AU2015234384B2 - Influenza viruses able to infect canids, uses thereof - Google Patents

Abstract

The subject invention pertains to isolated influenza virus that is capable of infecting canids and causing respiratory disease in the canid. The subject invention also pertains to compositions and methods for inducing an immune response against an influenza virus of the present invention. The subject invention also pertains to compositions and methods for identifying a virus of the invention and diagnosing infection of an animal with a virus of the invention.

Description

DESCRIPTION

MATERIALS AND METHODS FOR RESPIRATORY DISEASE

CONTROL IN CANINES

CROSS-REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of application U.S. Serial No. 11/409,416, filed April 21, 2006; and this application claims priority to U.S. Serial Nos. 60/728,449, filed October 19, 2005; 60/754,881, filed December 29, 2005; 60/759,162, filed January 14, 2006; 0 60/761,451, filed January 23, 2006; and 60/779,080, filed March 3, 2006, the disclosure of each of which is hereby incorporated by reference herein in its entirety, including any brief summary, detailed descriptions of the invention, examples, claims, abstract, figures, tables, nucleic acid sequences, amino acid sequences, and drawings.

BACKGROUND OF THE INVENTION “Kennel cough” or infectious tracheobronchitis (ITB) is an acute, contagious respiratory infection in dogs characterized mainly by coughing (Ford et al, 1998). Canine ITB is considered one of the most prevalent infectious respiratory diseases of dogs worldwide, and outbreaks can reach epidemic proportions when dogs are housed in highΌ density population environments such as kennels. Most outbreaks are due to direct dog-todog contact or aerosolization of respiratory secretions (Ford et al, 1998). The clinical signs are caused by infection with one 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 canine adenoviruses-1 and -2 (CAV-1, CAV-2), along with bacteria such as Streptococcus sp., Pasteurella multicoda and Escherichia coli, can influence the clinical course and outcome (Ford et al, 1998). While outbreaks occur most efficiently and rapidly in high-density populations with high morbidity, complicated respiratory infections and death are uncommon. Although life-threatening secondary bacterial pneumonia can develop, the majority of ITB cases are self-limiting and resolve without any treatment (Ford et al, 1998).

In July 1992, a respiratory infection presumed to be “kennel cough” became epidemic at several greyhound tracks in New England, Florida, West Virginia, Wisconsin, Kansas,

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Colorado, Oklahoma and Arizona. According to veterinarians, most of the affected dogs had a mild cough that resolved, but more than a dozen greyhounds developed an acute hemorrhagic pneumonia followed by rapid death (Greyhound Daily News, 1999).

In late 1998 to early 1999, several outbreaks of “kennel cough” occurred in racing 5 greyhound kennels across the country, resulting in mandatory closure of tracks and quarantine of all racing greyhounds in the U.S. for several weeks (Greyhound Daily News, 1999). At one track in Florida (Palm Beach Kennel Club), coughing was recorded in nearly 40% of the dog population on a single day (Personal communication from Dr. William Duggar). Similar to the outbreak in 1992, the coughing resolved in most greyhounds, but 10 0 dogs in Florida died from 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 U.S. The outbreak is believed to have originated in kennels at four tracks in Florida and caused the suspension of racing and quarantine of dogs for almost three weeks. Nearly 25% of the dogs at the track in West Palm Beach were affected, while almost 50% of the 1400 dogs at Derby Fane in St. Petersburg developed coughing. Again, most dogs recovered, but several dogs have died from the respiratory infection. The estimated economic impact of the respiratory outbreak at the Derby Fane track alone was $2 million.

There are no published reports documenting the etiology or clinicopathology of the

Ό “kennel cough” epidemics in racing greyhound kennels in 1992, 1998-1999, or 2003. The assumption has been that the infections were due to CPiV and/or B. bronchiseptica, the two most common causes of kennel cough. Unsubstantiated communications such as web sites have attributed the fatal hemorrhagic pneumonias reported in some of the coughing dogs to infection with β-hemolytic Streptococcus equi subspecies zooepidemicus, and refer to the syndrome as “canine streptococcal toxic shock.”

Transmission of virus from one host species to another is a crucial feature of the ecology and epidemiology of influenza virus (Webster, 1998). Two basic mechanisms of interspecies transmission of influenza virus are possible (Webster et al., 1992; Fipatov et al., 2004). One is the direct transfer of an essentially unaltered virus from one species to another.

Examples of this mechanism include the recent human infections with the H5N1 subtype of avian influenza virus (Subbarao et al., 1998; Peiris et al., 2004; Guan et al., 2004) and possibly the pandemic of 1918, known as Spanish flu (Reid et al., 2004). The second mechanism is a consequence of the segmented nature of the influenza genome. Co-infection

2015234384 02 Oct 2015 of a host with viruses from different species can result in reassortment of the segmented viral genes and the generation of a recombinant with the ability to infect other species. For example, novel viruses generated by gene reassortment between avian and human influenza viruses resulted in human influenza pandemics in 1957 and 1968 (Webster et al., 1992; 5 Lipatov et al., 2004; Kawaoka et al., 1989).

Most direct transmissions of unaltered influenza viruses from the natural host species to a different species are terminal events because sustained transmission between individuals of the new species fails to occur. Multiple virus-host interactions are necessary for replication and horizontal transmission and provide a formidable barrier to perpetuation of 0 influenza viruses in the new host (Webby et al., 2004). Therefore, establishment of new host-specific lineages of influenza virus is uncommon and has only occurred in domestic poultry, pigs, horses, and humans (Webster et al., 1992; Lipatov et al., 2004).

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

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to isolated influenza virus that is capable of infecting canids and causing respiratory disease in the canid. The subject invention also pertains to compositions and methods for inducing an immune response against an influenza virus of the

Ό present invention. The subject invention also pertains to compositions and methods for identifying a virus of the invention and diagnosing infection of an animal with a virus of the invention.

One aspect of the invention relates to vaccines and methods for protecting canines from canine influenza, kits comprising such vaccines, and methods for using such vaccines.

This protection includes preventing, reducing the risk of, delaying the onset of, reducing the spread of, ameliorating, suppressing, and/or eradicating the influenza and/or one or more (typically two or more) of its symptoms. It is believed that the vaccines, kits, and methods of this invention are generally suitable for use with canines. Canines include wild, zoo, and domestic canines, such as wolves, coyotes, and foxes. Canines also include dogs, particularly domestic dogs, such as, for example, pure-bred and/or mongrel companion dogs, show dogs, working dogs, herding dogs, hunting dogs, guard dogs, police dogs, racing dogs, and/or laboratory dogs.

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This invention is also directed, in part, to a method for protecting a canine from an influenza virus infection (i.e., preventing, reducing the risk of, delaying the onset of, suppressing, ameliorating, or eradicating an influenza virus infection). The method comprises administering a therapeutically effective amount of a vaccine that comprises at 5 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 is directed, in part, to a method for protecting a canine from respiratory lesions (i.e., preventing, reducing the risk of, delaying the onset of, suppressing, ameliorating, or eradicating respiratory lesions) caused by canine influenza virus. The 0 method comprises administering to the canine 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 is directed, in part, to a method for protecting a cainine from having canine influenza virus in nasal or oral secretion (i.e., preventing, reducing the risk of, 5 delaying the onset of, suppressing, ameliorating, or eradicating canine influenza virus in nasal or oral secretion) caused by canine influenza virus infection. The method comprises administering to the canine 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.

Ό This invention also is directed, 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 also is directed, in part, to a kit for protecting a canine from influenza virus infection. The kit comprises 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. In addition, the kit comprises at least one of the following:

an apparatus for administering the vaccine to the canine, a pharmaceutically acceptable excipient that aids in administering the vaccine to the canine, a pharmaceutically acceptable excipient that enhances the canine's immune response to the vaccine,

2015234384 02 Oct 2015 a food to be consumed by the canine simultaneously with the vaccine, and/or a treat to be consumed by the canine simultaneously with the vaccine.

Further benefits of Applicants’ invention will be apparent to one skilled in the art from reading this specification.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1B show phylogenetic relationships among the hemagglutinin genes. Figure 1A shows a tree of HA genes from representative canine, human, avian, swine, and equine isolates, including A/Budgerigar/Hokkaido/1/77 (H4) as outgroup. Figure IB shows 0 a tree of the canine influenza virus HA genes with contemporary and older equine HA genes, using A/Duck/Ukraine/63 (H3) as outgroup. Phylogenetic trees were inferred from nucleotide sequences by the neighbor joining method and bootstrap analysis values >90% are shown. The bar denotes the number of nucleotide changes per unit length of the horizontal tree branches.

Figures 2A-2B show immunohistochemical detection of influenza H3 antigen in the lungs. Lung tissue sections were probed with a mouse monoclonal antibody to H3 hemagglutinin and binding was detected by immunoperoxidase reaction (brown precipitate). Figure 2A shows bronchial epithelium from a greyhound with spontaneous disease. Viral H3 antigen was detected in bronchial epithelial cell cytoplasm and in macrophages in airway

Ό lumens and in alveolar spaces. Figure 2B shows bronchial epithelium from a dog 5 days after inoculation with A/canine/Florida/43/2004 (H3N8). Viral H3 antigen was detected in bronchial epithelial cell cytoplasm. Scale bar, 66 pm.

Figure 3 shows the characteristic histological changes in the bronchi of greyhounds that died from hemorrhagic pneumonia associated with influenza virus infection. The tissues are stained with H&E. Upper panel: Normal bronchus with ciliated epithelial cells, mucous cells, and basal cells. Lower panel: Bronchus from a greyhound with spontaneous influenza. There is necrosis and erosion of the bronchial ciliated epithelial cells. Scale bar, 100 pm.

Figures 4A-4B shows phylogenetic relationships among the H3 hemagglutinin genes. Figure 4A shows a phylogenetic tree of the canine influenza virus HA genes with contemporary and older equine HA genes. Figure 4B shows a phylogenetic tree of the canine influenza virus HA protein with contemporary and older equine HA. Phylogenetic trees were inferred from genetic or amino acid sequences by the neighbor joining method and

2015234384 02 Oct 2015 bootstrap analysis values >80% are shown. The bar denotes the number of amino acid changes per unit length of the horizontal tree branches.

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

Figures 6A-6D show amplification plots of H3 and Matrix genes (Figure 6A and

Figure 6B) obtained from the amplification of 10-fold serially diluted in vitro transcribed RNA standards. Standard curves of H3 and Matrix genes (Figure 6C and Figure 6D) constructed by plotting the log of starting RNA concentrations against the threshold cycle (Ct) obtained from each dilution.

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

BRIEF DESCRIPTION OF THE SEQUENCES

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

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

SEQ ID NO: 3 is a nucleotide sequence of a canine influenza virus (Florida/43/04) encoding a PB1 protein that can be used according to the present invention.

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

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

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

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

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

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

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

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

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

SEQ ID NO: 13 is a nucleotide sequence of a canine influenza virus (Florida/43/04) encoding an MA protein that can be used according to the present invention.

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

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

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

SEQ ID NO: 17 is a nucleotide sequence of a canine influenza virus (FL/242/03) encoding a PB2 protein that can be used according to the present invention.

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

SEQ ID NO: 19 is a nucleotide sequence of a canine influenza virus (FL/242/03) 5 encoding a PB1 protein that can be used according to the present invention.

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

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

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

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

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

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

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

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

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

SEQ ID NO: 29 is a nucleotide sequence of a canine influenza virus (FL/242/03) 30 encoding an MA protein that can be used according to the present invention.

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

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

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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 wherein the N-terminal 16 amino acid signal sequence has been removed.

SEQ ID NO: 34 is the mature form of the HA protein shown in SEQ ID NO: 32 5 wherein the N-terminal 16 amino acid signal sequence has been removed.

SEQ ID NO: 35 is an oligonucleotide that can be used according to the present invention.

SEQ ID NO: 36 is an oligonucleotide that can be used according to the present invention.

SEQ ID NO: 37 is an oligonucleotide that can be used according to the present invention.

SEQ ID NO: 38 is an oligonucleotide that can be used according to the present invention.

SEQ ID NO: 39 is an oligonucleotide that can be used according to the present 5 invention.

SEQ ID NO: 41 is an oligonucleotide that can be used according to the present invention.

SEQ ID NO: 42 is an oligonucleotide that can be used according to the present invention.

Ό SEQ ID NO: 43 is an oligonucleotide that can be used according to the present invention.

SEQ ID NO: 44 is an oligonucleotide that can be used according to the present invention.

SEQ ID NO: 45 is an oligonucleotide that can be used according to the present 25 invention.

SEQ ID NO: 46 is an oligonucleotide that can be used according to the present invention.

SEQ ID NO: 47 is a nucleotide sequence of a canine influenza virus (Miami/2005) encoding a PB2 protein that can be used according to the present invention.

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

SEQ ID NO: 49 is a nucleotide sequence of a canine influenza virus (Miami/2005) encoding a PB1 protein that can be used according to the present invention.

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

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

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

SEQ ID NO: 53 is a nucleotide sequence of a canine influenza virus (Miami/2005) 5 encoding an NS protein that can be used according to the present invention.

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

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

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

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

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

SEQ ID NO: 59 is a nucleotide sequence of a canine influenza virus (Miami/2005) encoding an MA protein that can be used according to the present invention.

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

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

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

SEQ ID NO: 63 is a nucleotide sequence of a canine influenza virus Ό (Jacksonville/2005) encoding a PB2 protein that can be used according to the present invention.

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

SEQ ID NO: 65 is a nucleotide sequence of a canine influenza virus (Jacksonville/2005) encoding a PB1 protein that can be used according to the present invention.

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

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

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

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

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

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

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

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

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

SEQ ID NO: 75 is a nucleotide sequence of a canine influenza virus (Jacksonville/2005) encoding an MA protein that can be used according to the present invention.

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

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

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

SEQ invention.

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SEQ ID NO: 87 is an oligonucleotide that can be used according to the present invention.

SEQ ID NO: 88 is an oligonucleotide that can be used according to the present invention.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns isolated influenza virus that is capable of infecting canids and causing respiratory disease. In one embodiment, an influenza virus of the invention comprises a polynucleotide which encodes a protein having an 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. In a specific embodiment, the polynucleotide comprises 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. Influenza virus of the present invention can have an HA subtype of Hl, H2, H3, H4, H5, H6, H7, H8, and H9, H10, Hll, H12, H13, H14, H15, or H16 or an NA subtype of Nl, N2, N3, N4, N5, N6, N7, N8, OR N9. In a specific embodiment, an influenza virus of the present invention is a subtype H3. Virus can be isolated from infected dogs and cultured in cells or eggs according to methods described herein. In an exemplified embodiment, the influenza virus is an influenza A virus.

The subject invention also concerns polynucleotides that comprise 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 the invention comprises an influenza hemagglutinin (HA) gene, neuraminidase (NA) gene, nucleoprotein (NP) gene, matrix protein (MA or M) gene, polymerase basic (PB) protein gene, polymerase acidic (PA) protein gene, non-structural (NS) protein gene, or a functional fragment or variant of any of these genes. In a specific embodiment, a polynucleotide of the invention comprises the hemagglutinin (HA) gene, or a functional fragment or variant thereof. In a further embodiment, the HA gene encodes a hemagglutinin protein having one or more of the following: a serine at position 83; a leucine at position 222; a threonine at position 328; and/or a threonine at position 483, versus the amino acid sequence of equine H3 consensus sequence. In one embodiment, the HA gene encodes a polypeptide having an amino acid sequence shown in SEQ ID NOs: 16, 32, 62, or 78, or a functional and/or immunogenic fragment or variant thereof. In a specific

2015234384 02 Oct 2015 embodiment, the HA gene comprises a nucleotide sequence shown in SEQ ID NOs: 15, 31, 61, or 77.

In one embodiment, a polynucleotide of the 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 or variant thereof. In a specific embodiment, the 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, comprises 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 subject invention concerns polynucleotide sequences 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, of 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. In a further specific embodiment, a polynucleotide of the invention can comprise: Nucleotides 1-2271 of SEQ ID NO: 3; Nucleotides 1-2148 of SEQ ID NO: 5; Nucleotides 1-657 of SEQ ID NO: 7; Nucleotides 11494 of SEQ ID NO: 9; Nucleotides 1-1410 of SEQ ID NO: 11; Nucleotides 1-756 of SEQ ID NO: 13; Nucleotides 1-1695 of SEQ ID NO: 15; Nucleotides 1-2271 of SEQ ID NO: 19; Nucleotides 1-2148 of SEQ ID NO: 21; Nucleotides 1-657 of SEQ ID NO: 23; Nucleotides 1-1494 of SEQ ID NO: 25; Nucleotides 1-756 of SEQ ID NO: 29; Nucleotides 1-1695 of SEQ ID NO: 31; Nucleotides 1-2277 of SEQ ID NO: 47; Nucleotides 1-2271 of SEQ ID NO: 49; Nucleotides 1-2148 of SEQ ID NO: 51; Nucleotides 1-690 of SEQ ID NO: 53; Nucleotides 1-1494 of SEQ ID NO: 55; Nucleotides 1-1410 of SEQ ID NO: 57; Nucleotides 1-756 of SEQ ID NO: 59; Nucleotides 1-1695 of SEQ ID NO: 61; Nucleotides 1-2277 of SEQ ID NO: 63; Nucleotides 1-2271 of SEQ ID NO: 65; Nucleotides 1-2148 of SEQ ID NO: 67; Nucleotides 1-690 of SEQ ID NO: 69; Nucleotides 1-1494 of SEQ ID NO: 71; Nucleotides 1-1410 of SEQ ID NO: 73; Nucleotides 1-756 of SEQ ID NO: 75; and Nucleotides 1-1695 of SEQ ID NO: 77. Nucleotide and amino acid sequences of viral polynucleotide and polypeptide sequences contemplated within the scope of the present

2015234384 02 Oct 2015 invention have also been deposited with GenBank at accession Nos. DQ124147 through DQ124161 and DQ124190, the disclosure of which is incorporated herein by reference.

The subject invention also concerns polypeptides encoded by polynucleotides of an influenza virus of the present invention. The subject invention also concerns functional and/or immunogenic fragments and variants of the subject polypeptides. Polypeptides contemplated include HA protein, NA protein, NS protein, nucleoprotein, polymerase basic protein, polymerase acidic protein, and matrix protein of an influenza virus of the invention. In an exemplified embodiment, a polypeptide of the invention has an 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 subject invention also concerns polynucleotide expression constructs comprising a polynucleotide sequence of the present invention. In one embodiment, an expression construct of the invention comprises a polynucleotide sequence encoding a polypeptide comprising an 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. In a specific embodiment, the 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 comprises 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 subject invention concerns expression constructs comprising 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, of 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. In a preferred embodiment, an expression construct of the present invention provides for overexpression of an operably linked polynucleotide of the invention.

Expression constructs of the invention generally include regulatory elements that are functional in the intended host cell in which the expression construct is to be expressed.

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Thus, a person 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, the regulatory elements are ones that are functional in canine cells. Regulatory elements include promoters, transcription termination 5 sequences, translation termination sequences, enhancers, and polyadenylation elements. As used herein, the term “expression construct” refers to a combination of nucleic acid sequences that provides for transcription of an operably linked nucleic acid sequence. As used herein, the term “operably linked” refers to a juxtaposition of the components described wherein the components are in a relationship that permits them to function in their intended 0 manner. In general, operably linked components are in contiguous relation.

An expression construct of the invention can comprise a promoter sequence operably linked to a polynucleotide sequence encoding a polypeptide of the invention. Promoters can be incorporated into a polynucleotide using standard techniques known in the art. Multiple copies of promoters or multiple promoters can be used in an expression construct of the 5 invention. In a preferred embodiment, a promoter can be positioned about the same distance from the transcription start site in the expression construct as it is from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without substantial decrease in promoter activity. A transcription start site is typically included in the expression construct. Preferably, the promoter associated with an expression construct of the Ό invention provides for overexpression of an operably linked polynucleotide of the invention.

Promoters for use with an expression construct of the invention in eukaryotic cells can be 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, desmin gene promoter 25 and actin gene promoter Promoters suitable for use with an expression construct of the invention in yeast cells include, but are not limited to, 3-phosphoglycerate kinase promoter, glyceraldehyde-3-phosphate dehydrogenase promoter, metallothionein promoter, alcohol dehydrogenase-2 promoter, and hexokinase promoter.

If the expression construct is to be provided in or introduced into a plant cell, then 30 plant viral promoters, such as, for example, a cauliflower mosaic virus (CaMV) 35S (including the enhanced CaMV 35S promoter (see, for example U.S. Patent No. 5,106,739 and An, 1997)) or a CaMV 19S promoter can be used. Other promoters that can be used for expression constructs in plants include, for example, prolifera promoter, Ap3 promoter, heat

2015234384 02 Oct 2015 shock promoters, T-DNA T- or 2'-promoter of A tumefaciens, polygalacturonase promoter, chalcone synthase A (CHS-A) promoter from petunia, tobacco PR-la promoter, ubiquitin promoter, actin promoter, alcA gene promoter, pin2 promoter (Xu et al., 1993), maize Wipl promoter, maize trpA gene promoter (U.S. Patent No. 5,625,136), maize CDPK gene 5 promoter, and RUBISCO SSU promoter (U.S. Patent No. 5,034,322) can also be used. Rootspecific promoters, such as any of the promoter sequences described in U.S. Patent No. 6,455,760 or U.S. Patent No. 6,696,623, or in published U.S. patent application Nos. 20040078841; 20040067506; 20040019934; 20030177536; 20030084486; or 20040123349, can be used with an expression construct of the invention. Constitutive promoters (such as 0 the CaMV, ubiquitin, actin, or NOS promoter), developmentally-regulated promoters, and inducible promoters (such as those promoters than can be induced by heat, light, hormones, or chemicals) are also contemplated for use with polynucleotide expression constructs of the invention. Tissue-specific promoters, for example fruit-specific promoters, such as the E8 promoter of tomato (accession number: AF515784; Good et al. (1994)) can also be used. 5 Seed-specific promoters such as the promoter from a β-phaseolin gene (for example, of kidney bean) or a glycinin gene (for example, of soybean), and others, can also be used.

For expression in prokaryotic systems, an expression construct of the invention can comprise promoters such as, for example, alkaline phosphatase promoter, tryptophan (trp) promoter, lambda Pl promoter, β-lactamase promoter, lactose promoter, pho A promoter, T3 Ό promoter, T7 promoter, or tac promoter (de Boer et al., 1983).

Expression constructs of the invention may optionally contain a transcription termination sequence, a translation termination sequence, a sequence encoding a signal peptide, and/or enhancer elements. Transcription termination regions can typically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription 25 termination sequences can be positioned downstream of a coding sequence to provide for efficient termination. A signal peptide sequence is a short amino acid sequence typically present at the amino terminus of a protein that is responsible for the relocation of an operably linked mature polypeptide to a wide range of post-translational cellular destinations, ranging from a specific organelle compartment to sites of protein action and the extracellular 30 environment. Targeting gene products to an intended cellular and/or extracellular destination through the use of an operably linked signal peptide sequence is contemplated for use with the polypeptides of the invention. Classical enhancers are cis-acting elements that increase gene transcription and can also be included in the expression construct. Classical enhancer

2015234384 02 Oct 2015 elements are known in the art, and include, but are not limited to, the CaMV 35S enhancer element, cytomegalovirus (CMV) early promoter enhancer element, and the SV40 enhancer element. Intron-mediated enhancer elements that enhance gene expression are also known in the art. These elements must be present within the transcribed region and are orientation 5 dependent.

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

Expression constructs can also include one or more dominant selectable marker genes, 0 including, for example, genes encoding antibiotic resistance and/or herbicide-resistance for selecting transformed cells. Antibiotic-resistance genes can provide for resistance to one or more of the following antibiotics: hygromycin, kanamycin, bleomycin, G418, streptomycin, paromomycin, neomycin, and spectinomycin. Kanamycin resistance can be provided by neomycin phosphotransferase (NPT II). Herbicide-resistance genes can provide for 5 resistance to phosphinothricin acetyltransferase or glyphosate. Other markers used for cell transformation screening include, but are not limited to, genes encoding β-glucuronidase (GUS), β-galactosidase, luciferase, nopaline synthase, chloramphenicol acetyltransferase (CAT), green fluorescence protein (GFP), or enhanced GFP (Yang et at., 1996).

The subject invention also concerns polynucleotide vectors comprising a Ό polynucleotide sequence of the invention that encodes a polypeptide of the invention. Unique restriction enzyme sites can be included at the 5' and 3' ends of an expression construct or polynucleotide of the invention to allow for insertion into a polynucleotide vector. As used herein, the term “vector” refers to any genetic element, including for example, plasmids, cosmids, chromosomes, phage, virus, and the like, which is capable of replication when 25 associated with proper control elements and which can transfer polynucleotide sequences between cells. Vectors contain a nucleotide sequence that permits the vector to replicate in a selected host cell. A number of vectors are available for expression and/or cloning, and include, but are not limited to, pBR322, pUC series, M13 series, pGEM series, and pBLUESCRIPT vectors (Stratagene, La Jolla, CA and Pro mega, Madison, WI).

The subject invention also concerns oligonucleotide probes and primers, such as polymerase chain reaction (PCR) primers, that can hybridize to a coding or non-coding sequence of a polynucleotide of the present invention. Oligonucleotide probes of the invention can be used in methods for detecting influenza virus nucleic acid sequences.

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Oligonucleotide primers of the invention can be used in PCR methods and other methods involving nucleic acid amplification. In a preferred embodiment, a probe or primer of the invention can hybridize to a polynucleotide of the invention under stringent conditions. Probes and primers of the invention can optionally comprise a detectable label or reporter 5 molecule, such as fluorescent molecules, enzymes, radioactive moiety, and the like. Probes and primers of the invention can be of any suitable length for the method or assay in which they are being employed. Typically, probes and primers of the invention will be 10 to 500 or more nucleotides in length. Probes and primers that are 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 81 to 90, 91 to 100, or 101 or more nucleotides in length are 0 contemplated within the scope of the invention. In one embodiment, 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 in length. Probes and primers of the invention can have complete (100%) nucleotide sequence identity with the polynucleotide sequence, or the sequence identity can be less than 100%. For example, sequence identity between a probe or primer and a sequence 5 can be 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70% or any other percentage sequence identity so long as the probe or primer can hybridize under stringent conditions to a nucleotide sequence of a polynucleotide of the invention. Exemplified probes and primers of the invention include those 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 Ό 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 of the SEQ ID NOs: 35-46.

As used herein, the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” refer to a deoxyribonucleotide, ribonucleotide, or a mixed deoxyribonucleotide and ribonucleotide polymer in either single- or double-stranded form, and unless otherwise 25 limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally-occurring nucleotides. Polynucleotide sequences include the DNA strand sequence that can be transcribed into RNA and the RNA strand that can be translated into protein. The complementary sequence of any nucleic acid, polynucleotide, or oligonucleotide of the present invention is also contemplated within the scope of the 30 invention. Polynucleotide sequences also include both full-length sequences as well as shorter sequences derived from the full-length sequences. The subject invention also encompasses those polynucleotides that are complementary in sequence to the

2015234384 02 Oct 2015 polynucleotides disclosed herein. Polynucleotides and polypeptides of the invention can be provided in purified or isolated form.

Because of the degeneracy of the genetic code, a variety of different polynucleotide sequences can encode a polypeptide of the present invention. A table showing all possible 5 triplet codons (and where U also stands for T) and the amino acid encoded by each codon is described in Lewin (1985). In addition, it is well within the skill of a person trained in the art to create alternative polynucleotide sequences encoding the same, or essentially the same, polypeptides of the subject invention. These degenerate variant and alternative polynucleotide sequences are within the scope of the subject invention. As used herein, 0 references to “essentially the same” sequence refers to sequences which encode amino acid substitutions, deletions, additions, or insertions which do not materially alter the functional and/or immunogenic activity of the polypeptide encoded by the polynucleotides of the present invention.

The subject invention also concerns variants of the polynucleotides of the present 5 invention that encode polypeptides of the invention. Variant sequences include those sequences wherein one or more nucleotides of the sequence have been substituted, deleted, and/or inserted. The nucleotides that can be substituted for natural nucleotides of DNA have a base moiety that can include, but is not limited to, inosine, 5-fluorouracil, 5-bro mo uracil, hypoxanthine, 1-methylguanine, 5-methylcytosine, and tritylated bases. The sugar moiety of

Ό the nucleotide in a sequence can also be modified and includes, but is not limited to, arabinose, xylulose, and hexose. In addition, the adenine, cytosine, guanine, thymine, and uracil bases of the nucleotides can be modified with acetyl, methyl, and/or thio groups. Sequences containing nucleotide substitutions, deletions, and/or insertions can be prepared and tested using standard techniques known in the art.

Substitution of amino acids other than those specifically exemplified or naturally present in a polypeptide of the invention are also contemplated within the scope of the present invention. For example, non-natural amino acids can be substituted for the amino acids of a polypeptide, so long as the polypeptide having the substituted amino acids retains substantially the same functional activity as the polypeptide in which amino acids have not been substituted. Examples of non-natural amino acids include, but are not limited to, ornithine, citrulline, hydroxyproline, homo serine, phenylglycine, taurine, iodo tyro sine, 2,4diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, 2-amino butyric acid, γamino butyric acid, ε-amino hexanoic acid, 6-amino hexanoic acid, 2-amino isobutyric acid,

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3-amino propionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, τbutylglycine, τ-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogues in general. Non-natural amino acids also include amino acids having derivatized side groups. Furthermore, any of the amino acids in the protein can be of the D (dextrorotary) form or L (levorotary) form. Allelic variants of a protein sequence of a polypeptide of the present invention are also encompassed within the scope of the invention.

Amino acids can be generally categorized in 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 another amino acid of the same class fall within the scope of the subject invention so long as the polypeptide having the substitution still retains substantially the same functional activity as the polypeptide that does not have the substitution. Polynucleotides encoding a polypeptide having one or more amino acid substitutions in the sequence are contemplated within the scope of the present invention. Table 11 below provides a listing of examples of amino acids belonging to each class. Single letter amino acid abbreviations are defined in Table 12.

Fragments and variants of polypeptides of influenza virus of the present invention can be generated using standard methods known in the art and tested for the presence of function or immunogenecity using standard techniques known in the art. For example, for testing fragments and/or variants of a neuraminidase polypeptide of the invention, enzymatic activity can be assayed. Thus, an ordinarily skilled artisan can readily prepare and test fragments and variants of a polypeptide of the invention and determine whether the fragment or variant retains activity relative to full-length or a non-variant polypeptide.

Polynucleotides and polypeptides contemplated within the scope of the subject invention can also be defined in terms of more particular identity and/or similarity ranges with those sequences of the 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 can be greater than 95%. The identity and/or similarity of a sequence 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% as compared to a sequence exemplified herein. Unless otherwise specified, as used herein percent sequence identity and/or similarity

2015234384 02 Oct 2015 of two sequences can be determined using the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990). BLAST searches can be performed with the NBLAST program, score = 100, wordlength = 12, to obtain sequences 5 with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used. See NCBI/NIH website.

The subject invention also contemplates those polynucleotide molecules having 0 sequences which are sufficiently homologous with the polynucleotide sequences exemplified herein so as to permit hybridization with that sequence under standard stringent conditions and standard methods (Maniatis et al., 1982). As used herein, “stringent” conditions for hybridization refers to conditions wherein hybridization is typically carried out overnight at 20-25 C below the melting temperature (Tm) of the DNA hybrid in 6x SSPE, 5x Denhardt’s 5 solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature, 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/length of duplex in base pairs.

Washes are typically carried out as follows:

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

(2) Once at Tm-20 C for 15 minutes in 0.2x SSPE, 0.1% SDS (moderate stringency wash).

The subject invention also concerns viral proteins and peptides encoded by the genes 25 of an influenza virus of the present invention. In one embodiment, the viral protein is a mature HA protein. In a specific embodiment, the mature HA protein comprises one or more of the following: a serine at position 82; a leucine at position 221; a threonine at position 327; and/or a threonine at position 482. In an exemplified embodiment, the mature HA protein has an amino acid sequence shown in SEQ ID NO: 33 or SEQ ID NO: 34, or a functional and/or immunogenic fragment or variant of SEQ ID NO: 33 or SEQ ID NO: 34. In another embodiment, the viral protein is an NA protein, NS protein, PB protein, PA protein, or MA protein. Viral proteins and peptides of the invention can be used to generate

2015234384 02 Oct 2015 antibodies that bind specifically to the protein or peptide. Viral proteins and peptides of the present invention can also be used as immunogens and in vaccine compositions.

The subject invention also concerns compositions and methods for inducing an immune response against an influenza virus that is capable of infecting a susceptible host 5 animal and causing respiratory disease. The invention can be used to induce an immune response against an influenza virus of any subtype in a susceptible host animal. For example, the influenza virus can be an HA subtype of Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hll, H12, H13, H14, H15, or H16, and an NA subtype of Nl, 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 0 is N7 or N8. In a specific embodiment, an immune response is induced against an influenza virus of subtype H3N8. In one embodiment, the host animal is a canid. Canines include wild, zoo, and domestic canines, such as wolves, coyotes, and foxes. Canines also include dogs, particularly domestic dogs, such as, for example, pure-bred and/or mongrel companion dogs, show dogs, working dogs, herding dogs, hunting dogs, guard dogs, police dogs, racing 5 dogs, and/or laboratory dogs. In a specific embodiment, the host animal is a domesticated dog, such as a greyhound. In one embodiment, an animal is administered an effective amount of an immunogenic composition of the present invention sufficient to induce an immune response against an influenza virus of the invention. The immune response can be a humoral and/or cellular immune response. In a specific embodiment, the immune response is a Ό protective immune response that is capable of preventing or minimizing viral infection in the immunized host animal for some period of time subsequent to the immunization. Thus, the subject invention also concerns vaccine compositions and methods that can provide a vaccinated animal with a protective immune response to a virus of the present invention.

As described herein, the vaccine or immunogenic compositions of the subject 25 invention may comprise cell-free whole virus, including attenuated or inactivated virus, or portions of the virus, including subvirion particles (including “split vaccine” wherein a virion is treated to remove some or all viral lipids), viral proteins (including individual proteins and macromolecular complexes of multiple proteins), polypeptides, and peptides, as well as virusinfected cell lines, or a combination of any of these. Vaccine or immunogenic compositions 30 comprising virus-infected cell lines may comprise multiple cell lines, each infected with a different viral strain.

In one embodiment of the invention, a canine may be immunized with one or more inactivated (i.e., killed) and/or live attenuated influenza virus vaccines or vaccines

2015234384 02 Oct 2015 comprising one or a multiplicity of 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% 5 or more amino acid sequence identity with a canine influenza virus polypeptide. In one embodiment, an influenza antigen used in a vaccine of the present invention has at least about 96% sequence identity with an HA antigen and/or NA antigen of a canine influenza virus.

An example of an inactivated vaccine is EQUICINE II™, which has been marketed by Intervet Inc. (Millsboro, DE, USA) as a liquid vaccine. EQUICINE II™ contains 0 inactivated A/Pennsylvania/63 influenza virus (A/Pa/63) and A/equine/Kentucky/93 influenza virus (A/KY/93) with carbopol (i.e., HAVLOGEN® (Intervet Inc.)). More specifically, a dose of EQUICINE II™ contains: inactivated A/Pa/63 at IO⁶⁰ EID50, inactivated A/KY/93 at 10⁶⁷ EID50, 0.25% by volume carbopol, and sufficient PBS to create a total volume of 1 ml.

Another example of an inactivated vaccine is equine flu virus A/equine/Ohio/03 (Ohio 03). In some embodiments, such a vaccine contains CARBIGEN™, which is an emulsified polymer-based adjuvant commercially available from MVP Laboratories, Inc. (Ralston, NE). In such vaccines, a dosage unit typically comprises at least about 250 HA units of the virus, from about 250 to about 12,500 HA units of the virus, or from about 1000

Ό to about 6200 HA units of the virus. The recommended concentration of CARBIGEN™ is from about 5 to about 30% (by mass).

An example of a live attenuated vaccine is modified live equine/Kentucky/91 (A/KY/91) influenza in the form of a freeze-dried vaccine that may be reconstituted with water. In some embodiments, this reconstitution is conducted using vaccine-grade water sufficient to bring the vaccine dosage to a total volume of 1 ml. Aspects of such vaccines are discussed in, for example, U.S. Patent Nos. 6,436,408; 6,398,774; and 6,177,082, which are incorporated by reference in their entirety into this patent. When reconstituted, a dose of such a vaccine may, for example, contain A/KY/91 at 10 TCID50 per ml, 0.015 grams N-Z AMINE AS™ per ml, 0.0025 grams gelatin per ml, and 0.04 grams D lactose per ml. N-Z

AMINE AS™ is a refined source of amino acids and peptides produced by enzymatic hydrolysis of casein. N-Z AMINE AS™ is marketed by Kerry Bio-Science (Norwich, NY, USA).

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In a preferred embodiment, the vaccine comprises an H3 influenza antigen having at least about 93% homology with 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, for example, the equine/Alaska/1/91 and equine/Santiago/85 strains. In the examples that 5 follow, the equine/Kentucky/ 91, equine-2/Kentucky/93, equine-l/Pennsylvania/63, and equine Ohio/03 influenza antigens were incorporated into vaccines. Preferred vaccines also include vaccines comprising equine/Wisconsin/03, equine/Kentucky/02, equine/Kentucky/93, and equine/New Market 2/93. In the examples that follow, H3N8 viruses were used. It is believed, however, that other H3 influenza viruses can be used in accordance with this 0 invention.

Live attenuated vaccines can be prepared by conventional means. Such means generally include, for example, modifying pathogenic strains by in vitro passaging, cold adaptation, modifying the pathogenicity of the organism by genetic manipulation, preparation of chimeras, insertion of antigens into viral vectors, selecting non-virulent wild type strains, etc.

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

Ό In some embodiments, the live attenuated virus strain is prepared by co-infection of permissible cells with an attenuated mutant virus and pathogenic virus. The desired resultant recombinant virus has the safety of the attenuated virus with genes coding for protective antigens from the pathogenic virus.

In some embodiments, the live attenuated virus strain is prepared by cold adaptation.

A cold-adapted virus has an advantage of replicating only at the temperature found in upper respiratory tract. A method of generation of a cold-adapted equine influenza virus has been described in U.S. Patent No. 6,177,082. A desired resulting cold-adapted virus confers one or more of the following phenotypes: cold adaptation, temperature sensitivity, dominant interference, and/or attenuation.

In some embodiments, the live attenuated virus strain is prepared by molecular means, such as point mutation, deletion, or insertion to convert a pathogenic virus to a nonpathogenic or less-pathogenic virus compared to the original virus, while preserving the protective properties of the original virus.

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In some embodiments, the live attenuated virus is prepared by cloning the candidate of genes of protective antigens into a genome of a non-pathogenic or less-pathogenic virus or other organism.

Inactivated (i.e., killed) virus vaccines may be prepared by inactivating the virus 5 using conventional methods. Typically, such vaccines include excipients that may enhance an immune response, as well as other excipients that are conventionally used in vaccines. For example, in the examples that follow, EQUICINE II™ comprises HAVLOGEN®. Inactivation of the virus can be accomplished by treating the virus with inactivation chemicals (e.g., formalin, beta propiolactone (BPL), bromoethylamine (BEA), and binary 0 ethylenimine (BEI)) or by non-chemical methods (e.g., heat, freeze/thaw, or sonication) to disable the replication capacity of the virus.

In the examples that follow, equine/Ohio/03 was used as a challenge virus. It is known to have about 99% homology with Florida/43/04 isolates, and has been shown to induce symptoms of infection and seroconversion in dogs. Example 18 illustrates the 5 efficacy of equine influenza vaccine in dogs, showing hemagglutination inhibition (or HI or HAI) titers in dogs vaccinated with inactivated Ohio 03 antigen in a vaccine composition comprising CARBIGEN™ adjuvant. Table 29 shows titers pre-vaccination, postvaccination, and post-second vaccination, as well as post-challenge. The results indicate HI titers at each stage post-vaccination for the vaccinated dogs, with little or no increase for Ό controls. Table 30 illustrates the clinical signs, virus isolation, and histopathology results from the same study. Although challenged animals did not show clinical signs, virus shedding, or positive histopathology, the positive HI titers (Table 29) indicate significant antibody titers in immunized animals.

It should be noted that other H3 influenza virus antigen vaccines are encompassed by 25 this invention as well. Those described in this specification and the following examples are provided to illustrate the invention and its preferred embodiments, and not to limit the scope of the invention claimed.

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

In general, the vaccine is administered in a therapeutically effective amount. A therapeutically effective amount is an amount sufficient to induce a protective response in

2015234384 02 Oct 2015 the canine patient against the target virus. Typically, a dosage is therapeutically effective if it prevents, reduces the risk of, delays the onset of, reduces the spread of, ameliorates, suppresses, or eradicates the influenza or one or more (typically two or more) of its symptoms. Typical influenza symptoms include, for example, fever (for dogs, typically 5 >103.0°F; >39.4°C), cough, sneezing, histopathological lesions, ocular discharge, nasal discharge, vomiting, diarrhea, depression, weight loss, gagging, hemoptysis, and/or audible rales. Other often more severe symptoms may include, for example, hemorrhage in the lungs, mediastanum, or pleural cavity; tracheitis; bronchitis; bronchiolitis; supportive bronchopneumonia; and/or infiltration of the epithelial lining and airway lumens of the lungs 0 with neutrophils and/or macrophages.

The vaccine may be administered as part of a combination therapy, i.e., a therapy that includes, in addition to the vaccine itself, administering one or more additional active agents, adjuvants, therapies, etc. In that instance, it should be recognized 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 the vaccine were to be administered alone. Other therapies may include those known in the art, such as, for example, anti-viral medications, analgesics, fever-reducing medications, expectorants, anti-inflammation medications, antihistamines, antibiotics to treat bacterial infection that results from the influenza virus infection, rest, and/or administration of fluids. In some embodiments, the

Ό vaccine of this invention is administered in combination with a bordetella vaccine, adenovirus vaccine, and/or parainfluenza virus vaccine.

In some embodiments, for example, a typical dose for a live attenuated vaccine is at least about 10 pfu/canine, and more typically from about 10 to about 10 pfu/canine. In this patent, pfu means plaque forming units. In some embodiments, a typical dose for a live attenuated vaccine is at least about 10 TCIDso/canine, and more typically from about 10 to about 10⁹ TCIDso/canine. In some embodiments, a typical dose for a live attenuated vaccine is at least about 10 EIDso/canine, and more typically from about 10 to about 10 EIDso/canine. In some embodiments, a typical dose for a killed vaccine is at least about 40 HA units, typically from about 40 to about 10,000 HA units, and more typically from about

500 to about 6200 HA units. In some embodiments, the dose is from about 6100 to about

6200 HA units.

In some preferred embodiments, the vaccine comprises a live attenuated vaccine at a concentration which is at least about IO^{0 5} pfu/canine greater than the immunogenicity level.

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In some preferred embodiments, the vaccine comprises a live attenuated vaccine at a concentration which is at least about IO⁰⁵ TCIDWcanine greater than the immunogenicity level. In some preferred embodiments, the vaccine comprises a live attenuated vaccine at a concentration which is at least about IO⁰⁵ EIDso/canine greater than the immunogenicity 5 level.

The immunogenicity level may be determined experimentally by challenge dose titration study techniques generally known in the art. Such techniques typically include vaccinating a number of canines with the vaccine at different dosages, and then challenging the canines with the virulent virus to determine the minimum protective dose.

Factors affecting the preferred dosage regimen may include, for example, the type (e.g., species and breed), age, weight, sex, diet, activity, lung size, and condition of the subject; the route of administration; the efficacy, safety, and duration-of-immunity profiles of the particular vaccine used; whether a delivery system is used; and whether the vaccine is administered as part of a drug and/or vaccine combination. Thus, the dosage actually employed can vary for specific animals, and, therefore, can deviate from the typical dosages set forth above. Determining such dosage adjustments is generally within the skill of those in the art using conventional means. It should further be noted that live attenuated viruses are generally self-propagating; thus, the specific amount of such a virus administered is not necessarily critical.

Ό It is contemplated that the vaccine may be administered to the canine patient a single time; or, alternatively, two or more times over days, weeks, months, or years. In some embodiments, the vaccine is administered at least two times. In some such embodiments, for example, the vaccine is administered twice, with the second dose (e.g., the booster) being administered at least about 2 weeks after the first. In some embodiments, the vaccine is administered twice, with the second dose being administered no greater than 8 weeks after the first. In some embodiments, the second dose is administered at from about 2 weeks to about 4 years after the first dose, from about 2 to about 8 weeks after the first dose, or from about 3 to about 4 weeks after the first dose. In some embodiments, the second dose is administered about 4 weeks after the first dose. In the above embodiments, the first and subsequent dosages may vary, such as, for example, in amount and/or form. Often, however, the dosages are the same as to amount and form. When only a single dose is administered, the amount of vaccine in that dose alone generally comprises a therapeutically effective

2015234384 02 Oct 2015 amount of the vaccine. When, however, more than one dose is administered, the amounts of vaccine in those doses together may constitute a therapeutically effective amount.

In some embodiments, the vaccine is administered before the canine recipient is infected with influenza. In such embodiments, the vaccine may, for example, be 5 administered to prevent, reduce the risk of, or delay the onset of influenza or one or more (typically two or more) influenza symptoms.

In some embodiments, the vaccine is administered after the canine recipient is infected with influenza. In such embodiments, the vaccine may, for example, ameliorate, suppress, or eradicate the influenza or one or more (typically two or more) influenza 0 symptoms.

The preferred composition of the vaccine depends on, for example, whether the vaccine is an inactivated vaccine, live attenuated vaccine, or both. It also depends on the method of administration of the vaccine. It is contemplated that the vaccine will comprise one or more conventional pharmaceutically acceptable carriers, adjuvants, other immune 5 response enhancers, and/or vehicles (collectively referred to as excipients). Such excipients are generally selected to be compatible with the active ingredient(s) in the vaccine. Use of excipients is generally known to those skilled in the art.

The term “pharmaceutically acceptable” is used adjectivally to mean that the modified noun is appropriate for use in a pharmaceutical product. When it is used, for example, to

Ό describe an excipient in a pharmaceutical vaccine, it characterizes the excipient as being compatible with the other ingredients of the composition and not disadvantageously deleterious to the intended recipient canine.

The vaccines may be administered by conventional means, including, for example, mucosal administration, (such as intranasal, oral, intratracheal, and ocular), and parenteral administration. Mucosal administration is often particularly advantageous for live attenuated vaccines. Parenteral administration is often particularly advantageous for inactivated vaccines.

Mucosal vaccines may be, for example, liquid dosage forms, such as pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. Excipients suitable for such vaccines include, for example, inert diluents commonly used in the art, such as, water, saline, dextrose, glycerol, lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate,

2015234384 02 Oct 2015 polyvinylpyrrolidone, and/or polyvinyl alcohol. Excipients also can comprise various wetting, emulsifying, suspending, flavoring (e.g., sweetening), and/or perfuming agents.

Oral mucosal vaccines also may, for example, be tableted or encapsulated for convenient administration. Such capsules or tablets can contain a controlled-release 5 formulation. In the case of capsules, tablets, and pills, the dosage forms also can comprise buffering agents, such as sodium citrate, or magnesium or calcium carbonate or bicarbonate. Tablets and pills additionally can be prepared with enteric coatings.

It is contemplated that the vaccine may be administered via the canine patient's drinking water and/or food. It is further contemplated that the vaccine may be administered 0 in the form of a treat or toy.

Parenteral administration includes subcutaneous injections, submucosal injections, intravenous injections, intramuscular injections, intrasternal injections, transcutaneous injections, and infusion. Injectable preparations (e.g., sterile injectable aqueous or oleaginous suspensions) can be formulated according to the known art using suitable excipients, such as vehicles, solvents, dispersing, wetting agents, emulsifying agents, and/or suspending agents. These typically include, for example, water, saline, dextrose, glycerol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, benzyl alcohol, 1,3-butanediol, Ringer's solution, isotonic sodium chloride solution, bland fixed oils (e.g., synthetic mono- or diglycerides), fatty acids (e.g., oleic acid), dimethyl acetamide, surfactants (e.g., ionic and

Ό non-ionic detergents), propylene glycol, and/or polyethylene glycols. Excipients also may include small amounts of other auxiliary substances, such as pH buffering agents.

The vaccine may include one or more excipients that enhance a canine patient's immune response (which may include an antibody response, cellular response, or both), thereby increasing the effectiveness of the vaccine. Use of such excipients (or adjuvants) may be particularly beneficial when using an inactivated vaccine. The adjuvant(s) may be a substance that has a direct (e.g., cytokine or Bacille Calmette-Guerin (BCG)) or indirect effect (liposomes) on cells of the canine patient's immune system. Examples of often suitable adjuvants include oils (e.g., mineral oils), metallic salts (e.g., aluminum hydroxide or aluminum phosphate), bacterial components (e.g., bacterial liposaccharides, Freund’s adjuvants, and/or MDP), plant components (e.g., Quil A), and/or one or more substances that have a carrier effect (e.g., bentonite, latex particles, liposomes, and/or Quil A, ISCOM). As noted above, adjuvants also include, for example, CARBIGEN™ and carbopol. It should be

2015234384 02 Oct 2015 recognized that this invention encompasses both vaccines that comprise an adjuvant(s), as well as vaccines that do not comprise any adjuvant.

It is contemplated that the vaccine may be freeze-dried (or otherwise reduced in liquid volume) for storage, and then reconstituted in a liquid before or at the time of administration. 5 Such reconstitution may be achieved using, for example, vaccine-grade water.

The present invention further comprises kits that are suitable for use in performing the methods described above. The kit comprises a dosage form comprising a vaccine described above. The kit also comprises at least one additional component, and, typically, instructions for using the vaccine with the additional component(s). The additional component(s) may, 0 for example, be one or more additional ingredients (such as, for example, one or more of the excipients discussed above, food, and/or a treat) that can be mixed with the vaccine before or during administration. The additional component(s) may alternatively (or additionally) comprise one or more apparatuses for administering the vaccine to the canine patient. Such an apparatus may be, for example, a syringe, inhaler, nebulizer, pipette, forceps, or any 5 medically acceptable delivery vehicle. In some embodiments, the apparatus is suitable for subcutaneous administration of the vaccine. In some embodiments, the apparatus is suitable for intranasal administration of the vaccine.

Other excipients and modes of administration known in the pharmaceutical or biologies arts also may be used.

Ό The vaccine or immunogenic compositions of the subject invention also encompass recombinant viral vector-based constructs that may comprise, for example, genes encoding HA protein, NA protein, nucleoprotein, polymerase basic protein, polymerase acidic protein, and/or matrix protein of an influenza virus of the present invention. Any suitable viral vector that can be used to prepare a recombinant vector/virus construct is contemplated for use with the subject invention. For example, viral vectors derived from adenovirus, avipox, herpesvirus, vaccinia, canarypox, entomopox, swinepox, West Nile virus and others known in the art can be used with the compositions and methods of the present invention. Recombinant polynucleotide vectors that encode and express components can be constructed using standard genetic engineering techniques known in the art. In addition, the various vaccine compositions described herein can be used separately and in combination with each other. For example, primary immunizations of an animal may use recombinant vector-based constructs, having single or multiple strain components, followed by secondary boosts with vaccine compositions comprising inactivated virus or inactivated virus-infected cell lines.

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Other immunization protocols with the vaccine compositions of the invention are apparent to persons skilled in the art and are contemplated within the scope of the present invention.

The subject invention also concerns reassortant virus comprising at least one gene or genomic segment of an influenza virus of the present invention and the remainder of viral 5 genes or genomic segments from a different influenza virus of the invention or from an influenza virus other than a virus of the present invention. Reassortant virus can be produced by genetic reassortant of nucleic acid of a donor influenza virus of the present invention with nucleic acid of a recipient influenza virus and then selecting for reassortant virus that comprises the nucleic acid of the donor virus. Methods to produce and isolate reassortant 0 virus are well known in the art (Fields et al., 1996). In one embodiment, a reassortant virus of the invention comprises genes or genomic segments of a human, avian, swine, or equine influenza virus. A reassortant virus of the present invention can include any combination of nucleic acid from donor and recipient influenza virus so long as the reassortant virus comprises at least one gene or genomic segment from a donor influenza virus of the present 5 invention. In one embodiment, a recipient influenza virus can be an equine influenza virus.

Natural, recombinant or synthetic polypeptides of viral proteins, and peptide fragments thereof, can also be used as vaccine compositions according to the subject methods. In one embodiment, a vaccine composition comprises a polynucleotide or a polypeptide of a canine influenza virus. In one embodiment, a vaccine composition Ό comprises 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 a functional and/or immunogenic fragment or variant thereof. In a specific embodiment, the 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, 25 30, 32, 33, 34, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78, comprises 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, 30 62, 64, 66, 68, 70, 72, 74, 76, or 78. In a further specific embodiment, a polynucleotide of the invention can comprise: Nucleotides 1-2271 of SEQ ID NO: 3; Nucleotides 1-2148 of SEQ ID NO: 5; Nucleotides 1-657 of SEQ ID NO: 7; Nucleotides 1-1494 of SEQ ID NO: 9; Nucleotides 1-1410 of SEQ ID NO: 11; Nucleotides 1-756 of SEQ ID NO: 13; Nucleotides

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1-1695 of SEQ ID NO: 15; Nucleotides 1-2271 of SEQ ID NO: 19; Nucleotides 1-2148 of SEQ ID NO: 21; Nucleotides 1-657 of SEQ ID NO: 23; Nucleotides 1-1494 of SEQ ID NO: 25; Nucleotides 1-756 of SEQ ID NO: 29; Nucleotides 1-1695 of SEQ ID NO: 31; Nucleotides 1-2277 of SEQ ID NO: 47; Nucleotides 1-2271 of SEQ ID NO: 49; Nucleotides 5 1-2148 of SEQ ID NO: 51; Nucleotides 1-690 of SEQ ID NO: 53; Nucleotides 1-1494 of

SEQ ID NO: 55; Nucleotides 1-1410 of SEQ ID NO: 57; Nucleotides 1-756 of SEQ ID NO: 59; Nucleotides 1-1695 of SEQ ID NO: 61; Nucleotides 1-2277 of SEQ ID NO: 63; Nucleotides 1-2271 of SEQ ID NO: 65; Nucleotides 1-2148 of SEQ ID NO: 67; Nucleotides 1-690 of SEQ ID NO: 69; Nucleotides 1-1494 of SEQ ID NO: 71; Nucleotides 1-1410 of 0 SEQ ID NO: 73; Nucleotides 1-756 of SEQ ID NO: 75; and Nucleotides 1-1695 of SEQ ID NO: 77. In another embodiment, a vaccine composition comprises 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 or variant thereof. In a further embodiment, a 5 vaccine composition comprises a polynucleotide or a polypeptide of an equine influenza virus wherein the polynucleotide or polypeptide has at least about 90%, or at least about 95%, or at least about 96%, or 97%, or 98%, or 99% or more sequence identity with a canine influenza polynucleotide or polypeptide. In one embodiment, viral polypeptides derived from multiple strains can be combined in a vaccine composition and are used to vaccinate a host animal. Ό For example, polypeptides based on the viral HA protein from at least two different strains of influenza virus of the invention can be combined in the vaccine. The polypeptides may be homologous to one strain or may comprise Ahybrid@ or Achimeric@ polypeptides whose amino acid sequence is derived from joining or linking polypeptides from at least two distinct strains. Procedures for preparing viral polypeptides are well known in the art. For example, 25 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 techniques wherein a polynucleotide molecule encoding an viral protein or peptide is expressed in a host cell, such as bacteria, yeast, or mammalian cell lines, and the expressed protein purified using standard techniques of the art.

Vaccine compositions of the present invention also include naked nucleic acid compositions. In one embodiment, a nucleic acid may comprise a nucleotide sequence encoding an HA and/or an NA protein of an influenza virus of the present invention. Methods for nucleic acid vaccination are known in the art and are described, for example, in

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U.S. Patent Nos. 6,063,385 and 6,472,375. The nucleic acid can 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, that 5 can be used in accordance with the present invention can be provided with a pharmaceutically-acceptable carrier or diluent. Compounds and compositions useful in the subject invention can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, 0 Remington’s Pharmaceutical Science by E.W. Martin, Easton Pennsylvania, Mack Publishing Company, 19^th ed., 1995, describes formulations which can be used in connection with the subject invention. In general, the compositions of the subject invention will be formulated such that an effective amount of an immunogen is combined with a suitable carrier in order to facilitate effective administration of the composition. The compositions 5 used in the present methods can also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharmaceutically acceptable carriers and Ό diluents which are known to those skilled in the art. Examples of carriers or diluents for use with the subject peptidomimetics include, but are not limited to, water, saline, oils including mineral oil, ethanol, dimethyl sulfoxide, gelatin, cyclodextrans, magnesium stearate, dextrose, cellulose, sugars, calcium carbonate, glycerol, alumina, starch, and equivalent carriers and diluents, or mixtures of any of these. Formulations of an immunogen of the 25 invention can also comprise suspension agents, protectants, lubricants, buffers, preservatives, and stabilizers. To provide for the administration of such dosages for the desired therapeutic treatment, pharmaceutical compositions of the invention will advantageously comprise between about 0.1% and 45%, and especially, 1 and 15% by weight of the immunogen or immunogens based on the weight of the total composition including carrier or diluent.

The vaccine and immunogenic compositions of the subject invention can be prepared by procedures well known in the art. For example, the vaccine or immunogens are typically prepared as injectables, e.g., liquid solutions or suspensions. The vaccine or immunogens are administered in a manner that is compatible with dosage formulation, and in such amount as

2015234384 02 Oct 2015 will be therapeutically effective and immunogenic in the recipient. The optimal dosages and administration patterns for a particular vaccine or immunogens formulation can be readily determined by a person skilled in the art.

Peptides and/or polypeptides of the present invention can also be provided in the form 5 of a multiple antigenic peptide (MAP) construct. The preparation of MAP constructs has been described in Tam (1988). MAP constructs utilize a core matrix of lysine residues onto which multiple copies of an immunogen are synthesized (Posnett et al., 1988). Multiple MAP constructs, each containing the same or different immunogens, can be prepared and administered in a vaccine composition in accordance with methods of the present invention. 0 In one embodiment, a MAP construct is provided with and/or administered with one or more adjuvants. Influenza polypeptides of the invention can also be produced and administered as macro molecular protein structures comprising one or more polypeptides. Published U.S. Patent Application US2005/0009008 describes methods for producing virus-like particles as a vaccine for influenza virus.

According to the methods of the subject invention, the vaccine and immunogenic compositions described herein are administered to susceptible hosts, typically canids, and more typically domesticated dogs, in an effective amount and manner to induce protective immunity against subsequent challenge or infection of the host by virus. In one embodiment, the host animal is a canid. Canines include wild, zoo, and domestic canines, such as wolves,

Ό coyotes, and foxes. Canines also include dogs, particularly domestic dogs, such as, for example, pure-bred and/or mongrel companion dogs, show dogs, working dogs, herding dogs, hunting dogs, guard dogs, police dogs, racing dogs, and/or laboratory dogs. In a specific embodiment, the host animal is a domesticated dog, such as a greyhound. The vaccines or immunogens are typically administered parenterally, by injection, for example, either subcutaneously, intraperitoneally, or intramuscularly. Other suitable modes of administration include oral or nasal administration. Usually, the vaccines or immunogens are administered to an animal at least two times, with an interval of one or more weeks between each administration. However, other regimens for the 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.

Virus and virus-infected cells in a vaccine formulation may 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 propio lactone

2015234384 02 Oct 2015 (BPL), bromoethylamine (BEA), binary ethylenimine (BEI), phenol, UV light, elevated temperature, freeze thawing, sonication (including ultrasonication), and the like. The amount of cell-free whole virus in a vaccine dose can be in the range from about 0.1 mg to about 5 mg, and more usually being from about 0.2 mg to about 2 mg. The dosage for vaccine 5 formulations comprising virus-infected cell lines will usually contain from about 10⁶ to about 10 cells per dose, and more usually from about 5x10 to about 7.5 x 10 cells per dose. The amount of protein or peptide immunogen in a dose for an animal can vary from about 0.1 pg to 10000 pg, or about 1 pg to 5000 pg, or about 10 pg to 1000 pg, or about 25 pg to 750 pg, or about 50 pg to 500 pg, or 100 pg to 250 pg, depending upon the size, age, etc., of the 0 animal receiving the dose.

An immunogenic or vaccine composition of the invention, such as virus or virusinfected cells or viral proteins or peptides, can be combined with an adjuvant, typically just prior to administration. Adjuvants contemplated for use in the vaccine formulations include threonyl muramyl dipeptide (MDP) (Byars et al., 1987), saponin, Cornebacterium parvum,

Freund’s complete and Fruend’s incomplete adjuvants, aluminum, or a mixture of any of these. A variety of other adjuvants suitable for use with the methods and vaccines of the subject invention, such as alum, are well known in the art and are contemplated for use with the subject invention.

The subject invention also concerns antibodies that bind specifically to a protein or a

Ό peptide of the present invention. Antibodies of the subject invention include monoclonal and polyclonal antibody compositions. Preferably, the antibodies of the subject invention are monoclonal antibodies. Whole antibodies and antigen binding fragments thereof are contemplated in the present invention. Thus, for example, suitable antigen binding fragments include Fab2, Fab and Fv antibody fragments. Antibodies of the invention can be labeled with a detectable moiety, such as a fluorescent molecule (e.g., fluorescein or an enzyme).

The subject invention also concerns methods and compositions for detection and identification of an influenza virus of the invention and for diagnosis of infection of an animal with an influenza virus of the present invention. The methods of the invention include detection of the presence of canine influenza, in a biological sample from an animal. The detection of canine influenza in a sample, is useful to diagnose canine influenza in an animal. In turn, this information can provide the ability to determine the prognosis of an animal based on distinguishing levels of canine influenza present over time, and can assist in selection of therapeutic agents and treatments for the animal, and assist in monitoring therapy. The

2015234384 02 Oct 2015 method also provides the ability to establish the absence of canine influenza in an animal tested.

The ability to detect canine influenza in an animal permits assessment of outbreaks of canine influenza in different geographical locations. This information also permits early 5 detection so that infected animals can be isolated, to limit the spread of disease, and allows early intervention for treatment options. In addition, having this information available can provide direction to medical personnel for preparing to treat large numbers of ill animals, including assembling medical supplies, and, if available, vaccines.

In one embodiment, a method of the present invention involves the collection of a 0 biological sample from a test animal, such as a canine. The biological sample may be any biological material, including, cells, tissue, hair, whole blood, serum, plasma, nipple aspirate, lung lavage, cerebrospinal fluid, saliva, sweat and tears.

The animal test sample may come from an animal suspected of having canine influenza virus, whether or not the animal exhibits symptoms of the disease. Control samples 5 can also be provided or collected from animals known to be free of canine influenza. Additional controls may be provided, e.g., to reduce false positive and false negative results, and verify that the reagents in the assay are actively detecting canine influenza A virus.

In addition to detecting the presence or absence of canine influenza in a biological sample, the methods of detection used in the invention can detect mutations in canine Ό influenza virus, such as changes in nucleic acid sequence, that may result from the environment, drug treatment, genetic manipulations or mutations, injury, change in diet, aging, or any other characteristic(s) of an animal. Mutations may also cause canine influenza A to become resistant to a drug that was formerly effective, or to enable the virus to infect and propagate in a different species of animal, or human. For example, avian influenza A 25 virus has been shown to infect other animals and humans.

In one embodiment for detecting an influenza virus in an animal, diagnosis is facilitated by the collection of high-quality specimens, their rapid transport to a testing facility, and appropriate storage, before laboratory testing. Virus is best detected in specimens containing infected cells and secretions. In one embodiment, specimens for the 30 direct detection of viral antigens and/or for nucleic acids and/or virus isolation in cell cultures are taken during the first 3 days after onset of clinical symptoms. A number of types of specimens are suitable to diagnose virus infections of the upper respiratory tract, including, but not limited to, nasal swab, nasopharyngeal swab, nasopharyngeal aspirate, nasal wash and

2015234384 02 Oct 2015 throat swabs. In addition to swabs, samples of tissue or serum may be taken, and invasive procedures can also be performed.

In one embodiment, respiratory specimens are collected and transported in 1 -5 ml of virus transport media. A number of media that are satisfactory for the recovery of a wide 5 variety of viruses are commercially available. Clinical specimens are added to transport medium. Nasal or nasopharyngeal swabs can also be transported in the virus transport medium. One example of a transport medium is 10 gm of veal infusion broth and 2 gm of bovine albumin fraction V, added to sterile distilled water to 400 m. Antibiotics such as 0.8 ml gentamicin sulfate solution (50 mg/ml) and 3.2 ml amphotericin B (250 pg/ml) can also 0 be added. The medium is preferably sterilized by filtration. Nasal washes, such as sterile saline (0.85% NaCl), can also be used to collect specimens of respiratory viruses.

In one embodiment, sera is collected in an amount of from 1-5 ml of whole blood from an acute-phase animal, soon after the onset of clinical symptoms, and preferably not later than 7 days. A convalescent-phase serum specimen can also be collected, for example 5 at about 14 days after onset of symptoms. Serum specimens can be useful for detecting antibodies against respiratory viruses in a neutralization test.

In some instances, samples may be collected from individual animals over a period of time (e.g., once a day, once a week, once a month, biannually or annually). Obtaining numerous samples from an individual animal, over a period of time, can be used to verify Ό results from earlier detections, and/or to identify response or resistance to a specific treatment, e.g., 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 the pathological agent can be used, including, but not limited to, antibody assays including enzyme-linked immunosorbent assays (ELISAs), indirect fluorescent antibody (IFA) tests, hemagglutinating, and inhibition of hemagglutination (HI) assays, and Western Blot. Known cell-culture methods can also be used. Positive cultures can be further identified using immunofluorescence of cell cultures or HI assay of the cell culture medium (supernatant).

In addition, methods for detecting nucleic acid (DNA or RNA) or protein can be used.

Such methods include, but are not limited to, polymerase chain reaction (PCR), and reverse transcriptase (RT) PCR tests and real time tests, and quantitative nuclease protection assays.

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There are commercially available test kits available to perform these assays. For example, QIAGEN (Valencia, CA) sells a one step RT-PCR kit, and viral RNA extraction kit.

In one embodiment, the method utilizes an antibody specific for a virus or viral protein of the invention. In a specific embodiment, an antibody specific for an HA protein of 5 a virus of the invention is utilized. In another embodiment, an antibody specific for an NP protein of a virus of the invention is used. A suitable sample, such as from the nasal or nasopharyngeal region, is obtained from an animal and virus or viral protein is isolated therefrom. The viral components are then screened for binding of an antibody specific to a protein, such as HA or NP, of a virus of the invention. In another embodiment, a serum 0 sample (or other antibody containing sample) is obtained from an animal and the serum screened for the presence of antibody that binds to a protein of a virus of the invention. For example, an EFISA assay can be performed where the plate walls have HA and/or NP protein, or a peptide fragment thereof, bound to the wall. The plate wall is then contacted with serum or antibody from a test animal. The presence of antibody in the animal that binds 5 specifically to the HA and/or NP protein is indicative that the test animal is infected or has been infected with an 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 the agent, in a biological sample. It can take some time (e.g. months) after an animal is infected before antibodies can be detected in a Ό blood test. Once formed, antibodies usually persist for many years, even after successful treatment of the disease. Finding antibodies to canine influenza A may not indicate whether the infection was recent, or sometime in the past.

Antibody testing can also be done on fluid(s). Antibody assays include enzymelinked immunosorbent assays (EFISAs), indirect fluorescent antibody (IFA) assays, and 25 Western Blot. Preferably, antibody testing is done using multiple assays, for example EFISA or IFA followed by Western blot. Antibody assays can be done in a two-step process, using either an EFISA or IFA assay, followed by a Western blot assay. EFISA is considered a more reliable and accurate assay than IFA, but IFA may be used if EFISA is not available. The Western blot test (which is a more specific test) can also be done in all animals, 30 particularly those that have tested positive or borderline positive (equivocal) in an EFISA or IFA assay.

Other antibody-based tests that can be used for detection of influenza virus include hemagglutination inhibition assays. Hemagglutination activity can be detected in a biological

2015234384 02 Oct 2015 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 or an HA protein or peptide of the invention is contacted with a test sample containing serum or antibody. Red blood cells (RBC) from an animal, such as a bird, are then added. If antibody to HA is present, then 5 the RBC will not agglutinate. If antibody to HA is not present, the RBC will agglutinate in the presence of HA. Variations and modifications to standard hemagglutination inhibition assays are known in the art and contemplated within the scope of the present invention.

Infection of an animal can also be determined by isolation of the virus from a sample, such as a nasal or nasopharyngeal swab. Viral isolation can be performed using standard 0 methods, including cell culture and egg inoculation.

In a further embodiment, a nucleic acid-based assay can be used for detection of a virus of the present invention. In one embodiment, a nucleic acid sample is obtained from an animal and the nucleic acid subjected to PCR using primers that will generate an amplification product if the nucleic acid contains a sequence specific to an influenza virus of 5 the present invention. In a specific embodiment, RT-PCR is used in an assay for the subject virus. In an exemplified embodiment, real-time RT-PCR is used to assay for an influenza virus of the invention. PCR, RT-PCR and real-time PCR methods are known in the art and have been described in U.S. Patent Nos. 4,683,202; 4,683,195; 4,800,159; 4,965,188; 5,994,056; 6,814,934; and in Saiki et al. (1985); Sambrook et al. (1989); Lee et al. (1993); Ό and Livak et al. (1995). In one embodiment, the PCR assay uses oligonucleotides specific for an influenza matrix (MA) gene and/or HA gene. The amplification product can also be sequenced to determine if the product has a sequence of an influenza virus of the present invention. Other nucleic acid-based assays can be used for detection and diagnosis of viral infection by a virus of the invention and such assays are contemplated within the scope of the 25 present invention. In one embodiment, a sample containing a nucleic acid is subjected to a PCR-based amplification using forward and reverse primers where the primers are specific for a viral polynucleotide or gene sequence. If the nucleic acid in the sample is RNA, then RT-PCR can be performed. For real-time PCR, a detectable probe is utilized with the primers.

Primer sets specific for the hemagglutinin (HA) gene of many of the circulating influenza viruses are known, and are continually being developed. The influenza virus genome is single-stranded RNA, and a DNA copy (cDNA) must be made using a reverse transcriptase (RT) polymerase. The amplification of the RNA genome, for example using

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RT-PCR, requires a pair of oligonucleotide primers, typically designed on the basis of the known HA sequence of influenza A subtypes and of neuraminadase (NM)-l. The primers can be selected such that they will specifically amplify RNA of only one virus subtype. DNAs generated by using subtype-specific primers can be further analyzed by molecular genetic techniques such as sequencing. The test is preferably run with a positive control, or products are confirmed by sequencing and comparison with known sequences. The absence of the target PCR products (i.e, a “negative” result) may not rule out the presence of the virus. Results can then be made available within a few hours from either clinical swabs or infected cell cultures. PCR and RT-PCR tests for influenza A virus are described by Fouchier et al.,

2000 and Maertzdorf et al., 2004.

The subject invention also concerns methods for screening for compounds or drugs that have antiviral activity against a virus of the present invention. In one embodiment, cells infected with a virus of the invention are contacted with a test compound or drug. The amount of virus or viral activity following contact is then determined. Those compounds or drugs that exhibit antiviral activity can be selected for further evaluation.

The subject invention also concerns isolated cells infected with an influenza virus of the present invention. In one embodiment, the cell is a canine cell, such as canine kidney epithelial cells.

The subject invention also concerns cells transformed with a polynucleotide of the

Ό present invention encoding a polypeptide of the invention. Preferably, the polynucleotide sequence is provided in an expression construct of the invention. More preferably, the expression construct provides for overexpression in the cell of an operably linked polynucleotide of the invention. In one embodiment, the cell is transformed with a polynucleotide sequence comprising 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, the cell is transformed with 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 comprises 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 fragment or variant of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12,

2015234384 02 Oct 2015

74, 76, or 78. Thus, the subject invention concerns cells transformed with 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, of 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.

The transformed cell can be a eukaryotic cell, for example, a plant cell, including protoplasts, or the transformed cell can be a prokaryotic cell, for example, a bacterial cell such as E. coli or B. subtilis. Animal cells include human cells, mammalian cells, partially canine cells, avian cells, and insect cells. Plant cells include, but are not limited to, dicotyledonous, monocotyledonous, and conifer cells.

The subject invention also concerns plants, including transgenic plants that express and produce a viral protein or polypeptide of the present invention. Plants, plant tissues, and plant cells transformed with or bred to contain a polynucleotide of the invention are contemplated by the present invention. Preferably, the polynucleotide of the invention is overexpressed in the plant, plant tissue, or plant cell. Plants can be used to produce influenza vaccine compositions of the present invention and the vaccines can be administered through consumption of the plant (see, for example, U.S. Patent Nos. 5,484,719 and 6,136,320).

The subject invention also concerns kits for detecting a virus or diagnosing an infection by a virus of the present invention. In one embodiment, a kit comprises an antibody of the invention that specifically binds to an influenza virus of the present invention, or an antigenic portion thereof. In another embodiment, a kit comprises one or more polypeptides or peptides of the present invention. In a specific embodiment, the polypeptides have an 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. In a further embodiment, a kit comprises one or more polynucleotides or oligonucleotides of the present invention. In a specific embodiment, the polynucleotides have a 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. A kit may optionally comprise one or more control antibody, control polypeptide or peptide, and/or control polynucleotide or oligonucleotide. The antibody, polypeptides, peptides, polynucleotides, and/or oligonucleotides of the kit can be provided in a suitable container or package.

2015234384 02 Oct 2015

The subject application also concerns the use of mongrel dogs as a model for infection and pathogenesis of influenza virus. In one embodiment, a mongrel dog is inoculated with an influenza virus, such as a canine influenza virus of the present invention. Optionally, the dog can be administered therapeutic agents subsequent to inoculation. The dog can also have 5 been administered a composition for generating an immune response against an influenza virus prior to inoculation with virus. Tissue, blood, serum, and other biological samples can be obtained before and/or after inoculation and examined for the presence of virus and pathogenesis of tissue using methods known in the art including, but not limited to, PCR, RTPCR, nucleic acid sequencing, and immunohistochemistry.

Canine influenza virus strains (designated as “A/canine/Florida/43/2004” and “A/canine/Florida/242/2003”) were deposited with American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, on October 9, 2006. Canine influenza virus strains (designated as “canine/Jax/05” and “canine/Miami/05”), were deposited with American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, on

October 17, 2006. The subject virus strains have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposit will be available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed.

Ό However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Further, the subject virus deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., it will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the culture. The depositor acknowledges the duty to replace the deposit should the depository be unable to furnish a sample when requested, due to the condition of the deposit. All restrictions on the availability to the public of the subject culture deposit will be irrevocably removed upon the granting of a patent disclosing it.

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

Any element of any embodiment disclosed herein can be combined with any other element or embodiment disclosed herein and such combinations are specifically contemplated within the scope of the present invention.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, 0 to the extent they are not inconsistent with the explicit teachings of this specification.

MATERIALS AND METHODS FOR EXAMPLES 1-6

Blood and Nasal Swab Collection from Greyhounds.

Acute and convalescent blood samples were collected by jugular venipuncture from clinically diseased or normal greyhounds in racing kennels experiencing outbreaks of respiratory disease. Convalescent samples were collected 4 to 12 weeks after the acute sample. Serum was harvested and stored at -80°C. Nasal swabs were collected and placed in Amies transport medium with charcoal (Becton Dickinson Biosciences) pending submission

Ό for bacterial isolation.

Postmortem examination of greyhounds.

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

Serological tests for canine viral respiratory pathogens.

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Paired acute and convalescent serum samples were submitted 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 were expressed as the last dilution of serum that 5 inhibited viral infection of cell cultures. Seroconversion, defined as a > 4-fold increase in antibody titer between the acute and convalescent sample, indicated viral infection. No seroconversions to these viral pathogens were detected.

Microbial tests for canine bacterial respiratory pathogens.

Paired nasal swabs and postmortem tissues were submitted to the Diagnostic Clinical

Microbiology/Parasitology/Serology Service at the UF CVM for bacterial isolation and identification. The samples were cultured on nonselective media as well as media selective for Bordetella species (Regan-Lowe; Remel) and Mycoplasma species (Remel). All cultures were held for 21 days before reporting no growth. Nasal swabs from some of the greyhounds 5 were also submitted to the Department of Diagnostic Medicine/Pathobiology at the Kansas State University College of Veterinary Medicine for bacterial culture. Of 70 clinically diseased dogs tested, Bordetella bronchiseptica was isolated from the nasal cavity of 1 dog, while Mycoplasma spp. were recovered from the nasal cavity of 33 dogs. Pasteurella multocida was commonly recovered from the nasal cavity of dogs with purulent nasal Ό discharges. Two of the dogs that died in the January 2004 outbreak had scant growth of Escherichia coli in the lungs postmortem, one dog had scant growth of E. coli and Streptococcus canis, and another had scant growth of Pseudomonas aeruginosa and a yeast. Neither Bordetella bronchiseptica nor Mycoplasma was isolated from the trachea or lungs of dogs that died.

Virus isolation from postmortem tissues.

Frozen tissues were thawed and homogenized in 10 volumes of minimum essential medium (MEM) supplemented with 0.5% bovine serum albumin (BSA) and antibiotics. Solid debris was removed by centrifugation and supernatants were inoculated onto cultured cells or into 10-day old embryonated chicken eggs. Tissue homogenates from greyhounds that died were inoculated into diverse cell cultures that supported the replication of a broad range of viral pathogens. The cell cultures included Vero (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),

2015234384 02 Oct 2015 primary canine kidney epithelial cells (AHDL, Cornell University), primary canine lung epithelial cells (AHDL), and primary bovine testicular cells (AHDL). MDCK and HRT cells were cultured in MEM supplemented with 2.5 ug/mL TPCK-treated trypsin (Sigma); the remaining cell lines were cultured in MEM supplemented with 10% fetal calf serum and 5 antibiotics. Cells were grown in 25 cm flasks at 37°C in a humidified atmosphere containing 5% CO2. A control culture was inoculated with the supplemented MEM. The cultures were observed daily for morphologic changes and harvested at 5 days post inoculation. The harvested fluids and cells were clarified by centrifugation and inoculated onto fresh cells as described for the initial inoculation; two blind passages were performed. Hemagglutination 0 activity in the clarified supernatants was determined using chicken or turkey red blood cells as described (Burleson et al., 1992; Kendal et al., 1982). For virus isolation in chicken embryos, 0.1 mL of tissue homogenate was inoculated into the allantoic sac and incubated for 48 hours at 35°C. After two blind passages, the hemagglutination activity in the allantoic fluids was determined as described (Burleson et al., 1992; Kendal et al., 1982).

RT-PCR, nucleotide sequencing, and phylogenetic analyses.

Total RNA was extracted from tissue culture supernatant or allantoic fluid using the

RNeasy kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. The total RNA (10 ng) was reverse transcribed to cDNA using a one-step RT-PCR Kit (Qiagen, Valencia,

Ό CA) according to manufacturer’s instructions. PCR amplification of the coding region of the 8 influenza viral genes in the cDNA was performed as previously described (Klimov et al., 1992a), using universal gene-specific primer sets. The resulting DNA amplicons were used as templates for automated sequencing on an Applied Biosystems 3100 automated DNA sequencer using cycle sequencing dye terminator chemistry (ABI). Nucleotide sequences were analyzed using the GCG Package®, Version 10.0 (Accelyrs) (Womble, 2000). The Phytogeny Inference Package Version 3.5 was used to estimate phytogenies and calculate bootstrap values from the nucleotide sequences (Felsenstein, 1989). Phylogenetic trees were compared to those generated by neighbor-joining analysis with the Tamura-Nei gamma model implemented in the MEGA program (Kumar et al., 2004) and confirmed by the ©

PAUP 4.0 Beta program (Sinauer Associates).

Experimental inoculation of dogs.

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Four 6-month old specific pathogen-free beagles [(2 males and 2 females (Liberty Research)] were used. Physical examination and baseline blood tests including complete blood cell count/differential, serum chemistry panel, and urinalysis determined that the animals were healthy. They were housed together in a BSL 2-enhanced facility accredited by 5 the Association for Assessment and Accreditation of Laboratory Animal Care. Baseline rectal temperatures were recorded twice daily for 7 days. The dogs were anesthetized by intravenous injection of propofol (Diprivan®, Zeneca Pharmaceuticals, 0.4 mg/kg body weight to effect) for intubation with endotracheal tubes. Each dog was inoculated with a total dose of 10⁶⁶ median tissue culture infectious doses (TCID50) of A/Canine/Florida/43/2004 0 (Canine/FL/04) (H3N8) virus with half the dose administered into the distal trachea through the endotracheal tube and the other half administered into the deep nasal passage through a catheter. Physical examinations and rectal temperature recordings were performed twice daily for 14 days post inoculation (p.i.). Blood samples (4 mL) were collected by jugular venipuncture on days 0, 3, 5, 7, 10, and 14 p.i. Nasal and oropharyngeal specimens were 5 collected with polyester swabs (Fisher Scientific) from each dog on days 0 to 5, 7, 10, and 14 p.i. The swabs were placed in viral transport medium (Remel) and stored at -80°C. Two dogs (1 male and 1 female) were euthanatized by intravenous inoculation of Beuthanasia-D® solution (1 mL/5 kg body weight; Schering-Plough Animal Health Corp) on day 5 p.i. and the remaining 2 dogs on day 14 for postmortem examination. Tissues for histological analysis Ό were processed as described. Tissues for virus culture were stored at -80°C. This study was approved by the University of Florida Institutional Animal Care and Use Committee.

Virus shedding from experimentally inoculated dogs.

Serial dilutions of lung homogenates and swab extracts, prepared by clarification of 25 the swab transport media by centrifugation, were set up 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 plates. Inoculated cell monolayers were overlaid with supplemented MEM containing 0.8% agarose and 1.5 ug/mL of TPCKtrypsin. Cells were cultured for 72 hours at 37°C in a humidified atmosphere containing 5%

CO2 prior to fixation and staining with crystal violet. Virus concentration was expressed as plaque forming units (PFU) per gram of tissue or per swab.

Immunohistochemistry.

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Deparaffinized and rehydrated 5-pm lung tissue sections from the greyhounds and beagles were mounted on Bond-Rite™ slides (Richard-Allan Scientific, Kalamazoo, MI) and subsequently treated with proteinase K (DakoCytomation, Carpenteria, CA) followed by peroxidase blocking reagent (Dako® EnVision™ Peroxidase Kit, Dako Corp.). The sections 5 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 H3 (Chemicon International, Inc.) for 2 hours at room temperature. Controls included incubation of the same sections with mouse IgG (1 mg/mL, Serotec, Inc.), and incubation of the monoclonal antibodies with normal canine lung sections. Following 0 treatment with the primary antibodies, the sections were incubated with secondary immunoperoxidase and peroxidase substrate reagents (Dako® EnVision™ Peroxidase Kit, Dako Corp.) according to the manufacturer’s instructions. The sections were counterstained with hematoxylin, treated with Clarifier #2 and Bluing Reagent (Richard-Allan Scientific, Kalamazoo, MI), dehydrated, and coverslips applied with Permount (ProSciTech).

Hemagglutination inhibition (HI) assay.

Serum samples were incubated with receptor destroying enzyme (RDE, Denka) (1 part serum: 3 parts RDE) for 16 hours at 37°C prior to heat inactivation for 60 minutes at 56°C. Influenza A/Canine/FL/04 (H3N8) virus was grown in MDCK cells for 36-48 hr at

Ό 37°C. Virus culture supernatants were harvested, clarified by centrifugation, and stored at 80°C. The HI assay was performed as described previously (Kendal et al., 1982). Briefly, 4 hemagglutinating units of virus in 25pl 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 turkey erythrocytes was added and the hemagglutination titers were estimated visually after 30 minutes. The endpoint HI titer was defined as the last dilution of serum that completely inhibited hemagglutination. Seroconversion was defined as > 4-fold increase in HI titer between paired acute and convalescent samples. Seropositivity of a single sample was defined as a HI antibody titer >1:32.

Micro neutralization (MN) assay.

Neutralizing serum antibody responses to A/Canine/FL/04 (H3N8) were detected by a MN assay as described previously (Rowe et al., 1999) except that canine sera were RDE47

2015234384 02 Oct 2015 treated as described above prior to the assay. The endpoint titer was defined as the highest dilution of serum that gave 50% neutralization of 100 TCID50 of virus. Seroconversion was defined as > 4-fold increase in MN titer between paired acute and convalescent samples. Seropositivity of a single sample was defined as a MN titer >1:80.

Following are examples which illustrate procedures for practicing the invention.

These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1

In January 2004, an outbreak of respiratory disease occurred in 22 racing greyhounds housed in 2 kennels at a Florida track and the local farm that supplied dogs to these kennels. There were approximately 60 dogs in each kennel building and 300 dogs at the farm. The outbreak occurred over a 6-day period after which no new cases were identified. Fourteen of the 22 dogs had fevers of 39.5 to 41.5°C, a soft, gagging cough for 10 to 14 days, and eventual recovery. Of the remaining 8 dogs, 6 apparently healthy dogs died unexpectedly with hemorrhage from the mouth and nose. Two other dogs were euthanatized within 24 hours of onset of hemorrhage from the mouth and nose due to rapid deterioration. Both of these dogs had fevers of 41°C. Four of the 8 deaths occurred in the kennel buildings and 4 occurred at the farm. Fifty percent of the deaths occurred on day 3 of the outbreak. The 22

Ό dogs ranged in age from 17 months to 4 years, but 73% were 17 to 33 months old.

Two clinical syndromes were evident: a milder illness characterized by initial fever and then cough for 10-14 days (14 dogs) with subsequent recovery, or a peracute death associated with hemorrhage in the respiratory tract (8 dogs for a mortality rate of 36%). Postmortem examinations were performed on 6 of the 8 fatal cases. All dogs had extensive 25 hemorrhage in the lungs, mediastinum, and pleural cavity. Histological examination of the respiratory tract revealed that in addition to pulmonary hemorrhage, all dogs had tracheitis, bronchitis, bronchiolitis, and suppurative bronchopneumonia (Figure 3). The epithelial lining and airway lumens in these tissues were infiltrated by neutrophils and macrophages. Lung homogenates prepared from these dogs were inoculated into a variety of monkey, human, 30 bovine, and canine cell lines for virus culture. The lung homogenate from one dog caused cytopathic effects in Madin-Darby canine kidney epithelial cells (MDCK) cultured in the presence of trypsin, and the cell culture supernatant agglutinated chicken red blood cells. Preliminary evidence of an influenza type A virus was provided by a commercial ELISA for

2015234384 02 Oct 2015 detection of the nucleoprotein of influenza A and B viruses, and by PCR analysis using primers specific for the matrix gene of influenza A viruses. In addition, the hemagglutinating activity was inhibited by reference antisera to the equine influenza A H3 subtype, but not by antisera specific for human influenza A subtypes Hl-Hll and H13 (Table 3). To 5 characterize the molecular properties of the virus, we determined the nucleotide sequences of the 8 RNA segments of the viral genome. Sequence comparisons with known influenza virus genes and phylogenetic analyses indicated that the 8 genes of the canine isolate were most similar to those from contemporary equine influenza A (H3N8) viruses, with which they shared ^96-97% sequence identity (Figure 1A, Table 4). In contrast, representative genes 0 from avian, swine, and human influenza A isolates had <94% identity with the canine isolate (Table 4). These data identified the canine isolate A/Canine/Florida/43/2004 (Canine/FL/04) as an influenza A H3N8 virus closely related to contemporary lineages of equine influenza viruses. Since all genes of the canine isolate were of equine influenza virus origin, we concluded that the entire genome of an equine influenza virus had been transmitted to the 5 dog.

EXAMPLE 2

To investigate the role of the Canine/FL/04 virus in the clinical and pathological observations in the greyhounds, we performed immunohistochemical staining (IHC) on lung

Ό tissues using a monoclonal antibody to influenza A H3. Viral H3 antigen was consistently detected in the cytoplasm of bronchial and bronchiolar epithelial cells, bronchial gland epithelial cells, and macrophages in airway lumens and alveolar spaces (Figure 2A). These data support a diagnosis of pulmonary infection with influenza virus of the H3 subtype in multiple dogs.

EXAMPLE 3

To determine involvement of a Canine/FL/04-like virus in the etiology of the respiratory disease outbreak, we analyzed paired acute and convalescent sera from 11 sick dogs and 16 asymptomatic contacts by hemagglutination inhibition (HI) and microneutralization (MN). Seroconversion, defined as a > 4-fold rise in antibody titer to Canine/FL/04 from the acute to convalescent phase, occurred in 8 of 11 (73%) sick dogs in both assays (Table 1). Seroconversion occurred in 6 of 16 (38%) asymptomatic contacts in the HI assay, while 8 of 16 (50%) seroconverted in the MN assay (Table 1). The

2015234384 02 Oct 2015 seroconversion data demonstrated infection of the dogs with a Canine/FL/04-like virus which coincided temporally with the onset of respiratory disease in most animals.

Single serum samples were collected 3 months after the outbreak from an additional 46 asymptomatic dogs housed with the sick dogs. Of these, 43 (93%) were seropositive in 5 both assays. For the total population of 73 dogs tested, 93% were seropositive in both assays, including 82% (9/11) of the sick dogs and 95% (59/62) of the healthy contacts. The high seroprevalence in dogs with no history of respiratory disease indicates that most infections with canine influenza virus are subclinical and suggest efficient spread of the virus among dogs. It is not known if subclinical infections contribute to the spread of the virus.

EXAMPLE 4

To better understand the capacity of the Canine/FL/04 virus to infect dogs, four 6month old purpose-bred beagles were each inoculated with 10⁶⁶ median tissue culture infectious doses (TCID50) by the intratracheal and intranasal routes. All dogs developed a fever (rectal temperature ^39°C) for the first 2 days postinoculation (p.i.), but none exhibited respiratory symptoms such as cough or nasal discharge over a 14 day observation period. Virus shedding was examined by quantification of virus in nasal and oropharyngeal swabs. Only 2 of the 4 dogs shed detectable amounts of virus. One dog shed virus on days 1 and 2 p.i. (1.0-2.5 logio PFU per swab), whereas the other dog shed virus for 4 consecutive days

Ό after inoculation (1.4-4.5 logio PFU per swab). Postmortem examination of 2 dogs on day 5 p.i. revealed necrotizing and hyperplastic tracheitis, bronchitis, and bronchiolitis similar to that found in the spontaneous disease in greyhounds, but there was no pulmonary hemorrhage or bronchopneumonia. Viral H3 antigen was detected in the cytoplasm of epithelial cells of bronchi, bronchioles, and bronchial glands by IHC (Figure 2B). Infectious virus was recovered from the lung tissue of one of the dogs. Postmortem examination of the remaining 2 dogs on day 14 p.i. showed minimal histological changes in respiratory tissues, no viral H3 antigen by IHC, and no recovery of virus from lung homogenates. Seroconversion in these latter 2 dogs was detected in MN assays by day 7 p.i., with a further 2-to 3-fold increase in antibody titers by day 14. These results established the susceptibility of dogs to infection with Canine/FL/04, as evidenced by the febrile response, presence of viral antigen and infectious virus in the lung parenchyma, histopathological findings typical for influenza, and seroconversion. The failure to reproduce severe disease and death in the experimentally

2015234384 02 Oct 2015 inoculated beagles is not surprising since a large proportion of the naturally infected greyhounds were asymptomatic.

EXAMPLE 5

To investigate whether a Canine/FL/04-like influenza virus had circulated among greyhound populations in Florida prior to the January 2004 outbreak, archival sera from 65 racing greyhounds were tested for the presence of antibodies to Canine/FL/04 using the 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). The seropositive dogs were located at Florida tracks involved in outbreaks of respiratory disease of unknown etiology from 1999 to 2003, suggesting that a Canine/FL/04-like virus may have been the causative agent of those outbreaks. To investigate this possibility further, we examined archival tissues from greyhounds that died from hemorrhagic bronchopneumonia in March 2003. Lung homogenates inoculated into MDCK cells and chicken embryos from one dog yielded H3N8 influenza virus, termed A/Canine/Florida/242/2003 (Canine/FL/03). Sequence analysis of the complete genome of Canine/FL/03 revealed >99% identity to Canine/FL/04 (Table 4), indicating that Canine/FL/04-like viruses had infected greyhounds prior to 2004.

Ό EXAMPLE 6

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

Officials at some of these tracks estimated that at least 80% of their dog population 25 had clinical disease. Most of the dogs had clinical signs of fever (> 3 9 °C) and cough similar to the dogs in the January 2004 outbreak, but many dogs also had a mucopurulent nasal discharge. Multiple deaths were reported but an accurate mortality rate could not be determined.

We collected paired acute and convalescent sera from 94 dogs located at 4 Florida 30 tracks: 56% of these dogs had ^4-fold rises in antibody titers to Canine/FL/04, and 100% were seropositive (Table 6). Convalescent sera from 29 dogs in West Virginia and Kansas also had antibodies to Canine/FL/04. We isolated influenza A (H3N8) virus from the lungs of a greyhound that died of hemorrhagic bronchopneumonia at a track in Texas. Sequence

2015234384 02 Oct 2015 analysis of the entire genome 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 fatal canine cases over a 13-month period and from different geographic locations, together with the substantial serological evidence of widespread 5 infection among racing greyhounds, suggested sustained circulation of a Canine/FL/04-like virus in the dog population.

Phylogenetic analysis of the HA genes of Canine/FL/03, Canine/FL/04, and Canine/TX/04 showed that they constitute a monophyletic group with robust bootstrap support that was clearly distinct from contemporary H3 genes of equine viruses isolated in 0 2002 and 2003 (Figure IB). Phylogentic analysis and pairwise nucleotide sequence comparisons of the other 7 genomic segments supported the segregation of the canine genes as a distinct sub-lineage most closely related to the equine virus lineage (data not shown, and Table 4). The clustering of the canine influenza genes as a monophyletic group separate from equine influenza is also supported by the presence of 4 signature amino acid changes in the 5 HA (Table 2). Together with the serological results from 2003 and 2004, these data are consistent with a single virus transmission event from horses to dogs with subsequent horizontal spread of the virus in the greyhound population. However, repeated introductions of this unique lineage of influenza virus from an unidentified reservoir species can not be formally excluded, unlikely as it may be.

Ό The viral HA is a critical determinant of host species specificity of influenza virus (Suzuki et al., 2000). To identify residues within HA that may be associated with adaptation to the canine host, we compared the deduced amino acid sequence of canine HAs to those of contemporary equine viruses. Four amino acid changes differentiate the equine and canine mature HA consensus amino acid sequences: N83S, W222L, I328T, and N483T (see Table

2). The canine viruses have an amino acid deletion when compared to the consensus equine sequences. Therefore, amino acid position 7 in the HA equine sequence is position 6 in the HA canine sequence, amino acid position 29 in the HA equine sequence is position 28 in the HA canine sequence, amino acid position 83 in the HA equine sequence is position 82 in the HA canine sequence, etc. Thus, the four substituted amino acids are at position 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 consensus sequence position 83 is a change of unknown functional significance since various polar residues are found in H3 molecules from other species. The strictly conserved isoleucine at consensus sequence position 328 near the

2015234384 02 Oct 2015 cleavage site of the H3 HA has been replaced by threonine. The pivotal role of HA cleavage by host proteases in pathogenesis suggests that this change merits further study. The substitution of leucine for tryptophan at consensus sequence position 222 is quite remarkable because it represents a non-conservative change adjacent to the sialic acid binding pocket 5 which could modulate receptor function (Weis et al., 1988). Interestingly, leucine at position 222 is not unique to canine H3 HA since it is typically found in the H4, H8, H9, and H12 HA subtypes (Nobusawa et al., 1991; Kovacova et al., 2002). The leucine substitution may be more compatible with virus specificity for mammalian hosts since infections of swine with subtype H4 (Karasin et al., 2000) and humans and swine with subtype H9 (Peiris et al., 1999) 0 viruses have been reported. The substitution of asparagine with threonine at consensus sequence position 483 resulted in the loss of a glycosylation site in the HA2 subunit that is conserved in all HA subtypes (Wagner et al., 2002). Although the importance of these amino acid changes in the HA for adaptation of an equine virus to dogs remains to be determined, similar amino acid changes have been observed previously in association with interspecies 5 transfer (Vines et al., 1998; Matrosovich et al., 2000). Amino acid differences between other influenza viral proteins of the invention and equine consensus sequence are shown in Tables 19 to 25.

The source of the equine influenza virus that initially infected racing greyhounds remains speculative. Kennels at greyhound racetracks are not located near horses or horse

Ό racetracks, suggesting that contact between greyhounds and shedding horses is not a sufficient explanation for the multiple outbreaks in different 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 packing houses that render carcasses, including horses which could carry influenza. Precedents for this mode of infection include reports of interspecies transmission of H5N1 avian influenza virus to pigs and zoo felids fed infected chicken carcasses (Webster, 1998; Keawcharoen et al., 2004; Kuiken et al., 2004). Although this is a plausible route for the initial introduction of equine influenza into dogs, it does not explain the recent multiple influenza outbreaks in thousands of dogs in different states. Our experimental inoculation study demonstrated the presence of virus in the nasal passages and oropharynx of dogs, albeit at modest titers. Nevertheless, these results indicate that shedding is possible, and that dog-to-dog transmission of virus by large droplet aerosols, fomites, or direct mucosal contact could play a role in the epizootiology of the disease.

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The interspecies transfer of a whole mammalian influenza virus to an unrelated mammal species is a rare event. Previous studies have 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 was no evidence of sustained circulation in the canine host. Although direct transfer of swine influenza viruses from pigs to people 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 the swine viruses in human hosts. In this report, we provide virological, serological, and molecular evidence for interspecies transmission of an entire equine influenza A (H3N8) virus to another mammalian species, the dog. Unique amino acid substitutions in the canine virus HA, coupled with serological confirmation of infection of dogs in multiple states in the U.S., suggest adaptation of the virus to the canine host. Since dogs are a primary companion animal for humans, these findings have implications for public health; dogs may provide a new source for transmission of novel influenza A viruses to humans.

Table 1. Antibody response to A/Canine/Florida/43/2004 (H3N8).
	Sick Dogs (ll)a	Healthy Contacts (16)b
Response	HIC	SNd	HI	SN
Seroconversion (%)e	73	73	38	50
Seropositive (%)*	82	82	100	100
Geometric mean titerg	329	424	268	431

^a Number of dogs with clinical signs of disease.

^b Number of asymptomatic dogs housed in contact with clinically diseased dogs.

^c Hemagglutination-inhibition (HI) assay using A/Canine/Florida/43/2004 virus.

^d Micro neutralization (MN) assay using A/Canine/Florida/43/2004 virus.

^e Percentage of dogs with at least a 4-fold increase in antibody titer in paired acute and convalescent sera.

£

Percentage of dogs with a positive antibody titer (HI titer ^32: MN titer >80) in the convalescent sera.

^g Geometric mean antibody titer for the convalescent sera.

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Table 2. Amino acid differences between the canine and equine H3 hemagglutinins.
Equine H3 consensus	Can/FL/03	Can/FL/04	Can/TX/04	Potential functional significance
G7*	D	. t	-	D also found in duck and human H3 HA
129	-	M	M	I is conserved in H3 HAs from all species
N83	S	S	S	Various polar amino acids present at this position in H3 HAs of other species
S92	-	N	-	N is present in some duck H3 HAs
L118	-	-	V	L is conserved in all H3 HAs
W222	L	L	L	W is conserved in most H3 HAs of all species; located near the receptor binding site
A272	V	A	V	V is present in some recent equine isolates
1328	T	T	T	T is strictly conserved in all avian, swine or humans H3 HAs
N483	T	T	T	N occurs in all H3 and other HA subtypes. Replacement results in loss of a glycosylation site.
K541	-	R	-	Basic amino acid conservative change

* Amino acid residue (single letter code) and position in the mature H3 HA. The amino acid code is: 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 . t Denotes no change from the consensus equine H3 HAs.

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Table 3. Hemagglutination inhibition of a virus isolate by reference antisera to different HA subtypes.
Reference Antisera	HA Specificity	HI titer3
Puerto Rico/8/34	Hl	5
Swine/Iowal5/30	Hl	5
Singapore/01/57	H2	5
Shanghai/11/87	H3b	5
Equine/Miami/1/63	H3	160
Duck/Czecho slo vakia/5 6	H4	5
Tern/South Africa/61	H5	5
T urkey/Mas sachu ssetts/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/5 6	HU	5
Giill/Maryland/704/77	H13	5
Normal sheep serum	-	5
Normal ferret serum	-	5

^a Hemagglutination inhibition titer to virus isolate from dog # 43.

^b Polyclonal antisera were produced in ferrets, whereas all other antisera were produced in 5 sheep or goats.

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Table 4. Sequence homology of A/Canine/Florida/43/2004 (H3N8) genes to equine, avian, swine, and human strains of influenza A.
Gene	Equine	Avian	Swine	Human
PB2 DQ124147	96.9 (98.7)a Eq/Kentucky/2/8 M73526	88.6 (96.8) Mall/Albci'ta/98/85 AY633315	87.9 (96.8) Sw/Ontario/ 01911-1/99 AF285892	86.2 (96.4) PR/8/34 (HK/213/03) AF389115 (AY576381)
PB1 DQ124148	97.1 (98.8) Eq/Tennessee/5/86 M25929	83.9 (97.1) Ck/BritishColumbia/04 (Gull/Md/704/77) AY61675 (M25933)	83.9 (97.1) Sw/Korea/S 109/04 (Sw/Saskatch/ 18789/02) AY790287 (AY619955)	83.9 (97.1) WSN/33 (Sing/1/57) J02178 (M25924)
PA DQ124149	96.3 (97.5) M26082 Eq/T ennesee/5/8 6	87.0 (94.3) Ck/Chile/4591/02 (Ostrich/SA/08103/95) AY303660(AF508662)	84.3 (94.6) Sw/Hong Kong/ 126/02 M26081	83.8 (93.4) Taiwan/2/70 (Viet Nam/ 1203/04) AY210199 (AY818132)
HA (H3) DQ124190	97.4 (97.1) Eq/FL/1/93 L39916	80.7 (89.0) Dk/Norway/1/03 AJ841293	80.0 (87.7) Sw/Ontario/42729a/01 AY619977	81.8 (87.9) HK/1/68 AF348176
NP DQ124150	96.6 (97.9) Eq/T ennesee/5/8 6 M30758	87.9 (95.1) Ck/Chile/176822/02 AY303658	85.4 (93.5) Sw/Ontario/42729a/01 (Sw/Fujian/1/2003) AY619974 (AY747611)	84.7 (93.0) HK/1073/99 (Hong Kong /538/97) AF255742 (AF255751)
ΝΑ (N8) DQ124151	96.8 (97.0) Eq/T ennesee/5/8 6 L06583	84.0 (85.2) Dk/NJ/2000 L06583	b na	b na
M DQ124152	97.9 (95.7) Eq/T ennesee/5/8 6 (Eq/Kentucky/92) M63529 (AF001683)	94.1 (94.0) Tky/Mn/833/80 AF001683	93.7 (93.5) Sw/Saskatchewan/ 18789/02 M63527	91.2 (95.4) WSN/33 (Hong Kong/ 1073/99) J02177 (AJ278646)
NS DQ124153	97.5 (95.7) Eq/Tn/5/86 (Eq/Kentucky/92) M80973 (AF001671)	92.0 (90.4) Mal/N Y/6750/78 M80945	91.1 (89.1) Sw/China/8/78 (Sw/Korea/s452/04) M80968 (AY790309)	91.4 (90.0) Brevig Mission/1/18 AF333238

^a Percent nucleotide and amino acid (in parentheses) sequence identity of A/Canine/Florida/43/2004 (H3N8) genes to the most homologous gene of influenza virus virus isolates from the species, followed by their Genbank sequence database accession numbers.

^b Not applicable: N8 neuraminidase was never reported in human or swine viruses.

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Table 5. Antibody titers to A/canine/Florida/43/2004 (H3N8) in greyhound serum collected from 1996 to 2003.
	Yeara
1996	1997	1998	2000	2002	2003
No. of dogs tested	8	6	19	4	6	22
No. of seropositive dogs	0	0	0	1	2	6
Antibody titersb				512	232, 524	280-2242

^aThe year of serum sample collection from racing greyhounds in Florida.

^b Microneutralization assay antibody titers for seropositive dogs, including the range for the six 2003 seropositive dogs.

Table 6. Antibody response to A/canine/Florida/43/2004 (H3N8) in racing greyhounds at 4 Florida tracks in June 2004.
Response	Track A	Track B	Track C	Track D
Number of dogs testeda	37	10	22	25
Seroconversion (%)b	46	90	100	64
Seropositive (%)c	100	100	100	100
Geometric mean titerd	401	512	290	446

^a Number of clinically diseased dogs tested by HI using A/canine/Florida/43/2004 (H3N8). ^b Percentage of dogs with >4-fold increase in antibody titer between acute and convalescent sera.

^c Percentage of dogs with a positive antibody titer (HI titer>16) in the convalescent sera.

^d Geometric mean antibody titer for the convalescent sera.

MATERIALS AND METHODS FOR EXAMPLES 7-11

Canine tissues

Postmortem 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 a shelter facility in northeast Florida, and on a pet Yorkshire Terrier dog that died in May 2005 during an influenza outbreak in a

2015234384 02 Oct 2015 veterinary clinic in southeast Florida. Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and 5-pm sections were stained with hematoxylin and eosin for histopathologic diagnosis. Unfixed tissues were stored at -80°C pending virological analyses.

RNA extraction from canine tissue samples

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

Real-time RT-PCR

A single-step quantitative real-time RT-PCR was performed on total RNA extracted from the canine tissue samples using the QuantiTect® Probe RT-PCR Kit containing ROX as a passive reference dye (QIAGEN Inc., Valencia, CA). Briefly, 2 primer-probe sets were used for detection of influenza A sequences in each sample (Table 7). One primer-probe set was selective for canine hemagglutinin (H3) gene sequences. The other primer-probe set

Ό targeted a highly conserved region of the matrix (M) gene of type A influenza virus. For each real-time RT-PCR reaction, 5 pL of extracted total RNA were added to a reaction mixture containing 12.5 pL of 2X QuantiTech® Probe RT-PCR Master Mix, 0.25 pL of QuantiTech® RT Mix, forward and reverse primers (0.4 pM final concentration for each), probe (0.1 pM final concentration) and RNase-free water in a final volume of 25 pL. The

TaqMan® Ribosomal RNA Control Reagents (Applied Biosystems, Foster City, CA) were used according to manufacturer’s instructions for detection of 18S rRNA as an endogenous internal control for the presence of RNA extracted from the canine tissue samples.

Quantitative one-step real-time RT-PCR was performed on the reaction mixtures in a 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 the HotStarTaq® DNA polymerase, and amplification for 40 cycles. Each amplification cycle included denaturation at 94°C for 15 seconds followed by

2015234384 02 Oct 2015 annealing/extension at 60°C for 1 minute. The FAM (emission wavelength 518 nm) and VIC (emission wavelength 554 nm) fluorescent signals 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 program (Stratagene, La 5 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 smaller than the Ct for lung tissues from dogs without respiratory disease. The positive control consisted of amplification of RNA extracted from A/canine/FL/242/03 (H3N8) virus.

Virus isolation in MDCK cells

Frozen lung tissues from each of the 7 dogs were thawed and homogenized in 10 volumes of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 0.5% (BSA) and antibiotics (gentamycin and ciprofloxacin). Solid debris was removed by centrifugation and supernatants were inoculated onto Madin-Darby canine kidney (MDCK) cells cultured in

DMEM supplemented with 1 pg/mL TPCK-treated trypsin (Sigma-Aldrich Corp., St. Louis, MO) and antibiotics (gentamycin and ciprofloxacin). Cells were grown in 25 cm flasks at 37°C in a humidified atmosphere containing 5% CO2. The cultures were observed daily for morphologic changes and harvested at 5 days post inoculation. The harvested cultures were clarified by centrifugation and the supernatants inoculated onto fresh MDCK cells as

Ό described for the initial inoculation; two additional passages were performed for samples that did not show evidence of influenza virus by hemagglutination or RT-PCR. Hemagglutination activity in the clarified supernatants was determined using 0.5% turkey red blood cells as previously described (Burleson, F. et al., 1992; Kendal, P. et al., 1982). RT-PCR was performed as described below.

Virus isolation in embryonated chicken eggs

Homogenates were prepared from frozen lung tissues as described above for inoculation of MDCK cells. The homogenates (0.2 mL) were inoculated into the allantoic sac of 10-day old embryonated chicken eggs. After 48 hours of incubation at 35°C, the eggs were chilled at 4°C overnight before harvesting the allantoic fluid. Hemagglutination activity in the clarified supernatants was determined using 0.5% turkey red blood cells as previously described (Burleson, F. et al., 1992; Kendal, P. et al., 1982). RT-PCR was performed as

2015234384 02 Oct 2015 described below. Two additional passages in embryonated eggs were performed for samples that did not show evidence of influenza virus after the initial inoculation.

RT-PCR, nucleotide sequencing, and phylogenetic analyses 5 Viral RNA was extracted from MDCK supernatant or allantoic fluid using the

QIAamp® Viral RNA Mini Kit (QIAGEN Inc., Valencia, CA) according to manufacturer’s instructions. The viral RNA was reverse transcribed to cDNA using the QIAGEN® OneStep RT-PCR Kit (QIAGEN Inc., Valencia, CA) according to manufacturer’s instructions. PCR amplification of the coding region of the 8 influenza viral genes in the cDNA was performed 0 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 the ABI PRISM® 3100 automated DNA sequencer using cycle sequencing dye terminator chemistry (Applied Biosystems, Foster City, CA). Nucleotide sequences were analyzed using the Lasergene 6 Package® (DNASTAR, Inc., 5 Madison, WI). The PHYLIP Version 3.5 software program was used to estimate phytogenies and calculate bootstrap values from the nucleotide sequences (Felsenstein, J., 1989). Phylogenetic trees were compared to those generated by neighbor-joining analysis with the Tamura-Nei model implemented in the MEGA program (Kumar, S. et al., 2004) and confirmed by the PAUP 4.0 Beta program (Sinauer Associates, Inc., Sunderland, MA).

Ό

Hemagglutination inhibition (HI) assay

Serum samples were incubated with receptor destroying enzyme (RDE, DENKA SEIKEN Co., Ltd., Tokyo, Japan) (1 part serum: 3 parts RDE) for 16 hours at 37°C prior to heat inactivation for 30 minutes at 56°C. Influenza A/Canine/Jacksonville/05 (H3N8) virus was grown in MDCK cells for 72 hrs at 37°C in 5% CO2. Virus culture supernatants were harvested, clarified by centrifugation, and stored at -80°C. All other viruses used in the HI assay were grown in 10-day old embryonated chicken eggs from which allantoic fluid was collected and stored at -80°C. The HI assay was performed as described previously (Kendal, P. et al., 1982). Briefly, 4 hemagglutinating units of virus in 25pl were added to an equal volume of serially diluted serum in 96-well plastic plates and incubated at room temperature for 30 minutes. An equal volume of 0.5% turkey erythrocytes was added and the hemagglutination titers were estimated visually after 30 minutes. The endpoint HI titer was defined as the last dilution of serum that completely inhibited hemagglutination.

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EXAMPLE 7-CLINICAL CASES

In April and May 2005, a previously described (Crawford, P.C. et al., 2005) respiratory disease outbreak occurred in dogs housed in a shelter facility in northeast Florida. 5 The outbreak involved at least 58 dogs ranging in age from 3 months to 9 years, and included purebred dogs as well as mixed breeds. The most common clinical signs were purulent nasal discharge and a cough for 7 to 21 days. Of the 43 dogs that had clinical disease for >7 days, 41 had HI antibody titers to canine/FL/04 (H3N8) ranging from 32 to >1024. At least 10 dogs progressed to pneumonia, of which 6 were euthanized. These 6 mixed breed dogs 0 included 3 males and 3 females ranging in age from 4 months to 3 years. The duration of clinical signs ranged from 2 to 10 days at the time of euthanasia. On postmortem examination, these dogs had pulmonary congestion and edema. Histological examination of the respiratory tract revealed rhinitis, tracheitis, bronchitis, bronchiolitis, and suppurative bronchopneumonia. There was epithelial cell necrosis and erosion in the trachea, bronchi, 5 bronchioles, and bronchial glands. The respiratory tissues were infiltrated by 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 a cough for 10 to 30 days. Of the 40 dogs, 17 were seropositive for canine/FL/04 (H3N8)

Ό with HI antibody tiers ranging from 32 to >1024. Seroconversion occurred in 10 dogs for which paired acute and convalescent sera were available. Three dogs progressed to pneumonia. One of these dogs, a 9-year old male Yorkshire Terrier, died 3 days after onset of clinical signs. This dog had tracheobronchitis, pulmonary edema and congestion, and severe bronchopneumonia. Similar to the 6 shelter dogs, there was epithelial cell necrosis and erosion of the airways and neutrophilic infiltrates in the tissues.

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

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

2015234384 02 Oct 2015 embryonated chicken eggs, influenza A subtype H3N8 virus was recovered from the lungs of the pet dog that also died after 3 days of pneumonia. This virus was named A/canine/Miami//05 (H3N8) (canine/Miami/05).

EXAMPLE 9—GENETIC ANALYSES OF THE CANINE INFLUENZA A H3N8

ISOLATES

Sequence analyses of canine/Jax/05 and canine/Miami/05 revealed that their hemagglutinin (HA) genes were 98% identical to the canine/FL/04, canine/TX/04, and canine/Iowa/05 isolates recovered from the lungs of racing greyhounds that died of 0 pneumonia during influenza outbreaks at tracks in 2004 and 2005 (Crawford, P.C. et al., 2005; Yoon K-Y. et al., 2005). In addition, the HA genes of canine/Jax/05 and canine/Miami/05 were 98% identical to contemporary equine influenza viruses isolated after the year 2000. Phylogenetic comparisons of the HA genes showed that the canine/Jax/05 and canine/Miami/05 viruses were clustered with the canine/FL/04, canine/TX/04, and 5 canine/Iowa/05 greyhound isolates and contemporary equine isolates, forming a distinct group from the older equine viruses isolated in the early 1990’s (Figure 4). Furthermore, the canine/Jax/05, canine/Miami/05, and canine/Iowa/05 isolates were more closely related to canine/Tx/04 than to either canine/FL/04 or canine/FL/03. The 2005 isolates formed a subgroup that appears to branch off from the earlier 2003 and 2004 canine viruses with Ό differences at approximately 10 parsimony-informative sites. These differences support the hypothesis that canine influenza virus is being transmitted horizontally from dog-to-dog as opposed to being reintroduced periodically from an outside source. The accumulation of mutations from 2003 to 2005 illustrates the ongoing process of adaptation that the virus must undergo after being transmitted to a new host, as is expected to have happened for the canine 25 influenza viruses.

EXAMPLE 10—AMINO ACID ANALYSES OF THE CANINE INFLUENZA A H3N8

ISOLATES.

There were conserved amino acid substitutions in all 6 canine isolates that 30 differentiated them from contemporary equine influenza viruses (Table 9). These conserved 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 (Figure 4). There were 3 amino

2015234384 02 Oct 2015 acid changes (L118V, K261N, and G479E) that differentiated this subgroup from the other canine viruses (Table 9). There were 2 amino acid changes (F79L and G218E) that differentiated the 2005 isolates from their canine/TX/04 root. Furthermore, the 2005 isolates from non-greyhound dogs, canine/Jax/05 and canine/Miami/05, differed from the 5 canine/Iowa/05 greyhound isolate by one amino acid change, R492K. Finally, canine/Jax/05 differed from canine/Miami/05 at a single amino acid, S107P. In all other H3N8 equine and canine viruses, S is conserved at position 107 except for A/Equine/Jilin/1/89 which has a T (Guo Y. et al., 1992).

EXAMPLE 11—ANTIGENIC ANALYSES OF THE CANINE INFLUENZA A H3N8

ISOLATES

Hemagglutination inhibition (HI) tests were performed using an antigen panel of older and contemporary equine influenza viruses and the canine influenza viruses, and serum collected in 2005 from horses and dogs that had been infected with influenza virus (Table

10). Serum from ferrets immunized against canine/FL/04 was also included in the analyses.

The HI antibody titers in equine serum were 8 to 16-fold higher when tested with contemporary equine viruses compared to older isolates, but decreased by at least 4-fold when tested with the canine viruses. The canine serum was nonreactive with the older equine viruses, but the antibody titers increased 4-fold when tested with contemporary equine

Ό isolates and canine isolates. This was also observed for the serum from ferrets immunized against canine influenza virus. These seroreactivity patterns demonstrated the antigenic similarity between the canine influenza viruses and contemporary equine influenza viruses and were consistent with the phylogenetic analyses. The antibody titers in equine, canine, and ferret sera to the canine/Miami/05 isolate were similar to those for the 2003 and 2004 canine isolates. However, the titers were 2 to 4-fold lower for the canine/Jax/05 isolate. This suggests that canine/Jax/05 is antigenically distinct from the other canine isolates, which may in part be related to the single amino acid change at position 107 in the mature HA.

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Underlined letter r represents nucleotide a or g and underlined letter k represents nucleotide g or t. Uppercase letters represent locked nucleic acid residues.

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Table 8. Quantitative real-time RT-PCR and viral isolation performed on lung tissues from dogs that died from pneumonia during respiratory disease outbreaks in a shelter and veterinary clinic in Florida.
Dog ID	Location	Duration of clinical disease	Real-time I	tT-PCR	Virus Isolation
M (Ct)	HA (Ct)
A/canine/FF/242/03 positive control	28.15	27.36
1079	Shelter (NE FL)	2 days	29.81	28.84	none
1078	Shelter (NE FL)	3 days	30.37	29.71	MDCK 3rd passage
318	Shelter (NE FL)	9 days	33.89	32.97	none
320	Shelter (NE FL)	10 days	39.44	37.09	none
319	Shelter (NE FL)	6 days	33.87	32.23	none
1080	Shelter (NE FL)	6 days	38.87	38.23	none
374	Veterinary clinic (SE FL)	3 days	24.05	22.65	Egg 2nd passage

2015234384 02 Oct 2015 <C

SC

Table 9. Amino acid comparison of the mature HA for canine influenza viruses and contemporary equine influenza viruses.	Amino Acid	541					&
492	&
483	Z,				H			H	H
479	O					ω	ω	ω	ω
328				H	H
261						z	z	z	z
222	£			J	J	J	J	J	J
218	ϋ						ω	ω	ω
159	z	vi	vi	vi	vi	Vi	Vi	Vi	Vi
oo	ζ					>	>	>	>
107	co
(N OS	v				%
50	Z,			Vi	Vi	Vi	Vi	Vi	Vi
σ\ Γ-							Z	Z	Z
OO	>	<	<	<	<	<	<	<	<
	Z
Γ-	ϋ		Q
		A/equine/KY/5/02	A/equine/MA/213/03	A/equine/OH/1/03	A/canine/FL/242/03	A/canine/FL/43/04	A/canine/TX/1/04	A/canine/Iowa/05	A/canine/Miami/05	A/canine/Jacksonville/05

2015234384 02 Oct 2015

Table 10. Antibody titers in equine, canine, and ferret serum to older and contemporary equine influenza viruses and canine influenza viruses.
	Serum	antibody	titers a
Antigens	Equine	Canine	Ferretb
equine/Miami/63	40	<10	16
equine/Ky/86	40	40	32
equine/KY/92	40	<10	32
equine/NY/99	320	40	128
equine/KY/05/02	320	160	256
equine/M A/213/03	640	160	512
equine/OH/01/03	640	160	512
canine/FL/03	160	160	512
canine/FL/04	160	80	512
canine/Tx/04	160	160	512
canine/Miami/05	160	80	256
canine/Jax/05	40	40	128

^a Antibody titers were determined in a hemagglutination inhibition assay 25 performed with serial dilutions of equine, canine, or ferret serum and the viruses listed in the antigen column. ^b Serum from ferrets immunized with canine/FL/04 virus.

MATERIALS AND EXAMPLES METHODS FOR EXAMPLES 12-15

Canine influenza virus inoculum.

The virus inoculum was prepared by inoculation of Madin-Darby canine kidney (MDCK) epithelial cells with a stock of A/canine/FL/43/04 (H3N8) representing passage 3 of

2015234384 02 Oct 2015 the original isolate previously described (Crawford et al., 2005). The inoculated MDCK cells in Dulbecco’s Minimal Essential Media (DMEM) supplemented with 1 pg/mL TPCK-treated trypsin (Sigma-Aldrich Corp., St. Louis, MO) and antibiotics (gentamycin and ciprofloxacin) were grown in 250 cm flasks at 37°C in a humidified atmosphere containing 5% CO2. The 5 cultures were observed daily for morphologic 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 dogs. An aliquot of supernatant was used for determination of virus titer by the Reed and Muench method. The titer was 10 median tissue culture infectious doses (TCIDso)of A/canine/Florida/43/2004 (canine/FL/04) per mL.

Experimental inoculation.

Eight 4-month old colony bred mongrel dogs (Marshall BioResources, North Rose, NY) (4 males and 4 females) were used for the experimental inoculation study approved by the University of Florida Institutional Animal Care and Use Committee. The dogs’ body weights ranged from 13 to 17 kg. The dogs were healthy based on physical examinations, baseline blood tests, and recording of body temperatures for 2 weeks prior to inoculation. All dogs were free from prior exposure to canine influenza virus based on serology tests performed on paired serum samples collected at the time of arrival into the facility and 2 weeks later. The dogs were anesthetized by intravenous injection of propofol (Diprivan®,

Ό Zeneca Pharmaceuticals, 0.4 mg/kg body weight to effect) for intubation with endotracheal tubes. Six dogs (3 males and 3 females) were each inoculated with 10 TCID50 of canine/FL/04 virus in 5 mL of sterile saline administered 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 sham-inoculated control dogs were housed in a different room from the virus-inoculated dogs and cared for by different personnel. Physical examinations and rectal temperature recordings were performed twice daily for 6 days post inoculation (p.i.).

Pharyngeal and rectal swab collection.

To monitor for virus shedding, oropharyngeal specimens were collected twice daily from each dog on days 0 to 6 p.i. using polyester swabs (Fisher Scientific International Inc., Pittsburgh, PA). The swabs were placed in 1 mL of sterile phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA). Rectal swabs were collected from each dog

2015234384 02 Oct 2015 daily from days 0 to 6. Swab extracts were prepared by clarification of the swab transport media by centrifugation. An aliquot of swab extract was tested immediately for influenza A virus nucleoprotein using the Directigen™ commercial immunoassay kit (BD, Franklin Lakes, NJ) according to the manufacturer’s instructions. The remaining extract was stored at 5 -80°C pending other virological assays.

Postmortem examinations.

On day 1 p.i., one sham-inoculated dog and one virus-inoculated dog were euthanatized by intravenous inoculation of Beuthanasia-D® solution (1 mL/5 kg body weight; 0 Schering-Plough Animal Health Corp). One virus-inoculated dog was similarly euthanatized each day from days 2 to 5 p.i. On day 6 p.i., the remaining sham-inoculated and virusinoculated dog were euthanatized. Complete postmortem examinations were performed by one of the investigators (WLC). Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and 5-pm sections were either stained with hematoxylin and eosin for 5 histopathologic diagnosis or processed for immunohistochemistry as described below. Unfixed lung tissues were submitted to the Diagnostic Clinical Microbiology/Parasitology/ Serology Service at the University of Florida College of Veterinary Medicine for bacterial isolation and identification. The samples were cultured on nonselective media as well as media selective for Bordetella species (Regan-Lowe; Remel, Lenexa, KS) and Mycoplasma Ό species (Remel). All cultures were held for 21 days before reporting no growth. Unfixed tissues were also stored at -80°C pending virological analyses.

Immunohistochemistry.

Deparaffinized and rehydrated 5-pm trachea and lung tissue sections were mounted 25 on Bond-Rite™ slides (Richard-Allan Scientific, Kalamazoo, MI) and subsequently treated with proteinase K (DAKOCytomation Inc., 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 monoclonal antibody to influenza A H3 (Chemicon International, Inc., Temecula, CA) for 2 hours at room temperature. Controls included incubation of the same sections with mouse IgG (1 mg/mL, Serotec, Inc. Raleigh, NC), and incubation of the monoclonal antibody with normal canine lung sections. Following treatment with the primary antibody, the sections were incubated with secondary immunoperoxidase and peroxidase substrate reagents (Dako® EnVision™ Peroxidase Kit,

2015234384 02 Oct 2015

Dako Corp.) according to the manufacturer’s instructions. The sections were counterstained with hematoxylin, treated with Clarifier #2 and Bluing Reagent (Richard-Allan Scientific, Kalamazoo, MI), dehydrated, and coverslips applied with Permount (ProSciTech, Queensland, Australia).

RNA extraction from swabs and tissues.

Lung and tracheal tissues from each dog were thawed and homogenized in minimum essential medium (MEM) supplemented with 0.5% bovine serum albumin (BSA) and antibiotics (gentamycin and ciprofloxacin) using a disposable tissue grinder (Kendall, 0 Lifeline Medical Inc., Danbury, CT). Total RNA was extracted from the tissue homogenates as well as orpharyngeal and rectal swab extracts using a commercial kit (RNeasy® Mini Kit, QIAGEN Inc., Valencia, CA) according to manufacturer’s instructions and eluted in a final volume of 60 qL of buffer.

Real-time RT-PCR.

A single-step quantitative real-time RT-PCR was performed on the total RNA using the QuantiTect® Probe RT-PCR Kit containing ROX as a passive reference dye (QIAGEN Inc., Valencia, CA) and a primer-probe set that targeted a highly conserved region of the matrix (M) gene of type A influenza virus (Payungporn S. et al., 2006a; Payungporn S. et al.,

Ό 2006b). For each real-time RT-PCR reaction, 5 μΕ of extracted total RNA were added to a reaction mixture containing 12.5 μΕ of 2X QuantiTech® Probe RT-PCR Master Mix, 0.25 pL of QuantiTech® RT Mix, forward and reverse primers (0.4 μΜ final concentration for each), probe (0.1 μΜ final concentration) and RNase-free water in a final volume of 25 μΕ. The TaqMan® GAPDH Control Reagents (Applied Biosystems, Foster City, CA) were used according to manufacturer’s instructions for detection of GAPDH as an endogenous internal control for the presence of RNA extracted from the swab and tissue samples and as a normalization control.

Quantitative one-step real-time RT-PCR was performed on the reaction mixtures in a 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 the HotStarTaq® DNA polymerase, and amplification for 40 cycles. Each amplification cycle included denaturation at 94°C for 15 seconds followed by

2015234384 02 Oct 2015 annealing/extension at 60°C for 1 minute. The FAM (emission wavelength 518 nm) and VIC (emission wavelength 554 nm) fluorescent signals 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 program (Stratagene, La 5 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 M Ct value by the corresponding GAPDH Ct value for each sample.

Virus re-isolation 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 debris was removed by centrifugation and supernatants were inoculated onto MDCK cells cultured in DMEM supplemented with 1 pg/mL TPCK-treated trypsin (Sigma-Aldrich Corp.,

St. Louis, MO) and antibiotics as described above. Cells were grown in 25 cm flasks at 37°C in a humidified atmosphere containing 5% CO2. The cultures were observed daily for morphologic changes and harvested at 5 days post inoculation. The harvested cultures were clarified by centrifugation and the supernatants inoculated onto fresh MDCK cells as described for the initial inoculation; two additional passages were performed for samples that

Ό did not show evidence of influenza virus by hemagglutination or RT-PCR. Hemagglutination activity in the clarified supernatants was determined using 0.5% turkey red blood cells as previously described (Crawford et al., 2005). RT-PCR was performed as described below.

RT-PCR, nucleotide sequencing, and phylogenetic analyses.

Viral RNA was extracted from MDCK supernatant using the QIAamp® Viral RNA

Mini Kit (QIAGEN Inc., Valencia, CA) according to manufacturer’s instructions. The viral RNA was reverse transcribed to cDNA using the QIAGEN® OneStep RT-PCR Kit (QIAGEN Inc., Valencia, CA) according to manufacturer’s instructions. PCR amplification of the coding region of the 8 influenza viral genes in the cDNA was performed as previously described (Crawford et al., 2005), using universal gene-specific primer sets (primer sequences available on request). The resulting DNA amplicons were used as templates for automated sequencing in the ABI PRISM® 3100 automated DNA sequencer using cycle

2015234384 02 Oct 2015 sequencing dye terminator chemistry (Applied Biosystems, Foster City, CA). Nucleotide sequences were analyzed using the Fasergene 6 Package® (DNASTAR, Inc., Madison, WI). The nucleotide sequences for viruses recovered from infected dogs were compared to the sequences of the virus in the inoculum to determine if any changes had occurred during 5 replication in the respiratory tract.

EXAMPFE 12—CFINICAF DISEASE

All 6 virus-inoculated dogs developed a transient fever (rectal temperature >39°C) for the first 2 days p.i., but none exhibited respiratory signs such as cough or nasal discharge 0 over the 6-day observation period. The sham-inoculated dogs remained clinically healthy.

EXAMPFE 13—VIRUS SHEDDING

Influenza A nucleoprotein was detected in the oropharyngeal swab collected from one of the virus-inoculated dogs at 24 hours p.i. The oropharyngeal swabs collected from one dog at 72, 84, and 120 hours p.i., and another dog at 108, 120, and 132 hours p.i., were positive for virus by quantitative real-time RT-PCR (Table 11). The absolute number of influenza M gene copies per μΕ of swab extract increased with time from 3 to 6 days p.i. No virus was detected in the rectal swabs.

Ό EXAMPFE 14—POSTMORTEM EXAMINATIONS

In contrast to the previous experimental infection using specific pathogen-free

Beagles (Crawford et at., 2005), the virus-inoculated mongrel dogs had pneumonia as evidenced by gross and histological analyses of the lungs from days 1 to 6 p.i. In addition to pneumonia, the dogs had rhinitis, tracheitis, bronchitis, and bronchiolitis similar to that described in naturally infected dogs (Crawford et at., 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 (Figure 5, upper panels). Immunohistochemistry detected viral H3 antigen in the epithelial cells of bronchi, bronchioles, and bronchial glands (Figure 5, lower panels). No bacterial superinfection was present. The respiratory tissues from the 2 sham-inoculated dogs were normal.

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EXAMPLE 15—VIRUS REPLICATION IN TRACHEA AND LUNGS

The trachea and lungs were positive for virus by quantitative real-time RT-PCR in all dogs from 1 to 6 days p.i. (Table 12). The absolute number of influenza M gene copies per pL of trachea homogenate increased from 1 to 5 days p.i., then decreased on day 6. The absolute number of M gene copies per pL of lung homogenate decreased from 1 to 6 days p.i.

In general, the trachea contained > one logio more virus than the lung on each of the 6 days p.i.

Table 11. Detection of virus shedding in the oropharynx of mongrel dogs inoculated with canine influenza virus by quantitative real-time RT-PCR.

Dog ID	Time p.i. (hours)3	M/GAPDH ratio”	Matrix gene (copies / uL)c
860	72	1.20	1.57E+02
84	1.30	8.25E+02
120	1.23	1.47E+03
894	108	1.17	1.17E+02
120	1.41	1.37E+02
132	1.27	3.74E+02

^aTime that oropharyngeal swabs were collected from the dogs following inoculation with A/canine/FL/43/04 (H3N8) virus.

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

^c The absolute number of matrix gene copies per uL of swab extract.

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Table 12. Detection of virus replication in the trachea and lung of
mongrel dogs	inoculated with canine real-time RT	ϊ influenza virus by quantitative -PCR.
Dog ID	Time p.i. (hours)3	M/GAPDH ratiob	Matrix gene (copies / uL)c
Lung	Trachea	Lung	Trachea
797	24	1.20	1.43	8.22E+05	3.11E+04
801	48	1.33	0.99	1.15E+05	6.52E+06
789	72	1.44	1.12	2.39E+04	1.56E+05
819	96	1.40	1.27	3.19E+04	1.43E+05
860	120	1.59	1.04	3.48E+03	1.17E+06
894	144	1.70	1.15	4.78E+02	1.50E+03

^aTime that tissues were collected from the dogs following inoculation with A/canine/FL/43/04 (H3N8) virus.

^b Normalization ratios were calculated by dividing the M (Ct) by the GAPDH (Ct) for each tissue homogenate.

^cThe absolute number of matrix gene copies per uL of tissue homogenate.

MATERIALS AND EXAMPLES METHODS FOR EXAMPLE 16

Virus strains

Canine influenza virus strains as well as those of avian, equine and human origin (listed in Table 15) were propagated in embryonated eggs or MDCK cells and their infectivity was titrated by endpoint dilution in chicken embryos, or plaque assay. Rapid virus quantification was performed by hemagglutination assay using turkey red blood cell erythrocytes.

Diagnostic specimens

A Total of 60 canine’s lung tissues collected from suspect cases of viral respiratory disease 20 during the year of 2005 were tested for the presence of canine influenza virus.

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RNA extraction from canine tissue samples

Blocks of lung tissue weighing between 20 and 30 mg were homogenized in a disposable tissue grinder (Kendal). Total RNA was extracted using a commercial kit (RNeasy Mini Kit, Qiagen, Valencia, CA) and eluted in a final volume of 60 pL, following 5 the manufacturer’s recommendations.

Primers and probes design

Multiple sequence alignments of the H3 and M genes from various subtypes and from diverse species were performed using the CLUSTAL X program (Version 1.8). Matrix (M) 0 primers and probe were selected from the conserved regions of over the known sequences corresponding to different subtypes of influenza A virus, whereas the H3 hemagglutinin gene-specific primers and probe set were selected to specifically match equine and canine influenza A virus genes and mismatch the homologous avian and human genes (Table 13). Primer design software (OLIGOS Version 9.1) and the web based analysis tools provided by 5 EXIQON (http://lnatools.com) was used for Tm calculation and prediction of secondary structure as well as self hybridization. A conserved region of an 18S rRNA gene was used as endogenous internal control for the presence of RNA extracted from canine tissue sample. The Pre-Developed TaqMan® Assay Reagents for Eukaryotic 18S rRNA (VIC/TAMRA) (Applied Biosystems) was used for the real-time detection of 18S rRNA in tissue samples.

Ό

Real-time RT-PCR condition

A single-step real-time RT-PCR was performed by using the Quantitect Probe RTPCR Kit containing ROX as a passive reference dye (Qiagen, Valencia, CA). In each realtime RT-PCR reaction, 5 pL of RNA sample were used as a template to combine with a reaction mixture containing 10 pL of 2X QuantiTech Probe RT-PCR Master Mix, 0.2 pL of QuantiTech RT Mix, primers (0.4 pM final cone, for H3 gene or 0.6 pM final cone, for M gene), probe (0.1 pM final cone, for H3 gene or 0.2 pM final cone, for M gene) and RNasefree water in a final volume of 20 pL. One-step real-time RT-PCR was performed in the Mx3005P Real-Time 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 in order to activate the HotStarTaq DNA polymerase, amplification was performed during 40 cycles including denaturation (94°C for 15 seconds) and annealing/extension (60°C

2015234384 02 Oct 2015 for 30 seconds). The FAM (emission wavelength 516 nm for H3 and M detection) and VIC (emission wavelength 555 nm for 18S rRNA detection) fluorescent signals were obtained once per cycle at the end of the extension step. Data acquisition and analysis of the real-time PCR assay were performed using the Mx3005P software version 2.02 (Stratagene).

Specificity of H3 primers/ probe set for canine influenza (H3N8) and universality of M primers/probe set for type A influenza virus

In order to test the specificity of each primers/probe set, RNA extracted from several known subtypes of influenza A viruses were used as a template in the real-time RT-PCR 0 assay (Table 15).

RNA standard for determination of the real-timer RT-PCR performance

The genes of canine influenza A virus (A/canine/Florida/242/2003(H3N8)) were used to generate the PCR amplicons for H3 (nt 1-487) and M (nt 1-276) by using primers linked 5 with T7 promoter (Table 13). Then the purified PCR amplicons of H3 and M genes were used as templates for in vitro transcription by using Riboprobe in vitro Transcription SystemT7 (Promega) following the manufacturer’s recommendations. The concentration of the transcribed RNAs was calculated by measuring absorbance at 260 nm. The RNAs were then o

serially diluted 10-fold, ranging from 10 to 10 copies/ qL to perform sensitivity tests.

Ό Moreover, a standard curve was generated by plotting the log of initial RNA template concentrations (copies/ qL) against the threshold cycle (Ct) obtained from each dilution in order to determine the overall performance of real-time RT-PCR.

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

Stock viruses of two viral strains including A/Wyoming/3/2003 (H3N2) at 10⁶'⁶⁷

EIDso/mL (HA=64) and A/canine/Florida/242/2003(H3N8) at 10⁷¹⁷ EID₅₀/mL (HA=16) were used for the detection threshold assay. Logarithmic dilution of specimens in phosphatebuffered saline (PBS) (125 qL) were used in a rapid influenza A antigen detection kit, Directigen Flu A, (Becton, Dickinson and Company) following the manufacturer’s instructions. Each Directigen Flu A test device has an H1N1 influenza antigen spot in the center of the membrane which develops as a purple dot and indicates the integrity of the test, which is based on a monoclonal antibody to the nucleoprotein (NP). The development of a

2015234384 02 Oct 2015 purple triangle surrounding the dot is indicative of the presence of influenza NP in the tested specimen. The intensity of the purple signal from the triangle was scored as + (outline of triangle), ++ (lightly colored triangle), +++ (dark-purple triangle) and ++++ (very darkpurple triangle). Viral RNA was extracted 125 pL aliquots of each virus dilution by using 5 QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA) and eluting in a final volume of 50 pL. A volume of 5 uL of the extracted viral RNAs were tested by real-time RT-PCR for comparative sensitivity test with Directigen Flu A kit.

EXAMPLE 16

The real-time RT-PCR assay for canine influenza relies on information from three molecular probes which target 18S rRNA from host cell was well as M and H3 from the influenza A virus genome (Table 14). Amplification of the host gene is a reporter of specimen quality and integrity. Clinical, necropsy or laboratory samples containing canine influenza (H3N8) virus are expected to yield amplification signal with the three probes.

Specimens yielding amplification signal with M and 18S rRNA probes but negative for H3 would be indicative of an influenza virus subtype H3 from human, swine or avian origin or from non-H3 subtypes. These rare cases could be resolved by RT-PCR using HA universal primers to generate amplicon cDNA that can be analyzed by sequencing. Properly collected and handled specimens lacking influenza A virus yield 18S rRNA amplification signal only.

Ό Situations in which only the 18S rRNA probe and the H3 probes yield amplification signal are indicative of faulty technique, unless proven otherwise; either a false negative with M probes or false positive for H3 need to be demostrated. Finally, specimens failing to yield amplification signals with the three probes are indicative of defective sample collection, degradation, faulty RNA extraction or the presence of inhibitors the polymerases used in 25 PCR.

In order to test the specificity of the H3 primers/probe set for canine influenza A virus (H3N8) and the universality of M primers/probe set for type A influenza, several subtypes of influenza A viruses were tested by real-time RT-PCR. The results show that H3 primers/probe set yielded a positive amplification signal only with canine influenza (H3N8).

No significant false positive or non-specific amplification signals were observed in other subtypes or human H3 strains. The M primers/probe set yielded positive amplification signal with all of the strains tested (Table 15). These results indicated that H3 primers/probe

2015234384 02 Oct 2015 specifically detects canine influenza A virus (H3N8) whereas M primers/probe detect multiple subtypes of type A influenza viruses.

The performance of real-time RT-PCR assays was evaluated by endpoint dilution of M and H3 in vitro transcribed RNAs. As expected, the threshold cycle (Ct) increased in 5 direct correlation with the dilution of the RNA standards. The fluorescent signals can be detected at RNA standard dilutions of M and H3 as low as 10 and 10 copies/qL, respectively (Figure 6A and 6B). The standard curves of M and H3 genes were constructed by plotting the log of starting RNA concentrations against the threshold cycle (Ct) obtained from each dilution (Figure 6C and 6D). The slope of the standard curve is used to determine 0 the PCR reaction efficiency, which is theoretically exponential; 100% amplification efficiency would imply doubling of amplicon cocentration each cycle. The standard curves with a slope between approximately -3.1 and -3.6 are typically acceptable for most applications requiring accurate quantification (90-110 % reaction efficiency). An Rsq value is the fit of all data to the standard curve plot. If all the data lie perfectly on the line, the Rsq 5 will be 1.00. As the data fall further from the line, the Rsq decreases. An Rsq value > 0.985 is acceptable for most assays. The M standard curve revealed a slope of -3.576 (efficiency= 90.4 %) and Rsq= 1.00 whereas H3 standard curve yielded a slope of -3.423 (efficiency= 95.9%) and Rsq= 0.999. These values indicate satisfactory amplification efficiency and overall performance of the real-time RT-PCR assays. We attribute the lower efficiency and Ό sensitivity of M primers/probe set as compared to H3 primers/probe set to the N-fold degeneracy of M primer sequences required to ensure broad coverage of M gene sequences variability across viruses of multiple subtypes, hosts and lineages.

The sensitivity of real-time RT-PCR assay was also compared with the commercial rapid antigen detection assay (Directigen Flu A). Logarithmic dilutions of 25 A/Wyoming/3/2003 (H3N2) and A/canine/Florida/242/2003(H3N8) were analyzed with Directigen Flu A and by real-time RT-PCR. The results of Directigen Flu A showed that the sensitivities against both viral strains are approximately 100-fold dilution from the stock viruses used in these experiments (Figure 7). The signals (purple color) generated by the canine virus (A/canine/Florida/242/2003: 10^6x PFU/ml) samples were much weaker than 30 those found in human virus (A/Wyoming/3/2003: 10^7x PFU/ml), in agreement with the lower virus concentration in these samples. Alternatively, lower signal for canine influenza could

2015234384 02 Oct 2015 be attributed to the molecular specificity of monoclonal antibodies against the NP; i.e. poor conservation of the amino acids within the NP epitope of canine influenza A viruses.

Real-time RT-PCR of the M gene yielded Ct values above threshold with virus 10 and 30 PFU equivalents per reaction of A/canine/Florida/242/2003 and A/Wyoming/3/2003, 5 respectively (Table 16). The differences between the sensitivity value of 2 viral strains because the differences in the original viral titers. The H3 gene detection comparison between canine and human influenza viruses was not performed because the H3 primers/probe in our realtime RT-PCR assay amplifies exclusively canine influenza A virus. RT-PCR was 10⁵ times more sensitive than the rapid antigen detection kit.

To evaluate the performance of the RT-PCR test in necropsy specimens from dogs with acute respiratory disease, sixty canine lung tissue samples submitted during the year of 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 with both M and H3 genes whereas the remaining 48 samples yielded negative result for both M and H3 gene. Virus isolation 5 attempts were conducted by egg and MDCK cell inoculation, to evaluate the specificity of the realtime assay; 2 out 12 samples that were positive for canine influenza by RT-PCR yielded canine influenza virus (data not shown, manuscript in preparation). Although all of the tissues were collected from dogs with a history of severe respiratory disease, most of the samples yielded no canine influenza virus by either realtime PCR or conventional isolation, Ό suggesting a high incidence of other respiratory pathogens such as Bordetella bronchiseptica, canine distemper or parainfluenza virus. The single step real-time RT-PCR assay herein provides a rapid, sensitive and cost-effective approach for canine influenza A virus (H3N8) detection. Rapid laboratory diagnosis of canine influenza A virus (H3N8) infections in the early stage of the disease can yield information relevant to clinical patient and facility 25 management.

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Note: Uppercases = LNA (Locked Nucleic Acid) residues, r = a or g, k= g or t, underline= T7 promoter sequence

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Table 14: Interpretation of the real-time RT-PCR assay
Interpretation	Results
M	H3	18S rRNA
Positive for canine influenza A virus (H3N8)	+	+	+
Positive for influenza A virus (unknown subtype)	+	-	+
Negative for influenza A virus	-	-	+
Error in RNA extraction or presence of PCR inhibitor	-	-	-

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Table 15: Specificity test of canine H3 primers/probe set and universality test of M primers/probe set with several subtypes of influenza A viruses

Subtypes	Strain Name	Host	Real-time RT-PCR detection
H3 gene (Ct)	M gene (Ct)
Hl	A/Ohio/1983	Human	No Ct	15.40
A/WSN/1933	Human	No Ct	20.09
H3	A/Wyo ming/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	Avian	No Ct	17.49
Clinical sample*	Avian	No Ct	20.87
H5	AChicken/Thailand/CUK2/2004	Avian	No Ct	20.13
A/Pheasant/NJ/1335/1998	Avian	No Ct	16.64
H6	Clinical sample*	Avian	No Ct	19.52
H10	Clinical sample*	Avian	No Ct	25.64
Clinical sample*	Avian	No Ct	19.59
Hll	Clinical sample*	Avian	No Ct	15.72
Clinical sample*	Avian	No Ct	24.55

* Note that subtypes of clinical samples were confirmed by nucleotide sequencing.

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Table 16: Comparative sensitivity tests for influenza A virus detection between real-time
	RT-PCR and Directigen Flu A
Virus	Directigen Flu A	Real-time RT-PCR of M (Ct)
dilutions	A/canine/242/03	A/W yoming/3/03	A/canine/242/03	A/Wyoming/3/2003
10’1	+ +	+ + + +	22.42	19.48
kF	+	+ + +	25.85	22.66
kF	-	-	29.27	25.76
ΙΟ’4	Not done	Not done	32.66	28.66
kF	Not done	Not done	35.48	33.14
kF	Not done	Not done	37.51	35.06
ΙΟ’7	Not done	Not done	39.09	36.44
kF	Not done	Not done	No Ct	38.93

Table 17.
Class of Amino Acid	Examples of Amino Acids
Nonpolar	Ala, Val, Leu, He, Pro, Met, Phe, Trp
Uncharged Polar	Gly, Ser, Thr, Cys, Tyr, Asn, Gin
Acidic	Asp, Glu
Basic	Lys, Arg, His

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Table 18.
Letter Symbol	Amino Acid	Letter Symbol	Amino Acid
A	Alanine	M	Methionine
B	Asparagine or aspartic acid	N	Asparagine
C	Cysteine	P	Proline
D	Aspartic Acid	Q	Glutamine
E	Glutamic Acid	R	Arginine
F	Phenylalanine	S	Serine
G	Glycine	T	Threonine
H	Histidine	V	Valine
I	Isoleucine	w	Tryptophan
K	Lysine	Y	Tyrosine
L	Leucine	z	Glutamine or glutamic acid

Table 19. Amino acid differences between PB2 proteins of H3N8 equine and canine influenza viruses
Position	Equine Consensus *	Canine/FL/03	Canine/FL/04
5	K	K	E
12	s	L	L
37	G	G	E
175	R	R	I
374	L	I	I
375	R	R	K
447	Q	Q	H

Table 20. Amino acid differences between PB1 proteins of H3N8 equine and canine
	influenza viruses
Position	Equine Consensus *	Canine/FL/03	Canine/FL/04
114	V	I	I
154	D	G	G
221	A	T	T
317	M	I	I
459	I	I	V
682	I	I	V

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Table 21. Amino acid differences between PA proteins of H3N8 equine and canine influenza viruses
Position	Equine Consensus *	Canine/FL/03	Canine/FL/04
27	D	N	N
62	I	V	V
213	R	K	K
337	A	T	T
343	A	E	E
345	L	I	I
353	K	R	R
400	T	T	A
450	V	I	I
460	M	M	I
673	R	R	K
675	N	D	D

*Based on available genes of viruses isolated between 1963 and 1998.

Table 22. Amino acid differences between NP proteins of H3N8 equine and canine influenza viruses
Position	Equine Consensus *	Canine/FL/03	Canine/FL/04
16	G	D	D
157	A	T	T
214	R	R	K
285	V	V	I
286	A	T	T
359	A	T	T
375	D	D	N
384	R	K	K
452	R	K	K

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Table 23. Amino acid differences between NA proteins of H3N8 equine and canine influenza viruses
Position	Equine Consensus *	Canine/FL/03	Canine/FL/04
9	A/T	T	A
12	S	F	F
20	L	I	I
40	G	R	R
42	G	D	D
46	N	K	K
52	E	E	K
61	R	K	K
69	N	S	S
72	E	K	K
201	V	I	I
261	I	V	V
301	I	I	V
396	N	D	D
397	L	P	P

Table 24. Amino acid differences between Ml proteins of H3N8 equine and canine influenza viruses
Position	Equine Consensus *	Canine/FL/03	Canine/FL/04
Ml 161	S	S	A
Ml 208	K/Q	R	R

*Based on available genes of viruses isolated between 1963 and 1998. 5

Table 25. Amino acid differences between NS1 proteins of H3N8 equine and canine influenza viruses
Position	Equine Consensus *	Canine/FL/03	Canine/FL/04
44	K	R	R
59	R	H	H
71	E	K	K
86	A	T	T
88	R	R	L
140	R	G	G
216	P	S	S

* Based on available genes of viruses isolated between 1963 and 1998.

2015234384 02 Oct 2015

EXAMPLE 17 - CANINE INFLUENZA CHALLENGE MODEL DEVELOPMENT.

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

Procedure:

Ten 13-week-old beagles of mixed sex were obtained from a commercial supplier, and housed in individual cages in a BSL-2 facility. The dogs were randomly assigned to two groups of 5 dogs each. As shown in Table 26, one group was subjected to a intratracheal challenge, and the other group was subjected to an oronasal challenge. The dogs were challenged at 14 weeks-of-age.

Table 26: Experimental Design
Group	Number of Dogs	Challenge Route
1	5	Intratracheal
2	5	Oronasal

A cell culture grown equine flu virus A/equine/Ohio/03 was used as the challenge virus. For intratracheal challenge, the challenge virus was administered via a delivery tube, which consisted of a cuffed tracheal tube (Size 4.0/4.5, Sheridan, USA) and feeding tube (size 5Fr, 1.7 mm, /16 inches in length, Kendall, USA) in 0.5 to 1.0 ml volume. For oronasal challenge, the challenge virus (10 to 10 TCID50 per dog) was administered as a mist using a nebulizer (DeVilbiss Ultra-Neb®99 ultrasonic nebulizer, Sunrise Medical, USA) in a 2 to 3 ml volume.

The dogs were observed for flu related clinical signs for 14 days post-challenge.

Serum samples were collected from each dog on day zero (before challenge), and days 7 and 14 post-challenge for determining the HI titer using a H3N8 equine influenza virus with a 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.

2015234384 02 Oct 2015

Results:

The results of this experiment are summarized in Table 27. Influenza related clinical signs were observed in a few dogs after challenge. These signs included fever (>103°F;

>39.4°C) and cough. Two of 5 dogs (i.e., 40%) had fevers (>103°F; >39.4oC) in Group 1, compared to 1 of 5 (i.e., 20%) dogs in Group 2. One dog from the oronasal challenge group had sneezing, and another had cough following the challenge. An HI titer range from 10 to 80, with a geometric mean titer (GMT) of 20, was observed for Group 1. A titer range of 40 to 160, with a GMT of 86, was observed for Group 2. One dog from each group had histopathological lesions compatible with or pathognomic for influenza.

2015234384 02 Oct 2015

Table 27. Canine flu challenge - clinical signs, virus isolation, histopathology results and serology results

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2015234384 02 Oct 2015

EXAMPLE 18 - EFFICACY OF AN EQUINE INFLUENZA VIRUS VACCINE FOR

DOGS.

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

Procedure:

Nine 7-week-old beagles of mixed sex were obtained from a commercial supplier, and housed in individual cages in a BSL-2 facility. These dogs were randomly assigned to two groups, as summarized in Table 28:

Table 28: Experimental Design
Group	Number of Dogs	Treatment
1	5	Vaccine
2	4	Control

The first group consisted of 5 dogs, which were vaccinated with an inactivated, CARBIGEN™ adjuvanted, equine flu virus A/equine/Ohio/03 vaccine at 8 and 12 weeks-ofage via subcutaneous (SQ) route. The A/equine/Ohio/03 was inactivated by binary ethylenimine (BEI) using a standard method. Each dose of the vaccine contained 5% by mass CARBIGEN™, 4096 HA units of the inactivated virus, sufficient PBS to bring the total volume of the dose to 1 ml, and sufficient NaOH to adjust the pH to between 7.2 and 7.4. Serum samples were collected from all dogs on the day of first and second vaccination and day 7 and 14, post-first and -second vaccination, and at pre-challenge for determining the HI titer using a H3N8 equine influenza virus a standard protocol (SAM 124, CVB, USDA, Ames, IA). At 3 weeks post-second vaccination, the 5 vaccinated dogs and the second group (i.e., the control group) consisting of 4 age-matched dogs were challenged oronasally with a cell-culture-grown equine influenza virus A/equine/Ohio/03 (10 to 10 TCID50 per dog) in a 1-2 ml volume per dose. The challenge virus was administered to the dogs as a mist using a nebulizer (DeVilbiss Ultra-Neb®99 ultrasonic nebulizer, Sunrise Medical, USA).

2015234384 02 Oct 2015

The dogs were observed for flu-related clinical signs for 14 days post-challenge. 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 for collection of lung tissues in 10% buffered formalin for histopathological evaluation.

Results:

The results of this experiment are summarized in Tables 29 and 30. All vaccinated dogs seroconverted following the vaccination. An HI titer range from 40 to 640, with the GMT of 129, was observed during the post-vaccination period with equine influenza virus

A/equine/Ohio/03, and a HI titer of 160 to 320, with a geometric mean titer of 211, was observed with canine flu isolate, A/canine/Florida/242/03. Two of 6 vaccinates had a fever of >103°F (>39.4°C) for one day and no other clinical signs were observed in any of the dogs following challenge.

Conclusion:

All the vaccinated dogs responded to the inactivated, CARBIGEN™ adjuvanted equine influenza vaccine. The HI titer results with a canine influenza virus isolate suggest that the inactivated equine influenza vaccine did induce a detectable level of cross reactive antibody to canine influenza virus. Even though the challenge virus used in this did not

Ό induce any noticeable clinical disease in beagle dogs, based on the HI titer with a canine influenza virus isolate, it was concluded that inactivated equine vaccine could be used in dogs to induce cross reactive antibodies, which could potentially protect dogs against “canine flu” disease caused by H3N8 type canine influenza viruses.

2015234384 02 Oct 2015

Table 29. Serology - Pre- and post-vaccination and post-challenge HI titers

HI titers ||

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2015234384 02 Oct 2015

EXAMPLE 19 - EFFICACY OF AN EQUINE INFLUENZA VIRUS VACCINE FOR DOGS.

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

Procedure:

Approximately 16 month old, 20 mongrels and 20 beagles of mixed sex were used in the study. The 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™, Intervet Inc., Millsboro, DE) at 16 and 17 months of age via subcutaneous (SQ) route. The dogs in groups 2 and 5 were vaccinated with a modified live equine/Kentucky/91 influenza vaccine in a 1 ml volume via intranasal route (single nostril). Blood samples were collected on the day of vaccination, day 7 and 14 post first vaccination (groups 1, 2, 4, and 5) and post second vaccination (groups 1 and 4) for determining the HI titer using an H3N8 equine influenza virus and a canine influenza virus using per a standard protocol (SAM 124, CVB, USDA, Ames, IA).

Vaccinates (at 72 days post final vaccination) and the controls were challenged 7 0 oronasally with a cell-culture grown equine influenza virus strain A/equine/Ohio/03 (10 ο n to 10 TCID50 per dog) in a 1-2 ml volume. The challenge virus was administered to the dogs as mist using a nebulizer (DeVilbiss Ultra-Neb®99 ultrasonic nebulizer, Sunrise Medical, USA). The dogs were observed for influenza-related clinical signs for 12 days post-challenge. The nasal and oropharyngeal swabs were collected in Earl’s MEM medium with antibiotics (neomycin and polymyxin B) daily from day -1 to day 12 post challenge for virus isolation. The presence of virus in the 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 tissues were collected in 10% buffered formalin for histopathological evaluation.

2015234384 02 Oct 2015

Table 31. Experimental design
Group	Number of dogs	Breed	Treatment	Number of doses	Route of vaccination
1	7	Beagle	EQUICINE II™**	2	Subcutaneous
2	7	Beagle	A/KY/91***	1	Intranasal
3	6	Beagle	Control	N/A*	N/A*
4	7	Mongrel	EQUICINE II™	2	Subcutaneous
5	7	Mongrel	A/KY/91	1	Intranasal
6	6	Mongrel	Control	N/A*	N/A*

* Not applicable ** EQUICINE II™ is marketed by Intervet Inc. as a liquid vaccine. EQUICINE II™ contains inactivated A/Pennsylvania/63 influenza (or A/Pa/63) virus and A/equine/Kentucky/93 influenza (or A/KY/93) virus with carbopol (i.e., HAVLOGEN® (Intervet Inc.)). More specifically, a dose of EQUICINE II™ contains: inactivated A/Pa/63 at 10^{6 0} EID50, inactivated A/KY/93 at 10⁶⁷ EID50, 0.25% by volume carbopol, and sufficient PBS to create a total volume of 1 ml.

*** A/KY/91 is a freeze-dried vaccine that was reconstituted with water. Such reconstitution was conducted using vaccine-grade water sufficient to bring the vaccine dosage to a total volume of 1 ml. The vaccine contained equine/Kentucky/91 influenza (or A/KY/91) virus, and is discussed in, for example, U.S. Patent Nos. 6,436,408;

6,398,774; and 6,177,082, which are incorporated by reference in their entirety into this patent. When reconstituted, a dose of the vaccine contained A/KY/91 at 10 TCID50 per ml, 0.015 grams N-Z AMINE AS™ per ml, 0.0025 grams gelatin per ml, and 0.04 grams

D lactose per ml. N-Z AMINE AS™ is a refined source of amino acids and peptides produced by enzymatic hydrolysis of casein. N-Z AMINE AS™ is marketed by Kerry Bio-Science (Norwich, NY, USA).

Results:

All vaccinated dogs seroconverted following the vaccination and the HI titers ranged from 10 to 80 for EQUICINE II™ vaccine group dogs compared to 10 to 40 for the A/KY/91 vaccine group dogs using an equine influenza virus (H3N8 type).

The samples collected at 2 weeks post vaccination (post second vaccination for EQUICINE II™ vaccine) were analyzed for HI titer determination with a canine influenza as well as with an equine influenza virus (H3N8 type). The HI results are shown in Table 32. The clinical signs include fever (>103°F; >39.4oC), occasional cough, and mild nasal discharge observed following the challenge.

2015234384 02 Oct 2015

Table 32. Serology - HI titers at 2 weeks post vaccination
Group	Number of dogs	Breed	Treatment	HI titer with
				Equine influenza virus	Canine influenza virus
				Range	GMT	Range	GMT
1	7	Beagle	Equicine II1M	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	Mongrel	Equicine II1M	40-80	54	40-80	50
5	7	Mongrel	A/KY/91	10-40	24	40-80	49
6	6	Mongrel	Control	N/A*	N/A*	N/A*	N/A*

* Not applicable

Among beagles, 2 of 6 dogs in the EQUICINE II ™ vaccine group (Group 1), 1 of 5 7 dogs in the A/KY/91 vaccine group (Group 2) and 2 of 6 dogs in the control group (Group 3) had fever. One of 6 dogs in Group 3 (control) was positive for virus in the cell culture supernatant of nasal swab material by hemagglutination assay with 0.25% chicken red blood cells (CRBC). One of 6 dogs in the control group (Group 3) and 1 of 7 dogs in the A/KY/91 vaccine group (Group 2) had mild nasal discharge during the post challenge observation period. There was no statistical significant difference (P > 0.05) between control and vaccine groups for beagle dogs.

Among mongrels, 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 fever. One dog from each of Group 4 and 6 had a 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 the nasal swab by HA assay. None of the dogs from the A/KY/91 group (Group 5) were positive for influenza virus in the nasal swab materials.

Conclusion:

By serology, it was demonstrated that vaccination of dogs with commercially available equine influenza vaccines stimulated a moderate level influenza antibody response. There may be some breed difference in development of influenza-related clinical signs in dogs following a challenge with H3N8 type influenza virus. The live attenuated equine influenza vaccine (A/KY/91) provided a significant (P< 0.05) protection

2015234384 02 Oct 2015 from clinical disease development in rectal temperature in mongrels. Also, the live attenuated viral vaccine prevented the shedding of influenza virus in the nasal secretions.

EXAMPLE 20 - CANINE INFLUENZA CHALLENGE MODEL DEVELOPMENT

In view of reports that inducing disease in canines for purposes of study had not proven successful, the potential for using a canine influenza virus, H3N8, to develop a canine influenza challenge model in dogs was investigated in the following study.

Procedure:

Ten mongrels of mixed sex were obtained from a commercial supplier, and housed in cages in a BSL-2 facility. The dogs were randomly assigned to two groups of 5 dogs each. As shown in Table 33, one group was subjected to an intratracheal/intranasal challenge, and the other group was subjected.

Table 33. Experimental design
Group	Number of dogs	Challenge route
1	5	Intratracheal/intranasal
2	5	Oronasal

The dogs were challenged at approximately 12 weeks-of-age. Embryonated-chicken-egg grown canine influenza virus (A/canine/Florida/242/03) virus was used as challenge virus. Each dog received a total of approximately 10 TCID50 of virus in either 2 ml (for oronasal route) or 4 ml (intratracheal/intranasal route) volume.

For intratracheal/intranasal challenge, 3 ml of the challenge virus was administered into the trachea first, followed by 5 ml of PBS using a delivery tube, which consisted of a cuffed tracheal tube (Size 4.5/5.0, Sheridan, USA) and feeding tube (size 5Fr, 1.7 mm; 16 inches (41 cm) in length, Kendall, USA), and a 1 ml challenge virus, followed by 3 ml of atmospheric air was administered into nostrils using a syringe.

For oronasal challenge, the challenge virus was administered as a mist using a nebulizer (Nebulair™, DVM Pharmaceuticals, Inc., Miami, FL) in approximately 2 ml volume. The dogs were observed for flu-related clinical signs for 14 days post-challenge. The dogs were euthanized at day 14 post challenge, and tissue (lung and trachea) samples were collected in 10% buffered formalin for histopathological examination.

2015234384 02 Oct 2015

Results:

All dogs in groups 1 and 2 developed canine influenza clinical signs within 24 to 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, gagging, hemoptysis, and audible rales. Lung tissues from 5 of 5 dogs from group 1 and 4 of 5 dogs from group 2 had histopathological lesions which included one or more of the following: diffuse suppurative bronchopneumonia, bronchitis/bronchoiolitis with plugs of neutrophilic exudate in the lumina and marked mononuclear cell aggregation in mucosa and peribronchiolar tissue, mixed exudate within alveoli with large numbers of foamy macrophages, lymphocellular and plasma cellular as well as granulocytic cell infiltration, and thickening of alveolar septa with proliferation of type II pneumocytes compatible with or pathognomic to an influenza virus infection. The trachea tissue samples were normal.

Conclusion:

An H3N8 canine influenza isolate such as the one used in this study may be used for inducing canine influenza disease in dogs using one of the methods described in this study or a similar method.

EXAMPLE 21 - CANINE INFLUENZA CHALLENGE MODEL DEVELOPMENT.

The potential for using a canine influenza virus, H3N8, to develop a canine influenza challenge model in dogs was further investigated in the following study.

Procedure:

Fifteen 17- to 18-week-old mongrels and five 15-week-old beagles were obtained from commercial suppliers, and were housed in cages in a BSL-2 facility. The mongrels were randomly assigned to 3 groups (Groups 1 to 3) of 5 dogs each. All beagles were assigned to one group (Group 4) as shown in Table 34:

Table 34. Experimental design
Group	Breed	Number of dogs	Challenge virus dose
1	Mongrels	5	1068TCID50
2	Mongrels	5	IO5’8 TCIDso
3	Mongrels	5	IO4'8 TCID5o
4	Beagles	5	106 8 TCID50

2015234384 02 Oct 2015

The dogs were challenged oronasally with a virulent canine influenza virus, A/Canine/Florida/242/2003 (isolated from lung of a greyhound dog with canine influenza disease (provided by Dr. Cynda Crawford at the University of Florida)). The challenge virus was administered as a mist using a nebulizer (Nebulair™) in approximately 2 ml volume. The dogs were observed for flu-related clinical signs for 14 days post-challenge.

Results:

Eighty percent (4 of 5) of the dogs in Group 1 and 4, 100% of the dogs in Group 2 10 and 3, developed canine influenza clinical signs 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 ocular discharge, serous or mucopurulent nasal discharge, vomiting, diarrhea, depression, weight loss, gagging, and rales. The clinical signs observed in beagles were generally milder and short-course compared to mongrels.

Conclusion:

An H3N8 canine influenza isolate such as the one used in this study may be used for inducing canine-influenza-like or kennel-cough-like disease in dogs using method described in this study or a similar method with a challenge dose range from 10^{4 8} to 10^{6 8}

TCID50. There were some differences in clinical signs observed in mongrels and beagles. In general, beagles tend to have milder flu-related clinical signs compared to mongrels.

EXAMPLE 22 - CANINE INFLUENZA VACCINE EFFICACY STUDY.

The following study was conducted to assess the efficacy of an H3N8 equine 25 influenza vaccine in dogs against canine influenza virus.

Procedure:

Seventeen 14-week-old mongrels and ten 8-week-old beagles were obtained from commercial suppliers. The dogs were randomly assigned to 5 groups as shown in Table

35, and housed in a research facility.

100

2015234384 02 Oct 2015

Table 35. Experimental design
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 a HAVLOGEN®-adjuvanted, inactivated equine influenza virus (A/equine/KY/02) vaccine. To prepare this vaccine, the virus was inactivated by binary ethylenimine (BEI) using a standard method. Each vaccine dose contained HAVLOGEN® (10% v/v), 6144 HA units of the inactivated virus, 0.1% (v/v) of 10% thimerosal, 0.1 % (v/v) of phenol red, sufficient NaOH to adjust the pH to from 6.8 to 7.2, and sufficient PBS to bring the total dose volume to 1 ml.

The dogs in Groups 1 and 4 were vaccinated with 2 doses of the vaccine. The 10 second dose (i.e., the booster) was administered 4 weeks after the first. The dogs in Group 2 were vaccinated with 1 dose at 18 weeks-of-age. Blood samples were collected to assess HI titer using a standard protocol (e.g., SAM 124, CVB, USDA, Ames, IA) with an H3N8 canine influenza isolate on days zero (before vaccination), 7, and 14 post first and second vaccinations. Approximately 5 days before challenge, the dogs were moved to a BSL-2 facility and housed in individual cages.

All vaccinates and age-matched control dogs were challenged oronasally with a virulent canine influenza virus (10^{7 7} TCID50 of A/Canine/Florida/242/2003 per dog) at 2 weeks post second vaccination of Groups 1 and 4 and first vaccination of Group 2. The challenge virus was administered as a mist using a nebulizer (Nebulair™) at 2 ml per dog.

The dogs were observed for influenza-related clinical signs for 17 days post-challenge. Nasal and oropharygeal swabs were collected in tubes containing 2 ml of virus transport medium for virus isolation from day -1 (i.e., one day before challenge) to day 17 days post-challenge. All dogs were euthanized at day 17 post-challenge and lung and tracheal samples were collected in 10% buffered formalin for histopathology. Blood samples were collected on days 7 and 14 post challenge for HI titer determination. The clinical sign score assignments used for the post challenge observation are shown in Table 36.

101

2015234384 02 Oct 2015

Results:

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

Virus isolation results are shown in Table 39. Among 14-week-old dogs, canine influenza 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 1-dose vaccine group (Group 2), and 5 of 5 dogs (100%) from the control group (Group 3). Among 8-week-old dogs, the 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 reduction (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 reduction in the number of dogs (60% vs. 100%) positive for canine influenza virus in swab samples between 1-dose vaccine group (Group 2) and the control group (Group 3), the difference was not statistically significant (P = 0.222).

Histopathological evaluation of lung and tracheal tissue samples for lesions was conducted to identify lesions compatible with or pathognomic to canine influenza disease. This includes, for example, determination of whether one or more of the following exist: areas with suppurative bronchopneumonia; peribronchitis/peribronchiolitis with mononuclear cell aggregation (lymphocytes, plasma cells); presence of plugs of granulocytic cellular debris in the lumina; hyperplasia of respiratory epithelium; mixed exudate in the alveoli with large amount of granulocytic cells and cell debris; aggregates of (foamy) macrophages, plasma cells, and lymphocytes; and thickening of alveolar septa with proliferation of type II pneumocytes.

102

2015234384 02 Oct 2015

Table 40 provides a summary of the extent of lesions in this experiment for the dogs. Among 14-week-old dogs, the 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 controls dogs (Group 3) had severe and extensive lesions suggestive of no protection. There was no difference in tracheal lesions due to 1 or 2-dose vaccination among 14- week-old dogs. Among 8-week-old dogs, there was no difference in lung lesions between 2-dose vaccinates and control dogs. None of the dogs had any tracheal lesions.

Conclusion:

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

Table 36. Clinical signs and scoring system
Clinical signs	Score per day
Temp
<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
Coughing
No coughing	0
Occasional	2
Paroxysmal	4
Sneezing
No sneezing	0
Occasional	1
Paroxysmal	2
Nasal discharge
No discharge	0
Serous -slight	1
Serous -copious	1
Mucopurulent-slight	2
Mucopurulent-copious	3

103

2015234384 02 Oct 2015

Table 36. Clinical signs and scoring system
Clinical signs	Score per day
Ocular discharge
No discharge	0
Serous -slight	1
Serous -copious	1
Mucopurulent-slight	2
Mucopurulent-copious	3
Hemoptysis
No	0
Yes	5
Depression
No	0
Yes	1
Anorexia
No	0
Yes	1
Respiratory signs
None	0
Rales	3
Dyspnea	4
Gasping	5
Mucous expectorate
No	0
Yes	2
Vomiting
No	0
Yes	1
Fecal abnormalities
No	0
Yes	1

2015234384 02 Oct 2015

Table 37. Serology - Hemagglutination inhibition titers

HI titer

S ω O 60 Cl G

| 320 1

| >640 1

| >640 1

| 320 1

| 320 1

| 320 1

| >640 1

| >640 1

| >640 1

| >640 1

| 320 1

| >640 |

O SO H

| 320 1

o so H

o so H

o so H

| >640 1

| >640 1

| >640 1

| >640 1

| >640 |

o so H

1 80 1

1 40 1

o so H

o SO H

Days chall

Γ-

o so

80

80

o so H

o so H

o so H

80

>640

320

320

o so H

320

40

40

40

40

40

o so H

320

o so H

o so H

o so H

80

80

20

80

40

Days post first vaccination of Groups 1 and 4

* * * C4

| 20 I

| 80 1

| 20 1

| 40 I

| 40 I

| 40 I

| 40 1

| < io |

| < 10 1

| < 10 1

I < io |

I < io |

I < io |

| < io |

| < io |

| < io |

I < io |

| 80 1

| 40 1

| 80 1

o so H

o so H

| < io |

| < io |

| < io |

I < io |

I < io |

| 35 1

| 40 I

| 40 1

| 20 1

| 40 I

| 20 I

| 40 I

| 20 1

| < io |

| < 10 1

| < 10 1

| < io |

I < io |

I < io |

| < io |

| < io |

| < io |

I < io |

| 80 1

| 40 1

| 20 1

| 80 1

| 80 |

| < io |

| < io |

| < io |

I < io |

I < io |

28** |

| 20 I

| 40 |

o H

o H

o H

| 20 I

o H

| <io |

| <10 |

| <10 |

| <io |

I <io |

I <io |

| <io |

| <io |

| <io |

I <io |

| 40 1

| 40 1

| 20 1

| 40 1

| 40 1

| <io |

| <io |

| <io |

I <io |

I <io |

o

| <10 |

| <10 |

| <10 1

| <10 1

| <10 |

| <10 |

| <io |

| <10 |

| <10 |

| <io |

I <io |

I <io |

| <io |

| <io |

| <io |

I <io |

o H

| 20 1

o H

o H

I <10 |

| <io |

| <io |

| <io |

I <io |

I <io |

Γ-

| <10 1

o H V

| <10 |

| <10 |

| <10 |

| <10 1

| <10 |

| <io |

| <10 |

| <10 |

| <io |

I <io |

I <io |

| <io |

| <io |

| <io |

I <io |

| <10 |

| 20 1

| <10 |

| <10 |

I <io |

o H V

| <io |

| <io |

I <io |

I <io |

* o

| < 10

| < 10

| < 10

| < 10

| < 10

| < 10

| < 10

| < io

| < 10

| < 10

| < io

I < io

I < io

| < io

| < io

| < io

I < io

| < io

I < io

| < io

| < io

I < io

I < io

| < io

| < io

I < io

I < io

Number of doses

04

04

04

04

04

04

04

H

H

H

H

H

N/A |

N/A |

N/A |

N/A |

N/A |

04

04

04

04

04

N/A |

N/A |

N/A |

N/A |

N/A |

Treatment

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Control

Control

Control

Control

Control

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Control

Control

Control

Control

Control

Age (week)

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

H

oo

CO

CO

CO

CO

CO

CO

CO

CO

CO

Dog ID

H 04 Os

| 926 1

H CO Os

| 955 1

H H o

| 013 1

| 019 1

922 |

| 953 1

| 015 1

| 016 1

| 017 1

923 |

| 012 I

| 014 I

| 018 1

| 01A 1

406 |

407 |

| 504 1

704 |

| 705 |

404 |

| 405 1

| 610 1

c4 O r-

703 |

Group No

H

H

H

H

H

H

H

04

04

04

04

04

CO

CO

CO

co

CO

to

to

*First vaccination - Groups 1 and 4 **Second vaccination - Groups 1 and 4; First vaccination - Group 2 ***Day of challenge

2015234384 02 Oct 2015

Table 38. Analysis of total canine influenza disease clinical scores	P-value* 1		0.003 (Group 1 vs. 3)	0.294 (Group 2 vs. 3)	1 1	0.040 (Group 4 vs. 5)	1 1
Average total	Score per dog	8.7	21.8	25.4	2.0	5.4
Age at first vaccination	of Groups 1 and 4	14 weeks	14 weeks (these dogs were vaccinated once, when they were 18 weeks old)	14 weeks (these dogs were not vaccinated)	8 weeks	8 weeks (these dogs were not vaccinated)
Number of doses	of vaccine	C4	-	1 1	04	1 1
Treatment		Vaccinate	Vaccinate	Control	Vaccinate	Control
1 Group	1	-	04	CO		MO

<υ

-w a

cn cd

S-H

C o

X o

cj <u

-w

W) c

O cj <u ¢3 cn

C4

O

S-H

W) <u c

CJ (J cd >

o

4= <N oo c

o >

©

CO <

CO <υ

S-H =3 <υ <υ ο

S-H

Οη

Ζ <

<

Οη

Ζ

OQ

C ζΛ ο

<υ

Ν rZ* ’cd

C <

*

2015234384 02 Oct 2015

Table 39. Virus shedding

Days post-challenge

o

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

se

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

Nt

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

CM

z

z

z

z

z

z

z

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z

z

z

z

z

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z

z

z

z

z

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z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

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z

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z

z

z

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z

z

z

z

z

z

z

z

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O

z

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z

z

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z

z

z

O\

z

z

z

z

z

z

z

z

z

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z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

so

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

r~

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

sc

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

m

z

z

z

z

z

z

z

z

z

9i

9

z

9i

z

9

z

9

z

z

z

z

z

9

9

z

z

Nt

z

z

z

z

/O

z

z

z

:0/

:O/

(Ο

z

/0/

/0/

:O/

z

z

z

z

z

z

z

z

z

z

en

z

z

z

z

z

z

z

z

z

z

z

9

z

z

z

9

9

z

z

z

z

z

z

z

z

z

z

CM

z

z

z

z

z

z

z

z

z

z

z

9

z

9

z

z

z

z

z

z

z

z

z

z

:Rii

z

z

z

z

z

z

z

z

z

9

z

z

z

z

z

z

z

9i

z

z

z

z

o

z

z

z

z

Z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

Z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

z

Number of vaccine doses

CM

CM

CM

CM

CM

CM

CM

i—H

i—H

i—H

i—H

N/A I

N/A |

N/A |

N/A |

N/A |

CM

CM

CM

CM

CM

N/A |

N/A |

N/A 1

N/A |

N/A I

Treatment

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Control

Control

Control

Control

Control

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Vaccinate

Control

Control

Control

Control

Control

Age (week)

Nt i—H

Nt i—H

Nt i—H

Nt i—H

Nt i—H

Nt i—H

Nt i—H

Nt i—H

Nt i—H

Nt i—H

Nt i—H

Nt i-H

Nt i—H

Nt i—H

Nt 1—H

Nt 1—H

Nt 1—H

CO

CO

CO

CO

CO

CO

CO

CO

CO

CO

w> — © Q Q M

i—H CM 05

| 926 1

i—H m 05

| 955 1

i—H i—H o

I 013 I

| 019 1

922 |

| 953 1

| 015 1

| 016 |

| 017 I

923 |

| 012 I

| 014 1

| 018 |

| 01A I

406 |

407 |

| 504 1

704 |

| 705 1

404 |

| 405 1

| 610 |

702 |

703 |

Group No

i—H

i—H

i—H

i—H

i—H

i—H

i—H

CM

CM

CM

CM

CM

cc

cc

cc

cc

cc

Nt

Nt

Nt

Nt

Nt

in

in

in

in

in

107

2015234384 02 Oct 2015

Table 40. Histopathological evaluation of tissue samples
Group No	Dog ID	Age (week)	Treatment	Number of doses	Microscopic lesion (Histopathology)
Lungs	Trachea
1	921	14	Vaccinate	2	+/-	-
1	926	14	Vaccinate	2	-	+/-
1	931	14	Vaccinate	2	-	-
1	955	14	Vaccinate	2	+/-	-
1	Oil	14	Vaccinate	2	+/-	-
1	013	14	Vaccinate	2	+/-	+/-
1	019	14	Vaccinate	2	+/-	+/-
2	922	14	Vaccinate	1	+/-	-
2	953	14	Vaccinate	1	+/-	+/-
2	015	14	Vaccinate	1	+/-	+
2	016	14	Vaccinate	1	-	-
2	017	14	Vaccinate	1	+/-	+/-
3	923	14	Control	N/A	+	+/-
3	012	14	Control	N/A	+	-
3	014	14	Control	N/A	+	-
3	018	14	Control	N/A	+	-
3	01A	14	Control	N/A	+	+/-
4	406	8	Vaccinate	2	+/-	-
4	407	8	Vaccinate	2	-	-
4	504	8	Vaccinate	2	+/-	-
4	704	8	Vaccinate	2	-	-
4	705	8	Vaccinate	2	-	-
5	404	8	Control	N/A	-	-
5	405	8	Control	N/A	-	-
5	610	8	Control	N/A	+/-	-
5	702	8	Control	N/A	+/-	-
5	703	8	Control	N/A	-	-

“+” Severe lesion consistent or pathognomic to an influenza infection 5 “+/-“ Mild lesion (inconclusive)

Normal

EXAMPLE 23 - CANINE INFLUENZA VACCINE EFFICACY STUDY

The following study was conducted to determine the efficacy of a multivalent

H3N8 equine influenza vaccine against canine influenza virus in dogs.

Procedure:

108

2015234384 02 Oct 2015

Seventeen 15-week-old beagles were obtained from a commercial supplier. The dogs were randomly assigned to 3 groups as shown in Table 41, and housed in a research facility.

Table 41. Experimental design
Group	Number of dogs	Treatment	Number of doses	Age at Vaccination (weeks)
1	7	Vaccinate	2	15 & 19
2	5	Vaccinate	1	19
3	5	Control	—	—

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

The dogs in Group 1 were vaccinated with 2 doses of the vaccine. The second (i.e., booster) dose was administered 4 weeks after the first dose. The dogs in Group 2 were vaccinated with 1 dose of vaccine at 19 weeks-of-age. Blood samples were collected to assess HI titer using a standard protocol with an H3N8 canine influenza isolate on days zero (before vaccination), 7, and 14 post first and second vaccinations. Seven days before challenge, the dogs were moved to a BSL-2 facility and housed in individual cages.

All vaccinates and age-matched control dogs were challenged oronasally with a virulent canine influenza virus (10^{7 3} TCID50 of A/Canine/Florida/242/2003 per dog) at 2 weeks post second vaccination of Group 1 and first vaccination of Group 2. The challenge virus was administered as a mist using a nebulizer (Nebulair™) at 2 ml per dog. The dogs were observed for influenza-related clinical signs for 14 days post challenge. All dogs were euthanized at day 14 post-challenge, and lung and trachea samples were collected in

10% buffered formalin for histopathology. Blood samples were collected on days 7 and post challenge for HI titer determination. The clinical sign score assignments used for the post challenge observation are shown in Table 42.

109

2015234384 02 Oct 2015

Results:

All vaccinated dogs developed HI antibody titer responses to the canine influenza virus isolate (Table 43). Following the challenge, approximately a 4 fold increase in HI titer on day 14 post challenge compared to the pre-challenge HI titer in all groups indirectly indicate that all dogs were exposed to the challenge virus. All dogs exhibited signs canine influenza disease with each dog demonstrating one 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, and dyspnea. Vaccinates had less severe clinical signs, compared to age-matched controls (Table 44). There was a significant (P = 0.028) reduction in clinical signs due to the 2dose vaccination in dogs (Group 1). One dose vaccination did not provide a significant (P = 0.068) reduction in clinical signs (Group 2).

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

Conclusion:

The results from this study demonstrate that 1) inactivated H3N8 equine influenza virus can induce canine influenza virus cross reactive HI antibody responses in vaccinated dogs, and 2) Use of a H3N8 equine influenza virus vaccine can reduce the severity of canine influenza virus disease in dogs.

Table 42. Clinical signs anc	scoring system
Clinical signs	Score ner dav
Temp
<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

110

2015234384 02 Oct 2015

Table 42. Clinical signs anc	scoring system
Clinical signs	Score per day
Coughing
No coughing	0
Occasional	2
Paroxysmal	4
Sneezing
No sneezing	0
Occasional	1
Paroxysmal	2
Nasal discharge
No discharge	0
Serous -slight	1
Serous -copious	1
Mucopurulent-slight	2
Mucopurulent-copious	3
Ocular discharge
No discharge	0
Serous -slight	1
Serous -copious	1
Mucopurulent-slight	2
Mucopurulent-copious	3
Hemoptysis
No	0
Yes	5
Depression
No	0
Yes	1
Anorexia
No	0
Yes	1
Respiratory signs
None	0
Rales	3
Dyspnea	4
Gasping	5
Mucous expectorate
No	0
Yes	2
Vomiting
No	0
Yes	1
Fecal abnormalities
No	0
Yes	1

2015234384 02 Oct 2015

Table 43. Serology - Hemagglutination inhibition titers

HI titer

ω o W) ci. fi O

TO

320

320

80

160

320

160

160

o

80

320

80

320

160

160

80

80

80

.5 Λ Q υ

Γ-

o o

o o

o o

o o

o o

o 00

o TO

o <N cn

>640

>640

o <N cc

o <N cc

o TO

o 00

o <N

o 00

o TO

Days post first vaccination of Group 1

* * * TO

o TO

o TO

o TO

o TO

o <N

o TO

O TO

o

o <N

o

o

o

o V

o V

o V

o V

o V

m CO

O 00

O

O TO

O TO

O TO

o TO

O TO

o V

o V

o V

o V

o V

o V

o V

o V

o V

o V

* * 00

o

O

O <N

o

o

O <N

O TO

o V

o V

o V

o V

o V

o V

o V

o V

o V

o V

TO

o

o

O <N

o

o

o

O TO

o V

o V

o V

o V

o V

o V

o V

o V

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2015234384 02 Oct 2015

Table 44. Analysis of total canine influenza disease clinical scores	P-value*	0.028 (Group 1 vs. 3)	0.068 (Group 2 vs. 3)	1 1
Average total Score per dog	6.3	14.2	24.4
Age at first vaccination of Group 1	15 weeks	15 weeks (these dogs were vaccinated once, when they were 19 weeks old)	15 weeks (these dogs were not vaccinated)
Number of doses		-	1 1
Treatment	Vaccinate	Vaccinate	Control
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2015234384 02 Oct 2015

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Normal

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114

2015234384 02 Oct 2015

EXAMPLE 24 - CANINE INFLUENZA VACCINE EFFICACY STUDY

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

Procedure:

Thirty 10-week old mongrels were obtained from a commercial supplier. The dogs were randomly assigned to 6 groups as shown in Table 46, and housed in a research facility.

Table 46. Experimental design
Group	Number of dogs	Treatment	Route 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 (VAX-1, VAX-2, and VAX-3) were used. The VAX-1 was a HAVLOGEN®-adjuvanted, inactivated equine influenza virus (A/equine/KY/02) monovalent vaccine, and each dose contained HAVLOGEN® (10% v/v), 6144 HA units of the inactivated virus, 0.1% (v/v) of 10% thimerosal, 0.1 % (v/v) of phenol red, sufficient NaOH to adjust the pH to 6.8 to 7.2, and sufficient PBS to bring the total dose volume to 1 ml. The VAX-2 was a HAVLOGEN®-adjuvanted, inactivated equine influenza virus (A/equine/KY/02) monovalent vaccine, and each dose of vaccine contained HAVLOGEN® (10% v/v), 4096 HA units of the inactivated virus, 0.1% (v/v) of

10% thimerosal, 0.1 % (v/v) of phenol red, sufficient NaOH to adjust the pH to 6.8 to 7.2, and sufficient PBS to bring the total dose volume to 1 ml. The VAX-3 was a HAVLOGEN®-adjuvanted, inactivated equine influenza (A/equine/KY/02, A/equine/KY/93, and A/equine/NM/2/93 ) multivalent vaccine, and contained HAVLOGEN® (10% v/v), 2048 HA units of inactivated virus per strain, 0.1% (v/v) of

10% thimerosal, 0.1 % (v/v) of phenol red, sufficient NaOH to adjust the pH to 6.8 to 7.2, and sufficient PBS to bring the total dose volume to 1 ml. All influenza viruses used for

115

2015234384 02 Oct 2015 the vaccine formulation were inactivated by binary ethylenimine (BEI) using a standard method.

The vaccines and routes of administration for each group are described in Table 46. All dogs in the vaccinated groups were vaccinated either via the intranasal (IN) or the subcutaneous (SQ) route, and each dog received 2 doses. The second (i.e., booster) dose was administered 4 weeks after the first dose. Blood samples were collected to assess HI titer using a standard protocol with an H3N8 canine influenza isolate on days zero (before vaccination), 7, and 14 post first and second vaccinations. Seven days before challenge, the dogs were moved to a BSL-2 facility and housed in individual cages.

All vaccinates and age-matched control dogs were challenged oronasally with a virulent canine influenza virus (10^{7 4} TCID50 of A/Canine/Florida/242/2003 per dog) at 2 weeks post second vaccination. The challenge virus was administered as a mist using a nebulizer (Nebulair™) in a 2 ml volume per day. The dogs were observed for influenzarelated clinical signs for 14 days post-challenge. Blood samples were collected on days 7 and 14 post challenge for HI titer determination. All dogs were euthanized at day 14 postchallenge, and lung and trachea samples were collected in 10% buffered formalin for histopathology. The clinical sign score assignments used for the post challenge observation are shown in Table 47.

Results:

All dogs vaccinated via the SQ route developed HI antibody titer responses to the canine influenza virus isolate, regardless of the vaccine type (Table 48). None of the dogs from the IN vaccination groups (i.e., Groups 1, 3, and 5) developed HI antibody titer responses to the canine influenza virus isolate, regardless of the vaccine type, during the post vaccination period. There was, however, a 4-fold increase in titer by day 14 post challenge in all dogs indirectly, 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 ocular discharge, serous or mucopurulent nasal discharge, vomiting, diarrhea, depression, weight loss, and dyspnea. Vaccinates had less severe clinical signs, compared to age-matched controls (Table 49). There was a significant reduction in clinical signs in dogs vaccinated with VAX-3 via the SQ route (Group 4). In this experiment, IN administration of either VAX-1, VAX-2, or VAX-3 did not provide a significant reduction in clinical signs of canine influenza virus.

116

2015234384 02 Oct 2015

As in Examples 22 and 23, histopathological evaluation of lung and tracheal tissue samples for lesions was conducted to identify lesions compatible with or pathognomic to canine influenza disease. Table 50 provides a summary of the extent of lesions in this experiment for the dogs. Five of 5 control dogs (Group 6) had lung lesions consistence with an influenza infection. Two of 5 dogs vaccinated with VAX-2 via the SC route (Group 2) and 3 of 5 dogs vaccinated with VAX-3 via the SC route (Group 4) were free of any influenza-related lung lesions. All the dogs that received the vaccine via the intranasal route, irrespective of the vaccine type, had severe lung lesions consistent with an influenza infection. The trachea lesions observed in this study were very mild.

Conclusion:

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

117

2015234384 02 Oct 2015

Table 47. Clinical signs and scoring system
Clinical signs	Score per day
Temp
<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
Coughing
No coughing	0
Occasional	2
Paroxysmal	4
Sneezing
No sneezing	0
Occasional	1
Paroxysmal	2
Nasal discharge
No discharge	0
Serous -slight	1
Serous -copious	1
Mucopurulent-slight	2
Mucopurulent-copious	3
Ocular discharge
No discharge	0
Serous -slight	1
Serous -copious	1
Mucopurulent-slight	2
Mucopurulent-copious	3
Hemoptysis
No	0
Yes	5
Depression
No	0
Yes	1
Anorexia
No	0
Yes	1
Respiratory signs
None	0
Rales	3
Dyspnea	4
Gasping	5
Mucous expectorate
No	0
Yes	2

118

2015234384 02 Oct 2015

Table 47. Clinical signs and scoring system
Clinical signs	Score per day
Vomiting
No	0
Yes	1
Fecal abnormalities
No	0
Yes	1

2015234384 02 Oct 2015

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2015234384 02 Oct 2015

Table 49. Analysis of total canine influenza disease clinical scores	P-value*	0.500 (Group 1 vs. 6)	0.345 (Group 2 vs. 6)	0.631 (Group 3 vs. 6)	0.016 (Group 4 vs. 6)	0.790 (Group 4 vs. 6)	1 1
Average total Score per dog	35.2	31.0	39.4	13.0	42.6	36.8
Route of vaccination	Z	SQ	Z	SQ	Z	1 1
Treatment	VAX-1	VAX-2	VAX-2	VAX-3	VAX-3	Control
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2015234384 02 Oct 2015

Table 50. Histopathological evaluation of tissue samples
Group No	Dog ID	Treatment	Route of vaccination	Number of doses	Microscopic lesion (Histopathology)
Lung	Trachea
1	248	Vaccinate	IN	2	+	-
1	501	Vaccinate	IN	2	+	-
1	502	Vaccinate	IN	2	+	-
1	469	Vaccinate	IN	2	+	+
1	46A	Vaccinate	IN	2	+	+
2	232	Vaccinate	SQ	2	+	-
2	511	Vaccinate	SQ	2	+	-
2	514	Vaccinate	SQ	2	-	-
2	461	Vaccinate	SQ	2	+	-
2	463	Vaccinate	SQ	2	-	-
3	246	Vaccinate	IN	2	+	-
3	505	Vaccinate	IN	2	+	-
3	506	Vaccinate	IN	2	+	+
3	464	Vaccinate	IN	2	+	-
3	465	Vaccinate	IN	2	+	+
4	23B	Vaccinate	SQ	2	-	-
4	247	Vaccinate	SQ	2	+/-	-
4	508	Vaccinate	SQ	2	-	-
4	512	Vaccinate	SQ	2	-	+/-
4	516	Vaccinate	SQ	2	+	+
5	503	Vaccinate	IN	2	+	+/-
5	513	Vaccinate	IN	2	+	+
5	462	Vaccinate	IN	2	+	+/-
5	466	Vaccinate	IN	2	+	+
5	46B	Vaccinate	IN	2	+	-
6	236	Control	—	2	+	-
6	504	Control	—	2	+	+
6	507	Control	—	2	+	+
6	515	Control	—	2	+	+/-
6	468	Control	—	2	+	+

“+” Severe lesion consistent or pathognomic to an influenza infection “+/-“ Mild lesion (inconclusive)

Normal

EXAMPLE 25 - CANINE INFLUENZA VACCINE EFFICACY STUDY

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

123

2015234384 02 Oct 2015

Approximately 80% of exposed dogs develop clinical signs. In the following study the efficacy of an inactivated H3N8 equine influenza virus vaccine and a canine influenza virus vaccine were determined.

Procedure:

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

Table 51. Experimental design
Group	Treatment	Vaccination route	Number of dogs	Age at vaccination (weeks)	Challenge
1	VAX-1	IM	15	8 & 12	Yes
2	VAX-2	SC	5	8 & 12	Yes
3	Control	N/A	20	N/A	Yes

The dogs in Groups 1 and 2 were vaccinated with either VAX-1 or VAX-2 (Table 51). VAX-1 was a HAVLOGEN® adjuvanted, inactivated equine influenza virus (A/equine/KY/02) vaccine. For vaccine preparation, the vaccine virus was inactivated by binary ethylenimine (BEI) using a standard method. Each dose of vaccine contained HAVLOGEN® (10% v/v), 6144 HA units of the inactivated virus, 0.1% (v/v) of 10% thimerosal, 0.1 % (v/v) of phenol red and sufficient PBS to bring the total dose volume to 1 ml and sufficient NaOH to adjust the pH to 6.8 to 7.2.

VAX-2 was an inactivated, CARBIGENTM adjuvanted, canine influenza antigen vaccine (A/canine/Fl/43/2004). The A/canine/Fl/43/2004 was inactivated by binary ethylenimine (BEI) using a standard method. Each dose of the vaccine contained 5% by mass CARBIGENTM, approximately 1280 HA units of the inactivated virus, sufficient PBS to bring the total volume of the dose to 1 ml, and sufficient NaOH to adjust the pH to between 7.2 and 7.4. Serum samples were collected from all dogs on the day of first and second vaccination, days 7 and 14 post first and second vaccinations, and at pre-challenge to determine the HI titers using an H3N8 equine influenza virus standard protocol (SAM 124, CVB, USDA, Ames, IA). Seven days before challenge, the dogs were moved to a ABSL-2 facility and housed in individual cages.

124

2015234384 02 Oct 2015

All vaccinates and age-matched control dogs were challenged oronasally with virulent canine influenza virus (10^{7 2} TCID50 of A/Canine/Florida/242/2003 per dog) at 2 weeks post second vaccination. The challenge virus was administered as a mist (2ml/dog) using a nebulizer (Nebulair™). The dogs were observed for influenza-related clinical signs for 14 days post-challenge. Nasal and oropharyngeal swabs were collected daily in tubes containing 2 ml of virus transport medium for virus isolation from day -1 (i.e., one day before challenge) through day 14 post-challenge. Blood samples were collected on days 7 and 14 post challenge for HI titer determination. The clinical sign score assignments used for post challenge observation are shown in Table 52.

Results:

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

Virus isolation results are shown in Tables 55 and 56. Following a virulent canine influenza virus challenge, the 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 inactivated equine influenza vaccine (VAX-1) and canine influenza virus (VAX-2) vaccinates demonstrated a significant (P = 0.004) reduction in virus shedding in nasal or oral secretions or both (Table 55) compared to controls.

Conclusion:

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

125

2015234384 02 Oct 2015

Table 52. Clinical signs and scoring system
Clinical signs	Score per day
Temp
<103.0°F (<39.4°C)	0
103.0- 103.9°F (39.4-	2
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Occasional	1
Paroxysmal	2
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No discharge	0
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Serous -copious	1
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Mucopurulent-copious	3
Ocular discharge
No discharge	0
Serous -slight	1
Serous -copious	1
Mucopurulent-slight	2
Mucopurulent-copious	3
Hemoptysis
No	0
Yes	5
Depression
No	0
Yes	1
Anorexia
No	0
Yes	1
Respiratory signs
None	0
Rales	3
Dyspnea	4
Gasping	5
Mucous expectorate
No	0
Yes	2
Vomiting
No	0
Yes	1

126

2015234384 02 Oct 2015

Table 52. Clinical signs and scoring system
Clinical signs	Score per day
Fecal abnormalities
No	0
Yes	1

2015234384 02 Oct 2015

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129

2015234384 02 Oct 2015

Table 54.	Analysis of total canine influenza disease clinical scores
Group	Treatment	Average total Score 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 a NPARIWAY procedure of SAS® Version 9.1 (the vaccine groups were compared using the GLM procedure)

Table 55.	’ost-challenge virus shedding
Group	Treatment	Percent dogs excreted 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 a FREQ procedure of SAS® (Version 9.1) and P-value associated with Fisher’s exact test

2015234384 02 Oct 2015

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132

2015234384 02 Oct 2015

Table 57. Hemagglutinin, neuraminidase and nucleoprotein gene amino acid sequence similarities among influenza viruses
Gene (Canine /Florida/43/2004)	Amino acid sequence similarity	Gene of influenza virus used for comparison
Hemagglutinin	88	equine/Algiers/72
HA	90	equine/Sao paulo/6/69
HA	91	equine/Miami/1/63
HA	93	equine/Newmarket/79
HA	94	equine/Kentucky/1/81
HA	95	Equi-2/Ludhiana/87
HA	96	Equine/Alaska/1/91
HA	97	equine/Tennessee/5/86
HA	98	equine/Kentucky/5/02
HA	99	equine/Ohio/1/2003
HA	99	canine/Florida/242/2003
Neuraminidase	88	Eq/Algiers/72
NA	90	equine/Sao Paulo/6/69
NA	91	equine/Miami/1/63
NA	93	equine/Newmarket/79
NA	94	equine/Kentucky/1/81
NA	95	Equi-2/Ludhiana/87
NA	96	eq uine/S antiago/8 5
NA	97	equine/Tennessee/5/86
NA	98	equine/Kentucky/5/2002
NA	99	eq uine/Ohio/1/2003
NA	99	canine/Florida/242/2003
Nucleoprotein (NP)	94	equi/Miami/1/63
NP	97	equine/Kentucky/1/81
NP	99	equine/Kentucky/5/02
NP	99	equine/Ohio/1/2003
NP	99	canine/Florida/242/2003

The words comprise, comprises, and “comprising in this patent (including the 5 claims) are to be interpreted inclusively rather than exclusively. This interpretation is intended to be the same as the interpretation that these words are given under United States patent law.

The above detailed description of preferred embodiments is intended only to acquaint others skilled in the art with the invention, its principles, and its practical application so that

133

2015234384 02 Oct 2015 others skilled in the art may adapt and apply the invention in its numerous forms, as they may be best suited to the requirements of a particular use. This invention, therefore, is not limited to the above embodiments, and may be variously modified.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

134

2015234384 02 Oct 2015

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Claims (6)

1. The claims defining the invention are as follows.

   1. An isolated type A canine influenza virus that is capable of infecting a canid 5 animal, comprising a polypeptide having the amino acid sequence shown in SEQ ID NO:78 or a mature sequence thereof wherein the N-terminus amino acid signal sequence of the full length sequence has been removed.
2. 2. The type A canine influenza virus of claim 1 further comprising a polypeptide 0 having 98% or greater sequence identity with the amino acid sequence shown in any of SEQ

   ID NOs: 64, 66, 68, 70, 72, 74 or 76; or a polypeptide having the amino acid sequence shown in any of SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, or a functional and/or immunogenic fragment thereof.

   5 3. The type A canine influenza virus according to claim 1 or claim 2, wherein said type A canine influenza virus comprises a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 77.

   4. The type A canine influenza virus according to claim 2 or claim 3, wherein

   Ό said influenza virus further comprises a polypeptide encoded by the nucleotide sequence shown in any of SEQ ID NOs: 63, 65, 67, 69, 71, 73 or 75, or a polynucleotide having 98% or greater sequence identity with the nucleotide sequence shown in any of SEQ ID NOs: 63, 65,67, 69,71,73 or 75.

   25 5. The type A canine influenza virus according to any one of claims 1 to 4, wherein said influenza virus is inactivated or attenuated.

   6. The type A canine influenza virus according to any one of claims 1 to 5, wherein said influenza virus is provided in a pharmaceutically acceptable carrier or diluent.

   7. An isolated recombinant polynucleotide comprising a polynucleotide which encodes a canine influenza A virus polypeptide as defined in claim 1 and a polynucleotide sequence heterologous to said influenza virus.

   141

   2015234384 16 Jan 2018

   8. A recombinant chimeric polynucleotide, comprising a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 77 and a polynucleotide having 98% or greater sequence identity with the nucleotide sequence shown in any of SEQ ID NOs: 63, 65,

   5 67, 69, 71,73 or 75 .

   9. The recombinant chimeric polynucleotide according to claim 7 or claim 8, wherein said polynucleotide is formulated in a pharmaceutically acceptable carrier or diluent.

   0 10. A polynucleotide expression construct comprising the recombinant chimeric polynucleotide according to claim 7 or claim 8.

   11. An isolated polypeptide having the amino acid sequence shown in SEQ ID NO:78 or a mature sequence thereof wherein the N-terminus amino acid signal sequence of

   5 the full length sequence has been removed, or having the amino acid sequence shown in any of SEQ ID NOs: 64, 66, 68, 70, 72, 74 or 76 or a polypeptide having 98% or greater sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 64, 66, 68, 70, 72 or 76.

   12. The polypeptide according to claim 11, wherein said polypeptide is selected

   Ό from the group consisting of a hemagglutinin protein, a neuraminidase protein, a nucleoprotein, a matrix protein, a polymerase basic protein, a polymerase acidic protein, and a non-structural protein.

   13. The polypeptide according to claim 11 or 12, wherein said polypeptide is 25 formulated in a pharmaceutically acceptable carrier or diluent.

   14. A composition comprising the canineinfluenza type A virus of any one of claims 1 to 5, or an immunogen of the canine influenza type A virus of any one of claims 1 to 5, optionally further comprising an adjuvant, wherein said immunogen is capable of inducing

   30 an immune response against an influenza virus that is capable of infecting a canid animal and wherein said immunogen comprises a canine influenza virus polypeptide as defined in claim 11.

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   142

   15. The composition according to claim 14, wherein said composition is formulated in a pharmaceutically acceptable carrier or diluent.

   16. A method for inducing an immune response in a canid animal against an

   5 influenza virus capable of infecting a canid animal, said method comprising administering to said canid animal an effective amount of a composition according to claims 14 or claim 15 or the recombinant polynucleotide of any one of claims 7 to 9, or the polynucleotide expression construct of claim 10.

   0 17. The method according to claim 16, wherein said composition comprises a viral polypeptide, having the amino acid sequence shown in SEQ ID NO:78 or a mature sequence thereof wherein the N-terminus amino acid signal sequence of the full length sequence has been removed, or having the amino acid sequence shown in any of SEQ ID NOs: 64, 66, 68, 70, 72, 74 or 76 or a polypeptide having 98% or greater sequence identity with the amino

   5 acid sequence shown in any of SEQ ID NOs: 64, 66, 68, 70, 72 or 76.

   18. The method according to claim 16 or 17, wherein said immune response is against the viral isolate designated as canine/Jax/05, or a portion thereof.

   Ό 19. The method according to claim 16 or 17, wherein said immune response is a protective immune response that prevents or inhibits infection of said animal by an influenza virus.

   20. The method according to claim 16 or 17, wherein said influenza virus has an

   25 HA serotype selected from H3.

   21. The method according to claim 16 or 17, wherein said influenza virus has the serotype H3N8.

   30 22. The method according to claim 16 or 17, wherein said composition further comprises an adjuvant.

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   143

   23. The method according to claim 16 or 17, wherein said canid animal is a domesticated dog.

   24. The method according to claim 17, wherein said composition is administered 5 parenterally.

   25. The method according to claim 24, wherein said composition is administered subcutaneously, intraperitoneally, or intramuscularly.

   0 26. The method according to claim 16 or 17, wherein said composition is administered nasally or orally.

   27. The method according to claim 16, wherein said influenza virus is inactivated or attenuated.

   28. A cell comprising a type A canine influenza virus according to any one of claims 1 to 5 or a polypeptide having the sequence shown in SEQ ID NO: 78 and a polypeptide having 98% or greater sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 64, 66, 68, 70, 72, 74 or 76 or the expression construct of claim 10.

   Ό

   29. The cell according to claim 28, wherein said cell is a canid animal cell.

   30. A method for screening for a compound that possesses antiviral activity against canine influenza viral isolate canine/Jax/05, said method comprising contacting a cell

   25 infected with or comprising a virus of claim 1 with a test compound and determining whether said test compound inhibits activity of said virus in said cell.

   31. A reassortant virus comprising at least a canine influenza A virus polynucleotide encoding a polypeptide according to claim 1.

   32. The reassortant virus according to claim 31, wherein said reassortant virus is formulated in a pharmaceutically acceptable carrier or diluent.

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   144

   33. The canine influenza virus according to claim 1 further comprising one or more polynucleotides wherein said one or more polynucleotides encode a canine influenza virus protein selected from the group consisting of HA, NA, NP, MA, PB1, PB2, PA, and NS, or a functional or immunogenic fragment thereof.

   34. The virus according to claim 33, wherein said one or more polynucleotides comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 65, 67, 69,71,73 or 75.

   0 35. The virus according to claim 33 or 34, wherein said virus is provided in a pharmaceutically acceptable carrier or diluent.

   36. A method for protecting a canine from an H3 influenza virus infection, wherein the method comprises administering to the canine a therapeutically effective

   5 amount of a vaccine comprising:

   at least one H3 influenza virus antigen and/or at least one H7 influenza virus antigen, and at least one pharmaceutically acceptable excipient; and one or more antigens from canine influenza viral isolate canine/Jax/05, wherein said wherein said antigen comprises a

   Ό canine influenza virus polypeptide as defined in claim 1 or claim 2.

   37. The method according to claim 36, wherein the vaccine comprises at least one antigen from an H7 influenza virus.

   25 38. The method according to claim 36, wherein the H3 influenza virus antigen(s) comprises an inactivated virus(s).

   39. The method according to claim 36, wherein the H3 influenza virus antigen(s) comprises a live attenuated virus(s).

   40. The method according to claim 36, wherein the vaccine comprises antigens from more than one canine influenza virus isolate.

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   145

   41. The method according to claim 36, wherein the vaccine is administered subcutaneously.

   42. The method according to claim 36, wherein the vaccine is administered 5 intranasally.

   43. The method according to claim 36, wherein the vaccine is administered to the canine once.

   44. The method according to claim 36, wherein the vaccine is administered to the canine at least two times.

   45. The method according to claim 44, wherein a second dose of the vaccine is administered to the canine at from about 2 to about 8 weeks after the first dose.

   46. The method according to claim 36, wherein the vaccine is administered in combination with at least one vaccine selected from the group consisting of a bordetella vaccine, adenovirus vaccine, and parainfluenza virus vaccine.

   Ό 47. A canine influenza vaccine, wherein the vaccine comprises:

   a therapeutically effective amount of at least one H3 influenza virus antigen and/or at least one H7 influenza virus antigen, and at least one pharmaceutically acceptable excipient; and one or more antigens from canine influenza viral isolate canine/Jax/05, wherein said wherein said antigen comprises a

   25 canine influenza virus polypeptide as defined in claim 1 or claim 2.

   48. The vaccine according to claim 47, wherein the virus antigen/s) comprises an inactivated virus(s).

   30 49. The vaccine according to claim 47, wherein the virus antigen(s) comprises a live attenuated virus(s).

   2015234384 16 Jan 2018

   146

   50. A vaccine comprising an influenza virus of any of claims 1 to 5, or a polynucleotide of any one of claims 7 to 10 , or a polynucleotide expression construct of claim 10, or a composition of claims 14 or 15, or a polypeptide of any one of claims 11 to 13.

   5 51. The vaccine according to claims 47 to 49, wherein the vaccine comprises a pharmaceutically acceptable carrier or diluent.

   52. The vaccine according to claims 47 to 49, wherein the vaccine comprises an adjuvant.

   53. A method for inducing an immune response in a canid animal against an influenza virus capable of infecting a canid animal, said method comprising administering to said canid animal an effective amount of the vaccine of any one of claims 50 to 52

   5 54. A reassortant virus comprising a polynucleotide sequence shown in SEQ ID

   NO: 77 and a polynucleotide having 98% or greater sequence identity with the nucleotide sequence shown in any of SEQ ID NOs: 63, 65, 67, 69, 71, 73 or 75, or a polypeptide having the sequence shown in SEQ ID NO: 78 and a polypeptide having 98% or greater sequence identity with the amino acid sequence shown in any of SEQ ID NOs: 64, 66, 68, 70, 72, 74 or

   Ό 76.

   55. A canine influenza vaccine, wherein the vaccine comprises:

   a therapeutically effective amount of an antigen of at least one influenza virus of claim 1, and at least one pharmaceutically acceptable excipient, wherein said antigen comprises:

   25 (a) an HA polypeptide as defined in claim 1; and/or (b) a polynucleotide encoding an HA polypeptide as defined in claim 1.

   56. The vaccine according to claim 58, wherein the virus antigen comprises an inactivated virus or a live attenuated virus.

   57. A recombinant viral vector comprising a polynucleotide encoding a hemagglutinin (HA) comprising the amino acid sequence of SEQ ID NO:78, or a mature sequence thereof where the N-terminal 16 amino acid signal sequence of the full-length

   2015234384 16 Jan 2018

   147 sequence has been removed, wherein the mature sequence of said HA protein comprises a serine at position 82, a leucine at position 221, a threonine at position 327, and a threonine at position 482 of the amino acid sequence.

   5 58. The recombinant viral vector according to claim 57, wherein said polynucleotide comprises the nucleotide sequence of SEQ ID NO:77.

   59. The recombinant viral vector according to any of claims 57 or claim 58, wherein said viral vector is from adenovirus, avipox, herpesvirus, vaccinia virus, canarypox,

   0 entomopox, swinepox, or West Nile virus.

   60. The recombinant viral vector according to any of claims 57 to 59, wherein said viral vector is a canarypox viral vector.

   5 61. A polynucleotide vector comprising a polynucleotide encoding a hemagglutinin (HA) comprising the amino acid sequence of SEQ ID NO:78, or a mature sequence thereof where the N-terminal 16 amino acid signal sequence of the full-length sequence has been removed, or the mature sequence thereof, wherein the mature sequence of said HA protein comprises a serine at position 82, a leucine at position 221, a threonine at position 327, and a

   Ό threonine at position 482 of the amino acid sequence.

   62. The polynucleotide vector according to claim 61, wherein said polynucleotide encodes a hemagglutinin (HA) that comprises the amino acid sequence of SEQ ID NO:78, or a mature sequence thereof where the N-terminal 16 amino acid signal sequence of the full25 length sequence has been removed.

   63. The polynucleotide vector according to claim 61, wherein said polynucleotide comprises the nucleotide sequence of SEQ ID NO:77.

   30 64. The polynucleotide vector according to claim 61, wherein said polynucleotide vector is a viral vector.

   WO 2007/047938

   PCT/US2006/041061

   2015234384 02 Oct 2015

   1/9

   H4

   H3 HA

   I-1

   100 nt loo

   96|

   100 r A/Eq u in e/Kentu cky/15/02

   F A/Canine/Florida/43/04 |r A/Equine/Ohio/1/03 * A/Eq u in e/Wisco n s in/0 3 *- A/Equine/N Y/99

   Equine |r A/Eq u in e/Kentu cky/1 /94 T· A/E q U in e/F Io rid a/1 /93 — A/Eq u ine/Ke ntucky/1 /31 -: A/Equ jne/Miarni/1/63

   A/Mallard/Alberta/127/00 A/Pintail/Alberta/37/99

   - A/Duck/U kraine/1 /63 — A/Goose/HK/I0/76

   -A/Sydney/5/97

   A/Bangkok/1/79 A/Memphis/102/72 *—^; A/HOng Kong/1/68

   FIG. 1A

   Avian

   Human

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   2/9

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   10 nt

   I— Cahine/Fexas/1/04 98J— Canine/Florida/43/04 ^_L Canine/Florida/242/03 97 r— Eq/Ohio/03 πΓε

   Eq/Wisconsin/03 Eq/Massach usetts/213/03 t- Eq/California/191/03' L Eq/Kentucky/5/02 L Eq/New York/1/99 j- Eq/Florida/1/93 Eq/Kentuc.ky/1/94 — Eq/Afgentina/1/93 Eq/Kentucky/1 /90 Eq/SuffO|k/89 Eq/Saht,iagQ/1/85

   H3^ avian

   Eq/Kentucky/1/81 — Eq/NeWrnarket/76 Eq/Franee/i/76

   -Eq/Uruguay/1/63

   Eq/Miami/1/63

   FIG. IB

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   2015234384 02 Oct 2015

   FIG. 2A

   FIG. 2B
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   2015234384 02 Oct 2015
5. 5/9

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   Γ A/canine/Jax/05 A/canine/Miami/05 — A/canine/lowa/05 A/canine/TX/04 A/canine/FL/43/04 ^L A/canine/FL/242/03 A/equine/Massachusetts/213/03 - A/equine/Ohio/1/03 ^L A/equine/Kentucky/5/02 A/equine/New York/99 A/equine/Kentucky/92

   100

   A/equine/Alaska/1/91 A/equine/Kentucky/2/36

   A/equine/Miami/63

   0.01

   FIG. 4A

   A/canine/Miami/05 A/canine/TX/1/04

   -A/canine/lowa/05 *— A/canine/Jax/05 h- A/canine/FL/43/04 A/canine/FL/242/03

   A/equine/Massachusetts/213/03 A/equine/Ohio/1/03 j A/equine/New York/99 ⁸²' A/equine/Kentucky/5/02 A/equine/Alaska/1/91 -A/equine/Kentucky/92

   A/equine/Kentucky/2/86

   A/equine/Miami/63

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   0.01

   FIG. 4B

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6. 6/9

   FIG. 5

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   2015234384 02 Oct 2015

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   A/camne/Florida/242/2003(H3W8)

   A/Wyoming/3/2003 (Ή3Ν2)

   IO³

   FIG. 7

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   SEQUENCE LISTING <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> Materials and Methods for Respiratory Disease Control in Canines <130> UF-445XC2 <150> US 11/409,416 <151> 2006-04-21 <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 <151> 2006-03-03 <160> 88 <170> Patentln version 3.3 <210> 1 <211> 2277 <212> DNA <213> Influenza virus <220>

   <221> CDS <222> (1)..(2277) <400> 1

   atg Met 1 gag Glu aga Arg ata gaa gaa lie Glu Glu 5 ctg Leu aga gat ctg atg tta caa Gin tcc Ser cgc Arg 15 acc Thr 48 Arg Asp Leu 10 Met Leu cgc gag ata eta aca aaa act act gtg gac cac atg gcc ata ate aag 96 Arg Glu lie Leu Thr Lys Thr Thr Val Asp His Met Ala He lie Lys 20 25 30 aaa tac aca tea gaa aga caa gag aag aac cct gca ett agg atg aaa 144 Lys Tyr Thr Ser Glu Arg Gin Glu Lys Asn Pro Ala Leu Arg Met Lys

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   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 lie Thr Ala Asp Lys Arg He Met

   50 55 60 gag atg att cct gag aga aat gaa cag ggt caa acc ctt tgg age aaa 240

   Glu Met lie Pro Glu Arg Asn Glu Gin Gly Gin Thr Leu Trp Ser Lys

   65 70 75 80 aeg aac gat get ggc tea gac ege gta atg gta tea 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 aeg age aca att cat tat cca 336

   Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr He 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 act ttt ggc ccc gtt cat ttt agg aat caa gtc aag ata aga ega aga 432

   Thr Phe Gly Pro Val His Phe Arg Asn Gin Val Lys He Arg Arg Arg

   130 135 140 gtt gat gta aac cct ggt cac geg gac etc agt gee aaa gaa gca caa 480

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

   145 150 155 160 gat gtg ate atg gaa gtt gtt ttc cca aat gaa gtg gga gee ata att 528

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

   165 170 175 eta aca teg gaa tea caa eta aca ata acc aaa gag aaa aag gaa gaa 576

   Leu Thr Ser Glu Ser Gin Leu Thr He Thr Lys Glu Lys Lys Glu Glu

   180 185 190 ctt cag gac tgc aaa att get ccc ttg atg gta gca tac atg eta gaa 624

   Leu Gin Asp Cys Lys He Ala Pro Leu Met Val Ala Tyr Met Leu Glu

   195 200 205 aga gag ttg gtc ega aaa aca agg ttc etc 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 age agt gta tac att gaa gtg ttg cat ctg act cag gga aca tgc tgg 720

   Ser Ser Val Tyr He Glu Val Leu His Leu Thr Gin 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 Gin Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp He Asp

   245 250 255 caa agt tta att att gca gee egg aac ata gtg aga aga geg aca gta 816

   Gin Ser Leu He He Ala Ala Arg Asn He Val Arg Arg Ala Thr Val

   260 265 270 tea gca gat cca eta gca tee eta ctg gaa atg tgc cac agt aca cag 864

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

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   275 280 285

   att lie ggt Gly 290 gga Gly ata agg atg He Arg Met gta gac ate He ett Leu aag Lys cag aat cca Pro aca Thr gag Glu 912 Val 295 Asp Gin 300 Asn gaa caa get gtg gat ata tgc aaa gca gca atg gga ttg aga att age 960 Glu Gin Ala Val Asp He Cys Lys Ala Ala Met Gly Leu Arg He Ser 305 310 315 320 tea tea ttc age ttt ggt gga ttc acc ttc aaa aga aca agt gga tea 1008 Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser 325 330 335 tea gtc aag aga gaa gaa gaa atg ett aeg ggc aac ett caa aca ttg 1056 Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gin Thr Leu 340 345 350 aaa ata aga gtg cat gag ggc tat gaa gaa ttc aca atg gtc gga aga 1104 Lys lie Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg 355 360 365 aga gca aca gcc att ate aaa aag gca acc aga aga ttg att caa ttg 1152 Arg Ala Thr Ala He He Lys Lys Ala Thr Arg Arg Leu He Gin Leu 370 375 380 ata gta agt ggg aga gat gaa caa tea att get gaa gca ata att gta 1200 He Val Ser Gly Arg Asp Glu Gin Ser He Ala Glu Ala He lie Val 385 390 395 400 gee atg gtg ttt teg caa gaa gat tgc atg ata aaa gca gtt ega ggc 1248 Ala Met Val Phe Ser Gin Glu Asp Cys Met He 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 Gin Arg Leu Asn Pro Met His 420 425 430 caa etc ttg agg cat ttc caa aaa gat gca aaa gtg ett ttc cac aat 1344 Gin Leu Leu Arg Hi s Phe Gin Lys Asp Ala Lys Val Leu Phe His Asn 435 440 445 tgg gga att gaa ccc ate gac aat gta atg ggg atg att gga ata ttg 1392 Trp Gly He Glu Pro He Asp Asn Val Met Gly Met He Gly lie Leu 450 455 460 cct gac atg acc cca age acc gag atg tea 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 age aaa atg gga gtg gat gag tac tcc age 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 age att gac cgt ttt tta aga gtt egg gat caa agg gga aac ata eta 1536 Ser He Asp Arg Phe Leu Arg Val Arg Asp Gin Arg Gly Asn He Leu 500 505 510 ctg tec cct gaa gaa gtc agt gaa aca caa gga aeg gaa aag ctg aca 1584 Leu Ser Pro Glu Glu Val Ser Glu Thr Gin Gly Thr Glu Lys Leu Thr

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   ) 515 520 525 ata att tat teg tea tea atg atg tgg gag att aat ggt ccc gaa tea 1632 lie He 530 Tyr Ser Ser Ser Met 535 Met Trp Glu He Asn 540 Gly Pro Glu Ser gtg ttg gtc aat act tat caa tgg ate ate agg aac tgg gaa att gta 1680 Val 545 Leu Val Asn Thr Tyr 550 Gin Trp He lie Arg 555 Asn Trp Glu He Val 560 aaa att cag tgg tea cag gac ccc aca atg tta tac aat aag ata gaa 1728 Lys He Gin Trp Ser 565 Gin Asp Pro Thr Met 570 Leu Tyr Asn Lys He 575 Glu ttt gag cca ttc caa tee ctg gtc cct agg gee ace aga age caa tac 1776 Phe Glu Pro Phe 580 Gin Ser Leu Val Pro 585 Arg Ala Thr Arg Ser 590 Gin Tyr age ggt ttc gta aga ace ctg tt't cag caa atg ega gat gta ett gga 1824 Ser Gly Phe 595 Val Arg Thr Leu Phe 600 Gin Gin Met Arg Asp 605 Val Leu Gly aca ttt gat act get caa ata ata aaa etc etc cct ttt gee get get 1872 Thr Phe 610 Asp Thr Ala Gin He 615 He Lys Leu Leu Pro 620 Phe Ala Ala Ala cct ccg gaa cag agt agg atg cag ttc tet tet ttg act gtt aat gta 1920 Pro 625 Pro Glu Gin Ser Arg 630 Met Gin Phe Ser Ser 635 Leu Thr Val Asn Val 64 0 aga ggt teg gga atg agg ata ett gta aga ggc aat tee cca gtg ttc 1968 Arg Gly Ser Gly Met 645 Arg He Leu Val Arg 650 Gly Asn Ser Pro Val 655 Phe aac tac aat aaa gee act aaa agg etc aca gtc etc gga aag gat gca 2016 Asn Tyr Asn Lys 660 Ala Thr Lys Arg Leu 665 Thr Val Leu Gly Lys 670 Asp Ala ggt geg ett act gag gac cca gat gaa ggt aeg get gga gta gaa tet 2064 Gly Ala Leu 675 Thr Glu Asp Pro Asp 680 Glu Gly Thr Ala Gly 685 Val Glu Ser get gtt eta aga ggg ttt etc att tta ggt aaa gag aac aag aga tat 2112 Ala Val 690 Leu Arg Gly Phe Leu 695 He Leu Gly Lys Glu 700 Asn Lys Arg Tyr ggc cca gca eta age ate aat gaa eta age aaa ett gca aaa ggg gag 2160 Gly 705 Pro Ala Leu Ser He 710 Asn Glu Leu Ser Lys 715 Leu Ala Lys Gly Glu 720 aaa gee aat gta eta att ggg caa ggg gac gta gtg ttg gta atg aaa 2208 Lys Ala Asn Val Leu 725 He Gly Gin Gly Asp 730 Val Val Leu Val Met 735 Lys egg aaa cgt gac tet age ata ett act gac age cag aca geg acc aaa 2256 Arg Lys Arg Asp 740 Ser Ser He Leu Thr 745 Asp Ser Gin Thr Ala 750 Thr Lys

   2277 agg att egg atg gee ate aat Arg He Arg Met Ala lie Asn

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   755 <210> 2 <211> 759 <212> PRT <213> Influenza virus <400> 2

   Met Glu Arg lie Glu Glu Leu Arg Asp Leu Met Leu Gin Ser Arg Thr 15 10 15

   Arg Glu lie Leu Thr Lys Thr Thr Val Asp His Met Ala lie lie Lys 20 25 30

   Lys Tyr Thr Ser Glu Arg Gin Glu Lys Asn Pro Ala Leu Arg Met Lys 35 40 45

   Trp Met Met Ala Met Lys Tyr Pro lie Thr Ala Asp Lys Arg lie Met 50 55 60

   Glu Met lie Pro Glu Arg Asn Glu Gin Gly Gin 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 lie 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 Gin Val Lys lie Arg Arg Arg 130 135 140

   Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gin 145 150 155 160

   Asp Val lie Met Glu Val Val Phe Pro Asn Glu Val Gly Ala lie lie 165 170 175

   Leu Thr Ser Glu Ser Gin Leu Thr lie Thr Lys Glu Lys Lys Glu Glu 180 185 190

   Leu Gin Asp Cys Lys lie Ala Pro Leu Met Val Ala Tyr Met Leu Glu 195 200 205

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   Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr 210 215 220

   Ser Ser Val Tyr He Glu Val Leu His Leu Thr Gin Gly Thr Cys Trp 225 230 235 240

   Glu Gin Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp He Asp 245 250 255

   Gin Ser Leu lie He Ala Ala Arg Asn lie Val Arg Arg Ala Thr Val 260 265 270

   Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gin 275 280 285

   He Gly Gly He Arg Met Val Asp He Leu Lys Gin Asn Pro Thr Glu 290 295 300

   Glu Gin Ala Val Asp He Cys Lys Ala Ala Met Gly Leu Arg He 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 Gin Thr Leu 340 345 350

   Lys He Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg 355 360 365

   Arg Ala Thr Ala He He Lys Lys Ala Thr Arg Arg Leu He Gin Leu 370 375 380 lie Val Ser Gly Arg Asp Glu Gin Ser He Ala Glu Ala He He Val 385 390 395 400

   Ala Met Val Phe Ser Gin Glu Asp Cys Met lie Lys Ala Val Arg Gly 405 410 415

   Asp Leu Asn Phe Val Asn Arg Ala Asn Gin Arg Leu Asn Pro Met His 420 425 430

   Gin Leu Leu Arg His Phe Gin Lys Asp Ala Lys Val Leu Phe His Asn 435 440 445

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   Trp Gly He Glu Pro lie Asp Asn Val Met Gly Met He Gly lie 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 He Asp Arg Phe Leu Arg Val Arg Asp Gin Arg Gly Asn He Leu 500 505 510

   Leu Ser Pro Glu Glu Val Ser Glu Thr Gin Gly Thr Glu Lys Leu Thr 515 520 525

   He He Tyr Ser Ser Ser Met Met Trp Glu He Asn Gly Pro Glu Ser 530 535 540

   Val Leu Val Asn Thr Tyr Gin Trp He He Arg Asn Trp Glu He Val 545 550 555 560

   Lys He Gin Trp Ser Gin Asp Pro Thr Met Leu Tyr Asn Lys He Glu 565 570 575

   Phe Glu Pro Phe Gin Ser Leu Val Pro Arg Ala Thr Arg Ser Gin Tyr 580 585 590

   Ser Gly Phe Val Arg Thr Leu Phe Gin Gin Met Arg Asp Val Leu Gly 595 600 605

   Thr Phe Asp Thr Ala Gin He He Lys Leu Leu Pro Phe Ala Ala Ala 610 615 620

   Pro Pro Glu Gin Ser Arg Met Gin Phe Ser Ser Leu Thr Val Asn Val 625 630 635 640

   Arg Gly Ser Gly Met Arg He 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

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   Ala Val 690 Leu Arg Gly Phe Leu 695 He Leu Gly Lys Glu 700 Asn Lys Arg Tyr Gly 705 Pro Ala Leu Ser He 710 Asn Glu Leu Ser Lys 715 Leu Ala Lys Gly Glu 720 Lys Ala Asn Val Leu 725 He Gly Gin Gly Asp 730 Val Val Leu Val Met 735 Lys Arg Lys Arg Asp 740 Ser Ser He Leu Thr 745 Asp Ser Gin Thr Ala 750 Thr Lys Arg lie Arg Met Ala He Asn

   755 <210> 3 <211> 2274 <212> DNA <213> Influenza virus <220>

   <221> CDS

   <222 !> (1) - - (2274) <400> 3 atg gat gtc aat ecg 'act eta etc ttc tta aag gtg cca geg cag aat 48 Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gin Asn 1 5 10 15 get ata age aca aca ttc cct tat act gga gat cct ccc tac agt cat 96 Ala He 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 Gin 35 40 45 tat tea 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 He Gly Ala Pro 50 55 60 caa ctt aat cca ate gat gga cca ctt cct gaa gac aat gaa cca age 240 Gin Leu Asn Pro He 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 get ttc ctt gaa 288 Gly Tyr Ala Gin Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 85 90 95 gaa tcc cat cca gga ate ttt gaa aat teg tgt ctt gaa aeg atg gag 336 Glu Ser His Pro Gly He Phe Glu Asn Ser Cys Leu Glu Thr Met Glu 100 105 110

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   gtg Val att lie cag Gin 115 cag Gin aca Thr aga gtg gac Asp 120 aaa Lys eta Leu aca Thr caa ggc Gin Gly 125 ega Arg caa act Gin Thr 384 Arg Val tat gat tgg ace ttg aat agg aat caa cct gcc gca aca gca ett get 432 Tyr Asp Trp Thr Leu Asn Arg Asn Gin Pro Ala Ala Thr Ala Leu Ala 130 135 140 aat aeg att gaa gta ttc aga tea aat ggt ctg act tcc aat gaa teg 480 Asn Thr lie Glu Val Phe Arg Ser Asn Gly Leu Thr Ser Asn Glu Ser 145 150 155 160 ggg aga ttg atg gac ttc etc 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 egg aag aga aga gta aga 576 Glu Glu Met Glu lie Thr Thr His Phe Gin 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 Gin Arg Thr lie Gly Lys Lys 195 200 205 aaa caa ega tta aac aga aag age tat eta ate aga aca tta acc eta 672 Lys Gin Arg Leu Asn Arg Lys Ser Tyr Leu lie Arg Thr Leu Thr Leu 210 215 220 aac aca atg acc aag gac get gag aga ggg aaa ttg aaa ega ega gca 720 Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala 225 230 235 240 ate get ace cca ggg atg cag ata aga ggg ttt gta tat ttt gtt gaa 768 lie Ala Thr Pro Gly Met Gin lie Arg Gly Phe Val Tyr Phe Val Glu 245 250 255 aca eta get ega aga ata tgt gaa aag ett gaa caa tea gga ttg cca 816 Thr Leu Ala Arg Arg lie Cys Glu Lys Leu Glu Gin Ser Gly Leu Pro 260 265 270 gtt ggc ggt aat gag aaa aag gcc aaa ctg get 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 etc tcc ttc acc ate act ggg 912 Met Met Thr Asn Ser Gin Asp Thr Glu Leu Ser Phe Thr lie Thr Gly 290 295 300 gac aat ace aaa tgg aat gaa aat cag aac cca ege ata ttc ctg gca 960 Asp Asn Thr Lys Trp Asn Glu Asn Gin Asn Pro Arg He Phe Leu Ala 305 310 315 320 atg ate aca tac ata act aga aac cag cca gaa tgg ttc aga aat gtt 1008 Met lie Thr Tyr lie Thr Arg Asn Gin Pro Glu Trp Phe Arg Asn Val 325 330 335 eta age att gca ccg att atg ttc tea aat aaa atg gca aga ctg ggg 1056 Leu Ser lie Ala Pro lie Met Phe Ser Asn Lys Met Ala Arg Leu Gly 340 345 350

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   aaa gga tat Tyr 355 atg Met ttt Phe gaa Glu age Ser aaa Lys 360 agt atg aaa Lys ttg Leu aga Arg 365 act Thr caa Gin ata He 1104 Lys Gly Ser Met cca gca gaa atg eta gca age att gac eta aaa tat ttc aat gat tea 1152 Pro Ala Glu Met Leu Ala Ser He Asp Leu Lys Tyr Phe Asn Asp Ser 370 375 380 aca aaa aag aaa att gag aag ata ega cca ett ctg gtt gac ggg act 1200 Thr Lys Lys Lys He Glu Lys He Arg Pro Leu Leu Val Asp Gly Thr 385 390 395 400 get tea ctg agt cct ggc atg atg atg gga atg ttc aac atg ttg age 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 He Leu Asn Leu Gly Gin Arg Lys Tyr Thr 420 425 430 aag ace aca tac tgg tgg gat ggt ctg caa tea tcc gat gac ttt get 1344 Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gin Ser Ser Asp Asp Phe Ala 435 440 445 ttg ata gtg aat geg cct aat cat gaa gga gta caa get gga gta gac 1392 Leu lie Val Asn Ala Pro Asn His Glu Gly Val Gin Ala Gly Val Asp 450 455 460 aga ttc tat aga act tgc aaa ctg gtc ggg ate aac atg age aaa aag 1440 Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly He Asn Met Ser Lys Lys 465 470 475 480 aag tec tac ata aat aga act gga aca ttc gaa ttc aca age ttt ttc 1488 Lys Ser Tyr lie Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe 485 490 495 tac egg tat ggt ttt gta gcc aat ttc age atg gaa eta 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 tet gca gac atg age att gga gtg aca 1584 Gly Val Ser Gly He Asn Glu Ser Ala Asp Met Ser He Gly Val Thr 515 520 525 gtc ate aaa aac aac atg ata aat aat gat etc ggt cct gcc aeg gca 1632 Val He Lys Asn Asn Met He Asn Asn Asp Leu Gly Pro Ala Thr Ala 530 535 540 caa atg gca etc caa etc ttc att aag gat tat egg tac aca tac egg 1680 Gin Met Ala Leu Gin Leu Phe He Lys Asp Tyr Arg Tyr Thr Tyr Arg 545 550 555 560 tgc cat aga ggt gat acc cag ata caa acc aga aga tet ttt gag ttg 1728 Cys His Arg Gly Asp Thr Gin He Gin Thr Arg Arg Ser Phe Glu Leu 565 570 575 aag aaa ctg tgg gaa cag act ega tea aag act ggt eta ctg gta tea 1776 Lys Lys Leu Trp Glu Gin Thr Arg Ser Lys Thr Gly Leu Leu Val Ser 580 585 590

   PCT/US2006/041061

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   gat Asp ggg Gly ggt Gly 595 cca Pro aac Asn eta tat aac Asn 600 ate He aga Arg aac Asn eta Leu cac His 605 ate He ccg Pro gaa Glu 1824 Leu Tyr gtc tgt tta aaa tgg gag eta atg gat gaa gat tat aag ggg agg eta 1872 Val cys 610 Leu Lys Trp Glu Leu 615 Met Asp Glu Asp Tyr 620 Lys Gly Arg Leu tgc aat cca ttg aat cct ttc gtt agt cac aaa gaa att gaa tea gtc 1920 Cys 625 Asn Pro Leu Asn Pro 630 Phe Val Ser His Lys 635 Glu He Glu Ser Val 640 aac agt gca gta gta atg cct geg cat ggc cct gcc aaa age atg gag 1968 Asn Ser Ala Val Val 645 Met Pro Ala His Gly 650 Pro Ala Lys Ser Met 655 Glu tat gat get gtt gca aca aca cat tet tgg ate ccc aag agg aac egg 2016 Tyr Asp Ala Val 660 Ala Thr Thr His Ser 665 Trp He Pro Lys Arg 670 Asn Arg tec ata ttg aac aca age caa agg gga gta etc gaa gat gag cag atg 2064 Ser He Leu 675 Asn Thr Ser Gin Arg 680 Gly Val Leu Glu Asp 685 Glu Gin Met tat cag aaa tgc tgc aac ctg ttt gaa aaa ttc ttc ccc age age tea 2112 Tyr Gin 690 Lys Cys Cys Asn Leu 695 Phe Glu Lys Phe Phe 700 Pro Ser Ser Ser tac aga aga cca gtc gga att tet agt atg gtt gag gcc atg gtg tcc 2160 Tyr 705 Arg Arg Pro Val Gly 710 He Ser Ser Met Val 715 Glu Ala Met Val Ser 720 agg gee ege att gat gca ega att gac ttc gaa tet gga egg ata aag 2208 Arg Ala Arg He Asp 725 Ala Arg He Asp Phe 730 Glu Ser Gly Arg He 735 Lys aag gat gag ttc get gag ate atg aag ate tgt tcc acc att gaa gag 2256 Lys Asp Glu Phe 740 Ala Glu He Met Lys 745 lie Cys Ser Thr He 750 Glu Glu

   etc aga egg caa aaa tag 2274

   Leu Arg Arg Gin 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 Gin Asn 1 5 10 15

   Ala lie Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His 20 25 30

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   Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gin 35 40 45

   Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu He Gly Ala Pro 50 55 60

   Gin Leu Asn Pro He Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser 65 70 75 80

   Gly Tyr Ala Gin Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 85 90 95

   Glu Ser His Pro Gly He Phe Glu Asn Ser Cys Leu Glu Thr Met Glu 100 105 110

   Val He Gin Gin Thr Arg Val Asp Lys Leu Thr Gin Gly Arg Gin Thr 115 120 125

   Tyr Asp Trp Thr Leu Asn Arg Asn Gin Pro Ala Ala Thr Ala Leu Ala 130 135 140

   Asn Thr He 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 He Thr Thr His Phe Gin Arg Lys Arg Arg Val Arg 180 185 190

   Asp Asn Met Thr Lys Arg Met Val Thr Gin Arg Thr He Gly Lys Lys 195 200 205

   Lys Gin Arg Leu Asn Arg Lys Ser Tyr Leu He 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

   He Ala Thr Pro Gly Met Gin He Arg Gly Phe Val Tyr Phe Val Glu 245 250 255

   Thr Leu Ala Arg Arg He Cys Glu Lys Leu Glu Gin Ser Gly Leu Pro 260 265 270

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   Val Gly Gly Asn Glu Lys Lys Ala 275 280

   Met Met Thr Asn Ser Gin Asp Thr 290 295

   Asp Asn Thr Lys Trp Asn Glu Asn 305 310

   Met lie Thr Tyr He Thr Arg Asn 325

   Leu Ser He Ala Pro He Met Phe 340

   Lys Leu Ala Asn Val Val Arg Lys 285

   Glu Leu Ser Phe Thr He Thr Gly 300

   Gin Asn Pro Arg He Phe Leu Ala 315 320

   Gin Pro Glu Trp Phe Arg Asn Val 330 335

   Ser Asn Lys Met Ala Arg Leu Gly 345 350

   Lys Gly Tyr Met Phe Glu Ser Lys 355 350

   Ser Met Lys Leu Arg Thr Gin He 365

   Pro Ala Glu Met Leu Ala Ser He 370 375

   Asp Leu Lys Tyr Phe Asn Asp Ser 380

   Thr Lys Lys Lys He Glu Lys He 385 390

   Arg Pro Leu Leu Val Asp Gly Thr 395 400

   Ala Ser Leu Ser Pro Gly Met Met 405

   Met Gly Met Phe Asn Met Leu Ser 410 415

   Thr Val Leu Gly Val Ser He Leu 420

   Asn Leu Gly Gin Arg Lys Tyr Thr 425 430

   Lys Thr Thr Tyr Trp Trp Asp Gly 435 440

   Leu Gin Ser Ser Asp Asp Phe Ala 445

   Leu He Val Asn Ala Pro Asn His 450 455

   Glu Gly Val Gin Ala Gly Val Asp 460

   Arg Phe Tyr Arg Thr Cys Lys Leu 465 470

   Val Gly He Asn Met Ser Lys Lys 475 480

   Lys Ser Tyr He Asn Arg Thr Gly 485

   Thr Phe Glu Phe Thr Ser Phe Phe 490 495

   Tyr Arg Tyr Gly Phe Val Ala Asn 500

   Phe Ser Met Glu Leu Pro Ser Phe 505 510

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   Gly Val Ser Gly lie Asn Glu Ser Ala Asp Met Ser lie Gly Val Thr 515 520 525

   Val lie Lys Asn Asn Met lie Asn Asn Asp Leu Gly Pro Ala Thr Ala 530 535 540

   Gin Met Ala Leu Gin Leu Phe He Lys Asp Tyr Arg Tyr Thr Tyr Arg 545 550 555 560

   Cys His Arg Gly Asp Thr Gin lie Gin Thr Arg Arg Ser Phe Glu Leu 565 570 575

   Lys Lys Leu Trp Glu Gin Thr Arg Ser Lys Thr Gly Leu Leu Val Ser 580 585 590

   Asp Gly Gly Pro Asn Leu Tyr Asn He Arg Asn Leu His He 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 He 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 He Pro Lys Arg Asn Arg 660 665 670

   Ser He Leu Asn Thr Ser Gin Arg Gly Val Leu Glu Asp Glu Gin Met 675 680 685

   Tyr Gin Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser 690 695 700

   Tyr Arg Arg Pro Val Gly lie Ser Ser Met Val Glu Ala Met Val Ser 705 710 715 720

   Arg Ala Arg He Asp Ala Arg He Asp Phe Glu Ser Gly Arg lie Lys 725 730 735

   Lys Asp Glu Phe Ala Glu He Met Lys He Cys Ser Thr He Glu Glu 740 745 750

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   Leu Arg Arg Gin Lys 755 <210> 5 <211> 2151 <212> DNA <213> Influenza virus <220>

   <221> CDS <222> (1)..(2151) <400> 5

   atg Met 1 gaa Glu gac Asp ttt Phe gtg Val 5 ega Arg cag Gin tgc Cys ttc Phe aat Asn 10 cca Pro atg Met ate He gtc Val gag Glu 15 ett Leu 48 gcg gaa aag gca atg aaa gaa tat gga gag aac ccg aaa ate gaa aca 96 Ala Glu Lys Ala 20 Met Lys Glu Tyr Gly 25 Glu Asn Pro Lys lie 30 Glu Thr aac aaa ttt gca gca ata tgc act cac ttg gaa gtc tgc ttc atg tac 144 Asn Lys Phe 35 Ala Ala lie Cys Thr 40 His Leu Glu Val Cys 45 Phe Met Tyr teg gat ttt cac ttt att aat gaa ctg ggt gag tea gtg gtc ata gag 192 Ser Asp 50 Phe His Phe He Asn 55 Glu Leu Gly Glu Ser 60 Val Val lie Glu tet ggt gac cca aat get ett ttg aaa cac aga ttt gaa ate att gag 240 Ser 65 Gly Asp Pro Asn Ala 70 Leu Leu Lys His Arg 75 Phe Glu He He Glu 80 ggg aga gat ega aca atg gca tgg aca gta gta aac age ate tgc aac 288 Gly Arg Asp Arg Thr 85 Met Ala Trp Thr Val 90 Val Asn Ser lie Cys 95 Asn acc aca aga get gaa aaa cct aaa ttt ett cca gat tta tac gac tat 336 Thr Thr Arg Ala 100 Glu Lys Pro Lys Phe 105 Leu Pro Asp Leu Tyr 110 Asp Tyr aag gag aac aga ttt gtt gaa att ggt gtg aca agg aga gaa gtt cac 384 Lys Glu Asn 115 Arg Phe Val Glu He 120 Gly Val Thr Arg Arg 125 Glu Val His ata tac tac ctg gag aag gcc aac aaa ata aag tet gag aaa aca cat 432 lie Tyr 130 Tyr Leu Glu Lys Ala 135 Asn Lys He Lys Ser 140 Glu Lys Thr His ate cac att ttc tea ttt aca gga gag gaa atg get aca aaa gcg gac 480 He 145 His lie Phe Ser Phe 150 Thr Gly Glu Glu Met 155 Ala Thr Lys Ala Asp 160 tat act ett gat gaa gag agt aga gcc agg ate aag acc aga eta ttc 528 Tyr Thr Leu Asp Glu 165 Glu Ser Arg Ala Arg 170 lie Lys Thr Arg Leu 175 Phe

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   acc Thr ata He aga Arg caa Gin 180 gaa Glu atg Met gee Ala agt Ser aga ggc etc Leu tgg Trp gat Asp tee Ser 190 ttt Phe cgt Arg 576 Arg 185 Gly cag tec gag aga ggc gaa gag aca att gaa gaa aga ttt gaa ata aca 624 Gin Ser Glu 195 Arg Gly Glu Glu Thr 200 He Glu Glu Arg Phe 205 Glu He Thr ggg aeg atg ege aag ett gee aat tac agt etc cca ccg aac ttc tee 672 Gly Thr 210 Met Arg Lys Leu Ala 215 Asn Tyr Ser Leu Pro 220 Pro Asn Phe Ser age ett gaa aat ttt aga gtc tat gtg gat gga ttc gaa ccg aac ggc 720 Ser 225 Leu Glu Asn Phe Arg 230 Val Tyr Val Asp Gly 235 Phe Glu Pro Asn Gly 240 tgc att gag agt aag ett tet caa atg tee aaa gaa gta aat gee aga 768 Cys lie Glu Ser Lys 245 Leu Ser Gin Met Ser 250 Lys Glu Val Asn Ala, 255 Arg ate gaa cca ttt tea aag aca aca ccc ega cca etc aaa atg cca ggt 816 lie Glu Pro Phe 260 Ser Lys Thr Thr Pro 265 Arg Pro Leu Lys Met 270 Pro Gly ggt cca ccc tgc cat cag ega tet aaa ttc ttg eta atg gat get ctg 864 Gly Pro Pro 275 Cys His Gin Arg Ser 280 Lys Phe Leu Leu Met 285 Asp Ala Leu aaa ctg age att gag gac cca agt cac gag gga gag gga ata cca eta 912 Lys Leu 290 Ser He Glu Asp Pro 295 Ser His Glu Gly Glu 300 Gly He Pro Leu tat gat gca ate aaa tgc atg aaa act ttc ttt gga tgg aaa gag ccc 960 Tyr 305 Asp Ala He Lys Cys 310 Met Lys Thr Phe Phe 315 Gly Trp Lys Glu Pro 320 agt att gtt aaa cca cat gaa aag ggt ata aac ccg aac, tat etc caa 1008 Ser He Val Lys Pro 325 His Glu Lys Gly He 330 Asn Pro Asn Tyr Leu 335 Gin act tgg aag caa gta tta gaa gaa ata caa gac ett gag aac gaa gaa 1056 Thr Trp Lys Gin 340 Val Leu Glu Glu He 345 Gin Asp Leu Glu Asn 350 Glu Glu agg acc ccc aag acc aag aat atg aaa aaa aca age caa ttg aaa tgg 1104 Arg Thr Pro 355 Lys Thr Lys Asn Met 360 Lys Lys Thr Ser Gin 365 Leu Lys Trp gca eta ggt gaa aat atg gca cca gag aaa gtg gat ttt gag gat tgt 1152 Ala Leu 370 Gly Glu Asn Met Ala 375 Pro Glu Lys Val Asp 380 Phe Glu Asp Cys aaa gac ate aat gat tta aaa caa tat gac agt gat gag cca gaa gca 1200 Lys 385 Asp He Asn Asp Leu 390 Lys Gin Tyr Asp Ser 395 Asp Glu Pro Glu Ala 400 agg tet ett gca agt tgg att caa agt gag ttc aac aaa get tgt gag 1248 Arg Ser Leu Ala Ser 405 Trp lie Gin Ser Glu 410 Phe Asn Lys Ala Cys 415 Glu

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   ctg Leu aca Thr gat Asp tea Ser 420 age Ser tgg Trp ata He gag Glu etc Leu 425 gat Asp gaa Glu att lie ggg Gly gag Glu 430 gat Asp gtc Val 1296 gcc cca ata gaa tac att geg age atg agg aga aat tat ttt act get 1344 Ala Pro He Glu Tyr He 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 He Ser His Cys Arg Ala Thr Glu Tyr He He Lys Gly Val Tyr 450 455 460 ate aac act get eta etc aat gca tcc tgt get geg atg gat gaa ttt 1440 lie 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 Gin Leu He Pro Met lie 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 lie He Lys Gly Arg Ser His Leu Arg 500 505 510 aat gat act gac gtg gtg aac ttt gta agt atg gaa ttt tet etc 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 eta 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 ett eta aga act get gta ggt caa gtg tea aga ccc 1680 He Gly Asp Met Leu Leu Arg Thr Ala Val Gly Gin Val Ser Arg Pro 545 550 555 560 atg ttt ttg tat gta agg aca aat gga acc tet aaa att aaa atg aaa 172 8 Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys He Lys Met Lys 565 570 575 tgg gga atg gaa atg agg ege tgc etc ett cag tet ctg caa cag att 1776 Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gin Ser Leu Gin Gin He 580 585 590 gaa age atg ate gaa get gag tcc tea gtc aaa gaa aag gac atg acc 1824 Glu Ser Met He Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr 595 600 605 aaa gaa ttt ttt gag aac aaa tea gag aca tgg cct ata gga gag tcc 1872 Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro He Gly Glu Ser 610 615 620 ccc aaa gga gtg gaa gag ggc tea ate ggg aag gtt tgc agg acc tta 1920 Pro Lys Gly Val Glu Glu Gly Ser He Gly Lys Val Cys Arg Thr Leu 625 630 635 640 tta gca aaa tet gtg ttt aac agt tta tat gca tet cca caa ctg gaa 1968 Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gin Leu Glu 645 650 655

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   IS

   ggg ttt tea Ser get Ala 660 gaa Glu tet Ser agg aaa tta Leu 665 ett Leu etc Leu att He gtt Val cag Gin 670 get Ala ett Leu Gly Phe Arg Lys aag gat gac ctg gaa cct gga ace ttt gat att ggg ggg tta tat gaa Lys Asp Asp Leu Glu Pro Gly Thr Phe Asp lie Gly Gly Leu Tyr Glu 675 680 685 tea att gag gag tgc ctg att aat gat ccc tgg gtt ttg ett aat gca Ser He Glu Glu Cys Leu He Asn Asp Pro Trp Val Leu Leu Asn Ala 690 695 700 tet tgg ttc aac tec ttc ett aca cat gca ctg aag tag Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Lys 705 710 715

   2016

   2064

   2112

   2151 <210> 6 <211> 716 <212> PRT <213> Influenza virus <400> 6

   Met 1 Glu Asp Phe Val 5 Arg Gin Cys Ala Glu Lys Ala 20 Met Lys Glu Tyr Asn Lys Phe 35 Ala Ala He Cys Thr 40 Ser Asp 50 Phe His Phe He Asn 55 Glu Ser 65 Gly Asp Pro Asn Ala 70 Leu Leu Gly Arg Asp Arg Thr 85 Met Ala Trp Thr Thr Arg Ala 100 Glu Lys Pro Lys Lys Glu Asn 115 Arg Phe Val Glu He 120 lie Tyr 130 Tyr Leu Glu Lys Ala 135 Asn

   Phe Asn Pro Met He Val Glu Leu 10 15

   Gly Glu Asn Pro Lys lie Glu Thr 25 30

   His Leu Glu Val Cys Phe Met Tyr 45

   Leu Gly Glu Ser Val Val He Glu 60

   Lys His Arg Phe Glu He He Glu 75 80

   Thr Val Val Asn Ser He Cys Asn 90 95

   Phe Leu Pro Asp Leu Tyr Asp Tyr 105 110

   Gly Val Thr Arg Arg Glu Val His 125

   Lys He Lys Ser Glu Lys Thr His 140

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   2015234384 02 Oct 2015 lie His He 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 lie Lys Thr Arg Leu Phe 165 170 175

   Thr lie Arg Gin Glu Met Ala Ser Arg Gly Leu Trp Asp Ser Phe Arg 180 185 190

   Gin Ser Glu Arg Gly Glu Glu Thr He Glu Glu Arg Phe Glu He 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 He Glu Ser Lys Leu Ser Gin Met Ser Lys Glu Val Asn Ala Arg 245 250 255 lie Glu Pro Phe Ser Lys Thr Thr Pro Arg Pro Leu Lys Met Pro Gly 260 265 270

   Gly Pro Pro Cys His Gin Arg Ser Lys Phe Leu Leu Met Asp Ala Leu 275 280 285

   Lys Leu Ser He Glu Asp Pro Ser His Glu Gly Glu Gly He Pro Leu 290 295 300

   Tyr Asp Ala He Lys Cys Met Lys Thr Phe Phe Gly Trp Lys Glu Pro 305 310 315 320

   Ser He Val Lys Pro His Glu Lys Gly He Asn Pro Asn Tyr Leu Gin 325 330 335

   Thr Trp Lys Gin Val Leu Glu Glu He Gin Asp Leu Glu Asn Glu Glu 340 345 350

   Arg Thr Pro Lys Thr Lys Asn Met Lys Lys Thr Ser Gin 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

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   Lys Asp lie Asn Asp Leu Lys Gin 385 390

   Arg Ser Leu Ala Ser Trp He Gin 405

   Leu Thr Asp Ser Ser Trp He Glu 420

   Ala Pro He Glu Tyr He Ala Ser 435 440

   Glu He Ser His Cys Arg Ala Thr 450 455

   Tyr Asp Ser Asp Glu Pro Glu Ala 395 400

   Ser Glu Phe Asn Lys Ala Cys Glu 410 415

   Leu Asp Glu He Gly Glu Asp Val 425 430

   Met Arg Arg Asn Tyr Phe Thr Ala 445

   Glu Tyr lie He Lys Gly Val Tyr 460

   He Asn Thr Ala Leu Leu Asn Ala 465 470

   Ser Cys Ala Ala Met Asp Glu Phe ' 475 480

   Gin Leu He Pro Met lie Ser Lys 485

   Cys Arg Thr Lys Glu Gly Arg Arg 490 495

   Lys Thr Asn Leu Tyr Gly Phe He 500

   He Lys Gly Arg Ser His Leu Arg 505 510

   Asn Asp Thr Asp Val Val Asn Phe 515 520

   Val Ser Met Glu Phe Ser Leu Thr 525

   Asp Pro Arg Phe Glu Pro His Lys 530 535

   Trp Glu Lys Tyr Cys Val Leu Glu 540

   He Gly Asp Met Leu Leu Arg Thr 545 550

   Ala Val Gly Gin Val Ser Arg Pro 555 560

   Met Phe Leu Tyr Val Arg Thr Asn 565

   Gly Thr Ser Lys He Lys Met Lys 570 575

   Trp Gly Met Glu Met Arg Arg Cys 580

   Leu Leu Gin Ser Leu Gin Gin He 585 590

   Glu Ser Met He Glu Ala Glu Ser 595 600

   Ser Val Lys Glu Lys Asp Met Thr 605

   Lys Glu Phe Phe Glu Asn Lys Ser 610 615

   Glu Thr Trp Pro He Gly Glu Ser 620

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   Pro Lys Gly Val Glu Glu Gly Ser lie Gly Lys Val Cys Arg Thr Leu 625 630 635 640 Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gin Leu Glu 645 650 655 Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu lie Val Gin Ala Leu 660 665 670 Lys Asp Asp Leu Glu Pro Gly Thr Phe Asp lie Gly Gly Leu Tyr Glu 675 680 685 Ser lie Glu Glu Cys Leu lie 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 (657) <222 !> (1) .. <400> ' 7 ¹ atg gat tcc aac act gtg tea age ttt cag gta gac tgt ttt ctt tgg 48 Met Asp Ser Asn Thr Val Ser Ser Phe Gin Val Asp Cys Phe Leu Trp 1 5 10 15 cat gtc cgc aaa ega ttc gca gac caa gaa ctg ggt gat gcc cca ttc 96 His Val Arg Lys Arg Phe Ala Asp Gin Glu Leu Gly Asp Ala Pro Phe 20 25 30 ctt gac egg ctt cgc ega gac cag aag tcc eta agg gga aga ggt age 144 Leu Asp Arg Leu Arg Arg Asp Gin Lys Ser Leu Arg Gly Arg Gly Ser 35 40 45 act ctt ggt ctg gac ate gaa aca gcc act cat gca gga aag cag ata 192 Thr Leu Gly Leu Asp He Glu Thr Ala Thr His Ala Gly Lys Gin He 50 55 60 gtg gag cag att ctg gaa aag gaa tea gat gag gca ctt aaa atg acc 240 Val Glu Gin lie Leu Glu Lys Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 ¹ 75 80 att gcc tet gtt cct act tea etc tac tta act gac atg act ctt gat 288 He Ala Ser Val Pro Thr Ser Leu Tyr Leu Thr Asp Met Thr Leu Asp 85 90 95 gag atg tea aga gac tgg ttc atg etc atg ccc aag caa aaa gta aca 336

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   Glu Met Ser Arg Asp 100 Trp Phe Met Leu 105 22 Met Pro Lys Gin Lys 110 Val Thr ggc tcc eta tgt ata aga atg gac cag gca ate atg gat aag aac ate 384 Gly Ser Leu Cys He Arg Met Asp Gin Ala He Met Asp Lys Asn He 115 120 125 ata ett aaa gca aac ttt agt gtg att ttc gaa ggg ctg gaa aca eta 432 He Leu Lys Ala Asn Phe Ser Val He Phe Glu Gly Leu Glu Thr Leu 130 135 140 ata eta ett aga gcc ttc ace gaa gaa gga gca gtc gtt ggc gaa att 480 He Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu He 145 150 155 160 tea cca tta cct tet ett 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 etc ate gga gga ett aaa tgg aat gat aat aeg gtt 576 Ala He Gly Val Leu He Gly Gly Leu Lys Trp Asn Asp Asn Thr Val 180 185 190 aga ate tet gaa act eta cag aga ttc get tgg aga age agt cat gag 624 Arg lie Ser Glu Thr Leu Gin Arg Phe Ala Trp Arg Ser Ser His Glu 195 200 205 aat ggg aga cct tea ttc cct tea aag cag aaa tgaaaaatgg ; agagaacaat 677 Asn Gly Arg Pro Ser Phe Pro Ser Lys Gin 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 Gin Val Asp Cys Phe Leu Trp 1 5 10 15

   His Val Arg Lys Arg Phe Ala Asp Gin Glu Leu Gly Asp Ala Pro Phe 20 25 30

   Leu Asp Arg Leu Arg Arg Asp Gin Lys Ser Leu Arg Gly Arg Gly Ser 35 40 45

   Thr Leu Gly Leu Asp lie Glu Thr Ala Thr His Ala Gly Lys Gin He 50 55 60

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   Val Glu Gin He Leu Glu Lys Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80 lie 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 Gin Lys Val Thr 100 105 110 Gly Ser Leu Cys He Arg Met Asp Gin Ala He Met Asp Lys Asn He 115 120 125 He Leu Lys Ala Asn Phe Ser Val He Phe Glu Gly Leu Glu Thr Leu 13 0 135 140 He Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu lie 145 150 155 160 Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn 165 170 175 Ala He Gly Val Leu lie Gly Gly Leu Lys Trp Asn Asp Asn Thr Val 180 185 190 Arg lie Ser Glu Thr Leu Gin Arg Phe Ala Trp Arg Ser Ser His Glu 195 200 205 Asn Gly Arg Pro Ser Phe Pro Ser Lys Gin 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 Gin Gly Thr Lys Arg Ser Tyr Glu Gin Met Glu Thr Asp 1 5 10 15 ggg gaa cgc cag aat gca act gaa ate aga gca tct gtc gga agg atg 96 Gly Glu Arg Gin Asn Ala Thr Glu He Arg Ala Ser Val Gly Arg Met 20 25 30

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   gtg Val gga Gly gga Gly 35 ate lie ggc Gly ega Arg ttt Phe tat Tyr 40 gtt Val cag Gin atg Met tgt Cys act Thr 45 gag Glu ctt Leu aaa Lys 144 eta aac gac cat gaa ggg egg ctg att cag aac age ata aca ata gaa 192 Leu Asn 50 Asp His Glu Gly Arg 55 Leu He Gin Asn Ser 60 He Thr He Glu agg atg gta ctt teg gca ttc gac gaa aga aga aac aag tat etc gag 240 Arg 65 Met Val Leu Ser Ala 70 Phe Asp Glu Arg Arg 75 Asn Lys Tyr Leu Glu 80 gag cat ccc agt get ggg aaa gac cct aag aaa aeg gga ggc ccg ata 288 Glu His Pro Ser Ala 85 Gly Lys Asp Pro Lys 90 Lys Thr Gly Gly Pro 95 lie tac aga agg aaa gat ggg aaa tgg atg agg gaa etc ate etc cat gat 336 Tyr Arg Arg Lys 100 Asp Gly Lys Trp Met 105 Arg Glu Leu He Leu 110 His Asp aaa gaa gaa ate atg aga ate tgg cgt cag gcc aac aat ggt gaa gac 384 Lys Glu Glu 115 He Met Arg lie Trp 120 Arg Gin Ala Asn Asn 125 Gly Glu Asp get act get ggt ctt act cat atg atg ate tgg cac tec aat etc aat 432 Ala Thr 130 Ala Gly Leu Thr His 135 Met Met He Trp His 140 Ser Asn Leu Asn gac acc aca tac caa aga aca agg get ctt gtt egg act ggg atg gat 480 Asp 145 Thr Thr Tyr Gin Arg 150 Thr Arg Ala Leu Val 155 Arg Thr Gly Met Asp 160 ccc aga atg tgc tet ctg atg caa ggc tea acc etc cca egg aga tet 528 Pro Arg Met Cys Ser 165 Leu Met Gin Gly Ser 170 Thr Leu Pro Arg Arg 175 Ser gga gcc get ggt get gca gta aaa ggt gtt gga aca atg gta atg gaa 576 Gly Ala Ala Gly 180 Ala Ala Val Lys Gly 185 Val Gly Thr Met Val 190 Met Glu etc ate aga atg ate aaa ege gga ata aat gat egg aat ttc tgg aga 624 Leu lie Arg 195 Met He Lys Arg Gly 200 He Asn Asp Arg Asn 205 Phe Trp Arg ggt gaa aat ggt ega aaa acc aga att get tat gaa aga atg tgc aat 672 Gly Glu 210 Asn Gly Arg Lys Thr 215 Arg lie Ala Tyr Glu 220 Arg Met Cys Asn ate etc aaa ggg aaa ttt cag aca gca gca caa egg get atg atg gac 720 lie 225 Leu Lys Gly Lys Phe 230 Gin Thr Ala Ala Gin 235 Arg Ala Met Met Asp 240 cag gtg agg gaa ggc ege aat cct gga aac get gag att gag gat etc 768 Gin Val Arg Glu Gly 245 Arg Asn Pro Gly Asn 250 Ala Glu He Glu Asp 255 Leu att ttc ttg gca ega tea gca ctt att ttg aga gga tea gta gee cat 816 He Phe Leu Ala 260 Arg Ser Ala Leu He 265 Leu Arg Gly Ser Val 270 Ala His

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   aaa Lys tea Ser tgc Cys 275 eta cct Leu Pro gcc Ala tgt Cys gtt Val 280 tat Tyr ggc Gly ett gca Leu Ala ata He 285 acc Thr agt Ser ggg Gly 864 tat gac ttt gag aag gaa gga tac tet ctg gtt gga att gat cct ttc 912 Tyr Asp Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly He Asp Pro Phe 290 295 300 aaa eta etc cag aac agt caa att ttc agt eta ate aga cca aaa gaa 960 Lys Leu Leu Gin Asn Ser Gin lie Phe Ser Leu He Arg Pro Lys Glu 305 310 315 320 aac cca gca cac aag age cag ttg gtg tgg atg gca tgc cat tet gca 1008 Asn Pro Ala His Lys Ser Gin 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 lie Arg Gly Thr Lys Val 340 345 350 ate cca aga gga cag tta aca acc aga gga gtt caa att get tea aat 1104 lie Pro Arg Gly Gin Leu Thr Thr Arg Gly Val Gin He Ala Ser Asn 355 360 365 gaa aac atg gag aca ata aat tet age aca ett gaa ctg aga age aaa 1152 Glu Asn Met Glu Thr He Asn Ser Ser Thr Leu Glu Leu Arg Ser Lys 370 375 380 tat tgg gca ata agg acc aga age gga gga aac acc agt caa cag aga 1200 Tyr Trp Ala He Arg Thr Arg Ser Gly Gly Asn Thr Ser Gin Gin Arg 385 390 395 400 gca tet gca gga cag ata agt gtg caa cct act ttc tea gta cag aga 1248 Ala Ser Ala Gly Gin He Ser Val Gin Pro Thr Phe Ser Val Gin Arg 405 410 415 aat eta ccc ttt gag aga geg acc att atg get gca ttc act ggt aac 1296 Asn Leu Pro Phe Glu Arg Ala Thr lie Met Ala Ala Phe Thr Gly Asn 420 • 425 430 act gaa ggg agg act tec gac atg aga aeg gaa ate ata agg atg atg 1344 Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu He He Arg Met Met 435 440 445 gaa aat gee aaa tea gaa gat gtg tet ttc cag ggg egg gga gtc ttc 1392 Glu Asn Ala Lys Ser Glu Asp Val Ser Phe Gin Gly Arg Gly Val Phe 450 455 460 gag etc teg gac gaa aag gca aeg aac ecg ate gtg cct tec ttt gac 1440 Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro lie Val Pro Ser Phe Asp 465 470 475 480 atg age aat gaa ggg tet tat ttc ttc gga gac aat get gag gag ttt 1488 Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Phe 485 490 495

   1497 gac agt taa Asp Ser

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   26 <210> 10 <211> 498 <212> PRT <213> Influenza . virus <400> 10 Met Ala Ser Gin Gly Thr Lys Arg Ser Tyr Glu Gin Met Glu Thr Asp 1 5 10 15 Gly Glu Arg Gin Asn Ala Thr Glu He Arg Ala Ser Val Gly Arg Met 20 25 30 Val Gly Gly He Gly Arg Phe Tyr Val Gin Met Cys Thr Glu Leu Lys 35 40 45 Leu Asn Asp His Glu Gly Arg Leu He Gin Asn Ser lie Thr lie 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 He 85 90 95 Tyr Arg Arg Lys Asp Gly Lys Trp Met Arg Glu Leu lie Leu His Asp 100 105 110 Lys Glu Glu He Met Arg He Trp Arg Gin Ala Asn Asn Gly Glu Asp 115 120 125 Ala Thr Ala Gly Leu Thr His Met Met He Trp His Ser Asn Leu Asn 130 135 140 Asp Thr Thr Tyr Gin Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp 145 150 155 160 Pro Arg Met Cys Ser Leu Met Gin 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 He Arg Met lie Lys Arg Gly He Asn Asp Arg Asn Phe Trp Arg 195 200 205

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   Gly Glu Asn Gly Arg Lys Thr Arg 210 215 lie Leu Lys Gly Lys Phe Gin Thr 225 230

   Gin Val Arg Glu Gly Arg Asn Pro 245

   He Phe Leu Ala Arg Ser Ala Leu 260

   Lys Ser Cys Leu Pro Ala Cys Val 275 > 280

   He Ala Tyr Glu Arg Met Cys Asn 220

   Ala Ala Gin Arg Ala Met Met Asp 235 240

   Gly Asn Ala Glu He Glu Asp Leu 250 255

   He Leu Arg Gly Ser Val Ala His 265 270

   Tyr Gly Leu Ala lie Thr Ser Gly 285

   Tyr Asp Phe Glu Lys Glu Gly Tyr 290 295

   Ser Leu Val Gly He Asp Pro Phe 300

   Lys Leu Leu Gin Asn Ser Gin He 305 310

   Phe Ser Leu He Arg Pro Lys Glu 315 320

   Asn Pro Ala His Lys Ser Gin Leu 325

   Val Trp Met Ala Cys His Ser Ala 330 335

   Ala Phe Glu Asp Leu Arg Val Leu 340

   Asn Phe He Arg Gly Thr Lys Val 345 350

   He Pro Arg Gly Gin Leu Thr Thr 355 360

   Arg Gly Val Gin He Ala Ser Asn 365

   Glu Asn Met Glu Thr He Asn Ser 370 375

   Ser Thr Leu Glu Leu Arg Ser Lys 380

   Tyr Trp Ala He Arg Thr Arg Ser 385 390

   Gly Gly Asn Thr Ser Gin Gin Arg 395 400

   Ala Ser Ala Gly Gin He Ser Val 405

   Gin Pro Thr Phe Ser Val Gin Arg 410 415

   Asn Leu Pro Phe Glu Arg Ala Thr 420

   He Met Ala Ala Phe Thr Gly Asn 425 430

   Thr Glu Gly Arg Thr Ser Asp Met 435 440

   Arg Thr Glu He lie Arg Met Met 445

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   Glu Asn 450 Ala Lys Ser Glu Asp 455 Val Ser Glu 465 Leu Ser Asp Glu Lys 470 Ala Thr Asn Met Ser Asn Glu Gly 485 Ser Tyr Phe Phe Asp Ser

   Phe Gin Gly Arg Gly Val Phe 460

   Pro lie Val Pro Ser Phe Asp 475 480

   Gly Asp Asn Ala Glu Glu Phe 490 495

   <210> 11 <211> 1413 <212> DNA <213> Influenza l virus <220> <221> CDS <222> (1)..(1413) <400> 11 atg aat cca aat caa aag ata ata gca Met Asn Pro Asn Gin Lys lie lie Ala 1 5 ata tta ate att aat gtc att etc cat lie Leu lie lie Asn Val lie Leu His 20 25 gta ctg gtc etc aat aac aat aga aca Val Leu Val Leu Asn Asn Asn Arg Thr 35 40 ate ata aga aag tac aat gaa aca gta lie lie Arg Lys Tyr Asn Glu Thr Val 50 55 tgg tat aat acc agt aca att aag tac Trp Tyr Asn Thr Ser Thr lie Lys Tyr 65 70 tac tac atg aac aac act gaa cca ett Tyr Tyr Met Asn Asn Thr Glu Pro Leu 85 cca ttt tcc aaa gat aat gga ata ega Pro Phe Ser Lys Asp Asn Gly lie Arg 100 105 ttt.gtg ata aga gaa cct ttt gta tea phe Val lie Arg Glu Pro Phe Val Ser 115 12 0 acc ttt ttc etc aca cag ggc tea tta

   att gga ttt gca tea ttg ggg 48 lie Gly Phe Ala Ser Leu Gly 10 15 gta gtc age att ata gta aca 96

   Val Val Ser lie lie Val Thr gat ctg aac tgc aaa ggg aeg 144

   Asp Leu Asn Cys Lys Gly Thr aga gta gaa aaa att act caa 192

   Arg Val Glu Lys lie Thr Gin ata gag aga cct tea aat gaa 240 lie Glu Arg Pro Ser Asn Glu

   75 80 tgt gag gee caa ggc ttt gca 288

   Cys Glu Ala Gin Gly Phe Ala 90 95 att ggg teg aga ggc cat gtt 336 lie Gly Ser Arg Gly His Val

   110 tgt teg ccc tea gaa tgt aga 384

   Cys Ser Pro Ser Glu Cys Arg

   125 etc aat gac aaa cat tet aac 432

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   Thr Phe 130 Phe Leu Thr Gin 29 Gly Ser Leu Leu Asn Asp Lys His Ser Asn 135 140 ggc aca gta aag gac ega agt ccg tat agg act ttg atg agt gtc aaa 480 Gly 145 Thr Val Lys Asp Arg 150 Ser Pro Tyr Arg Thr 155 Leu Met Ser Val Lys 160 ata ggg caa tea cct aat gta tat caa get aga ttt gaa teg gta gca 528 lie Gly Gin Ser Pro 165 Asn Val Tyr Gin Ala 170 Arg Phe Glu Ser Val 175 Ala tgg tea gca aca gca tgc cat gat gga aaa aaa tgg atg aca gtt gga 576 Trp Ser Ala Thr 180 Ala Cys His Asp Gly 185 Lys Lys Trp Met Thr 190 Val Gly gtc aca ggg ccc gac aat caa gca att gca gta gtg aac tat gga ggt 624 Val Thr Gly 195 Pro Asp Asn Gin Ala 200 He Ala Val Val Asn 205 Tyr Gly Gly gtt ccg gtt gat att att aat tea tgg gca ggg gat att tta aga acc 672 Val Pro 210 Val Asp He He Asn 215 Ser Trp Ala Gly Asp 220 He Leu Arg Thr caa gaa tea tea tgc acc tgc att aaa gga gac tgt tat tgg gta atg 720 Gin 225 Glu Ser Ser Cys Thr 230 Cys He Lys Gly Asp 235 Cys Tyr Trp Val Met 240 act gat gga ccg gca aat agg caa get aaa tat agg ata ttc aaa gca 768 Thr Asp Gly Pro Ala 245 Asn Arg Gin Ala Lys 250 Tyr Arg lie Phe Lys 255 Ala aaa gat gga aga gta att gga cag act gat ata agt ttc aat ggg gga 816 Lys Asp Gly Arg 260 Val He Gly Gin Thr 265 Asp lie Ser Phe Asn 270 Gly Gly cac ata gag gag tgt tet tgt tac ccc aat gaa ggg aag gtg gaa tgc 864 His He Glu 275 Glu Cys Ser Cys Tyr 280 Pro Asn Glu Gly Lys 285 Val Glu Cys ata tgc agg gac aat tgg act gga aca aat aga cca gtt ctg gta ata 912 lie Cys 290 Arg Asp Asn Trp Thr 295 Gly Thr Asn Arg Pro 300 Val Leu Val He tet tet gat eta teg tac aca gtt gga tat ttg tgt get ggc att ccc 960 Ser 305 Ser Asp Leu Ser Tyr 310 Thr Val Gly Tyr Leu 315 Cys Ala Gly He Pro 320 act gac act cct agg gga gag gat agt caa ttc aca ggc tea tgt aca 1008 Thr Asp Thr Pro Arg 325 Gly Glu Asp Ser Gin 330 Phe Thr Gly Ser Cys 335 Thr agt cct ttg gga aat aaa gga tac ggt gta aaa ggt ttc ggg ttt ega 1056 Ser Pro Leu Gly 340 Asn Lys Gly Tyr Gly 345 Val Lys Gly Phe Gly 350 Phe Arg caa gga act gac gta tgg gcc gga agg aca att agt agg act tea aga 1104 Gin Gly Thr 355 Asp Val Trp Ala Gly 360 Arg Thr lie Ser Arg 365 Thr Ser Arg tea gga ttc gaa ata ata aaa ate agg aat ggt tgg aca cag aac agt 1152

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   Ser Gly Phe Glu He He Lys He Arg Asn Gly Trp Thr Gin Asn Ser 370 375 380 aaa gac caa ate agg agg caa gtg att ate gat gac cca aat tgg tea Lys Asp Gin He Arg Arg Gin Val He He Asp Asp Pro Asn Trp Ser 385 I 390 395 400 gga tat age ggt tet ttc aca ttg ccg gtt gaa eta aca aaa aag gga 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 Cys Leu Val Pro Cys Phe Trp Val Glu Met He Arg Gly Lys Pro Glu 420 425 430 gaa aca aca ata tgg acc tet agt age tcc att gtg atg tgt gga gta Glu Thr Thr He Trp Thr Ser Ser Ser Ser He Val Met Cys Gly Val 435 440 445 gat cat aaa att gcc agt tgg tea tgg cac gat gga get att ett ccc Asp His Lys He Ala Ser Trp Ser Trp His Asp Gly Ala He Leu Pro 450 455 460 ttt gac ate gat aag atg taa Phe Asp He Asp Lys Met 465 470 <210> 12 <211> - 470 <212> PRT <213> Influenza virus <400> 12 Met Asn Pro Asn Gin Lys He He Ala He Gly Phe Ala Ser Leu Gly 1 5 10 15 He Leu He He Asn Val He Leu His Val Val Ser He He Val Thr 20 25 30 Val Leu Val Leu Asn Asn Asn Arg Thr Asp Leu Asn Cys Lys Gly Thr 35 40 45 He He Arg Lys Tyr Asn Glu Thr Val Arg Val Glu Lys He Thr Gin 50 55 60 Trp Tyr Asn Thr Ser Thr He Lys Tyr He Glu Arg Pro Ser Asn Glu 65 70 75 80 Tyr Tyr Met Asn Asn Thr Glu Pro Leu Cys Glu Ala Gin Gly Phe Ala 85 90 95

   1200

   1248

   1296

   1344

   1392

   1413

   Pro Phe Ser Lys Asp Asn Gly lie Arg He Gly Ser Arg Gly His Val

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   100

   Phe Val lie Arg Glu Pro Phe Val 115 120

   Thr Phe Phe ,Leu Thr Gin Gly Ser 130 135

   Gly Thr Val Lys Asp Arg Ser Pro 145 150

   He Gly Gin Ser Pro Asn Val Tyr 165

   Trp Ser Ala Thr Ala Cys His Asp 180

   105 110

   Ser Cys Ser Pro Ser Glu Cys Arg 125

   Leu Leu Asn Asp Lys His Ser Asn 140

   Tyr Arg Thr Leu Met Ser Val Lys 155 160

   Gin Ala Arg Phe Glu Ser Val Ala 170 175

   Gly Lys Lys Trp Met Thr Val Gly 185 190

   Val Thr Gly Pro Asp Asn Gin Ala 195 200

   He Ala Val Val Asn Tyr Gly Gly 205

   Val Pro Val Asp lie He Asn Ser 210 215

   Trp Ala Gly Asp lie Leu Arg Thr 220

   Gin Glu Ser Ser Cys Thr Cys He 225 230

   Lys Gly Asp Cys Tyr Trp Val Met 235 240

   Thr Asp Gly Pro Ala Asn Arg Gin 245

   Ala Lys Tyr Arg He Phe Lys Ala 250 255

   Lys Asp Gly Arg Val He Gly Gin 260

   Thr Asp He Ser Phe Asn Gly Gly 265 , 270

   His He Glu Glu Cys Ser Cys Tyr 275 280

   Pro Asn Glu Gly Lys Val Glu Cys 285

   He Cys Arg Asp Asn Trp Thr Gly 290 295

   Thr Asn Arg Pro Val Leu Val lie 300

   Ser Ser Asp Leu Ser Tyr Thr Val 305 310

   Gly Tyr Leu Cys Ala Gly He Pro 315 320

   Thr Asp Thr Pro Arg Gly Glu Asp 325

   Ser Gin Phe Thr Gly Ser Cys Thr 330 335

   Ser Pro Leu Gly Asn Lys Gly Tyr Gly Val Lys Gly Phe Gly Phe Arg

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   340 345 350

   Gin Gly Thr Asp Val Trp Ala Gly 360 Arg Thr He Ser Arg 365 Thr Ser Arg 355 Ser Gly Phe Glu He He Lys lie Arg Asn Gly Trp Thr Gin Asn Ser 370 3 75 380 Lys Asp Gin He Arg Arg Gin Val He He 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 He Arg Gly Lys Pro Glu 420 425 430 Glu Thr Thr He Trp Thr Ser Ser Ser Ser He Val Met Cys Gly Val 435 440 445 Asp His Lys lie Ala Ser Trp Ser Trp His Asp Gly Ala He Leu Pro 450 455 460 Phe Asp lie Asp Lys Met

   465 470 <210> 13 <211> 982 <212> DNA

   <213> Influenza virus <220> <221> CDS <222> (1) · , (756) <400> 13 atg agt ett eta acc gag gtc gaa aeg tac gtt⁽ctc tet ate gta cca 48 Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser He Val Pro 1 5 10 15 tea ggc ccc etc aaa gcc gag ate geg cag aga ett gaa gat gtc ttt 96 Ser Gly Pro Leu Lys Ala Glu He Ala Gin Arg Leu Glu Asp Val Phe 20 25 30 gca gga aag aac acc gat ett gag gca etc atg gaa tgg eta aag aca 144 Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr 35 40 45 aga cca ate ctg tea cct ctg act aaa ggg att tta gga ttt gta ttc 192 Arg Pro lie Leu Ser Pro Leu Thr Lys Gly He Leu Gly Phe Val Phe

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   50 55 60

   acg etc Thr Leu 65 acc Thr gtg Val ccc Pro agt Ser 70 gag Glu ega gga Arg Gly ctg Leu cag Gin 75 cgt Arg aga Arg ege Arg ttt Phe gtc Val 80 240 caa aat gee ett agt gga aac gga gat cca aac aac atg gac aga gca 288 Gin Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala , 85 90 95 gta aaa ctg tac agg aag ett aaa aga gaa ata aca ttc cat ggg gca 336 Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu lie Thr Phe His Gly Ala 100 105 110 aaa gag gta gca etc age tat tee act ggt gca eta gcc age tgc atg 384 Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met 115 12 0 125 gga etc ata tac aac aga atg gga act gtt aca acc gaa gtg gca ttt 432 Gly Leu lie Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe 130 135 140 ggc ctg gta tgc gee aca tgt gaa cag att get gat tee cag cat ega 480 Gly Leu Val Cys Ala Thr Cys Glu Gin He Ala Asp Ser Gin His Arg 145 150 155 160 get cac agg cag atg gtg aca aca acc aac cca ttg ate aga cat gaa 528 Ala His Arg Gin Met Val Thr Thr Thr Asn Pro Leu lie Arg His Glu 165 170 175 aac aga atg gta' tta gcc agt acc acg get aaa gcc atg gaa cag atg 576 Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gin Met 180 185 190 gca gga teg agt gag cag gca gca gag gcc atg gag gtt get agt agg 624 Ala Gly Ser Ser Glu Gin Ala Ala Glu Ala Met Glu Val Ala Ser Arg 195 200 205 get agg cag atg gta cag gca atg aga acc att ggg acc cac cct age 672 Ala Arg Gin Met Val Gin Ala Met Arg Thr He Gly Thr His Pro Ser 210 215 220 tee agt gcc ggt ttg aaa gat gat etc ett gaa aat tta cag gee tac 720 Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gin Ala Tyr 225 230 235 240 cag aaa egg atg gga gtg caa atg cag ega ttc aag tgatcctctc 766 Gin Lys Arg Met Gly Val Gin Met Gin Arg Phe Lys 245 250

   gttattgcag caagtatcat tgggatcttg cacttgatat tgtggattct tgategtett 826 ttcttcaaat teatttateg tegeettaaa tacgggttga aaagagggcc ttctacggaa 886 ggagtacctg agtetatgag ggaagaatat cggcaggaac ageagaatge tgtggatgtt 946 gacgatggtc attttgtcaa catagagctg gagtaa 982

   <210> 14

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   34 <211> 252 <212> PRT <213> Influenza virus <400> 14 Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser He Val Pro 1 5 10 15 Ser Gly Pro Leu Lys Ala Glu lie Ala Gin 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 lie Leu Ser Pro Leu Thr Lys Gly He Leu Gly Phe Val Phe 50 55 60 Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gin Arg Arg Arg Phe Val 65 70 75 80 Gin 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 He 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 He Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe 13 0 135 14 0 Gly Leu Val Cys Ala Thr Cys Glu Gin He Ala Asp Ser Gin His Arg 145 150 155 160 Ala His Arg Gin Met Val Thr Thr Thr Asn Pro Leu He Arg His Glu 165 170 175 Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gin Met 180 185 190 Ala Gly Ser Ser Glu Gin Ala Ala Glu Ala Met Glu Val Ala Ser Arg 195 200 205 Ala Arg Gin Met Val Gin Ala Met Arg Thr He Gly Thr His Pro Ser 210 215 220

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   Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gin Ala Tyr 225 230 235 240

   Gin Lys Arg Met Gly Val Gin Met Gin Arg Phe Lys 245 250 <210> 15 <211> 1698 <212> DNA <213> Influenza virus <220>

   <221> CDS <222> (1)..(1698) <400> 15

   atg Met 1 aag Lys aca Thr acc Thr att He 5 att He ttg Leu ata lie eta Leu eta Leu 10 acc Thr cat His tgg Trp gcc Ala tac Tyr 15 agt Ser 48 caa aac cca ate agt ggc aac aac aca gcc aca ctg tgt ctg gga cac 96 Gin Asn Pro He 20 Ser Gly Asn Asn Thr 25 Ala Thr Leu Cys Leu 30 Gly His cat gca gta gca aat gga aca ttg gta aaa aca atg agt gat gat caa 144 His Ala Val 35 Ala Asn Gly Thr Leu 40 Val Lys Thr Met Ser 45 Asp Asp Gin att gag gtg aca aat get aca gaa tta gtt cag age att tea atg ggg 192 lie Glu 50 Val Thr Asn Ala Thr 55 Glu 'Leu Val Gin Ser 60 He Ser Met Gly aaa ata tgc aac aaa tea tat aga att eta gat gga aga aat tgc aca 240 Lys 65 He Cys Asn Lys Ser 70 Tyr Arg He Leu Asp 75 Gly Arg Asn Cys Thr 80 tta ata gat gca atg eta gga gac ccc cac tgt gac gcc ttt cag tat 288 Leu lie Asp Ala Met 85 Leu Gly Asp Pro His 90 Cys Asp Ala Phe Gin 95 Tyr gag agt tgg gac etc ttc ata gaa aga age aac get ttc age aat tgc 336 Glu Ser Trp Asp 100 Leu Phe lie Glu Arg 105 Ser Asn Ala Phe Ser 110 Asn Cys tac cca tat gac ate cct gac tat gca teg etc ega tcc att gta gca 384 Tyr Pro Tyr 115 Asp He Pro Asp Tyr 120 Ala Ser Leu Arg Ser 125 lie Val Ala tcc tea gga aca ttg gaa ttc aca gca gag gga ttc aca tgg aca ggt 432 Ser Ser 130 Gly Thr Leu Glu Phe 135 Thr Ala Glu Gly Phe 14 0 Thr Trp Thr Gly gtc act caa aac gga aga agt gga gcc tgc aaa agg gga tea gcc gat 480 Val 145 Thr Gin Asn Gly Arg 150 Ser Gly Ala Cys Lys 155 Arg Gly Ser Ala Asp 160

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   I

   agt Ser ttc Phe ttt Phe age Ser cga Arg 165 ctg Leu aat Asn tgg Trp eta Leu aca Thr 170 aaa Lys tct Ser gga Gly age Ser tct Ser 175 tac Tyr 528 ccc aca ttg aat gtg aca atg cct aac aat aaa aat ttc gac aag eta 576 Pro Thr Leu Asn 18Ό Val Thr Met Pro Asn 185 Asn Lys Asn Phe Asp 190 Lys Leu tac ate tgg ggg att cat cac ccg age tea aat caa gag cag aca aaa 624 Tyr He Trp 195 Gly He His His Pro 200 Ser Ser Asn Gin Glu 205 Gin Thr Lys ttg tac ate caa gaa tea gga cga gta aca gtc tea aca aaa aga agt 672 Leu Tyr 210 He Gin Glu Ser Gly 215 Arg Val Thr Val Ser 220 Thr Lys Arg Ser caa caa aca ata ate cct aac ate gga tct aga ccg ttg gtc aga ggt 720 Gin 225 Gin Thr lie lie Pro 230 Asn He Gly Ser Arg 235 Pro Leu Val Arg Gly 240 caa tea ggc agg ata age ata tac tgg acc att gta aaa cct gga gat 768 Gin Ser Gly Arg He 245 Ser He Tyr Trp Thr 250 He Val Lys Pro Gly 255 Asp ate eta atg ata aac agt aat ggc aac tta gtt gca ccg egg gga tat 816 lie Leu Met He 260 Asn Ser Asn Gly Asn 265 Leu Val Ala Pro Arg 270 Gly Tyr ttt aaa ttg aaa aca ggg aaa age tct gta atg aga tea gat gca ccc 864 Phe Lys Leu 275 Lys Thr Gly Lys Ser 280 Ser Val Met Arg Ser 285 Asp Ala Pro ata gac att tgt gtg tct gaa tgt att aca cca aat gga age ate tcc 912 He Asp 290 He Cys Val Ser Glu 295 Cys He Thr Pro Asn 300 Gly Ser He Ser aac gac aag cca ttc caa aat gtg aac aaa gtt aca tat gga aaa tgc 960 Asn 305 Asp Lys Pro Phe Gin 310 Asn Val Asn Lys Val 315 Thr Tyr Gly Lys Cys 320 cct aag tat ate agg caa aac act tta aag ctg gcc act ggg atg agg 1008 Pro Lys Tyr He Arg 325 Gin Asn Thr Leu Lys 330 Leu Ala Thr Gly Met 335 Arg aat gta cca gaa aag caa acc aga gga ate ttt gga gca ata gcg gga 1056 Asn Val Pro Glu 340 Lys Gin Thr Arg Gly 345 He Phe Gly Ala He 350 Ala Gly ttc ate gaa aac ggc tgg gaa gga atg gtt gat ggg tgg tat ggg ttc 1104 Phe lie Glu 355 Asn Gly Trp Glu Gly 360 Met Val Asp Gly Trp 365 Tyr Gly Phe cga tat caa aac tct gaa gga aca ggg caa get gca gat eta aag age 1152 Arg Tyr 370 Gin Asn Ser Glu Gly 375 Thr Gly Gin Ala Ala 380 Asp Leu Lys Ser act caa gca gcc ate gac cag att aat gga aag tta aac aga gtg att 1200 Thr 385 Gin Ala Ala lie Asp 390 Gin He Asn Gly Lys 395 Leu Asn Arg Val He 400

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   gaa Glu aga Arg acc Thr aat Asn gag Glu 405 aaa Lys ttc Phe cat His caa Gin ata He 410 gag Glu aag Lys gaa ttc tea Ser 415 gaa Glu 1248 Glu Phe gta gaa gga aga att cag gac ctg gag aaa tat gta gaa gac acc aaa 1296 Val Glu Gly Arg He Gin Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys 420 425 430 ata gac eta tgg tcc tac aat gca gaa ttg ctg gtg get eta gaa aat 1344 lie. 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 Gin His Thr He Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu 450 455 460 aag act aga ege cag tta aga gaa aac gca gaa gac atg gga ggt gga 1440 Lys Thr Arg Arg Gin 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 tea ata 1488 Cys Phe Lys He Tyr His Lys Cys Asp Asn Ala Cys He Gly Ser He 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 He Tyr Arg Asp Glu Ala Leu Asn 500 505 510 aac cga ttt cag ate aaa ggt gta gag ttg aaa tea ggc tac aaa gat 1584 Asn Arg Phe Gin He Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp 515 520 525 tgg ata ttg tgg att tea ttc gcc ata tea tgc ttc tta att tgc gtt 1632 Trp He Leu Trp He Ser Phe Ala He Ser Cys Phe Leu He Cys Val 530 535 540 gtt eta ttg ggt ttc att atg tgg get tgc caa aga ggc aac ate aga 1680 Val Leu Leu Gly Phe lie Met Trp Ala Cys Gin Arg Gly Asn He Arg 545 550 555 560 tgc aac att tgc att tga 1698 Cys' Asn lie Cys He 565

   <210> 16 <211> 565 <212> PRT <213> Influenza virus <400> 16 1 Met Lys ! Thr Thr He He Leu He Leu Leu Thr His Trp Ala Tyr Ser 1 5 10 15

   Gin Asn Pro He Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His 20 25 30

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   His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Met Ser Asp Asp Gin 35 40 45 lie Glu Val Thr Asn Ala Thr Glu Leu Val Gin Ser He Ser Met Gly 50 55 60

   Lys He Cys Asn Lys Ser Tyr Arg He Leu Asp Gly Arg Asn Cys Thr 65 70 75 80

   Leu He Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gin Tyr 85 , 90 95

   Glu Ser Trp Asp Leu Phe He Glu Arg Ser Asn Ala Phe Ser Asn Cys 100 105 110

   Tyr Pro Tyr Asp He Pro Asp Tyr Ala Ser Leu Arg Ser He 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 Gin 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 He Trp Gly He His His Pro Ser Ser Asn Gin Glu Gin Thr Lys 195 200 205

   Leu Tyr He Gin Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser 210 215 220

   Gin Gin Thr He He Pro Asn He Gly Ser Arg Pro Leu Val Arg Gly 225 230 235 240

   Gin Ser Gly Arg He Ser He Tyr Trp Thr He Val Lys Pro Gly Asp 245 250 255

   He Leu Met He Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr 260 265 270

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   Phe Lys Leu Lys Thr Gly Lys Ser Ser Val Met Arg Ser Asp Ala Pro 275 280 285 lie Asp He Cys Val Ser Glu Cys He Thr Pro Asn Gly Ser He Ser 290 295 300

   Asn Asp Lys Pro Phe Gin Asn Val Asn Lys Val Thr Tyr Gly Lys Cys

   305 310 ' ¹ 315 320

   Pro Lys Tyr He Arg Gin Asn Thr Leu Lys Leu Ala Thr Gly Met Arg 325 330 335

   Asn Val Pro Glu Lys Gin Thr Arg Gly He Phe Gly Ala He Ala Gly 340 345 350

   Phe He Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe 355 360 365

   Arg Tyr Gin Asn Ser Glu Gly Thr Gly Gin Ala Ala Asp Leu Lys Ser 370 375 380

   Thr Gin Ala Ala He Asp Gin He Asn Gly Lys Leu Asn Arg Val He 385 390 395 400

   Glu Arg Thr Asn Glu Lys Phe His Gin He Glu Lys Glu Phe Ser Glu 405 410 415

   Val Glu Gly Arg He Gin Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys 420 425 430

   He Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val 'Ala Leu Glu Asn 435 440 445

   Gin His Thr He Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu 450 455 460

   Lys Thr Arg Arg Gin Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly 465 470 475 480

   Cys Phe Lys lie Tyr His Lys Cys Asp Asn Ala Cys He Gly Ser He 485 490 495

   Arg Thr Gly Thr Tyr Asp His Tyr lie Tyr Arg Asp Glu Ala Leu Asn 500 505 510

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   Asn Arg Phe Gin lie Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp 515 520 525

   Trp He Leu Trp He Ser Phe Ala He Ser Cys Phe Leu He Cys Val 530 535 540

   Val Leu Leu Gly Phe He Met Trp Ala Cys Gin Arg Gly Asn lie Arg 545 550 555 560

   Cys Asn He Cys He 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 He <400> 17

   atg gag aga ata aaa gaa ctg aga gat ctg atg tta caa tcc cgc acc Met 1 Glu Arg He Lys 5 Glu Leu Arg Asp Leu 10 Met Leu Gin Ser Arg 15 Thr cgc gag ata eta aca aaa act act gtg gac cac atg gcc ata ate aag Arg Glu He Leu 20 Thr Lys Thr Thr Val 25 Asp His Met Ala He 30 lie Lys aaa tac aca tea gga aga caa gag aag aac cct gca ctt agg atg aaa Lys Tyr Thr 35 Ser Gly Arg Gin Glu 40 Lys Asn Pro Ala Leu 45 Arg Met Lys tgg atg atg gca atg aaa tac cca att aca gca gat aag agg ata atg Trp Met 50 Met Ala Met Lys Tyr 55 Pro He Thr Ala Asp 60 Lys Arg He Met gag atg att cct gag aga aat gaa cag gga caa acc ctt tgg age aaa Glu 65 Met lie Pro Glu Arg 70 Asn Glu Gin Gly Gin 75 Thr Leu Trp Ser Lys 80 acg aac gat get ggc tea gac cgc gta atg gta tea cct ctg gca gtg Thr Asn Asp Ala Gly 85 Ser Asp Arg Val Met 90 Val Ser Pro Leu Ala 95 Val aca tgg tgg aat agg aat gga cca aca acg age aca att cat tat cca Thr Trp Trp Asn 100 Arg Asn Gly Pro Thr 105 Thr Ser Thr He His 110 Tyr Pro

   144

   192

   240

   288

   336

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   aaa Lys gtc Val tac Tyr 115 aaa Lys act Thr tat Tyr ttt Phe gaa Glu 120 aag Lys gtt gaa aga Arg ttg Leu 125 aaa Lys cac His gga Gly 384 Val Glu acc ttt ggc ccc gtt cat ttt agg aat caa gtc aag ata aga ega aga 432 Thr Phe 130 Gly Pro Val His Phe 135 Arg Asn Gin Val Lys 140 He Arg Arg Arg gtt gat gta aac cct ggt cac geg gac etc agt gee aaa gaa gca caa 480 Val 145 Asp Val Asn Pro Gly 150 His Ala Asp Leu Ser 155 Ala Lys Glu Ala Gin 160 gat gtg ate atg gaa gtt gtt ttc cca aat gaa gtg gga gcc aga att 528 Asp Val He Met Glu 165 Val Val Phe Pro Asn 170 Glu Val Gly Ala Arg 175 He eta aca teg gaa tea caa eta aca ata acc aaa gag aaa aag gaa gaa 576 Leu Thr Ser Glu 180 Ser Gin Leu Thr He 185 Thr Lys Glu Lys Lys 190 Glu Glu ett cag gac tgc aaa att get ccc ttg atg gta gca tac atg eta gaa 624 Leu Gin Asp 195 Cys Lys He Ala Pro 200 Leu Met Val Ala Tyr 205 Met Leu Glu aga gag ttg gtc ega aaa aca agg ttc etc cca gta gca ggc gga aca 672 Arg Glu 210 Leu Val Arg Lys Thr 215 Arg Phe Leu Pro Val 220 Ala Gly Gly Thr age agt gta tac att gaa gtg ttg cat ctg act cag gga aca tgc tgg 720 Ser 225 Ser Val Tyr He Glu 230 Val Leu His Leu Thr 235 Gin Gly Thr Cys Trp 240 gag caa atg tac acc cca gga gga gaa gtt aga aac gat gat att gat 768 Glu Gin Met Tyr Thr 245 Pro Gly Gly Glu Val 250 Arg Asn Asp Asp He 255 Asp caa agt tta att att gca gcc egg aac ata gtg aga aga geg aca gta 816 Gin Ser Leu He 260 He Ala Ala Arg Asn 265 He Val Arg Arg Ala 270 Thr Val tea gca gat cca eta gca tcc eta ctg gaa atg tgc cac agt aca cag 864 Ser Ala Asp 275 Pro Leu Ala Ser Leu 280 Leu Glu Met Cys His, 285 Ser Thr Gin att ggt gga ata agg atg gta gac ate ett aag cag aat cca aca gag 912 lie Gly 290 Gly He Arg Met Val 295 Asp He Leu Lys Gin 300 Asn Pro Thr piu gaa caa get gtg gat ata tgc aaa gca gca atg gga ttg aga att age 960 Glu 305 Gin Ala Val Asp He 310 Cys Lys Ala Ala Met 315 Gly Leu Arg He Ser 320 tea tea ttc age ttt ggt gga ttc acc ttc aaa aga aca agt gga tea 1008 Ser Ser Phe Ser Phe 325 Gly Gly Phe Thr Phe 330 Lys Arg Thr Ser Gly 335 Ser tea gtc aag aga gaa gaa gaa atg ett aeg ggc aac ett caa aca ttg 1056 Ser 1 Val Lys Arg 340 Glu Glu Glu Met Leu 345 Thr Gly Asn Leu Gin 350 Thr Leu

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   aaa Lys ata He aga gtg Arg Val 355 cat His gag Glu ggc Gly tat Tyr 360 gaa Glu gaa Glu ttc Phe aca Thr atg gtc gga Gly aga Arg 1104 Met 365 Val aga gca aca gcc att ate aga aag gca acc aga aga ttg att caa ttg 1152 Arg Ala Thr Ala lie He Arg Lys Ala Thr Arg Arg Leu He Gin Leu 370 375 380 ata gta agt ggg aga gat gaa caa tea att get gaa gca ata att gta 1200 lie Val Ser Gly Arg Asp Glu Gin Ser He Ala Glu Ala He He Val 385 390 395 400 gcc atg gtg ttt teg caa gaa gat tgc atg ata aaa gca gtt ega ggc 1248 Ala Met Val Phe Ser Gin Glu Asp Cys Met He 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 Gin Arg Leu Asn Pro Met His 420 425 430 caa etc ttg agg cat ttc caa aaa gat gca aaa gtg ett ttc caa aat 1344 Gin Leu Leu Arg His Phe Gin Lys Asp Ala Lys Val Leu Phe Gin Asn 435 440 445 tgg gga att gaa ccc ate gac aat gta atg ggg atg att gga ata ttg 1392 Trp Gly He Glu Pro He Asp Asn Val Met Gly Met lie Gly He Leu 450 455 460 cct gac atg acc cca 1 age acc gag atg tea 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 age aaa atg gga gtg gat gag tac tcc age 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 age att gac cgt ttt tta aga gtt egg gat caa agg gga aac ata eta 1536 Ser He Asp Arg Phe Leu Arg Val Arg Asp Gin Arg Gly Asn lie Leu 500 505 510 ctg tcc cct gaa gaa gtc agt gaa aca caa gga aeg gaa aag ctg aca 1584 Leu Ser Pro Glu Glu Val Ser Glu Thr Gin Gly Thr Glu Lys Leu Thr 515 520 525 ata att tat teg tea tea atg atg tgg gag att aat ggt ccc gaa tea 1632 He He Tyr Ser Ser Ser Met Met Trp Glu He Asn Gly Pro Glu Ser 530 535 540 gtg ttg gtc aat act tat caa tgg ate ate agg aac tgg gaa att gta 1680 Val Leu Val Asn Thr Tyr Gin Trp He He Arg Asn Trp Glu He Val 545 550 555 560 aaa att cag tgg tea cag gac ccc aca atg tta tac aat aag ata gaa 1728 Lys He Gin Trp Ser Gin Asp Pro Thr Met Leu Tyr Asn Lys He Glu 565 570 575 ttt gag cca ttc caa tcc ctg gtc cct agg gcc acc aga age caa tac 1776 Phe Glu Pro Phe Gin Ser Leu Val Pro Arg Ala Thr Arg Ser Gin Tyr 580 585 590

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   age Ser ggt Gly ttc Phe 595 gta Val aga Arg acc Thr ctg Leu ttt Phe 600 cag caa Gin Gin atg Met ega Arg gat Asp 605 gta Val ett Leu gga Gly 1824 aca ttt gat act get caa ata ata aaa etc etc cct ttt gcc get get 1872 Thr Phe Asp Thr Ala Gin lie He Lys Leu Leu Pro Phe Ala Ala Ala 610 615 620 cct ccg gaa cag agt agg atg cag ttc tet tet ttg act gtt aat gta 1920 Pro Pro Glu Gin Ser Arg Met Gin Phe Ser Ser Leu Thr Val Asn Val 625 63 0 635 640 aga ggt teg gga atg agg ata ett gta aga ggc aat tee cca gtg ttc 1968 Arg Gly Ser Gly Met Arg lie Leu Val Arg Gly Asn Ser Pro Val Phe 645 650 655 aac tac aat aaa gcc act aaa agg etc aca gtc etc 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 geg ett act gag gac cca gat gaa ggt aeg get gga gta gaa tet 2064 Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser 675 680 685 get gtt eta aga ggg ttt etc att tta ggt aaa gaa aac aag aga tat 2112 Ala Val Leu Arg Gly Phe Leu He Leu Gly Lys Glu Asn Lys Arg Tyr 690 695 700 ggc cca gca eta age ate aat gaa eta age aaa ett gca aaa ggg gag 2160 Gly Pro Ala Leu Ser He Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu 705 710 715 720 aaa gcc aat gta eta att ggg caa ggg gac rta gtg ttg gta atg aaa 2208 Lys Ala Asn Val Leu He Gly Gin Gly Asp Xaa Val Leu Val Met Lys 725 73 0 735 egg aaa cgt gac tet age ata ett act gac age cag aca geg acc aaa 2256 Arg Lys Arg Asp Ser Ser lie Leu Thr Asp Ser Gin Thr Ala Thr Lys 740 745 750 agg att egg atg gcc ate aat 2277 Arg He Arg Met Ala He 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 lie.

   <400> 18

   Met Glu Arg He Lys Glu Leu Arg Asp Leu Met Leu Gin Ser Arg Thr 15 10 15

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   Arg Glu lie Leu Thr Lys Thr Thr Val Asp His Met Ala lie lie Lys 20 25 30

   Lys Tyr Thr Ser Gly Arg Gin Glu Lys Asn Pro Ala Leu Arg Met Lys 35 40 45

   Trp Met Met Ala Met Lys Tyr Pro lie Thr Ala Asp Lys Arg lie Met 50 55 60

   Glu Met He Pro Glu Arg Asn Glu Gin Gly Gin 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 lie 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 Gin Val Lys He Arg Arg Arg 130 135 140

   Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gin 145 150 155 160

   Asp Val He Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg He 165 170 175

   Leu Thr Ser Glu Ser Gin Leu Thr He Thr Lys Glu Lys Lys Glu Glu 180 185 190

   Leu Gin Asp Cys Lys He 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 He Glu Val Leu His Leu Thr Gin Gly Thr Cys Trp 225 230 235 240

   Glu Gin Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp He Asp 245 250 255

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   Gin Ser Leu. He lie Ala Ala Arg Asn He Val Arg Arg Ala Thr Val 260 265 270

   Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gin 275 280 285 lie Gly Gly He Arg Met Val Asp He Leu Lys Gin Asn Pro Thr Glu 290 295 300

   Glu Gin Ala Val Asp He Cys Lys Ala Ala Met Gly Leu Arg He 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 Gin Thr Leu 340 345 350

   Lys He Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg 355 ' 360 365

   Arg Ala Thr Ala He He Arg Lys Ala Thr Arg Arg Leu He Gin Leu 370 375 380 lie Val Ser Gly Arg Asp Glu Gin Ser lie Ala Glu Ala He He Val 385 390 395 400

   Ala Met Val Phe Ser Gin Glu Asp Cys Met He Lys Ala Val Arg Gly 405 410 415

   Asp Leu Asn Phe Val Asn Arg Ala Asn Gin Arg Leu Asn Pro Met His 420 425 430

   Gin Leu Leu Arg His Phe Gin Lys Asp Ala Lys Val Leu Phe Gin Asn 435 440 445

   Trp Gly He Glu Pro lie Asp Asn Val Met Gly Met He Gly He 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

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   Ser lie Asp Arg Phe Leu Arg Val Arg Asp Gin Arg Gly Asn He Leu 500 505 510

   Leu Ser Pro Glu Glu Val Ser Glu Thr Gin Gly Thr Glu Lys Leu Thr 515 520 525

   He He Tyr Ser Ser Ser Met Met Trp Glu He Asn Gly Pro Glu Ser 530 535 540

   Val Leu Val Asn Thr Tyr Gin Trp He He Arg Asn Trp Glu He Val 545 550 555 560

   Lys He Gin Trp Ser Gin Asp Pro Thr Met Leu Tyr Asn Lys He Glu 565 570 575

   Phe Glu Pro Phe Gin Ser Leu Val Pro Arg Ala Thr Arg Ser Gin Tyr 580 585 590

   Ser Gly Phe Val Arg Thr Leu Phe Gin Gin Met Arg Asp Val Leu Gly 595 600 605

   Thr Phe Asp Thr Ala Gin He He Lys Leu Leu Pro Phe Ala Ala Ala 610 615 620

   Pro Pro Glu Gin Ser Arg Met Gin Phe Ser Ser Leu Thr Val Asn Val 625 630 635 640

   Arg Gly Ser Gly Met Arg He 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 He Leu Gly Lys Glu Asn Lys Arg Tyr 690 695 700

   Gly Pro Ala Leu Ser He Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu 705 710 715 720

   Lys Ala Asn Val Leu He Gly Gin Gly Asp Xaa Val Leu Val Met Lys 725 730 735

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   Arg Lys Arg Asp Ser Ser lie Leu Thr Asp Ser Gin Thr Ala Thr Lys 740 745 750

   Arg He Arg Met Ala He Asn 755 <210> 19 <211> 2274 <212> DNA <213> Influenza virus <220>

   <221> CDS <222> (1)..(2274) <400> 19

   atg Met 1 gat Asp gtc Val aat Asn ccg Pro 5 act Thr eta Leu ett Leu ttc Phe tta Leu 10 aag Lys gtg Val cca Pro geg Ala caa Gin 15 aat Asn 48 get ata age aca aca ttc cct tat act gga gat cct ccc tac agt cat 96 Ala He Ser Thr 20 Thr Phe Pro Tyr Thr 25 Gly Asp Pro Pro Tyr 30 Ser His gga aca ggg aca gga tac acc atg gat act gtc aac aga aca cac caa 144 Gly Thr Gly 35 Thr Gly Tyr Thr Met 40 Asp Thr Val Asn Arg 45 Thr His Gin tat tea gaa aaa ggg aaa tgg aca aca aac act gag att gga gca cca 192 Tyr Ser 50 Glu Lys Gly Lys Trp 55 Thr Thr Asn Thr Glu 60 lie Gly Ala Pro caa ett aat cca ate gat gga cca ett cct gaa gac aat gaa cca agt 240 Gin 65 Leu Asn Pro He Asp 70 Gly Pro Leu Pro Glu 75 Asp Asn Glu Pro Ser 80 ggg tac gcc caa aca gat tgt gta ttg gaa gca atg get ttc ett gaa 288 Gly Tyr Ala Gin Thr 85 Asp Cys Val Leu Glu 90 Ala Met Ala Phe Leu 95 Glu gaa tcc cat ccc gga ate ttt gaa aat teg tgt ett gaa aeg atg gag 336 Glu Ser His Pro 100 Gly He Phe Glu Asn 105 Ser Cys Leu Glu Thr 110 Met Glu gtg att cag cag aca aga gtg gac aaa eta aca caa ggc ega caa act 384 Val He Gin 115 Gin Thr Arg Val Asp 120 Lys Leu Thr Gin Gly 125 Arg Gin Thr tat gat tgg acc ttg aat agg aat caa cct gcc gca aca gca ett get 432 Tyr Asp 13 0 Trp Thr Leu Asn Arg 135 Asn Gin Pro Ala Ala 140 Thr Ala Leu } Ala aat aeg att gaa gta ttc aga tea aat ggt ctg act tcc aat gaa teg 480 Asn 145 Thr He Glu Val Phe 150 Arg Ser Asn Gly Leu 155 Thr Ser Asn Glu Ser 160

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   ggg Gly aga Arg ttg Leu atg Met gac Asp 165 ttc Phe etc Leu aaa Lys gat Asp gtc Val 170 atg Met gag Glu tcc Ser atg Met aac Asn 175 aag Lys 528 gaa gaa atg gaa ata aca aca cac ttc caa egg aag aga aga gta aga 576 Glu Glu Met Glu 180 He Thr Thr His Phe 185 Gin Arg Lys Arg Ω Arg 190 Val Arg gac aac atg aca aag aga atg gta aca cag aga acc ata ggg aag aaa 624 Asp Asn Met 195 Thr Lys Arg Met Val 200 Thr Gin Arg Thr He 205 Gly Lys Lys aaa caa ega tta aac aga aag age tat eta ate aga aca tta acc eta 672 Lys Gin 210 Arg Leu Asn Arg Lys 215 Ser Tyr Leu He Arg 220 Thr Leu Thr Leu aac aca atg acc aag gac get gag aga ggg aaa ttg aaa ega ega gca 720 Asn 225 Thr Met Thr Lys Asp 230 Ala Glu Arg Gly Lys 235 Leu Lys Arg Arg Ala 240 ate get acc cca ggg atg cag ata aga ggg ttt gta tat ttt gtt gaa 768 lie Ala Thr Pro Gly 245 Met Gin He Arg Gly 250 Phe Val Tyr Phe Val 255 Glu aca eta gee ega aga ata tgt gaa aag ett gaa caa tea gga ttg cca 816 Thr Leu Ala Arg 260 Arg He Cys Glu Lys 265 Leu Glu Gin Ser Gly 270 Leu Pro gtt ggc ggt aat gag aaa aag gcc aaa ctg get aat gtc gtc aga aaa 864 Val Gly Gly 275 Asn Glu Lys Lys Ala 280 Lys Leu Ala Asn Val 285 Val Arg Lys atg atg act aat tcc caa gac act gaa etc tcc ttc acc ate act ggg 912 Met Met 290 Thr Asn Ser Gin Asp 295 Thr Glu Leu Ser Phe 300 Thr He Thr Gly gac aat acc aaa tgg aat gaa aat cag aac cca ege ata ttc ctg gca 960 Asp 305 Asn Thr Lys Trp Asn 310 Glu Asn Gin Asn Pro 315 Arg He Phe Leu Ala 320 atg ate aca tac ata act aga aac cag cca ( gaa tgg ttc aga aat gtt 1008 Met He Thr Tyr He 325 Thr Arg Asn Gin Pro 330 Glu Trp Phe Arg Asn 335 Val eta age att gca ccg att atg ttc tea aat aaa atg gca aga ctg ggg 1056 Leu Ser He Ala 340 Pro He Met Phe Ser 345 Asn Lys Met Ala Arg 350 Leu Gly aaa gga tat atg ttt gaa age aaa agt atg aaa ttg aga act caa ata 1104 Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gin He

   355 360 365

   cca gca gaa atg eta gca age att gac eta aaa tat ttc aat gat tea Pro Ala 370 Glu Met Leu Ala Ser ⁽375 He Asp Leu Lys Tyr 380 Phe Asn Asp Ser aca aaa aag aaa att gaa aag ata ega cca ett ctg gtt gac ggg act Thr 385 Lys Lys Lys He Glu 390 Lys He Arg Pro Leu 395 Leu Val Asp Gly Thr 400

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   get Ala tea Ser ctg Leu agt Ser cct Pro 405 ggc Gly atg atg atg gga atg ttc aac Asn atg Met ttg Leu 415 age Ser 1248 Met Met Met Gly 410 Met Phe act gtg ctg ggt gta tcc ata tta aac ctg ggc cag agg aaa tac aca 1296 Thr Val Leu Gly Val Ser He Leu Asn Leu Gly Gin Arg Lys Tyr Thr 420 425 430 aag acc aca tac tgg tgg gat ggt ctg caa tea tcc gat gac ttt get 1344 Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gin Ser Ser Asp Asp Phe Ala 435 440 445 ttg ata gtg aat geg cct aat cat gaa gga ata caa get gga gta gac 1392 Leu lie Val Asn Ala Pro Asn His Glu Gly He Gin Ala Gly Val Asp 450 455 460 aga ttc tat aga act tgc aaa ctg gtc ggg ate aac atg age aaa aag 1440 Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly He Asn Met Ser Lys Lys 465 470 475 480 aaa tcc tac ata aat aga act gga aca ttc gaa ttc aca age ttt ttc 1488 Lys Ser Tyr lie Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe 485 490 495 tac egg tat ggt ttt gta gcc aat ttc age atg gaa eta 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 tet gca gac atg age att gga gtg aca 1584 Gly Val Ser Gly He Asn Glu Ser Ala Asp Met Ser lie Gly Val Thr 515 520 525 gtc ate aaa aac aac atg ata aat aat gat etc ggt cct gcc aeg gca 1632 Val He Lys Asn Asn Met He Asn Asn Asp Leu Gly Pro Ala Thr Ala 530 535 540 caa atg gca etc caa etc ttc att aag gat tat egg tac aca tac egg 1680 Gin Met Ala Leu Gin Leu Phe He Lys Asp Tyr Arg Tyr Thr Tyr Arg 545 550 555 560 tgc cat aga ggt gat acc cag ata caa acc aga aga tet ttt gag ttg 1728 Cys His Arg Gly Asp Thr Gin He Gin Thr Arg Arg Ser Phe Glu Leu 565 570 575 aag aaa ctg tgg gaa cag act ega tea aag act ggt eta ctg gta tea 1776 Lys Lys Leu Trp Glu Gin Thr Arg Ser Lys Thr Gly Leu Leu Val Ser 580 585 590 gat ggg ggt cca aac eta tat aac ate aga aac eta cac ate ccg gaa 1824 Asp Gly Gly Pro Asn Leu Tyr Asn He Arg Asn Leu His He Pro Glu 595 600 605 gtc tgt tta aaa tgg gag eta atg gat gaa gat tat aag ggg agg eta 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 tea gtc 1920 Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu He Glu Ser Val 625 630 635 640

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   50 aac agt gca gta gta atg cct geg cat ggc cct gcc aaa age atg gag 1968 Asn Ser Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu 645 650 655 tat gat get gtt gca aca aca cat tet tgg ate ccc aag agg aac egg 2016 Tyr Asp Ala Val Ala Thr Thr His Ser Trp lie Pro Lys Arg Asn Arg 660 665 670 tcc ata ttg aac aca age caa agg gga ata etc gaa gat gag cag atg 2064 Ser lie Leu Asn Thr Ser Gin Arg Gly He Leu Glu Asp Glu Gin Met 675 680 685 tat cag aaa tgc tgc aac ctg ttt gaa aaa ttc ttc ccc age age tea 2112 Tyr Gin Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser 690 • 695 700 tac aga aga cca gtc gga att tet agt atg gtt gag gcc atg gtg tcc 2160 Tyr Arg Arg Pro Val Gly He Ser Ser Met Val Glu Ala Met Val Ser 705 710 715 720 agg gcc ege att gat gca ega att gac ttc gaa tet gga egg ata aag 2208 Arg Ala Arg He Asp Ala Arg He Asp Phe Glu Ser Gly Arg He Lys 725 730 735 aag gat gag ttc get gag ate atg aag ate tgt tcc acc att gaa gag 2256 Lys Asp Glu Phe Ala Glu lie Met Lys He Cys Ser Thr He Glu Glu 740 745 750 etc aga egg caa aaa tag 2274 Leu Arg Arg Gin 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 Gin Asn 1 5 10 15 Ala He 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 Gin 35 40 45 Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu He Gly Ala Pro 50 55 60 Gin Leu Asn Pro He Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser 65 70 75 80

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   Gly Tyr Ala Gin Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 85 90 95

   Glu Ser His Pro Gly lie Phe Glu Asn Ser Cys Leu Glu Thr Met Glu 100 105 110

   Val He Gin Gin Thr Arg Val Asp Lys Leu Thr Gin Gly Arg Gin Thr 115 120 125

   Tyr Asp Trp Thr Leu Asn Arg Asn Gin Pro Ala Ala Thr Ala Leu Ala 130 135 140

   Asn Thr He 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 He Thr Thr His Phe Gin Arg Lys Arg Arg Val Arg 180 185 190

   Asp Asn Met Thr Lys Arg Met Val Thr Gin Arg Thr He Gly Lys Lys 195 200 205

   Lys Gin Arg Leu Asn Arg Lys Ser Tyr Leu He 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

   He Ala Thr Pro Gly Met Gin He Arg Gly Phe Val Tyr Phe Val Glu 245 250 255

   Thr Leu Ala Arg Arg He Cys Glu Lys Leu Glu Gin 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 Gin Asp Thr Glu Leu Ser Phe Thr He Thr Gly 290 295 300

   Asp Asn Thr Lys Trp Asn Glu Asn Gin Asn Pro Arg He Phe Leu Ala 305 310 315 320

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   Met lie Thr Tyr lie Thr Arg Asn Gin Pro Glu Trp Phe Arg Asn Val 325 330 335

   Leu Ser He Ala Pro lie 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 Gin He 355 360 365

   Pro Ala Glu Met Leu Ala Ser He Asp Leu Lys Tyr Phe Asn Asp Ser 370 375 380

   Thr Lys Lys Lys He Glu Lys He 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 He Leu Asn Leu Gly Gin Arg Lys Tyr Thr 420 425 430

   Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gin Ser Ser Asp Asp Phe Ala 435 , 440 445

   Leu He Val Asn Ala Pro Asn His Glu Gly He Gin Ala Gly Val Asp 450 455 460

   Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly He Asn Met Ser Lys Lys 465 470 475 480

   Lys Ser Tyr He 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 He Asn Glu Ser Ala Asp Met Ser He Gly Val Thr 515 520 525

   Val He Lys Asn Asn Met He Asn Asn Asp Leu Gly Pro Ala Thr Ala 530 535 540

   Gin Met Ala Leu Gin Leu Phe He Lys Asp Tyr Arg Tyr Thr Tyr Arg 545 550 555 560

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   Cys His Arg Gly Asp Thr Gin lie Gin Thr Arg Arg Ser Phe Glu Leu 565 570 575

   Lys Lys Leu Trp Glu Gin Thr Arg Ser Lys Thr Gly Leu Leu Val Ser 580 585 590

   Asp Gly Gly Pro Asn Leu Tyr Asn He Arg Asn Leu His He 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 He 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 He Pro Lys Arg Asn Arg 660 665 670

   Ser He Leu Asn Thr Ser Gin Arg Gly He Leu Glu Asp Glu Gin Met 675 680 685

   Tyr Gin Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser 690 695 700

   Tyr Arg Arg Pro Val Gly He Ser Ser Met Val Glu Ala Met Val Ser 705 710 715 720

   Arg Ala Arg He Asp Ala Arg He Asp Phe Glu Ser Gly Arg He Lys 725 730 735

   Lys Asp Glu Phe Ala Glu He Met Lys He Cys Ser Thr He Glu Glu 740 745 ' 750

   Leu Arg Arg Gin Lys

   755 <210> 21 <211> 2151 <212> DNA <213> Influenza virus

   <220>

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   2015234384 02 Oct 2015 <221> CDS <222> (1) . . (2151) <400> 21

   atg gaa gac ttt gtg ega cag tgc ttc aat cca atg ate gtc gag ett 48 Met 1 Glu Asp Phe Val 5 Arg Gin Cys Phe Asn 10 Pro Met He Val Glu 15 Leu gcg gaa aag gca atg aaa gaa tat gga gag aac ecg aaa ate gaa aca 96 Ala Glu Lys Ala 20 Met Lys Glu Tyr Gly 25 Glu Asn Pro Lys He 30 Glu Thr aac aaa ttt gca gca ata tgc act cac ttg gaa gtc tgc ttc atg tac 144 Asn Lys Phe 35 Ala Ala lie Cys Thr 40 His Leu Glu Val Cys 45 Phe Met Tyr teg gat ttt cac ttt att aat gaa ctg ggt gag tea gtg gtc ata gag 192 Ser Asp 50 Phe His Phe lie Asn 55 Glu Leu Gly Glu Ser 60 Val Val He Glu tet ggt gac cca aat get ett ttg aaa cac aga ttt gaa ate att gag 240 Ser 65 Gly Asp Pro Asn Ala 70 Leu Leu Lys His Arg 75 Phe Glu He He Glu 80 ggg aga gat ega aca atg gca tgg aca gta gta aac age ate tgc aac 288 Gly Arg Asp Arg Thr 85 Met Ala Trp Thr Val 90 Val Asn Ser He Cys 95 Asn acc aca aga get gaa aaa cct aaa ttt ett cca gat tta tac gac tat 336 Thr Thr Arg Ala 100 Glu Lys Pro Lys Phe 105 Leu Pro Asp Leu Tyr 110 Asp Tyr aag gag aac aga ttt gtt gaa att ggt gtg aca agg aga gaa gtt cac 384 Lys Glu Asn 115 Arg Phe Val Glu He 120 Gly Val Thr Arg Arg 125 Glu Val His ata tac tac ctg gag aag gee aac aaa ata aag tet gag aaa aca cat 432 lie Tyr 13 0 Tyr Leu Glu Lys Ala 135 Asn Lys He Lys Ser 140 Glu Lys Thr His ate cac att ttc tea ttt aca gga gag gaa atg get aca aaa geg gac 480 lie 145 His lie Phe Ser Phe 150 Thr Gly Glu Glu Met 155 Ala Thr Lys Ala Asp 160 tat act ett gat gaa gag agt aga gee agg ate aag acc aga eta ttc 528 Tyr Thr Leu Asp Glu 165 Glu Ser Arg Ala Arg 170 He Lys Thr Arg Leu 175 Phe act ata aga caa gaa atg gee agt aga ggc etc tgg gat tee ttt cgt 576 Thr lie Arg Gin 180 Glu Met Ala Ser Arg 185 Gly Leu Trp Asp Ser 190 Phe Arg cag tec gag aga ggc gaa gag aca att gaa gaa aga ttt gaa ate aca 624 Gin Ser Glu 195 Arg Gly Glu Glu Thr 2*00 He Glu Glu Arg Phe 205 Glu He Thr ggg aeg atg ege aag ett gee aat tac agt etc cca ccg aac ttc tee 672 Gly Thr 210 Met Arg Lys Leu Ala 215 Asn Tyr Ser Leu Pro 220 Pro Asn Phe Ser

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   age Ser 225 ett Leu gaa Glu aat Asn ttt Phe aga Arg 230 gtc Val tat Tyr gtg Val gat Asp gga Gly 235 ttc Phe gaa Glu ccg Pro aac Asn ggc Gly 240 720 tgc att gag agt aag ett tet caa atg tcc aaa gaa gta aat gcc aga 768 Cys He Glu Ser Lys 245 Leu Ser Gin Met Ser 250 Lys Glu Val Asn Ala 255 Arg ate gaa cca ttt tea aag aca aca ccc ega cca etc aaa atg cca ggt 816 lie Glu Pro Phe 260 Ser Lys Thr Thr Pro 265 Arg Pro Leu Lys Met 270 Pro Gly ggt cca ccc tgc cat cag ega tet aaa ttc ttg eta atg gat get ctg 864 Gly Pro Pro 275 Cys His Gin Arg Ser 280 Lys Phe Leu Leu Met 285 Asp Ala Leu aaa ctg age att gag gac cca agt cac gag gga gag gga ata cca eta 912 Lys Leu 290 Ser lie Glu Asp Pro 295 Ser His Glu Gly Glu 300 Gly lie Pro Leu tat gat gca ate aaa tgc atg aaa act ttc ttt gga tgg aaa gag ccc 960 Tyr 305 Asp Ala He Lys Cys 310 Met Lys Thr Phe Phe 315 Gly Trp Lys Glu Pro 320 agt att gtt aaa cca cat gaa aag ggt ata aac ccg aac tat etc caa 1008 Ser He Val Lys Pro 325 His Glu Lys Gly He 330 Asn Pro Asn Tyr Leu 335 Gin act tgg aag caa gta tta gaa gaa ata caa gac ett gag aac gaa gaa 1056 Thr Trp Lys Gin 340 Val Leu Glu Glu He 345 Gin Asp Leu Glu Asn 350 Glu Glu agg acc ccc aag acc aag aat atg aaa aaa aca age caa ttg aaa tgg 1104 Arg Thr Pro 355 Lys Thr Lys Asn Met 360 Lys Lys Thr Ser Gin 365 Leu Lys Trp gca eta ggt gaa aat atg gca cca gag aaa gtg gat ttt gag gat tgt 1152 Ala Leu 370 Gly Glu Asn Met Ala 375 Pro Glu Lys Val Asp 380 Phe Glu Asp Cys aaa gac ate aat gat tta aaa caa tat gac agt gat gag cca gaa aca 1200 Lys 385 Asp He Asn Asp Leu 390 Lys Gin Tyr Asp Ser 395 Asp Glu Pro Glu Thr 400 agg tet ett gca agt tgg att caa agt gag ttc aac aaa get tgt gag 1248 Arg Ser Leu Ala Ser 405 Trp He Gin Ser Glu 410 Phe Asn Lys Ala Cys 415 Glu ctg aca gat tea age tgg ata gag etc gat gaa att ggg gag gat gtc 1296 Leu Thr Asp Ser 420 Ser Trp He Glu Leu 425 Asp Glu He Gly Glu 430 Asp Val gcc cca ata gaa tac att geg age atg agg aga aat tat ttt act get 1344 Ala Pro He 435 Glu Tyr He Ala Ser 440 Met Arg Arg Asn Tyr 445 Phe Thr Ala gag att tee cat tgt aga gca aca gaa tat ata atg aaa gga gtg tac 1392 Glu He Ser His Cys Arg Ala Thr Glu Tyr He Met Lys Gly Val Tyr

   450 455 460

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   ate He 465 aac Asn act Thr get Ala eta Leu etc Leu 470 aat Asn gca Ala tcc Ser tgt Cys get Ala 475 geg Ala atg Met gat Asp gaa Glu ttt Phe 480 1440 caa tta att ccg atg ata agt aaa tgc agg acc aaa gaa ggg aga agg 1488 Gin Leu He Pro Met He 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 He lie Lys Gly Arg Ser His Leu Arg 500 505 510 aat gat act gac gtg gtg aac ttt gta agt atg gaa ttt tot etc 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 eta 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 eta aga act get gta ggt caa gtg tea aga ccc 1680 He Gly Asp Met Leu Leu Arg Thr Ala Val Gly Gin Val Ser Arg Pro 545 550 555 560 atg ttt ttg tat gta agg aca aat gga acc tet aaa att aaa atg aaa 1728 Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys He Lys Met Lys 565 570 575 tgg gga atg gaa atg agg ege tgc etc ctt cag tet ctg caa cag att 1776 Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gin Ser Leu Gin Gin He 580 585 590 gaa age atg ate gaa get gag tcc tea gtc aaa gaa aag gac atg acc 1824 Glu Ser Met He Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr 595 600 605 aaa gaa ttt ttt gag aac aaa tea gag aca tgg cct ata gga gag tcc 1872 Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro He Gly Glu Ser 610 615 620 ccc aaa gga gtg gaa gag ggc tea ate ggg aag gtt tgc agg acc tta 1920 Pro Lys Gly Val Glu Glu Gly Ser He Gly Lys Val Cys Arg Thr Leu 625 630 635 640 tta gca aaa tot gtg ttt aac agt tta tat gca tet cca caa ctg gaa 1968 Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gin Leu Glu

   645 650 655 ggg ttt tea get gaa tet agg aaa tta ctt etc att gtt cag get ctt 2016

   Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu lie Val Gin Ala Leu

   660 665 670

   agg Arg gat gac Asp Asp 675 ctg gaa Leu Glu cct Pro gga Gly acc Thr 680 ttt Phe gat Asp att lie ggg Gly ggg Gly 685 tta Leu ) tat Tyr gaa Glu 2064 tea att gag gag tgc ctg att aat gat ccc tgg gtt ttg ctt aat gca 2112 Ser He Glu Glu Cys Leu He Asn Asp Pro Trp Val Leu Leu Asn Ala 690 695 700

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   tct tgg ttc aac tcc ttc ctt aca cat gca ctg aag tag 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 Gin Cys Phe Asn Pro Met He Val Glu Leu 1 5 10 15 Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asn Pro Lys He Glu Thr 20 25 30 Asn Lys Phe Ala Ala lie Cys Thr His Leu Glu Val Cys Phe Met Tyr 35 40 45 Ser Asp Phe His Phe lie Asn Glu Leu Gly Glu Ser Val Val He Glu 50 55 60 Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu lie He Glu 65 70 75 80 Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser lie 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 He Gly Val Thr Arg Arg Glu Val His 115 120 125 lie Tyr Tyr Leu Glu Lys Ala Asn Lys lie Lys Ser Glu Lys Thr His 130 135 140 lie His He 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 He Lys Thr Arg Leu Phe 165 170 175 Thr He Arg Gin Glu Met Ala Ser Arg Gly Leu Trp Asp Ser Phe Arg

   180

   185

   190

   2151

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   Gin Ser Glu Arg Gly Glu Glu Thr lie Glu Glu Arg Phe Glu lie 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 He Glu Ser Lys Leu Ser Gin Met Ser Lys Glu Val Asn Ala Arg 245 250 255

   He Glu Pro Phe Ser Lys Thr Thr Pro Arg Pro Leu Lys Met Pro Gly 260 ’ 265 270

   Gly Pro Pro Cys His Gin Arg Ser Lys Phe Leu Leu Met Asp Ala Leu 275 280 285

   Lys Leu Ser He Glu Asp Pro Ser His Glu Gly Glu Gly He Pro Leu 290 295 300

   Tyr Asp Ala He Lys Cys Met Lys Thr Phe Phe Gly Trp Lys Glu Pro 305 310 315 320

   Ser He Val Lys Pro His Glu Lys Gly He Asn Pro Asn Tyr Leu Gin 325 330 335

   Thr Trp Lys Gin Val Leu Glu Glu He Gin Asp Leu Glu Asn Glu Glu 340 345 350

   Arg Thr Pro Lys Thr Lys Asn Met Lys Lys Thr Ser Gin 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 lie Asn Asp Leu Lys Gin Tyr Asp Ser Asp Glu Pro Glu Thr 385 390 395 400

   Arg Ser Leu Ala Ser Trp He Gin Ser Glu Phe Asn Lys Ala Cys Glu 405 410 415

   Leu Thr Asp Ser Ser Trp He Glu Leu Asp Glu He Gly Glu Asp Val 420 425 430

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   Ala Pro lie Glu Tyr He Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala 435 440 445

   Glu He Ser His Cys Arg Ala Thr Glu Tyr He Met Lys Gly Val Tyr 450 455 460

   He Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Glu Phe 465 470 475 480

   Gin Leu He Pro Met He Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg 485 490 495

   Lys Thr Asn Leu Tyr Gly Phe He He 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

   He Gly Asp Met Leu Leu Arg Thr Ala Val Gly Gin Val Ser Arg Pro 545 550 555 560

   Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys He Lys Met Lys 565 570 575

   Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gin Ser Leu Gin Gin He 580 585 590

   Glu Ser Met He 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 He Gly Glu Ser 610 615 620

   Pro Lys Gly Val Glu Glu Gly Ser He Gly Lys Val Cys Arg Thr Leu 625 630 635 640

   Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gin Leu Glu 645 . 650 655

   Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu He Val Gin Ala Leu 660 665 670

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   Arg Asp Asp Leu Glu Pro Gly Thr Phe Asp He Gly Gly Leu Tyr Glu 675 680 685 Ser lie Glu Glu Cys Leu He 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> : InfluenzE i virus <220> <221> CDS <222> (1) - · . (657) <400> 23 atg gat tcc aac act gtg tea age ttt cag gta gac tgt ttt ett tgg 48 Met Asp Ser Asn Thr Val Ser Ser Phe Gin Val Asp Cys Phe Leu Trp 1 5 10 15 cat gtc ege aaa ega ttc gca gac caa gaa ctg ggt gat gcc cca ttc 96 His Val Arg Lys Arg Phe Ala Asp Gin Glu Leu Gly Asp Ala Pro Phe 20 25 30 ett gac egg ett ege ega gac cag aag tcc eta agg gga aga ggt age 144 Leu Asp Arg Leu Arg Arg Asp Gin Lys Ser Leu Arg Gly Arg Gly Ser 35 40 45 act ett ggt ctg gac ate gaa aca gcc act cat gca gga aag cag ata 192 Thr Leu Gly Leu Asp He Glu Thr Ala Thr His Ala Gly Lys Gin He 50 55 60 gtg gag cag att ctg gaa aag gaa tea gat gag gca ett aaa atg acc 240 Val Glu Gin He Leu Glu Lys Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80 att gcc tet gtt cct act tea ege tac tta act gac atg act ett gat 288 lie Ala Ser Val Pro Thr Ser Arg Tyr Leu Thr Asp Met Thr Leu Asp 85 90 95 gag atg tea aga gac tgg ttc atg etc atg ccc aag caa aaa gta aca 336 Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gin Lys Val Thr 100 105 110 ggc tcc eta tgt ata aga atg gac cag gca ate atg gat aag aac ate 384 Gly Ser Leu Cys He Arg Met Asp Gin Ala He Met Asp Lys Asn He 115 120 125 ata ett aaa gca aac ttt agt gtg att ttc gaa ggg ctg gaa aca eta 432 He Leu Lys Ala Asn Phe Ser Val He Phe Glu Gly Leu Glu Thr Leu 130 135 140 ata eta ett aga gcc ttc acc gaa gaa gga gca gtc gtt ggc gaa att 480

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   He Leu Leu 145 Arg Ala Phe 150 61 Thr Glu Glu Gly Ala Val 155 Val Gly Glu lie 160 tea cca tta cct tet 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 etc ate gga gga ctt aaa tgg aat gat aat acg gtt 576 Ala He Gly Val Leu He Gly Gly Leu Lys Trp Asn Asp Asn Thr Val 180 185 190 aga ate tet gaa act eta cag aga ttc get tgg aga age agt cat gag 624 Arg He Ser Glu Thr Leu Gin Arg Phe Ala Trp Arg Ser Ser His Glu 195 200 205 aat ggg aga cct tea ttc cct tea aag cag aaa tgaaaaatgg agagaacaat 677 Asn Gly Arg Pro Ser Phe Pro Ser Lys Gin Lys 210 215

   taagccagaa atttgaagaa ataagatggt tgattgaaga agtgcgacat agattgaaaa 737 atacagaaaa tagttttgaa caaataacat ttatgeaage cttacaacta ttgettgaag 797 tagaacaaga gataagaact ttctcgtttc agettattta a 838

   <210> 24 <211> 219 <212> PRT <213> Influenza virus <400> 24

   Met Asp Ser Asn Thr Val Ser Ser Phe Gin Val Asp Cys Phe Leu Trp 15 10 15

   His Val Arg Lys Arg Phe Ala Asp Gin Glu Leu Gly Asp Ala Pro Phe 20 25 30

   Leu Asp Arg Leu Arg Arg Asp Gin Lys Ser Leu Arg Gly Arg Gly Ser 35 40 45

   Thr Leu Gly Leu Asp lie Glu Thr Ala Thr His Ala Gly Lys Gin He 50 55 ' 60

   Val Glu Gin He Leu Glu Lys Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80

   He 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 Gin Lys Val Thr 100 105 110

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   Gly Ser Leu Cys He Arg Met Asp 120 Gin Ala lie Met Asp 125 Lys Asn lie 115 lie Leu Lys Ala Asn Phe Ser Val He Phe Glu Gly Leu Glu Thr Leu 13 0 135 140 lie Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu He 145 150 155 160 Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn 165 170 175 Ala lie Gly Val Leu He Gly Gly Leu Lys Trp Asn Asp Asn Thr Val 180 185 190 Arg He Ser Glu Thr Leu Gin Arg Phe Ala Trp Arg Ser Ser His Glu 195 200 205 Asn Gly Arg Pro Ser Phe Pro Ser Lys Gin Lys

   210 215 <210> 25

   <2ii> : 1497 <212> DNA <213> : Influenza virus <220> <221> CDS <222> (1) - · .(1497) <4oo> : 25 atg geg tet caa ggc acc aaa ega tcc tat gaa cag atg gaa act gat 48 Met Ala Ser Gin Gly Thr Lys Arg Ser Tyr Glu Gin Met Glu Thr Asp 1 5 10 15 ggg gaa ege cag aat gca act gaa ate aga gca tet gtc gga agg atg 96 Gly Glu Arg Gin Asn Ala Thr Glu He Arg Ala Ser Val Gly Arg Met 20 25 30 gtg gga gga ate ggc egg ttt tat gtt cag atg tgt act gag ett aaa 144 Val Gly Gly He Gly Arg Phe Tyr Val Gin Met Cys Thr Glu Leu Lys 35 40 45 eta aac gac cat gaa ggg egg ctg att cag aac age ata aca ata gaa 192 Leu Asn Asp His Glu Gly Arg Leu He Gin Asn Ser He Thr He Glu 50 55 60 agg atg gta ett teg gca ttc gac gaa aga aga aac aag tat etc gag 240 Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu 65 70 75 80

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   gag Glu cat His ccc Pro agt Ser get Ala 85 ggg aaa Gly Lys gac Asp cct Pro aag Lys 90 aaa aeg gga ggc ccg Pro 95 ata He 288 Lys Thr Gly Gly tac aga agg aaa gat ggg aaa tgg atg agg gaa etc ate etc cat gat 336 Tyr Arg Arg Lys Asp Gly Lys Trp Met Arg Glu Leu He Leu His Asp 100 105 110 aaa gaa gaa ate atg aga ate tgg cgt cag gcc aac aat ggt gaa gac 384 Lys Glu Glu He Met Arg He Trp Arg Gin Ala Asn Asn Gly Glu Asp 115 120 125 get act get ggt ett act cat atg atg ate tgg cac tec aat etc aat 432 Ala Thr Ala Gly Leu Thr His Met Met He Trp His Ser Asn Leu Asn 13 0 135 140 gac acc aca tac caa aga aca agg get ett gtt egg act ggg atg gat 480 Asp Thr Thr Tyr Gin Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp 145 150 155 160 ccc aga atg tgc tet ctg atg caa ggc tea acc etc cca egg aga tet 528 Pro Arg Met Cys Ser Leu Met Gin Gly Ser Thr Leu Pro Arg Arg Ser 165 170 175 gga gcc get ggt get 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 etc ate aga atg ate aaa cgc gga ata aat gat egg aat ttc tgg aga 624 Leu lie Arg Met He Lys Arg Gly lie Asn Asp Arg Asn Phe Trp Arg 195 200 205 ggt gaa aat ggt ega aga acc aga att get tat gaa aga atg tgc aat 672 Gly Glu Asn Gly Arg Arg Thr Arg He Ala Tyr Glu Arg Met Cys Asn 210 215 220 ate etc aaa ggg aaa ttt cag aca gca gca caa egg get atg atg gac 720 lie Leu Lys Gly Lys Phe Gin Thr Ala Ala Gin Arg Ala Met Met Asp 225 230 235 240 cag gtg agg gaa ggc cgc aat cct gga aac get gag att gag gat etc 768 Gin Val Arg Glu Gly Arg Asn Pro Gly Asn Ala Glu He Glu Asp Leu 245 250 255 1 att ttc ttg gca ega tea gca ett att ttg aga gga tea gta gcc cat 816 He Phe Leu Ala Arg Ser Ala Leu He Leu Arg Gly Ser Val Ala His 260 265 270 aaa tea tgc eta cct gee tgt gtt tat ggc ett 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 tet ctg gtt gga att gat cct ttc 912 Tyr Asp Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly He Asp Pro Phe 290 295 300 ( aaa eta etc cag aac agt caa att ttc agt eta ate aga cca aaa gaa 960 Lys Leu Leu Gin Asn Ser Gin He Phe Ser Leu He Arg Pro Lys Glu 305 310 315 320

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   aac Asn cca Pro gca Ala cac His aag Lys 325 age Ser cag Gin ttg Leu gtg Val tgg Trp 330 atg Met gca Ala tgc Cys cat His tct Ser 335 gca Ala 1008 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 He Arg Gly Thr Lys Val 340 345 350 ate cca aga gga cag tta aca acc aga gga gtt caa att get tea aat 1104 lie Pro Arg Gly Gin Leu Thr Thr Arg Gly Val Gin He Ala Ser Asn 355 360 365 gaa aac atg gag aca ata gat tct age aca ett gaa ctg aga age aaa 1152 Glu Asn Met Glu Thr He Asp Ser Ser Thr Leu Glu Leu Arg Ser Lys 370 375 380 tat tgg gca ata agg acc aga age gga gga aac acc agt caa cag aga 1200 Tyr Trp Ala He Arg Thr Arg Ser Gly Gly Asn Thr Ser Gin Gin Arg 385 390 395 400 gca tct gca gga cag ata agt gtg caa cct act ttc tea gta cag aga 1248 Ala Ser Ala Gly Gin He Ser Val Gin Pro Thr Phe Ser Val Gin Arg 405 410 415 aat ett ccc ttt gag aga gca acc att atg get gca ttc act ggt aac 1296 Asn Leu Pro Phe Glu Arg Ala Thr He Met Ala Ala Phe Thr Gly Asn 420 425 430 act gaa ggg agg act tcc gac atg aga aeg gaa ate ata agg atg atg 1344 Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu He He Arg Met Met 435 440 445 gaa aat gcc aaa tea gaa gat gtg tct ttc cag ggg egg gga gtc ttc 1392 Glu Asn Ala Lys Ser Glu Asp Val Ser Phe Gin Gly Arg Gly Val Phe 450 455 460 gag etc teg gac gaa aag gca aeg aac ccg ate gtg cct tcc ttt gac 1440 Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro He Val Pro Ser Phe Asp 465 470 475 480 atg age aat gaa ggg tct tat ttc ttc gga gac aat get 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 Gin Gly Thr Lys Arg Ser Tyr Glu Gin Met Glu Thr Asp 1 5 10 15

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   Gly Glu Arg Gin Asn Ala Thr Glu lie Arg Ala Ser Val Gly Arg Met 20 25 30

   Val Gly Gly He Gly Arg Phe Tyr Val Gin Met Cys Thr Glu Leu Lys 35 40 45

   Leu Asn Asp His Glu Gly Arg Leu He Gin Asn Ser He Thr He 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 He 85 90 95

   Tyr Arg Arg Lys Asp Gly Lys Trp Met Arg Glu Leu He Leu His Asp 100 105 110

   Lys Glu Glu He Met Arg He Trp Arg Gin Ala Asn Asn Gly Glu Asp 115 120 125

   Ala Thr Ala Gly Leu Thr His Met Met He Trp His Ser Asn Leu Asn 130 135 140

   Asp Thr Thr Tyr Gin Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp 145 150 155 160

   Pro Arg Met Cys Ser Leu Met Gin 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 He Arg Met He Lys Arg Gly He Asn Asp Arg Asn Phe Trp Arg 195 200 205

   Gly Glu Asn Gly Arg Arg Thr Arg He Ala Tyr Glu Arg Met Cys Asn 210 215 220

   He Leu Lys Gly Lys Phe Gin Thr Ala Ala Gin Arg Ala Met Met Asp 225 230 235 240

   Gin Val Arg Glu Gly Arg Asn Pro Gly Asn Ala Glu He Glu Asp Leu 245 250 255

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   Lys Ser Cys Leu Pro Ala Cys Val 275 280

   Tyr Asp Phe Glu Lys Glu Gly Tyr 290 295

   Lys Leu Leu Gin Asn Ser Gin He 305 310

   Asn Pro Ala His Lys Ser Gin Leu 325

   He Leu Arg Gly Ser Val Ala His 265 270

   Tyr Gly Leu Ala Val Thr Ser Gly 285

   Ser Leu Val Gly He Asp Pro Phe 300

   Phe Ser Leu He Arg Pro Lys Glu 315 320

   Val Trp Met Ala Cys His Ser Ala 330 335

   Ala Phe Glu Asp Leu Arg Val Leu 340

   Asn Phe He Arg Gly Thr Lys Val 345 350

   He Pro Arg Gly Gin Leu Thr Thr 355 360

   Arg Gly Val Gin He Ala Ser Asn 365

   Glu Asn Met Glu Thr He Asp Ser 370 375

   Ser Thr Leu Glu Leu Arg Ser Lys 380

   Tyr Trp Ala He Arg Thr Arg Ser 385 390

   Gly Gly Asn Thr Ser Gin Gin Arg 395 400

   Ala Ser Ala Gly Gin lie Ser Val 405

   Gin Pro Thr Phe Ser Val Gin Arg 410 415

   Asn Leu Pro Phe Glu Arg Ala Thr 420

   He Met Ala Ala Phe Thr Gly Asn 425 430

   Thr Glu Gly Arg Thr Ser Asp Met 435 440

   Arg Thr Glu He He Arg Met Met 445

   Glu Asn Ala Lys Ser Glu Asp Val 450 455

   Ser Phe Gin Gly Arg Gly Val Phe 460

   Glu Leu Ser Asp Glu Lys Ala Thr 465 470

   Asn Pro He Val Pro Ser Phe Asp 475 480

   Met Ser Asn Glu Gly Ser Tyr Phe 485

   Phe Gly Asp Asn Ala Glu Glu Phe 490 495

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   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 Met 1 Asn Pro Asn Gin 5 Lys He He Thr ata tta ate att aat gtc att etc cat He Leu He He 20 Asn Val lie Leu His 25 gta ctg gtc etc aat aac aat aga aca Val Leu Val 35 Leu Asn Asn Asn Arg 40 Thr ate ata aga gag tac aat gaa aca gta He He 50 Arg Glu Tyr Asn Glu 55 Thr Val tgg tat aat acc agt aca att aag tac Trp 65 Tyr Asn Thr Ser Thr 70 He Lys Tyr tac tac atg aac aac act gaa cca ett Tyr Tyr Met Asn Asn 85 Thr Glu Pro Leu cca ttt tcc aaa gat aat gga ata cga Pro Phe Ser Lys 100 Asp Asn Gly He Arg 105 ttt gtg ata aga gaa cct ttt gta tea Phe Val He 115 Arg Glu Pro Phe Val 12 0 Ser acc ttt ttc etc aca cag ggc tea tta Thr Phe 130 Phe Leu Thr Gin Gly 135 Ser Leu ggc aca gta aag gac cga agt ccg tat Gly 145 Thr Val Lys Asp Arg 150 Ser Pro Tyr ata ggg caa tea cct aat gta tat caa He Gly Gin Ser Pro 165 Asn Val Tyr Gin tgg tea gca aca gca tgc cat gat gga

   att gga ttt gca tea ttg ggg 48 lie Gly Phe Ala Ser Leu Gly 10 15 gta gtc age att ata gta aca 96

   Val Val Ser He He Val Thr gat ctg aac tgc aaa ggg aeg 144

   Asp Leu Asn Cys Lys Gly Thr aga gta gaa aaa att act caa 192

   Arg Val Glu Lys He Thr Gin ata gag aga cct tea aat gaa 240

   He Glu Arg Pro Ser Asn Glu

   75 80 tgt gag gcc caa ggc ttt gca 288

   Cys Glu Ala Gin Gly Phe Ala 90 95 att ggg teg aga ggc cat gtt 336

   He Gly Ser Arg Gly His Val

   110 tgt teg ccc tea gaa tgt aga 384

   Cys Ser Pro Ser Glu Cys Arg

   125 etc aat gac aaa cat tet aac 432

   Leu Asn Asp Lys His Ser Asn

   140 agg act ttg atg agt gtc aaa 480

   Arg Thr Leu Met Ser Val Lys

   155 160 get agg ttt gaa teg gtg gca 528

   Ala Arg Phe Glu Ser Val Ala 170 175 aaa aaa tgg atg aca gtt gga 576

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   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. Gin Ala lie Ala Val Val Asn Tyr Gly Gly

   195 200 205 gtt ccg gtt gat att att aat tea tgg gca ggg gat att tta aga acc 672

   Val Pro Val Asp He He Asn Ser Trp Ala Gly Asp He Leu Arg Thr

   210 215 220 caa gaa tea tea tgc acc tgc att aaa gga gac tgt tat tgg gta atg 720

   Gin Glu Ser Ser Cys Thr Cys He Lys Gly Asp Cys Tyr Trp Val Met

   225 230 235 240 act gat gga ccg gca aat agg caa get aaa tat agg ata ttc aaa gca 768

   Thr Asp Gly Pro Ala Asn Arg Gin Ala Lys Tyr Arg He 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 He Gly Gin Thr Asp He Ser Phe Asn Gly Gly

   260 265 270 cac ata gag gag tgt tet tgt tac ccc aat gaa ggg aag gtg gaa tgc 864

   His He 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

   He Cys Arg Asp Asn Trp Thr Gly Thr Asn Arg Pro He Leu Val He

   290 295 300 tet tet gat eta teg tac aca gtt gga tat ttg tgt get ggc att ccc 960

   Ser Ser Asp Leu Ser Tyr Thr Val Gly Tyr Leu Cys Ala Gly He Pro

   305 310 315 320 act gac act cct agg gga gag gat agt caa ttc aca ggc tea tgt aca 1008

   Thr Asp Thr Pro Arg. Gly Glu Asp Ser Gin Phe Thr Gly Ser Cys Thr

   325 330 335 agt cct ttg gga aat aaa gga tac ggt gta aaa ggt ttc ggg ttt ega 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 tea aga 1104

   Gin Gly Thr Asp Val Trp Ala Gly Arg Thr He Ser Arg Thr Ser Arg

   355 360 365 tea gga ttc gaa ata ata aaa ate agg aat ggt tgg aca cag aac agt 1152

   Ser Gly Phe Glu He He Lys He Arg Asn Gly Trp Thr Gin Asn Ser

   370 . 375 I 380 aaa gac caa ate agg agg caa gtg att ate gat gac cca aat tgg tea 1200

   Lys Asp Gin He Arg Arg Gin Val He He Asp Asp Pro Asn Trp Ser

   385 390 395 400 gga tat age ggt tet ttc aca ttg ccg gtt gaa eta 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

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   Cys Leu Val Pro 420 Cys Phe Trp Val Glu 425 Met He Arg Gly Lys 430 Pro Glu gaa aca aca ata tgg acc tet age age tcc att gtg atg tgt gga gta Glu Thr Thr He Trp Thr Ser Ser Ser Ser He Val Met Cys Gly Val 435 440 445 gat cat aaa att gcc agt tgg tea tgg cac gat gga get att ett ccc Asp His Lys He Ala Ser Trp Ser Trp His Asp Gly Ala He Leu Pro 450 455 460 ttt gac ate gat aag atg Phe Asp lie Asp Lys Met

   465 470 <210> 28 <211> 470 <212> PRT

   <213> Influenza virus <400> 28 Met 1 Asn Pro Asn Gin 5 Lys He lie Thr lie 10 Gly Phe Ala Ser Leu 15 Gly lie Leu lie lie 20 Asn Val He Leu His 25 Val Val Ser He He 30 Val Thr Val Leu Val 35 Leu Asn Asn Asn Arg 40 Thr Asp Leu Asn Cys 45 Lys Gly Thr He lie 50 Arg Glu Tyr Asn Glu 55 Thr Val Arg Val Glu 60 Lys He Thr Gin Trp 65 1 Tyr Asn Thr Ser Thr 70 lie Lys Tyr He Glu 75 Arg Pro Ser Asn Glu 80 Tyr Tyr Met Asn Asn 85 Thr Glu Pro Leu Cys 90 Glu Ala Gin Gly Phe 95 Ala Pro Phe Ser Lys 100 Asp Asn Gly lie Arg 105 He Gly Ser Arg Gly 110 His Val Phe Val He 115 Arg Glu Pro Phe Val 120 Ser Cys Ser Pro Ser 125 Glu Cys Arg Thr Phe 130 Phe Leu Thr Gin Gly 135 Ser Leu Leu Asn Asp 140 Lys His Ser Asn

   Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser Val Lys

   1344

   1392

   1410

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   145 150 155 160 lie Gly Gin Ser Pro Asn Val Tyr Gin 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 Gin Ala He Ala Val Val Asn Tyr Gly Gly 195 200 205

   Val Pro Val Asp He He Asn Ser Trp Ala Gly Asp He Leu Arg Thr 210 215 220

   Gin Glu Ser Ser Cys Thr Cys He Lys Gly Asp Cys Tyr Trp Val Met 225 230 235 240

   Thr Asp Gly Pro Ala Asn Arg Gin Ala Lys Tyr Arg He Phe Lys Ala 245 250 255

   Lys Asp Gly Arg Val He Gly Gin Thr Asp He Ser Phe Asn Gly Gly 260 265 270

   His He Glu Glu Cys Ser Cys Tyr Pro Asn Glu Gly Lys Val Glu Cys 275 280 285 lie Cys Arg Asp Asn Trp Thr Gly Thr Asn Arg Pro He Leu Val lie 290 295 300

   Ser Ser Asp Leu Ser Tyr Thr Val Gly Tyr Leu Cys Ala Gly He Pro 305 310 315 320

   Thr Asp Thr Pro Arg Gly Glu Asp Ser Gin 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

   Gin Gly Thr Asp Val Trp Ala Gly Arg Thr He Ser Arg Thr Ser Arg 355 360 365

   Ser Gly Phe Glu lie He Lys He Arg Asn Gly Trp Thr Gin Asn Ser 370 375 380

   Lys Asp Gin He Arg Arg Gin Val He lie Asp Asp Pro Asn Trp Ser

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   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 lie Arg Gly Lys Pro Glu 420 425 430

   Glu Thr Thr He Trp Thr Ser Ser Ser Ser He Val Met Cys Gly Val 435 440 445

   Asp His Lys He Ala Ser Trp Ser Trp His Asp Gly Ala He Leu Pro 450 455 460

   Phe Asp He 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 eta acc gag gtc gaa acg tac gtt etc tet ate gta cca

   Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser He Val Pro

   15 10 15 tea ggc ccc etc aaa gcc gag ate geg cag aga ctt gaa gat gtc ttt

   Ser Gly Pro 'Leu Lys Ala Glu lie Ala Gin Arg Leu Glu Asp Val Phe

   20 25 30 gca ggg aag aac acc gat ctt gag gca etc atg gaa tgg eta aag aca

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

   35 40 45 aga cca ate ctg tea cct ctg act aaa ggg att tta gga ttt gta ttc

   Arg Pro He Leu Ser Pro Leu Thr Lys Gly He Leu Gly Phe Val Phe

   50 55 60 acg etc acc gtg ccc agt gag ega gga ctg cag cgt aga cgc ttt gtc

   Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gin 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

   Gin 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

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

   144

   192

   240 /

   288

   336

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   100 105 110

   aaa Lys gag Glu gtg gca Val Ala 115 etc Leu age Ser tat Tyr tcc Ser 120 act Thr ggt Gly gca Ala eta Leu gcc Ala 125 age Ser tgc Cys atg Met 384 gga etc ata tac aac aga atg gga act gtt aca acc gaa gtg gca ttt 432 Gly Leu He Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe 13 0 135 140 ggc ctg gta tgc gcc aca tgt gaa cag att get gat tcc cag cat ega 480 Gly Leu Val Cys Ala Thr Cys Glu Gin He Ala Asp Ser Gin His Arg 145 150 155 160 tet cac agg cag atg gtg aca aca acc aac cca tta ate aga cat gaa 528 Ser His Arg Gin Met Val Thr Thr Thr Asn Pro Leu He Arg His Glu 165 170 175 aac aga atg gta tta gcc agt acc aeg get aaa gcc atg gaa cag atg 576 Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gin Met 180 185 190 gca gga teg agt gag cag gca gca gag gcc atg gag gtt get agt agg 624 Ala Gly Ser Ser Glu Gin Ala Ala Glu Ala Met Glu Val Ala Ser Arg 195 200 205 get agg cag atg gta cag gca atg aga acc att ggg acc cac cct age 672 Ala Arg Gin Met Val Gin Ala Met Arg Thr lie Gly Thr His Pro Ser 210 215 220 tcc agt gcc ggt ttg aaa gat gat etc ett gaa aat tta cag gcc tac 720 Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gin Ala Tyr 225 230 235 240 cag aaa egg atg gga gtg caa atg cag ega ttc aag tgatcctctc 766 Gin Lys Arg Met Gly Val Gin Met Gin Arg Phe Lys 245 250

   gttattgcag caagtatcat tgggatcttg cacttgatat tgtggattct tgategtett 826 ttcttcaaat teatttateg tegeettaaa tacgggttga aaagagggcc ttctacggaa 886 ggagtacctg agtetatgag ggaagaatat cggcaggaac ageagaatge 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 lie Val Pro 1 5 10 15

   Ser Gly Pro Leu Lys Ala Glu He Ala Gin Arg Leu Glu Asp Val Phe 20 25 30

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   Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr 35 40 45

   Arg Pro lie Leu Ser Pro Leu Thr Lys Gly He Leu Gly Phe Val Phe 50 55 60

   Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gin Arg Arg Arg Phe Val 65 70 75 80

   Glh 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 He 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 He Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe 130 135 140

   Gly Leu Val Cys Ala Thr Cys Glu Gin He Ala Asp' Ser Gin His Arg 145 150 155 160

   Ser His Arg Gin Met Val Thr Thr Thr Asn Pro Leu He Arg His Glu 165 170 175

   Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gin Met 180 185 190

   Ala Gly Ser Ser Glu Gin Ala Ala Glu Ala Met Glu Val Ala Ser Arg 195 200 205

   Ala Arg Gin Met Val Gin Ala Met Arg Thr He Gly Thr His Pro Ser 210 215 220

   Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gin Ala Tyr 225 230 235 240

   Gin Lys Arg Met Gly Val Gin Met Gin Arg Phe Lys 245 250 <210> 31 <211> 1698

   WO 2007/047938

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   2015234384 02 Oct 2015 <212 > DNA <213> Influenza virus <220>

   <221> CDS <222> (1) . . (1698) <400> 31

   atg aag aca acc att att ttg ata eta ctg acc cat tgg gcc tac agt Met 1 Lys Thr Thr He 5 He Leu He Leu Leu 10 Thr His Trp Ala Tyr 15 Ser caa aac cca ate agt gac aac aac aca gcc aca ctg tgt ctg gga cac Gin Asn Pro He 20 Ser Asp Asn Asn Thr 25 Ala Thr Leu Cys Leu 30 Gly His cat gca gta gca aat gga aca ttg gta aaa aca ata agt gat gat caa His Ala Val 35 Ala Asn Gly Thr Leu 40 Val Lys Thr He Ser 45 Asp Asp Gin att gag gtg aca aat get aca gaa tta gtt cag age att tea atg ggg lie Glu 50 Val Thr Asn Ala Thr 55 Glu Leu Val Gin Ser 60 He Ser Met Gly aaa ata tgc aac aaa tea tat aga att eta gat gga aga aat tgc aca Lys 65 He Cys Asn Lys Ser 70 Tyr Arg He Leu Asp 75 Gly Arg Asn Cys Thr 80 tta ata gat gca atg eta gga gac ccc cac tgt gac gcc ttt cag tat Leu He Asp Ala Met 85 Leu Gly Asp Pro His 90 Cys Asp Ala Phe Gin 95 Tyr gag agt tgg gac etc ttt ata gaa aga age age get ttc age aat tgc Glu Ser Trp Asp 100 Leu Phe He Glu Arg 105 Ser Ser Ala Phe Ser 110 Asn Cys tac cca tat gac ate cct gac tat gca teg etc ega tcc att gta gca Tyr Pro Tyr 115 Asp He Pro Asp Tyr 120 Ala Ser Leu Arg Ser 125 He Val Ala tcc tea gga aca ttg gaa ttc aca gca gag gga ttc aca tgg aca ggt Ser Ser 130 Gly Thr Leu Glu Phe 135 Thr Ala Glu Gly Phe 140 Thr Trp Thr Gly gtc act caa aac gga aga agt gga gcc tgc aaa agg gga tea gcc gat Val 145 Thr Gin Asn Gly Arg 150 Ser Gly Ala Cys Lys 155 Arg Gly Ser Ala Asp 160 agt ttc ttt age ega ctg aat tgg eta aca aaa tet gga age tet tac Ser Phe Phe Ser Arg 165 Leu Asn Trp Leu Thr 170 Lys Ser Gly Ser Ser 175 Tyr ccc aca ttg aat gtg aca atg cct aac aat aaa aat ttc gac aag eta Pro Thr Leu Asn 180 Val Thr Met Pro Asn 185 Asn Lys Asn Phe Asp 190 Lys Leu tac ate tgg ggg att cat cac ccg age tea aat caa gag cag aca aaa Tyr He Trp 195 Gly lie His His Pro 200 Ser Ser Asn Gin Glu 205 Gin Thr Lys

   144

   192

   240

   288

   336

   384

   432

   480

   528

   576

   624

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   ttg tac Leu Tyr ate He caa gaa tea Ser gga Gly 215 ega Arg gta aca gtc Val tea Ser 220 aca Thr aaa Lys aga Arg agt Ser 672 Gin Glu Val Thr 210 caa caa aca ata ate cct aac ate gga tet aga ccg ttg gtc aga ggt 720 Gin 225 Gin Thr He He Pro 230 Asn He Gly Ser Arg 235 Pro Leu Val Arg Gly 240 caa tea ggc agg ata age ata tac tgg acc att gta aaa cct gga gat 768 Gin Ser Gly Arg He 245 Ser lie Tyr Trp Thr 250 He Val Lys Pro Gly 255 Asp ate eta atg ata aac agt aat ggc aac tta gtt gca ccg egg gga tat 816 lie Leu Met lie 260 Asn Ser Asn Gly Asn 265 Leu Val Ala Pro Arg 270 Gly Tyr ttt aaa ttg aaa aca ggg aaa age tet gta atg aga tea gat gta ccc 864 Phe Lys Leu 275 Lys Thr Gly Lys Ser 280 Ser Val Met Arg Ser 285 Asp Val Pro ata gac att tgt gtg tet gaa tgt att aca cca aat gga age ate tcc 912 He Asp 290 He Cys Val Ser Glu 295 Cys He Thr Pro Asn 300 Gly Ser He Ser aac gac aag cca ttc caa aat gtg aac aaa gtt aca tat gga aaa tgc 960 Asn 305 Asp Lys Pro Phe Gin 310 Asn Val Asn Lys Val 315 Thr Tyr Gly Lys Cys 320 ccc aag tat ate agg caa aac act tta aag ctg gcc act ggg atg agg 1008 Pro Lys Tyr He Arg 325 Gin Asn Thr Leu Lys 330 Leu Ala Thr Gly Met 335 Arg aat gta cca gaa aag caa acc aga gga ate ttt gga gca ata geg gga 1056 Asn Val Pro Glu 340 Lys Gin Thr Arg Gly 345 He Phe Gly Ala He 350 Ala Gly ttc ate gaa aac ggc tgg gaa gga atg gtt gat ggg tgg tat ggg ttc 1104 Phe He Glu 355 Asn Gly Trp Glu Gly 360 Met Val Asp Gly Trp 365 Tyr Gly Phe ega tat caa aac tet gaa gga aca ggg caa get gca gat eta aag age 1152 Arg Tyr 370 Gin Asn Ser Glu Gly 375 Thr Gly Gin Ala Ala 380 Asp Leu Lys Ser act caa gca gcc ate gac cag att aat gga aag tta aac aga gtg att i 1200 Thr 385 Gin Ala Ala He Asp 390 Gin He Asn Gly Lys 395 Leu Asn Arg Val He 400 gaa aga acc aat gag aaa ttc cat caa ata gag aag gaa ttc tea gaa 1248 Glu Arg Thr Asn Glu 405 Lys Phe His Gin He 410 Glu Lys Glu Phe Ser 415 Glu gta gaa gga aga att cag gac ttg gag aaa tat gta gaa gac acc aaa 1296 Val Glu Gly Arg 420 He Gin Asp Leu Glu 425 Lys Tyr Val Glu Asp 430 Thr Lys ( ata gac eta tgg tcc tac aat gca gaa ttg ctg gtg get eta gaa aat 1344 He Asp Leu 435 Trp Ser Tyr Asn Ala 440 Glu Leu Leu Val Ala 445 Leu Glu Asn

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   caa Gin cat His 450 aca Thr att gac tta Leu aca gat Thr Asp 455 gca Ala gaa Glu atg Met aat Asn 460 aaa Lys tta Leu ttt Phe gag Glu lie Asp aag act aga ege cag tta aga gaa aac gca gaa gac atg gga ggt gga Lys Thr Arg Arg Gin 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 tea ata Cys Phe Lys He Tyr His Lys Cys Asp Asn Ala Cys He Gly Ser He 485 490 495 aga act ggg aca tat gac cat tac ata tac aga gat gaa gca tta aac Arg Thr Gly Thr Tyr Asp His Tyr lie Tyr Arg Asp Glu Ala Leu Asn 500 505 510 aac ega ttt cag ate aaa ggt gta gag ttg aaa tea ggc tac aaa gat Asn Arg Phe Gin lie Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp 515 520 525 tgg ata ctg tgg att tea ttc gcc ata tea tgc ttc tta att tgc gtt Trp He Leu Trp lie Ser Phe Ala He Ser Cys Phe Leu He Cys Val 530 535 540 gtt eta ttg ggt ttc att atg tgg get tgc caa aaa ggc aac ate aga Val Leu Leu Gly Phe He Met Trp Ala Cys Gin Lys Gly Asn lie Arg 545 550 555 560 tgc aac att tgc att tga Cys Asn He Cys He 565 <210> 32 <211> 565 <212> PRT <213> Influenza virus <400> 32 Met Lys Thr Thr lie lie Leu lie Leu Leu Thr His Trp Ala Tyr Ser 1 5 10 15 Gin Asn Pro He 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 He Ser Asp Asp Gin 35 40 45 He Glu Val Thr Asn Ala Thr Glu Leu Val Gin Ser He Ser Met Gly 50 55 60

   1392

   1440

   1488

   1536

   1584

   1632

   1680

   1698

   Lys lie Cys Asn Lys Ser Tyr Arg lie Leu Asp Gly Arg Asn Cys Thr 65 70 75 80

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   Leu lie Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gin Tyr 85 90 95

   Glu Ser Trp Asp Leu Phe He Glu Arg Ser Ser Ala Phe Ser Asn Cys 100 105 110

   Tyr Pro Tyr Asp He Pro Asp Tyr Ala Ser Leu Arg Ser He 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 Gin 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 He Trp Gly He His His Pro Ser Ser Asn Gin Glu Gin Thr Lys 195 200 205

   Leu Tyr He Gin Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser 210 215 220

   Gin Gin Thr lie He Pro Asn He Gly Ser Arg Pro Leu Val Arg Gly 225 230 235 240

   Gin Ser Gly Arg He Ser He Tyr Trp Thr He Val Lys Pro Gly Asp 245 250 255

   He Leu Met lie 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

   He Asp He Cys Val Ser Glu Cys lie Thr Pro Asn Gly Ser He Ser 290 295 300

   Asn Asp Lys Pro Phe Gin Asn Val Asn Lys Val Thr Tyr Gly Lys Cys 305 310 315 320

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   Pro Lys Tyr He Arg 325 Gin Asn Thr Leu Lys 330 Leu Ala Thr Gly Met 335 Arg Asn Val Pro Glu 340 Lys Gin Thr Arg Gly 345 He Phe Gly Ala He 350 Ala Gly Phe He Glu 355 Asn Gly Trp Glu Gly 360 Met Val Asp Gly Trp 365 Tyr Gly Phe Arg Tyr 3 70' Gin Asn Ser Glu Gly 375 Thr Gly Gin Ala Ala 380 Asp Leu Lys Ser Thr 385 Gin Ala Ala He Asp 390 Gin He Asn Gly Lys 395 Leu Asn Arg Val lie 400 Glu Arg Thr Asn Glu 405 Lys Phe His Gin He 410 Glu Lys Glu Phe Ser 415 Glu Val Glu Gly Arg 420 He Gin Asp Leu Glu 425 Lys Tyr Val Glu Asp 430 Thr Lys lie Asp Leu 435 Trp Ser Tyr Asn Ala 440 Glu Leu Leu Val Ala 445 Leu Glu Asn Gin His 450 Thr He Asp Leu Thr 455 Asp Ala Glu Met Asn 460 Lys Leu Phe Glu Lys 465 Thr Arg Arg Gin Leu 470 Arg Glu Asn Ala Glu 475 Asp Met Gly Gly Gly 480 Cys Phe Lys He Tyr 485 His Lys Cys Asp Asn 490 Ala Cys lie Gly Ser 495 He Arg Thr Gly Thr 500 Tyr Asp His Tyr He 505 Tyr Arg Asp Glu Ala 510 Leu Asn Asn Arg Phe 515 Gin He Lys Gly Val 520 Glu Leu Lys Ser Gly 525 Tyr Lys Asp Trp He 530 Leu Trp He Ser Phe 535 Ala He Ser Cys Phe 540 Leu He Cys Val Val 545 Leu Leu Gly Phe He 550 Met Trp Ala Cys Gin 555 Lys Gly Asn lie Arg 560

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   Cys Asn lie Cys He

   565 <210> 33 <211> 549 <212> PRT <213> Influenza virus <400> 33 Gin Asn Pro He 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 Gin 20 25 30 lie Glu Val Thr Asn Ala Thr Glu Leu Val Gin Ser He Ser Met Gly 35 40 45 Lys He Cys Asn Lys Ser Tyr Arg lie Leu Asp Gly Arg Asn Cys Thr 50 55 60 Leu lie Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gin Tyr 65 70 75 80 Glu Ser Trp Asp Leu Phe He Glu Arg Ser Asn Ala Phe Ser Asn Cys 85 90 95 Tyr Pro Tyr Asp He Pro Asp Tyr Ala Ser Leu Arg Ser He 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 Gin 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 He Trp Gly He His His Pro Ser Ser Asn Gin Glu Gin Thr Lys 180 185 190

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   Leu Tyr lie Gin Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser 195 200 205

   Gin Gin Thr He He Pro Asn He Gly Ser Arg Pro Leu Val Arg Gly 210 ' 215 220

   Gin Ser Gly Arg He Ser He Tyr Trp Thr He Val Lys Pro Gly Asp 225 230 235 240

   He Leu Met He 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 lie Asp He Cys Val Ser Glu Cys He Thr Pro Asn Gly Ser He Ser 275 280 285

   Asn Asp Lys Pro Phe Gin Asn Val Asn Lys Val Thr Tyr Gly Lys Cys 290 295 300

   Pro Lys Tyr He Arg Gin Asn Thr Leu Lys Leu Ala Thr Gly Met Arg 305 310 315 320

   Asn Val Pro Glu Lys Gin Thr Arg Gly He Phe Gly Ala He Ala Gly 325 330 335

   Phe lie Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe 340 345 350

   Arg Tyr Gin Asn Ser Glu Gly Thr Gly Gin Ala Ala Asp Leu Lys Ser 355 360 365

   Thr Gin Ala Ala He Asp Gin lie Asn Gly Lys Leu Asn Arg Val He 370 375 380

   Glu Arg Thr Asn Glu Lys Phe His Gin He Glu Lys Glu Phe Ser Glu 385 390 395 400

   Val Glu Gly Arg He Gin Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys 405 410 415

   He Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn 420 425 430

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   Gin His Thr He Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu 435 440 445 Lys Thr Arg Arg Gin Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly 450 455 460 Cys Phe Lys He Tyr His Lys Cys Asp Asn Ala Cys He Gly Ser He 465 470 475 480 Arg Thr Gly Thr Tyr Asp His Tyr He Tyr Arg Asp Glu Ala Leu Asn 485 490 495 Asn Arg Phe Gin He Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp 500 505 510 Trp He Leu Trp He Ser Phe Ala He Ser Cys Phe Leu He Cys Val 515 520 525 Val Leu Leu Gly Phe He Met Trp Ala Cys Gin Arg Gly Asn He Arg 530 535 540 Cys Asn He Cys He 545 <210> 34 <211> i 549 <212> : PRT <213> Influenza virus <400> 34 Gin Asn Pro He 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 He Ser Asp Asp Gin 20 25 30 He Glu Val Thr Asn Ala Thr Glu Leu Val Gin Ser He Ser Met Gly 35 40 45 Lys He Cys Asn Lys Ser Tyr Arg He Leu Asp Gly Arg Asn Cys Thr 50 55 60 Leu He Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gin Tyr 65 70 75 80

   Glu Ser Trp Asp Leu Phe lie Glu Arg Ser Ser Ala Phe Ser Asn Cys

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   85 90 95

   Tyr Pro Tyr Asp lie Pro Asp Tyr Ala Ser Leu Arg Ser He 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 Gin 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 He Trp Gly He His His Pro Ser Ser Asn Gin Glu Gin Thr Lys 180 185 190

   Leu Tyr lie Gin Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser 195 200 205

   Gin Gin Thr He He Pro Asn He Gly Ser Arg Pro Leu Val Arg Gly 210 215 220

   Gin Ser Gly Arg He Ser He Tyr Trp Thr lie Val Lys Pro Gly Asp 225 230 235 240

   He Leu Met He 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 lie Asp He Cys Val Ser Glu Cys lie Thr Pro Asn Gly Ser He Ser 275 280 285

   Asn Asp Lys Pro Phe Gin Asn Val Asn Lys Val Thr Tyr Gly Lys Cys 290 295 300

   Pro Lys Tyr He Arg Gin Asn Thr Leu Lys Leu Ala Thr Gly Met Arg 305 310 315 320

   Asn Val Pro Glu Lys Gin Thr Arg Gly He Phe Gly Ala He Ala Gly

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   325

   Phe lie Glu Asn Gly Trp Glu Gly 340

   Arg Tyr Gin Asn Ser Glu Gly Thr 355 360

   Thr Gin Ala Ala He Asp Gin lie 370 375

   Glu Arg Thr Asn Glu Lys Phe His 385 390

   Val Glu Gly Arg He Gin Asp Leu 405

   330 335

   Met Val Asp Gly Trp Tyr Gly Phe 345 350

   Gly Gin Ala Ala Asp Leu Lys Ser 365

   Asn Gly Lys Leu Asn Arg Val He 380

   Gin He Glu Lys Glu Phe Ser Glu 395 400

   Glu Lys Tyr Val Glu Asp Thr Lys 410 415

   He Asp Leu Trp Ser Tyr Asn Ala 420

   Glu Leu Leu Val Ala Leu Glu Asn 425 430

   Gin His Thr He Asp Leu Thr Asp 435 440

   Ala Glu Met Asn Lys Leu Phe Glu 445

   Lys Thr Arg Arg Gin Leu Arg Glu 450 455

   Asn Ala Glu Asp Met Gly Gly Gly 460

   Cys Phe Lys He Tyr His Lys Cys 465 470

   Asp Asn Ala Cys He Gly Ser He 475 480

   Arg Thr Gly Thr Tyr Asp His Tyr 485

   He Tyr Arg Asp Glu Ala Leu Asn 490 495

   Asn Arg Phe Gin He Lys Gly Val 500

   Glu Leu Lys Ser Gly Tyr Lys Asp 505 510

   Trp He Leu Trp He Ser Phe Ala 515 520

   He Ser Cys Phe Leu He Cys Val 525

   Val Leu Leu Gly Phe He Met Trp 530 535

   Ala Cys Gin Lys Gly Asn He Arg 540

   Cys Asn He Cys lie 545 <210> 35

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   2015234384 02 Oct 2015 <211> 9 <212> DNA <213> Influenza virus <400> 35 gagagttgg 9

   <210> <211> <212> <213> 36 9 DNA Influenza virus

   <400> 36 ccgttggtc 9

   <210> <211> <212> <213> 37 9 1 DNA , Influenza virus ,

   <400> 37 caaaccaga 9

   <210> <211> <212> <213> 38 9 DNA Influenza virus

   <400> 38 agaactggg 9

   <210> <211> <212> <213> 39 15 DNA Influenza virus

   <400> 39 tatgagagtt gggac 15

   <210> <211> <212> <213> 40 15 DNA Influenza virus

   <400> 40 agaccgttgg tcaga 15

   <210> <211> <212> <213> 41 15 DNA Influenza virus <400> 41

   aagcaaacca gagga 15

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   <210> 42 <211> 15 <212> DNA <213> Influenza virus <400> 42 ataagaactg ggaca <210> 43 <211> 9 <212> DNA <213> Influenza virus <400> 43 acaatgagt <210> 44 <211> 15 <212> DNA <213> Influenza virus <400> 44 aaaacaatga gtgat ( <210> 45 <211> 9 <212> DNA <213> Influenza virus <400> 45 gatgtaccc <210> 46 <211> 15 <212> DNA <213> Influenza virus <400> 46 tcagatgtac ccata <210> 47 <211> 2280 <212> DNA <213> Influenza virus <220> <221> CDS <222> (1)..(2280) <400> 47 atg gag aga ata aaa gaa Met Glu Arg lie ' Lys Glu

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   15 10 15

   cgc Arg gag Glu ata lie eta aca aaa Lys act Thr act Thr gtg gac cac His atg Met gcc Ala ata He 30 ate He aag Lys 96 Leu 20 Thr Val 25 Asp aaa tac aca tea gga aga caa gag aag aac cct gca ctt agg atg aaa 144 Lys Tyr Thr 35 Ser Gly Arg Gin Glu 40 Lys Asn Pro Ala Leu 45 Arg Met Lys tgg atg atg gca atg aaa tac cca att aca gca gat aag agg ata atg 192 Trp Met 50 Met Ala Met Lys Tyr 55 Pro He Thr Ala Asp 60 Lys Arg He Met gag atg att cct gag aga aat gaa cag gga caa acc ctt tgg age aaa 240 Glu 55 Met He Pro Glu Arg 70 Asn Glu Gin Gly Gin 75 Thr Leu Trp Ser Lys 80 acg aac gat get ggc tea gac cgc gta atg gta tea cct ctg gca gtg 288 Thr Asn Asp Ala Gly 85 Ser Asp Arg Val Met 90 Val Ser Pro Leu Ala 95 Val aca tgg tgg aat agg aat gga cca aca acg aac aca att cat tat cca 336 Thr Trp Trp Asn 100 Arg Asn Gly Pro Thr 105 Thr Asn Thr He His 110 Tyr Pro aaa gtc tac aaa act tat ttt gaa aag gtt gaa aga ttg aaa cac gga 384 Lys Val Tyr 115 Lys Thr Tyr Phe Glu 120 Lys Val Glu Arg Leu 125 Lys His Gly acc ttt ggc ccc gtt cat ttt agg aat caa gtc aag ata aga ega aga 432 Thr Phe 13 0 Gly Pro Val His Phe 135 Arg Asn Gin Val Lys 140 He Arg Arg Arg gtt gat gta aac cct ggt cac geg gac etc agt get aaa gaa gca caa 480 Val 145 Asp Val Asn Pro Gly 150 His Ala Asp Leu Ser 155 Ala Lys Glu Ala Gin 160 gat gtg ate atg gaa gtt gtt ttc cca aat gaa gtg gga gcc aga att 528 Asp Val He Met Glu 165 Val Val Phe Pro Asn 170 Glu Val Gly Ala Arg 175 He eta aca tea gaa tea caa eta aca ata acc aaa gag aaa aag gaa gaa 576 Leu Thr Ser Glu 180 Ser Gin Leu Thr lie 185 Thr Lys Glu Lys Lys 190 Glu Glu ctt cag gac tgc aaa att get ccc ttg atg gta gca tac atg eta gaa 624 Leu Gin Asp 195 Cys Lys He Ala Pro 200 Leu Met Val Ala Tyr 205 Met Leu Glu aga gag ttg gtc ega aaa aca agg ttc etc cca gta gta ggc gga aca 672 Arg Glu 210 Leu Val Arg Lys Thr 215 Arg Phe Leu Pro Val 220 Val Gly Gly Thr age agt gta tac att gaa gtg ttg cat ctg act cag gga aca tgc tgg 720 Ser 225 Ser Val Tyr He Glu 230 Val Leu His Leu Thr 235 Gin Gly Thr Cys Trp 240 gag caa atg tac acc cca gga gga aaa gtt aga aac gat gat att gat 768 Glu Gin Met Tyr Thr Pro Gly Gly Lys Val Arg Asn Asp Asp He Asp

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   245 250 255

   caa agt Gin Ser tta Leu att He 260 att He gca gcc Ala Ala egg aac Arg Asn 265 ata He gtg Val aga Arg aga Arg gca Ala 270 aca Thr gta Val 816 tea gca gat cca eta gca tcc eta ctg gaa atg tgc cac agt aca cag 864 Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gin 275 280 285 att ggt gga aca agg atg gta gac ate ctt aag cag aac cca aca gag 912 lie Gly Gly Thr Arg Met Val Asp He Leu Lys Gin Asn Pro Thr Glu 290 295 300 gaa caa get gtg gat ata tgc aaa gca gca atg gga ttg aga att age 960 Glu Gin Ala Val Asp He Cys Lys Ala Ala Met Gly Leu Arg lie Ser 305 310 315 1 320 tea tea ttc age ttt ggt gga ttc acc ttc aaa agg aca agt gga tea 1008 Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser 325 330 335 tea gtc aag aga gaa gaa gaa atg ctt aeg ggc aac ctt caa aca ttg 1056 Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gin Thr Leu 340 345 350 aaa ata aga gtg cat gag ggc tat gaa gaa ttc aca atg gtc gga aga 1104 Lys He Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg 355 360 365 aga gca aca gcc att ate aga aag gca acc aga aga ttg att caa ttg 1152 Arg Ala Thr Ala He lie Arg Lys Ala Thr Arg Arg Leu He Gin Leu 370 375 380 ata gta agt ggg a'ga gat gaa caa tea att get gaa gca ata att gta 1200 He Val Ser Gly Arg Asp Glu Gin Ser He Ala Glu Ala He He Val 385 390 395 400 gee atg gtg ttt teg caa gaa gat tgc atg ata aaa gca gtt ega ggc 1248 Ala Met Val Phe Ser Gin Glu Asp Cys Met He 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 Gin Arg Leu Asn Pro Met His 420 425 430 caa etc ttg agg cat ttc caa aaa gat gca aaa gtg ctt ttc caa aat 1344 Gin Leu Leu Arg His Phe Gin Lys Asp Ala Lys Val Leu Phe Gin Asn 435 440 445 tgg gga att gaa ccc ate gac aat gta atg ggg atg att gga ata ttg 1392 Trp Gly He Glu Pro He Asp Asn Val Met Gly Met He Gly He Leu 450 455 460 cct gac atg acc cca age acc gag atg tea 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 age aaa atg gga gtg gat gag tac tcc age act gag aga gtg gtg gtg 1488 Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val

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   485 490 495 age att gac cgt ttt tta aga gtt egg gat caa agg gga aac ata eta 1536

   Ser lie Asp Arg Phe Leu Arg Val Arg Asp Gin Arg Gly Asn He Leu

   500 505 510 ctg tcc cct gaa gaa gtc agt gaa aca caa gga aeg gaa aag ctg aca 1584

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

   515 520 525 ata att tat tog tea tea atg atg tgg gag att aat ggt ccc gaa tea 1632

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

   530 535 540 gtg ttg gtc aat act tat caa tgg ate ate aga aac tgg gaa att gta 1680

   Val Leu Val Asn Thr Tyr Gin Trp He He Arg Asn Trp Glu He Val

   545 550 555 560 aaa att cag tgg tea cag gac ccc aca atg tta tac aat aag ata gaa 1728

   Lys He Gin Trp Ser Gin Asp Pro Thr Met Leu Tyr Asn Lys He Glu

   565 570 575 ttt gaa cca ttc caa tcc ctg gtc cct agg gcc acc aga age caa tac 1776

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

   580 585 590 age ggt ttc gta aga acc ctg ttt cag caa atg ega gat gta ctt gga 1824

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

   595 600 605 aca ttt gat act get caa ata ata aaa etc etc cct ttt gcc get get 1872

   Thr Phe Asp Thr Ala Gin He He Lys Leu Leu Pro Phe Ala Ala Ala

   610 615 620 cct ccg gaa cag agt agg atg cag ttc tet tet ttg act gtt aat gta 1920

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

   625 630 635 640 aga ggt teg gga atg agg ata ctt gta aga ggc aat tcc cca gtg ttc 1968

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

   645 650 655 aac tac aat aaa gtc act aaa agg etc aca gtc etc 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 geg ctt act gag gac cca gat gaa ggt aeg get gga gta gag tet 2064

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

   675 680 685 get gtt eta aga ggg ttt etc att tta ggt aaa gaa aac aag aga tat 2112

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

   690 695 700 ggc cca gca eta age ate aat gaa ctt age aaa ctt gca aaa ggg gag 2160

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

   705 710 715 720 aaa gcc aat gta eta att ggg caa ggg gac gta gtg ttg gta atg aaa 2208

   Lys Ala Asn Val Leu He Gly Gin Gly Asp Val Val Leu Val Met Lys

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   725

   730

   735

   egg aaa cgt gac tct age ata ett act gac age cag aca gcg acc aaa Arg Lys Arg Asp Ser Ser He Leu Thr Asp Ser Gin Thr Ala Thr Lys 740 745 750 agg att egg atg gcc ate aat tag Arg lie Arg Met Ala lie Asn 755 <210> 48 <211> 759 <212> PRT <213> Influenza virus <400> 48 Met Glu Arg He Lys Glu Leu Arg Asp Leu Met Leu Gin Ser Arg Thr 1 5 10 15 Arg Glu He Leu Thr Lys Thr Thr Val Asp His Met Ala He He Lys 20 25 30 Lys Tyr Thr Ser Gly Arg Gin Glu Lys Asn Pro Ala Leu Arg Met Lys 35 40 45 Trp Met Met Ala Met Lys Tyr Pro He Thr Ala Asp Lys Arg He Met 50 55 60 Glu Met He Pro Glu Arg Asn Glu Gin Gly Gin 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 He 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 Gin Val Lys He Arg. Arg Arg 130 135 140 Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gin 145 150 155 160 Asp Val He Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg He 165 170 175

   2256

   2280

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   Leu Thr Ser Glu Ser Gin Leu Thr 180

   Leu Gin Asp Cys Lys lie Ala Pro 195 200

   Arg Glu Leu Val Arg Lys Thr Arg 210 215

   Ser Ser Val Tyr He Glu Val Leu 225 230

   Glu Gin Met Tyr Thr Pro Gly Gly 245

   He Thr Lys Glu Lys Lys Glu Glu 185 , 190

   Leu Met Val Ala Tyr Met Leu Glu 205

   Phe Leu Pro Val Val Gly Gly Thr 220

   His Leu Thr Gin Gly Thr Cys Trp 235 240

   Lys Val Arg Asn Asp Asp He Asp 250 255

   Gin Ser Leu He He Ala Ala Arg 260

   Asn lie Val Arg Arg Ala Thr Val 265 270

   Ser Ala Asp Pro Leu Ala Ser Leu 275 280

   Leu Glu Met Cys His Ser Thr Gin 285

   He Gly Gly Thr Arg Met Val Asp 290 295

   He Leu Lys Gin Asn Pro Thr Glu 300

   Glu Gin Ala Val Asp He Cys Lys 305 310

   Ala Ala Met Gly Leu Arg He Ser 315 320

   Ser Ser Phe Ser Phe Gly Gly Phe 325

   Thr Phe Lys Arg Thr Ser Gly Ser 330 335

   Ser Val Lys Arg Glu Glu Glu Met 340

   Leu Thr Gly Asn Leu Gin Thr Leu 345 350

   Lys lie Arg Val His Glu Gly Tyr 355 f 360

   Glu Glu Phe Thr Met Val Gly Arg 365

   Arg Ala Thr Ala He He Arg Lys 370 375

   Ala Thr Arg Arg Leu lie Gin Leu 380

   He Val Ser Gly Arg Asp Glu Gin 385 390

   Ser He Ala Glu Ala He He Val 395 400

   Ala Met Val Phe Ser Gin Glu Asp 405

   Cys Met lie Lys Ala Val Arg Gly 410 415

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   Asp Leu Asn Phe Val Asn Arg Ala Asn Gin Arg Leu Asn Pro Met His 420 425 430

   Gin Leu Leu Arg His Phe Gin Lys Asp Ala Lys Val Leu Phe Gin Asn 435 440 445

   Trp Gly lie Glu Pro He Asp Asn Val Met Gly Met He Gly He 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 He Asp Arg Phe Leu Arg Val Arg Asp Gin Arg Gly Asn He Leu 500 505 510

   Leu Ser Pro Glu Glu Val Ser Glu Thr Gin Gly Thr Glu Lys Leu Thr 515 520 525

   He lie Tyr Ser Ser Ser Met Met Trp Glu lie Asn Gly Pro Glu Ser 530 535 540

   Val Leu Val Asn Thr Tyr Gin Trp He He Arg Asn Trp Glu lie Val 545 550 555 560

   Lys He Gin Trp Ser Gin Asp Pro Thr Met Leu Tyr Asn Lys He Glu 565 570 575

   Phe Glu Pro Phe Gin Ser Leu Val Pro Arg Ala Thr Arg Ser Gin⁷Tyr 580 5‘85 590

   Ser Gly Phe Val Arg Thr Leu Phe Gin Gin Met Arg Asp Val Leu Gly 595 600 605

   Thr Phe Asp Thr Ala Gin He He Lys Leu Leu Pro Phe Ala Ala Ala 610 615 620

   Pro Pro Glu Gin Ser Arg Met Gin Phe Ser Ser Leu Thr Val Asn Val 625 630 635 640

   Arg Gly Ser Gly Met Arg He Leu Val Arg Gly Asn Ser Pro Val Phe 645 650 655

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   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 lie Leu Gly Lys Glu Asn Lys Arg Tyr 690 695 700 Gly Pro Ala Leu Ser He Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu 705 710 715 720 Lys Ala Asn Val Leu He Gly Gin Gly Asp Val Val Leu Val Met Lys 725 730 735 Arg Lys Arg Asp Ser Ser He Leu Thr Asp Ser Gin Thr Ala Thr Lys 740 745 750 Arg Tie Arg Met Ala He Asn

   755 <210> 49 <211> 2274 <212> DNA <213> Influenza virus <220>

   <221> CDS <222> (1) . . (2274) <400> 49

   atg Met 1 gat Asp gtc Val aat Asn ccg Pro 5 act Thr eta Leu ett Leu ttc Phe tta Leu 10 aag gtg Lys Val cca Pro gcg Ala caa Gin 15 aat Asn 48 get ata age aca aca ttc cct tat act gga gat cct ccc tac agt cat 96 Ala He 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 Gin 35 40 45 tat tea 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 He Gly Ala Pro 50 55 60 caa ett aat cca ate gat gga cca ett cct gaa gac aat gaa cca agt 240 Gin Leu Asn Pro He Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser 65 70 75 80

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   ggg tac gee caa aca gat tgt gta ttg gaa gca atg get ttc ett gaa Gly Tyr Ala Gin Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 85 90 95 gaa tcc cat ccc gga ate ttt gaa aat teg tgt ett gaa aeg atg gag Glu Ser His Pro Gly He Phe Glu Asn Ser Cys Leu Glu Thr Met Glu 100 105 110 gtg att cag cag aca aga gtg gac aaa eta aca caa ggc ega caa act Val He Gin Gin Thr Arg Val Asp Lys Leu Thr Gin Gly Arg Gin Thr 115 12 0 125 tat gat tgg acc ttg aat agg aat caa cct gcc gca aca gca ett get Tyr Asp Trp Thr Leu Asn Arg Asn Gin Pro Ala Ala Thr Ala Leu Ala 130 135 140 aat aeg att gaa gta ttc aga tea aat ggt ctg acc tcc aat gaa teg Asn Thr He Glu Val Phe Arg Ser Asn Gly Leu Thr Ser Asn Glu Ser 145 150 155 160 ggg aga ttg atg gac ttc etc aaa gat gtc atg gag tcc atg aac aag 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 egg aag aga aga gta aga Glu Glu Met Glu He Thr Thr His Phe Gin 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 Asp Asn Met Thr Lys Arg Met He Thr Gin Arg Thr He Gly Lys Lys 195 200 205 aaa caa ega tta age aga aag age tat eta ate aga aca tta acc eta Lys Gin Arg Leu Ser Arg Lys Ser Tyr Leu lie Arg Thr Leu Thr Leu 210 215 220 aac aca atg acc aag gac get gag aga ggg aaa ttg aaa ega ega gca Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala 225 230 235 240 ate get acc cca ggg atg cag ata aga gga ttt gta tat ttt gtt gaa lie Ala Thr- Pro Gly Met Gin He Arg Gly Phe Val Tyr Phe Val Glu 245 250 255 aca eta get ega aga ata tgt gaa aag ett gaa caa tea gga ttg cca Thr Leu Ala Arg Arg lie Cys Glu Lys Leu Glu Gin Ser Gly Leu Pro 260 265 270 gtt ggc ggt aat gag aaa aag gee aaa ctg get aat gtc gtc aga aaa 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 etc tcc ttc acc ate act ggg Met Met Thr Asn Ser Gin Asp Thr Glu Leu Ser Phe Thr He Thr Gly 290 295 300 gac aat acc aaa tgg aat gaa aat cag aac cca ege ata ttc ctg gca Asp Asn Thr Lys Trp Asn Glu Asn Gin Asn Pro Arg lie Phe Leu Ala 305 310 315 320

   288

   336

   384

   432

   488

   528

   576

   624

   672

   720

   768

   816

   864

   912

   960

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   atg Met ate lie aca Thr tac Tyr ata act He Thr 325 aga Arg gat Asp cag Gin cca Pro 330 gaa Glu tgg Trp ttc Phe aga Arg aat Asn 335 gtt Val 1008 eta age att gca ccg att atg ttc tea aat aaa atg gca aga ctg ggg 1056 Leu Ser He Ala Pro He Met Phe Ser Asn Lys Met Ala Arg Leu Gly 340 345 350 aaa gga tat atg ttt gaa age aaa agt atg aaa ttg aga act caa ata 1104 Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gin He 355 360 365 cca gca gaa atg eta gca age att gac eta aaa tat ttc aat gat tea 1152 Pro Ala Glu Met Leu Ala Ser lie Asp Leu Lys Tyr Phe Asn Asp Ser 370 375 380 aca aaa aag aaa att gaa aag ata ega cca etc ctg gtt gac ggg act 1200 Thr Lys Lys Lys He Glu Lys lie Arg Pro Leu Leu Val Asp Gly Thr 385 390 395 400 get tea ctg agt cct ggc atg atg atg gga atg ttc aac atg ttg age 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 He Leu Asn Leu Gly Gin Arg Lys Tyr Thr 420 425 430 aag acc aca tac tgg tgg gat ggt ctg caa tea tcc gat gac ttt get 1344 Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gin Ser Ser Asp Asp Phe Ala 435 440 445 ttg ata gtg aat geg cct aat cat gaa gga ata caa get gga gta gac 1392 Leu He Val Asn Ala Pro Asn His Glu Gly He Gin Ala Gly Val Asp 450 455 460 aga ttc tat aga act tgc aaa ctg gtc ggg ate aac atg age aaa aag 1440 Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly He Asn Met Ser Lys Lys 465 470 475 480 aag tec tac ata aat aga act gga aca ttc gaa ttc aca age ttt ttc 1488 Lys Ser Tyr He Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe 485 490 495 tac egg tat ggt ttt gta gcc aat ttc age atg gaa eta 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 tec gga ata aat gaa tet gca gac atg age att gga gtg aca 1584 Gly Val Ser Gly He Asn Glu Ser Ala Asp Met Ser He Gly Val Thr 515 520 525 gtc ate aaa aac aac atg ata aat aat gat etc ggt cct gcc aeg gca 1632 Val He Lys Asn Asn Met He Asn Asn Asp Leu Gly Pro Ala Thr Ala 530 535 540 1 caa atg gca etc caa etc ttc att aag gat tat egg tac aca tac egg 1680 Gin Met Ala Leu Gin Leu Phe He Lys Asp Tyr Arg Tyr Thr Tyr Arg 545 550 555 560

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   tgc cat aga ggt gat acc cag ata caa acc aga aga tet ttt gag ttg Cys His Arg Gly Asp 565 Thr Gin He Gin Thr 570 Arg Arg Ser Phe Glu 575 Leu aag aaa ctg tgg gaa cag act ega tea aag act ggt eta ctg gta tea Lys Lys Leu Trp 580 Glu Gin Thr Arg Ser 585 Lys Thr Gly Leu Leu 590 Val Ser gat ggg ggt cca aac eta tat aac ate aga aac eta cac ate ccg gaa Asp Gly Gly 595 Pro Asn Leu Tyr Asn 600 He Arg Asn Leu His 605 He Pro Glu gtc tgt tta aaa tgg gag eta atg gat gaa gat tat aag ggg agg eta Val Cys 610 Leu Lys Trp Glu Leu 615 Met Asp Glu Asp Tyr 620 Lys Gly Arg Leu tgc aat cca ttg aat cct ttc gtt agt cac aaa gaa att gaa tea gtc Cys 625 Asn Pro Leu Asn Pro 630 Phe Val Ser His Lys 635 Glu lie Glu Ser Val 640 aac agt gca gta gta atg cct get cat ggc cct gcc aaa age atg gag Asn Ser Ala Val Val 645 Met Pro Ala His Gly 650 Pro Ala Lys Ser Met 655 Glu tat gat get gtt gca aca aca cat tet tgg ate ccc aag agg aac egg Tyr Asp Ala Val 660 Ala Thr Thr His Ser 665 Trp He Pro Lys Arg 670 Asn Arg tec ata ttg aac aca age caa agg gga ata eta gaa gat gag cag atg Ser lie Leu 675 Asn Thr Ser Gin Arg 680 Gly He Leu Glu Asp 685 Glu Gin Met tat cag aaa tgc tgc aac ctg ttt gaa aaa ttc ttc ccc age age tea Tyr Gin 690 Lys Cys Cys Asn Leu 695 Phe Glu Lys Phe Phe 700 Pro Ser Ser Ser tac aga aga cca gtc gga att tet agt atg gtt gag gcc atg gta tec Tyr 705 Arg Arg Pro Val Gly 710 He Ser Ser Met Val 715 Glu Ala Met Val Ser 720 agg gcc ege att gat gca ega att gac ttc gaa tet gga egg ata aag Arg Ala Arg He Asp 725 Ala Arg He Asp Phe 730 Glu Ser Gly Arg He 735 Lys aag gat gag ttc get gag ate atg aag ate tgt tec acc att gaa gag Lys Asp Glu Phe 740 Ala Glu He Met Lys 745 lie Cys Ser Thr He 750 Glu Glu

   1728

   1776

   1824

   1872

   1920

   1968

   2016

   2064

   2112

   2160

   2208

   2256 etc aga egg caa aaa tag Leu Arg Arg Gin Lys

   755 <210> 50 <211> 757 <212> PRT <213> Influenza virus <400> 50

   2274

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   Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gin Asn 15 10 15

   Ala lie 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 Gin 35 40 45

   Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu He Gly Ala Pro 50 55 60

   Gin Leu Asn Pro He Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser 65 70 75 80

   Gly Tyr Ala Gin Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 85 90, 95

   Glu Ser His Pro Gly He Phe Glu Asn Ser Cys Leu Glu Thr Met Glu 100 105 110

   Val lie Gin Gin Thr Arg Val Asp Lys Leu Thr Gin Gly Arg Gin Thr 115 120 125

   Tyr Asp Trp Thr Leu Asn Arg Asn Gin Pro Ala Ala Thr Ala Leu Ala 130 135 140

   Asn Thr He 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 He Thr Thr His Phe Gin Arg Lys Arg Arg Val Arg 180 185 190

   Asp Asn Met Thr Lys Arg Met lie Thr Gin Arg Thr He Gly Lys Lys 195 200 205

   Lys Gin Arg Leu Ser Arg Lys Ser Tyr Leu He Arg Thr Leu Thr Leu 210 215 220

   Asn Thr Met Thr Lys 225

   Asp Ala Glu Arg Gly Lys 230 235

   Leu

   Lys

   Arg Arg Ala 240

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   Thr Leu Ala Arg Arg He Cys Glu Lys Leu Glu Gin 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 Gin Asp Thr Glu Leu Ser Phe Thr He Thr Gly 290 295 300

   Asp Asn Thr Lys Trp Asn Glu Asn Gin Asn Pro Arg He Phe Leu Ala 305 310 315 320

   Met He Thr Tyr He Thr Arg Asp Gin Pro Glu Trp Phe Arg Asn Val 325 330 335

   Leu Ser He Ala Pro He 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 Gin He 355 360 365

   Pro Ala Glu Met Leu Ala Ser He Asp Leu Lys Tyr Phe Asn Asp Ser 370 375 380

   Thr Lys Lys Lys He Glu Lys He 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 He Leu'Asn Leu Gly Gin Arg Lys Tyr Thr 420 425 430

   Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gin Ser Ser Asp Asp Phe Ala 435 440 ’ 445

   Leu He Val Asn Ala Pro Asn His Glu Gly He Gin Ala Gly Val Asp 450 455 460

   Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly He Asn Met Ser Lys Lys 465 470 475 480

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   Lys Ser Tyr lie 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 He Asn Glu Ser Ala Asp Met Ser He Gly Val Thr 515 520 525

   Val He Lys Asn Asn Met lie Asn Asn Asp Leu Gly Pro Ala Thr Ala 530 535 540

   Gin Met Ala Leu Gin Leu Phe He Lys Asp Tyr Arg Tyr Thr Tyr Arg 545 550 555 560

   Cys His Arg Gly Asp Thr Gin He Gin Thr Arg Arg Ser Phe Glu Leu 565 570 575

   Lys Lys Leu Trp Glu Gin Thr Arg Ser Lys Thr Gly Leu Leu Val Ser 580 585 590

   Asp Gly Gly Pro Asn Leu Tyr Asn He Arg Asn Leu His He 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 He 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 He Pro Lys Arg Asn Arg 660 665 670

   Ser He Leu Asn Thr Ser Gin Arg Gly He Leu Glu Asp Glu Gin Met 675 680 685

   Tyr Gin Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser 690 695 700

   Tyr Arg Arg Pro Val Gly He Ser Ser Met Val Glu Ala Met Val Ser 705 710 715 720

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   Arg Ala Arg lie Asp Ala Arg He Asp Phe Glu Ser Gly Arg He Lys 725 730 735

   Lys Asp Glu Phe Ala Glu He Met Lys He Cys Ser Thr He Glu Glu 740 745 750

   Leu Arg Arg Gin Lys 755 <210> 51 <211> 2151 <212> DNA <213> Influenza virus <220>

   <221>

   <222>

   CDS (1)..(2151) <400> 51

   atg gaa gac ttt gtg ega cag tgc ttc aat cca atg ate gtc gag ett 48 Met Glu Asp Phe Val Arg Gin Cys Phe Asn Pro Met He Val Glu Leu 1 5 10 15 geg gaa aag gca atg aaa gaa tat gga gag aac ccg aaa ate gaa aca 96 Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asn Pro Lys He 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 lie Cys Thr His Leu Glu Val Cys Phe Met Tyr 35 40 45 teg gat ttc cac ttt ata aat gaa ctg ggt gag tea gtg gtc ata gag 192 Ser Asp Phe His Phe He Asn Glu Leu Gly Glu Ser Val Val He Glu 50 55 60 tet ggt gac cca aat get ett ttg aaa cac aga ttt gaa ate att gag 240 Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu He He Glu 65 70 75 80 ggg aga gat ega aca atg gca tgg aca gta gta aac age ate tgc aac 288 Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser He Cys Asn 85 90 95 acc aca aga get gaa aaa cct aaa ttt ett 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 He Gly Val Thr Arg Arg Glu Val His 115 120 125 ata tac tac ctg gag aag gcc aac aaa ata aag tet gag aaa aca cat 432 He Tyr Tyr Leu Glu Lys Ala Asn Lys He Lys Ser Glu Lys Thr His 130 135 14 0

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   ate lie 145 cac His att He ttc Phe tea Ser ttt Phe 150 aca Thr gga Gly gaa Glu gaa Glu atg Met 155 get Ala aca Thr aaa Lys geg Ala gac Asp 160 480 tat act ett gat gaa gag agt aga gcc agg ate aag acc aga eta ttc 528 Tyr Thr Leu Asp Glu 165 Glu Ser Arg Ala Arg 170 He Lys Thr Arg Leu 175 Phe act ata aga caa gaa atg gcc agt aga ggc etc tgg gat tcc ttt cgt 576 Thr He Arg Gin 180 Glu Met Ala Ser Arg 185 Gly Leu Trp Asp Ser 190 Phe Arg cag tcc gag aga ggc gaa gag aca att gaa gaa aga ttt gaa ate aca 624 Gin Ser Glu 195 Arg Gly Glu Glu Thr 200 He Glu Glu Arg Phe 205 Glu He Thr ggg aeg atg ege aag ett gcc aat tac agt etc cca ccg aac ttc tcc 672 Gly Thr 210 Met Arg Lys Leu Ala 215 Asn Tyr Ser Leu Pro 220 Pro Asn Phe Ser age ett gaa aat ttt aga gtc tat ata gat gga ttc gaa ccg aac ggc 720 Ser 225 Leu Glu Asn Phe Arg 230 Val Tyr He Asp Gly 235 Phe Glu Pro Asn Gly 240 tgc att gag agt aag ett tet caa atg tcc aaa gaa gta aat gcc aaa 768 Cys He Glu Ser Lys 245 Leu Ser Gin Met Ser 250 Lys Glu Val Asn Ala 255 Lys ata gaa cca ttt tea aag aca aca ccc ega cca etc aaa atg cca ggt 816 lie Glu Pro Phe 260 Ser Lys Thr Thr Pro 265 Arg Pro Leu Lys Met 270 Pro Gly ggt cca ccc tgc cat cag ega tcc aaa ttc ttg eta atg gat get ctg 864 Gly Pro Pro 275 Cys His Gin Arg Ser 280 Lys Phe Leu Leu Met 285 Asp Ala Leu aaa ctg age att gag gac cca agt cac gag gga gag ggg ata cca eta 912 Lys Leu 290 Ser He Glu Asp Pro 295 Ser His Glu Gly Glu 300 Gly He Pro Leu tat gat gca ate aaa tgc atg aaa act ttc ttt gga tgg aaa gag ccc 960 Tyr 305 Asp Ala He Lys Cys 310 Met Lys Thr Phe Phe 315 Gly Trp Lys Glu Pro 320 agt att gtt aaa cca cat aaa aag ggt ata aac ccg aac tat etc caa 1008 Ser He Val Lys Pro 325 His Lys Lys Gly He 330 Asn Pro Asn Tyr Leu 335 Gin act tgg aag caa gta tta gaa gaa ata caa gac ett gag aac gaa gaa 1056 Thr Trp Lys Gin 340 Val Leu Glu Glu He 345 Gin Asp Leu Glu Asn 350 Glu Glu agg acc ccc aag acc aag aat atg aaa aaa aca age caa ttg aaa tgg 1104 Arg Thr Pro 355 Lys Thr Lys Asn Met 360 Lys Lys Thr Ser Gin 365 Leu Lys Trp gca eta ggt gaa aat atg gca cca gag aaa gtg gat ttt gag gat tgt 1152 Ala Leu 370 Gly Glu Asn Met Ala 375 Pro Glu Lys Val Asp 380 Phe Glu Asp Cys

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   aaa Lys 385 gac Asp ate He aat Asn gat Asp tta aaa caa Gin tat Tyr gac Asp agt Ser 395 gat Asp gag Glu cca Pro gaa Glu gca Ala 400 1200 Leu 390 Lys agg tet ett gca agt tgg att caa agt gag ttc aac aag get tgt gag 1248 Arg Ser Leu Ala Ser Trp He Gin Ser Glu Phe Asn Lys Ala Cys Glu 405 410 415 ctg aca gat tea age tgg ata gag etc gat gaa att ggg gag gat gtc 1296 Leu Thr Asp Ser Ser Trp lie Glu Leu Asp Glu He Gly Glu Asp Val •420 425 430 gcc cca ata gaa tac att geg age atg agg aga aat tat ttt act get 1344 Ala Pro He Glu Tyr He 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 He Ser His Cys Arg Ala Thr Glu Tyr He Met Lys Gly Val Tyr 450 455 460 ate aac act get eta etc aat gca tcc tgt get geg atg gat gaa ttt 1440 lie 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 Gin Leu He Pro Met lie 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 He lie Lys Gly Arg Ser His Leu Arg 500 505 510 aat gat act gac gtg gtg aac ttt gta agt atg gaa ttt tet etc 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 eta 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 ett tta aga act get gta ggt caa gtg tea aga ccc 1680 lie Gly Asp Met Leu Leu Arg Thr Ala Val Gly Gin Val Ser Arg Pro 545 550 ¹ 555 560 atg ttt ttg tat gta agg aca aat gga acc tet aaa att aaa atg aaa 1728 Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys lie Lys Met Lys 565 570 575 tgg gga atg gaa atg agg ege tgc etc ett cag tet ctg caa cag att 1776 Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gin Ser Leu Gin Gin • He 580 585 590 gaa age atg ate gaa get gag tcc tea gtc aaa gaa aag gac atg acc 1824 Glu Ser Met He Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr 595 600 605 aaa gaa ttt ttt gag aac aaa tea gag aca tgg cct ata gga gag tcc 1872 Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro lie Gly Glu Ser 610 615 620

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   ccc Pro 625 aaa gga gtg Val gaa Glu gag ggc tea Ser ate He ggg aag gtt Gly Lys Val 635 tgc Cys agg Arg acc Thr tta Leu 640 1920 Lys Gly Glu 630 Gly tta gca aaa tet gtg ttt aac agt tta tat gca tet cca caa ctg gaa 1968 Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gin Leu Glu 645 650 655 gga ttt tea get gaa tet agg aaa tta Ctt etc att gtt cag get ctt 2016 Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu He Val Gin 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 lie Gly Gly Leu Tyr Glu 675 680 685 tea att gag gag tgc ctg att aat gat ccc tgg gtt ttg ctt aat gca 2112 Ser He Glu Glu Cys Leu He Asn Asp Pro Trp Val Leu Leu Asn Ala 690 695 700 tet tgg ttc aac tcc ttc etc 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 Gin Cys Phe Asn Pro Met lie Val Glu Leu 15 10 15

   Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asn Pro Lys He Glu Thr 20 25 30

   Asn Lys Phe Ala Ala He Cys Thr His Leu Glu Val Cys Phe Met Tyr 35 40 45

   Ser Asp Phe His Phe He Asn Glu Leu Gly Glu Ser Val Val He Glu 50 55 60

   Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu He He Glu 65 70 75 80

   Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser He Cys Asn 85 90 95

   Thr Thr Arg Ala Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr 100 105 110

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   Lys Glu Asn Arg Phe Val 115 lie Tyr Tyr Leu Glu Lys 130

   He His He Phe Ser Phe 145 150

   Tyr Thr Leu Asp Glu Glu 165

   Thr He Arg Gin Glu Met 180

   Gin Ser Glu Arg Gly Glu 195

   Gly Thr Met Arg Lys Leu 210

   Ser Leu Glu Asn Phe Arg 225 230

   Cys He Glu Ser Lys Leu 245

   He Glu Pro Phe Ser Lys 260

   Gly Pro Pro Cys His Gin 275

   Lys Leu Ser He Glu Asp 290

   Tyr Asp Ala He Lys Cys 305 310

   Ser He Val Lys

   103

   Glu He Gly Val Thr 120

   Ala Asn Lys He Lys 135

   Thr Gly Glu Glu Met 155

   Ser Arg Ala Arg He 170

   Ala Ser Arg Gly Leu 185

   Glu Thr lie Glu Glu 200

   Ala Asn Tyr Ser Leu 215

   Val Tyr He Asp Gly 235

   Ser Gin Met Ser Lys 250

   Thr Thr Pro Arg Pro 265

   Arg Ser Lys Phe Leu 280

   Pro Ser His Glu Gly 295

   Met Lys Thr Phe Phe 315

   Pro His Lys Lys 325

   Gly He Asn 330

   Arg Arg Glu Val His 125

   Ser Glu Lys Thr His 140

   Ala Thr Lys Ala Asp 160

   Lys Thr Arg Leu Phe 175

   Trp Asp Ser Phe Arg 190

   Arg Phe Glu He Thr 205

   Pro Pro Asn Phe Ser 220

   Phe Glu Pro Asn Gly 240

   Glu Val Asn Ala Lys 255

   Leu Lys Met Pro Gly 270

   Leu Met Asp Ala Leu 285

   Glu Gly He Pro Leu 300

   Gly Trp Lys Glu Pro 320

   Pro Asn Tyr Leu Gin 335

   Thr Trp Lys Gin Val Leu Glu Glu He Gin Asp Leu Glu Asn Glu Glu 340 345 350

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   Arg Thr Pro Lys Thr Lys Asn Met 355 360

   Ala Leu Gly Glu Asn Met Ala Pro 370 375

   Lys Asp lie Asn Asp Leu Lys Gin 385 390

   Arg Ser Leu Ala Ser Trp He Gin 405

   Leu Thr Asp Ser Ser Trp He Glu 420'

   104

   Lys Lys Thr Ser Gin Leu Lys Trp 365

   Glu Lys Val Asp Phe Glu Asp Cys 380

   Tyr Asp Ser Asp Glu Pro Glu Ala 395 400

   Ser Glu Phe Asn Lys Ala Cys Glu 410 415

   Leu Asp Glu He Gly Glu Asp Val 425 430

   Ala Pro He Glu Tyr He Ala Ser 435 440

   Met Arg Arg Asn Tyr Phe Thr Ala 445

   Glu He Ser His Cys Arg Ala Thr 450 455

   Glu Tyr He Met Lys Gly Val Tyr 460

   He Asn Thr Ala Leu Leu Asn Ala 465 470

   Ser Cys Ala Ala Met Asp Glu Phe 475 480

   Gin Leu He Pro Met He Ser Lys 485

   Cys Arg Thr Lys Glu Gly Arg Arg 490 495

   Lys Thr Asn Leu Tyr Gly Phe He 500

   He Lys Gly Arg Ser His Leu Arg 505 510

   Asn Asp Thr Asp Val Val Asn Phe 515 520

   Val Ser Met Glu Phe Ser Leu Thr 525

   Asp Pro Arg Phe Glu Pro His Lys 530 535

   Trp Glu Lys Tyr Cys Val Leu Glu 540

   He Gly Asp Met Leu Leu Arg Thr 545 550

   Ala Val Gly Gin Val Ser Arg Pro 555 560

   Met Phe Leu Tyr Val Arg Thr Asn 565

   Gly Thr Ser Lys He Lys Met Lys 570 ' 575

   Trp Gly Met Glu Met Arg Arg Cys 580

   Leu Leu Gin Ser Leu Gin Gin He 585 590

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   Glu Ser Met lie Glu Ala Glu Ser 595 500

   Lys Glu Phe Phe Glu Asn Lys Ser 610 615

   Pro Lys Gly Val Glu Glu Gly Ser 625 630

   Leu Ala Lys Ser Val Phe Asn Ser 645

   Gly Phe Ser Ala Glu Ser Arg Lys 660

   Arg Asp Asp Leu Glu Pro Gly Thr 675 680

   Ser lie Glu Glu Cys Leu lie Asn 690 695

   Ser Trp Phe Asn Ser Phe Leu Thr 705 710 <210> 53 <211> 844 <212> DNA <213> Influenza virus

   105

   Ser Val Lys Glu Lys Asp Met Thr 605

   Glu Thr Trp Pro lie Gly Glu Ser 620 lie Gly Lys Val Cys Arg Thr Leu 635 640

   Leu Tyr Ala Ser Pro Gin Leu Glu 650 655

   Leu Leu Leu lie Val Gin Ala Leu 665 670

   Phe Asp lie Gly Gly Leu Tyr Glu 685

   Asp Pro Trp Val Leu Leu Asn Ala 700

   His Ala Leu Lys 715 <220>

   <221> CDS <222> (1)..(690) <400> 53 atg gat tcc aac act gtg tea age ttt cag gta gac tgt ttt ett tgg

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

   15 10 15 cat gtc cgt aaa ega ttc gca gac caa gaa ctg ggt gat gcc cca ttc

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

   20 25 30 ett gac egg ett ege ega gac cag aag tcc eta agg gga aga ggt age

   Leu Asp Arg Leu Arg Arg Asp Gin Lys Ser Leu Arg Gly Arg Gly Ser

   35 40 45 act ett ggt ctg gac ate gaa aca gcc act cat gca gga aag cag ata

   Thr Leu Gly Leu Asp lie Glu Thr Ala Thr His Ala Gly Lys Gin lie

   50 55 60 gtg gag cag att ctg gaa aag gaa tea gat gag gca ett aaa atg acc

   144

   192

   240

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   Val 65 Glu Gin He Leu Glu 70 Lys Glu Ser Asp Glu 75 Ala Leu Lys Met Thr 80 att gcc tet gtt cct get tea ege tac tta act gac atg act ett gat 288 He Ala Ser Val Pro 85 Ala Ser Arg Tyr Leu 90 Thr Asp Met Thr Leu 95 Asp gag atg tea aga gac tgg ttc atg etc atg ccc aag caa aaa gta aca 336 Glu Met Ser Arg 100 Asp Trp Phe Met Leu 105 Met Pro Lys Gin Lys 110 Val Thr ggc tcc eta tgt ata aga atg gac cag gca ate atg gat aag aac ate 384 Gly Ser Leu 115 Cys lie Arg Met Asp 120 Gin Ala He Met Asp 125 Lys Asn He ata ett aaa gca aac ttt agt gtg att ttc gaa agg ctg gaa aca eta 432 He Leu 130 Lys Ala Asn Phe Ser 135 Val He Phe Glu Arg 140 Leu Glu Thr Leu ata eta ett aga gcc ttc acc gaa gaa gga gca gtc gtt ggc gaa att 480 He 145 Leu Leu Arg Ala Phe 150 Thr Glu Glu Gly Ala 155 Val Val Gly Glu He 160 tea cca tta cct tet ett cca gga cat act aat gag gat gtc aaa aat 528 Ser Pro Leu Pro Ser 165 Leu Pro Gly His Thr 170 Asn Glu Asp Val Lys 175 Asn gca att ggg gtc etc ate gga gga ett aaa tgg aat gat aat aeg gtt 576 Ala He Gly Val 180 Leu He Gly Gly Leu 185 Lys Trp Asn Asp Asn 190 Thr Val aga ate tet gaa act eta cag aga ttc get tgg aga age agt cat gaa 624 Arg lie Ser 195 Glu Thr Leu Gin Arg 200 Phe Ala Trp Arg Ser 205 Ser His Glu aat ggg aga cct tea ttc cct tea aaa cag aaa ega aaa atg gag aga 672 Asn Gly 210 Arg Pro Ser Phe Pro 215 Ser Lys Gin Lys Arg 220 Lys Met Glu Arg

   aca att aag cca gaa att tgaagaaata agatggttga ttgaagaagt 720

   Thr lie Lys Pro Glu He 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 Gin Val Asp Cys Phe Leu Trp 15 10 15

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   His Val Arg Lys Arg Phe Ala Asp Gin Glu Leu Gly Asp Ala Pro Phe 20 25 30

   Leu Asp Arg Leu Arg Arg Asp Gin Lys Ser Leu Arg Gly Arg Gly Ser 35 40 45

   Thr Leu Gly Leu Asp lie Glu Thr Ala Thr His Ala Gly Lys Gin He 50 55 60

   Val Glu Gin He Leu Glu Lys Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80

   He 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 Gin Lys Val Thr 100 105 110

   Gly Ser Leu Cys He Arg Met Asp Gin Ala He Met Asp Lys Asn He 115 120 125 lie Leu Lys Ala Asn Phe Ser Val He Phe Glu Arg Leu Glu Thr Leu 130 135 140

   He Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu He 145 150 155 160

   Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn 165 170 175

   Ala He Gly Val Leu He Gly Gly Leu Lys Trp Asn Asp Asn Thr Val 180 185 190

   Arg He Ser Glu Thr Leu Gin Arg Phe Ala Trp Arg Ser Ser His Glu 195 200 205

   Asn Gly Arg Pro Ser Phe Pro Ser Lys Gin Lys Arg Lys Met Glu Arg 210 215 220

   Thr He Lys Pro Glu He 230

   225 <210> 55 <211> 1497 <212> DNA

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   108 <213> Influenza virus

   <220> <221> CDS <222> (1).. <400> 55 (1497) atg geg tet caa ggc acc aaa ega tcc tat gaa cag atg gaa act gat Met 1 Ala Ser Gin Gly 5 Thr Lys Arg Ser Tyr 10 Glu Gin Met Glu Thr 15 Asp ggg gaa ege cag aat gca act gaa ate aga gca tet gtc gga agg atg Gly Glu Arg Gin 20 Asn Ala Thr Glu He 25 Arg Ala Ser Val Gly 30 Arg Met gtg gga gga ate gga egg ttt tat gtc cag atg tgt act gag ett aaa Val Gly Gly 35 He Gly Arg Phe Tyr 40 Val Gin Met Cys Thr 45 Glu Leu Lys eta aac gac cat gaa ggg egg ctg att cag aac age ata aca ata gaa Leu Asn 50 Asp His Glu Gly Arg 55 Leu He Gin Asn Ser 60 He Thr lie Glu agg atg gtg ett teg gca ttc gac gaa aga aga aac aag tat etc gag Arg 65 Met Val Leu Ser Ala 70 Phe Asp Glu Arg Arg 75 Asn Lys Tyr Leu Glu 80 gag cat ccc agt get ggg aaa gac cct aag aaa aeg gga ggc ccg ata Glu His Pro Ser Ala 85 Gly Lys Asp Pro Lys 90 Lys Thr Gly Gly Pro 95 lie tac aga aga aaa gat ggg aaa tgg atg agg gaa etc ate etc cat gat Tyr Arg Arg Lys 100 Asp Gly Lys Trp Met 105 Arg Glu Leu He Leu 110 His Asp aaa gaa gaa ate atg aga ate tgg cgt cag gee aac aat ggt gaa gac Lys Glu Glu 115 He Met Arg He Trp 120 Arg Gin Ala Asn Asn 125 Gly Glu Asp get act get ggt ett act cat atg atg ate tgg cac tcc aat etc aat Ala Thr 130 Ala Gly Leu Thr His 135 Met Met lie Trp His 140 Ser Asn Leu Asn gac acc aca tac caa aga aca agg get ett gtt egg act ggg atg gat Asp 145 Thr Thr Tyr Gin Arg 150 Thr Arg Ala Leu Val 155 Arg Thr Gly Met Asp 160 CCC aga atg tgc tet ctg atg caa ggc tea acc etc cca egg aga tet Pro Arg Met Cys Ser 165 Leu Met Gin Gly Ser 170 Thr Leu Pro Arg Arg 175 Ser gga gcc get ggt get gca gta aaa ggc gtt gga aca atg gta atg gaa Gly Ala Ala Gly 180 Ala Ala Val Lys Gly 185 Val Gly Thr Met Val 190 Met Glu etc ate aga atg ate aag ege gga ata aat gat egg aat ttc tgg aga Leu lie Arg 195 Met He Lys Arg Gly 200 He Asn Asp Arg Asn 205 Phe Trp Arg

   144

   192

   240

   288

   336

   384

   432

   480

   528

   576

   624

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   ggt Gly gaa Glu 210 aat Asn ggt Gly ega Arg aga Arg acc Thr 215 aga Arg att lie get Ala tat Tyr gaa Glu 220 aga Arg atg Met tgc Cys aat Asn 672 ate etc aaa ggg aaa ttt cag aca gca gca caa egg get atg atg gac 720 lie 225 Leu Lys Gly Lys Phe 230 Gin Thr Ala Ala Gin 235 Arg Ala Met Met Asp 240 cag gtg agg gaa ggc cgc aat cct gga aac get gag att gag gat etc 768 Gin Val Arg Glu Gly 245 Arg Asn Pro Gly Asn 250 Ala Glu lie Glu Asp 255 Leu att ttc ttg gca ega tea gca ett att ttg aga gga tea gta gcc cat 816 He Phe Leu Ala 260 Arg Ser Ala Leu He 265 Leu Arg Gly Ser Val 270 Ala His aaa tea tgc eta cct gcc tgt gtt tat ggc ett gca gta acc agt ggg 864 Lys Ser Cys 275 Leu Pro Ala Cys Val 280 Tyr Gly Leu Ala Val 285 Thr Ser Gly tat gac ttt gag aag gaa gga tac tet ctg gtt gga att gat cct ttc 912 Tyr Asp 290 Phe Glu Lys Glu Gly 295 Tyr Ser Leu Val Gly 300 He Asp Pro Phe aaa eta etc cag aac agt caa att ttc agt eta ate aga cca aaa gaa 960 Lys 305 Leu Leu Gin Asn Ser 310 Gin He Phe Ser Leu 315 He Arg Pro Lys Glu 320 aac cca gca cac aaa age cag ttg gtg tgg atg gca tgc cat tet gca 1008 Asn Pro Ala His Lys 325 Ser Gin Leu Val Trp 330 Met Ala Cys His Ser 335 Ala gca ttt gag gat ctg aga gtt tta aat ttc att aga gga acc aaa gta 1056 Ala Phe Glu Asp 340 Leu Arg Val Leu Asn 345 Phe He Arg Gly Thr 350 Lys Val ate cca aga gga cag tta aca acc aga gga gtt caa att get tea aat 1104 He Pro Arg 355 Gly Gin Leu Thr Thr 36& Arg Gly Val Gin He 365 Ala Ser Asn gaa aac atg gag aca ata aat tet age aca ett gaa ctg aga age aaa 1152 Glu Asn 370 Met Glu Thr He Asn 375 Ser Ser Thr Leu Glu 380 Leu Arg Ser Lys tat tgg gca ata agg acc aga age gga gga aac acc agt caa cag aga 1200 Tyr 385 Trp Ala He Arg Thr 390 Arg Ser Gly Gly Asn 395 Thr Ser Gin Gin Arg 400 gca tet gca gga cag ata agt gtg caa cct act ttc tea gta cag aga 1248 Ala Ser Ala Gly Gin 405 lie Ser Val Gin Pro 410 Thr Phe Ser Val Gin 415 Arg aat ett ccc ttt gag aga gca acc att atg get gca ttc act ggt aac 1296 Asn Leu Pro Phe 420 Glu Arg Ala Thr He 425 Met Ala Ala Phe Thr 430 Gly Asn act gaa gga agg act tec gac atg aga aeg gaa ate ata agg atg atg 1344 Thr Glu Gly 435 Arg Thr Ser Asp Met 440 Arg Thr Glu He He 445 Arg Met Met

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   gaa Glu aat Asn 450 gcc Ala aaa Lys tea Ser gaa Glu gat Asp 455 gtg Val tct Ser gag etc teg gac gaa aag gca aeg aac Glu Leu Ser Asp Glu Lys Ala Thr Asn 465 470 atg age aat gaa ggg tct tat ttc ttc Met Ser Asn Glu Gly Ser Tyr Phe Phe 485 gac agt taa Asp Ser

   ttc Phe cag Gin ggg Gly 460 egg Arg gga Gly gtc Val ttc Phe 1392 ccg ate gtg cct tcc ttt gac 1440 Pro He Val Pro Ser Phe Asp 475 480 gga gac aat get gag gag ttt 1488 Gly Asp Asn Ala Glu Glu Phe 490 495

   1497

   <210> 56 <211> 498 <212> PRT <213> Influenza virus <400> 56 Met 1 Ala Ser Gin Gly 5 Thr Lys Arg Ser Gly Glu Arg Gin 20 Asn Ala Thr Glu He 25 Val Gly Gly He 35 Gly Arg Phe Tyr 40 Val Leu Asn 50 Asp His Glu Gly Arg 55 Leu He Arg 65 Met Val Leu Ser Ala 70 Phe Asp Glu Glu His Pro Ser Ala 85 Gly Lys Asp Pro Tyr Arg Arg Lys 100 Asp Gly Lys Trp Met 105 Lys Glu Glu He 115 Met Arg He Trp 12 0 Arg Ala Thr 130 Ala Gly Leu Thr His 135 Met Met

   Tyr Glu Gin Met Glu Thr Asp 10 15

   Arg Ala Ser Val Gly Arg Met 30

   Gin Met Cys Thr Glu Leu Lys 45

   Gin Asn Ser lie Thr He Glu SO

   Arg Arg Asn Lys Tyr Leu Glu 75 80

   Lys Lys Thr Gly Gly Pro He 90 95

   Arg Glu Leu He Leu His Asp 110

   Gin Ala Asn Asn Gly Glu Asp 125

   He Trp His Ser Asn Leu Asn 140

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   Asp Thr Thr Tyr Gin Arg Thr Arg 145 150

   Pro Arg Met Cys Ser Leu Met Gin 165

   Gly Ala Ala Gly Ala Ala Val Lys 180

   Leu lie Arg Met He Lys Arg Gly 195 200

   Ύ'/ Gly Glu Asn Gly Arg Arg Thr Arg

   37¹ 210 215

   O <N

   He Leu Lys Gly Lys Phe Gin Thr 225 230

   111

   Ala Leu Val Arg Thr Gly Met Asp 155 160

   Gly Ser Thr Leu Pro Arg Arg Ser 170 175

   Gly Val Gly Thr Met Val Met Glu 185 190

   He Asn Asp Arg Asn Phe Trp Arg 205

   He Ala Tyr Glu Arg Met Cys Asn 220

   Ala Ala Gin Arg Ala Met Met Asp 235 240

   Gin Val Arg Glu Gly Arg Asn Pro 245

   Gly Asn Ala Glu He Glu Asp Leu 250 255

   He Phe Leu Ala Arg Ser Ala Leu 260

   He Leu Arg Gly Ser Val Ala His 265 270

   Lys Ser Cys Leu Pro Ala Cys Val 275 280

   Tyr Gly Leu Ala Val Thr Ser Gly 285

   Tyr Asp Phe Glu Lys Glu Gly Tyr 290 295

   Ser Leu Val Gly He Asp Pro Phe 300

   Lys Leu Leu Gin Asn Ser Gin He 305 310

   Phe Ser Leu He Arg Pro Lys Glu 315 320

   Asn Pro Ala His Lys Ser Gin Leu 325

   Val Trp Met Ala Cys His Ser Ala 330 335

   Ala Phe Glu Asp Leu Arg Val Leu 340

   Asn Phe He Arg Gly Thr Lys Val 345 350

   He Pro Arg Gly Gin Leu Thr Thr 355 360

   Arg Gly Val Gin He Ala Ser Asn 365

   Glu Asn Met Glu Thr He Asn Ser Ser Thr Leu Glu Leu Arg Ser Lys 370 375 380

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   Tyr Trp 385 Ala Tie Arg Thr Arg Ser 390 Gly Gly Asn 395 Thr Ser Gin Gin Arg 400 Ala Ser Ala Gly Gin He Ser Val Gin Pro Thr Phe Ser Val Gin Arg 405 410 415 Asn Leu Pro Phe Glu Arg Ala Thr He Met Ala Ala Phe Thr Gly Asn 420 425 430 Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu He He Arg Met Met 435 440 445 Glu Asn Ala Lys Ser Glu Asp Val Ser Phe Gin Gly Arg Gly Val Phe 450 455 460 Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro He 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 <22: >> (1) - · . (1413) <400> 57 atg aat cca aat caa aag ata ata gca att gga ttt gca tea ttg ggg 48 Met Asn Pro Asn Gin Lys lie He Ala He Gly Phe Ala Ser Leu Gly 1 5 10 15 ata tta ate att aat gtc att etc cat gta gtc age att ata gta aca 96 He Leu lie lie Asn Val lie Leu His Val Val Ser lie He Val Thr 20 25 30 gta ctg gtc etc aat aac aat aga aca gat ctg aac tgc aaa ggg aeg 144 Val Leu Val Leu Asn Asn Asn Arg Thr Asp Leu Asn Cys Lys Gly Thr 35 40 45 ate ata aga gaa tac aat gaa aca gta aga gta gaa aaa ett act caa 192 lie lie Arg Glu Tyr Asn Glu Thr Val Arg Val Glu Lys Leu Thr Gin 50 55 60 tgg tat aat acc agt aca att aag tac ata gag aga cct tea aat gaa 240

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   Trp 65 Tyr Asn Thr Ser Thr 70 He Lys Tyr He Glu 75 Arg Pro Ser Asn Glu 80 tac tac atg aat aac act gaa cca ett tgt gag gcc caa ggc ttt gca 288 Tyr Tyr Met Asn Asn 85 Thr Glu Pro Leu Cys 90 Glu Ala Gin Gly Phe 95 Ala cca ttt tcc aaa gat aat gga ata ega att ggg teg aga ggc cat gtt 336 Pro Phe Ser Lys 100 Asp Asn Gly He Arg 105 He Gly Ser Arg Gly 110 His Val ttt gtg ata aga gaa cct ttt gta tea tgt teg ccc tea gaa tgt aga 384 Phe Val He 115 Arg Glu Pro Phe Val 120 Ser Cys Ser Pro Ser 125 Glu Cys Arg 1 acc ttt ttc etc aca cag ggc tea tta etc aat gac aaa cat tct aac 432 Thr Phe 130 Phe Leu Thr Gin Gly 135 Ser Leu Leu Asn Asp 140 Lys His Ser Asn ggc aca ata aag gat ega agt ccg tat agg act ttg atg agt gtc aaa 480 Gly 145 Thr lie Lys Asp Arg 150 Ser Pro Tyr Arg Thr 155 Leu Met Ser Val Lys 160 ata ggg caa tea cct aat gta tat caa get agg ttt gaa teg gtg gca 528 lie Gly Gin Ser Pro 165 Asn Val Tyr Gin Ala 170 Arg Phe Glu Ser Val 175 Ala tgg tea gca aca gca tgc cat gat gga aaa aaa tgg atg aca gtt gga 576 Trp Ser Ala Thr 180 Ala Cys His Asp Gly 185 Lys Lys Trp Met Thr 190 Val Gly gtc aca ggg ccc gac aat caa gca att gca gta gtg aac tat gga ggt 624 Val Thr Gly 195 Pro Asp Asn Gin Ala 200 He Ala Val Val Asn 205 Tyr Gly Gly gtt ccg gtt gat att att aat tea tgg gca ggg gat att tta aga acc 672 Val Pro 210 Val Asp He lie Asn 215 Ser Trp Ala Gly Asp 220 He Leu Arg Thr caa gaa tea tea tgc acc tgc att aaa gga gac tgt tat tgg gta atg 720 Gin 225 Glu Ser Ser Cys Thr 230 Cys lie Lys Gly Asp 235 Cys Tyr Trp Val Met 240 act gat gga ccg gca aat agg caa get aaa tat agg ata ttc aaa gca 768 Thr Asp Gly Pro Ala 245 Asn Arg Gin Ala Lys 250 Tyr Arg He Phe Lys 255 Ala aaa gat gga aga gta att gga caa act gat ata agt ttc aat ggg gga 816 Lys Asp Gly Arg 260 Val He Gly Gin Thr 265 Asp He Ser Phe Asn 270 Gly Gly cac ata gag gag tgt tct tgt tac ccc aat gaa ggg aag gtg gaa tgc 864 His lie Glu 275 Glu Cys Ser Cys Tyr 280 Pro Asn Glu Gly Lys 285 Val Glu Cys ata tgc agg gac aat tgg act gga aca aat aga cca att ctg gta ata 912 lie Cys 290 Arg Asp Asn Trp Thr 295 Gly Thr Asn Arg Pro 300 He Leu Val He tct tct gat eta teg tac aca gtt gga tat ttg tgt get ggc att ccc 960

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   Ser 305 Ser Asp Leu Ser Tyr Thr Val 310 Gly Tyr Leu Cys Ala 315 Gly He Pro 320 act gac act cct agg gga gag gat agt caa ttc aca ggc tea tgt aca 1008 Thr Asp Thr Pro Arg Gly Glu Asp Ser Gin Phe Thr Gly Ser Cys Thr 325 330 335 agt cct ttg gga aat aaa gga tac ggt gta aaa ggc ttc ggg ttt ega 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 tea aga 1104 Gin Gly Thr Asp Val Trp Ala Gly Arg Thr lie Ser Arg Thr Ser Arg 355 360 365 tea gga ttc gaa ata ata aaa ate agg aat ggt tgg aca cag aac agt 1152 Ser Gly Phe Glu lie He Lys He Arg Asn Gly Trp Thr Gin Asn Ser 370 375 380 aag gac caa ate agg agg caa gtg att ate gat gac cca aat tgg tea 1200 Lys Asp Gin He Arg Arg Gin Val He He Asp Asp Pro Asn Trp Ser 385 390 395 400 gga tat age ggt tet 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 He Arg Gly Lys Pro Glu 420 425 430 gaa aca aca ata tgg acc tet age age tcc att gtg atg tgt gga gta 1344 Glu Thr Thr He Trp Thr Ser Ser Ser Ser He Val Met Cys Gly Val 435 440 445 gat cat aaa att gcc agt tgg tea tgg cac gat gga get att ett ccc 1392 Asp His Lys He Ala Ser Trp Ser Trp His Asp Gly Ala lie Leu Pro 450 455 460 ttt gac ate gat aag atg taa 1413 Phe Asp He Asp Lys Met

   465 470 <210> 58 <211> 470 ,<212> PRT <213> Influenza virus <400> 58

   Met Asn Pro Asn Gin Lys lie He Ala He Gly Phe Ala Ser Leu Gly 15 10 15

   He Leu lie He Asn Val He Leu His Val Val Ser He He Val Thr 20 25 30

   Val Leu Val Leu Asn Asn Asn Arg Thr Asp Leu Asn Cys Lys Gly Thr

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   35 40 45 lie lie Arg Glu Tyr Asn Glu Thr Val Arg Val Glu Lys Leu Thr Gin 50 ’ 55 , 60

   Trp Tyr Asn Thr Ser Thr He Lys Tyr He Glu Arg Pro Ser Asn Glu 65 70 75 80

   Tyr Tyr Met Asn Asn Thr Glu Pro Leu Cys Glu Ala Gin Gly Phe Ala 85 90 95

   Pro Phe Ser Lys Asp Asn Gly He Arg He Gly Ser Arg Gly His Val 100 105 110

   Phe Val He Arg Glu Pro Phe Val Ser Cys Ser Pro Ser Glu Cys Arg 115 120 125

   Thr Phe Phe Leu Thr Gin Gly Ser Leu Leu Asn Asp Lys His Ser Asn 130 135 140

   Gly Thr He Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser Val Lys 145 150 155 160

   He Gly Gin Ser Pro Asn Val Tyr Gin 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 Gin Ala He Ala Val Val Asn Tyr Gly Gly 195 200 205

   Val Pro Val Asp He He Asn Ser Trp Ala Gly Asp He Leu Arg Thr 210 215 220

   Gin Glu Ser Ser Cys Thr Cys He Lys Gly Asp Cys Tyr Trp Val Met 225 230 235 240

   Thr Asp Gly Pro Ala Asn Arg Gin Ala Lys Tyr Arg He Phe Lys Ala 245 250 255

   Lys Asp Gly Arg Val He Gly Gin Thr Asp He Ser Phe Asn Gly Gly 260 265 270

   His lie Glu Glu Cys Ser Cys Tyr Pro Asn Glu Gly Lys Val Glu Cys

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   275 280 285 lie Cys Arg Asp Asn Trp Thr Gly Thr Asn Arg Pro He Leu Val He 290 295 300

   Ser Ser Asp Leu Ser Tyr Thr Val Gly Tyr Leu Cys Ala Gly He Pro 305 310 315 320

   Thr Asp Thr Pro Arg Gly Glu Asp Ser Gin 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

   Gin Gly Thr Asp Val Trp Ala Gly Arg Thr lie Ser Arg Thr Ser Arg 355 360 365

   Ser Gly Phe Glu He lie Lys He Arg Asn Gly Trp Thr Gin Asn Ser 370 375 380

   Lys Asp Gin lie Arg Arg Gin Val He lie 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 i 410 415

   Cys Leu Val Pro Cys Phe Trp Val Glu Met He Arg Gly Lys Pro Glu 420 425 430

   Glu Thr Thr He Trp Thr Ser Ser Ser Ser He Val Met Cys Gly Val 435 440 445

   Asp His Lys He Ala Ser Trp Ser Trp His Asp Gly Ala He Leu Pro 450 455 460

   Phe Asp He Asp Lys Met 465 470 <210> 59 <211> 981 <212.> DNA <213> Influenza virus <220>

   <221> CDS <222> (1) . . (756)

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   <400> 59 gtc Val gaa Glu acg Thr tac Tyr 10 gtt Val etc Leu tet Ser ate He gta Val 15 cca Pro 48 atg Met 1 agt Ser ctt Leu eta Leu acc Thr 5 gag Glu tea ggc ccc etc aaa gcc gag ate geg cag aga ctt gaa gat gtc ttt 96 Ser Gly Pro Leu 20 Lys Ala Glu He Ala 25 Gin Arg Leu Glu Asp 30 Val Phe geg gga aag aac acc gat ctt gag gca etc atg gaa tgg eta aag aca 144 Ala Gly Lys 35 Asn Thr Asp Leu Glu 40 Ala Leu Met Glu Trp 45 Leu Lys Thr aga cca ate ctg tea cct ctg act aaa ggg att tta gga ttt gta ttc 192 Arg Pro 50 lie Leu Ser Pro Leu 55 Thr Lys Gly He Leu 60 Gly Phe Val Phe acg etc acc gtg ccc agt gag ega gga ctg cag cgt aga cgc ttt gtc 240 Thr 65 Leu Thr Val Pro Ser 70 Glu Arg Gly Leu Gin 75 Arg Arg Arg Phe Val 80 caa aat gee ctt agt gga aac gga gat cca aac aac atg gac aga gca 288 Gin Asn Ala Leu Ser 85 Gly Asn Gly Asp Pro 90 Asn Asn Met Asp Arg 95 Ala gta aaa ctg tac agg aag ctt aaa aga gaa ata aca ttc cat ggg gca 336 Val Lys Leu Tyr 100 Arg Lys Leu Lys Arg 105 Glu He Thr Phe His 110 Gly Ala aaa gag gtg gca etc age tat tcc act ggt gca eta gcc age tgc atg 384 Lys Glu Val 115 Ala Leu Ser Tyr Ser 120 Thr Gly Ala Leu Ala 125 Ser Cys Met gga etc ata tac aac aga atg gga act gtt aca acc gaa gtg gca ttt 432 Gly Leu 130 lie Tyr Asn Arg Met 135 Gly Thr Val Thr Thr 14 0 Glu Val Ala Phe ggc ctg gta tgc gee aca tgt gaa cag att get gat tcc cag cat ega 480 Gly 145 Leu •Val Cys Ala Thr 150 Cys Glu Gin He Ala 155 Asp Ser Gin His Arg 160 tet cac agg cag atg gtg aca aca acc aac cca tta ate aga cat gaa 528 Ser His Arg Gin Met 165 Val Thr Thr Thr Asn 170 Pro Leu He Arg His 175 Glu aac aga atg gta tta gcc agt acc acg get aaa gee atg gaa cag atg 576 Asn Arg Met Val 180 Leu Ala Ser Thr Thr 185 Ala Lys Ala Met Glu 190 Gin Met gca gga teg agt gag cag gca gca gag gcc atg gag gtt get agt agg 624 Ala Gly Ser 195 Ser Glu Gin Ala Ala 200 Glu Ala Met Glu Val 205 Ala Ser Arg get agg cag atg gta cag gca atg aga acc att ggg acc cac cct age 672 Ala Arg 210 Gin Met Val' Gin Ala 215 Met Arg Thr He Gly 220 Thr His Pro Ser tcc agt gee ggt ttg aaa gat gat etc ctt gaa aat tta cag gcc tac 720 Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gin Ala Tyr

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   225 230 235 240 cag aaa egg atg gga gtg caa atg cag ega ttc aag tgatcctctc 766 Gin Lys Arg Met Gly Val Gin Met Gin Arg Phe Lys 245 250

   gtcattgcag caagtatcat tgggatcttg cacttgatat tgtggattct tgategtett 826 ttcttcaaat teatttateg tegeettaaa tacgggttga aaagagggcc ttctacggaa 886 ggagtacctg agtetatgag ggaagaatat cggcaggaac ageagaatge 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 lie Val Pro 15 10 15

   Ser Gly Pro Leu Lys Ala Glu He Ala Gin 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 He Leu Ser Pro Leu Thr Lys Gly He Leu Gly Phe Val Phe 50 55 60

   Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gin Arg Arg Arg Phe Val 65 70 75 80

   Gin 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 He Thr Phe His Gly Ala 100 105 HO

   Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met 115 120 125

   Gly Leu He Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val' Ala Phe 130 135 140

   Gly Leu Val Cys Ala Thr Cys Glu Gin He Ala Asp Ser Gin His Arg 145 150 155 160

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   Ser His Arg Gin Met Val Thr Thr Thr Asn Pro Leu He Arg His Glu 165 170 175 ¹ Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gin Met 180 185 190 Ala Gly Ser Ser Glu Gin Ala Ala Glu Ala Met Glu Val Ala Ser Arg 195 200 205 Ala Arg Gin Met Val Gin Ala Met Arg Thr lie Gly Thr His Pro Ser 210 215 220 Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gin Ala Tyr 225 230 235 240 Gin Lys Arg Met Gly Val Gin Met Gin 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 eta ctg acc cat tgg gcc tac agt 48 Met Lys Thr Thr He He Leu He Leu Leu Thr His Trp Ala Tyr Ser 1 5 10 15 caa aac cca ate agt ggc aat aac aca gcc aca ctg tgt ctg gga cac 96 Gin Asn Pro He 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 Gin 35 40 45 att gag gtg aca aat get aca gaa tta gtt cag age att tea atg ggg 192 lie Glu Val Thr Asn Ala Thr Glu Leu Val Gin Ser He Ser Met Gly 50 55 60 aaa ata tgc aac aaa tea tat aga att eta gat gga aga aat tgc aca 240 Lys He Cys Asn Lys Ser Tyr Arg He Leu Asp Gly Arg Asn Cys Thr 65 70 75 80 tta ata gat gca atg eta gga gac ccc cac tgt gac gcc ttt cag tat 288 Leu He Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gin Tyr 85 90 95

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   gag Glu agt Ser tgg Trp gac Asp 100 etc Leu ttt Phe ata He gaa Glu aga Arg 105 age Ser age Ser get Ala ttc Phe age Ser 110 aat Asn tgc Cys 336 tac cca tat gac ate cct gac tat gca teg etc ega tcc att gta gca 384 Tyr Pro Tyr 115 Asp He Pro Asp Tyr 120 Ala Ser Leu Arg Ser 125 He Val Ala tcc tea ggg aca gtg gaa ttc aca gca gag gga ttc aca tgg aca ggt 432 Ser Ser 130 Gly Thr Val Glu Phe 135 Thr Ala Glu Gly Phe 140 Thr Trp Thr Gly gta act caa aac gga aga agt gga gcc tgc aaa agg gga tea gcc gat 480 Val 145 Thr Gin Asn Gly Arg 150 Ser Gly Ala Cys Lys 155 Arg Gly Ser Ala Asp 160 agt ttc ttt age ega ctg aat tgg eta aca aaa tct gga age tct tac 528 Ser Phe Phe Ser Arg 165 Leu Asn Trp Leu Thr 170 Lys Ser Gly Ser Ser 175 Tyr ccc aca ttg aat gtg aca Thr atg cct aac aat aaa aat ttc gac aag eta 576 Pro Thr Leu Asn 180 Val ^lMet Pro Asn 185 Asn Lys Asn Phe Asp 190 Lys Leu tac ate tgg ggg att cat cac ccg age tea aat caa gag cag aca aaa 624 Tyr He Trp 195 Gly He His His Pro 200 Ser Ser Asn Gin Glu 205 Gin Thr Lys ttg tac ate caa gaa tea gga ega gta aca gtc tea aca aaa aga agt 672 Leu Tyr 210 He Gin Glu Ser Gly 215 Arg Val Thr Val Ser 220 Thr Lys Arg Ser caa caa aca ata ate cct aac ate gga tct aga ccg ttg gtc aga ggt 720 Gin 225 Gin Thr He He Pro 230 Asn He Gly Ser Arg 235 Pro Leu Val Arg Gly 240 caa tea ggc agg ata age ata tac tgg acc att gta aaa cct gga gat 768 Gin Ser Gly Arg He 245 Ser He Tyr Trp Thr 250 He Val Lys Pro Gly 255 Asp ate eta atg ata aac agt aat ggc aac tta gtt gca ccg egg gga tat 816 lie Leu Met He 260 Asn Ser Asn Gly Asn 265 Leu Val Ala Pro Arg 270 Gly Tyr ttt aaa ttg aac aca ggg aaa age tct gta atg aga tcc gat gta ccc 864 Phe Lys Leu 275 Asn Thr Gly Lys Ser 280 Ser Val Met Arg Ser 285 Asp Val Pro ata gac att tgt gtg tct gaa tgt att aca cca aat gga age ate tcc 912 He Asp 290 lie Cys Val Ser Glu 295 Cys He Thr Pro Asn 300 Gly Ser He Ser aac gac aag cca ttc caa aat gtg aac aaa gtt aca tat gga aaa tgc 960 Asn 305 Asp Lys Pro Phe Gin 310 Asn Val Asn Lys Val 315 Thr Tyr Gly Lys Cys 320 ccc aag tat ate agg caa aac act tta aag ctg gcc act ggg atg agg 1008 Pro Lys Tyr He Arg Gin Asn Thr Leu Lys Leu Ala Thr Gly Met Arg

   325 330 335

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   aat Asn gta Val cca gaa Pro Glu 340 aag Lys caa Gin acc Thr aga Arg gga Gly 345 ate He ttt Phe gga Gly gca ata geg gga 1056 Ala He 350 Ala Gly ttc ate gaa aac ggc tgg gaa gga atg gtt gat ggg tgg tat ggg ttc 1104 Phe He Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe 355 360 365 ega tat caa aac tet gaa gga aca ggg caa get gca gat eta aag age 1152 Arg Tyr Gin Asn Ser Glu Gly Thr Gly Gin Ala Ala Asp Leu Lys Ser 370 375 380 act caa gca gcc ate gac cag att aat gga aag tta aac aga gtg att 1200 Thr Gin Ala Ala He Asp Gin He Asn Gly Lys Leu Asn Arg Val He 385 390 395 400 gaa aga acc aat gag aaa ttc cat caa ata gag aag gaa ttc tea gaa 1248 Glu Arg Thr Asn Glu Lys Phe His Gin He 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 He Gin Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys 420 425 430 ata gac eta tgg tcc tac aat gca gaa ttg ctg gtg get eta gaa aat 1344 He 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 Gin His Thr lie Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu 450 455 460 aag act aga ege cag tta aga gaa aac gca gaa gac atg gga ggt gga 1440 Lys Thr Arg Arg Gin 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 tea ata 1488 Cys Phe Lys He Tyr His Lys Cys Asp Asn Ala Cys He Glu Ser He 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 He Tyr Lys Asp Glu Ala Leu Asn 500 505 510 aat ega ttt cag ate aaa ggt gta gag ttg aaa tea ggc tac aaa gat 1584 Asn Arg Phe Gin He Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp 515 520 525 tgg ata ctg tgg att tea ttc gcc ata tea tgc ttc tta att tgc gtt 1632 Trp He Leu Trp He Ser Phe Ala He Ser Cys Phe Leu He Cys Val 530 535 540 gtt eta ttg ggt ttc att atg tgg get tgc caa aaa ggc aac ate aga 1680 Val Leu Leu Gly Phe He Met Trp Ala Cys Gin Lys Gly Asn lie Arg 545 550 555 560

   tgc aac att tgc att tga Cys Asn lie Cys He ' 565

   1698

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   Oct 2015 <N

   <210> 62 <211> 565 <212> PRT <213> Influenza virus <400> 62 Met Lys Thr Thr He 11'

   io oo

   Xj Gin Asn Pro lie Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His ΓΠ 20 25 30 <N

   IT) o

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   His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Met Ser Asp Asp Gin 35 40 45

   He Glu Val Thr Asn Ala Thr Glu Leu Val Gin Ser He Ser Met Gly 50 55 60

   Lys He Cys Asn Lys Ser Tyr Arg He Leu Asp Gly Arg Asn Cys Thr 65 70 75 80

   Leu He Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gin Tyr 85 90 95

   Glu Ser Trp Asp Leu Phe He Glu Arg Ser Ser Ala Phe Ser Asn Cys 100 105 110

   Tyr Pro Tyr Asp He Pro Asp Tyr Ala Ser Leu Arg Ser lie 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 Gin 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 He Trp Gly He His His Pro Ser Ser Asn Gin Glu Gin Thr Lys 195 200 205

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   Leu Tyr lie Gin Glu Ser Gly Arg 210 215

   Gin Gin Thr He He Pro Asn He 225 230

   Gin Ser Gly Arg He Ser He Tyr 245

   He Leu Met He Asn Ser Asn Gly 260

   Phe Lys Leu Asn Thr Gly Lys Ser 275 280

   Val Thr Val Ser Thr Lys Arg Ser 220

   Gly Ser Arg Pro Leu Val Arg Gly 235 240

   Trp Thr He Val Lys Pro Gly Asp 250 255

   Asn Leu Val Ala Pro Arg Gly Tyr 265 270

   Ser Val Met Arg Ser Asp Val Pro 285

   He Asp He Cys Val Ser Glu Cys 290 295

   He Thr Pro Asn Gly Ser He Ser 300

   Asn Asp Lys Pro Phe Gin Asn Val 305 310

   Asn Lys Val Thr Tyr Gly Lys Cys 315 320

   Pro Lys Tyr He Arg Gin Asn Thr 325

   Leu Lys Leu Ala Thr Gly Met Arg 330 335

   Asn Val Pro Glu Lys Gin Thr Arg 340

   Gly He Phe Gly Ala He Ala Gly 345 350

   Phe lie Glu Asn Gly Trp Glu Gly 355 360

   Met Val Asp Gly Trp Tyr Gly Phe 365

   Arg Tyr Gin Asn Ser Glu Gly Thr 370 375

   Gly Gin Ala Ala Asp Leu Lys Ser 380

   Thr Gin Ala Ala He Asp Gin He 385 390

   Glu Arg Thr Asn Glu Lys Phe His 405 t

   Asn Gly Lys Leu Asn Arg Val He 395 400

   I

   Gin He Glu Lys Glu Phe Ser Glu 410 415

   Val Glu Gly Arg He Gin Asp Leu 420

   Glu Lys Tyr Val Glu Asp Thr Lys 425 430

   He Asp Leu Trp Ser Tyr Asn Ala 435 440

   Glu Leu Leu Val Ala Leu Glu Asn 445

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   Gin His 450 Thr He Asp Leu Thr 455 Asp Ala Glu Met Asn 460 Lys Leu Phe Glu Lys Thr Arg Arg Gin Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly 465 470 475 480 Cys Phe Lys He Tyr His Lys Cys Asp Asn Ala Cys He Glu Ser He 485 490 495 Arg Thr Gly Thr Tyr Asp His Tyr He Tyr Lys Asp Glu Ala Leu Asn 500 505 510 Asn Arg Phe Gin lie Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp 515 520 525 Trp lie Leu Trp He Ser Phe Ala He Ser Cys Phe Leu lie Cys Val 530 535 540 Val Leu Leu Gly Phe He Met Trp Ala Cys Gin Lys Gly Asn He Arg 545 550 555 560 Cys Asn He Cys He

   565 <210> 63 <211> 2280 <212> DNA

   <213> : Influenza virus <220> <221> CDS <222> (1) .. . (2280) <400> i S3 atg gag aga ata aaa gaa ctg aga gat ctg atg tta caa tcc cgc acc 48 Met Glu Arg He Lys Glu Leu Arg Asp Leu Met Leu Gin Ser Arg Thr 1 5 10 15 cgc gag ata eta aca aaa act act gtg gac cac atg gcc ata ate aag 96 Arg Glu He Leu Thr Lys Thr Thr Val Asp His Met Ala He He Lys 20 25 30 aaa tac aca tea gga aga caa gag aag aac cct gca ett agg atg aaa 144 Lys Tyr Thr Ser Gly Arg Gin 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 He Thr Ala Asp Lys Arg He Met

   50 55 60

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   gag Glu 65 atg Met att He cct Pro gag Glu aga Arg 70 aat Asn gaa Glu cag gga caa Gin 75 acc Thr ett Leu tgg Trp age Ser aaa Lys 80 240 Gin Gly aeg aac gat get ggc tea gac ege gta atg gta tea cct ctg gca gtg 288 Thr Asn Asp Ala Gly 85 Ser Asp Arg Val Met 90 Val Ser Pro Leu Ala 95 Val aca tgg tgg aat agg aat gga cca aca aeg aac aca att cat tat cca 336 Thr Trp Trp Asn 100 Arg Asn Gly Pro Thr 105 Thr Asn Thr lie His 110 Tyr Pro aaa gtc tac aaa act tat ttt gaa aag gtt gaa aga ttg aaa cac gga 384 Lys Val Tyr 115 Lys Thr Tyr Phe Glu 120 Lys Val Glu Arg Leu 125 Lys His Gly acc ttt ggc ccc gtt cat ttt agg aat caa gtc aag ata aga ega aga 432 Thr Phe 130 Gly Pro Val His Phe 135 Arg Asn Gin Val Lys 14 0 He Arg Arg Arg gtt gat gta aac cct ggt cac geg gac etc agt get aaa gaa gca caa 480 Val 145 Asp Val Asn Pro Gly 150 His Ala Asp Leu Ser 155 Ala Lys Glu Ala Gin 160 gat gtg ate atg gaa gtt gtt ttc cca aat gaa gtg gga gee aga att 528 Asp Val lie Met Glu 165 Val Val Phe Pro Asn 170 Glu Val Gly Ala Arg 175 He eta aca tea gaa tea caa eta aca ata acc aaa gag aaa aag gaa gaa 576 Leu Thr Ser Glu 180 Ser Gin Leu Thr He 185 Thr Lys Glu Lys Lys 190 Glu Glu ett cag gac tgc aaa att get ccc ttg atg gta gca tac atg eta gaa 624 Leu Gin Asp 195 Cys Lys He Ala Pro 200 Leu Met Val Ala Tyr 205 Met Leu Glu aga gag ttg gtc ega aaa aca agg ttc etc cca gta gta ggc gga aca 672 Arg Glu 210 Leu Val Arg Lys Thr 215 Arg Phe Leu Pro Val 220 Val Gly Gly Thr age agt gta tac att gaa gtg ttg cat ctg act cag gga aca tgc tgg 720 Ser 225 Ser Val Tyr lie Glu 230 Val Leu His Leu Thr 235 Gin Gly Thr Cys Trp 240 gag caa atg tac acc cca gga gga aaa gtt aga aac gat gat att gat 768 Glu Gin Met Tyr Thr 245 Pro Gly Gly Lys Val 250 Arg Asn Asp Asp He 255 Asp caa agt tta att att gca gcc egg aac ata gtg aga aga gca aca gta 816 Gin Ser Leu He 260 He Ala Ala Arg Asn 265 He Val Arg Arg Ala 270 Thr Val tea gca gat cca eta gca tcc eta ctg gaa atg tgc cac agt aca cag 864 Ser Ala Asp 275 Pro Leu Ala Ser Leu 280 Leu Glu Met Cys His 285 Ser Thr Gin att ggt gga aca agg atg gta gac ate ett aag cag aac cca aca gag 912 lie Gly 290 Gly Thr Arg Met Val 295 Asp He Leu Lys Gin 300 Asn Pro Thr Glu

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   gaa Glu 305 caa Gin get Ala gtg gat ata tgc aaa Lys gca Ala gca atg gga ttg aga att He age Ser 320 960 Val Asp lie 310 Cys Ala Met 315 Gly Leu Arg tea tea ttc age ttt ggt gga ttc acc ttc aaa agg aca agt gga tea 1008 Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser 325 330 335 tea gtc aag aga gaa gaa gaa atg ett aeg ggc aac ett caa aca ttg 1056 Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gin Thr Leu 340 345 350 aaa ata aga gtg cat gag ggc tat gaa gaa ttc aca atg gtc gga aga 1104 Lys He Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg 355 360 365 aga gca aca gcc att ate aga aag gca acc aga aga ttg att caa ttg 1152 Arg Ala Thr Ala He He Arg Lys Ala Thr Arg Arg Leu lie Gin Leu 370 375 380 ata gta agt ggg aga gat gaa caa tea att get gaa gca ata att gta 1200 lie Val Ser Gly Arg Asp Glu Gin Ser He Ala Glu Ala He He Val 385 390 395 400 gcc atg gtg ttt teg caa gaa gat tgc atg ata aaa gca gtt ega ggc 1248 Ala Met Val Phe Ser Gin Glu Asp Cys Met He 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 Gin Arg Leu Asn Pro Met His 420 425 430 caa etc ttg agg cat ttc caa aaa gat gca aaa gtg ett ttc caa aat 1344 Gin Leu Leu Arg His Phe Gin Lys Asp Ala Lys Val Leu Phe Gin Asn 435 440 445 tgg gga att gaa ccc ate gac aat gta atg ggg atg att gga ata ttg 1392 Trp Gly He Glu Pro He Asp Asn Val Met Gly Met He Gly He Leu 450 455 460 cct gac atg acc cca age acc gag atg tea 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 age aaa atg gga gtg gat gag tac tcc age 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 age att gac cgt ttt tta aga gtt egg gat caa agg gga aac ata eta 1536 Ser He Asp Arg Ρΐΐβ Leu Arg Val Arg Asp Gin Arg Gly Asn He Leu 500 505 510 ctg tcc cct gaa gaa gtc agt gaa aca caa gga aeg gaa aag ctg aca 1584 Leu Ser Pro Glu Glu Val Ser Glu Thr Gin Gly Thr Glu Lys Leu Thr 515 520 525 ata att tat teg tea tea atg atg tgg gag att aat ggt ccc gaa tea 1632 He He Tyr Ser Ser Ser Met Met Trp Glu He Asn Gly Pro Glu Ser 530 535 540

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   gtg ttg gtc aat Asn act Thr tat Tyr 550 caa tgg ate ate He aga Arg 555 aac Asn tgg Trp gaa Glu att lie gta Val 560 1680 Val 545 Leu Val Gin Trp He aaa att cag tgg tea cag gac ccc aca atg tta tac aat aag ata gaa 1728 Lys lie Gin Trp Ser Gin Asp Pro Thr Met Leu Tyr Asn Lys He Glu 565 570 575 ttt gaa cca ttc caa tcc ctg gtc cct agg gcc acc aga age caa tac 1776 Phe Glu Pro Phe Gin Ser Leu Val Pro Arg Ala Thr Arg Ser Gin Tyr 580 585 590 age ggt ttc gta aga acc ctg ttt cag caa atg cga gat gta ett gga 1824 Ser Gly Phe Val Arg Thr Leu Phe Gin Gin Met Arg Asp Val Leu Gly 595 600 605 aca ttt gat act get caa ata ata aaa etc etc cct ttt gcc get get 1872 Thr Phe Asp Thr Ala Gin He He Lys Leu Leu Pro Phe Ala Ala Ala 610 615 620 cct ccg gaa cag agt agg atg cag ttc tet tet ttg act gtt aat gta 1920 Pro Pro Glu Gin Ser Arg Met Gin Phe Ser Ser Leu Thr Val Asn Val 625 630 635 640 aga ggt teg gga atg agg ata ett gta aga ggc aat tcc cca gtg ttc 1968 Arg Gly Ser Gly Met Arg lie Leu Val Arg Gly Asn Ser Pro Val Phe 645 650 655 aac tac aat aaa gtc act aaa agg etc aca gtc etc 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 geg ett act gag gac cca gat gaa ggt aeg get gga gta gag tet 2064 Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser 675 680 685 get gtt eta aga ggg ttt etc att tta ggt aaa gaa aac aag aga tat 2112 Ala Val Leu Arg Gly Phe Leu He Leu Gly Lys Glu Asn Lys Arg Tyr 690 695 700 ggc cca gca eta age ate aat gaa ett age aaa ett gca aaa ggg gag 2160 Gly Pro Ala Leu Ser He Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu 705 710 715 720 aaa gcc aat gta eta att ggg caa ggg gac gta gtg ttg gta atg aaa 2208 Lys Ala Asn Val Leu He Gly Gin Gly Asp Val Val Leu Val Met Lys 725 730 735 egg aaa cgt gac tet age ata ett act gac age cag aca geg acc aaa 2256 Arg Lys Arg Asp Ser Ser He Leu Thr Asp Ser Gin Thr Ala Thr Lys 740 745 750 agg att egg atg gcc ate aat tag 2280 Arg He Arg Met Ala He Asn

   <210> 64 <211> 759 <212> PRT

   755

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   128 <213> Influenza virus <400> 64

   Met Glu Arg lie Lys Glu Leu Arg Asp Leu Met Leu Gin Ser Arg Thr

   Ο 1 5 10 15 <N ©

   Arg Glu He Leu Thr Lys Thr Thr Val Asp His Met Ala He He Lys 20 25 30

   V oo

   Lys Tyr Thr Ser Gly Arg Gin Glu Lys Asn Pro Ala Leu Arg Met Lys Xt 35 40 45 m

   <N

   Trp Met Met Ala Met Lys Tyr Pro He Thr Ala Asp Lys Arg He Met θ 50 55 60 <N

   Glu Met He Pro Glu Arg Asn Glu Gin Gly Gin 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 He 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 Gin Val Lys He Arg Arg Arg 130 135 140

   Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gin 145 150 155 160

   Asp Val He Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg He 165 170 175

   Leu Thr Ser Glu Ser Gin Leu Thr He Thr Lys Glu Lys Lys Glu Glu 180 185 190

   Leu Gin Asp Cys Lys He 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

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   Ser Ser Val Tyr lie Glu Val Leu His Leu Thr Gin Gly Thr Cys Trp 225 230 235 240

   Glu Gin Met Tyr Thr Pro Gly Gly Lys Val Arg Asn Asp Asp He Asp 245 250 255

   Gin Ser Leu He He Ala Ala Arg Asn He Val Arg Arg Ala Thr Val 260 265 270

   Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gin 275 280 285

   He Gly Gly Thr Arg Met Val Asp He Leu Lys Gin Asn Pro Thr Glu 290 295 300

   Glu Gin Ala Val Asp He Cys Lys Ala Ala Met Gly Leu Arg He 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 Gin Thr Leu 340 345 , 350

   Lys He Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg 355 360 365

   Arg Ala Thr Ala He He Arg Lys Ala Thr Arg Arg Leu He Gin Leu 370 375 380 lie Val Ser Gly Arg Asp Glu Gin Ser He Ala Glu Ala He He Val 385 390 395 400

   Ala Met Val Phe Ser Gin Glu Asp Cys Met He Lys Ala Val Arg Gly 405 410 415

   Asp Leu Asn Phe Val Asn Arg Ala Asn Gin Arg Leu Asn Pro Met His 420 425 430

   Gin Leu Leu Arg His Phe Gin Lys Asp Ala Lys Val Leu Phe Gin Asn 435 440 445

   Trp Gly lie Glu Pro He Asp Asn Val Met Gly Met He Gly He Leu 450 455 460

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   Pro Asp Met Thr Pro Ser Thr 465 470

   Ser Lys Met Gly Val Asp Glu 485

   Ser lie Asp Arg Phe Leu Arg 500

   OO

   Leu Ser Pro Glu Glu Val Ser 515 <N

   He He Tyr Ser Ser Ser Met θ 530 535 <N

   Val Leu Val Asn Thr Tyr Gin 545 550

   Lys He Gin Trp Ser Gin Asp 565

   Phe Glu Pro Phe Gin Ser Leu 580

   Ser Gly Phe Val Arg Thr Leu 595

   Thr Phe Asp Thr Ala Gin He 610 615

   Pro Pro Glu Gin Ser Arg Met 625 630

   Arg Gly Ser Gly Met Arg He 645

   Asn Tyr Asn Lys Val Thr Lys 660

   Gly Ala Leu Thr Glu Asp Pro 675

   Ala Val Leu Arg Gly Phe Leu 690 695

   130

   Glu Met Ser Leu Arg Gly Val Arg Val 475 480

   Tyr Ser Ser Thr Glu Arg Val Val Val 490 495

   Val Arg Asp Gin Arg Gly Asn He Leu 505 510

   Glu Thr Gin Gly Thr Glu Lys Leu Thr 520 525

   Met Trp Glu He Asn Gly Pro Glu Ser 540

   Trp He He Arg Asn Trp Glu He Val 555 560

   Pro Thr Met Leu Tyr Asn Lys He Glu 570 575

   Val Pro Arg Ala Thr Arg Ser Gin Tyr 585 590

   Phe Gin Gin Met Arg Asp Val Leu Gly 600 605

   He Lys Leu Leu Pro Phe Ala Ala Ala 620

   Gin Phe Ser Ser Leu Thr Val Asn Val 635 640

   Leu Val Arg Gly Asn Ser Pro Val Phe 650 655

   Arg Leu Thr Val Leu Gly Lys Asp Ala 665 670

   Asp Glu Gly Thr Ala Gly Val Glu Ser 680 685

   He Leu Gly Lys Glu Asn Lys Arg Tyr 700

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   Gly 705 Pro Ala Leu Ser He 710 Asn Glu Leu Ser Lys 715 Leu Ala Lys Gly Glu 720 Lys Ala Asn Val Leu 725 He Gly Gin Gly Asp 730 Val Val Leu Val Met 735 Lys Arg Lys Arg Asp 740 Ser Ser lie Leu Thr 745 Asp Ser Gin Thr Ala 750 Thr Lys Arg lie Arg Met Ala He 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 eta ett ttc tta aag gtg cca geg caa aat 48 Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gin Asn 1 5 10 15 get ata age aca aca ttc cct tat act gga gat cct ccc tac agt cat 96 Ala He 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 Gin 35 40 45 tat tea 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 He Gly Ala Pro 50 55 60 caa ett aat cca ate gat gga cca ett cct gaa gac aat gaa cca agt 240 Gin Leu Asn Pro He 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 get ttc ett gaa 288 Gly Tyr Ala Gin Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 85 90 95 gaa tcc cat ccc gga ate ttt gaa aat teg tgt ett gaa aeg atg gag 336 Glu Ser His Pro Gly He Phe Glu Asn Ser Cys Leu Glu Thr Met Glu 100 105 110 gtg att cag cag aca aga gtg gac aaa eta aca caa ggc ega caa act 384 Val He Gin Gin Thr Arg Val Asp Lys Leu Thr Gin Gly Arg Gin Thr 115 120 125 tat gat tgg acc ttg aat agg aat caa cct gcc gca aca gca ett get 432

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   Tyr Asp 130 Trp Thr Leu Asn Arg 135 Asn Gin Pro Ala Ala 140 Thr Ala Leu Ala aat aeg att gaa gta ttc aga tea aat ggt ctg acc tcc aat gaa teg 480 Asn 145 Thr lie Glu Val Phe 150 Arg Ser Asn Gly Leu 155 Thr Ser Asn Glu Ser 160 ggg aga ttg atg gac ttc etc aaa gat gtc atg gag tcc atg aac aag 528 Gly Arg Leu Met Asp 165 Phe Leu Lys Asp Val 170 Met Glu Ser Met Asn 175 Lys gag gaa atg gaa ata aca aca cac ttc caa egg aag aga aga gta aga 576 Glu Glu Met Glu 180 lie Thr Thr His Phe 185 Gin Arg Lys Arg Arg 190 Val Arg gac aac atg aca aag aga atg ata aca cag aga acc ata gga aag aaa 624 Asp Asn Met 195 Thr Lys Arg Met lie 200 Thr Gin Arg Thr He 205 Gly Lys Lys aaa caa ega tta age aga aag age tat eta ate aga aca tta acc eta 672 Lys Gin 210 Arg Leu Ser Arg Lys 215 Ser Tyr Leu He Arg 220 Thr Leu Thr Leu aac aca atg acc aag gac get gag aga ggg aaa ttg aaa ega ega gca 720 Asn 225 Thr Met Thr Lys Asp 230 Ala Glu Arg Gly Lys 235 Leu Lys Arg Arg Ala 240 ate get acc cca ggg atg cag ata aga gga ttt gta tat ttt gtt gaa 768 lie Ala Thr Pro Gly 245 Met Gin He Arg Giy 250 Phe Val Tyr Phe Val 255 Glu aca eta get ega aga ata tgt gaa aag ett gaa caa tea gga ttg cca 816 Thr Leu Ala Arg 260 Arg He Cys Glu Lys 265 Leu Glu Gin Ser Gly 270 Leu Pro gtt ggc ggt aat gag aaa aag gcc aaa ctg get aat gtc gtc aga aaa 864 Val Gly Gly 275 Asn Glu Lys Lys Ala 280 Lys Leu Ala Asn Val 285 Val Arg Lys atg atg act aat tcc caa gac act gaa etc tcc ttc acc ate act ggg 912 Met Met 290 Thr Asn Ser Gin Asp 295 Thr Glu Leu Ser Phe 300 Thr He Thr Gly gac aat acc aaa tgg aat gaa aat cag aac cca ege ata ttc ctg gca 960 Asp 305 Asn Thr Lys Trp Asn 310 Glu Asn Gin Asn Pro 315 Arg He Phe Leu Ala 320 atg ate aca tac ata act aga gat cag cca gaa tgg ttc aga aat gtt 1008 Met lie Thr Tyr lie 325 Thr Arg Asp Gin Pro 330 Glu Trp Phe Arg Asn 335 Val eta age att gca ccg att atg ttc tea aat aaa atg gca aga ctg ggg 1056 Leu Ser He Ala 340 Pro He Met Phe Ser 345 Asn Lys Met Ala Arg 350 Leu Gly aaa gga tat atg ttt gaa age aaa agt atg aaa ttg aga act caa ata 1104 Lys Gly Tyr 355 Met Phe Glu Ser Lys 360 Ser Met Lys Leu Arg 365 Thr Gin He cca gca gaa atg eta gca age att gac eta aaa tat ttc aat gat tea 1152

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   Pro Ala 370 Glu Met Leu Ala Ser 375 He Asp Leu Lys Tyr 380 Phe Asn Asp Ser aca aaa aag aaa att gaa aag ata ega cca etc ctg gtt gac ggg act 1200 Thr 385 Lys Lys Lys He Glu 390 Lys He Arg Pro Leu 395 Leu Val Asp Gly Thr 400 get tea ctg agt cct ggc atg atg atg gga atg ttc aac atg ttg age 1248 Ala Ser Leu Ser Pro 405 Gly Met Met Met Gly 410 Met Phe Asn Met Leu 415 Ser act gtg ctg ggt gta tcc ata tta aac ctg ggc cag agg aaa tat aca 1296 Thr Val Leu Gly 420 Val Ser He Leu Asn 425 Leu Gly Gin Arg Lys 430 Tyr Thr aag acc aca tac tgg tgg gat ggt ctg caa tea tcc gat gac ttt get 1344 Lys Thr Thr 435 Tyr Trp Trp Asp Gly 440 Leu Gin Ser Ser Asp 445 Asp Phe Ala ttg ata gtg aat geg cct aat cat gaa gga ata caa get gga gta gac 1392 Leu lie 450 Val Asn Ala Pro Asn 455 His Glu Gly lie Gin 460 Ala Gly Val Asp aga ttc tat aga act tgc aaa ctg gtc ggg ate aac atg age aaa aag 1440 Arg 465 Phe Tyr Arg Thr Cys 470 Lys Leu Val Gly He 475 Asn Met Ser Lys Lys 480 aag tcc tac ata aat aga act gga aca ttc gaa ttc aca age ttt ttc 1488 Lys Ser Tyr He Asn 485 Arg Thr Gly Thr Phe 490 Glu Phe Thr Ser Phe 495 Phe tac egg tat ggt ttt gta gcc aat ttc age atg gaa eta ccc agt ttt 1536 Tyr Arg Tyr Gly 500 Phe Val Ala Asn Phe 505 Ser Met Glu Leu Pro 510 Ser Phe ggg gtt tcc gga ata aat gaa tet gca gac atg age att gga gtg aca 1584 Gly Val Ser 515 Gly He Asn Glu Ser 520 Ala Asp Met Ser lie 525 Gly Val Thr gtc ate aaa aac aac atg ata aat aat gat etc ggt cct gcc aeg gca 1632 Val lie 530 Lys Asn Asn Met He 535 Asn Asn Asp Leu Gly 540 Pro Ala Thr Ala caa atg gca etc caa etc ttc att aag gat tat egg tac aca tac egg 1680 Gin 545 Met Ala Leu Gin Leu 550 Phe lie Lys Asp Tyr 555 Arg Tyr Thr Tyr Arg 560 tgc cat aga ggt gat acc cag ata caa acc aga aga tet ttt gag ttg 1728 Cys His Arg Gly Asp 565 Thr Gin He Gin Thr 570 Arg Arg Ser Phe Glu 575 Leu aag aaa ctg tgg gaa cag act ega tea aag act ggt eta ctg gta tea 1776 Lys Lys Leu Trp 580 Glu Gin Thr Arg Ser 585 Lys Thr Gly Leu Leu 590 Val Ser gat ggg ggt cca aac eta tat aac ate aga aac eta cac ate ccg gaa 1824 Asp Gly Gly 595 Pro Asn Leu Tyr Asn 600 He Arg Asn Leu His 605 He Pro Glu gtc tgt tta aaa tgg gag eta atg gat gaa gat tat aag ggg agg eta 1872

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   Val Cys 610 Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Lys Gly Arg Leu 615 620 tgc aat cca ttg aat cct ttc gtt agt cac aaa gaa att gaa tea gtc Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu He Glu Ser Val 625 630 635 640 aac agt gca gta gta atg cct get cat ggc cct gcc aaa age atg gag Asn Ser Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu 645 650 655 tat gat get gtt gca aca aca cat tet tgg ate ccc, aag agg aac egg Tyr Asp Ala Val Ala Thr Thr His Ser Trp He Pro Lys Arg Asn Arg 660 665 670 tcc ata ttg aac aca age caa agg gga ata eta gaa gat gag cag atg Ser lie Leu Asn Thr Ser Gin Arg Gly lie Leu Glu Asp Glu Gin Met 675 680 685 tat cag aaa tgc tgc aac ctg ttt gaa aaa ttc ttc ccc age age tea Tyr Gin Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser 690 695 700 tac aga aga cca gtc gga att tet agt atg gtt gag gcc atg gta tcc Tyr Arg Arg Pro Val Gly lie Ser Ser Met Val Glu Ala Met Val Ser 705 710 715 720 agg gcc ege att gat gca ega att gac ttc gaa tet gga egg ata aag Arg Ala Arg He Asp Ala Arg He Asp Phe Glu Ser Gly Arg He Lys 725 730 735 aag gat gag ttc get gag ate atg aag ate tgt tcc acc att gaa gag Lys Asp Glu Phe Ala Glu He Met Lys He Cys Ser Thr He Glu Glu 740 745 750 etc aga egg caa aaa tag Leu Arg Arg Gin Lys

   755

   1920

   1968

   2016

   2064

   2112

   2160

   2208

   2256

   2274 <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 Gin Asn 15 10 15

   Ala lie 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 Gin 35 40 45

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

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   Gin Leu Asn Pro lie Asp 65 70

   Gly Tyr Ala Gin Thr Asp 85

   Glu Ser His Pro Gly He 100

   Val He Gin Gin Thr Arg 115

   Tyr Asp Trp Thr Leu Asn 130

   Asn Thr He Glu Val Phe 145 150

   Gly Arg Leu Met Asp Phe 165

   Glu Glu Met Glu He Thr 180

   Asp Asn Met Thr Lys Arg 195

   Lys Gin Arg Leu Ser Arg 210

   Asn Thr Met Thr Lys Asp 225 230 lie Ala Thr Pro Gly Met 245

   Thr Leu Ala Arg Arg He 260

   Val Gly Gly Asn Glu Lys 275

   Gly Pro Leu Pro Glu 75

   Cys Val Leu Glu Ala 90

   Phe Glu Asn Ser Cys 105

   Val Asp Lys Leu Thr 120

   Arg Asn Gin Pro Ala 135

   Arg Ser Asn Gly Leu 155

   Leu Lys Asp Val Met 170

   Thr His Phe Gin Arg 185

   Met He Thr Gin Arg 200

   Lys Ser Tyr Leu He 215

   Ala Glu Arg Gly Lys 235

   Gin He Arg Gly Phe 250

   Cys Glu Lys Leu Glu 265

   Lys Ala Lys Leu Ala 280

   Asp Asn Glu Pro Ser 80

   Met Ala Phe Leu Glu 95

   Leu Glu Thr Met Glu 110

   Gin Gly Arg Gin Thr 125

   Ala Thr Ala Leu Ala 140

   Thr Ser Asn Glu Ser 160

   Glu Ser Met Asn Lys 175

   Lys Arg Arg Val Arg 190

   Thr He Gly Lys Lys 205

   Arg Thr Leu Thr Leu 220

   Leu Lys Arg Arg Ala 240

   Val Tyr Phe Val Glu 255

   Gin Ser Gly Leu Pro 270

   Asn Val Val Arg Lys 285

   Met Met Thr Asn Ser Gin Asp Thr Glu Leu Ser Phe Thr lie Thr Gly

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   290 295

   Asp Asn Thr Lys Trp Asn Glu Asn 305 310

   Met lie Thr Tyr He Thr Arg Asp 325

   Leu Ser He Ala Pro He Met Phe 340

   Lys Gly Tyr Met Phe Glu Ser Lys 355 360

   Pro Ala Glu Met Leu Ala Ser He 370 375

   300

   Gin Asn Pro Arg He Phe Leu Ala 315 320

   Gin Pro Glu Trp Phe Arg Asn Val 330 335

   Ser Asn Lys Met Ala Arg Leu Gly 345 350

   Ser Met Lys Leu Arg Thr Gin He 365

   Asp Leu Lys Tyr Phe Asn Asp Ser 380

   Thr Lys Lys Lys He Glu Lys He 385 390

   Arg Pro Leu Leu Val Asp Gly Thr 395 400

   Ala Ser Leu Ser Pro Gly Met Met 405

   Met Gly Met Phe Asn Met Leu Ser 410 415

   Thr Val Leu Gly Val Ser lie Leu 420

   Asn Leu Gly Gin Arg Lys Tyr Thr 425 430

   Lys Thr Thr Tyr Trp Trp Asp Gly 435 440

   Leu Gin Ser Ser Asp Asp Phe Ala 445

   Leu He Val Asn Ala Pro Asn His 450 455

   Glu Gly He Gin Ala Gly Val Asp 460

   Arg Phe Tyr Arg Thr Cys Lys Leu 465 470

   Val Gly He Asn Met Ser Lys Lys 475 480

   Lys Ser Tyr He Asn Arg Thr Gly 485

   Thr Phe Glu Phe Thr Ser Phe Phe 490 495

   Tyr Arg Tyr Gly Phe Val Ala Asn 500

   Phe Ser Met Glu Leu Pro Ser Phe 505 510

   Gly Val Ser Gly He Asn Glu Ser 515 520

   Ala Asp Met Ser lie Gly Val Thr 525

   Val lie Lys Asn Asn Met He Asn Asn Asp Leu Gly Pro Ala Thr Ala

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   530 535

   Gin Met Ala Leu Gin Leu Phe lie 545 550

   Cys His Arg Gly Asp Thr Gin He 565

   Lys Lys Leu Trp Glu Gin Thr Arg 580

   Asp Gly Gly Pro Asn Leu Tyr Asn 595 600

   Val Cys Leu Lys Trp Glu Leu Met 610 615

   Cys Asn Pro Leu Asn Pro Phe Val 625 630

   Asn Ser Ala Val Val Met Pro Ala 645

   Tyr Asp Ala Val Ala Thr Thr His 660

   Ser He Leu Asn Thr Ser Gin Arg 675 680

   Tyr Gin Lys Cys Cys Asn Leu Phe 690 695

   Tyr Arg Arg Pro Val Gly He Ser 705 710

   Arg Ala Arg lie Asp Ala Arg lie 725

   Lys Asp Glu Phe Ala Glu He Met 740

   540

   Lys Asp Tyr Arg Tyr Thr Tyr Arg 555 560

   Gin Thr Arg Arg Ser Phe Glu Leu 570 575

   Ser Lys Thr Gly Leu Leu Val Ser 585 590 lie Arg Asn Leu His He Pro Glu 605

   Asp Glu Asp Tyr Lys Gly Arg Leu 620

   Ser His Lys Glu He Glu Ser Val 635 640

   His Gly Pro Ala Lys Ser Met Glu 650 655

   Ser Trp He Pro Lys Arg Asn Arg 665 670

   Gly He Leu Glu Asp Glu Gin Met 685

   Glu Lys Phe Phe Pro Ser Ser Ser 700

   Ser Met Val Glu Ala Met Val Ser 715 720

   Asp Phe Glu Ser Gly Arg He Lys 730 735

   Lys He Cys Ser Thr He Glu Glu 745 750

   Leu Arg Arg Gin Lys 755 <210> 67

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   2015234384 02 Oct 2015 <211> 2151 <212> DNA <213> Influenza virus <220>

   <221> CDS <222> (1) . . (2151) <400> 67 atg gaa gac ttt gtg ega cag tgc ttc aat cca atg ate gtc gag ett

   Met Glu Asp Phe Val Arg Gin Cys Phe Asn Pro Met lie Val Glu Leu

   15 10 15 geg gaa aag gca atg aaa gaa tat gga gag aac ccg aaa ate gaa aca

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

   20 25 30 aac aaa ttt gca gca ata tgc act cac ttg gaa gtc tgc ttc atg tac

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

   35 40 45 teg gat ttc cac ttt ata aat gaa ctg ggt gag tea gtg gtc ata gag

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

   50 55 60 tet ggt gac cca aat get ett ttg aaa cac aga ttt gaa ate att gag

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

   65 70 75 80 ggg aga gat ega aca atg gca tgg aca gta gta aac age ate tgc aac

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

   85 90 95 acc aca aga get gaa aaa cct aaa ttt ett cca gat tta tac gac tat

   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

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

   115 120 125 ata tac tac ctg gag aag gcc aac aaa ata aag tet gag aaa aca cat

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

   130 135 140 ate cac att ttc tea ttt aca gga gaa gaa atg get aca aaa geg gac

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

   145 150 155 ' 160 tat act ett gat gaa gag agt aga gcc agg ate aag acc aga eta ttc

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

   165 170 175 act ata aga caa gaa atg gcc agt aga ggc etc tgg gat tcc ttt cgt

   Thr lie Arg Gin 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 ate aca

   Gin Ser Glu Arg Gly Glu Glu Thr He Glu Glu Arg Phe Glu He Thr

   144

   192

   240

   288

   336

   384

   432

   480

   528

   576

   624

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   139

   195 200 205

   ggg Gly acg Thr 210 atg Met ege Arg aag Lys ett Leu gcc Ala 215 aat Asn tac Tyr agt Ser etc Leu cca Pro 220 ccg Pro aac Asn ttc Phe tcc Ser 672 age ett gaa aat ttt aga gtc tat ata gat gga ttc gaa ccg aac ggc 720 Ser 225 Leu Glu Asn Phe Arg 230 Val Tyr He Asp Gly 235 Phe Glu Pro Asn Gly 240 tgc att gag agt aag ett tot caa atg tcc aaa gaa gta aat gcc aaa 768 Cys He Glu Ser Lys 245 Leu Ser Gin Met Ser 250 Lys Glu Val Asn Ala 255 Lys ata gaa cca ttt tea aag aca aca ccc ega cca etc aaa atg cca ggt 816 lie Glu Pro Phe 260 Ser Lys Thr Thr Pro 265 Arg Pro Leu Lys Met 270 Pro Gly ggt cca ccc tgc cat cag ega tcc aaa ttc ttg eta atg gat get ctg 864 Gly Pro Pro 275 Cys His Gin Arg Ser 280 Lys Phe Leu Leu Met 285 Asp Ala Leu aaa ctg age att gag gac cca agt cac gag gga gag ggg ata cca eta 912 Lys Leu 290 Ser lie Glu Asp Pro 295 Ser His Glu Gly Glu 300 Gly He Pro Leu tat gat gca ate aaa tgc atg aaa act ttc ttt gga tgg aaa gag ccc 960 Tyr 305 Asp Ala He Lys Cys 310 Met Lys Thr Phe Phe 315 Gly Trp Lys Glu Pro 320 agt att gtt aaa cca cat aaa aag ggt ata aac ccg aac tat etc caa 1008 Ser He Val Lys Pro 325 His Lys Lys Gly He 330 Asn Pro Asn Tyr Leu 335 Gin act tgg aag caa gta tta gaa gaa ata caa gac ett gag aac gaa gaa 1056 Thr Trp Lys Gin 340 Val Leu Glu Glu He 345 Gin Asp Leu Glu Asn 350 Glu Glu agg acc ccc aag acc aag aat atg aaa aaa aca age caa ttg aaa tgg 1104 Arg Thr Pro 355 Lys Thr Lys Asn Met 360 Lys Lys Thr Ser Gin 365 Leu Lys Trp gca eta ggt gaa aat atg gca cca gag aaa gtg gat ttt gag gat tgt 1152 Ala Leu 370 Gly Glu Asn Met Ala 375 Pro Glu Lys Val Asp 380 Phe Glu Asp Cys aaa gac ate aat gat tta aaa caa tat gac agt gat gag cca gaa gca 1200 Lys 385 Asp He Asn Asp Leu 390 Lys Gin Tyr Asp Ser 395 Asp Glu Pro Glu Ala 400 agg tet ett gca agt tgg att caa agt gag ttc aac aag get tgt gag 1248 Arg Ser Leu Ala Ser 405 Trp lie Gin Ser Glu 410 Phe Asn Lys Ala Cys 415 Glu ctg aca gat tea age tgg ata gag etc gat gaa att ggg gag gat gtc 1296 Leu Thr Asp Ser 420 Ser Trp He Glu Leu 425 Asp Glu He Gly Glu 430 Asp Val gcc cca ata gaa tac att gcg age atg agg aga aat tat ttt act get 1344 Ala Pro He Glu Tyr He Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala

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   435 440 445 gag att tcc cat tgt aga gca aca gaa tat ata atg aaa gga gtg tac Glu He Ser His Cys Arg Ala Thr Glu Tyr He Met Lys Gly Val Tyr 450 455 460 ate aac act get eta etc aat gca tcc tgt get geg atg gat gaa ttt lie 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 Gin Leu He Pro Met lie 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 Lys Thr Asn Leu Tyr Gly Phe Tie He Lys Gly Arg Ser His Leu Arg 500 505 510 aat gat act gac gtg gtg aac ttt gta agt atg gaa ttt tct etc act 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 eta gaa Asp Pro Arg Phe Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu 530 535 540 att gga gac atg ett tta aga act get gta ggt caa gtg tea aga ccc He Gly Asp Met Leu Leu Arg Thr Ala Val Gly Gin Val Ser Arg Pro 545 550 555 560 atg ttt ttg tat gta agg aca aat gga acc tct aaa att aaa atg aaa Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys lie Lys Met Lys 565 570 575 tgg gga atg gaa atg agg ege tgc etc ett cag tct ctg caa cag att Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gin Ser Leu Gin Gin He 580 585 590 gaa age atg ate gaa get gag tcc tea gtc aaa gaa aag gac atg acc Glu Ser Met He Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr 595 600 605 aaa gaa ttt ttt gag aac aaa tea gag aca tgg cct ata gga gag tcc Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro lie Gly Glu Ser 610 615 620 ccc aaa gga gtg gaa gag ggc tea ate ggg aag gtt tgc agg acc tta Pro Lys Gly Val Glu Glu Gly Ser lie Gly Lys Val Cys Arg Thr Leu 625 630 635 64 0 tta gca aaa tct gtg ttt aac agt tta tat gca tct cca caa ctg gaa Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gin Leu Glu 645 650 655 gga ttt tea get gaa tct agg aaa tta ett etc att gtt cag get ett Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu He Val Gin Ala Leu 660 665 670 aga gat gac ctg gaa cct gga acc ttt gat att ggg ggg tta tat gaa Arg Asp Asp Leu Glu Pro Gly Thr Phe Asp He Gly Gly Leu Tyr Glu

   1392

   1440

   1488

   1536

   1584

   1632

   1680

   1728

   1776

   1824

   1872

   1920

   1968

   2016

   2064

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   675

   680

   685

   tea att gag gag tgc ctg att aat gat ccc tgg gtt ttg ett aat gca Ser He Glu Glu Cys Leu He Asn Asp Pro Trp Val Leu Leu Asn Ala 690 695 700 tet tgg ttc aac tcc ttc etc aca cat gca ctg aag tag 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 Gin Cys Phe Asn Pro Met He Val Glu Leu 1 5 10 15 Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asn Pro Lys He Glu Thr 20 25 30 Asn Lys Phe Ala Ala He Cys Thr His Leu Glu Val Cys Phe Met Tyr 35 40 45 Ser Asp Phe His Phe He Asn Glu Leu Gly Glu Ser Val Val He Glu 50 55 60 Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu He lie Glu 65 70 75 80 Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser He 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 He Gly Val Thr Arg Arg Glu Val His 115 120 125 He Tyr Tyr Leu Glu Lys Ala Asn Lys He Lys Ser Glu Lys Thr His 130 135 140 He His He Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys Ala Asp 145 150 155 160

   2112

   2151

   Tyr Thr Leu Asp Glu Glu Ser Arg Ala Arg lie Lys Thr Arg Leu Phe 165 170 175

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   Thr He Arg Gin Glu Met Ala Ser 180

   Gin Ser Glu Arg Gly Glu Glu Thr 195 200

   Gly Thr Met Arg Lys Leu Ala Asn 210 215

   Ser Leu Glu Asn Phe Arg Val Tyr 225 230

   Cys He Glu Ser Lys Leu Ser Gin 245

   Arg Gly Leu Trp Asp Ser Phe Arg 185 190 lie Glu Glu Arg Phe Glu He Thr 205

   Tyr Ser Leu Pro Pro Asn Phe Ser 220

   He Asp Gly Phe Glu Pro Asn Gly 235 240

   Met Ser Lys Glu Val Asn Ala Lys 250 255

   He Glu Pro Phe Ser Lys Thr Thr 260

   Pro Arg Pro Leu Lys Met Pro Gly 265 270

   Gly Pro Pro Cys His Gin.Arg Ser 275 280

   Lys Phe Leu Leu Met Asp Ala Leu 285

   Lys Leu Ser He Glu Asp Pro Ser 290 295

   His Glu Gly Glu Gly He Pro Leu 300

   Tyr Asp Ala He Lys Cys Met Lys 305 310

   Thr Phe Phe Gly Trp Lys Glu Pro 315 320

   Ser He Val Lys Pro His Lys Lys 325

   Gly lie Asn Pro Asn Tyr Leu Gin 330 335

   Thr Trp Lys Gin Val Leu Glu Glu 340

   He Gin Asp Leu Glu Asn Glu Glu 345 350

   Arg Thr Pro Lys Thr Lys Asn Met 355 360

   Lys Lys Thr Ser Gin Leu Lys Trp 365

   Ala Leu Gly Glu Asn Met Ala Pro 370 375

   Glu Lys Val Asp Phe Glu Asp Cys 380

   Lys Asp He Asn Asp Leu Lys Gin 385 390

   Tyr Asp Ser Asp Glu Pro Glu Ala 395 400

   Arg Ser Leu Ala Ser Trp lie Gin 405

   Ser Glu Phe Asn Lys Ala Cys Glu 410 415

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   Leu Thr Asp Ser Ser Trp lie Glu Leu Asp Glu lie Gly Glu Asp Val 420 425 430

   Ala Pro He Glu Tyr He Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala 435 440 445

   Glu He Ser His Cys Arg Ala Thr Glu Tyr He Met Lys Gly Val Tyr 450 455 460

   He Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Glu Phe 465 470 475 480

   Gin Leu He Pro Met He Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg 485 490 495

   Lys Thr Asn Leu Tyr Gly Phe He He 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

   He Gly Asp Met Leu Leu Arg Thr Ala Val Gly Gin Val Ser Arg Pro 545 550 555 560

   Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys He Lys Met Lys 565 570 575

   Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gin Ser Leu Gin Gin He 580 585 590

   Glu Ser Met He Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr 595 600 605

   Lys Glu Phe 610

   Phe Glu Asn Lys 615

   Ser Glu Thr Trp

   Pro

   620

   He Gly Glu Ser

   Pro Lys Gly Val 625

   Glu Glu Gly Ser 630

   He Gly Lys 635

   Val

   Cys Arg Thr Leu 640

   Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gin Leu Glu 645 650 655

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   1 144 Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu He Val Gin Ala Leu 660 665 670 Arg Asp Asp Leu Glu Pro Gly Thr Phe Asp He Gly Gly Leu Tyr Glu 675 680 685 Ser lie Glu Glu Cys Leu He Asn Asp Pro 1 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).. <400> 69 (690) atg gat tcc aac act gtg tea age ttt cag gta gac tgt ttt ett tgg 48 Met 1 Asp Ser Asn Thr 5 Val Ser Ser Phe Gin 10 Val Asp Cys Phe Leu 15 Trp cat gtc cgt aaa ega ttc gca gac caa gaa ctg ggt gat gcc cca ttc 96 His Val Arg Lys 20 Arg Phe Ala Asp Gin 25 Glu Leu Gly Asp Ala 30 Pro Phe ett gac egg ett ege ega gac cag aag tcc eta agg gga aga ggt age 144 Leu Asp Arg 35 Leu Arg Arg Asp Gin 40 Lys Ser Leu Arg Gly 45 Arg Gly Ser act ett ggt ctg gac ate gaa aca gcc act cat gca gga aag cag ata 192 Thr Leu 50 Gly Leu Asp He Glu 55 Thr Ala Thr His Ala 60 Gly Lys Gin He gtg gag cag att ctg gaa aag gaa tea gat gag gca ett aaa atg acc 240 Val 65 Glu Gin He Leu Glu 70 Lys Glu Ser Asp Glu 75 Ala Leu Lys Met Thr 80 att gcc tet gtt cct get tea ege tac tta act gac atg act ett gat 288 He Ala Ser Val Pro 85 Ala Ser Arg Tyr Leu 90 Thr Asp Met Thr Leu 95 Asp gag atg tea aga gac tgg ttc atg etc atg ccc aag caa aaa gta aca 336 Glu Met Ser Arg 100 Asp Trp Phe Met Leu 105 Met Pro Lys Gin Lys 110 Val Thr ggc tcc eta tgt ata aga atg gac cag gca ate atg gat aag aac ate 384 Gly Ser Leu 115 Cys He Arg Met Asp 120 Gin Ala lie Met Asp 125 Lys Asn lie

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   ata He ett Leu 130 aaa Lys gca Ala aac Asn ttt Phe agt Ser 135 gtg Val att He ttc Phe gaa Glu agg Arg 140 ctg Leu gaa Glu aca Thr eta Leu 432 ata eta ett aga gcc ttc acc gaa gaa gga gca gtc gtt ggc gaa att 480 He 145 Leu Leu Arg Ala Phe 150 Thr Glu Glu Gly Ala 155 Val Val Gly Glu lie 160 tea cca tta cct tet ett cca gga cat act aat gag gat gtc aaa aat 528 Ser Pro Leu Pro Ser 165 Leu Pro Gly His Thr 170 Asn Glu Asp Val Lys 175 Asn gca att ggg gtc etc ate gga gga ett aaa tgg aat gat aat aeg gtt 576 Ala He Gly Val 180 Leu He Gly Gly Leu 185 Lys Trp Asn Asp Asn 190 Thr Val aga ate tet gaa act eta cag aga ttc get tgg aga age agt cat gaa 624 Arg He Ser 195 Glu Thr Leu Gin Arg 200 Phe Ala Trp Arg Ser 205 Ser His Glu aat ggg aga cct tea ttc cct tea aaa cag aaa ega aaa atg gag aga 672 Asn Gly 210 Arg Pro Ser Phe Pro 215 Ser Lys Gin Lys Arg 220 Lys Met Glu Arg

   aca att aag cca gaa att tgaagaaata agatggttga ttgaagaagt 720

   Thr lie Lys Pro Glu He 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 Gin Val Asp Cys Phe Leu Trp 15 10 15

   His Val Arg Lys Arg Phe Ala Asp Gin Glu Leu Gly Asp Ala Pro Phe 20 25 30

   Leu Asp Arg Leu Arg Arg Asp Gin Lys Ser Leu Arg Gly Arg Gly Ser 35 40 45

   Thr Leu Gly Leu Asp 50

   He

   Glu Thr Ala Thr His Ala 55 60

   Gly Lys

   Gin He

   Val Glu Gin lie Leu Glu Lys Glu Ser Asp Glu Ala Leu Lys Met Thr

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   146

   65 70 75 80

   lie 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 Gin Lys Val Thr 100 105 110 Gly Ser Leu Cys He Arg Met Asp Gin Ala He Met Asp Lys Asn lie 115 120 125 He Leu Lys Ala Asn Phe Ser Val He Phe Glu Arg Leu Glu Thr Leu 130 135 140 He Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu lie 145 150 155 160 Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn 165 170 175 Ala He Gly Val Leu He Gly Gly Leu Lys Trp Asn Asp Asn Thr Val 180 185 190 Arg lie Ser Glu Thr Leu Gin Arg Phe Ala Trp Arg Ser Ser His Glu 195 200 205 Asn Gly Arg Pro Ser Phe Pro Ser Lys Gin Lys Arg Lys Met Glu Arg 210 215 220 Thr He Lys Pro Glu He • 225 230 <210> 71 <211> 1497 <212> DNA <213> Influenza virus <220> <221> CDS <222> (1) - .(1497) <400> 71 atg gcg tet caa ggc acc aaa ega tcc tat gaa cag atg gaa act gat Met Ala Ser Gin Gly Thr Lys Arg Ser Tyr Glu Gin Met Glu Thr Asp 1 5 10 15 ggg gaa ege cag aat gca act gaa ate aga gca tet gtc gga agg atg Gly Glu Arg Gin Asn Ala Thr Glu lie Arg Ala Ser Val Gly Arg Met

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   20 25 30 gtg gga gga ate gga egg ttt tat gtc cag atg tgt act gag ctt aaa 144 Val Gly Gly 35 He Gly Arg Phe Tyr 40 Val Gin Met Cys Thr 45 Glu Leu Lys eta aac ’ gac cat gaa ggg egg ctg att cag aac age ata aca ata gaa 192 Leu Asn 50 Asp His Glu Gly Arg 55 Leu He Gin Asn Ser 60 He Thr He Glu agg atg gtg ctt teg gca ttc gac gaa aga aga aac aag tat etc gag 240 Arg 65 Met Val Leu Ser Ala 70 Phe Asp Glu Arg Arg 75 Asn Lys Tyr Leu Glu 80 gag cat ccc agt get ggg aaa gac cct aag aaa acg gga ggc ecg ata 288 Glu His Pro Ser Ala 85 Gly Lys Asp Pro Lys 90 Lys Thr Gly Gly Pro 95 He tac aga aga aaa gat ggg aaa tgg atg agg gaa etc ate etc cat gat 336 Tyr Arg Arg Lys 100 Asp Gly Lys Trp Met 105 Arg Glu Leu He Leu 110 His Asp aaa gaa gaa ate atg aga ate tgg cgt cag gcc aac aat ggt gaa gac 384 Lys Glu Glu 115 lie Met Arg He Trp 120 Arg Gin Ala Asn Asn 125 Gly Glu Asp get act get ggt ctt act cat atg atg ate tgg cac tcc aat etc aat 432 Ala Thr 130 Ala Gly Leu Thr His 135 Met Met lie Trp His 140 Ser Asn Leu Asn gac acc aca tac caa aga aca agg get ctt gtt egg act ggg atg gat 480 Asp 145 Thr Thr Tyr Gin Arg 150 Thr Arg Ala Leu Val 155 Arg Thr Gly Met Asp 160 ccc aga atg tgc tet ctg atg caa ggc tea acc etc cca egg aga tet 528 Pro Arg Met Cys Ser 165 Leu Met Gin Gly Ser 170 Thr Leu Pro Arg Arg 175 Ser gga gcc get ggt get gca gta aaa ggc gtt gga aca atg gta atg gaa 576 Gly Ala Ala Gly 180 Ala Ala Val Lys Gly 185 Val Gly Thr Met Val 190 Met Glu etc ate aga atg ate aag cgc gga ata aat gat egg aat ttc tgg aga 62,4 Leu He Arg 195 Met He Lys Arg Gly 200 He Asn Asp Arg Asn 2 05 Phe Trp Arg ggt gaa aat ggt ega aga acc aga att get tat gaa aga atg tgc aat 672 Gly Glu 210 Asn Gly Arg Arg Thr 215 Arg He Ala Tyr Glu 220 Arg Met Cys Asn ate etc aaa ggg aaa ttt cag aca gca gca caa egg get atg atg gac 720 lie 225 Leu Lys Gly Lys Phe 230 Gin Thr Ala Ala Gin 235 Arg Ala Met Met Asp 240 cag gtg agg gaa ggc cgc aat cct gga aac get gag att gag gat etc 768 Gin Val Arg Glu Gly 245 Arg Asn Pro Gly Asn 250 Ala Glu He Glu Asp 255 Leu att ttc ttg gca ega tea gca ctt att ttg aga gga tea gta gcc cat 816 lie Phe Leu Ala Arg Ser Ala Leu He Leu Arg Gly Ser Val Ala His

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   260 265 270

   aaa Lys tea tgc Ser Cys 275 eta Leu cct Pro gcc Ala tgt Cys gtt Val 280 tat Tyr ggc Gly ett gca eta acc agt Ser ggg Gly 864 Leu Ala Leu 285 Thr tat gac ttt gag aag gaa gga tac tet ctg gtt gga att gat cct ttc 912 Tyr Asp Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly He Asp Pro Phe 290 295 300 aaa eta etc cag aac agt caa att ttc agt eta ate aga cca aaa gaa 960 Lys Leu Leu Gin Asn Ser Gin lie Phe Ser Leu lie Arg Pro Lys Glu 305 310 315 320 aac cca gca cac aaa age cag ttg gtg tgg atg gca tgc cat tet gca 1008 Asn Pro Ala His Lys Ser Gin 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 He Arg Gly Thr Lys Val 340 345 350 ate cca aga gga cag tta aca acc aga gga gtt caa att get tea aat 1104 lie Pro Arg Gly Gin Leu Thr Thr Arg Gly Val Gin lie Ala Ser Asn 355 360 365* gaa aac atg gag aca ata aat tet age aca ett gaa ctg aga age aaa 1152 Glu Asn Met Glu Thr He Asn Ser Ser Thr Leu Glu Leu Arg Ser Lys 370 375 380 tat tgg gca ata agg acc aga age gga gga aac acc agt caa cag aga 1200 Tyr Trp Ala lie Arg Thr Arg Ser Gly Gly Asn Thr Ser Gin Gin Arg 385 390 395 400 gca tet gca gga cag ata agt gtg caa cct act ttc tea gta cag aga 1248 Ala Ser Ala Gly Gin He Ser Val Gin Pro Thr Phe Ser Val Gin Arg 405 410 415 aat ett ccc ttt gag aga gca acc att atg get gca ttc act ggt aac 1296 Asn Leu Pro Phe Glu Arg Ala Thr lie Met Ala Ala Phe Thr Gly Asn 420 425 430 act gaa gga agg act tcc gac atg aga aeg gaa ate ata agg atg atg 1344 Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu He He Arg Met Met 435 440 445 gaa aat gcc laaa tea gaa gat gtg tet ttc cag ggg egg gga gtc ttc 1392 Glu Asn Ala Lys Ser Glu Asp Val Ser Phe Gin Gly Arg Gly Val Phe 450 455 460 gag etc teg gac gaa aag gca aeg aac ccg ate gtg cct tcc ttt gac 1440 Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro He Val Pro Ser Phe Asp 465 470 475 480 atg age aat gaa ggg tet tat ttc ttc gga gac aat get 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 Asp Ser

   1497

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   CD

   TO

   CD

   Cd

   QD

   149 <210> 72 <211> 498 <212> PRT <213> Influenza virus <400> 72 Met Ala Ser Gin Gly Thr Lys Arg Ser Tyr Glu Gin Met Glu Thr Asp 1 5 10 15 Gly Glu Arg Gin Asn Ala Thr Glu He Arg Ala Ser Val Gly Arg Met 20 25 30 Val Gly Gly He Gly Arg Phe Tyr Val Gin Met Cys Thr Glu Leu Lys 35 40 45 Leu Asn Asp His Glu Gly Arg Leu He Gin Asn Ser He Thr He 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 He 85 90 95 Tyr Arg Arg Lys Asp Gly Lys Trp Met Arg Glu Leu He Leu His Asp 100 105 110 Lys Glu Glu He Met Arg He Trp Arg Gin Ala Asn Asn Gly Glu Asp 115 120 125 Ala Thr Ala Gly Leu Thr His Met Met He Trp His Ser Asn Leu Asn 130 135 140 Asp Thr Thr Tyr Gin Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp 145 150 155 160 Pro Arg Met Cys Ser Leu Met Gin 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 lie Arg Met He Lys Arg Gly He Asn Asp Arg Asn Phe Trp Arg 195 200 205

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   Gly Glu Asn Gly Arg Arg Thr Arg lie Ala Tyr Glu Arg Met Cys Asn 210 215 220

   He Leu Lys Gly Lys Phe Gin Thr Ala Ala Gin Arg Ala Met Met Asp 225 230 235 240

   Gin Val Arg Glu Gly Arg Asn Pro Gly Asn Ala Glu He Glu Asp Leu 245 250 255

   He Phe Leu Ala Arg Ser Ala Leu He 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 He Asp Pro Phe 290 ' 295 ¹ 300

   Lys Leu Leu Gin Asn Ser Gin He Phe Ser Leu He Arg Pro Lys Glu 305 310 315 320

   Asn Pro Ala His Lys Ser Gin Leu Val Trp Met Ala Cys His Ser Ala 325 330 335

   Ala Phe Glu Asp Leu Arg Val Leu Asn Phe He Arg Gly Thr Lys Val 340 345 350

   He Pro Arg Gly Gin Leu Thr Thr Arg Gly Val Gin He Ala Ser Asn 355 360 365

   Glu Asn Met Glu Thr He Asn Ser Ser Thr Leu Glu Leu Arg Ser Lys 370 375 380

   Tyr Trp Ala He Arg Thr Arg Ser Gly Gly Asn Thr Ser Gin Gin Arg 385 390 395 400

   Ala Ser Ala Gly Gin He Ser Val Gin Pro Thr Phe Ser Val Gin Arg 405 410 415

   Asn Leu Pro Phe Glu Arg Ala Thr He Met Ala Ala Phe Thr Gly Asn 420 425 430

   Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu He He Arg Met Met 435 440 445

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   Glu Asn Ala 450 Lys Ser Glu Asp 455 Val Ser Phe Gin Gly 460 Arg Gly Val Phe Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro lie 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).. <400> 73 (1413) atg aat cca aat caa aag ata ata gca att gga ttt gca tea ttg ggg Met 1 Asn Pro Asn Gin 5 Lys He He Ala He 10 Gly Phe Ala Ser Leu 15 Gly ata tta ate att aat gtc att etc cat gta gtc age att ata gta aca He Leu He lie 20 Asn Val He Leu His 25 Val Val Ser He He 30 Val Thr gta ctg gtc etc aat aac aat aga aca gat ctg aac tgc aaa ggg aeg Val Leu Val 35 Leu Asn Asn Asn Arg 40 Thr Asp Leu Asn Cys 45 Lys Gly Thr ate ata aga gaa tac aat gaa aca gta aga gta gaa aaa ctt act caa He He 50 Arg Glu Tyr Asn Glu 55 Thr Val Arg Val Glu 60 Lys Leu Thr Gin tgg tat aat acc agt aca att aag tac ata gag aga cct tea aat gaa Trp 65 Tyr Asn Thr Ser Thr 70 He Lys Tyr He Glu 75 Arg Pro Ser Asn Glu 80 tac tac atg aat aac act gaa cca ctt tgt gag gcc caa ggc ttt gca Tyr Tyr Met Asn Asn 85 Thr Glu Pro Leu Cys 90 Glu Ala Gin Gly Phe 95 Ala cca ttt tcc aaa gat aat gga ata ega att ggg teg aga ggc cat gtt Pro Phe Ser Lys 100 Asp Asn Gly He Arg 105 lie Gly Ser Arg Gly 110 His Val ttt gtg ata aga gaa cct ttt gta tea tgt teg ccc tea gaa tgt aga Phe Val lie 115 Arg Glu Pro Phe Val 120 Ser Cys Ser Pro Ser 125 Glu Cys Arg

   144

   192

   240

   288

   336

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   acc Thr ttt Phe 130 ttc Phe etc Leu aca Thr cag Gin ggc Gly 135 tea Ser tta Leu etc Leu aat Asn gac Asp 140 aaa Lys cat His tet Ser aac Asn 432 ggc aca ata aag gat ega agt ccg tat agg act ttg atg agt gtc aaa 480 Gly 145 Thr He Lys Asp Arg 150 Ser Pro Tyr Arg Thr 155 Leu Met Ser Val Lys 160 ata ggg caa tea cct aat gta tat caa get agg ttt gaa teg gtg gca 528 lie Gly Gin Ser Pro 165 Asn Val Tyr Gin Ala 170 Arg Phe Glu Ser Val 175 Ala tgg tea gca aca gca tgc cat gat gga aaa aaa tgg atg aca gtt gga 576 Trp Ser Ala Thr 180 Ala Cys His Asp Gly 185 Lys Lys Trp Met Thr 190 Val Gly gtc aca ggg ccc gac aat caa gca att gca gta gtg aac tat gga ggt 624 Val Thr Gly 195 Pro Asp Asn Gin Ala 200 He Ala Val Val Asn 205 Tyr Gly Gly gtt ccg gtt gat att att aat tea tgg gca ggg gat att tta aga acc 672 Val Pro 210 Val Asp He He Asn 215 Ser Trp Ala Gly Asp 220 He Leu Arg Thr caa gaa tea tea tgc acc tgc att aaa gga gac tgt tat tgg gta atg 720 Gin 225 Glu Ser Ser Cys Thr 230 Cys lie Lys Gly Asp 235 Cys Tyr Trp Val Met 240 act gat gga ccg gca aat agg caa get aaa tat agg ata ttc aaa gca 768 Thr Asp Gly Pro Ala 245 Asn Arg Gin Ala Lys 250 Tyr Arg He Phe Lys 255 Ala aaa gat gga aga gta att gga caa act gat ata agt ttc aat ggg gga 816 Lys Asp Gly Arg 260 Val He Gly Gin Thr 265 Asp He Ser Phe Asn 270 Gly Gly cac ata gag gag tgt tet tgt tac ccc aat gaa ggg aag gtg gaa tgc 864 His He Glu 275 Glu Cys Ser Cys Tyr 280 Pro Asn Glu Gly Lys 285 Val Glu Cys ata tgc agg gac aat tgg act gga aca aat aga cca att ctg gta ata 912 He Cys 290 Arg Asp Asn Trp Thr 295 Gly Thr Asn Arg Pro 300 He Leu Val He tet tet gat eta teg tac aca gtt gga tat ttg tgt get ggc att ccc 960 Ser 305 Ser Asp Leu Ser Tyr 310 Thr Val Gly Tyr Leu 315 Cys Ala Gly He Pro 320 act gac act cct agg gga gag gat agt caa ttc aca ggc tea tgt aca 1008 Thr Asp Thr Pro Arg 325 Gly Glu Asp Ser Gin 330 Phe Thr Gly Ser Cys 335 Thr agt cct ttg gga aat aaa gga tac ggt gta aaa ggc ttc ggg ttt ega 1056 Ser Pro Leu Gly 340 Asn Lys Gly Tyr Gly 345 Val Lys Gly Phe Gly 350 Phe Arg caa gga act gac gta tgg gcc gga agg aca att agt agg act tea aga 1104 Gin Gly Thr 355 Asp Val Trp Ala Gly 360 Arg Thr He Ser Arg 365 Thr Ser Arg

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   tea Ser gga ttc gaa Glu ata He ata He aaa Lys 375 ate lie agg Arg aat Asn ggt Gly tgg Trp 380 aca Thr cag Gin aac Asn agt Ser 1152 Gly 370 Phe aag gac caa ate agg agg caa gtg att ate gat gac cca aat tgg tea 1200 Lys Asp Gin lie Arg Arg Gin Val He He Asp Asp Pro Asn Trp Ser 385 390 395 400 gga tat age ggt tet 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 He Arg Gly Lys Pro Glu 420 425 430 gaa aca aca ata tgg acc tet age age tcc att gtg atg tgt gga gta 1344 Glu Thr Thr He Trp Thr Ser Ser Ser Ser lie Val Met Cys Gly Val 435 440 445 gat cat aaa att gcc agt tgg tea tgg cac gat gga get att ett ccc 1392 Asp His Lys He Ala Ser Trp Ser Trp His Asp Gly Ala He Leu Pro 450 455 460 ttt gac ate gat aag atg taa 1413 Phe Asp He Asp Lys Met 465 470 <210> ' 74 <211> 470 <212> PRT <213> Influenza virus <400> ¹ 74 Met Asn Pro Asn Gin Lys He He Ala He Gly Phe Ala Ser Leu Gly 1 5 10 15 lie Leu He He Asn Val He Leu His Val Val Ser Xie He Val Thr 20 25 30 Val Leu Val Leu Asn Asn Asn Arg Thr Asp Leu Asn Cys Lys Gly Thr 35 40 45 He He Arg Glu Tyr Asn Glu Thr Val Arg Val Glu Lys Leu Thr Gin 50 55 60 Trp Tyr Asn Thr Ser Thr He Lys Tyr He Glu Arg Pro Ser Asn Glu 65 70 75 80 Tyr Tyr Met Asn Asn Thr Glu Pro Leu Cys Glu Ala Gin Gly Phe Ala

   85 90 95

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   Pro Phe Ser Lys Asp Asn Gly lie Arg lie Gly Ser Arg Gly His Val 100 105 110

   Phe Val He Arg Glu Pro Phe Val Ser Cys Ser Pro Ser Glu Cys Arg 115 120 125

   Thr Phe Phe Leu Thr Gin Gly Ser Leu Leu Asn Asp Lys His Ser Asn 130 135 140

   Gly Thr He Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser Val Lys 145 150 155 160

   He Gly Gin Ser Pro Asn Val Tyr Gin 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 Gin Ala He Ala Val Val Asn Tyr Gly Gly 195 200 205

   Val Pro Val Asp He He Asn Ser Trp Ala Gly Asp He Leu Arg Thr 210 215 220

   Gin Glu Ser Ser Cys Thr Cys He Lys Gly Asp Cys Tyr Trp Val Met 225 230 235 240

   Thr Asp Gly Pro Ala Asn Arg Gin Ala Lys Tyr Arg He Phe Lys Ala 245 250 255

   Lys Asp Gly Arg Val lie Gly Gin Thr Asp He Ser Phe Asn Gly Gly 260 265 270

   His He Glu Glu Cys Ser Cys Tyr Pro Asn Glu Gly Lys Val Glu Cys 275 280 285

   He Cys Arg Asp Asn Trp Thr Gly Thr Asn Arg Pro He Leu Val He 290 295 300

   Ser Ser Asp Leu Ser Tyr Thr Val Gly Tyr Leu Cys Ala Gly He Pro 305 310 315 320

   Thr Asp Thr Pro Arg Gly Glu Asp Ser Gin Phe Thr Gly Ser Cys Thr 325 330 335

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   Ser Pro Leu Gly Asn 340 Lys Gly Tyr Gly Val Lys 345 Gly Phe Gly 350 Phe Arg Gin Gly Thr Asp Val Trp Ala Gly Arg Thr He Ser Arg Thr Ser Arg 355 360 365 Ser Gly Phe Glu He He Lys He Arg Asn Gly Trp Thr Gin Asn Ser 370 375 380 Lys Asp Gin He Arg Arg Gin Val He lie 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 lie Arg Gly Lys Pro Glu 420 425 430 Glu Thr Thr He Trp Thr Ser Ser Ser Ser He Val Met Cys Gly Val 435 440 445 Asp His Lys He Ala Ser Trp Ser Trp His Asp Gly Ala He Leu Pro 450 455 460 Phe Asp lie Asp Lys Met

   465 470 <210> 75 <211> 981 <212> DNA

   <213> Influenza virus <220> <221> CDS <222 > (1) - · . (756) <400> ' 75 atg agt ett eta acc gag gtc gaa aeg tac gtt etc tet ate gta cca 48 Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser lie Val Pro 1 5 10 15 tea ggc ccc etc aaa gcc gag ate geg cag aga ett gaa gat gtc ttt 96 Ser Gly Pro Leu Lys Ala Glu He Ala Gin Arg Leu Glu Asp Val Phe 20 25 30 geg gga aag aac acc gat ett gag gca etc atg gaa tgg eta aag aca 144 Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr 35 40 45

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   aga Arg cca Pro 50 ate lie ctg tea cct ctg Leu 55 act Thr aaa Lys ggg Gly att He tta gga ttt Leu Gly Phe 60 gta Val ttc Phe Leu Ser Pro aeg etc acc gtg ccc agt gag ega gga ctg cag cgt aga ege ttt gtc Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gin Arg Arg Arg Phe Val 65 70 75 80 caa aat gcc ett agt gga aac gga gat cca aac aac atg gac aga gca Gin Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala 85 90 95 gta aaa ctg tac agg aag ett aaa aga gaa ata aca ttc cat ggg gca Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu He Thr Phe His Gly Ala 100 105 110 aaa gag gtg gca etc age tat tcc act ggt gca eta gcc age tgc atg Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met 115 120 125 gga etc ata tac aac aga atg gga act gtt aca acc gaa gtg gca ttt Gly Leu He Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe 130 135 14 0 ggc ctg gta tgc gee aca tgt gaa cag att get gat tcc cag cat ega Gly Leu Val Cys Ala Thr Cys Glu Gin He Ala Asp Ser Gin His Arg 145 150 155 160 tet cac agg cag atg gtg aca aca acc aac cca tta ate aga cat gaa Ser His Arg Gin Met Val Thr Thr Thr Asn Pro Leu He Arg His Glu 165 170 175 aac aga atg gta tta gcc agt acc aeg get aaa gcc atg gaa cag atg Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gin Met 180 185 190 gca gga teg agt gag cag gca gca gag gcc atg gag gtt get agt agg Ala Gly Ser Ser Glu Gin Ala Ala Glu Ala Met Glu Val Ala Ser Arg 195 200 205 get agg cag atg gta cag gca atg aga acc att ggg acc cac cct age Ala Arg Gin Met Val Gin Ala Met Arg Thr He Gly Thr His Pro Ser 210 215 220 tcc agt gcc ggt ttg aaa gat gat etc ett gaa aat tta cag gcc tac Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gin Ala Tyr 225 230 235 240 cag aaa egg atg gga gtg caa atg cag ega ttc aag tgatcctctc Gin Lys Arg Met Gly Val Gin Met Gin Arg Phe Lys 245 250

   gtcattgcag ttcttcaaat ggagtacctg caagtatcat tcatttatcg agtctatgag tgggatcttg tcgccttaaa ggaagaatat cacttgatat tacgggttga cggcaggaac tgtggattct aaagagggcc agcagaatgc tgatcgtctt ttctacggaa tgtggatgtt

   192

   240

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   336

   384

   432

   480

   528

   576

   624

   672

   720

   766

   826

   886

   946 gacgatggtc attttgtcaa catagagctg gagta

   981

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   IT) ©

   <N

   157 <210> 76 <211> 252 <212> PRT <213> Influenza virus <400> 76 Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser He Val Pro 1 5 10 15 Ser Gly Pro Leu Lys Ala Glu He Ala Gin 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 Xie Leu Ser Pro Leu Thr Lys Gly He Leu Gly Phe Val Phe 50 55 60 Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gin Arg Arg Arg Phe Val 65 70 75 80 Gin Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala ¹ 85 90 95 Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu He 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 lie Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe 130 135 140 Gly Leu Val Cys Ala Thr Cys Glu Gin He Ala Asp Ser Gin His Arg 145 150 155 160 Ser His Arg Gin Met Val Thr Thr Thr Asn Pro Leu He Arg His Glu 165 170 175 Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gin Met 180 185 190 Ala Gly Ser Ser Glu Gin Ala Ala Glu Ala Met Glu Val Ala Ser Arg 195 200 205

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   Ala Arg Gin Met Val Gin Ala Met Arg Thr lie Gly Thr His Pro Ser 210 215 220

   Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gin Ala Tyr 225 230 235 240

   Gin Lys Arg Met Gly Val Gin Met Gin 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 eta ctg acc cat tgg gcc tac agt

   Met Lys Thr Thr He He Leu He Leu Leu Thr His Trp Ala Tyr Ser

   1 5 10 15 caa aac cca ate agt ggc aat aac aca gcc aca ctg tgt ctg gga cac

   Gin Asn Pro He 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

   His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Met Ser Asp Asp Gin

   35 40 45 att gag gtg aca aat get aca gaa tta gtt cag age att tea atg ggg

   He Glu Val Thr Asn Ala Thr Glu Leu Val Gin Ser He Ser Met Gly

   50 55 60 aaa ata tgc aac aaa tea tat aga att eta gat gga aga aat tgc aca

   Lys He Cys Asn Lys Ser Tyr Arg He Leu Asp Gly Arg Asn Cys Thr

   65 70 75 80 tta ata gat gca atg eta gga gac ccc cac tgt gac gcc ttt cag tat

   Leu He Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gin Tyr

   85 90 95 gag agt tgg gac etc ttt ata gaa aga age age get ttc age aat tgc

   Glu Ser Trp Asp Leu Phe He Glu Arg Ser Ser Ala Phe Ser Asn Cys

   100 105 110 tac cca tat gac ate cct gac tat gca ccg etc ega tcc att gta gca

   Tyr Pro Tyr Asp He Pro Asp Tyr Ala Pro Leu Arg Ser He Val Ala

   115 120 125 tcc tea ggg aca gtg gaa ttc aca gca gag gga ttc aca tgg aca ggt

   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 tea gcc gat

   144

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   432

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   Val 145 Thr Gin Asn Gly Arg 150 Ser Gly Ala Cys Lys 155 Arg Gly Ser Ala Asp 160 agt ttc ttt age ega ctg aat tgg eta aca aaa tet gga age tet tac 528 Ser Phe Phe Ser Arg 165 Leu Asn Trp Leu Thr 170 Lys Ser Gly Ser Ser 175 Tyr ccc aca ttg aat gtg aca atg cct aac aat aaa aat ttc gac aag eta 576 Pro Thr Leu Asn 180 Val Thr Met Pro Asn 185 Asn Lys Asn Phe Asp 190 Lys Leu tac ate tgg ggg att cat cac ccg age tea aat caa· gag cag aca aaa 624 Tyr He Trp 195 Gly He His His Pro 200 Ser Ser Asn Gin Glu 205 Gin Thr Lys ttg tac ate caa gaa tea gga ega gta aca gtc tea aca aaa aga agt 672 Leu Tyr 210 He Gin Glu Ser Gly 215 Arg Val Thr Val Ser 220 Thr Lys Arg Ser caa caa aca ata ate cct aac ate gga tet aga ccg ttg gtc aga ggt 720 Gin 225 Gin Thr He He Pro 230 Asn He Gly Ser Arg 235 Pro Leu Val Arg Gly 240 caa tea ggc agg ata age ata tac tgg acc att gta aaa cct gga gat 768 Gin Ser Gly Arg lie 245 Ser He Tyr Trp Thr 250 He Val Lys Pro Gly 255 Asp ate eta atg ata aac agt aat ggc aac tta gtt gca ccg egg gga tat 816 lie Leu Met He 260 Asn Ser Asn Gly Asn 265 Leu Val Ala Pro Arg 270 Gly Tyr ttt aaa ttg aac aca ggg aaa age tet gta atg aga tcc gat gta ccc 864 Phe Lys Leu 275 Asn Thr Gly Lys Ser 280 Ser Val Met Arg Ser 285 Asp Val Pro ata gac att tgt gtg tet gaa tgt att aca cca aat gga age ate tcc 912 lie Asp 290 He Cys Val Ser Glu 295 Cys He Thr Pro Asn 300 Gly Ser He Ser aac gac aag cca ttc caa aat gtg aac aaa gtt aca tat gga aaa tgc 960 Asn 305 Asp Lys Pro Phe Gin 310 Asn Val Asn Lys Val 315 Thr Tyr Gly Lys Cys 320 ccc aag tat ate agg caa aac act tta aag ctg gee act ggg atg agg 1008 Pro Lys Tyr He Arg 325 Gin Asn Thr Leu Lys 330 Leu Ala Thr Gly Met 335 Arg aat gta cca gaa aag caa acc aga gga ate ttt gga gca ata geg gga 1056 Asn Val Pro Glu 340 Lys Gin Thr Arg Gly 345 He Phe Gly Ala He 350 Ala Gly ttc ate gaa aac ggc tgg gaa gga atg gtt gat ggg tgg tat ggg ttc 1104 Phe He Glu 355 Asn Gly Trp Glu Gly 360 Met Val Asp Gly Trp 365 Tyr Gly Phe ega tat caa aac tet gaa gga aca ggg caa get gca gat eta aag age 1152 Arg Tyr 370 Gin Asn Ser Glu Gly 375 Thr Gly Gin Ala Ala 380 Asp Leu Lys Ser act caa gca gcc ate gac cag att aat gga aag tta aac aga gtg att 1200

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   Thr 385 Gin Ala Ala He Asp 390 Gin He Asn Gly Lys 395 Leu Asn Arg Val He 400 gaa aga acc aat gag aaa ttc cat caa ata gag aag gaa ttc tea gaa 1248 Glu Arg Thr Asn Glu 405 Lys Phe His Gin He 410 Glu Lys Glu Phe Ser 415 Glu gta gaa gga aga att cag gac ttg gag aaa tat gta gaa gac acc aaa 1296 Val Glu Gly Arg 420 He Gin Asp Leu Glu 425 Lys Tyr Val Glu Asp 430 Thr Lys ata gac eta tgg tcc tac aat gca gaa ttg ctg gtg get eta gaa aat 1344 He Asp Leu 435 Trp Ser Tyr Asn Ala 440 Glu Leu Leu Val Ala 445 Leu Glu Asn caa cat aca att gac tta aca gat gca gaa atg aat aaa tta ttt gag 1392 Gin His 450 Thr 1 He Asp Leu Thr 455 Asp Ala Glu Met Asn 460 Lys Leu Phe Glu aag act aga ege cag tta aga gaa aac gca gaa gac atg gga ggt gga 1440 Lys 465 Thr Arg Arg Gin Leu 470 Arg Glu Asn Ala Glu 475 Asp Met Gly Gly Gly 480 tgt ttc aag att tac cac aaa tgt gat aat gca tgc att gaa tea ata 1488 Cys Phe Lys He Tyr 485 His Lys Cys Asp Asn 490 Ala Cys He Glu Ser 495 He aga act ggg aca tat gac cat tac ata tac aaa gat gaa gca tta aac 1536 Arg Thr Gly Thr 500 Tyr Asp His Tyr He 505 Tyr Lys Asp Glu Ala 510¹ Leu Asn aat ega ttt cag ate aaa ggt gta gag ttg aaa tea ggc tac aaa gat 1584 Asn Arg Phe 515 Gin He Lys Gly Val 520 Glu Leu Lys Ser Gly 525 Tyr Lys Asp tgg ata ctg tgg att tea ttc gcc ata tea tgc ttc tta att tgc gtt 1632 Trp He 530 Leu Trp He Ser Phe 535 Ala He Ser Cys Phe 540 Leu lie Cys Val gtt eta ttg ggt ttc att atg tgg get tgc caa aaa ggc aac ate aga 1680 Val 545 Leu Leu. Gly Phe He 550 Met Trp Ala Cys Gin 555 Lys Gly Asn lie Arg 560

   tgc aac att tgc att tga 1698

   Cys Asn lie Cys He

   565 <210> 78 <211> 565 <212> PRT <213> Influenza virus <400> 78

   Met Lys Thr Thr He He Leu He Leu Leu Thr His Trp Ala Tyr Ser 15 10 15

   Gin Asn Pro He Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His

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   20 25 30

   His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Met Ser Asp Asp Gin 35 40 45 lie Glu Val Thr Asn Ala Thr Glu Leu Val Gin Ser He Ser Met Gly 50 55 60

   Lys lie Cys Asn Lys Ser Tyr Arg lie Leu Asp Gly Arg Asn Cys Thr 65 70 75 80

   Leu He Asp Ala Met Leu Gly Asp Pro His Cys Asp Ala Phe Gin Tyr 85 90 95

   Glu Ser Trp Asp Leu Phe He Glu Arg Ser Ser Ala Phe Ser Asn Cys 100 105 110

   Tyr Pro Tyr Asp He Pro Asp Tyr Ala Pro Leu Arg Ser He 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 Gin 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 He Trp Gly He His His Pro Ser Ser Asn Gin Glu Gin Thr Lys 195 200 205

   Leu Tyr 210

   He Gin Glu

   Ser Gly Arg Val 215

   Thr Val

   Ser Thr Lys Arg Ser 220

   Gin Gin Thr He 225

   He

   Pro Asn He 230

   Gly Ser Arg 235

   Pro

   Leu Val Arg Gly 240

   Gin Ser Gly Arg He Ser He Tyr Trp Thr He Val Lys 245 250

   Pro Gly Asp 255

   He Leu Met He Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr

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   260 265 270

   Phe Lys Leu Asn Thr Gly Lys Ser Ser Val Met Arg Ser Asp Val Pro 275 280 285 lie Asp He Cys Val Ser Glu Cys He Thr Pro Asn Gly Ser He Ser 290 295 300

   Asn Asp Lys Pro Phe Gin Asn Val Asn Lys Val Thr Tyr Gly Lys Cys 305 310 315 320

   Pro Lys Tyr He Arg Gin Asn Thr Leu Lys Leu Ala Thr Gly Met Arg 325 330 335

   Asn Val Pro Glu Lys Gin Thr Arg Gly He Phe Gly Ala He Ala Gly 340 345 350

   Phe He Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe 355 360 365

   Arg Tyr Gin Asn Ser Glu Gly Thr Gly Gin Ala Ala Asp Leu Lys Ser 370 375 380

   Thr Gin Ala Ala He Asp Gin He Asn Gly Lys Leu Asn Arg Val He 385 390 395 400

   Glu Arg Thr Asn Glu Lys Phe His Gin He Glu Lys Glu Phe Ser Glu 405 410 415

   Val Glu Gly Arg He Gin Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys 420 425 430

   He Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn 435 440 445

   Gin His Thr He Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu 450 455 460

   Lys

   465

   Thr Arg Arg

   Gin

   Leu Arg Glu Asn Ala 470

   Glu Asp 475

   Met

   Gly Gly Gly 480

   Cys Phe Lys lie Tyr His Lys 485

   Cys Asp Asn Ala Cys 490

   He Glu Ser He 495

   Arg Thr Gly Thr Tyr Asp His Tyr He Tyr Lys Asp Glu Ala Leu Asn

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   500

   505

   510

   Asn Arg Phe Gin He Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp 515 520 525

   Trp He Leu Trp He Ser Phe Ala He Ser Cys Phe Leu He Cys Val 530 535 540

   Val Leu Leu Gly Phe He Met Trp Ala Cys Gin Lys Gly Asn He Arg 545 550 555 560

   Cys Asn He Cys He 565 <210> 79 <211> 20 <212> DNA <213> Influenza virus <400> 79 tatgcatcgc tccgatccat <210> 80 <211> 21 <212> DNA <213> Influenza virus <400> 80 gctccacttc ttccgttttg a <210> 81 <211> 30 <212> DNA <213> Influenza virus <400> 81 aattcacagc agagggattc acatggacag <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

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   2015234384 02 Oct 2015 <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