aga 1200

Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Ser Gln Gln Arg

385 390 395 400

gca tct gca gga cag ata agt gtg caa cct act ttc tca gta cag aga 1248

Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg

405 410 415

aat ctt ccc ttt gag aga gca acc att atg gct gca ttc act ggt aac 1296

Asn Leu Pro Phe Glu Arg Ala Thr Ile Met Ala Ala Phe Thr Gly Asn

420 425 430

act gaa ggg agg act tcc gac atg aga acg gaa atc ata agg atg atg 1344

Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met

435 440 445

gaa aat gcc aaa tca gaa gat gtg tct ttc cag ggg cgg gga gtc ttc 1392

Glu Asn Ala Lys Ser Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe

450 455 460

gag ctc tcg gac gaa aag gca acg aac ccg atc gtg cct tcc ttt gac 1440

Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp

465 470 475 480

atg agc aat gaa ggg tct tat ttc ttc gga gac aat gct gag gag ttt 1488

Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Phe

485 490 495

gac agt taa 1497

Asp Ser

<210> 26

<211> 498

<212> PRT

<213> Influenza virus

<400> 26

Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp

1 5 10 15

Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Arg Met

20 25 30

Val Gly Gly Ile Gly Arg Phe Tyr Val Gln Met Cys Thr Glu Leu Lys

35 40 45

Leu Asn Asp His Glu Gly Arg Leu Ile Gln Asn Ser Ile Thr Ile Glu

50 55 60

Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu

65 70 75 80

Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile

85 90 95

Tyr Arg Arg Lys Asp Gly Lys Trp Met Arg Glu Leu Ile Leu His Asp

100 105 110

Lys Glu Glu Ile Met Arg Ile Trp Arg Gln Ala Asn Asn Gly Glu Asp

115 120 125

Ala Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn

130 135 140

Asp Thr Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp

145 150 155 160

Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser

165 170 175

Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu

180 185 190

Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg

195 200 205

Gly Glu Asn Gly Arg Arg Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn

210 215 220

Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp

225 230 235 240

Gln Val Arg Glu Gly Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu

245 250 255

Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His

260 265 270

Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala Val Thr Ser Gly

275 280 285

Tyr Asp Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe

290 295 300

Lys Leu Leu Gln Asn Ser Gln Ile Phe Ser Leu Ile Arg Pro Lys Glu

305 310 315 320

Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala

325 330 335

Ala Phe Glu Asp Leu Arg Val Leu Asn Phe Ile Arg Gly Thr Lys Val

340 345 350

Ile Pro Arg Gly Gln Leu Thr Thr Arg Gly Val Gln Ile Ala Ser Asn

355 360 365

Glu Asn Met Glu Thr Ile Asp Ser Ser Thr Leu Glu Leu Arg Ser Lys

370 375 380

Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Ser Gln Gln Arg

385 390 395 400

Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg

405 410 415

Asn Leu Pro Phe Glu Arg Ala Thr Ile Met Ala Ala Phe Thr Gly Asn

420 425 430

Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met

435 440 445

Glu Asn Ala Lys Ser Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe

450 455 460

Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp

465 470 475 480

Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Phe

485 490 495

Asp Ser

<210> 27

<211> 1410

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(1410)

<400> 27

atg aat cca aat caa aag ata ata aca att gga ttt gca tca ttg ggg 48

Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Phe Ala Ser Leu Gly

1 5 10 15

ata tta atc att aat gtc att ctc cat gta gtc agc att ata gta aca 96

Ile Leu Ile Ile Asn Val Ile Leu His Val Val Ser Ile Ile Val Thr

20 25 30

gta ctg gtc ctc aat aac aat aga aca gat ctg aac tgc aaa ggg acg 144

Val Leu Val Leu Asn Asn Asn Arg Thr Asp Leu Asn Cys Lys Gly Thr

35 40 45

atc ata aga gag tac aat gaa aca gta aga gta gaa aaa att act caa 192

Ile Ile Arg Glu Tyr Asn Glu Thr Val Arg Val Glu Lys Ile Thr Gln

50 55 60

tgg tat aat acc agt aca att aag tac ata gag aga cct tca aat gaa 240

Trp Tyr Asn Thr Ser Thr Ile Lys Tyr Ile Glu Arg Pro Ser Asn Glu

65 70 75 80

tac tac atg aac aac act gaa cca ctt tgt gag gcc caa ggc ttt gca 288

Tyr Tyr Met Asn Asn Thr Glu Pro Leu Cys Glu Ala Gln Gly Phe Ala

85 90 95

cca ttt tcc aaa gat aat gga ata cga att ggg tcg aga ggc cat gtt 336

Pro Phe Ser Lys Asp Asn Gly Ile Arg Ile Gly Ser Arg Gly His Val

100 105 110

ttt gtg ata aga gaa cct ttt gta tca tgt tcg ccc tca gaa tgt aga 384

Phe Val Ile Arg Glu Pro Phe Val Ser Cys Ser Pro Ser Glu Cys Arg

115 120 125

acc ttt ttc ctc aca cag ggc tca tta ctc aat gac aaa cat tct aac 432

Thr Phe Phe Leu Thr Gln Gly Ser Leu Leu Asn Asp Lys His Ser Asn

130 135 140

ggc aca gta aag gac cga agt ccg tat agg act ttg atg agt gtc aaa 480

Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser Val Lys

145 150 155 160

ata ggg caa tca cct aat gta tat caa gct agg ttt gaa tcg gtg gca 528

Ile Gly Gln Ser Pro Asn Val Tyr Gln Ala Arg Phe Glu Ser Val Ala

165 170 175

tgg tca gca aca gca tgc cat gat gga aaa aaa tgg atg aca gtt gga 576

Trp Ser Ala Thr Ala Cys His Asp Gly Lys Lys Trp Met Thr Val Gly

180 185 190

gtc aca ggg ccc gac aat caa gca att gca gta gtg aac tat gga ggt 624

Val Thr Gly Pro Asp Asn Gln Ala Ile Ala Val Val Asn Tyr Gly Gly

195 200 205

gtt ccg gtt gat att att aat tca tgg gca ggg gat att tta aga acc 672

Val Pro Val Asp Ile Ile Asn Ser Trp Ala Gly Asp Ile Leu Arg Thr

210 215 220

caa gaa tca tca tgc acc tgc att aaa gga gac tgt tat tgg gta atg 720

Gln Glu Ser Ser Cys Thr Cys Ile Lys Gly Asp Cys Tyr Trp Val Met

225 230 235 240

act gat gga ccg gca aat agg caa gct aaa tat agg ata ttc aaa gca 768

Thr Asp Gly Pro Ala Asn Arg Gln Ala Lys Tyr Arg Ile Phe Lys Ala

245 250 255

aaa gat gga aga gta att gga cag act gat ata agt ttc aat ggg gga 816

Lys Asp Gly Arg Val Ile Gly Gln Thr Asp Ile Ser Phe Asn Gly Gly

260 265 270

cac ata gag gag tgt tct tgt tac ccc aat gaa ggg aag gtg gaa tgc 864

His Ile Glu Glu Cys Ser Cys Tyr Pro Asn Glu Gly Lys Val Glu Cys

275 280 285

ata tgc agg gac aat tgg act gga aca aat aga cca att ctg gta ata 912

Ile Cys Arg Asp Asn Trp Thr Gly Thr Asn Arg Pro Ile Leu Val Ile

290 295 300

tct tct gat cta tcg tac aca gtt gga tat ttg tgt gct ggc att ccc 960

Ser Ser Asp Leu Ser Tyr Thr Val Gly Tyr Leu Cys Ala Gly Ile Pro

305 310 315 320

act gac act cct agg gga gag gat agt caa ttc aca ggc tca tgt aca 1008

Thr Asp Thr Pro Arg Gly Glu Asp Ser Gln Phe Thr Gly Ser Cys Thr

325 330 335

agt cct ttg gga aat aaa gga tac ggt gta aaa ggt ttc ggg ttt cga 1056

Ser Pro Leu Gly Asn Lys Gly Tyr Gly Val Lys Gly Phe Gly Phe Arg

340 345 350

caa gga act gac gta tgg gcc gga agg aca att agt agg act tca aga 1104

Gln Gly Thr Asp Val Trp Ala Gly Arg Thr Ile Ser Arg Thr Ser Arg

355 360 365

tca gga ttc gaa ata ata aaa atc agg aat ggt tgg aca cag aac agt 1152

Ser Gly Phe Glu Ile Ile Lys Ile Arg Asn Gly Trp Thr Gln Asn Ser

370 375 380

aaa gac caa atc agg agg caa gtg att atc gat gac cca aat tgg tca 1200

Lys Asp Gln Ile Arg Arg Gln Val Ile Ile Asp Asp Pro Asn Trp Ser

385 390 395 400

gga tat agc ggt tct ttc aca ttg ccg gtt gaa cta aca aaa aag gga 1248

Gly Tyr Ser Gly Ser Phe Thr Leu Pro Val Glu Leu Thr Lys Lys Gly

405 410 415

tgt ttg gtc ccc tgt ttc tgg gtt gaa atg att aga ggt aaa cct gaa 1296

Cys Leu Val Pro Cys Phe Trp Val Glu Met Ile Arg Gly Lys Pro Glu

420 425 430

gaa aca aca ata tgg acc tct agc agc tcc att gtg atg tgt gga gta 1344

Glu Thr Thr Ile Trp Thr Ser Ser Ser Ser Ile Val Met Cys Gly Val

435 440 445

gat cat aaa att gcc agt tgg tca tgg cac gat gga gct att ctt ccc 1392

Asp His Lys Ile Ala Ser Trp Ser Trp His Asp Gly Ala Ile Leu Pro

450 455 460

ttt gac atc gat aag atg 1410

Phe Asp Ile Asp Lys Met

465 470

<210> 28

<211> 470

<212> PRT

<213> Influenza virus

<400> 28

Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Phe Ala Ser Leu Gly

1 5 10 15

Ile Leu Ile Ile Asn Val Ile Leu His Val Val Ser Ile Ile Val Thr

20 25 30

Val Leu Val Leu Asn Asn Asn Arg Thr Asp Leu Asn Cys Lys Gly Thr

35 40 45

Ile Ile Arg Glu Tyr Asn Glu Thr Val Arg Val Glu Lys Ile Thr Gln

50 55 60

Trp Tyr Asn Thr Ser Thr Ile Lys Tyr Ile Glu Arg Pro Ser Asn Glu

65 70 75 80

Tyr Tyr Met Asn Asn Thr Glu Pro Leu Cys Glu Ala Gln Gly Phe Ala

85 90 95

Pro Phe Ser Lys Asp Asn Gly Ile Arg Ile Gly Ser Arg Gly His Val

100 105 110

Phe Val Ile Arg Glu Pro Phe Val Ser Cys Ser Pro Ser Glu Cys Arg

115 120 125

Thr Phe Phe Leu Thr Gln Gly Ser Leu Leu Asn Asp Lys His Ser Asn

130 135 140

Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser Val Lys

145 150 155 160

Ile Gly Gln Ser Pro Asn Val Tyr Gln Ala Arg Phe Glu Ser Val Ala

165 170 175

Trp Ser Ala Thr Ala Cys His Asp Gly Lys Lys Trp Met Thr Val Gly

180 185 190

Val Thr Gly Pro Asp Asn Gln Ala Ile Ala Val Val Asn Tyr Gly Gly

195 200 205

Val Pro Val Asp Ile Ile Asn Ser Trp Ala Gly Asp Ile Leu Arg Thr

210 215 220

Gln Glu Ser Ser Cys Thr Cys Ile Lys Gly Asp Cys Tyr Trp Val Met

225 230 235 240

Thr Asp Gly Pro Ala Asn Arg Gln Ala Lys Tyr Arg Ile Phe Lys Ala

245 250 255

Lys Asp Gly Arg Val Ile Gly Gln Thr Asp Ile Ser Phe Asn Gly Gly

260 265 270

His Ile Glu Glu Cys Ser Cys Tyr Pro Asn Glu Gly Lys Val Glu Cys

275 280 285

Ile Cys Arg Asp Asn Trp Thr Gly Thr Asn Arg Pro Ile Leu Val Ile

290 295 300

Ser Ser Asp Leu Ser Tyr Thr Val Gly Tyr Leu Cys Ala Gly Ile Pro

305 310 315 320

Thr Asp Thr Pro Arg Gly Glu Asp Ser Gln Phe Thr Gly Ser Cys Thr

325 330 335

Ser Pro Leu Gly Asn Lys Gly Tyr Gly Val Lys Gly Phe Gly Phe Arg

340 345 350

Gln Gly Thr Asp Val Trp Ala Gly Arg Thr Ile Ser Arg Thr Ser Arg

355 360 365

Ser Gly Phe Glu Ile Ile Lys Ile Arg Asn Gly Trp Thr Gln Asn Ser

370 375 380

Lys Asp Gln Ile Arg Arg Gln Val Ile Ile Asp Asp Pro Asn Trp Ser

385 390 395 400

Gly Tyr Ser Gly Ser Phe Thr Leu Pro Val Glu Leu Thr Lys Lys Gly

405 410 415

Cys Leu Val Pro Cys Phe Trp Val Glu Met Ile Arg Gly Lys Pro Glu

420 425 430

Glu Thr Thr Ile Trp Thr Ser Ser Ser Ser Ile Val Met Cys Gly Val

435 440 445

Asp His Lys Ile Ala Ser Trp Ser Trp His Asp Gly Ala Ile Leu Pro

450 455 460

Phe Asp Ile Asp Lys Met

465 470

<210> 29

<211> 982

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(756)

<400> 29

atg agt ctt cta acc gag gtc gaa acg tac gtt ctc tct atc gta cca 48

Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro

1 5 10 15

tca ggc ccc ctc aaa gcc gag atc gcg cag aga ctt gaa gat gtc ttt 96

Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe

20 25 30

gca ggg aag aac acc gat ctt gag gca ctc atg gaa tgg cta aag aca 144

Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr

35 40 45

aga cca atc ctg tca cct ctg act aaa ggg att tta gga ttt gta ttc 192

Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe

50 55 60

acg ctc acc gtg ccc agt gag cga gga ctg cag cgt aga cgc ttt gtc 240

Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val

65 70 75 80

caa aat gcc ctt agt gga aac gga gat cca aac aac atg gac aga gca 288

Gln Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala

85 90 95

gta aaa ctg tac agg aag ctt aaa aga gaa ata aca ttc cat ggg gca 336

Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala

100 105 110

aaa gag gtg gca ctc agc tat tcc act ggt gca cta gcc agc tgc atg 384

Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met

115 120 125

gga ctc ata tac aac aga atg gga act gtt aca acc gaa gtg gca ttt 432

Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe

130 135 140

ggc ctg gta tgc gcc aca tgt gaa cag att gct gat tcc cag cat cga 480

Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg

145 150 155 160

tct cac agg cag atg gtg aca aca acc aac cca tta atc aga cat gaa 528

Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu

165 170 175

aac aga atg gta tta gcc agt acc acg gct aaa gcc atg gaa cag atg 576

Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met

180 185 190

gca gga tcg agt gag cag gca gca gag gcc atg gag gtt gct agt agg 624

Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Arg

195 200 205

gct agg cag atg gta cag gca atg aga acc att ggg acc cac cct agc 672

Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser

210 215 220

tcc agt gcc ggt ttg aaa gat gat ctc ctt gaa aat tta cag gcc tac 720

Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr

225 230 235 240

cag aaa cgg atg gga gtg caa atg cag cga ttc aag tgatcctctc 766

Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys

245 250

gttattgcag caagtatcat tgggatcttg cacttgatat tgtggattct tgatcgtctt 826

ttcttcaaat tcatttatcg tcgccttaaa tacgggttga aaagaggggcc ttctacggaa 886

ggagtacctg agtctatgag ggaagaatat cggcaggaac agcagaatgc tgtggatgtt 946

gacgatggtc attttgtcaa catagagctg gagtaa 982

<210> 30

<211> 252

<212> PRT

<213> Influenza virus

<400> 30

Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro

1 5 10 15

Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe

20 25 30

Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr

35 40 45

Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe

50 55 60

Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val

65 70 75 80

Gln Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala

85 90 95

Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala

100 105 110

Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met

115 120 125

Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe

130 135 140

Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg

145 150 155 160

Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu

165 170 175

Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met

180 185 190

Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Arg

195 200 205

Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser

210 215 220

Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr

225 230 235 240

Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys

245 250

<210> 31

<211> 1698

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(1698)

<400> 31

atg aag aca acc att att ttg ata cta ctg acc cat tgg gcc tac agt 48

Met Lys Thr Thr Ile Ile Leu Ile Leu Leu Thr His Trp Ala Tyr Ser

1 5 10 15

caa aac cca atc agt gac aac aac aca gcc aca ctg tgt ctg gga cac 96

Gln Asn Pro Ile Ser Asp Asn Asn Thr Ala Thr Leu Cys Leu Gly His

20 25 30

cat gca gta gca aat gga aca ttg gta aaa aca ata agt gat gat caa 144

His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Ile Ser Asp Asp Gln

35 40 45

att gag gtg aca aat gct aca gaa tta gtt cag agc att tca atg ggg 192

Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ile Ser Met Gly

50 55 60

aaa ata tgc aac aaa tca tat aga att cta gat gga aga aat tgc aca 240

Lys Ile Cys Asn Lys Ser Tyr Arg Ile Leu Asp Gly Arg Asn Cys Thr

65 70 75 80

tta ata gat gca atg cta gga gac ccc cac tgt gac gcc ttt cag tat 288

Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gln Tyr

85 90 95

gag agt tgg gac ctc ttt ata gaa aga agc agc gct ttc agc aat tgc 336

Glu Ser Trp Asp Leu Phe Ile Glu Arg Ser Ser Ala Phe Ser Asn Cys

100 105 110

tac cca tat gac atc cct gac tat gca tcg ctc cga tcc att gta gca 384

Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Ser Leu Arg Ser Ile Val Ala

115 120 125

tcc tca gga aca ttg gaa ttc aca gca gag gga ttc aca tgg aca ggt 432

Ser Ser Gly Thr Leu Glu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly

130 135 140

gtc act caa aac gga aga agt gga gcc tgc aaa agg gga tca gcc gat 480

Val Thr Gln Asn Gly Arg Ser Gly Ala Cys Lys Arg Gly Ser Ala Asp

145 150 155 160

agt ttc ttt agc cga ctg aat tgg cta aca aaa tct gga agc tct tac 528

Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Lys Ser Gly Ser Ser Tyr

165 170 175

ccc aca ttg aat gtg aca atg cct aac aat aaa aat ttc gac aag cta 576

Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu

180 185 190

tac atc tgg ggg att cat cac ccg agc tca aat caa gag cag aca aaa 624

Tyr Ile Trp Gly Ile His Pro Ser Ser Asn Gln Glu Gln Thr Lys

195 200 205

ttg tac atc caa gaa tca gga cga gta aca gtc tca aca aaa aga agt 672

Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser

210 215 220

caa caa aca ata atc cct aac atc gga tct aga ccg ttg gtc aga ggt 720

Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Leu Val Arg Gly

225 230 235 240

caa tca ggc agg ata agc ata tac tgg acc att gta aaa cct gga gat 768

Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp

245 250 255

atc cta atg ata aac agt aat ggc aac tta gtt gca ccg cgg gga tat 816

Ile Leu Met Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr

260 265 270

ttt aaa ttg aaa aca ggg aaa agc tct gta atg aga tca gat gta ccc 864

Phe Lys Leu Lys Thr Gly Lys Ser Ser Val Met Arg Ser Asp Val Pro

275 280 285

ata gac att tgt gtg tct gaa tgt att aca cca aat gga agc atc tcc 912

Ile Asp Ile Cys Val Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Ser

290 295 300

aac gac aag cca ttc caa aat gtg aac aaa gtt aca tat gga aaa tgc 960

Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys

305 310 315 320

ccc aag tat atc agg caa aac act tta aag ctg gcc act ggg atg agg 1008

Pro Lys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg

325 330 335

aat gta cca gaa aag caa acc aga gga atc ttt gga gca ata gcg gga 1056

Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala Gly

340 345 350

ttc atc gaa aac ggc tgg gaa gga atg gtt gat ggg tgg tat ggg ttc 1104

Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe

355 360 365

cga tat caa aac tct gaa gga aca ggg caa gct gca gat cta aag agc 1152

Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser

370 375 380

act caa gca gcc atc gac cag att aat gga aag tta aac aga gtg att 1200

Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile

385 390 395 400

gaa aga acc aat gag aaa ttc cat caa ata gag aag gaa ttc tca gaa 1248

Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu

405 410 415

gta gaa gga aga att cag gac ttg gag aaa tat gta gaa gac acc aaa 1296

Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys

420 425 430

ata gac cta tgg tcc tac aat gca gaa ttg ctg gtg gct cta gaa aat 1344

Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn

435 440 445

caa cat aca att gac tta aca gat gca gaa atg aat aaa tta ttt gag 1392

Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu

450 455 460

aag act aga cgc cag tta aga gaa aac gca gaa gac atg gga ggt gga 1440

Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly

465 470 475 480

tgt ttc aag att tac cac aaa tgt gat aat gca tgc att gga tca ata 1488

Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile

485 490 495

aga act ggg aca tat gac cat tac ata tac aga gat gaa gca tta aac 1536

Arg Thr Gly Thr Tyr Asp His Tyr Ile Tyr Arg Asp Glu Ala Leu Asn

500 505 510

aac cga ttt cag atc aaa ggt gta gag ttg aaa tca ggc tac aaa gat 1584

Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp

515 520 525

tgg ata ctg tgg att tca ttc gcc ata tca tgc ttc tta att tgc gtt 1632

Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Ile Cys Val

530 535 540

gtt cta ttg ggt ttc att atg tgg gct tgc caa aaa ggc aac atc aga 1680

Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg

545 550 555 560

tgc aac att tgc att tga 1698

Cys Asn Ile Cys Ile

565

<210> 32

<211> 565

<212> PRT

<213> Influenza virus

<400> 32

Met Lys Thr Thr Ile Ile Leu Ile Leu Leu Thr His Trp Ala Tyr Ser

1 5 10 15

Gln Asn Pro Ile Ser Asp Asn Asn Thr Ala Thr Leu Cys Leu Gly His

20 25 30

His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Ile Ser Asp Asp Gln

35 40 45

Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ile Ser Met Gly

50 55 60

Lys Ile Cys Asn Lys Ser Tyr Arg Ile Leu Asp Gly Arg Asn Cys Thr

65 70 75 80

Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gln Tyr

85 90 95

Glu Ser Trp Asp Leu Phe Ile Glu Arg Ser Ser Ala Phe Ser Asn Cys

100 105 110

Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Ser Leu Arg Ser Ile Val Ala

115 120 125

Ser Ser Gly Thr Leu Glu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly

130 135 140

Val Thr Gln Asn Gly Arg Ser Gly Ala Cys Lys Arg Gly Ser Ala Asp

145 150 155 160

Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Lys Ser Gly Ser Ser Tyr

165 170 175

Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu

180 185 190

Tyr Ile Trp Gly Ile His Pro Ser Ser Asn Gln Glu Gln Thr Lys

195 200 205

Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser

210 215 220

Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Leu Val Arg Gly

225 230 235 240

Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp

245 250 255

Ile Leu Met Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr

260 265 270

Phe Lys Leu Lys Thr Gly Lys Ser Ser Val Met Arg Ser Asp Val Pro

275 280 285

Ile Asp Ile Cys Val Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Ser

290 295 300

Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys

305 310 315 320

Pro Lys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg

325 330 335

Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala Gly

340 345 350

Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe

355 360 365

Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser

370 375 380

Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile

385 390 395 400

Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu

405 410 415

Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys

420 425 430

Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn

435 440 445

Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu

450 455 460

Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly

465 470 475 480

Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile

485 490 495

Arg Thr Gly Thr Tyr Asp His Tyr Ile Tyr Arg Asp Glu Ala Leu Asn

500 505 510

Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp

515 520 525

Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Ile Cys Val

530 535 540

Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg

545 550 555 560

Cys Asn Ile Cys Ile

565

<210> 33

<211> 549

<212> PRT

<213> Influenza virus

<400> 33

Gln Asn Pro Ile Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His

1 5 10 15

His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Met Ser Asp Asp Gln

20 25 30

Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ile Ser Met Gly

35 40 45

Lys Ile Cys Asn Lys Ser Tyr Arg Ile Leu Asp Gly Arg Asn Cys Thr

50 55 60

Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gln Tyr

65 70 75 80

Glu Ser Trp Asp Leu Phe Ile Glu Arg Ser Asn Ala Phe Ser Asn Cys

85 90 95

Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Ser Leu Arg Ser Ile Val Ala

100 105 110

Ser Ser Gly Thr Leu Glu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly

115 120 125

Val Thr Gln Asn Gly Arg Ser Gly Ala Cys Lys Arg Gly Ser Ala Asp

130 135 140

Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Lys Ser Gly Ser Ser Tyr

145 150 155 160

Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu

165 170 175

Tyr Ile Trp Gly Ile His Pro Ser Ser Asn Gln Glu Gln Thr Lys

180 185 190

Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser

195 200 205

Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Leu Val Arg Gly

210 215 220

Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp

225 230 235 240

Ile Leu Met Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr

245 250 255

Phe Lys Leu Lys Thr Gly Lys Ser Ser Val Met Arg Ser Asp Ala Pro

260 265 270

Ile Asp Ile Cys Val Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Ser

275 280 285

Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys

290 295 300

Pro Lys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg

305 310 315 320

Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala Gly

325 330 335

Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe

340 345 350

Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser

355 360 365

Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile

370 375 380

Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu

385 390 395 400

Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys

405 410 415

Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn

420 425 430

Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu

435 440 445

Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly

450 455 460

Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile

465 470 475 480

Arg Thr Gly Thr Tyr Asp His Tyr Ile Tyr Arg Asp Glu Ala Leu Asn

485 490 495

Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp

500 505 510

Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Ile Cys Val

515 520 525

Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Arg Gly Asn Ile Arg

530 535 540

Cys Asn Ile Cys Ile

545

<210> 34

<211> 549

<212> PRT

<213> Influenza virus

<400> 34

Gln Asn Pro Ile Ser Asp Asn Asn Thr Ala Thr Leu Cys Leu Gly His

1 5 10 15

His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Ile Ser Asp Asp Gln

20 25 30

Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ile Ser Met Gly

35 40 45

Lys Ile Cys Asn Lys Ser Tyr Arg Ile Leu Asp Gly Arg Asn Cys Thr

50 55 60

Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gln Tyr

65 70 75 80

Glu Ser Trp Asp Leu Phe Ile Glu Arg Ser Ser Ala Phe Ser Asn Cys

85 90 95

Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Ser Leu Arg Ser Ile Val Ala

100 105 110

Ser Ser Gly Thr Leu Glu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly

115 120 125

Val Thr Gln Asn Gly Arg Ser Gly Ala Cys Lys Arg Gly Ser Ala Asp

130 135 140

Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Lys Ser Gly Ser Ser Tyr

145 150 155 160

Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu

165 170 175

Tyr Ile Trp Gly Ile His Pro Ser Ser Asn Gln Glu Gln Thr Lys

180 185 190

Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser

195 200 205

Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Leu Val Arg Gly

210 215 220

Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp

225 230 235 240

Ile Leu Met Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr

245 250 255

Phe Lys Leu Lys Thr Gly Lys Ser Ser Val Met Arg Ser Asp Val Pro

260 265 270

Ile Asp Ile Cys Val Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Ser

275 280 285

Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys

290 295 300

Pro Lys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg

305 310 315 320

Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala Gly

325 330 335

Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe

340 345 350

Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser

355 360 365

Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile

370 375 380

Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu

385 390 395 400

Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys

405 410 415

Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn

420 425 430

Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu

435 440 445

Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly

450 455 460

Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile

465 470 475 480

Arg Thr Gly Thr Tyr Asp His Tyr Ile Tyr Arg Asp Glu Ala Leu Asn

485 490 495

Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp

500 505 510

Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Ile Cys Val

515 520 525

Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg

530 535 540

Cys Asn Ile Cys Ile

545

<210> 35

<211> 9

<212> DNA

<213> Influenza virus

<400> 35

gagagttgg 9

<210> 36

<211> 9

<212> DNA

<213> Influenza virus

<400> 36

ccgttggtc 9

<210> 37

<211> 9

<212> DNA

<213> Influenza virus

<400> 37

caaaccaga 9

<210> 38

<211> 9

<212> DNA

<213> Influenza virus

<400> 38

agaactggg 9

<210> 39

<211> 15

<212> DNA

<213> Influenza virus

<400> 39

tatgagagtt gggac 15

<210> 40

<211> 15

<212> DNA

<213> Influenza virus

<400> 40

agaccgttgg tcaga 15

<210> 41

<211> 15

<212> DNA

<213> Influenza virus

<400> 41

aagcaaacca gagga 15

<210> 42

<211> 15

<212> DNA

<213> Influenza virus s

<400> 42

ataagaactg ggaca 15

<210> 43

<211> 9

<212> DNA

<213> Influenza virus

<400> 43

acaatgagt 9

<210> 44

<211> 15

<212> DNA

<213> Influenza virus

<400> 44

aaaacaatga gtgat 15

<210> 45

<211> 9

<212> DNA

<213> Influenza virus

<400> 45

gatgtaccc 9

<210> 46

<211> 15

<212> DNA

<213> Influenza virus

<400> 46

tcagatgtac ccata 15

<210> 47

<211> 2280

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(2280)

<400> 47

atg gag aga ata aaa gaa ctg aga gat ctg atg tta caa tcc cgc acc 48

Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Leu Gln Ser Arg Thr

1 5 10 15

cgc gag ata cta aca aaa act act gtg gac cac atg gcc ata atc aag 96

Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys

20 25 30

aaa tac aca tca gga aga caa gag aag aac cct gca ctt agg atg aaa 144

Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys

35 40 45

tgg atg atg gca atg aaa tac cca att aca gca gat aag agg ata atg 192

Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met

50 55 60

gag atg att cct gag aga aat gaa cag gga caa acc ctt tgg agc aaa 240

Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys

65 70 75 80

acg aac gat gct ggc tca gac cgc gta atg gta tca cct ctg gca gtg 288

Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val

85 90 95

aca tgg tgg aat agg aat gga cca aca acg aac aca att cat tat cca 336

Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Asn Thr Ile His Tyr Pro

100 105 110

aaa gtc tac aaa act tat ttt gaa aag gtt gaa aga ttg aaa cac gga 384

Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly

115 120 125

acc ttt ggc ccc gtt cat ttt agg aat caa gtc aag ata aga cga aga 432

Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg

130 135 140

gtt gat gta aac cct ggt cac gcg gac ctc agt gct aaa gaa gca caa 480

Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln

145 150 155 160

gat gtg atc atg gaa gtt gtt ttc cca aat gaa gtg gga gcc aga att 528

Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile

165 170 175

cta aca tca gaa tca caa cta aca ata acc aaa gag aaa aag gaa gaa 576

Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu

180 185 190

ctt cag gac tgc aaa att gct ccc ttg atg gta gca tac atg cta gaa 624

Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu

195 200 205

aga gag ttg gtc cga aaa aca agg ttc ctc cca gta gta gta ggc gga aca 672

Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Val Gly Gly Thr

210 215 220

agc agt gta tac att gaa gtg ttg cat ctg act cag gga aca tgc tgg 720

Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp

225 230 235 240

gag caa atg tac acc cca gga gga aaa gtt aga aac gat gat att gat 768

Glu Gln Met Tyr Thr Pro Gly Gly Lys Val Arg Asn Asp Asp Ile Asp

245 250 255

caa agt tta att att gca gcc cgg aac ata gtg aga aga gca aca gta 816

Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val

260 265 270

tca gca gat cca cta gca tcc cta ctg gaa atg tgc cac agt aca cag 864

Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln

275 280 285

att ggt gga aca agg atg gta gac atc ctt aag cag aac cca aca gag 912

Ile Gly Gly Thr Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu

290 295 300

gaa caa gct gtg gat ata tgc aaa gca gca atg gga ttg aga att agc 960

Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser

305 310 315 320

tca tca ttc agc ttt ggt gga ttc acc ttc aaa agg aca agt gga tca 1008

Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser

325 330 335

tca gtc aag aga gaa gaa gaa atg ctt acg ggc aac ctt caa aca ttg 1056

Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu

340 345 350

aaa ata aga gtg cat gag ggc tat gaa gaa ttc aca atg gtc gga aga 1104

Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg

355 360 365

aga gca aca gcc att atc aga aag gca acc aga aga ttg att caa ttg 1152

Arg Ala Thr Ala Ile Ile Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu

370 375 380

ata gta agt ggg aga gat gaa caa tca att gct gaa gca ata att gta 1200

Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val

385 390 395 400

gcc atg gtg ttt tcg caa gaa gat tgc atg ata aaa gca gtt cga ggc 1248

Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly

405 410 415

gat ttg aac ttt gtt aat aga gca aat cag cgt ttg aac ccc atg cat 1296

Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His

420 425 430

caa ctc ttg agg cat ttc caa aaa gat gca aaa gtg ctt ttc caa aat 1344

Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn

435 440 445

tgg gga att gaa ccc atc gac aat gta atg ggg atg att gga ata ttg 1392

Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu

450 455 460

cct gac atg acc cca agc acc gag atg tca ttg aga gga gtg aga gtc 1440

Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val

465 470 475 480

agc aaa atg gga gtg gat gag tac tcc agc act gag aga gtg gtg gtg 1488

Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val

485 490 495

agc att gac cgt ttt tta aga gtt cgg gat caa agg gga aac ata cta 1536

Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Ile Leu

500 505 510

ctg tcc cct gaa gaa gtc agt gaa aca caa gga acg gaa aag ctg aca 1584

Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr

515 520 525

ata att tat tcg tca tca atg atg tgg gag att aat ggt ccc gaa tca 1632

Ile Ile Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser

530 535 540

gtg ttg gtc aat act tat caa tgg atc atc aga aac tgg gaa att gta 1680

Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Ile Val

545 550 555 560

aaa att cag tgg tca cag gac ccc aca atg tta tac aat aag ata gaa 1728

Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Ile Glu

565 570 575

ttt gaa cca ttc caa tcc ctg gtc cct agg gcc acc aga agc caa tac 1776

Phe Glu Pro Phe Gln Ser Leu Val Pro Arg Ala Thr Arg Ser Gln Tyr

580 585 590

agc ggt ttc gta aga acc ctg ttt cag caa atg cga gat gta ctt gga 1824

Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly

595 600 605

aca ttt gat act gct caa ata ata aaa ctc ctc cct ttt gcc gct gct 1872

Thr Phe Asp Thr Ala Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala

610 615 620

cct ccg gaa cag agt agg atg cag ttc tct tct ttg act gtt aat gta 1920

Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val

625 630 635 640

aga ggt tcg gga atg agg ata ctt gta aga ggc aat tcc cca gtg ttc 1968

Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe

645 650 655

aac tac aat aaa gtc act aaa agg ctc aca gtc ctc gga aag gat gca 2016

Asn Tyr Asn Lys Val Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala

660 665 670

ggt gcg ctt act gag gac cca gat gaa ggt acg gct gga gta gag tct 2064

Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser

675 680 685

gct gtt cta aga ggg ttt ctc att tta ggt aaa gaa aac aag aga tat 2112

Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asn Lys Arg Tyr

690 695 700

ggc cca gca cta agc atc aat gaa ctt agc aaa ctt gca aaa ggg gag 2160

Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu

705 710 715 720

aaa gcc aat gta cta att ggg caa ggg gac gta gtg ttg gta atg aaa 2208

Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys

725 730 735

cgg aaa cgt gac tct agc ata ctt act gac agc cag aca gcg acc aaa 2256

Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys

740 745 750

agg att cgg atg gcc atc aat tag 2280

Arg Ile Arg Met Ala Ile Asn

755

<210> 48

<211> 759

<212> PRT

<213> Influenza virus

<400> 48

Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Leu Gln Ser Arg Thr

1 5 10 15

Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys

20 25 30

Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys

35 40 45

Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met

50 55 60

Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys

65 70 75 80

Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val

85 90 95

Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Asn Thr Ile His Tyr Pro

100 105 110

Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly

115 120 125

Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg

130 135 140

Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln

145 150 155 160

Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile

165 170 175

Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu

180 185 190

Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu

195 200 205

Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Val Gly Gly Thr

210 215 220

Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp

225 230 235 240

Glu Gln Met Tyr Thr Pro Gly Gly Lys Val Arg Asn Asp Asp Ile Asp

245 250 255

Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val

260 265 270

Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln

275 280 285

Ile Gly Gly Thr Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu

290 295 300

Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser

305 310 315 320

Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser

325 330 335

Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu

340 345 350

Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg

355 360 365

Arg Ala Thr Ala Ile Ile Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu

370 375 380

Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val

385 390 395 400

Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly

405 410 415

Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His

420 425 430

Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn

435 440 445

Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu

450 455 460

Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val

465 470 475 480

Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val

485 490 495

Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Ile Leu

500 505 510

Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr

515 520 525

Ile Ile Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser

530 535 540

Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Ile Val

545 550 555 560

Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Ile Glu

565 570 575

Phe Glu Pro Phe Gln Ser Leu Val Pro Arg Ala Thr Arg Ser Gln Tyr

580 585 590

Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly

595 600 605

Thr Phe Asp Thr Ala Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala

610 615 620

Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val

625 630 635 640

Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe

645 650 655

Asn Tyr Asn Lys Val Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala

660 665 670

Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser

675 680 685

Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asn Lys Arg Tyr

690 695 700

Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu

705 710 715 720

Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys

725 730 735

Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys

740 745 750

Arg Ile Arg Met Ala Ile Asn

755

<210> 49

<211> 2274

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(2274)

<400> 49

atg gat gtc aat ccg act cta ctt ttc tta aag gtg cca gcg caa aat 48

Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn

1 5 10 15

gct ata agc aca aca ttc cct tat act gga gat cct ccc tac agt cat 96

Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His

20 25 30

gga aca ggg aca gga tac acc atg gat act gtc aac aga aca cac caa 144

Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln

35 40 45

tat tca gaa aaa ggg aaa tgg aca aca aac act gag att gga gca cca 192

Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro

50 55 60

caa ctt aat cca atc gat gga cca ctt cct gaa gac aat gaa cca agt 240

Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser

65 70 75 80

ggg tac gcc caa aca gat tgt gta ttg gaa gca atg gct ttc ctt gaa 288

Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu

85 90 95

gaa tcc cat ccc gga atc ttt gaa aat tcg tgt ctt gaa acg atg gag 336

Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu

100 105 110

gtg att cag cag aca aga gtg gac aaa cta aca caa ggc cga caa act 384

Val Ile Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr

115 120 125

tat gat tgg acc ttg aat agg aat caa cct gcc gca aca gca ctt gct 432

Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala

130 135 140

aat acg att gaa gta ttc aga tca aat ggt ctg acc tcc aat gaa tcg 480

Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr Ser Asn Glu Ser

145 150 155 160

ggg aga ttg atg gac ttc ctc aaa gat gtc atg gag tcc atg aac aag 528

Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys

165 170 175

gag gaa atg gaa ata aca aca cac ttc caa cgg aag aga aga gta aga 576

Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg

180 185 190

gac aac atg aca aag aga atg ata aca cag aga acc ata gga aag aaa 624

Asp Asn Met Thr Lys Arg Met Ile Thr Gln Arg Thr Ile Gly Lys Lys

195 200 205

aaa caa cga tta agc aga aag agc tat cta atc aga aca tta acc cta 672

Lys Gln Arg Leu Ser Arg Lys Ser Tyr Leu Ile Arg Thr Leu Thr Leu

210 215 220

aac aca atg acc aag gac gct gag aga ggg aaa ttg aaa cga cga gca 720

Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala

225 230 235 240

atc gct acc cca ggg atg cag ata aga gga ttt gta tat ttt gtt gaa 768

Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu

245 250 255

aca cta gct cga aga ata tgt gaa aag ctt gaa caa tca gga ttg cca 816

Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro

260 265 270

gtt ggc ggt aat gag aaa aag gcc aaa ctg gct aat gtc gtc aga aaa 864

Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys

275 280 285

atg atg act aat tcc caa gac act gaa ctc tcc ttc acc atc act ggg 912

Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly

290 295 300

gac aat acc aaa tgg aat gaa aat cag aac cca cgc ata ttc ctg gca 960

Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Ile Phe Leu Ala

305 310 315 320

atg atc aca tac ata act aga gat cag cca gaa tgg ttc aga aat gtt 1008

Met Ile Thr Tyr Ile Thr Arg Asp Gln Pro Glu Trp Phe Arg Asn Val

325 330 335

cta agc att gca ccg att atg ttc tca aat aaa atg gca aga ctg ggg 1056

Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly

340 345 350

aaa gga tat atg ttt gaa agc aaa agt atg aaa ttg aga act caa ata 1104

Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile

355 360 365

cca gca gaa atg cta gca agc att gac cta aaa tat ttc aat gat tca 1152

Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser

370 375 380

aca aaa aag aaa att gaa aag ata cga cca ctc ctg gtt gac ggg act 1200

Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Val Asp Gly Thr

385 390 395 400

gct tca ctg agt cct ggc atg atg atg gga atg ttc aac atg ttg agc 1248

Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser

405 410 415

act gtg ctg ggt gta tcc ata tta aac ctg ggc cag agg aaa tat aca 1296

Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Arg Lys Tyr Thr

420 425 430

aag acc aca tac tgg tgg gat ggt ctg caa tca tcc gat gac ttt gct 1344

Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala

435 440 445

ttg ata gtg aat gcg cct aat cat gaa gga ata caa gct gga gta gac 1392

Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp

450 455 460

aga ttc tat aga act tgc aaa ctg gtc ggg atc aac atg agc aaa aag 1440

Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys

465 470 475 480

aag tcc tac ata aat aga act gga aca ttc gaa ttc aca agc ttt ttc 1488

Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe

485 490 495

tac cgg tat ggt ttt gta gcc aat ttc agc atg gaa cta ccc agt ttt 1536

Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe

500 505 510

ggg gtt tcc gga ata aat gaa tct gca gac atg agc att gga gtg aca 1584

Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr

515 520 525

gtc atc aaa aac aac atg ata aat aat gat ctc ggt cct gcc acg gca 1632

Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala

530 535 540

caa atg gca ctc caa ctc ttc att aag gat tat cgg tac aca tac cgg 1680

Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg

545 550 555 560

tgc cat aga ggt gat acc cag ata caa acc aga aga tct ttt gag ttg 1728

Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu

565 570 575

aag aaa ctg tgg gaa cag act cga tca aag act ggt cta ctg gta tca 1776

Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Thr Gly Leu Leu Val Ser

580 585 590

gat ggg ggt cca aac cta tat aac atc aga aac cta cac atc ccg gaa 1824

Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu

595 600 605

gtc tgt tta aaa tgg gag cta atg gat gaa gat tat aag ggg agg cta 1872

Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Lys Gly Arg Leu

610 615 620

tgc aat cca ttg aat cct ttc gtt agt cac aaa gaa att gaa tca gtc 1920

Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val

625 630 635 640

aac agt gca gta gta atg cct gct cat ggc cct gcc aaa agc atg gag 1968

Asn Ser Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu

645 650 655

tat gat gct gtt gca aca aca cat tct tgg atc ccc aag agg aac cgg 2016

Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg

660 665 670

tcc ata ttg aac aca agc caa agg gga ata cta gaa gat gag cag atg 2064

Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met

675 680 685

tat cag aaa tgc tgc aac ctg ttt gaa aaa ttc ttc ccc agc agc tca 2112

Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser

690 695 700

tac aga aga cca gtc gga att tct agt atg gtt gag gcc atg gta tcc 2160

Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser

705 710 715 720

agg gcc cgc att gat gca cga att gac ttc gaa tct gga cgg ata aag 2208

Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys

725 730 735

aag gat gag ttc gct gag atc atg aag atc tgt tcc acc att gaa gag 2256

Lys Asp Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu

740 745 750

ctc aga cgg caa aaa tag 2274

Leu Arg Arg Gln Lys

755

<210> 50

<211> 757

<212> PRT

<213> Influenza virus

<400> 50

Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn

1 5 10 15

Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His

20 25 30

Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln

35 40 45

Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro

50 55 60

Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser

65 70 75 80

Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu

85 90 95

Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu

100 105 110

Val Ile Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr

115 120 125

Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala

130 135 140

Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr Ser Asn Glu Ser

145 150 155 160

Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys

165 170 175

Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg

180 185 190

Asp Asn Met Thr Lys Arg Met Ile Thr Gln Arg Thr Ile Gly Lys Lys

195 200 205

Lys Gln Arg Leu Ser Arg Lys Ser Tyr Leu Ile Arg Thr Leu Thr Leu

210 215 220

Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala

225 230 235 240

Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu

245 250 255

Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro

260 265 270

Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys

275 280 285

Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly

290 295 300

Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Ile Phe Leu Ala

305 310 315 320

Met Ile Thr Tyr Ile Thr Arg Asp Gln Pro Glu Trp Phe Arg Asn Val

325 330 335

Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly

340 345 350

Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile

355 360 365

Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser

370 375 380

Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Val Asp Gly Thr

385 390 395 400

Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser

405 410 415

Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Arg Lys Tyr Thr

420 425 430

Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala

435 440 445

Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp

450 455 460

Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys

465 470 475 480

Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe

485 490 495

Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe

500 505 510

Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr

515 520 525

Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala

530 535 540

Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg

545 550 555 560

Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu

565 570 575

Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Thr Gly Leu Leu Val Ser

580 585 590

Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu

595 600 605

Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Lys Gly Arg Leu

610 615 620

Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val

625 630 635 640

Asn Ser Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu

645 650 655

Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg

660 665 670

Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met

675 680 685

Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser

690 695 700

Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser

705 710 715 720

Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys

725 730 735

Lys Asp Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu

740 745 750

Leu Arg Arg Gln Lys

755

<210> 51

<211> 2151

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(2151)

<400> 51

atg gaa gac ttt gtg cga cag tgc ttc aat cca atg atc gtc gag ctt 48

Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu

1 5 10 15

gcg gaa aag gca atg aaa gaa tat gga gag aac ccg aaa atc gaa aca 96

Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asn Pro Lys Ile Glu Thr

20 25 30

aac aaa ttt gca gca ata tgc act cac ttg gaa gtc tgc ttc atg tac 144

Asn Lys Phe Ala Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr

35 40 45

tcg gat ttc cac ttt ata aat gaa ctg ggt gag tca gtg gtc ata gag 192

Ser Asp Phe His Phe Ile Asn Glu Leu Gly Glu Ser Val Val Ile Glu

50 55 60

tct ggt gac cca aat gct ctt ttg aaa cac aga ttt gaa atc att gag 240

Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu

65 70 75 80

ggg aga gat cga aca atg gca tgg aca gta gta aac agc atc tgc aac 288

Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn

85 90 95

acc aca aga gct gaa aaa cct aaa ttt ctt cca gat tta tac gac tat 336

Thr Thr Arg Ala Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr

100 105 110

aaa gag aac aga ttt gtt gaa att ggt gtg aca agg aga gaa gtt cac 384

Lys Glu Asn Arg Phe Val Glu Ile Gly Val Thr Arg Arg Glu Val His

115 120 125

ata tac tac ctg gag aag gcc aac aaa ata aag tct gag aaa aca cat 432

Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Lys Thr His

130 135 140

atc cac att ttc tca ttt aca gga gaa gaa atg gct aca aaa gcg gac 480

Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys Ala Asp

145 150 155 160

tat act ctt gat gaa gag agt aga gcc agg atc aag acc aga cta ttc 528

Tyr Thr Leu Asp Glu Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe

165 170 175

act ata aga caa gaa atg gcc agt aga ggc ctc tgg gat tcc ttt cgt 576

Thr Ile Arg Gln Glu Met Ala Ser Arg Gly Leu Trp Asp Ser Phe Arg

180 185 190

cag tcc gag aga ggc gaa gag aca att gaa gaa aga ttt gaa atc aca 624

Gln Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr

195 200 205

ggg acg atg cgc aag ctt gcc aat tac agt ctc cca ccg aac ttc tcc 672

Gly Thr Met Arg Lys Leu Ala Asn Tyr Ser Leu Pro Pro Asn Phe Ser

210 215 220

agc ctt gaa aat ttt aga gtc tat ata gat gga ttc gaa ccg aac ggc 720

Ser Leu Glu Asn Phe Arg Val Tyr Ile Asp Gly Phe Glu Pro Asn Gly

225 230 235 240

tgc att gag agt aag ctt tct caa atg tcc aaa gaa gta aat gcc aaa 768

Cys Ile Glu Ser Lys Leu Ser Gln Met Ser Lys Glu Val Asn Ala Lys

245 250 255

ata gaa cca ttt tca aag aca aca ccc cga cca ctc aaa atg cca ggt 816

Ile Glu Pro Phe Ser Lys Thr Thr Pro Arg Pro Leu Lys Met Pro Gly

260 265 270

ggt cca ccc tgc cat cag cga tcc aaa ttc ttg cta atg gat gct ctg 864

Gly Pro Pro Cys His Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu

275 280 285

aaa ctg agc att gag gac cca agt cac gag gga gag ggg ata cca cta 912

Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu

290 295 300

tat gat gca atc aaa tgc atg aaa act ttc ttt gga tgg aaa gag ccc 960

Tyr Asp Ala Ile Lys Cys Met Lys Thr Phe Phe Gly Trp Lys Glu Pro

305 310 315 320

agt att gtt aaa cca cat aaa aag ggt ata aac ccg aac tat ctc caa 1008

Ser Ile Val Lys Pro His Lys Lys Gly Ile Asn Pro Asn Tyr Leu Gln

325 330 335

act tgg aag caa gta tta gaa gaa ata caa gac ctt gag aac gaa gaa 1056

Thr Trp Lys Gln Val Leu Glu Glu Ile Gln Asp Leu Glu Asn Glu Glu

340 345 350

agg acc ccc aag acc aag aat atg aaa aaa aca agc caa ttg aaa tgg 1104

Arg Thr Pro Lys Thr Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp

355 360 365

gca cta ggt gaa aat atg gca cca gag aaa gtg gat ttt gag gat tgt 1152

Ala Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Glu Asp Cys

370 375 380

aaa gac atc aat gat tta aaa caa tat gac agt gat gag cca gaa gca 1200

Lys Asp Ile Asn Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Ala

385 390 395 400

agg tct ctt gca agt tgg att caa agt gag ttc aac aag gct tgt gag 1248

Arg Ser Leu Ala Ser Trp Ile Gln Ser Glu Phe Asn Lys Ala Cys Glu

405 410 415

ctg aca gat tca agc tgg ata gag ctc gat gaa att ggg gag gat gtc 1296

Leu Thr Asp Ser Ser Ser Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val

420 425 430

gcc cca ata gaa tac att gcg agc atg agg aga aat tat ttt act gct 1344

Ala Pro Ile Glu Tyr Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala

435 440 445

gag att tcc cat tgt aga gca aca gaa tat ata atg aaa gga gtg tac 1392

Glu Ile Ser His Cys Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr

450 455 460

atc aac act gct cta ctc aat gca tcc tgt gct gcg atg gat gaa ttt 1440

Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Glu Phe

465 470 475 480

caa tta att ccg atg ata agt aaa tgc agg acc aaa gaa ggg aga agg 1488

Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg

485 490 495

aaa aca aat tta tat gga ttc ata ata aag gga agg tcc cat tta aga 1536

Lys Thr Asn Leu Tyr Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg

500 505 510

aat gat act gac gtg gtg aac ttt gta agt atg gaa ttt tct ctc act 1584

Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr

515 520 525

gat cca aga ttt gag cca cac aaa tgg gaa aaa tac tgc gtt cta gaa 1632

Asp Pro Arg Phe Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu

530 535 540

att gga gac atg ctt tta aga act gct gta ggt caa gtg tca aga ccc 1680

Ile Gly Asp Met Leu Leu Arg Thr Ala Val Gly Gln Val Ser Arg Pro

545 550 555 560

atg ttt ttg tat gta agg aca aat gga acc tct aaa att aaa atg aaa 1728

Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys

565 570 575

tgg gga atg gaa atg agg cgc tgc ctc ctt cag tct ctg caa cag att 1776

Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile

580 585 590

gaa agc atg atc gaa gct gag tcc tca gtc aaa gaa aag gac atg acc 1824

Glu Ser Met Ile Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr

595 600 605

aaa gaa ttt ttt gag aac aaa tca gag aca tgg cct ata gga gag tcc 1872

Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser

610 615 620

ccc aaa gga gtg gaa gag ggc tca atc ggg aag gtt tgc agg acc tta 1920

Pro Lys Gly Val Glu Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu

625 630 635 640

tta gca aaa tct gtg ttt aac agt tta tat gca tct cca caa ctg gaa 1968

Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu

645 650 655

gga ttt tca gct gaa tct agg aaa tta ctt ctc att gtt cag gct ctt 2016

Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu

660 665 670

aga gat gac ctg gaa cct gga acc ttt gat att ggg ggg tta tat gaa 2064

Arg Asp Asp Leu Glu Pro Gly Thr Phe Asp Ile Gly Gly Leu Tyr Glu

675 680 685

tca att gag gag tgc ctg att aat gat ccc tgg gtt ttg ctt aat gca 2112

Ser Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala

690 695 700

tct tgg ttc aac tcc ttc ctc aca cat gca ctg aag tag 2151

Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Lys

705 710 715

<210> 52

<211> 716

<212> PRT

<213> Influenza virus

<400> 52

Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu

1 5 10 15

Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asn Pro Lys Ile Glu Thr

20 25 30

Asn Lys Phe Ala Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr

35 40 45

Ser Asp Phe His Phe Ile Asn Glu Leu Gly Glu Ser Val Val Ile Glu

50 55 60

Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu

65 70 75 80

Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn

85 90 95

Thr Thr Arg Ala Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr

100 105 110

Lys Glu Asn Arg Phe Val Glu Ile Gly Val Thr Arg Arg Glu Val His

115 120 125

Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Lys Thr His

130 135 140

Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys Ala Asp

145 150 155 160

Tyr Thr Leu Asp Glu Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe

165 170 175

Thr Ile Arg Gln Glu Met Ala Ser Arg Gly Leu Trp Asp Ser Phe Arg

180 185 190

Gln Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr

195 200 205

Gly Thr Met Arg Lys Leu Ala Asn Tyr Ser Leu Pro Pro Asn Phe Ser

210 215 220

Ser Leu Glu Asn Phe Arg Val Tyr Ile Asp Gly Phe Glu Pro Asn Gly

225 230 235 240

Cys Ile Glu Ser Lys Leu Ser Gln Met Ser Lys Glu Val Asn Ala Lys

245 250 255

Ile Glu Pro Phe Ser Lys Thr Thr Pro Arg Pro Leu Lys Met Pro Gly

260 265 270

Gly Pro Pro Cys His Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu

275 280 285

Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu

290 295 300

Tyr Asp Ala Ile Lys Cys Met Lys Thr Phe Phe Gly Trp Lys Glu Pro

305 310 315 320

Ser Ile Val Lys Pro His Lys Lys Gly Ile Asn Pro Asn Tyr Leu Gln

325 330 335

Thr Trp Lys Gln Val Leu Glu Glu Ile Gln Asp Leu Glu Asn Glu Glu

340 345 350

Arg Thr Pro Lys Thr Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp

355 360 365

Ala Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Glu Asp Cys

370 375 380

Lys Asp Ile Asn Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Ala

385 390 395 400

Arg Ser Leu Ala Ser Trp Ile Gln Ser Glu Phe Asn Lys Ala Cys Glu

405 410 415

Leu Thr Asp Ser Ser Ser Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val

420 425 430

Ala Pro Ile Glu Tyr Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala

435 440 445

Glu Ile Ser His Cys Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr

450 455 460

Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Glu Phe

465 470 475 480

Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg

485 490 495

Lys Thr Asn Leu Tyr Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg

500 505 510

Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr

515 520 525

Asp Pro Arg Phe Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu

530 535 540

Ile Gly Asp Met Leu Leu Arg Thr Ala Val Gly Gln Val Ser Arg Pro

545 550 555 560

Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys

565 570 575

Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile

580 585 590

Glu Ser Met Ile Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr

595 600 605

Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser

610 615 620

Pro Lys Gly Val Glu Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu

625 630 635 640

Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu

645 650 655

Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu

660 665 670

Arg Asp Asp Leu Glu Pro Gly Thr Phe Asp Ile Gly Gly Leu Tyr Glu

675 680 685

Ser Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala

690 695 700

Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Lys

705 710 715

<210> 53

<211> 844

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(690)

<400> 53

atg gat tcc aac act gtg tca agc ttt cag gta gac tgt ttt ctt tgg 48

Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

cat gtc cgt aaa cga ttc gca gac caa gaa ctg ggt gat gcc cca ttc 96

His Val Arg Lys Arg Phe Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

ctt gac cgg ctt cgc cga gac cag aag tcc cta agg gga aga ggt agc 144

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

act ctt ggt ctg gac atc gaa aca gcc act cat gca gga aag cag ata 192

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr His Ala Gly Lys Gln Ile

50 55 60

gtg gag cag att ctg gaa aag gaa tca gat gag gca ctt aaa atg acc 240

Val Glu Gln Ile Leu Glu Lys Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

att gcc tct gtt cct gct tca cgc tac tta act gac atg act ctt gat 288

Ile Ala Ser Val Pro Ala Ser Arg Tyr Leu Thr Asp Met Thr Leu Asp

85 90 95

gag atg tca aga gac tgg ttc atg ctc atg ccc aag caa aaa gta aca 336

Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Thr

100 105 110

ggc tcc cta tgt ata aga atg gac cag gca atc atg gat aag aac atc 384

Gly Ser Leu Cys Ile Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile

115 120 125

ata ctt aaa gca aac ttt agt gtg att ttc gaa agg ctg gaa aca cta 432

Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Glu Arg Leu Glu Thr Leu

130 135 140

ata cta ctt aga gcc ttc acc gaa gaa gga gca gtc gtt ggc gaa att 480

Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile

145 150 155 160

tca cca tta cct tct ctt cca gga cat act aat gag gat gtc aaa aat 528

Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn

165 170 175

gca att ggg gtc ctc atc gga gga ctt aaa tgg aat gat aat acg gtt 576

Ala Ile Gly Val Leu Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val

180 185 190

aga atc tct gaa act cta cag aga ttc gct tgg aga agc agt cat gaa 624

Arg Ile Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser His Glu

195 200 205

aat ggg aga cct tca ttc cct tca aaa cag aaa cga aaa atg gag aga 672

Asn Gly Arg Pro Ser Phe Pro Ser Lys Gln Lys Arg Lys Met Glu Arg

210 215 220

aca att aag cca gaa att tgaagaaata agatggttga ttgaagaagt 720

Thr Ile Lys Pro Glu Ile

225 230

gcgacataga ttgaaaaata cagaaaatag ttttgaacaa ataacattta tgcaagcctt 780

acaactattg cttgaagtag aacaagagat aagaactttc tcgtttcagc ttatttaatg 840

ataa 844

<210> 54

<211> 230

<212> PRT

<213> Influenza virus

<400> 54

Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

His Val Arg Lys Arg Phe Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr His Ala Gly Lys Gln Ile

50 55 60

Val Glu Gln Ile Leu Glu Lys Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

Ile Ala Ser Val Pro Ala Ser Arg Tyr Leu Thr Asp Met Thr Leu Asp

85 90 95

Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Thr

100 105 110

Gly Ser Leu Cys Ile Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile

115 120 125

Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Glu Arg Leu Glu Thr Leu

130 135 140

Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile

145 150 155 160

Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn

165 170 175

Ala Ile Gly Val Leu Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val

180 185 190

Arg Ile Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser His Glu

195 200 205

Asn Gly Arg Pro Ser Phe Pro Ser Lys Gln Lys Arg Lys Met Glu Arg

210 215 220

Thr Ile Lys Pro Glu Ile

225 230

<210> 55

<211> 1497

<212> DNA

<213> Influenza virus s

<220>

<221> CDS

<222> (1)..(1497)

<400> 55

atg gcg tct caa ggc acc aaa cga tcc tat gaa cag atg gaa act gat 48

Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp

1 5 10 15

ggg gaa cgc cag aat gca act gaa atc aga gca tct gtc gga agg atg 96

Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Arg Met

20 25 30

gtg gga gga atc gga cgg ttt tat gtc cag atg tgt act gag ctt aaa 144

Val Gly Gly Ile Gly Arg Phe Tyr Val Gln Met Cys Thr Glu Leu Lys

35 40 45

cta aac gac cat gaa ggg cgg ctg att cag aac agc ata aca ata gaa 192

Leu Asn Asp His Glu Gly Arg Leu Ile Gln Asn Ser Ile Thr Ile Glu

50 55 60

agg atg gtg ctt tcg gca ttc gac gaa aga aga aac aag tat ctc gag 240

Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu

65 70 75 80

gag cat ccc agt gct ggg aaa gac cct aag aaa acg gga ggc ccg ata 288

Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile

85 90 95

tac aga aga aaa gat ggg aaa tgg atg agg gaa ctc atc ctc cat gat 336

Tyr Arg Arg Lys Asp Gly Lys Trp Met Arg Glu Leu Ile Leu His Asp

100 105 110

aaa gaa gaa atc atg aga atc tgg cgt cag gcc aac aat ggt gaa gac 384

Lys Glu Glu Ile Met Arg Ile Trp Arg Gln Ala Asn Asn Gly Glu Asp

115 120 125

gct act gct ggt ctt act cat atg atg atc tgg cac tcc aat ctc aat 432

Ala Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn

130 135 140

gac acc aca tac caa aga aca agg gct ctt gtt cgg act ggg atg gat 480

Asp Thr Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp

145 150 155 160

ccc aga atg tgc tct ctg atg caa ggc tca acc ctc cca cgg aga tct 528

Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser

165 170 175

gga gcc gct ggt gct gca gta aaa ggc gtt gga aca atg gta atg gaa 576

Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu

180 185 190

ctc atc aga atg atc aag cgc gga ata aat gat cgg aat ttc tgg aga 624

Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg

195 200 205

ggt gaa aat ggt cga aga acc aga att gct tat gaa aga atg tgc aat 672

Gly Glu Asn Gly Arg Arg Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn

210 215 220

atc ctc aaa ggg aaa ttt cag aca gca gca caa cgg gct atg atg gac 720

Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp

225 230 235 240

cag gtg agg gaa ggc cgc aat cct gga aac gct gag att gag gat ctc 768

Gln Val Arg Glu Gly Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu

245 250 255

att ttc ttg gca cga tca gca ctt att ttg aga gga tca gta gcc cat 816

Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His

260 265 270

aaa tca tgc cta cct gcc tgt gtt tat ggc ctt gca gta acc agt ggg 864

Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala Val Thr Ser Gly

275 280 285

tat gac ttt gag aag gaa gga tac tct ctg gtt gga att gat cct ttc 912

Tyr Asp Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe

290 295 300

aaa cta ctc cag aac agt caa att ttc agt cta atc aga cca aaa gaa 960

Lys Leu Leu Gln Asn Ser Gln Ile Phe Ser Leu Ile Arg Pro Lys Glu

305 310 315 320

aac cca gca cac aaa agc cag ttg gtg tgg atg gca tgc cat tct gca 1008

Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala

325 330 335

gca ttt gag gat ctg aga gtt tta aat ttc att aga gga acc aaa gta 1056

Ala Phe Glu Asp Leu Arg Val Leu Asn Phe Ile Arg Gly Thr Lys Val

340 345 350

atc cca aga gga cag tta aca acc aga gga gtt caa att gct tca aat 1104

Ile Pro Arg Gly Gln Leu Thr Thr Arg Gly Val Gln Ile Ala Ser Asn

355 360 365

gaa aac atg gag aca ata aat tct agc aca ctt gaa ctg aga agc aaa 1152

Glu Asn Met Glu Thr Ile Asn Ser Ser Thr Leu Glu Leu Arg Ser Lys

370 375 380

tat tgg gca ata agg acc aga agc gga gga aac acc agt caa cag aga 1200

Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Ser Gln Gln Arg

385 390 395 400

gca tct gca gga cag ata agt gtg caa cct act ttc tca gta cag aga 1248

Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg

405 410 415

aat ctt ccc ttt gag aga gca acc att atg gct gca ttc act ggt aac 1296

Asn Leu Pro Phe Glu Arg Ala Thr Ile Met Ala Ala Phe Thr Gly Asn

420 425 430

act gaa gga agg act tcc gac atg aga acg gaa atc ata agg atg atg 1344

Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met

435 440 445

gaa aat gcc aaa tca gaa gat gtg tct ttc cag ggg cgg gga gtc ttc 1392

Glu Asn Ala Lys Ser Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe

450 455 460

gag ctc tcg gac gaa aag gca acg aac ccg atc gtg cct tcc ttt gac 1440

Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp

465 470 475 480

atg agc aat gaa ggg tct tat ttc ttc gga gac aat gct gag gag ttt 1488

Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Phe

485 490 495

gac agt taa 1497

Asp Ser

<210> 56

<211> 498

<212> PRT

<213> Influenza virus

<400> 56

Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp

1 5 10 15

Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Arg Met

20 25 30

Val Gly Gly Ile Gly Arg Phe Tyr Val Gln Met Cys Thr Glu Leu Lys

35 40 45

Leu Asn Asp His Glu Gly Arg Leu Ile Gln Asn Ser Ile Thr Ile Glu

50 55 60

Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu

65 70 75 80

Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile

85 90 95

Tyr Arg Arg Lys Asp Gly Lys Trp Met Arg Glu Leu Ile Leu His Asp

100 105 110

Lys Glu Glu Ile Met Arg Ile Trp Arg Gln Ala Asn Asn Gly Glu Asp

115 120 125

Ala Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn

130 135 140

Asp Thr Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp

145 150 155 160

Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser

165 170 175

Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu

180 185 190

Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg

195 200 205

Gly Glu Asn Gly Arg Arg Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn

210 215 220

Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp

225 230 235 240

Gln Val Arg Glu Gly Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu

245 250 255

Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His

260 265 270

Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala Val Thr Ser Gly

275 280 285

Tyr Asp Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe

290 295 300

Lys Leu Leu Gln Asn Ser Gln Ile Phe Ser Leu Ile Arg Pro Lys Glu

305 310 315 320

Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala

325 330 335

Ala Phe Glu Asp Leu Arg Val Leu Asn Phe Ile Arg Gly Thr Lys Val

340 345 350

Ile Pro Arg Gly Gln Leu Thr Thr Arg Gly Val Gln Ile Ala Ser Asn

355 360 365

Glu Asn Met Glu Thr Ile Asn Ser Ser Thr Leu Glu Leu Arg Ser Lys

370 375 380

Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Ser Gln Gln Arg

385 390 395 400

Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg

405 410 415

Asn Leu Pro Phe Glu Arg Ala Thr Ile Met Ala Ala Phe Thr Gly Asn

420 425 430

Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met

435 440 445

Glu Asn Ala Lys Ser Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe

450 455 460

Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp

465 470 475 480

Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Phe

485 490 495

Asp Ser

<210> 57

<211> 1413

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(1413)

<400> 57

atg aat cca aat caa aag ata ata gca att gga ttt gca tca ttg ggg 48

Met Asn Pro Asn Gln Lys Ile Ile Ala Ile Gly Phe Ala Ser Leu Gly

1 5 10 15

ata tta atc att aat gtc att ctc cat gta gtc agc att ata gta aca 96

Ile Leu Ile Ile Asn Val Ile Leu His Val Val Ser Ile Ile Val Thr

20 25 30

gta ctg gtc ctc aat aac aat aga aca gat ctg aac tgc aaa ggg acg 144

Val Leu Val Leu Asn Asn Asn Arg Thr Asp Leu Asn Cys Lys Gly Thr

35 40 45

atc ata aga gaa tac aat gaa aca gta aga gta gaa aaa ctt act caa 192

Ile Ile Arg Glu Tyr Asn Glu Thr Val Arg Val Glu Lys Leu Thr Gln

50 55 60

tgg tat aat acc agt aca att aag tac ata gag aga cct tca aat gaa 240

Trp Tyr Asn Thr Ser Thr Ile Lys Tyr Ile Glu Arg Pro Ser Asn Glu

65 70 75 80

tac tac atg aat aac act gaa cca ctt tgt gag gcc caa ggc ttt gca 288

Tyr Tyr Met Asn Asn Thr Glu Pro Leu Cys Glu Ala Gln Gly Phe Ala

85 90 95

cca ttt tcc aaa gat aat gga ata cga att ggg tcg aga ggc cat gtt 336

Pro Phe Ser Lys Asp Asn Gly Ile Arg Ile Gly Ser Arg Gly His Val

100 105 110

ttt gtg ata aga gaa cct ttt gta tca tgt tcg ccc tca gaa tgt aga 384

Phe Val Ile Arg Glu Pro Phe Val Ser Cys Ser Pro Ser Glu Cys Arg

115 120 125

acc ttt ttc ctc aca cag ggc tca tta ctc aat gac aaa cat tct aac 432

Thr Phe Phe Leu Thr Gln Gly Ser Leu Leu Asn Asp Lys His Ser Asn

130 135 140

ggc aca ata aag gat cga agt ccg tat agg act ttg atg agt gtc aaa 480

Gly Thr Ile Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser Val Lys

145 150 155 160

ata ggg caa tca cct aat gta tat caa gct agg ttt gaa tcg gtg gca 528

Ile Gly Gln Ser Pro Asn Val Tyr Gln Ala Arg Phe Glu Ser Val Ala

165 170 175

tgg tca gca aca gca tgc cat gat gga aaa aaa tgg atg aca gtt gga 576

Trp Ser Ala Thr Ala Cys His Asp Gly Lys Lys Trp Met Thr Val Gly

180 185 190

gtc aca ggg ccc gac aat caa gca att gca gta gtg aac tat gga ggt 624

Val Thr Gly Pro Asp Asn Gln Ala Ile Ala Val Val Asn Tyr Gly Gly

195 200 205

gtt ccg gtt gat att att aat tca tgg gca ggg gat att tta aga acc 672

Val Pro Val Asp Ile Ile Asn Ser Trp Ala Gly Asp Ile Leu Arg Thr

210 215 220

caa gaa tca tca tgc acc tgc att aaa gga gac tgt tat tgg gta atg 720

Gln Glu Ser Ser Cys Thr Cys Ile Lys Gly Asp Cys Tyr Trp Val Met

225 230 235 240

act gat gga ccg gca aat agg caa gct aaa tat agg ata ttc aaa gca 768

Thr Asp Gly Pro Ala Asn Arg Gln Ala Lys Tyr Arg Ile Phe Lys Ala

245 250 255

aaa gat gga aga gta att gga caa act gat ata agt ttc aat ggg gga 816

Lys Asp Gly Arg Val Ile Gly Gln Thr Asp Ile Ser Phe Asn Gly Gly

260 265 270

cac ata gag gag tgt tct tgt tac ccc aat gaa ggg aag gtg gaa tgc 864

His Ile Glu Glu Cys Ser Cys Tyr Pro Asn Glu Gly Lys Val Glu Cys

275 280 285

ata tgc agg gac aat tgg act gga aca aat aga cca att ctg gta ata 912

Ile Cys Arg Asp Asn Trp Thr Gly Thr Asn Arg Pro Ile Leu Val Ile

290 295 300

tct tct gat cta tcg tac aca gtt gga tat ttg tgt gct ggc att ccc 960

Ser Ser Asp Leu Ser Tyr Thr Val Gly Tyr Leu Cys Ala Gly Ile Pro

305 310 315 320

act gac act cct agg gga gag gat agt caa ttc aca ggc tca tgt aca 1008

Thr Asp Thr Pro Arg Gly Glu Asp Ser Gln Phe Thr Gly Ser Cys Thr

325 330 335

agt cct ttg gga aat aaa gga tac ggt gta aaa ggc ttc ggg ttt cga 1056

Ser Pro Leu Gly Asn Lys Gly Tyr Gly Val Lys Gly Phe Gly Phe Arg

340 345 350

caa gga act gac gta tgg gcc gga agg aca att agt agg act tca aga 1104

Gln Gly Thr Asp Val Trp Ala Gly Arg Thr Ile Ser Arg Thr Ser Arg

355 360 365

tca gga ttc gaa ata ata aaa atc agg aat ggt tgg aca cag aac agt 1152

Ser Gly Phe Glu Ile Ile Lys Ile Arg Asn Gly Trp Thr Gln Asn Ser

370 375 380

aag gac caa atc agg agg caa gtg att atc gat gac cca aat tgg tca 1200

Lys Asp Gln Ile Arg Arg Gln Val Ile Ile Asp Asp Pro Asn Trp Ser

385 390 395 400

gga tat agc ggt tct ttc aca ttg ccg gtt gaa ctg aca aaa aag gga 1248

Gly Tyr Ser Gly Ser Phe Thr Leu Pro Val Glu Leu Thr Lys Lys Gly

405 410 415

tgt ttg gtc ccc tgt ttc tgg gtt gaa atg att aga ggt aaa cct gaa 1296

Cys Leu Val Pro Cys Phe Trp Val Glu Met Ile Arg Gly Lys Pro Glu

420 425 430

gaa aca aca ata tgg acc tct agc agc tcc att gtg atg tgt gga gta 1344

Glu Thr Thr Ile Trp Thr Ser Ser Ser Ser Ile Val Met Cys Gly Val

435 440 445

gat cat aaa att gcc agt tgg tca tgg cac gat gga gct att ctt ccc 1392

Asp His Lys Ile Ala Ser Trp Ser Trp His Asp Gly Ala Ile Leu Pro

450 455 460

ttt gac atc gat aag atg taa 1413

Phe Asp Ile Asp Lys Met

465 470

<210> 58

<211> 470

<212> PRT

<213> Influenza virus

<400> 58

Met Asn Pro Asn Gln Lys Ile Ile Ala Ile Gly Phe Ala Ser Leu Gly

1 5 10 15

Ile Leu Ile Ile Asn Val Ile Leu His Val Val Ser Ile Ile Val Thr

20 25 30

Val Leu Val Leu Asn Asn Asn Arg Thr Asp Leu Asn Cys Lys Gly Thr

35 40 45

Ile Ile Arg Glu Tyr Asn Glu Thr Val Arg Val Glu Lys Leu Thr Gln

50 55 60

Trp Tyr Asn Thr Ser Thr Ile Lys Tyr Ile Glu Arg Pro Ser Asn Glu

65 70 75 80

Tyr Tyr Met Asn Asn Thr Glu Pro Leu Cys Glu Ala Gln Gly Phe Ala

85 90 95

Pro Phe Ser Lys Asp Asn Gly Ile Arg Ile Gly Ser Arg Gly His Val

100 105 110

Phe Val Ile Arg Glu Pro Phe Val Ser Cys Ser Pro Ser Glu Cys Arg

115 120 125

Thr Phe Phe Leu Thr Gln Gly Ser Leu Leu Asn Asp Lys His Ser Asn

130 135 140

Gly Thr Ile Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser Val Lys

145 150 155 160

Ile Gly Gln Ser Pro Asn Val Tyr Gln Ala Arg Phe Glu Ser Val Ala

165 170 175

Trp Ser Ala Thr Ala Cys His Asp Gly Lys Lys Trp Met Thr Val Gly

180 185 190

Val Thr Gly Pro Asp Asn Gln Ala Ile Ala Val Val Asn Tyr Gly Gly

195 200 205

Val Pro Val Asp Ile Ile Asn Ser Trp Ala Gly Asp Ile Leu Arg Thr

210 215 220

Gln Glu Ser Ser Cys Thr Cys Ile Lys Gly Asp Cys Tyr Trp Val Met

225 230 235 240

Thr Asp Gly Pro Ala Asn Arg Gln Ala Lys Tyr Arg Ile Phe Lys Ala

245 250 255

Lys Asp Gly Arg Val Ile Gly Gln Thr Asp Ile Ser Phe Asn Gly Gly

260 265 270

His Ile Glu Glu Cys Ser Cys Tyr Pro Asn Glu Gly Lys Val Glu Cys

275 280 285

Ile Cys Arg Asp Asn Trp Thr Gly Thr Asn Arg Pro Ile Leu Val Ile

290 295 300

Ser Ser Asp Leu Ser Tyr Thr Val Gly Tyr Leu Cys Ala Gly Ile Pro

305 310 315 320

Thr Asp Thr Pro Arg Gly Glu Asp Ser Gln Phe Thr Gly Ser Cys Thr

325 330 335

Ser Pro Leu Gly Asn Lys Gly Tyr Gly Val Lys Gly Phe Gly Phe Arg

340 345 350

Gln Gly Thr Asp Val Trp Ala Gly Arg Thr Ile Ser Arg Thr Ser Arg

355 360 365

Ser Gly Phe Glu Ile Ile Lys Ile Arg Asn Gly Trp Thr Gln Asn Ser

370 375 380

Lys Asp Gln Ile Arg Arg Gln Val Ile Ile Asp Asp Pro Asn Trp Ser

385 390 395 400

Gly Tyr Ser Gly Ser Phe Thr Leu Pro Val Glu Leu Thr Lys Lys Gly

405 410 415

Cys Leu Val Pro Cys Phe Trp Val Glu Met Ile Arg Gly Lys Pro Glu

420 425 430

Glu Thr Thr Ile Trp Thr Ser Ser Ser Ser Ile Val Met Cys Gly Val

435 440 445

Asp His Lys Ile Ala Ser Trp Ser Trp His Asp Gly Ala Ile Leu Pro

450 455 460

Phe Asp Ile Asp Lys Met

465 470

<210> 59

<211> 981

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(756)

<400> 59

atg agt ctt cta acc gag gtc gaa acg tac gtt ctc tct atc gta cca 48

Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro

1 5 10 15

tca ggc ccc ctc aaa gcc gag atc gcg cag aga ctt gaa gat gtc ttt 96

Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe

20 25 30

gcg gga aag aac acc gat ctt gag gca ctc atg gaa tgg cta aag aca 144

Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr

35 40 45

aga cca atc ctg tca cct ctg act aaa ggg att tta gga ttt gta ttc 192

Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe

50 55 60

acg ctc acc gtg ccc agt gag cga gga ctg cag cgt aga cgc ttt gtc 240

Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val

65 70 75 80

caa aat gcc ctt agt gga aac gga gat cca aac aac atg gac aga gca 288

Gln Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala

85 90 95

gta aaa ctg tac agg aag ctt aaa aga gaa ata aca ttc cat ggg gca 336

Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala

100 105 110

aaa gag gtg gca ctc agc tat tcc act ggt gca cta gcc agc tgc atg 384

Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met

115 120 125

gga ctc ata tac aac aga atg gga act gtt aca acc gaa gtg gca ttt 432

Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe

130 135 140

ggc ctg gta tgc gcc aca tgt gaa cag att gct gat tcc cag cat cga 480

Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg

145 150 155 160

tct cac agg cag atg gtg aca aca acc aac cca tta atc aga cat gaa 528

Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu

165 170 175

aac aga atg gta tta gcc agt acc acg gct aaa gcc atg gaa cag atg 576

Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met

180 185 190

gca gga tcg agt gag cag gca gca gag gcc atg gag gtt gct agt agg 624

Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Arg

195 200 205

gct agg cag atg gta cag gca atg aga acc att ggg acc cac cct agc 672

Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser

210 215 220

tcc agt gcc ggt ttg aaa gat gat ctc ctt gaa aat tta cag gcc tac 720

Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr

225 230 235 240

cag aaa cgg atg gga gtg caa atg cag cga ttc aag tgatcctctc 766

Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys

245 250

gtcattgcag caagtatcat tgggatcttg cacttgatat tgtggattct tgatcgtctt 826

ttcttcaaat tcatttatcg tcgccttaaa tacgggttga aaagaggggcc ttctacggaa 886

ggagtacctg agtctatgag ggaagaatat cggcaggaac agcagaatgc tgtggatgtt 946

gacgatggtc attttgtcaa catagagctg gagta 981

<210> 60

<211> 252

<212> PRT

<213> Influenza virus

<400> 60

Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro

1 5 10 15

Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe

20 25 30

Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr

35 40 45

Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe

50 55 60

Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val

65 70 75 80

Gln Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala

85 90 95

Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala

100 105 110

Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met

115 120 125

Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe

130 135 140

Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg

145 150 155 160

Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu

165 170 175

Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met

180 185 190

Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Arg

195 200 205

Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser

210 215 220

Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr

225 230 235 240

Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys

245 250

<210> 61

<211> 1698

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(1698)

<400> 61

atg aag aca acc att att tta ata cta ctg acc cat tgg gcc tac agt 48

Met Lys Thr Thr Ile Ile Leu Ile Leu Leu Thr His Trp Ala Tyr Ser

1 5 10 15

caa aac cca atc agt ggc aat aac aca gcc aca ctg tgt ctg gga cac 96

Gln Asn Pro Ile Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His

20 25 30

cat gca gta gca aat gga aca ttg gta aaa aca atg agt gat gat caa 144

His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Met Ser Asp Asp Gln

35 40 45

att gag gtg aca aat gct aca gaa tta gtt cag agc att tca atg ggg 192

Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ile Ser Met Gly

50 55 60

aaa ata tgc aac aaa tca tat aga att cta gat gga aga aat tgc aca 240

Lys Ile Cys Asn Lys Ser Tyr Arg Ile Leu Asp Gly Arg Asn Cys Thr

65 70 75 80

tta ata gat gca atg cta gga gac ccc cac tgt gac gcc ttt cag tat 288

Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gln Tyr

85 90 95

gag agt tgg gac ctc ttt ata gaa aga agc agc gct ttc agc aat tgc 336

Glu Ser Trp Asp Leu Phe Ile Glu Arg Ser Ser Ala Phe Ser Asn Cys

100 105 110

tac cca tat gac atc cct gac tat gca tcg ctc cga tcc att gta gca 384

Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Ser Leu Arg Ser Ile Val Ala

115 120 125

tcc tca ggg aca gtg gaa ttc aca gca gag gga ttc aca tgg aca ggt 432

Ser Ser Gly Thr Val Glu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly

130 135 140

gta act caa aac gga aga agt gga gcc tgc aaa agg gga tca gcc gat 480

Val Thr Gln Asn Gly Arg Ser Gly Ala Cys Lys Arg Gly Ser Ala Asp

145 150 155 160

agt ttc ttt agc cga ctg aat tgg cta aca aaa tct gga agc tct tac 528

Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Lys Ser Gly Ser Ser Tyr

165 170 175

ccc aca ttg aat gtg aca atg cct aac aat aaa aat ttc gac aag cta 576

Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu

180 185 190

tac atc tgg ggg att cat cac ccg agc tca aat caa gag cag aca aaa 624

Tyr Ile Trp Gly Ile His Pro Ser Ser Asn Gln Glu Gln Thr Lys

195 200 205

ttg tac atc caa gaa tca gga cga gta aca gtc tca aca aaa aga agt 672

Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser

210 215 220

caa caa aca ata atc cct aac atc gga tct aga ccg ttg gtc aga ggt 720

Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Leu Val Arg Gly

225 230 235 240

caa tca ggc agg ata agc ata tac tgg acc att gta aaa cct gga gat 768

Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp

245 250 255

atc cta atg ata aac agt aat ggc aac tta gtt gca ccg cgg gga tat 816

Ile Leu Met Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr

260 265 270

ttt aaa ttg aac aca ggg aaa agc tct gta atg aga tcc gat gta ccc 864

Phe Lys Leu Asn Thr Gly Lys Ser Ser Val Met Arg Ser Asp Val Pro

275 280 285

ata gac att tgt gtg tct gaa tgt att aca cca aat gga agc atc tcc 912

Ile Asp Ile Cys Val Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Ser

290 295 300

aac gac aag cca ttc caa aat gtg aac aaa gtt aca tat gga aaa tgc 960

Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys

305 310 315 320

ccc aag tat atc agg caa aac act tta aag ctg gcc act ggg atg agg 1008

Pro Lys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg

325 330 335

aat gta cca gaa aag caa acc aga gga atc ttt gga gca ata gcg gga 1056

Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala Gly

340 345 350

ttc atc gaa aac ggc tgg gaa gga atg gtt gat ggg tgg tat ggg ttc 1104

Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe

355 360 365

cga tat caa aac tct gaa gga aca ggg caa gct gca gat cta aag agc 1152

Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser

370 375 380

act caa gca gcc atc gac cag att aat gga aag tta aac aga gtg att 1200

Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile

385 390 395 400

gaa aga acc aat gag aaa ttc cat caa ata gag aag gaa ttc tca gaa 1248

Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu

405 410 415

gta gaa gga aga att cag gac ttg gag aaa tat gta gaa gac acc aaa 1296

Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys

420 425 430

ata gac cta tgg tcc tac aat gca gaa ttg ctg gtg gct cta gaa aat 1344

Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn

435 440 445

caa cat aca att gac tta aca gat gca gaa atg aat aaa tta ttt gag 1392

Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu

450 455 460

aag act aga cgc cag tta aga gaa aac gca gaa gac atg gga ggt gga 1440

Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly

465 470 475 480

tgt ttc aag att tac cac aaa tgt gat aat gca tgc att gaa tca ata 1488

Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Glu Ser Ile

485 490 495

aga act ggg aca tat gac cat tac ata tac aaa gat gaa gca tta aac 1536

Arg Thr Gly Thr Tyr Asp His Tyr Ile Tyr Lys Asp Glu Ala Leu Asn

500 505 510

aat cga ttt cag atc aaa ggt gta gag ttg aaa tca ggc tac aaa gat 1584

Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp

515 520 525

tgg ata ctg tgg att tca ttc gcc ata tca tgc ttc tta att tgc gtt 1632

Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Ile Cys Val

530 535 540

gtt cta ttg ggt ttc att atg tgg gct tgc caa aaa ggc aac atc aga 1680

Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg

545 550 555 560

tgc aac att tgc att tga 1698

Cys Asn Ile Cys Ile

565

<210> 62

<211> 565

<212> PRT

<213> Influenza virus

<400> 62

Met Lys Thr Thr Ile Ile Leu Ile Leu Leu Thr His Trp Ala Tyr Ser

1 5 10 15

Gln Asn Pro Ile Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His

20 25 30

His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Met Ser Asp Asp Gln

35 40 45

Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ile Ser Met Gly

50 55 60

Lys Ile Cys Asn Lys Ser Tyr Arg Ile Leu Asp Gly Arg Asn Cys Thr

65 70 75 80

Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gln Tyr

85 90 95

Glu Ser Trp Asp Leu Phe Ile Glu Arg Ser Ser Ala Phe Ser Asn Cys

100 105 110

Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Ser Leu Arg Ser Ile Val Ala

115 120 125

Ser Ser Gly Thr Val Glu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly

130 135 140

Val Thr Gln Asn Gly Arg Ser Gly Ala Cys Lys Arg Gly Ser Ala Asp

145 150 155 160

Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Lys Ser Gly Ser Ser Tyr

165 170 175

Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu

180 185 190

Tyr Ile Trp Gly Ile His Pro Ser Ser Asn Gln Glu Gln Thr Lys

195 200 205

Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser

210 215 220

Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Leu Val Arg Gly

225 230 235 240

Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp

245 250 255

Ile Leu Met Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr

260 265 270

Phe Lys Leu Asn Thr Gly Lys Ser Ser Val Met Arg Ser Asp Val Pro

275 280 285

Ile Asp Ile Cys Val Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Ser

290 295 300

Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys

305 310 315 320

Pro Lys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg

325 330 335

Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala Gly

340 345 350

Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe

355 360 365

Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser

370 375 380

Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile

385 390 395 400

Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu

405 410 415

Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys

420 425 430

Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn

435 440 445

Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu

450 455 460

Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly

465 470 475 480

Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Glu Ser Ile

485 490 495

Arg Thr Gly Thr Tyr Asp His Tyr Ile Tyr Lys Asp Glu Ala Leu Asn

500 505 510

Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp

515 520 525

Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Ile Cys Val

530 535 540

Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg

545 550 555 560

Cys Asn Ile Cys Ile

565

<210> 63

<211> 2280

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(2280)

<400> 63

atg gag aga ata aaa gaa ctg aga gat ctg atg tta caa tcc cgc acc 48

Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Leu Gln Ser Arg Thr

1 5 10 15

cgc gag ata cta aca aaa act act gtg gac cac atg gcc ata atc aag 96

Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys

20 25 30

aaa tac aca tca gga aga caa gag aag aac cct gca ctt agg atg aaa 144

Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys

35 40 45

tgg atg atg gca atg aaa tac cca att aca gca gat aag agg ata atg 192

Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met

50 55 60

gag atg att cct gag aga aat gaa cag gga caa acc ctt tgg agc aaa 240

Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys

65 70 75 80

acg aac gat gct ggc tca gac cgc gta atg gta tca cct ctg gca gtg 288

Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val

85 90 95

aca tgg tgg aat agg aat gga cca aca acg aac aca att cat tat cca 336

Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Asn Thr Ile His Tyr Pro

100 105 110

aaa gtc tac aaa act tat ttt gaa aag gtt gaa aga ttg aaa cac gga 384

Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly

115 120 125

acc ttt ggc ccc gtt cat ttt agg aat caa gtc aag ata aga cga aga 432

Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg

130 135 140

gtt gat gta aac cct ggt cac gcg gac ctc agt gct aaa gaa gca caa 480

Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln

145 150 155 160

gat gtg atc atg gaa gtt gtt ttc cca aat gaa gtg gga gcc aga att 528

Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile

165 170 175

cta aca tca gaa tca caa cta aca ata acc aaa gag aaa aag gaa gaa 576

Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu

180 185 190

ctt cag gac tgc aaa att gct ccc ttg atg gta gca tac atg cta gaa 624

Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu

195 200 205

aga gag ttg gtc cga aaa aca agg ttc ctc cca gta gta gta ggc gga aca 672

Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Val Gly Gly Thr

210 215 220

agc agt gta tac att gaa gtg ttg cat ctg act cag gga aca tgc tgg 720

Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp

225 230 235 240

gag caa atg tac acc cca gga gga aaa gtt aga aac gat gat att gat 768

Glu Gln Met Tyr Thr Pro Gly Gly Lys Val Arg Asn Asp Asp Ile Asp

245 250 255

caa agt tta att att gca gcc cgg aac ata gtg aga aga gca aca gta 816

Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val

260 265 270

tca gca gat cca cta gca tcc cta ctg gaa atg tgc cac agt aca cag 864

Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln

275 280 285

att ggt gga aca agg atg gta gac atc ctt aag cag aac cca aca gag 912

Ile Gly Gly Thr Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu

290 295 300

gaa caa gct gtg gat ata tgc aaa gca gca atg gga ttg aga att agc 960

Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser

305 310 315 320

tca tca ttc agc ttt ggt gga ttc acc ttc aaa agg aca agt gga tca 1008

Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser

325 330 335

tca gtc aag aga gaa gaa gaa atg ctt acg ggc aac ctt caa aca ttg 1056

Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu

340 345 350

aaa ata aga gtg cat gag ggc tat gaa gaa ttc aca atg gtc gga aga 1104

Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg

355 360 365

aga gca aca gcc att atc aga aag gca acc aga aga ttg att caa ttg 1152

Arg Ala Thr Ala Ile Ile Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu

370 375 380

ata gta agt ggg aga gat gaa caa tca att gct gaa gca ata att gta 1200

Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val

385 390 395 400

gcc atg gtg ttt tcg caa gaa gat tgc atg ata aaa gca gtt cga ggc 1248

Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly

405 410 415

gat ttg aac ttt gtt aat aga gca aat cag cgt ttg aac ccc atg cat 1296

Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His

420 425 430

caa ctc ttg agg cat ttc caa aaa gat gca aaa gtg ctt ttc caa aat 1344

Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn

435 440 445

tgg gga att gaa ccc atc gac aat gta atg ggg atg att gga ata ttg 1392

Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu

450 455 460

cct gac atg acc cca agc acc gag atg tca ttg aga gga gtg aga gtc 1440

Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val

465 470 475 480

agc aaa atg gga gtg gat gag tac tcc agc act gag aga gtg gtg gtg 1488

Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val

485 490 495

agc att gac cgt ttt tta aga gtt cgg gat caa agg gga aac ata cta 1536

Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Ile Leu

500 505 510

ctg tcc cct gaa gaa gtc agt gaa aca caa gga acg gaa aag ctg aca 1584

Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr

515 520 525

ata att tat tcg tca tca atg atg tgg gag att aat ggt ccc gaa tca 1632

Ile Ile Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser

530 535 540

gtg ttg gtc aat act tat caa tgg atc atc aga aac tgg gaa att gta 1680

Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Ile Val

545 550 555 560

aaa att cag tgg tca cag gac ccc aca atg tta tac aat aag ata gaa 1728

Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Ile Glu

565 570 575

ttt gaa cca ttc caa tcc ctg gtc cct agg gcc acc aga agc caa tac 1776

Phe Glu Pro Phe Gln Ser Leu Val Pro Arg Ala Thr Arg Ser Gln Tyr

580 585 590

agc ggt ttc gta aga acc ctg ttt cag caa atg cga gat gta ctt gga 1824

Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly

595 600 605

aca ttt gat act gct caa ata ata aaa ctc ctc cct ttt gcc gct gct 1872

Thr Phe Asp Thr Ala Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala

610 615 620

cct ccg gaa cag agt agg atg cag ttc tct tct ttg act gtt aat gta 1920

Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val

625 630 635 640

aga ggt tcg gga atg agg ata ctt gta aga ggc aat tcc cca gtg ttc 1968

Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe

645 650 655

aac tac aat aaa gtc act aaa agg ctc aca gtc ctc gga aag gat gca 2016

Asn Tyr Asn Lys Val Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala

660 665 670

ggt gcg ctt act gag gac cca gat gaa ggt acg gct gga gta gag tct 2064

Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser

675 680 685

gct gtt cta aga ggg ttt ctc att tta ggt aaa gaa aac aag aga tat 2112

Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asn Lys Arg Tyr

690 695 700

ggc cca gca cta agc atc aat gaa ctt agc aaa ctt gca aaa ggg gag 2160

Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu

705 710 715 720

aaa gcc aat gta cta att ggg caa ggg gac gta gtg ttg gta atg aaa 2208

Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys

725 730 735

cgg aaa cgt gac tct agc ata ctt act gac agc cag aca gcg acc aaa 2256

Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys

740 745 750

agg att cgg atg gcc atc aat tag 2280

Arg Ile Arg Met Ala Ile Asn

755

<210> 64

<211> 759

<212> PRT

<213> Influenza virus

<400> 64

Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Leu Gln Ser Arg Thr

1 5 10 15

Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys

20 25 30

Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys

35 40 45

Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met

50 55 60

Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys

65 70 75 80

Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val

85 90 95

Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Asn Thr Ile His Tyr Pro

100 105 110

Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly

115 120 125

Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg

130 135 140

Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln

145 150 155 160

Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile

165 170 175

Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu

180 185 190

Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu

195 200 205

Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Val Gly Gly Thr

210 215 220

Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp

225 230 235 240

Glu Gln Met Tyr Thr Pro Gly Gly Lys Val Arg Asn Asp Asp Ile Asp

245 250 255

Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val

260 265 270

Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln

275 280 285

Ile Gly Gly Thr Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu

290 295 300

Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser

305 310 315 320

Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser

325 330 335

Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu

340 345 350

Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg

355 360 365

Arg Ala Thr Ala Ile Ile Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu

370 375 380

Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val

385 390 395 400

Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly

405 410 415

Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His

420 425 430

Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn

435 440 445

Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu

450 455 460

Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val

465 470 475 480

Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val

485 490 495

Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Ile Leu

500 505 510

Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr

515 520 525

Ile Ile Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser

530 535 540

Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Ile Val

545 550 555 560

Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Ile Glu

565 570 575

Phe Glu Pro Phe Gln Ser Leu Val Pro Arg Ala Thr Arg Ser Gln Tyr

580 585 590

Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly

595 600 605

Thr Phe Asp Thr Ala Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala

610 615 620

Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val

625 630 635 640

Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe

645 650 655

Asn Tyr Asn Lys Val Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala

660 665 670

Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser

675 680 685

Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asn Lys Arg Tyr

690 695 700

Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu

705 710 715 720

Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys

725 730 735

Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys

740 745 750

Arg Ile Arg Met Ala Ile Asn

755

<210> 65

<211> 2274

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(2274)

<400> 65

atg gat gtc aat ccg act cta ctt ttc tta aag gtg cca gcg caa aat 48

Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn

1 5 10 15

gct ata agc aca aca ttc cct tat act gga gat cct ccc tac agt cat 96

Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His

20 25 30

gga aca ggg aca gga tac acc atg gat act gtc aac aga aca cac caa 144

Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln

35 40 45

tat tca gaa aaa ggg aaa tgg aca aca aac act gag att gga gca cca 192

Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro

50 55 60

caa ctt aat cca atc gat gga cca ctt cct gaa gac aat gaa cca agt 240

Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser

65 70 75 80

ggg tac gcc caa aca gat tgt gta ttg gaa gca atg gct ttc ctt gaa 288

Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu

85 90 95

gaa tcc cat ccc gga atc ttt gaa aat tcg tgt ctt gaa acg atg gag 336

Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu

100 105 110

gtg att cag cag aca aga gtg gac aaa cta aca caa ggc cga caa act 384

Val Ile Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr

115 120 125

tat gat tgg acc ttg aat agg aat caa cct gcc gca aca gca ctt gct 432

Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala

130 135 140

aat acg att gaa gta ttc aga tca aat ggt ctg acc tcc aat gaa tcg 480

Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr Ser Asn Glu Ser

145 150 155 160

ggg aga ttg atg gac ttc ctc aaa gat gtc atg gag tcc atg aac aag 528

Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys

165 170 175

gag gaa atg gaa ata aca aca cac ttc caa cgg aag aga aga gta aga 576

Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg

180 185 190

gac aac atg aca aag aga atg ata aca cag aga acc ata gga aag aaa 624

Asp Asn Met Thr Lys Arg Met Ile Thr Gln Arg Thr Ile Gly Lys Lys

195 200 205

aaa caa cga tta agc aga aag agc tat cta atc aga aca tta acc cta 672

Lys Gln Arg Leu Ser Arg Lys Ser Tyr Leu Ile Arg Thr Leu Thr Leu

210 215 220

aac aca atg acc aag gac gct gag aga ggg aaa ttg aaa cga cga gca 720

Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala

225 230 235 240

atc gct acc cca ggg atg cag ata aga gga ttt gta tat ttt gtt gaa 768

Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu

245 250 255

aca cta gct cga aga ata tgt gaa aag ctt gaa caa tca gga ttg cca 816

Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro

260 265 270

gtt ggc ggt aat gag aaa aag gcc aaa ctg gct aat gtc gtc aga aaa 864

Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys

275 280 285

atg atg act aat tcc caa gac act gaa ctc tcc ttc acc atc act ggg 912

Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly

290 295 300

gac aat acc aaa tgg aat gaa aat cag aac cca cgc ata ttc ctg gca 960

Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Ile Phe Leu Ala

305 310 315 320

atg atc aca tac ata act aga gat cag cca gaa tgg ttc aga aat gtt 1008

Met Ile Thr Tyr Ile Thr Arg Asp Gln Pro Glu Trp Phe Arg Asn Val

325 330 335

cta agc att gca ccg att atg ttc tca aat aaa atg gca aga ctg ggg 1056

Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly

340 345 350

aaa gga tat atg ttt gaa agc aaa agt atg aaa ttg aga act caa ata 1104

Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile

355 360 365

cca gca gaa atg cta gca agc att gac cta aaa tat ttc aat gat tca 1152

Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser

370 375 380

aca aaa aag aaa att gaa aag ata cga cca ctc ctg gtt gac ggg act 1200

Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Val Asp Gly Thr

385 390 395 400

gct tca ctg agt cct ggc atg atg atg gga atg ttc aac atg ttg agc 1248

Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser

405 410 415

act gtg ctg ggt gta tcc ata tta aac ctg ggc cag agg aaa tat aca 1296

Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Arg Lys Tyr Thr

420 425 430

aag acc aca tac tgg tgg gat ggt ctg caa tca tcc gat gac ttt gct 1344

Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala

435 440 445

ttg ata gtg aat gcg cct aat cat gaa gga ata caa gct gga gta gac 1392

Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp

450 455 460

aga ttc tat aga act tgc aaa ctg gtc ggg atc aac atg agc aaa aag 1440

Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys

465 470 475 480

aag tcc tac ata aat aga act gga aca ttc gaa ttc aca agc ttt ttc 1488

Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe

485 490 495

tac cgg tat ggt ttt gta gcc aat ttc agc atg gaa cta ccc agt ttt 1536

Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe

500 505 510

ggg gtt tcc gga ata aat gaa tct gca gac atg agc att gga gtg aca 1584

Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr

515 520 525

gtc atc aaa aac aac atg ata aat aat gat ctc ggt cct gcc acg gca 1632

Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala

530 535 540

caa atg gca ctc caa ctc ttc att aag gat tat cgg tac aca tac cgg 1680

Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg

545 550 555 560

tgc cat aga ggt gat acc cag ata caa acc aga aga tct ttt gag ttg 1728

Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu

565 570 575

aag aaa ctg tgg gaa cag act cga tca aag act ggt cta ctg gta tca 1776

Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Thr Gly Leu Leu Val Ser

580 585 590

gat ggg ggt cca aac cta tat aac atc aga aac cta cac atc ccg gaa 1824

Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu

595 600 605

gtc tgt tta aaa tgg gag cta atg gat gaa gat tat aag ggg agg cta 1872

Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Lys Gly Arg Leu

610 615 620

tgc aat cca ttg aat cct ttc gtt agt cac aaa gaa att gaa tca gtc 1920

Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val

625 630 635 640

aac agt gca gta gta atg cct gct cat ggc cct gcc aaa agc atg gag 1968

Asn Ser Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu

645 650 655

tat gat gct gtt gca aca aca cat tct tgg atc ccc aag agg aac cgg 2016

Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg

660 665 670

tcc ata ttg aac aca agc caa agg gga ata cta gaa gat gag cag atg 2064

Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met

675 680 685

tat cag aaa tgc tgc aac ctg ttt gaa aaa ttc ttc ccc agc agc tca 2112

Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser

690 695 700

tac aga aga cca gtc gga att tct agt atg gtt gag gcc atg gta tcc 2160

Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser

705 710 715 720

agg gcc cgc att gat gca cga att gac ttc gaa tct gga cgg ata aag 2208

Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys

725 730 735

aag gat gag ttc gct gag atc atg aag atc tgt tcc acc att gaa gag 2256

Lys Asp Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu

740 745 750

ctc aga cgg caa aaa tag 2274

Leu Arg Arg Gln Lys

755

<210> 66

<211> 757

<212> PRT

<213> Influenza virus

<400> 66

Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn

1 5 10 15

Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His

20 25 30

Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln

35 40 45

Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro

50 55 60

Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser

65 70 75 80

Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu

85 90 95

Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu

100 105 110

Val Ile Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr

115 120 125

Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala

130 135 140

Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr Ser Asn Glu Ser

145 150 155 160

Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys

165 170 175

Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg

180 185 190

Asp Asn Met Thr Lys Arg Met Ile Thr Gln Arg Thr Ile Gly Lys Lys

195 200 205

Lys Gln Arg Leu Ser Arg Lys Ser Tyr Leu Ile Arg Thr Leu Thr Leu

210 215 220

Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala

225 230 235 240

Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu

245 250 255

Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro

260 265 270

Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys

275 280 285

Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly

290 295 300

Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Ile Phe Leu Ala

305 310 315 320

Met Ile Thr Tyr Ile Thr Arg Asp Gln Pro Glu Trp Phe Arg Asn Val

325 330 335

Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly

340 345 350

Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile

355 360 365

Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser

370 375 380

Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Val Asp Gly Thr

385 390 395 400

Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser

405 410 415

Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Arg Lys Tyr Thr

420 425 430

Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala

435 440 445

Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp

450 455 460

Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys

465 470 475 480

Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe

485 490 495

Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe

500 505 510

Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr

515 520 525

Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala

530 535 540

Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg

545 550 555 560

Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu

565 570 575

Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Thr Gly Leu Leu Val Ser

580 585 590

Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu

595 600 605

Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Lys Gly Arg Leu

610 615 620

Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val

625 630 635 640

Asn Ser Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu

645 650 655

Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg

660 665 670

Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met

675 680 685

Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser

690 695 700

Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser

705 710 715 720

Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys

725 730 735

Lys Asp Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu

740 745 750

Leu Arg Arg Gln Lys

755

<210> 67

<211> 2151

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(2151)

<400> 67

atg gaa gac ttt gtg cga cag tgc ttc aat cca atg atc gtc gag ctt 48

Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu

1 5 10 15

gcg gaa aag gca atg aaa gaa tat gga gag aac ccg aaa atc gaa aca 96

Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asn Pro Lys Ile Glu Thr

20 25 30

aac aaa ttt gca gca ata tgc act cac ttg gaa gtc tgc ttc atg tac 144

Asn Lys Phe Ala Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr

35 40 45

tcg gat ttc cac ttt ata aat gaa ctg ggt gag tca gtg gtc ata gag 192

Ser Asp Phe His Phe Ile Asn Glu Leu Gly Glu Ser Val Val Ile Glu

50 55 60

tct ggt gac cca aat gct ctt ttg aaa cac aga ttt gaa atc att gag 240

Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu

65 70 75 80

ggg aga gat cga aca atg gca tgg aca gta gta aac agc atc tgc aac 288

Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn

85 90 95

acc aca aga gct gaa aaa cct aaa ttt ctt cca gat tta tac gac tat 336

Thr Thr Arg Ala Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr

100 105 110

aaa gag aac aga ttt gtt gaa att ggt gtg aca agg aga gaa gtt cac 384

Lys Glu Asn Arg Phe Val Glu Ile Gly Val Thr Arg Arg Glu Val His

115 120 125

ata tac tac ctg gag aag gcc aac aaa ata aag tct gag aaa aca cat 432

Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Lys Thr His

130 135 140

atc cac att ttc tca ttt aca gga gaa gaa atg gct aca aaa gcg gac 480

Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys Ala Asp

145 150 155 160

tat act ctt gat gaa gag agt aga gcc agg atc aag acc aga cta ttc 528

Tyr Thr Leu Asp Glu Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe

165 170 175

act ata aga caa gaa atg gcc agt aga ggc ctc tgg gat tcc ttt cgt 576

Thr Ile Arg Gln Glu Met Ala Ser Arg Gly Leu Trp Asp Ser Phe Arg

180 185 190

cag tcc gag aga ggc gaa gag aca att gaa gaa aga ttt gaa atc aca 624

Gln Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr

195 200 205

ggg acg atg cgc aag ctt gcc aat tac agt ctc cca ccg aac ttc tcc 672

Gly Thr Met Arg Lys Leu Ala Asn Tyr Ser Leu Pro Pro Asn Phe Ser

210 215 220

agc ctt gaa aat ttt aga gtc tat ata gat gga ttc gaa ccg aac ggc 720

Ser Leu Glu Asn Phe Arg Val Tyr Ile Asp Gly Phe Glu Pro Asn Gly

225 230 235 240

tgc att gag agt aag ctt tct caa atg tcc aaa gaa gta aat gcc aaa 768

Cys Ile Glu Ser Lys Leu Ser Gln Met Ser Lys Glu Val Asn Ala Lys

245 250 255

ata gaa cca ttt tca aag aca aca ccc cga cca ctc aaa atg cca ggt 816

Ile Glu Pro Phe Ser Lys Thr Thr Pro Arg Pro Leu Lys Met Pro Gly

260 265 270

ggt cca ccc tgc cat cag cga tcc aaa ttc ttg cta atg gat gct ctg 864

Gly Pro Pro Cys His Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu

275 280 285

aaa ctg agc att gag gac cca agt cac gag gga gag ggg ata cca cta 912

Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu

290 295 300

tat gat gca atc aaa tgc atg aaa act ttc ttt gga tgg aaa gag ccc 960

Tyr Asp Ala Ile Lys Cys Met Lys Thr Phe Phe Gly Trp Lys Glu Pro

305 310 315 320

agt att gtt aaa cca cat aaa aag ggt ata aac ccg aac tat ctc caa 1008

Ser Ile Val Lys Pro His Lys Lys Gly Ile Asn Pro Asn Tyr Leu Gln

325 330 335

act tgg aag caa gta tta gaa gaa ata caa gac ctt gag aac gaa gaa 1056

Thr Trp Lys Gln Val Leu Glu Glu Ile Gln Asp Leu Glu Asn Glu Glu

340 345 350

agg acc ccc aag acc aag aat atg aaa aaa aca agc caa ttg aaa tgg 1104

Arg Thr Pro Lys Thr Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp

355 360 365

gca cta ggt gaa aat atg gca cca gag aaa gtg gat ttt gag gat tgt 1152

Ala Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Glu Asp Cys

370 375 380

aaa gac atc aat gat tta aaa caa tat gac agt gat gag cca gaa gca 1200

Lys Asp Ile Asn Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Ala

385 390 395 400

agg tct ctt gca agt tgg att caa agt gag ttc aac aag gct tgt gag 1248

Arg Ser Leu Ala Ser Trp Ile Gln Ser Glu Phe Asn Lys Ala Cys Glu

405 410 415

ctg aca gat tca agc tgg ata gag ctc gat gaa att ggg gag gat gtc 1296

Leu Thr Asp Ser Ser Ser Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val

420 425 430

gcc cca ata gaa tac att gcg agc atg agg aga aat tat ttt act gct 1344

Ala Pro Ile Glu Tyr Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala

435 440 445

gag att tcc cat tgt aga gca aca gaa tat ata atg aaa gga gtg tac 1392

Glu Ile Ser His Cys Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr

450 455 460

atc aac act gct cta ctc aat gca tcc tgt gct gcg atg gat gaa ttt 1440

Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Glu Phe

465 470 475 480

caa tta att ccg atg ata agt aaa tgc agg acc aaa gaa ggg aga agg 1488

Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg

485 490 495

aaa aca aat tta tat gga ttc ata ata aag gga agg tcc cat tta aga 1536

Lys Thr Asn Leu Tyr Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg

500 505 510

aat gat act gac gtg gtg aac ttt gta agt atg gaa ttt tct ctc act 1584

Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr

515 520 525

gat cca aga ttt gag cca cac aaa tgg gaa aaa tac tgc gtt cta gaa 1632

Asp Pro Arg Phe Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu

530 535 540

att gga gac atg ctt tta aga act gct gta ggt caa gtg tca aga ccc 1680

Ile Gly Asp Met Leu Leu Arg Thr Ala Val Gly Gln Val Ser Arg Pro

545 550 555 560

atg ttt ttg tat gta agg aca aat gga acc tct aaa att aaa atg aaa 1728

Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys

565 570 575

tgg gga atg gaa atg agg cgc tgc ctc ctt cag tct ctg caa cag att 1776

Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile

580 585 590

gaa agc atg atc gaa gct gag tcc tca gtc aaa gaa aag gac atg acc 1824

Glu Ser Met Ile Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr

595 600 605

aaa gaa ttt ttt gag aac aaa tca gag aca tgg cct ata gga gag tcc 1872

Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser

610 615 620

ccc aaa gga gtg gaa gag ggc tca atc ggg aag gtt tgc agg acc tta 1920

Pro Lys Gly Val Glu Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu

625 630 635 640

tta gca aaa tct gtg ttt aac agt tta tat gca tct cca caa ctg gaa 1968

Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu

645 650 655

gga ttt tca gct gaa tct agg aaa tta ctt ctc att gtt cag gct ctt 2016

Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu

660 665 670

aga gat gac ctg gaa cct gga acc ttt gat att ggg ggg tta tat gaa 2064

Arg Asp Asp Leu Glu Pro Gly Thr Phe Asp Ile Gly Gly Leu Tyr Glu

675 680 685

tca att gag gag tgc ctg att aat gat ccc tgg gtt ttg ctt aat gca 2112

Ser Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala

690 695 700

tct tgg ttc aac tcc ttc ctc aca cat gca ctg aag tag 2151

Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Lys

705 710 715

<210> 68

<211> 716

<212> PRT

<213> Influenza virus

<400> 68

Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu

1 5 10 15

Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asn Pro Lys Ile Glu Thr

20 25 30

Asn Lys Phe Ala Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr

35 40 45

Ser Asp Phe His Phe Ile Asn Glu Leu Gly Glu Ser Val Val Ile Glu

50 55 60

Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu

65 70 75 80

Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn

85 90 95

Thr Thr Arg Ala Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr

100 105 110

Lys Glu Asn Arg Phe Val Glu Ile Gly Val Thr Arg Arg Glu Val His

115 120 125

Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Lys Thr His

130 135 140

Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys Ala Asp

145 150 155 160

Tyr Thr Leu Asp Glu Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe

165 170 175

Thr Ile Arg Gln Glu Met Ala Ser Arg Gly Leu Trp Asp Ser Phe Arg

180 185 190

Gln Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr

195 200 205

Gly Thr Met Arg Lys Leu Ala Asn Tyr Ser Leu Pro Pro Asn Phe Ser

210 215 220

Ser Leu Glu Asn Phe Arg Val Tyr Ile Asp Gly Phe Glu Pro Asn Gly

225 230 235 240

Cys Ile Glu Ser Lys Leu Ser Gln Met Ser Lys Glu Val Asn Ala Lys

245 250 255

Ile Glu Pro Phe Ser Lys Thr Thr Pro Arg Pro Leu Lys Met Pro Gly

260 265 270

Gly Pro Pro Cys His Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu

275 280 285

Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu

290 295 300

Tyr Asp Ala Ile Lys Cys Met Lys Thr Phe Phe Gly Trp Lys Glu Pro

305 310 315 320

Ser Ile Val Lys Pro His Lys Lys Gly Ile Asn Pro Asn Tyr Leu Gln

325 330 335

Thr Trp Lys Gln Val Leu Glu Glu Ile Gln Asp Leu Glu Asn Glu Glu

340 345 350

Arg Thr Pro Lys Thr Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp

355 360 365

Ala Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Glu Asp Cys

370 375 380

Lys Asp Ile Asn Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Ala

385 390 395 400

Arg Ser Leu Ala Ser Trp Ile Gln Ser Glu Phe Asn Lys Ala Cys Glu

405 410 415

Leu Thr Asp Ser Ser Ser Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val

420 425 430

Ala Pro Ile Glu Tyr Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala

435 440 445

Glu Ile Ser His Cys Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr

450 455 460

Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Glu Phe

465 470 475 480

Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg

485 490 495

Lys Thr Asn Leu Tyr Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg

500 505 510

Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr

515 520 525

Asp Pro Arg Phe Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu

530 535 540

Ile Gly Asp Met Leu Leu Arg Thr Ala Val Gly Gln Val Ser Arg Pro

545 550 555 560

Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys

565 570 575

Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile

580 585 590

Glu Ser Met Ile Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr

595 600 605

Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser

610 615 620

Pro Lys Gly Val Glu Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu

625 630 635 640

Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu

645 650 655

Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu

660 665 670

Arg Asp Asp Leu Glu Pro Gly Thr Phe Asp Ile Gly Gly Leu Tyr Glu

675 680 685

Ser Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala

690 695 700

Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Lys

705 710 715

<210> 69

<211> 844

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(690)

<400> 69

atg gat tcc aac act gtg tca agc ttt cag gta gac tgt ttt ctt tgg 48

Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

cat gtc cgt aaa cga ttc gca gac caa gaa ctg ggt gat gcc cca ttc 96

His Val Arg Lys Arg Phe Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

ctt gac cgg ctt cgc cga gac cag aag tcc cta agg gga aga ggt agc 144

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

act ctt ggt ctg gac atc gaa aca gcc act cat gca gga aag cag ata 192

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr His Ala Gly Lys Gln Ile

50 55 60

gtg gag cag att ctg gaa aag gaa tca gat gag gca ctt aaa atg acc 240

Val Glu Gln Ile Leu Glu Lys Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

att gcc tct gtt cct gct tca cgc tac tta act gac atg act ctt gat 288

Ile Ala Ser Val Pro Ala Ser Arg Tyr Leu Thr Asp Met Thr Leu Asp

85 90 95

gag atg tca aga gac tgg ttc atg ctc atg ccc aag caa aaa gta aca 336

Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Thr

100 105 110

ggc tcc cta tgt ata aga atg gac cag gca atc atg gat aag aac atc 384

Gly Ser Leu Cys Ile Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile

115 120 125

ata ctt aaa gca aac ttt agt gtg att ttc gaa agg ctg gaa aca cta 432

Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Glu Arg Leu Glu Thr Leu

130 135 140

ata cta ctt aga gcc ttc acc gaa gaa gga gca gtc gtt ggc gaa att 480

Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile

145 150 155 160

tca cca tta cct tct ctt cca gga cat act aat gag gat gtc aaa aat 528

Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn

165 170 175

gca att ggg gtc ctc atc gga gga ctt aaa tgg aat gat aat acg gtt 576

Ala Ile Gly Val Leu Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val

180 185 190

aga atc tct gaa act cta cag aga ttc gct tgg aga agc agt cat gaa 624

Arg Ile Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser His Glu

195 200 205

aat ggg aga cct tca ttc cct tca aaa cag aaa cga aaa atg gag aga 672

Asn Gly Arg Pro Ser Phe Pro Ser Lys Gln Lys Arg Lys Met Glu Arg

210 215 220

aca att aag cca gaa att tgaagaaata agatggttga ttgaagaagt 720

Thr Ile Lys Pro Glu Ile

225 230

gcgacataga ttgaaaaata cagaaaatag ttttgaacaa ataacattta tgcaagcctt 780

acaactattg cttgaagtag aacaagagat aagaactttc tcgtttcagc ttatttaatg 840

ataa 844

<210> 70

<211> 230

<212> PRT

<213> Influenza virus

<400> 70

Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

His Val Arg Lys Arg Phe Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr His Ala Gly Lys Gln Ile

50 55 60

Val Glu Gln Ile Leu Glu Lys Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

Ile Ala Ser Val Pro Ala Ser Arg Tyr Leu Thr Asp Met Thr Leu Asp

85 90 95

Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Thr

100 105 110

Gly Ser Leu Cys Ile Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile

115 120 125

Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Glu Arg Leu Glu Thr Leu

130 135 140

Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile

145 150 155 160

Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn

165 170 175

Ala Ile Gly Val Leu Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val

180 185 190

Arg Ile Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser His Glu

195 200 205

Asn Gly Arg Pro Ser Phe Pro Ser Lys Gln Lys Arg Lys Met Glu Arg

210 215 220

Thr Ile Lys Pro Glu Ile

225 230

<210> 71

<211> 1497

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(1497)

<400> 71

atg gcg tct caa ggc acc aaa cga tcc tat gaa cag atg gaa act gat 48

Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp

1 5 10 15

ggg gaa cgc cag aat gca act gaa atc aga gca tct gtc gga agg atg 96

Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Arg Met

20 25 30

gtg gga gga atc gga cgg ttt tat gtc cag atg tgt act gag ctt aaa 144

Val Gly Gly Ile Gly Arg Phe Tyr Val Gln Met Cys Thr Glu Leu Lys

35 40 45

cta aac gac cat gaa ggg cgg ctg att cag aac agc ata aca ata gaa 192

Leu Asn Asp His Glu Gly Arg Leu Ile Gln Asn Ser Ile Thr Ile Glu

50 55 60

agg atg gtg ctt tcg gca ttc gac gaa aga aga aac aag tat ctc gag 240

Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu

65 70 75 80

gag cat ccc agt gct ggg aaa gac cct aag aaa acg gga ggc ccg ata 288

Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile

85 90 95

tac aga aga aaa gat ggg aaa tgg atg agg gaa ctc atc ctc cat gat 336

Tyr Arg Arg Lys Asp Gly Lys Trp Met Arg Glu Leu Ile Leu His Asp

100 105 110

aaa gaa gaa atc atg aga atc tgg cgt cag gcc aac aat ggt gaa gac 384

Lys Glu Glu Ile Met Arg Ile Trp Arg Gln Ala Asn Asn Gly Glu Asp

115 120 125

gct act gct ggt ctt act cat atg atg atc tgg cac tcc aat ctc aat 432

Ala Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn

130 135 140

gac acc aca tac caa aga aca agg gct ctt gtt cgg act ggg atg gat 480

Asp Thr Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp

145 150 155 160

ccc aga atg tgc tct ctg atg caa ggc tca acc ctc cca cgg aga tct 528

Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser

165 170 175

gga gcc gct ggt gct gca gta aaa ggc gtt gga aca atg gta atg gaa 576

Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu

180 185 190

ctc atc aga atg atc aag cgc gga ata aat gat cgg aat ttc tgg aga 624

Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg

195 200 205

ggt gaa aat ggt cga aga acc aga att gct tat gaa aga atg tgc aat 672

Gly Glu Asn Gly Arg Arg Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn

210 215 220

atc ctc aaa ggg aaa ttt cag aca gca gca caa cgg gct atg atg gac 720

Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp

225 230 235 240

cag gtg agg gaa ggc cgc aat cct gga aac gct gag att gag gat ctc 768

Gln Val Arg Glu Gly Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu

245 250 255

att ttc ttg gca cga tca gca ctt att ttg aga gga tca gta gcc cat 816

Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His

260 265 270

aaa tca tgc cta cct gcc tgt gtt tat ggc ctt gca cta acc agt ggg 864

Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala Leu Thr Ser Gly

275 280 285

tat gac ttt gag aag gaa gga tac tct ctg gtt gga att gat cct ttc 912

Tyr Asp Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe

290 295 300

aaa cta ctc cag aac agt caa att ttc agt cta atc aga cca aaa gaa 960

Lys Leu Leu Gln Asn Ser Gln Ile Phe Ser Leu Ile Arg Pro Lys Glu

305 310 315 320

aac cca gca cac aaa agc cag ttg gtg tgg atg gca tgc cat tct gca 1008

Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala

325 330 335

gca ttt gag gat ctg aga gtt tta aat ttc att aga gga acc aaa gta 1056

Ala Phe Glu Asp Leu Arg Val Leu Asn Phe Ile Arg Gly Thr Lys Val

340 345 350

atc cca aga gga cag tta aca acc aga gga gtt caa att gct tca aat 1104

Ile Pro Arg Gly Gln Leu Thr Thr Arg Gly Val Gln Ile Ala Ser Asn

355 360 365

gaa aac atg gag aca ata aat tct agc aca ctt gaa ctg aga agc aaa 1152

Glu Asn Met Glu Thr Ile Asn Ser Ser Thr Leu Glu Leu Arg Ser Lys

370 375 380

tat tgg gca ata agg acc aga agc gga gga aac acc agt caa cag aga 1200

Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Ser Gln Gln Arg

385 390 395 400

gca tct gca gga cag ata agt gtg caa cct act ttc tca gta cag aga 1248

Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg

405 410 415

aat ctt ccc ttt gag aga gca acc att atg gct gca ttc act ggt aac 1296

Asn Leu Pro Phe Glu Arg Ala Thr Ile Met Ala Ala Phe Thr Gly Asn

420 425 430

act gaa gga agg act tcc gac atg aga acg gaa atc ata agg atg atg 1344

Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met

435 440 445

gaa aat gcc aaa tca gaa gat gtg tct ttc cag ggg cgg gga gtc ttc 1392

Glu Asn Ala Lys Ser Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe

450 455 460

gag ctc tcg gac gaa aag gca acg aac ccg atc gtg cct tcc ttt gac 1440

Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp

465 470 475 480

atg agc aat gaa ggg tct tat ttc ttc gga gac aat gct gag gag ttt 1488

Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Phe

485 490 495

gac agt taa 1497

Asp Ser

<210> 72

<211> 498

<212> PRT

<213> Influenza virus

<400> 72

Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp

1 5 10 15

Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Arg Met

20 25 30

Val Gly Gly Ile Gly Arg Phe Tyr Val Gln Met Cys Thr Glu Leu Lys

35 40 45

Leu Asn Asp His Glu Gly Arg Leu Ile Gln Asn Ser Ile Thr Ile Glu

50 55 60

Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu

65 70 75 80

Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile

85 90 95

Tyr Arg Arg Lys Asp Gly Lys Trp Met Arg Glu Leu Ile Leu His Asp

100 105 110

Lys Glu Glu Ile Met Arg Ile Trp Arg Gln Ala Asn Asn Gly Glu Asp

115 120 125

Ala Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn

130 135 140

Asp Thr Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp

145 150 155 160

Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser

165 170 175

Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu

180 185 190

Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg

195 200 205

Gly Glu Asn Gly Arg Arg Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn

210 215 220

Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp

225 230 235 240

Gln Val Arg Glu Gly Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu

245 250 255

Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His

260 265 270

Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala Leu Thr Ser Gly

275 280 285

Tyr Asp Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe

290 295 300

Lys Leu Leu Gln Asn Ser Gln Ile Phe Ser Leu Ile Arg Pro Lys Glu

305 310 315 320

Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala

325 330 335

Ala Phe Glu Asp Leu Arg Val Leu Asn Phe Ile Arg Gly Thr Lys Val

340 345 350

Ile Pro Arg Gly Gln Leu Thr Thr Arg Gly Val Gln Ile Ala Ser Asn

355 360 365

Glu Asn Met Glu Thr Ile Asn Ser Ser Thr Leu Glu Leu Arg Ser Lys

370 375 380

Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Ser Gln Gln Arg

385 390 395 400

Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg

405 410 415

Asn Leu Pro Phe Glu Arg Ala Thr Ile Met Ala Ala Phe Thr Gly Asn

420 425 430

Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met

435 440 445

Glu Asn Ala Lys Ser Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe

450 455 460

Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp

465 470 475 480

Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Phe

485 490 495

Asp Ser

<210> 73

<211> 1413

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(1413)

<400> 73

atg aat cca aat caa aag ata ata gca att gga ttt gca tca ttg ggg 48

Met Asn Pro Asn Gln Lys Ile Ile Ala Ile Gly Phe Ala Ser Leu Gly

1 5 10 15

ata tta atc att aat gtc att ctc cat gta gtc agc att ata gta aca 96

Ile Leu Ile Ile Asn Val Ile Leu His Val Val Ser Ile Ile Val Thr

20 25 30

gta ctg gtc ctc aat aac aat aga aca gat ctg aac tgc aaa ggg acg 144

Val Leu Val Leu Asn Asn Asn Arg Thr Asp Leu Asn Cys Lys Gly Thr

35 40 45

atc ata aga gaa tac aat gaa aca gta aga gta gaa aaa ctt act caa 192

Ile Ile Arg Glu Tyr Asn Glu Thr Val Arg Val Glu Lys Leu Thr Gln

50 55 60

tgg tat aat acc agt aca att aag tac ata gag aga cct tca aat gaa 240

Trp Tyr Asn Thr Ser Thr Ile Lys Tyr Ile Glu Arg Pro Ser Asn Glu

65 70 75 80

tac tac atg aat aac act gaa cca ctt tgt gag gcc caa ggc ttt gca 288

Tyr Tyr Met Asn Asn Thr Glu Pro Leu Cys Glu Ala Gln Gly Phe Ala

85 90 95

cca ttt tcc aaa gat aat gga ata cga att ggg tcg aga ggc cat gtt 336

Pro Phe Ser Lys Asp Asn Gly Ile Arg Ile Gly Ser Arg Gly His Val

100 105 110

ttt gtg ata aga gaa cct ttt gta tca tgt tcg ccc tca gaa tgt aga 384

Phe Val Ile Arg Glu Pro Phe Val Ser Cys Ser Pro Ser Glu Cys Arg

115 120 125

acc ttt ttc ctc aca cag ggc tca tta ctc aat gac aaa cat tct aac 432

Thr Phe Phe Leu Thr Gln Gly Ser Leu Leu Asn Asp Lys His Ser Asn

130 135 140

ggc aca ata aag gat cga agt ccg tat agg act ttg atg agt gtc aaa 480

Gly Thr Ile Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser Val Lys

145 150 155 160

ata ggg caa tca cct aat gta tat caa gct agg ttt gaa tcg gtg gca 528

Ile Gly Gln Ser Pro Asn Val Tyr Gln Ala Arg Phe Glu Ser Val Ala

165 170 175

tgg tca gca aca gca tgc cat gat gga aaa aaa tgg atg aca gtt gga 576

Trp Ser Ala Thr Ala Cys His Asp Gly Lys Lys Trp Met Thr Val Gly

180 185 190

gtc aca ggg ccc gac aat caa gca att gca gta gtg aac tat gga ggt 624

Val Thr Gly Pro Asp Asn Gln Ala Ile Ala Val Val Asn Tyr Gly Gly

195 200 205

gtt ccg gtt gat att att aat tca tgg gca ggg gat att tta aga acc 672

Val Pro Val Asp Ile Ile Asn Ser Trp Ala Gly Asp Ile Leu Arg Thr

210 215 220

caa gaa tca tca tgc acc tgc att aaa gga gac tgt tat tgg gta atg 720

Gln Glu Ser Ser Cys Thr Cys Ile Lys Gly Asp Cys Tyr Trp Val Met

225 230 235 240

act gat gga ccg gca aat agg caa gct aaa tat agg ata ttc aaa gca 768

Thr Asp Gly Pro Ala Asn Arg Gln Ala Lys Tyr Arg Ile Phe Lys Ala

245 250 255

aaa gat gga aga gta att gga caa act gat ata agt ttc aat ggg gga 816

Lys Asp Gly Arg Val Ile Gly Gln Thr Asp Ile Ser Phe Asn Gly Gly

260 265 270

cac ata gag gag tgt tct tgt tac ccc aat gaa ggg aag gtg gaa tgc 864

His Ile Glu Glu Cys Ser Cys Tyr Pro Asn Glu Gly Lys Val Glu Cys

275 280 285

ata tgc agg gac aat tgg act gga aca aat aga cca att ctg gta ata 912

Ile Cys Arg Asp Asn Trp Thr Gly Thr Asn Arg Pro Ile Leu Val Ile

290 295 300

tct tct gat cta tcg tac aca gtt gga tat ttg tgt gct ggc att ccc 960

Ser Ser Asp Leu Ser Tyr Thr Val Gly Tyr Leu Cys Ala Gly Ile Pro

305 310 315 320

act gac act cct agg gga gag gat agt caa ttc aca ggc tca tgt aca 1008

Thr Asp Thr Pro Arg Gly Glu Asp Ser Gln Phe Thr Gly Ser Cys Thr

325 330 335

agt cct ttg gga aat aaa gga tac ggt gta aaa ggc ttc ggg ttt cga 1056

Ser Pro Leu Gly Asn Lys Gly Tyr Gly Val Lys Gly Phe Gly Phe Arg

340 345 350

caa gga act gac gta tgg gcc gga agg aca att agt agg act tca aga 1104

Gln Gly Thr Asp Val Trp Ala Gly Arg Thr Ile Ser Arg Thr Ser Arg

355 360 365

tca gga ttc gaa ata ata aaa atc agg aat ggt tgg aca cag aac agt 1152

Ser Gly Phe Glu Ile Ile Lys Ile Arg Asn Gly Trp Thr Gln Asn Ser

370 375 380

aag gac caa atc agg agg caa gtg att atc gat gac cca aat tgg tca 1200

Lys Asp Gln Ile Arg Arg Gln Val Ile Ile Asp Asp Pro Asn Trp Ser

385 390 395 400

gga tat agc ggt tct ttc aca ttg ccg gtt gaa ctg aca aaa aag gga 1248

Gly Tyr Ser Gly Ser Phe Thr Leu Pro Val Glu Leu Thr Lys Lys Gly

405 410 415

tgt ttg gtc ccc tgt ttc tgg gtt gaa atg att aga ggt aaa cct gaa 1296

Cys Leu Val Pro Cys Phe Trp Val Glu Met Ile Arg Gly Lys Pro Glu

420 425 430

gaa aca aca ata tgg acc tct agc agc tcc att gtg atg tgt gga gta 1344

Glu Thr Thr Ile Trp Thr Ser Ser Ser Ser Ile Val Met Cys Gly Val

435 440 445

gat cat aaa att gcc agt tgg tca tgg cac gat gga gct att ctt ccc 1392

Asp His Lys Ile Ala Ser Trp Ser Trp His Asp Gly Ala Ile Leu Pro

450 455 460

ttt gac atc gat aag atg taa 1413

Phe Asp Ile Asp Lys Met

465 470

<210> 74

<211> 470

<212> PRT

<213> Influenza virus

<400> 74

Met Asn Pro Asn Gln Lys Ile Ile Ala Ile Gly Phe Ala Ser Leu Gly

1 5 10 15

Ile Leu Ile Ile Asn Val Ile Leu His Val Val Ser Ile Ile Val Thr

20 25 30

Val Leu Val Leu Asn Asn Asn Arg Thr Asp Leu Asn Cys Lys Gly Thr

35 40 45

Ile Ile Arg Glu Tyr Asn Glu Thr Val Arg Val Glu Lys Leu Thr Gln

50 55 60

Trp Tyr Asn Thr Ser Thr Ile Lys Tyr Ile Glu Arg Pro Ser Asn Glu

65 70 75 80

Tyr Tyr Met Asn Asn Thr Glu Pro Leu Cys Glu Ala Gln Gly Phe Ala

85 90 95

Pro Phe Ser Lys Asp Asn Gly Ile Arg Ile Gly Ser Arg Gly His Val

100 105 110

Phe Val Ile Arg Glu Pro Phe Val Ser Cys Ser Pro Ser Glu Cys Arg

115 120 125

Thr Phe Phe Leu Thr Gln Gly Ser Leu Leu Asn Asp Lys His Ser Asn

130 135 140

Gly Thr Ile Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser Val Lys

145 150 155 160

Ile Gly Gln Ser Pro Asn Val Tyr Gln Ala Arg Phe Glu Ser Val Ala

165 170 175

Trp Ser Ala Thr Ala Cys His Asp Gly Lys Lys Trp Met Thr Val Gly

180 185 190

Val Thr Gly Pro Asp Asn Gln Ala Ile Ala Val Val Asn Tyr Gly Gly

195 200 205

Val Pro Val Asp Ile Ile Asn Ser Trp Ala Gly Asp Ile Leu Arg Thr

210 215 220

Gln Glu Ser Ser Cys Thr Cys Ile Lys Gly Asp Cys Tyr Trp Val Met

225 230 235 240

Thr Asp Gly Pro Ala Asn Arg Gln Ala Lys Tyr Arg Ile Phe Lys Ala

245 250 255

Lys Asp Gly Arg Val Ile Gly Gln Thr Asp Ile Ser Phe Asn Gly Gly

260 265 270

His Ile Glu Glu Cys Ser Cys Tyr Pro Asn Glu Gly Lys Val Glu Cys

275 280 285

Ile Cys Arg Asp Asn Trp Thr Gly Thr Asn Arg Pro Ile Leu Val Ile

290 295 300

Ser Ser Asp Leu Ser Tyr Thr Val Gly Tyr Leu Cys Ala Gly Ile Pro

305 310 315 320

Thr Asp Thr Pro Arg Gly Glu Asp Ser Gln Phe Thr Gly Ser Cys Thr

325 330 335

Ser Pro Leu Gly Asn Lys Gly Tyr Gly Val Lys Gly Phe Gly Phe Arg

340 345 350

Gln Gly Thr Asp Val Trp Ala Gly Arg Thr Ile Ser Arg Thr Ser Arg

355 360 365

Ser Gly Phe Glu Ile Ile Lys Ile Arg Asn Gly Trp Thr Gln Asn Ser

370 375 380

Lys Asp Gln Ile Arg Arg Gln Val Ile Ile Asp Asp Pro Asn Trp Ser

385 390 395 400

Gly Tyr Ser Gly Ser Phe Thr Leu Pro Val Glu Leu Thr Lys Lys Gly

405 410 415

Cys Leu Val Pro Cys Phe Trp Val Glu Met Ile Arg Gly Lys Pro Glu

420 425 430

Glu Thr Thr Ile Trp Thr Ser Ser Ser Ser Ile Val Met Cys Gly Val

435 440 445

Asp His Lys Ile Ala Ser Trp Ser Trp His Asp Gly Ala Ile Leu Pro

450 455 460

Phe Asp Ile Asp Lys Met

465 470

<210> 75

<211> 981

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(756)

<400> 75

atg agt ctt cta acc gag gtc gaa acg tac gtt ctc tct atc gta cca 48

Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro

1 5 10 15

tca ggc ccc ctc aaa gcc gag atc gcg cag aga ctt gaa gat gtc ttt 96

Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe

20 25 30

gcg gga aag aac acc gat ctt gag gca ctc atg gaa tgg cta aag aca 144

Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr

35 40 45

aga cca atc ctg tca cct ctg act aaa ggg att tta gga ttt gta ttc 192

Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe

50 55 60

acg ctc acc gtg ccc agt gag cga gga ctg cag cgt aga cgc ttt gtc 240

Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val

65 70 75 80

caa aat gcc ctt agt gga aac gga gat cca aac aac atg gac aga gca 288

Gln Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala

85 90 95

gta aaa ctg tac agg aag ctt aaa aga gaa ata aca ttc cat ggg gca 336

Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala

100 105 110

aaa gag gtg gca ctc agc tat tcc act ggt gca cta gcc agc tgc atg 384

Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met

115 120 125

gga ctc ata tac aac aga atg gga act gtt aca acc gaa gtg gca ttt 432

Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe

130 135 140

ggc ctg gta tgc gcc aca tgt gaa cag att gct gat tcc cag cat cga 480

Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg

145 150 155 160

tct cac agg cag atg gtg aca aca acc aac cca tta atc aga cat gaa 528

Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu

165 170 175

aac aga atg gta tta gcc agt acc acg gct aaa gcc atg gaa cag atg 576

Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met

180 185 190

gca gga tcg agt gag cag gca gca gag gcc atg gag gtt gct agt agg 624

Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Arg

195 200 205

gct agg cag atg gta cag gca atg aga acc att ggg acc cac cct agc 672

Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser

210 215 220

tcc agt gcc ggt ttg aaa gat gat ctc ctt gaa aat tta cag gcc tac 720

Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr

225 230 235 240

cag aaa cgg atg gga gtg caa atg cag cga ttc aag tgatcctctc 766

Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys

245 250

gtcattgcag caagtatcat tgggatcttg cacttgatat tgtggattct tgatcgtctt 826

ttcttcaaat tcatttatcg tcgccttaaa tacgggttga aaagaggggcc ttctacggaa 886

ggagtacctg agtctatgag ggaagaatat cggcaggaac agcagaatgc tgtggatgtt 946

gacgatggtc attttgtcaa catagagctg gagta 981

<210> 76

<211> 252

<212> PRT

<213> Influenza virus

<400> 76

Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro

1 5 10 15

Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe

20 25 30

Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr

35 40 45

Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe

50 55 60

Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val

65 70 75 80

Gln Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala

85 90 95

Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala

100 105 110

Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met

115 120 125

Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe

130 135 140

Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg

145 150 155 160

Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu

165 170 175

Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met

180 185 190

Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Arg

195 200 205

Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser

210 215 220

Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr

225 230 235 240

Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys

245 250

<210> 77

<211> 1698

<212> DNA

<213> Influenza virus

<220>

<221> CDS

<222> (1)..(1698)

<400> 77

atg aag aca acc att att tta ata cta ctg acc cat tgg gcc tac agt 48

Met Lys Thr Thr Ile Ile Leu Ile Leu Leu Thr His Trp Ala Tyr Ser

1 5 10 15

caa aac cca atc agt ggc aat aac aca gcc aca ctg tgt ctg gga cac 96

Gln Asn Pro Ile Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His

20 25 30

cat gca gta gca aat gga aca ttg gta aaa aca atg agt gat gat caa 144

His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Met Ser Asp Asp Gln

35 40 45

att gag gtg aca aat gct aca gaa tta gtt cag agc att tca atg ggg 192

Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ile Ser Met Gly

50 55 60

aaa ata tgc aac aaa tca tat aga att cta gat gga aga aat tgc aca 240

Lys Ile Cys Asn Lys Ser Tyr Arg Ile Leu Asp Gly Arg Asn Cys Thr

65 70 75 80

tta ata gat gca atg cta gga gac ccc cac tgt gac gcc ttt cag tat 288

Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gln Tyr

85 90 95

gag agt tgg gac ctc ttt ata gaa aga agc agc gct ttc agc aat tgc 336

Glu Ser Trp Asp Leu Phe Ile Glu Arg Ser Ser Ala Phe Ser Asn Cys

100 105 110

tac cca tat gac atc cct gac tat gca ccg ctc cga tcc att gta gca 384

Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Pro Leu Arg Ser Ile Val Ala

115 120 125

tcc tca ggg aca gtg gaa ttc aca gca gag gga ttc aca tgg aca ggt 432

Ser Ser Gly Thr Val Glu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly

130 135 140

gta act caa aac gga aga agt gga gcc tgc aaa agg gga tca gcc gat 480

Val Thr Gln Asn Gly Arg Ser Gly Ala Cys Lys Arg Gly Ser Ala Asp

145 150 155 160

agt ttc ttt agc cga ctg aat tgg cta aca aaa tct gga agc tct tac 528

Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Lys Ser Gly Ser Ser Tyr

165 170 175

ccc aca ttg aat gtg aca atg cct aac aat aaa aat ttc gac aag cta 576

Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu

180 185 190

tac atc tgg ggg att cat cac ccg agc tca aat caa gag cag aca aaa 624

Tyr Ile Trp Gly Ile His Pro Ser Ser Asn Gln Glu Gln Thr Lys

195 200 205

ttg tac atc caa gaa tca gga cga gta aca gtc tca aca aaa aga agt 672

Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser

210 215 220

caa caa aca ata atc cct aac atc gga tct aga ccg ttg gtc aga ggt 720

Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Leu Val Arg Gly

225 230 235 240

caa tca ggc agg ata agc ata tac tgg acc att gta aaa cct gga gat 768

Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp

245 250 255

atc cta atg ata aac agt aat ggc aac tta gtt gca ccg cgg gga tat 816

Ile Leu Met Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr

260 265 270

ttt aaa ttg aac aca ggg aaa agc tct gta atg aga tcc gat gta ccc 864

Phe Lys Leu Asn Thr Gly Lys Ser Ser Val Met Arg Ser Asp Val Pro

275 280 285

ata gac att tgt gtg tct gaa tgt att aca cca aat gga agc atc tcc 912

Ile Asp Ile Cys Val Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Ser

290 295 300

aac gac aag cca ttc caa aat gtg aac aaa gtt aca tat gga aaa tgc 960

Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys

305 310 315 320

ccc aag tat atc agg caa aac act tta aag ctg gcc act ggg atg agg 1008

Pro Lys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg

325 330 335

aat gta cca gaa aag caa acc aga gga atc ttt gga gca ata gcg gga 1056

Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala Gly

340 345 350

ttc atc gaa aac ggc tgg gaa gga atg gtt gat ggg tgg tat ggg ttc 1104

Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe

355 360 365

cga tat caa aac tct gaa gga aca ggg caa gct gca gat cta aag agc 1152

Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser

370 375 380

act caa gca gcc atc gac cag att aat gga aag tta aac aga gtg att 1200

Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile

385 390 395 400

gaa aga acc aat gag aaa ttc cat caa ata gag aag gaa ttc tca gaa 1248

Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu

405 410 415

gta gaa gga aga att cag gac ttg gag aaa tat gta gaa gac acc aaa 1296

Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys

420 425 430

ata gac cta tgg tcc tac aat gca gaa ttg ctg gtg gct cta gaa aat 1344

Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn

435 440 445

caa cat aca att gac tta aca gat gca gaa atg aat aaa tta ttt gag 1392

Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu

450 455 460

aag act aga cgc cag tta aga gaa aac gca gaa gac atg gga ggt gga 1440

Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly

465 470 475 480

tgt ttc aag att tac cac aaa tgt gat aat gca tgc att gaa tca ata 1488

Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Glu Ser Ile

485 490 495

aga act ggg aca tat gac cat tac ata tac aaa gat gaa gca tta aac 1536

Arg Thr Gly Thr Tyr Asp His Tyr Ile Tyr Lys Asp Glu Ala Leu Asn

500 505 510

aat cga ttt cag atc aaa ggt gta gag ttg aaa tca ggc tac aaa gat 1584

Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp

515 520 525

tgg ata ctg tgg att tca ttc gcc ata tca tgc ttc tta att tgc gtt 1632

Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Ile Cys Val

530 535 540

gtt cta ttg ggt ttc att atg tgg gct tgc caa aaa ggc aac atc aga 1680

Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg

545 550 555 560

tgc aac att tgc att tga 1698

Cys Asn Ile Cys Ile

565

<210> 78

<211> 565

<212> PRT

<213> Influenza virus

<400> 78

Met Lys Thr Thr Ile Ile Leu Ile Leu Leu Thr His Trp Ala Tyr Ser

1 5 10 15

Gln Asn Pro Ile Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His

20 25 30

His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Met Ser Asp Asp Gln

35 40 45

Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ile Ser Met Gly

50 55 60

Lys Ile Cys Asn Lys Ser Tyr Arg Ile Leu Asp Gly Arg Asn Cys Thr

65 70 75 80

Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gln Tyr

85 90 95

Glu Ser Trp Asp Leu Phe Ile Glu Arg Ser Ser Ala Phe Ser Asn Cys

100 105 110

Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Pro Leu Arg Ser Ile Val Ala

115 120 125

Ser Ser Gly Thr Val Glu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly

130 135 140

Val Thr Gln Asn Gly Arg Ser Gly Ala Cys Lys Arg Gly Ser Ala Asp

145 150 155 160

Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Lys Ser Gly Ser Ser Tyr

165 170 175

Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu

180 185 190

Tyr Ile Trp Gly Ile His Pro Ser Ser Asn Gln Glu Gln Thr Lys

195 200 205

Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser

210 215 220

Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Leu Val Arg Gly

225 230 235 240

Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp

245 250 255

Ile Leu Met Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr

260 265 270

Phe Lys Leu Asn Thr Gly Lys Ser Ser Val Met Arg Ser Asp Val Pro

275 280 285

Ile Asp Ile Cys Val Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Ser

290 295 300

Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys

305 310 315 320

Pro Lys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg

325 330 335

Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala Gly

340 345 350

Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe

355 360 365

Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser

370 375 380

Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile

385 390 395 400

Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu

405 410 415

Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys

420 425 430

Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn

435 440 445

Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu

450 455 460

Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly

465 470 475 480

Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Glu Ser Ile

485 490 495

Arg Thr Gly Thr Tyr Asp His Tyr Ile Tyr Lys Asp Glu Ala Leu Asn

500 505 510

Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp

515 520 525

Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Ile Cys Val

530 535 540

Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg

545 550 555 560

Cys Asn Ile Cys Ile

565

<210> 79

<211> 20

<212> DNA

<213> Influenza virus

<400> 79

tatgcatcgc tccgatccat 20

<210> 80

<211> 21

<212> DNA

<213> Influenza virus

<400> 80

gctccacttc ttccgttttg a 21

<210> 81

<211> 30

<212> DNA

<213> Influenza virus

<400> 81

aattcacagc agagggattc acatggacag 30

<210> 82

<211> 24

<212> DNA

<213> Influenza virus

<220>

<221> variation

<222>(7)..(7)

<223> r = a or g

<400> 82

catggartgg ctaaagacaa gacc 24

<210> 83

<211> 24

<212> DNA

<213> Influenza virus

<220>

<221> variation

<222> (18)..(18)

<223> k = g or t

<400> 83

agggcatttt ggacaaakcg tcta 24

<210> 84

<211> 18

<212> DNA

<213> Influenza virus

<400> 84

acgctcaccg tgcccagt 18

<210> 85

<211> 28

<212> DNA

<213> Influenza virus

<400> 85

tattcgtctc agggagcaaa agcagggg 28

<210> 86

<211> 42

<212> DNA

<213> Influenza virus

<400> 86

tgtaatacga ctcactatag ggctccactt cttccgtttt ga 42

<210> 87

<211> 30

<212> DNA

<213> Influenza virus

<400> 87

gatcgctctt cagggagcaa aagcaggtag 30

<210> 88

<211> 40

<212> DNA

<213> Influenza virus

<400> 88

tgtaatacga ctcactatag ggcattttgg acaaagcgtc 40

<---

Claims (16)

1. Canine influenza virus that induces an immune response against canine influenza virus, containing a polynucleotide encoding a hemagglutinin (HA) protein containing the amino acid sequence shown in SEQ ID NO: 62 or SEQ ID NO: 78, or its mature form, in which N -terminal 16 amino acids are deleted from SEQ ID NO: 62 or SEQ ID NO: 78, or an amino acid sequence having more than 95% amino acid sequence identity with said mature form, where said mature HA protein contains serine at position 82, leucine at position 221 , threonine at position 327 and threonine at position 482 of the amino acid sequence. 2. Canine influenza virus according to claim 1, wherein said polynucleotide encodes a hemagglutinin (HA) protein that contains the amino acid sequence shown in SEQ ID NO: 62 or SEQ ID NO: 78, or an amino acid sequence having more than 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 62 or SEQ ID NO: 78. 3. Canine influenza virus according to claim 1, wherein said polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 61 or SEQ ID NO: 77, or a nucleotide sequence having 98% or more sequence identity with SEQ ID NO: 61 or SEQ ID NO: 77. 4. A vaccine against an influenza virus that is capable of infecting an animal of the canine family, containing an effective amount of a canine influenza virus according to any one of paragraphs. 1-3. 5. The vaccine of claim 4, wherein the vaccine contains a pharmaceutically acceptable carrier or diluent. 6. The vaccine according to claim 4, where the vaccine contains an adjuvant. 7. A method of inducing an immune response in animals against canine influenza virus, comprising administering to said animal an effective amount of canine influenza virus according to any one of paragraphs. 1-3 or vaccines according to any one of paragraphs. 4-6. 8. The method of claim 7 wherein the immune response is a protective immune response that prevents or suppresses influenza virus infection of said animal. 9. A method of immunizing an animal against canine influenza virus infection, comprising administering to said animal an effective amount of canine influenza virus according to any one of paragraphs. 1-3 or vaccines according to any one of paragraphs. 4-6. 10. The method of claim 9, wherein said influenza virus has an HA serotype selected from H3 and H5. 11. The method of claim 9, wherein said influenza virus has the H3N8 serotype. 12. The method of claim 9, wherein said animal is a canine. 13. The method of claim 12 wherein said canine is a domestic dog. 14. The method of claim 12 wherein said canine influenza virus or vaccine is administered parenterally. 15. The method of claim 14 wherein said canine influenza virus or vaccine is administered subcutaneously, intraperitoneally, or intramuscularly. 16. The method of claim 9 wherein said canine influenza virus or vaccine is administered nasally or orally.