CA2529647C - Influenza hemagglutinin and neuraminidase variants - Google Patents

Influenza hemagglutinin and neuraminidase variants Download PDF

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CA2529647C
CA2529647C CA2529647A CA2529647A CA2529647C CA 2529647 C CA2529647 C CA 2529647C CA 2529647 A CA2529647 A CA 2529647A CA 2529647 A CA2529647 A CA 2529647A CA 2529647 C CA2529647 C CA 2529647C
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virus
influenza
sequence
sequences
polynucleotide
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CA2529647A1 (en
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Chin-Fen Yang
George Kemble
Chongguang G. Liu
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MedImmune LLC
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MedImmune Vaccines Inc
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Abstract

Polypeptides, polynucleotides, methods, compositions, and vaccines comprising influenza hemagglutinin and neuraminidase variants are provided.

Description

INFLUENZA HEMAGGLUTININ AND NEURAIVILNIDASE
VARIANTS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Pursuant to 35 U.S.C. 119(e), this application claims priority to and benefit of U.S. Provisional Patent Application Serial No. 60/479,078, filed on June 16, 2003.
BACKGROUND OF THE INVENTION
[0002] Vaccines against various and evolving strains of influenza are important from a community health standpoint, as well as commercially, since each year numerous individuals are infected with different strains and types of influenza virus.
Infants, the elderly, those without adequate health care and imrauno-compromised persons are at special risk of death from such infections. Compounding the problem of influenza infections is that novel influenza strains evolve readily and can spread between various species, thereby necessitating the continuous production of new vaccines.
[0003] Numerous vaccines capable of producing a protective immune response specific for different influenza viruses/virus strains have been produced for over 50 years and include whole virus vaccines, split virus vaccines, surface antigen vaccines and live attenuated virus vaccines. However, while appropriate formulations of any of these vaccine types are capable of producing a systemic immune response, live attenuated virus vaccines have the advantage of also being able to stimulate local mucosal immunity in the respiratory tract. 'Considerable work in the production of influenza viruses, and fragments thereof, for production of vaccines has been done by the present inventors and co-workers;
see, e.g., PCT publication WO 03/091401, US Published Patent Application No. US
2004/0029251, and PCT publication WO 2005/115448, all relating to "Multi-Plasmid System for the Production of Influenza Virus".
[0004] Because of the continual emergence (or re-emergence) or different influenza strains, new influenza vaccines are continually desired. Such vaccines typically are created using antigenic moieties of the newly emergent virus strains so, therefore, polypeptides and polynucleotides of novel, newly emergent, or newly re-emergent virus strains (especially sequences of antigenic genes) are highly desirable. Furthermore, such sequences within preferred vectors are also quite highly desired.
[0005] The present invention provides new and/or newly isolated influenza hemagglutinin and neuraminidase variants, optionally within preferred vectors, that are capable of use in production of numerous types of vaccines as well as in research, diagnostics, etc. Numerous other benefits will become apparent upon review of the following SUMMARY OF THE INVENTION
[0006] In some aspects herein, the invention comprises an isolated or recombinant polypeptide that is selected from: the polypeptides encoded by any one of the sequences of the sequence listing, e.g., SEQ ID NO:1 through SEQ ID NO:34, any one of the polypeptides encoded by the sequence listing, e.g., SEQ ED NO:35 through SEQ
ID NO:68;
any polypeptide that is encoded by a polynucleotide sequence which hybridizes under highly stringent conditions over substantially the entire length of a polynucleotide sequence of the sequence listing; and, a fragment of any of the above wherein the sequence comprises a hemagglutinin or neuraminidase polypeptide, or a fragment thereof. In various embodiments, the isolated or recombinant polypeptides of the invention are substantially identical to about 300 contiguous amino acid residues of any of the above polypeptides. In yet other embodiments, the invention comprises isolated or recombinant polypeptides (comprising hemagglutinin or fragments thereof), that comprise an amino acid sequence that is substantially identical over at least about 350 amino acids; over at least about 400 amino acids; over at least about 450 amino acids; over at least about 500 amino acids; over at least about 502 amino acids; over at least about 550 amino acids; over at least about 559 amino acids; over at least about 565 amino acids; or over at least about 566 amino acids contiguous of any of the polypeptides of claim of any of the above polypeptides. In yet other embodiments, the invention comprises isolated or recombinant polypeptides (e.g., comprising neuraminidase or fragments thereof), that comprise an amino acid sequence that is substantially identical over at least about 350 amino acids; over at least about 400 amino acids; over at least about 436 amino acids; over at least about 450 amino acids; over at least about 451 amino acids; over at least about 465 amino acids; over at least about 466 amino acids; over at least about 469 amino acids; or over at least about 470 amino acids contiguous of any of the polypeptides of any of the above polypeptides. Of course, in some embodiments, the polypeptide sequence (e.g., as listed in the sequence listing herein, e.g., SEQ ID NO:35 through SEQ ID NO:68) comprises less than 565, 559, etc. amino acids. In such embodiments, the shorter listed polypeptides optionally comprise less than 565, 559, etc. amino acids. In yet other embodiments, the polypeptides of the invention optionally comprise fusion proteins, proteins with a leader sequence, a precursor polypeptide, proteins with a secretion signal or a localization signal, or proteins with an epitope tag, an E-tag, or a His epitope tag, etc. In still other embodiments, the invention comprises a polypeptide comprising a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least 99.8%, or at least 99.9% sequence identity to at least one polypeptide listed above-. In some embodiments, such polypeptides are immunogenic. =
[0007] In other aspects, the invention comprises a composition with one or more polypeptide listed above, or fragments thereof. The invention also includes polypeptides that are specifically bound by a polyclonal antisera raised against at least 1 antigen that comprises at least one amino acid sequence described above, or a fragment thereof. Such antibodies specific for the polypeptides described above are also features of the invention.
The polypeptides of the invention are optionally immunogenic.
[0008] The invention also encompasses immunogenic compositions comprising an immunologically effective amount of one or more of any of the polypeptides described above as well as methods for stimulating the immune system of an individual to produce a protective immune response against influenza virus by administering to the individual an immunologically effective amount of any of the above polypeptides in a physiologically acceptable carrier.
[0009] Additionally, the invention has reassortant influenza virus that encode one or more of the polypeptides above, in addition to immunogenic compositions comprising an immunologically effective amount of such recombinant influenza virus. Methods for stimulating the immune system of an individual to produce a protective immune response against influenza virus, through administering an immunologically effective amount of such recombinant influenza virus in a physiologically acceptable carrier are also part of the invention. Such virus can optionally comprise a 6:2 reassortant virus with 6 genes encoding regions from one or more donor virus (e.g. A/AA/6/60, B/Ann Arbor/1/66, or A/Puerto Rico/8/34, which is more commonly known as PR8) and 2 gene encoding regions (typically and preferably encoding HA and NA or fragments thereof) selected from SEQ ID
NO:1 through SEQ ID NO:34 or from similar strains, as defined herein, to those having SEQ ID
NO:1-34, etc. Immunogenic compositions comprising such reassortant(recombinant) virus are also features of the invention.
[0010] In other aspects, the invention comprises an isolated or recombinant nucleic acid that is selected from: any one of the polynucleotide sequences of the sequence listing, e.g., SEQ ID NO:1 through SEQ ID NO:34 (or complementary sequences thereof), any one of the polynucleotide sequences encoding a polypeptide of the sequence listing, e.g., SEQ
ID NO:35 through SEQ NO:68 (or complementary polynucleotide sequences thereof), a polynucleotide sequence which hybridizes under highly stringent conditions over substantially the entire length of any of the above polynucleotide sequences, and a polynucleotide sequence comprising all or a fragment of any of the above polynucleotide sequences wherein the sequence encodes a hemagglutinin or neuraminidase polypeptide or a fragment thereof. Such nucleic acids can be DNA, RNA, cRNA, DNA:RNA hybrids, single stranded nucleic acid, double stranded nucleic acid, etc. The invention also includes an isolated or recombinant nucleic acid (e.g., comprising hemagglutinin or fragments thereof), that encodes an amino acid sequence which is substantially identical over at least about 300 amino acids of any of the above nucleic acids, or over at least about 350 amino acids; over at least about 400 amino acids; over at least about 450 amino acids; over at least about 500 amino acids; over at least about 502 amino acids; over at least about 550 amino acids; over at least about 559 amino acids; over at least about 565 amino acids; or over at least about 566 amino acids of any of the above nucleic acids. In yet other embodiments, the invention comprises isolated or recombinant nucleic acids (e.g., comprising neuraminidase or fragments thereof), that encode an amino acid sequence that is substantially identical over at least about 350 amino acids; over at least about 400 amino acids; over at least about 436 amino acids; over at least about 450 amino acids; over at least about 451 amino acids; over at least about 465 amino acids; over at least about 466 amino acids; over at least about 469 amino acids; or over at least about 470 amino acids contiguous of any of the polypeptides above. Again, in situations wherein the amino acid is less than, e.g., 566, 565, 559, etc. in., length (e.g., see, Sequence Listing in Figure 1) then it should be understood that the length is optionally less than 566, 565, 559, etc. The invention also includes any of the above nucleic acids that comprise a hemagglutinin or neuraminidase polypeptide, or fragment thereof. Other aspects of the invention include isolated or recombinant nucleic acids that encode a polypeptide (optionally a hemagglutinin or neuraminidase polypeptide) whose .
sequence has at least 95% identity, at least 96% identity, at least 97%
identity, at least 98%
identity, at least 98.5% identity, at least 99% identity, at least 99.2%
identity, at least 99.4%
identity, at least 99.6% identity, at least 99.8% identity, or at least 99.9%
identity to at least one of the above described polynucleotide. The invention also includes isolated or recombinant nucleic acids encoding a polypeptide of hemagglutinin or neuraminidase produced by mutating or recombining one or more above described polynucleotide sequence. The polynucleotide sequences of the invention can optionally comprise one or more of, e.g., a leader sequence, a precursor sequence, or an epitope tag sequence or the like, and can optionally encode a fusion protein (e.g., with one or more additional nucleic acid sequences). Such nucleic acids of the invention can optionally encode immunogenic polypeptides.
[0011] In yet other embodiments, the invention comprises a composition of matter having two or more above described nucleic acids or fragments thereof (e.g., a library comprising at least about 2, 5, 10, 50 or more nucleic acids). Such compositions can optionally be produced by cleaving one or more above described nucleic acid (e.g., mechanically, chemically, enzymatically with a restriction endonuclease/RNAse/DNAse, etc.). Other compositions of the invention include, e.g., compositions produced by incubating one or more above described nucleic acid in the presence of deoxyribonucleotide triphosphates and a thermostable nucleic acid polymerase. Immunogenic compositions having an immunologically effective amount of any of the above nucleic acids are also within the current invention.
[0012] Also within the invention are reassortant influenza virus comprising any of the above nucleic acids. Such reassortant viruses can (and preferably are) 6:2 reassortant viruses with 6 gene encoding regions from one or more donor virus (e.g., A/AAJ6/60, B/AA/1/66 (also sometimes referred to herein as B/Ann Arbor/1/66, or A/Puerto Rico/8/34) and 2 gene encoding regions from two sequences above (e.g., from SEQ lD NO:1-34, from similar strains to those encoded in SEQ ID NO:1-34, etc.). Preferably, such two regions encode hemagglutinin and/or neuraminidase. Immunogenic compositions with immunologically effective amounts of such reassortant/recombinant influenza virus are also within purview of the current invention.
[0013] Vectors comprising one or more nucleic acid from SEQ ID NO:1-34 (again, also from similar strains to those of the sequence identification numbers) or fragments thereof are also within the current invention. Such vectors (e.g., expression vectors) can optionally be plasmids, cosmids, phage, viruses, virus fragments, etc.
Especially preferred embodiments comprise plasmid vectors useful in plasmid rescue methods to produce virus (e.g., typically reassortant/recombinant virus for use in vaccines). Such plasmid systems are exampled in, e.g., PCT publication WO 03/091401, US Published Patent Application No. US 2004/0029251, and PCT publication WO 2005/115448, all relating to "Multi-Plasmid System for the Production of Influenza Virus"; Hoffmann, E., 2000, PNAS, 97(11):6108-6113; U.S. Published Patent Application No. 20020164770 to Hoffmann;
and U.S.P.N. 6,544,785 issued April 8,2003 to Palese, et al. Cells transduced, transformed, transfected, etc. with such vectors are also within the current invention.
[0014] The invention also encompasses cells comprising at least one above described nucleic acid, or a cleaved or amplified fragment or product thereof.
Such cells can optionally express a polypeptide encoded by such nucleic acid. Other embodiments of the invention include vectors (e.g., plasmids, cosmids, phage, viruses, virus fragments, etc.) comprising any of above described nucleic acid. Such vectors can optionally comprise an expression vector. Cells transduced by such vectors are also within the current invention.
[0015] In some embodiments, the invention encompasses a virus (e.g., an influenza virus) comprising one or more above described nucleic acid (e.g., from SEQ ID
NO:1-34 or from similar strains to such and optionally encoding hemagglutinin and/or neuraminidase), or one or more fragments thereof. Typically, such viruses are reassortant/recombinant viruses. Immunogenic compositions comprising such virus are also part of the current invention. Such viruses can comprises a reassortant virus such as a 6:2 reassortment virus (which comprises 6 gene encoding regions from one or more donor virus (e.g., a master donor virus or a backbone virus such as A/AA/6/60, B/AA/1/66, A/Puerto Rico/8/34, etc.) and 2 gene encoding regions from one or more above described nucleotide sequence, or one or more fragment thereof which can optionally comprise hemagglutinin and/or neuraminidase). Other reassortant/recombinant viruses can comprise 7:1 reassottments.

Reassortment viruses (optionally live viruses) of the invention can include donor viruses that are one or more of, e.g., temperature-sensitive (ts), cold-adapted (ca), or attenuated (att). For example, reassortment viruses can comprise, e.g., A/Ann Arbor/6/60, B/Ann Arbor/1/66, A/Puerto Rico/8/34, etc. In many embodiments, the produced viruses are live viruses (e.g., to be used in vaccines, etc.). Other embodiments include dead or inactivated viruses (e.g., also capable of use in vaccines, etc.). Cells comprising any of the above viruses are also products of the invention.
[0016] Methods of producing reassortant/recombinant influenza virus through culturing a host cell harboring an influenza virus in a suitable culture medium under conditions permitting expression of nucleic acid; and, isolating or recovering the recombinant influenza virus from one or more of the host cell or the medium are also part of the invention. Thus, introducing a plurality of vectors having an influenza virus genome into a population of host cells wherein the vectors comprise at least 6 internal genome segments of a first influenza strain (again, e.g., A/AA/6/60, B/AA/1/66, A/PR/8/34, etc.) and at least one (and preferably two) genome segments are selected from a second influenza strain (e.g., preferably one or more nucleic acid as described above, e.g., from SEQ lD
NO:1-34 or from a similar strain to such or optionally comprising a hemagglutinin and/or neuraminidase, etc.). is a feature of the invention. Preferably, the first strain of virus is cold-adapted and/or temperature sensitive and/or attenuated. Also preferably, such viruses are suitable for administration as part of an intranasal vaccine formulation.
Of course, other embodiments are suitable for administration as killed or inactivated vaccine formulations, live/attenuated nonnasal vaccine formulations, etc. The vectors in such methods can comprise influenza A viruses and/or influenza B viruses. Host cells for such methods can optionally comprise, e.g., Vero cells, PerC6 cells, MDCK cells, 293T cells, COS cells, etc.
Typical embodiments do not comprise helper viruses in the method and yet other typical embodiments comprise eight plasmid vectors to contain the influenza genome.
[0017] In other embodiments herein, the invention comprises immunogenic compositions having an immunologically effective amount of the above described recombinant influenza virus (e.g., a live virus). Other embodiments include methods for stimulating the immune system of an individual to produce a protective immune response against influenza virus by administering to the individual an immunologically effective ' amount of the recombinant influenza virus of described above (optionally in a physiologically effective carrier).
[0018] Other aspects of the invention include methods of producing an isolated or recombinant polypeptide by culturing any host cell above, in a.suitable culture medium under conditions permitting expression of nucleic acid and, isolating the polypeptide from one or more of the host cell or the medium in which it is grown.
[0019] Immunogenic compositions are also features of the invention.
For example, immunogenic compositions comprising one or more of the polypeptides and/or nucleic acids described above (e.g., a sequence from SEQ ID NO:1-68 or from similar strains to such, etc.) and, optionally, an excipient such as a pharmaceutically acceptable excipient or one or more pharmaceutically acceptable administration component. Immunogenic compositions of the invention can also comprise one or more above described virus as well (e.g., along with one or more pharmaceutically acceptable administration component).
[0020] Methods of producing an influenza virus vaccine are also included in the invention. For example, the invention includes introducing a plurality of vectors (e.g., plasmid vectors) comprising an influenza genome (e.g., influenza A or B) into a population of host cells that is capable of supporting replication of such virus, culturing the cells, recovering a plurality of influenza viruses and providing one or more pharmaceutically acceptable excipient with such virus to an individual (e.g., one in need of such treatment).
Such viruses can optionally be cold-adapted and/or temperature sensitive and/or attenuated and preferably are suitable for administration in an intranasal vaccine formulation. Such methods can include wherein the vectors have at least 6 internal genome segments of a first influenza strain and at least one genome segment (and preferably 2 segments) from another influenza strain (e.g., with sequence selected from SEQ D NO:1-34 or from similar strains to such, etc.) which segment optionally codes for an immunogenic influenza surface antigen of the second influenza strain.
[0021] Methods of producing immunogenic responses in a subject through administration of an effective amount of any of the above viruses to a subject' are also within the current invention. Additionally, methods of prophylactic or therapeutic treatment of a viral infection (e.g., viral influenza) in a subject through administration of one or more above described virus in an amount effective to produce an immunogenic response against the viral infection are also part of the current invention. Subjects for such treatment can include mammals (e.g., humans). Such methods can also comprise in vivo administration to the subject as well as in vitro or ex vivo administration to one or more cells of the subject.
Additionally, such methods can also comprise administration .of a composition of the virus and a pharmaceutically acceptable excipient that is administered to the subject in an amount effect to prophylactically or therapeutically treat the viral infection.
[0022] The invention also comprises compositions of matter having one or more sequence selected from SEQ ID NO:1 through SEQ ID NO:34, and a selected master donor virus, typically wherein the selected sequence and the master donor virus comprise a 6:2 reassortment, i.e., the HA and NA herein reassorted with the other six influenza genes from the donor virus. Such donor viruses are typically ca, att, ts influenza strains. For example, typically donor strains can include, e.g., A/Ann Arbor/6/60, B/Ann Arbor/1/66, or A/Puerto Rico/8/34 and variants thereof. Those of skill in the art will appreciate that typically donor strains can vary from reassortant to reassortant. Thus, those variations are also encompassed within the current invention. Another element of the invention comprises one or more live attenuated influenza vaccine comprising the such compositions, e.g., those having sequences herein reassorted in a 6:2 manner with a selected master donor virus.
[0023] Other aspects of the invention include, compositions of matter comprising a hemagglutinin polynucleotide and/or a neuraminidase polynucleotide reassorted with one or more master donor virus, again typically a ca, an, ts influenza virus, wherein the polynucleotide comprises a same virus strain as one or more virus strain of SEQ ID NO:1 through SEQ ID NO:34. Such hemagglutinin pd/or neuraminidase polynucleotide is typically determined to be "within the same strain" when it produces a titer that is within a four-fold range of another virus (e.g., ones having the sequences listed herein) as measured by a hemagglutinin inhibition assay. As described below, however, other common assays - can also be utilized to determine whether polynucleotides (i.e., viruses comprising such) are within the same strain.

[0023a] Specific aspects of the invention include:
a reassortant influenza virus comprising a genome segment encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 53;
an immunogenic composition comprising an immunologically effective amount of the reassortant influenza virus as described herein and a physiologically acceptable carrier;
a live attenuated influenza vaccine comprising the immunogenic composition as described herein;
a method for producing reassortant influenza viruses in cell culture, the method comprising: i) introducing a plurality of vectors comprising polynucleotides corresponding to an influenza virus genome into a population of host cells capable of supporting replication of influenza virus, which plurality of vectors comprises polynucleotides corresponding to at least 6 internal genome segments of a first influenza strain, and at least one genome segment of a second influenza strain, wherein the at least one genome segment of the second influenza strain comprises a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 53; ii) culturing the population of host cells; and iii) recovering a plurality of influenza viruses;
use of a reassortant virus for prophylactic or therapeutic treatment of an influenza virus infection in a subject, the reassortant virus comprising: a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 19; orb) a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 53, or a complementary polynucleotide sequence thereof;

in an amount effective to produce an immunogenic response against the viral infection;
an isolated or recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 53;
-9a-an isolated or recombinant polynucleotide, which polynucleotide is selected from the group consisting of: a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 19, or a complementary sequence thereof; and b) a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 53, or a complementary polynucleotide sequence thereof;
an immunogenic composition comprising an immunologically effective amount of the polynucleotide as described herein or the polypeptide as described herein and a physiologically acceptable carrier;
a vector comprising the polynucleotide as described herein; and a cell comprising the vector as described herein.
[0024] These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures appendix.
-9b-BRIEF DESCRIPTION OF THE FIGURES
[0025] Figure 1 displays the Sequence Listing of variant hemagglutinin and neuraminidase nucleic acids and polypeptides of the invention.
[0026] Figure 2 displays an alternative organization of variant hemagglutinin and neuraminidase sequences as found in Figure 1.
DETAILED DESCRIPTION
[0027] The present invention includes polypeptide and polynucleotide sequences of influenza hemagglutinin and neuraminidase as well as vectors, viruses, vaccines, compositions and the like comprising such sequences and methods of their use.
Additional features of the invention are described in more detail herein.
DEFINITIONS
[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. The following definitions supplement those in the art and are directed to the current application and are not necessarily to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.
Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Additional terms are defined and described throughout.
[0029] As used in this specification and the appended claims, the singular forms "a,"
"an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a virus" includes a plurality of viruses; reference to a "host cell"
includes mixtures of host cells, and the like.
[0030] The terms "nucleic acid," "polynucleotide," "polynucleotide sequence," and "nucleic acid sequence" refer to single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymers, chimeras or analogues thereof, or a character string representing such, depending on context. As used herein, the term optionally includes polymers of analogs of naturally occurring nucleotides having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). Unless otherwise indicated, a particular nucleic acid sequence of this invention optionally encompasses complementary sequences in addition to the sequence explicitly indicated. From any specified polynucleotide sequence, either the given nucleic acid or the complementary polynucleotide sequence (e.g., the complementary nucleic acid) can be determined.
[0031] The term "nucleic acid" or "polynucleotide" also encompasses any physical string of monomer units that can be corresponded to a string of nucleotides, including a polymer of nucleotides (e.g., a typical DNA or RNA polymer), PNAs, modified oligonucleotides (e.g., oligonucleotides comprising bases that are not typical to biological RNA or DNA in solution, such as 2'-0-methylated oligonucleotides), and the like. A
nucleic acid can be e.g., single-stranded or double-stranded.
[0032] A "subsequence" is any portion of an entire sequence, up to and including the complete sequence. Typically, a subsequence comprises less than the full-length sequence. A "unique subsequence" is a subsequence that is not found in any previously determined influenza polynucleotide or polypeptide sequence The phrase "substantially identical," in the context of two nucleic acids or polypeptides (e.g., DNAs encoding a HA or NA molecule, or the amino acid sequence of a HA or NA molecule) refers to two or more sequences or subsequences that have at least about 90%, preferably 91%, most preferably 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
[0033] The term "variant" with respect to a polypeptide refers to an amino acid sequence that is altered by one or more amino acids with respect to a reference sequence.
The variant can have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine.
Alternatively, a variant can have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan. Analogous minor variation can also include amino acid deletion or insertion, or both. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without eliminating biological or immunological activity can be found using computer programs well known in the art, for example, DNASTAR software.
Examples of conservative substitutions are also described herein.
[0034] The term "gene" is used broadly to refer to any nucleic acid associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. The term "gene" applies to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.
[0035] The "neuraminidase "polypeptides of the invention show immunological cross reactivity with one or more known neuraminidase molecule from an influenza virus.
The literature is replete with examples of such known neuraminidases (e.g., in GenBank, in publications from the CDC, etc.). Similarly, the "hemagglutinin" polypeptides of the invention show immunological cross-reactivity with one or more known hemagglutinin molecule from an influenza virus. Again, the literature is replete with examples of such known hemagglutinin molecules.
[0036] Genes also include non-expressed nucleic acid segments that, for example, form recognition sequences for other proteins. Non-expressed regulatory sequences include "promoters" and "enhancers," to which regulatory proteins such as transcription factors bind, resulting in transcription of adjacent or nearby sequences. A "tissue specific"
promoter or enhancer is one that regulates transcription in a specific tissue type or cell type, or types.
[0037] "Expression of a gene" or "expression of a nucleic acid" typically means transcription of DNA into RNA (optionally including modification of the RNA, e.g., splicing) or transcription of RNA into mRNA, translation of RNA into a polypeptide (possibly including subsequent modification of the polypeptide, e.g., post-translational modification), or both transcription and translation, as indicated by the context.
[0038] An "open reading frame" or "ORF" is a possible translational reading frame of DNA or RNA (e.g., of a gene), which is capable of being translated into a polypeptide.
That is, the reading frame is not interrupted by stop codons. However, it should be noted that the term ORF does not necessarily indicate that the polynucleotide is, in fact, translated into a polypeptide.
[0039] The term "vector" refers to the means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophages, pro-viruses, phagemids, transposons, artificial chromosomes, and the like, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA
polynucleotide, a polynucleotide composed of both DNA and.RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that is not autonomously replicating. In many, but not all, common embodiments, the vectors of the present invention are plasmids.
[0040] An "expression vector" is a vector, such as a plasmid that is capable of promoting expression, as well as replication of a nucleic acid incorporated therein.
Typically, the nucleic acid to be expressed is "operably linked" to a promoter and/or enhancer, and is subject to transcription regulatory control by the promoter and/or enhancer.
[0041] A "bi-directional expression vector" is characterized by two alternative promoters oriented in the opposite direction relative to a nucleic acid situated between the two promoters, such that expression can be initiated in both orientations resulting in, e.g., transcription of both plus (+) or sense strand, and negative (-) or antisense strand RNAs.
[0042] An "amino acid sequence" is a polymer of amino acid residues (a protein, polypeptide, etc.) or a character string representing an amino acid polymer, depending on context.
[0043] A "polypeptide" is a polymer comprising two or more amino acid residues (e.g., a peptide or a protein). The polymer can optionally comprise modifications such as glycosylation or the like. The amino acid residues of the polypeptide can be natural or non-natural and can be unsubstituted, unmodified, substituted or modified.
[0044] In the context of the invention, the term "isolated" refers to a biological material, such as a virus, a nucleic acid or a protein, which is substantially free from components that normally accompany or interact with it in its naturally occurring environment. The isolated biological material optionally comprises additional material not found with the biological material in its natural environment, e.g., a cell or wild-type virus.
For example, if the material is in its natural environment, such as a cell, the material can have been placed at a location in the cell (e.g., genome or genetic element) not native to such material found in that environment. For example, a naturally occurring nucleic acid (e.g., a coding sequence, a promoter, an enhancer, etc.) becomes isolated if it is introduced by non-naturally occurring means to a locus of the genome (e.g., a vector, such as a plasmid or virus vector, or amplicon) not native to that nucleic acid. Such nucleic acids are also referred to as "heterologous" nucleic acids. An isolated virus, for example, is in an environment (e.g., a cell culture system, or purified from cell culture) other than the native environment of wild-type virus (e.g., the nasopharynx of an infected individual).
[0045] The term "chimeric" or "chimera," when referring to a virus, indicates that the virus includes genetic and/or polypeptide components derived from more than one parental viral strain or source. Similarly, the term "chimeric" or "chimera,"
when referring to a viral protein, indicates that the protein includes polypeptide components (i.e., amino acid subsequences) derived from more than one parental viral strain or source.
As will be apparent herein, such chimeric viruses are typically reassortant/recombinant viruses. Thus, in some embodiments, a chimera can optionally include, e.g., a sequence (e.g., of HA and/or NA) from an A influenza virus placed into a backbone comprised of, or constructed/derived from a B influenza virus (e.g., B/AA/1/66, etc.) or a B influenza virus sequence placed into an A influenza virus backbone (i.e., donor virus) such as, e.g., A/AA/6/60, etc.
[0046] The term "recombinant" indicates that the material (e.g., a nucleic acid or protein) has been artificially or synthetically (non-naturally) altered by human intervention.
The alteration can be performed on the material within, or removed from, its natural environment or state. Specifically, e.g., an influenza virus is recombinant when it is produced by the expression of a recombinant nucleic acid. For example, a "recombinant nucleic acid" is one that is made by recombining nucleic acids, e.g., during cloning, DNA
shuffling or other procedures, or by chemical or other mutagenesis; a "recombinant polypeptide" or "recombinant protein" is a polypeptide or protein which is produced by expression of a recombinant nucleic acid; and a "recombinant virus," e.g., a recombinant influenza virus, is produced by the expression of a recombinant nucleic acid.
[0047] The term "reassortant," when referring to a virus (typically herein, an influenza virus), indicates that the virus includes genetic and/or polypeptide components derived from more than one parental viral strain or source. For example, a 7:1 reassortant includes 7 viral genomic segments (or gene segments) derived from a first parental virus, and a single complementary viral genomic segment, e.g., encoding a hemagglutinin or neuraminidase such as those listed in the SEQ ID Table herein. A 6:2 reassortant includes 6 genomic segments, most commonly the 6 internal genes from a first parental virus, and two complementary segments, e.g., hemagglutinin and neuraminidase, from one or more different parental virus. Reassortant viruses can also, depending upon context herein, be termed as "chimeric" and/or "recombinant."
[0048] The term "introduced" when referring to a heterologous or isolated nucleic acid refers to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid can be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondria' DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA). The term includes such methods as "infection,"
"transfection," "transformation," and "transduction." In the context of the invention a variety of methods can be employed to introduce nucleic acids into cells, including electroporation, calcium phosphate precipitation, lipid mediated transfection (lipofection), etc.
[0049] The term "host cell" means a cell that contains a heterologous nucleic acid, such as a vector or a virus, and supports the replication and/or expression of the nucleic acid. Host cells can be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, avian or mammalian cells, including human cells. Exemplary host cells can include, e.g., Vero (African green monkey kidney) cells, BHK (baby hamster kidney) cells, primary chick kidney (PCK) cells, Madin-Darby Canine Kidney (MDCK) cells, Madin-Darby Bovine Kidney (MDBK) cells, 293 cells (e.g., 293T cells), and COS
cells (e.g., COS 1, COS7 cells), etc. In other embodiments, host cells can optionally include eggs (e.g., hen eggs, embryonated hen eggs, etc.).
[0050] An "immunologically effective amount" of influenza virus is an amount sufficient to enhance an individual's (e.g., a human's) own immune response against a subsequent exposure to influenza virus. Levels of induced immunity can be monitored, e.g., by measuring amounts of neutralizing secretory and/or serum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent, or microneutralization assay.
[0051] A "protective immune response" against influenza virus refers to an immune response exhibited by an individual (e.g., a human) that is protective against disease when the individual is subsequently exposed to and/or infected with wild-type influenza virus. In some instances, the wild-type (e.g., naturally circulating) influenza virus can still cause infection, but it cannot cause a serious infection. Typically, the protective immune response results in detectable levels of host engendered serum and secretory antibodies that are capable of neutralizing virus of the same strain and/or subgroup (and possibly also of a different, non-vaccine strain and/or subgroup) in vitro and in yivo.
[0052] As used herein, an "antibody" is a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. A
typical immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 Id)) and one "heavy" chain (about 50-70 IcD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain ('VL) and variable heavy chain (YE) refer to these light and heavy chains respectively. Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases.
Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge region (see Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1999) for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA
methodology.
Thus, the term antibody, as used herein, includes antibodies or fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA
methodologies. Antibodies include, e.g., polyclonal antibodies, monoclonal antibodies, multiple or single chain antibodies, including single chain Fv (sFv or scFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide, and humanized or chimeric antibodies.
INFLUENZA VIRUS
[0053] The polypeptides and polynucleotides of the invention are variants of influenza HA and NA sequences. See, e.g., the Sequence Listing in Figure 1 below. In general, influenza viruses are made up of an internal ribonucleoprotein core containing a segmented single-stranded RNA genome and an outer lipoprotein envelope lined by a matrix protein. The genome of influenza viruses is composed of eight segments of linear (-) strand ribonucleic acid (RNA), encoding the immunogenic hemagglutinin (HA) and neuraminidase (NA) proteins, and six internal core polypeptides: the nucleocapsid nucleoprotein (NP); matrix proteins (M); non-structural proteins (NS); and 3 RNA
polymerase (PA, PB1, PB2) proteins. During replication, the genomic viral RNA
is transcribed into (+) strand messenger RNA and (-) strand genomic cRNA in the nucleus of the host cell. Each of the eight genomic segments is packaged into ribonucleoprotein complexes that contain, in addition to the RNA, NP and a polymerase complex (PB1, PB2, and PA). The hemagglutinin molecule consists of a surface glycoprotein and acts to bind to N-AcetylNeuraminic acid (NeuNAc), also known as sialic acid, on host cell surface receptors. In some embodiments herein, the polypeptides of the invention (and polypeptides encoded by the polynucleotides of the invention) can act to bind NeuNAc whether in vitro or in vivo. Such action can in some embodiments also be done by fragments of hemagglutinin which retain hemagglutinin activity. Hemagglutinin is made up of two subunits, HAI. and HA2 and the entire structure is about 550 amino acids in length _ and about 220 kD. NeuramMidase molecules cleave terminal sialic acid residues from cell surface receptors of influenza virus, thereby releasing virions from infected cells.
neuraminidase also removes sialic acid from newly made hemagglutinin and neuraminidase molecules. In some embodiments herein, the polypeptides of the invention (and polypeptides encoded by the polynucleotides of the invention) can act to cleave sialic acid residues whether in vitro or in vivo. This action can also be done in some embodiments by fragments of neuraminidase which retain neuraminidase activity. The neuraminidase polypeptides of the invention show immunological cross reactivity with one or more known neuraminidase molecule from an influenza virus. The literature is replete with examples Of such known neuraminidases (e.g., in GenBank, in publications from the CDC, etc.).

Similarly, the hemagglutinin polypeptides of the invention show immunological cross-reactivity with one or more known hemagglutinin molecule from an influenza virus. Again, the literature is replete with examples of such known hemagglutinin molecules.
[0054] Influenza is commonly grouped into influenza A and influenza B
categories, as well as a typically less important C category. Influenza A and influenza B
viruses each contain eight segments of single stranded RNA with negative polarity. The influenza A
genome encodes eleven polypeptides. Segments 1-3 encode three polypeptides, making up a RNA-dependent RNA polymerase. Segment 1 encodes the polymerase complex protein PB2. The remaining polymerase proteins PB1 and PA are encoded by segment 2 and segment 3, respectively. In addition, segment 1 of some influenza strains encodes a small protein, PB1-F2, produced from an alternative reading frame within the PB1 coding region.
Segment 4 encodes the hemagglutinin (HA) surface glycoprotein involved in cell attachment and entry during infection. Segment 5 encodes the nucleocapsid nucleoprotein (NP) polypeptide, the major structural component associated with viral RNA.
Segment 6 encodes a neuraminidase (NA) envelope glycoprotein. Segment 7 encodes two matrix proteins, designated M1 and M2, which are translated from differentially spliced mRNAs.
Segment 8 encodes NS1 and NS2, two nonstructural proteins, which are translated from alternatively spliced mRNA variants. The eight genome segments of influenza B
encode 11 proteins. The three largest genes code for components of the RNA polymerase, PB1, PB2 and PA. Segment 4 encodes the HA protein. Segment 5 encodes NP. Segment 6 encodes the NA protein and the NB protein. Both proteins, NB and NA, are translated from overlapping reading frames of a bicistronic mRNA. Segment 7 of influenza B
also encodes two proteins: M1 and BM2. The smallest segment encodes two products: NS1 is translated from the full length RNA, while NS2 is translated from a spliced mRNA variant.
[0055] Influenza types A and B are typically associated with influenza outbreaks in human populations. However, type A influenza also infects other creatures as well, e.g., birds, pigs, and other animals. The type A viruses are categorized into subtypes based upon differences within their hemagglutinin and neuraminidase surface glycoprotein antigens.
Hemagglutinin in type A viruses has 14 known subtypes and neuraminidase has 9 known subtypes. In humans, currently only about 3 different hemagglutinin and 2 different neuraminidase subtypes are known, e.g., H1, H2, 113, Ni, and N2. In particular, two major subtypes of influenza A have been active in humans, namely, H1N1 and H3N2.
HINZ, however has recently been of concern. Influenza B viruses are not divided into subtypes based upon their hemagglutinin and neuraminidase proteins. As will be appreciated, the sequences contained within the sequence listing in Figure 1 comprise a number of different subtypes of influenza. Thus, for example in the sequence listing A-H3N2 strains are exampled by ca A/Shandong/9/93, ca A/Johannesburg/33/94-like, ca A/Wuhan/395/95, ca A/Sydney/05/97, ca A/Panama/2007/99, ca A/Wyonaing/03/2003. A-H1N1 strains are shown in ca A/Texas/36/91, ca A/Shenzhen/227/95, ca A/Beijing/262/95, and ea A/New Caledonia/20/99, while B-HANA strains include ca B/Ann Arbor/1/94, ca B/Yamanashi/166/98, ca B/Johannesburg/5/99, ca B/Victoria/504/2000, ca B/Hong Kong/330/2001, ca B/Brisbane/32/2002, and ca Bailin/20/2003.
[0056] Different strains of influenza can be categorized based upon, e.g., the ability of influenza to agglutinate red blood cells (RBCs or erythrocytes). Antibodies specific for particular influenza strains can bind to the virus and, thus, prevent such agglutination.
Assays determining strain types based on such inhibition are typically known as hemagglutinin inhibition assays (H[ assays or HAT assays) and are standard and well known methods in the art to characterize influenza strains. Of course, those of skill in the art will be familiar with other assays, e.g., ELISA, indirect fluorescent antibody assays, immunohistochemistry, Western blot assays, etc. with which to characterize influenza strains and the use of and discussion herein of KC assays should not be necessarily construed as limiting.
[0057] Briefly, in typical HI assays, sera to be used for typing or categorization, which is often produced in ferrets, is added to erythrocyte samples in various dilutions, e.g., 2-fold, etc. Optical determination is then made whether the erythrocytes are clumped together (i.e., agglutinated) or are suspended (i.e., non-agglutinated). If the cells are not clumped, then agglutination did not occur due to the inhibition from antibodies in the sera that are specific for that influenza. Thus, the types of influenza are defined as being within the same strain. In some cases, one strain is described as being "like" the other, e.g., strain x is a "y-like" strain, etc. For example, if two samples are within four-fold titer of one another as measured by an III assay, then they can be described as belonging to the same strain (e.g., both belonging to the "New Caledonia" strain or both being "Moscow-like"
strains, etc.). In other words, strains are typically categorized based upon their immunologic or antigenic profile. An HAI titer is typically defined as the highest dilution of a serum that completely inhibits hemagglutination. See, e.g., Schild, et al., Bull. Wld filth Org., 1973, 48:269-278, etc. Again, those of skill in the art will be quite familiar with categorization and classification of influenza into strains and the methods to do so.
[0058] From the above it will be appreciated that the current invention not only comprises the specific sequences listed herein, but also such sequences within various vectors (e.g., ones used for plasmid reassortment and rescue, see below) as well as hemagglutinin and neuraminidase sequences within the same strains as the sequences listed herein. Also, such same strains that are within various vectors (e.g., typically ones used for plasmid reassortment and rescue such as A/Ann Arbor/6/60 or B/Ann Arbor/1/66, A/Puerto Rico/8/34, etc.) are also included.
[0059] As used herein, the term "similar strain" should be taken to indicate that a first influenza virus is of the same or related strain as a second influenza virus. In typical embodiments such relation is commonly determined through use of an HAI assay.
Influenza viruses that fall within a four-fold titer of one another in an HAI
assay are, thus, of a "similar strain." Those of skill in the art, however, will be familiar with other assays, etc. to determine similar strains, e.g., FRID, neutralization assays, etc. The current invention also comprises such similar strains (i.e., strains similar to the ones present in the sequence listing herein) in the various plasmids, vectors, viruses, methods, etc. herein.
Thus, unless the context clearly dictates otherwise, descriptions herein of particular sequences (e.g., those in the sequence listing) or fragments thereof also should be considered to include sequences from similar strains to those (i.e., similar strains to those strains having the sequences in those plasmids, vectors, viruses, etc.
herein). Also, it will be appreciated that the NA and HA polypeptides within such similar strains are, thus, "similar polypeptides" when compared between "similar strains."
INFLUENZA VIRUS VACCINES
[0060] The sequences, compositions and methods herein are primarily, but not solely, concerned with production of influenza viruses for vaccines.
Historically, influenza virus vaccines have primarily been produced in embryonated hen eggs using strains of virus selected or based on empirical predictions of relevant strains. More recently, reassortant viruses have been produced that incorporate selected hemagglutinin and/or neuraminidase., antigens in the context of an approved attenuated, temperature sensitive master strain.

Following culture of the virus through multiple passages in hen eggs, influenza viruses are recovered and, optionally, inactivated, e.g., using formaldehyde and/or P-propiolactone (or alternatively used in live attenuated vaccines). Thus, it will be appreciated that HA and NA
sequences (as in the current invention) are quite useful in constructing influenza vaccines.
[0061] Attempts at producing recombinant and reassortant vaccines in cell culture have been hampered by the inability of some of the strains approved for vaccine production to grow efficiently under standard cell culture conditions. However, prior work by the inventors and their coworkers provided a vector system, and methods for producing recombinant and reassortant viruses in culture, thus, making it possible to rapidly produce vaccines corresponding to one or many selected antigenic strains of virus, e.g., either A or B
strains, various subtypes or substrains, etc., e.g., comprising the HA and NA
sequences herein. See, PCT publication WO 03/091401, US Published Patent Application No. US 2004/0029251, and PCT publication WO 2005/115448, all relating to "Multi-Plasmid System for the Production of Influenza Virus". Typically, the cultures are maintained in a system, such as a cell culture incubator, under controlled humidity and CO2, at constant temperature using a temperature regulator, such as a thermostat to insure that the temperature does not exceed 35 C. Reassortant influenza viruses can be readily obtained by introducing a subset of vectors corresponding to genomic segments of a master influenza virus, in combination with complementary segments derived from strains of interest (e.g., HA and NA antigenic variants herein).
Typically, the master strains are selected on the basis of desirable properties relevant to vaccine administration.
For example, for vaccine production, e.g., for production of a live attenuated vaccine, the master donor virus strain may be selected for an attenuated phenotype, cold adaptation and/or temperature sensitivity. As explained elsewhere herein and, e.g., in U.S. Patent Application No. 10/423,828, etc., various embodiments of the invention utilize A/Ann Arbor (AA)/6/60 or B/Ann Arbor/1/66 or A/Puerto Rico/8/34 influenza strain as a = "backbone" upon which to add HA and/or NA genes (e.g., such as those sequences listed herein, etc.) to create desired reassortant viruses. Thus, for example, in a 6:2 reassortant, 2 genes (i.e., NA and HA) would be from the influenza strain(s) against which an immunogenic reaction is desired, while the other 6 genes would be from the Ann Arbor strain, or other backbone strain, etc. The Ann Arbor virus is useful for its cold adapted, attenuated, temperature sensitive attributes. Of course, it will be appreciated that the HA

and NA sequences herein are capable of reassortment with a number of other virus genes or virus types (e.g., a number of different "backbones" such as A/Puerto Rico/8/34, etc., containing the other influenza genes present in a reassortant, namely, the non-HA and non-NA genes). Live, attenuated influenza A virus vaccines against human influenza viruses were recently licensed in the United States. See above. Such vaccines are reassortant H1N1 and H1N2 viruses in which the internal protein genes of A/Ann Arbor (AA)/6/60 (H2N2) cold adapted (ca) virus confer the cold adapted, attenuation and temperature sensitive phenotypes of the AA ca virus on the reassortant viruses (i.e., the ones having the hemagglutinin and neuraminidase genes from the non:Ann Arbor strain). In some embodiments herein, the reassortants can also comprise 7:1 reassortants. In other words, only the HA or the NA is not from the backbone or MDV strain. Previous work has been reported with suitable backbone donor virus strains that optionally are within various embodiments of the current invention. See, e.g., PCT publication WO 03/091401, US Published Patent Application No. US 2004/0029251, and PCT publication WO 2005/115448, all relating to "Multi-Plasmid System for the Production of Influenza Virus"; Maassab et al., J. of Inf. Dis., 1982, 146:780-790; Cox, et al., Virology, 1988, 167:554-567; Wareing et al., Vaccine, 2001, 19:3320-3330;
Clements, et al., J Infect Dis., 1990, 161(5):869-77, etc., [0062] In some embodiments, the sequences herein can optionally have specific regions removed (both or either in the nucleic acid sequence or the amino acid sequence). For example, for those molecules having a polybasic cleavage site, such sites can optionally be removed.
Those of skill in the art will be familiar with various methods of removing such specific regions. The resulting shortened sequences are also contained within the.current invention.
See, e.g., Li et al., J. of Infectious Diseases, 179:1132-8, 1999 [0063] The terms "temperature sensitive," "cold adapted" and "attenuated"
as applied to viruses (typically used as vaccines or for vaccine production) which optionally encompass the current sequences, are well known in the art. For example, the term "temperature sensitive" (ts) indicates, e.g., that the virus exhibits a 100 fold or greater reduction in titer at 39 C relative to 33 C for influenza A strains, or that the virus exhibits a 100 fold or greater reduction in titer at 37 C relative to 33 C for influenza 13 strains. The term "cold adapted" (ca) indicates that the virus exhibits growth at 25 C
within 100 fold of its growth at 33 C, while the term "attenuated" (alt) indicates that the virus replicates in the upper airways of ferrets but is not detectable in their lung tissues, and does not cause influenza-like illness in the animal. It will be understood that viruses with intermediate phenotypes, i.e., viruses exhibiting titer reductions less than 100 fold at 39 C (for A strain viruses) or 37 C (for B strain viruses), or exhibiting growth at 25 C that is more than 100 fold than its growth at 33 C (e.g., within 200 fold, 500 fold, 1000 fold, 10,000 fold less), and/or exhibit reduced growth in the lungs relative to growth in the upper airways of ferrets (i.e., partially attenuated) and/or reduced influenza like illness in the animal, are also useful viruses and can be used in conjunction with the HA and NA sequences herein.
[0064] Thus, the present invention can utilize growth, e.g., in appropriate culture conditions, of virus strains (both A strain and B strain influenza viruses) with desirable properties relative to vaccine production (e.g., attenuated pathogenicity or phenotype, cold adaptation, temperature sensitivity, etc.) in vitro in cultured cells.
Influenza viruses can be produced by introducing a plurality of vectors incorporating cloned viral genome segments into host cells, and culturing the cells at a temperature not exceeding 35 C.
When vectors including an influenza virus genome are transfected, recombinant viruses suitable as vaccines can be recovered by standard purification procedures. Using the vector system and methods of the invention, reassortant viruses incorporating the six internal gene segments of a strain selected for its desirable properties with respect to vaccine production, and the immunogenic HA and NA segments from a selected, e.g., pathogenic strain such as those in the sequence listing herein, can be rapidly and efficiently produced in tissue culture. Thus, the system and methods described herein are useful for the rapid production in cell culture of recombinant and reassortant influenza A and B viruses, including viruses suitable for use _ .
as vaccines, including live attenuated vaccines, such as vaccines suitable for intranasal administration.
[0065] In such embodiments, typically, a single Master Donor Virus (MDV) strain is selected for each of the A and B subtypes. In the case of a live attenuated vaccine, the Master Donor Virus strain is typically chosen for its favorable properties, e.g., temperature sensitivity, cold adaptation and/or attenuation, relative to vaccine production. For example, exemplary Master Donor Strains include such temperature sensitive, attenuated and cold adapted strains of A/Ann Arbor/6/60 and B/Ann Arbor/1/66, respectively.
[0066] For example, a selected master donor type A virus ('MDV-A), or master donor type B virus (MDV-B), is produced from a plurality of cloned viral cDNAs constituting the viral genome. Embodiments include those wherein recombinant viruses are produced from eight cloned viral cDNAs. Eight viral cDNAs representing either the selected MDV-A or MDV-B sequences of PB2, PB1, PA, NP, HA, NA, M and NS are optionally cloned into a hi-directional expression vector, such as a plasmid (e.g., pAD3000), such that the viral genomic RNA can be transcribed from an RNA polyrnerase I
(poll) promoter from one strand and the viral niRNAs can be synthesized from an RNA
polymerase II (pol II) promoter from the other strand. Optionally, any gene segment can be modified, including the HA segment (e.g., to remove the multi-basic cleavage site).
[00671 Infectious recombinant MDV-A or MDV-B virus can be then recovered following transfection of plasmids bearing the eight viral cDNAs into appropriate host cells, e.g., Vero cells, co-cultured MDCK/293T or MDCK/COS7 cells. Using the plasmids and methods described herein and, e.g., in PCT publication WO 03/091401, US
Published Patent Application No. US 2004/0029251, and PCT publication WO 2005/115448, all relating to "Multi-Plasmid System for the Production of Influenza Virus"; Hoffmann, E., 2000, PNAS, 97(11):6108-6113; U.S. Published Patent Application No. 20020164770 to Hoffmann; and U.S.P.N. 6,544,785 issued April 8, 2003 to Palese, et al., the invention is useful, e.g., for generating 6:2 reassortant influenza vaccines by co-transfection of the 6 internal genes (PB1, PB2, PA, NP, M and NS) of the selected virus (e.g., MDV-A, MDV-B) together with the HA and NA derived from different corresponding type (A or B) influenza viruses e.g., as shown in the sequence listings herein.
For example, the HA segment is favorably selected from a pathogenically relevant111, H3 or B strain, as is routinely performed for vaccine production. Similarly, the HA segment can be selected from a strain with emerging relevance as a pathogenic strain such as those in the sequence listing herein. Reassortants incorporating seven genome segments of the MDV and either the HA or NA gene of a selected strain (7:1 reassortants) can also be =
produced. It will be appreciated, and as is detailed throughout, the molecules of the invention can optionally be combined in any desired combination. For example, the HA
and/or NA sequences herein can be placed, e.g., into a reassortant backbone such as A/AA/6/60, B/AA/1/66, A/Puerto Rico/8/34 (i.e., PR8), etc., in 6:2 reassortants or 7:1 reassortants, etc. Thus, as explained more fully below, there would be 6 backbone gene regions from the donor virus (again, e.g., A/AA/6/60, etc.) and 2 genes regions from a second strain (e.g., a wild-type strain, not the backbone donor virus). Such 2 gene regions are preferably the HA and NA genes. A similar situation arises for 7:1 reassortants, in which however, there are 7 gene regions from the background donor virus and 1 gene 25 [0068]
Again, the HA and NA sequences of the current invention are optionally utilized in such plasmid reassortment vaccines (and/or in other ts, cs, az, and/or att viruses and vaccines). However, it should be noted that the HA and NA sequences, etc.
of the invention are not limited to specific vaccine compositions or production methods, and can, thus, be utilized in substantially any vaccine type or vaccine production method which FLUMISTTm [0069] As mentioned previously, numerous examples and types of influenza vaccine exist. An exemplary influenza vaccine is FluMistTm (MeclImmune Vaccines Inc., Mt. View, CA) which is a live, attenuated vaccine that protects children and adults from influenza illness (Belshe et al. (1998) The efficacy of live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine in children N Engl J Med 338:1405-12;
Nichol et al.
(1999) Effectiveness of live, attenuated intranasal influenza virus vaccine in healthy, working adults: a randomized controlled trial JAMA 282:137-44). In typical, and preferred, embodiments, the methods and compositions of the current invention are preferably adapted to/used with production of FluMistTm vaccine. However, it will be appreciated by those skilled in the art that the sequences, methods, compositions, etc. herein are also adaptable to production of similar or even different viral vaccines.
[0070] FluMistTm vaccine strains contain, e.g., HA and NA gene segments derived from the wild-type strains to which the vaccine is addressed (or, in some instances, to related strains) along with six gene segments, PB1, PB2, PA, NP, M and NS, from a common master donor virus (MDV). The HA and NA sequences herein, thus, are optionally part of various FluMistTm formulations. The MDV for influenza A
strains of FluMistTm (MDV-A), was created by serial passage of the wild-type A/Ann Arbor/6/60 (A/AA/6/60) strain in primary chicken kidney tissue culture at successively lower temperatures (Maassab (1967) Adaptation and growth characteristics of influenza virus at degrees C Nature 213:612-4). MDV-A replicates efficiently at 25 C (ca, cold adapted), but its growth is restrictedat 38 and 39 C (ts, temperature sensitive).
Additionally, this - virus does not replicate in the lungs of infected ferrets (att, attenuation). The ts phenotype is believed to contribute to the attenuation of the vaccine in humans by restricting its 25 replication in all but the coolest regions of the respiratory tract. The stability of this property has been demonstrated in animal models and clinical studies. In contrast to the ts phenotype of influenza strains created by chemical mutagenesis, the ts property of MDV-A
does not revert following passage through infected hamsters or in shed isolates from children (for a recent review, see Murphy & Coelingh (2002) Principles underlying the development and use of live attenuated cold-adapted influenza A and B virus vaccines Viral Immunol 15:295-323).

[0071] Clinical studies in over 20,000 adults and children involving 12 separate 6:2 reassortant strains have shown that these vaccines are attenuated, safe and efficacious (Belshe et al. (1998) The efficacy of live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine in children N Engl J. Med 338:1405-12; Boyce et al.
(2000) Safety and immunogenicity of adjuvanted and unadjuvanted subunit influenza vaccines administered intranasally to healthy adults Vaccine 19:217-26; Edwards et al.
(1994) A
randomized controlled trial of cold adapted and inactivated vaccines for the prevention of influenza A disease J Infect Dis 169:68-76 ; Nichol et al. (1999) Effectiveness of live, attenuated intranasal influenza virus vaccine in healthy, working adults: a randomized controlled trial JAMA 282:137-44). Reassortants carrying the six internal genes of MDV-A and the two HA and NA gene segments of a wild-type virus (i.e., a 6:2 reassortant) consistently maintain ca,-ts and att phenotypes (Maassab et al. (1982) Evaluation of a cold-recombinant influenza virus vaccine in ferrets J. Infect. DiS. 146:780-900).
[0072] Production of such reassorted virus using B strains of influenza is more difficult, however, recent work (see, e.g., PCT publication WO 03/091401, US Published Patent Application No. US 2004/0029251, and PCT publication WO 2005/115448, all relating to "Multi-Plasmid System for the Production of Influenza Virus") has shown an eight plasmid system for the generation of influenza B virus entirely from cloned cDNA. Methods for the production of attenuated live influenza A and B virus suitable for vaccine formulations, such as live virus vaccine formulations useful for intranasal administration were also shown.
[0073] The system and methods described previously are useful for the rapid production in cell culture of recombinant and reassortant influenza A and B
viruses, including viruses suitable for use as vaccines, including live attenuated vaccines, such as vaccines suitable for intranasal administration. The sequences, methods, etc.
of the current invention, are optionally used in conjunction with, or in combination with, such previous work involving, e.g., reassorted influenza viruses for vaccine production to produce viruses for vaccines.

METHODS AND COMPOSITIONS FOR PROPHYLACTIC ADMINISTRATION OF
VACCINES
[0074] As stated above, alternatively, or in addition to, use in production of FluMisim vaccine, the current invention can be used in other vaccine formulations. In general, recombinant and reassortant viruses of the invention (e.g., those comprising polynucleotides of SEQ ID NO:1- 34 or polypeptides of SEQ ID NO:35-68, or similar strains of the virus sequences within SEQ ID NO:1-68, or fragments of any of the previous) can be administered prophylactically in an immunologically effective amount and in an appropriate carrier or excipient to stimulate an im_mune response specific for one or more strains of influenza virus as determined by the HA and/or NA sequence.
Typically, the carrier or excipient is a pharmaceutically acceptable carrier or excipient, such as sterile water, aqueous saline solution, aqueous buffered saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, ethanol, allantoic fluid from uninfected hen eggs (i.e., normal allantoic fluid or NAD), or combinations thereof. The preparation of such solutions insuring sterility, pH, isotonicity, and stability is effected according to protocols established in the art. Generally, a carrier or excipient is selected to minimize allergic and other undesirable effects, and to suit the particular route of administration, e.g., subcutaneous, intramuscular, intranasal, etc.
[0075] A related aspect of the invention provides methods for stimulating the immune system of an individual to produce a protective immune response against influenza virus. In the methods, an immunologically effective amount of a recombinant influenza virus (e.g., an HA and/or an NA molecule of the invention), an immunologically effective _ amount of a polypeptide of the invention, and/or an immunologically effective amount of a nucleic acid of the invention is administered to the individual in a physiologically acceptable carrier.
[0076] Generally, the influenza viruses of the invention are administered in a quantity sufficient to stimulate an immune response specific for one or more strains of influenza virus (i.e., against the HA and/or NA strains of the invention).
Preferably, administration of the influenza viruses elicits a protective immune response to such strains.
Dosages and methods for eliciting a protective immune response against one or more influenza strains are known to those of skill in the art. See, e.g., USPN
5,922,326; Wright, et al., Infect. Immun. 37:397-400 (1982); Kim et al., Pediatrics 52:56-63 (1973); and Wright et al., J. Pediatr. 88:931-936 (1976). For example, influenza viruses are provided in the range of about 1-1000 HID50 (human infectious dose), i.e., about 105 - 108 pfu (plaque forming units) per dose administered. Typically, the dose will be adjusted within this range based on, e.g., age, physical condition, body weight, sex, diet, time of administration, and other clinical factors. The prophylactic vaccine formulation is systemically administered, e.g., by subcutaneous or intramuscular injection using a needle and syringe, or a needle-less injection device. Alternatively, the vaccine formulation is administered intranasally, either by drops, large particle aerosol (greater than about 10 microns), or spray into the upper respiratory tract. While any of the above routes of delivery results in a protective systemic immune response, intranasal administration confers the added benefit of eliciting mucosal immunity at the site of entry of the influenza virus. For intranasal administration, attenuated live virus vaccines are often preferred, e.g., an attenuated, cold adapted and/or temperature sensitive recombinant or reassortant influenza virus. See above.
While stimulation of a protective immune response with a single dose is preferred, additional dosages can be administered, by the same or different route, to achieve the desired prophylactic effect.
[0077] Typically, the attenuated recombinant influenza of this invention as used in a vaccine is sufficiently attenuated such that symptoms of infection, or at least symptoms of serious infection, will not occur in most individuals immunized (or otherwise infected) with the attenuated influenza virus. In some instances, the attenuated influenza virus can still be capable of producing symptoms of mild illness (e.g., mild upper respiratory illness) and/or of dissemination to unvaccinated individuals. However, its virulence is sufficiently abrogated such that severe lower respiratory tract infections do not occur in the vaccinated or incidental host.
[0078] Alternatively, an immune response can be stimulated by ex vivo or in vivo targeting of dendritic cells with influenza viruses comprising the sequences herein. For example, proliferating dendritic cells are exposed to viruses in a sufficient amount and for a sufficient period of time to permit capture of the influenza antigens by the dendritic cells.
The cells are then transferred into a subject to be vaccinated by standard intravenous transplantation methods.
[0079] While stimulation of a protective immune response with a single dose is preferred, additional dosages can be administered, by the same or different route, to achieve the desired prophylactic effect. In neonates and infants, for example, multiple administrations may be required to elicit sufficient levels of immunity.
Administration can continue at intervals throughout childhood, as necessary to maintain sufficient levels of protection against wild-type influenza infection. Similarly, adults who are particularly susceptible to repeated or serious influenza infection, such as, for example, health care workers, day care workers, family members of young children, the elderly, and individuals with compromised cardiopulmonary function may require multiple immunizations to establish and/or maintain protective immune responses. Levels of induced immunity can be monitored, for example, by measuring amounts of neutralizing secretory and serum antibodies, and dosages adjusted or vaccinations repeated as necessary to elicit and maintain desired levels of protection.
[0080] Optionally, the formulation for prophylactic administration of the influenza viruses also contains one or more adjuvants for enhancing the immune response to the influenza antigens. Suitable adjuvants include: complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, bacille Calmette-Guerin (BCG), Corynebacterium parvum, and the synthetic adjuvants QS-21 and M.F59.
[0081] If desired, prophylactic vaccine administration of influenza viruses can be performed in conjunction with administration of one or more immunostimulatory molecules. Immunostimulatory molecules include various cytokin.es, lymphokines and chemokines with immunostimulatory, immunopotentiating, and pro-inflammatory activities, such as interleukins (e.g., IL-1, IL-2, IL-3, 1L-4, 11,-13); growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1;
B7.2, etc. The immunostimulatory molecules can be administered in the same formulation as the influenza viruses, or can be administered separately. Either the protein (e.g., an HA
and/or NA polypeptide of the invention) or an expression vector encoding the protein can be administered to produce an immunostimulatory effect.
[0082] The above described methods are useful for therapeutically and/or prophylactically treating a disease or disorder, typically influenza, by introducing a vector of the invention comprising a heterologous polynucleotide encoding a therapeutically or prophylactically effective HA and/or NA polypeptide (or peptide) or HA and/or NA RNA
(e.g., an antisense RNA or ribozyme) into a population of target cells in vitro, ex vivo or in vivo. Typically, the polynucleotide encoding the polypeptide (or peptide), or RNA, of interest is operably linked to appropriate regulatory sequences, e.g., as described herein.
Optionally, more than one heterologous coding sequence is incorporated into a single vector or virus. For example, in addition to a polynucleotide encoding a therapeutically or prophylactically active HA and/or NA polypeptide or RNA, the vector can also include additional therapeutic or prophylactic polypeptides, e.g., antigens, co-stimulatory molecules, cytoldnes, antibodies, etc., and/or markers; and the like.
[0083] Although vaccination of an individual with an attenuated influenza virus of a particular strain of a particular subgroup can induce cross-protection against influenza virus of different strains and/or subgroups, cross-protection can be enhanced, if desired, by vaccinating the individual with attenuated influenza virus from at least two strains, e.g., = each of which represents a different subgroup. Additionally, vaccine combinations can optionally include mixes of pandemic vaccines and non-pandemic strains.
Vaccine mixtures (or multiple vaccinations) can comprise components from human strains and/or non-human influenza strains (e.g., avian and human, etc.). Similarly, the attenuated influenza virus vaccines of this invention can optionally be combined with vaccines that induce protective immune responses against other infectious agents.
POLYNLTCLEOTIDES OF THE INVENTION
Probes [0084] The HA and NA polynucleotides of the invention, e.g., as shown in the sequences herein such as SEQ D NO:1 through SEQ ID NO:34, and fragments thereof, are optionally used in a number of different capacities alternative to, or in addition to, the vaccines described above. Other exemplary uses are described herein for illustrative purpose and not as limitations on the actual range of uses, etc. Different methods of construction, purification, and characterization of the nucleotide sequences of the invention are also described herein.
[0085] In some embodiments, nucleic acids including one or more polynucleotide sequence of the invention are favorably used as probes for the detection of corresponding or related nucleic acids in a variety of contexts, such as in nucleic hybridization experiments, e.g., to find and/or characterize homologous influenza variants (e.g., homologues to sequences herein, etc.) infecting other species or in different influenza outbreaks, etc. The probes can be either DNA or RNA molecules, such as restriction fragments of genomic or cloned DNA, cDNAs, PCR amplification products, transcripts, and oligonucleotides, and can vary in length from oligonucleotides as short as about 10 nucleotides in length to full length sequences or cDNAs in excess of 1 kb or more. For example, in some embodiments, a probe of the invention includes a polynucleotide sequence or subsequence selected, e.g., from among SEQ ID NO:1- SEQ ID NO:34, or sequences complementary thereto.
Alternatively, polynucleotide sequences that are variants of one of the above-designated sequences are used as probes. Most typically, such variants include one or a few conservative nucleotide variations. For example, pairs (or sets) of oligonucleotides can be selected, in which the two (or more) polynucleotide sequences are conservative variations of each other, wherein one polynucleotide sequence corresponds identically to a first variant or and the other(s) corresponds identically to additional variants. Such pairs of oligonucleotide probes are particularly useful, e.g., for specific hybridization experiments to detect polymorphic nucleotides or to, e.g., detect homologous influenza HA and NA
variants, e.g., homologous to the current LEA and NA sequences, infecting other species or present in different (e.g., either temporally and/or geographically different) influenza outbreaks. In other applications, probes are selected that are more divergent, that is probes that are at least about 91% (or about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 98.5%, about 98.7%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, or about 99.6% or more about 99.7%, about 99.8%, about 99.9% or more) identical are selected.
[0086] The probes of the invention, e.g., as exemplified by sequences derived from the sequences herein, can also be used to identify additional useful polynucleotide sequences according to procedures routine in the art. In one set of embodiments, one or more probes, as described above, are utilized to screen libraries of expression products or chromosomal segments (e.g., expression libraries or genomic libraries) to identify clones that include sequences identical to, or with significant sequence similarity to, e.g., one or more probe of, e.g., SEQ ID NO:1- SEQ ID NO:34, i.e., variants, homologues, etc. It will be understood that in addition to such physical methods as library screening, computer , assisted bioinformatic approaches, e.g., BLAST and other sequence homology search algorithms, and the like, can also be used for identifying related polynucleotide sequences.
Polynucleotide sequences identified in this manner are also a feature of the invention.
[0087] Oligonucleotide probes are optionally produced via a variety of methods well known to those skilled in the art. Most typically, they are produced by well known synthetic methods, such as the solid phase phosphoramidite triester method described by Beaucage and Caruthers (1981) Tetrahedron Letts 22(20):1859-1862, e.g., using an automated synthesizer, or as described in Needham-Van Devanter et al. (1984) Nucl Acids Res, 12:6159-6168. Oligonucleotides can also be custom made and ordered from a variety of commercial sources known to persons of skill. Purification of oligonucleotides, where necessary, is typically performed by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson and Regnier (1983) J Chrom 255:137-149.
The sequence of the synthetic oligonucleotides can be verified using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology 65:499-560. Custom oligos can also easily be ordered from a variety of commercial sources known to persons of skill.
[0088] In other circumstances, e.g., relating to attributes of cells or organisms expressing the polynucleotides and polypeptides of the invention (e.g., those harboring virus comprising the sequences of the invention), probes that are polypeptides, peptides or antibodies are favorably utilized. For example, isolated or recombinant polypeptides, polypeptide fragments and peptides derived from any of the amino acid sequences of the invention and/or encoded by polynucleotide sequences of the invention, e.g., selected from SEQ JD NO:1 through SEQ ID NO:34, are favorably used to identify and isolate antibodies, e.g., from phage display libraries, combinatorial libraries, polyclonal sera, and the like.
[0089] Antibodies specific for any a polypeptide sequence or subsequence, e.g., of SEQ ID NO:35 through SEQ lD NO:68, and/or encoded by polynucleotide sequences of the invention, e.g., selected from SEQ ID NO:1 through SEQ ID NO:34, are likewise valuable as probes for evaluating expression products, e.g., from cells or tissues. In addition, antibodies are particularly suitable for evaluating expression of proteins comprising amino acid subsequences, e.g., of those given herein, or encoded by polynucleotides sequences of the invention, e.g., selected from those shown herein, in situ, in a tissue array, in a cell, tissue or organism, e.g., an organism infected by an unidentified influenza virus or the like.
Antibodies can be directly labeled with a detectable reagent, or detected indirectly by labeling of a secondary antibody specific for the heavy chain constant region (i.e., isotype) of the specific antibody. Antibodies against specific amino acids sequences herein (e.g., SEQ lD NOs: 35-68) are also useful in determining whether other influenza viruses are within the same strain as the current sequences (e.g., through an HI assay, etc.). Additional details regarding production of specific antibodies are provided below.
Diagnostic Assays [0090] The nucleic acid sequences of the present invention can be used in diagnostic assays to detect influenza (and/or hemagglutinin and/or neuraminidase) in a sample, to detect hemagglutinin-like and/or neuraminidase-like sequences, and to detect strain differences in clinical isolates of influenza using either chemically synthesized or recombinant polynucleotide fragments, e.g., selected from the sequences herein. For example, fragments of the hemagglutinin and/or neuraminidase sequences comprising at least between 10 and 20 nucleotides can be used as primers to amplify nucleic acids using polymerase chain reaction (PCR) methods well known in the art (e.g., reverse transcription-PCR) and as probes in nucleic acid hybridization assays to detect target genetic material such as influenza RNA in clinical specimens.
[0091] The probes of the invention, e.g., as exemplified by unique subsequences selected from, e.g., SEQ ID NO:1 through SEQ ID NO:34, can also be used to identify additional useful polynucleotide sequences (such as to characterize additional strains of influenza) according to procedures routine in the art. In one set of preferred embodiments, one or more probes, as described above, are utilized to screen libraries of expression products or cloned viral nucleic acids (i.e., expression libraries or genomic libraries) to identify clones that include sequences identical to, or with significant sequence identity to the sequences herein. In turn, each of these identified sequences can be used to make probes, including pairs or sets of variant probes as described above. It will be understood that in addition to such physical methods as library screening, computer assisted bioinformatic approaches, e.g., BLAST and other sequence homology search algorithms, and the like, can also be used for identifying related polynucleotide sequences.
[0092] The probes of the invention are particularly useful for detecting the presence and for determining the identity of influenza nucleic acids in cells, tissues or other biological samples (e.g., a nasal wash or bronchial lavage). For example, the probes of the invention are favorably utilized to determine whether a biological sample, such as a subject (e.g., a human subject) or model system (such as a cultured cell sample) has been exposed to, or become infected with influenza, or particular strain(s) of influenza.
Detection of hybridization of the selected probe to nucleic acids originating in (e.g., isolated from) the biological sample or model system is indicative of exposure to. or infection with the virus (or a related virus) from which the probe polynucleotide is selected.
[0093] It will be appreciated that probe design is influenced by the intended application. For example, where several allele-specific probe-target interactions are to be detected in a single assay, e.g., on a single DNA chip, it is desirable to have similar melting temperatures for all of the probes. Accordingly, the lengths of the probes are adjusted so that the melting temperatures for all of the probes on the array are closely similar (it will be appreciated that different lengths for different probes may be needed to achieve a particular Tm where different probes have different GC contents). Although melting temperature is a primary consideration in probe design, other factors are optionally used to further adjust probe construction, such as selecting against primer self-complementarity and the like.
Vectors, Promoters and Expression Systems [0094] The present invention includes recombinant constructs incorporating one or more of the nucleic acid sequences described herein. Such constructs optionally include a vector, for example, a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), etc., into which one or more of the polynucleotide sequences of the invention, e.g., comprising any of SEQ ID NO:1 through SEQ ID NO:34, or a subsequence thereof etc., has been inserted, in a forward or reverse orientation. For example, the inserted nucleic acid can include a viral chromosomal sequence or cDNA including all or part of at least one of the polynucleotide sequences of the invention. In one embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.
[0095] The polynucleotides of the present invention can be included in any one of a variety of vectors suitable for generating sense or antisense RNA, and optionally, polypeptide (or peptide) expression products (e.g., a hemagglutinin and/or neuraminidase molecule of the invention, or fragments thereof). Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;
bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associated virus, retroviruses and many others (e.g., pCDL). Any vector that is capable of introducing genetic material into a cell, and, if replication is desired, which is replicable in the relevant host can be used.
[0096] In an expression vector, the HA and/or NA polynucleotide sequence of interest is physically arranged in proximity and orientation to an appropriate transcription control sequence (e.g., promoter, and optionally, one or more enhancers) to direct mRNA
synthesis. That is, the polynucleotide sequence of interest is operably linked to an appropriate transcription control sequence. Examples of such promoters include: LTR or SV40 promoter, E. coli lac or trp promoter, phage lambda Pi, promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
[0097] A variety of promoters are suitable for use in expression vectors for regulating transcription of influenza virus genome segments. In certain embodiments, the cytomegalovinis (CMV) DNA dependent RNA Polymerase II Pol II) promoter is utilized.
If desired, e.g., for regulating conditional expression, other promoters can be substituted which induce RNA transcription under the specified conditions, or in the specified tissues or cells. Numerous viral and mammalian, e.g., human promoters are available, or can be isolated according to the specific application contemplated. For example, alternative promoters obtained from the genomes of animal and human viruses include such promoters as the adenovirus (such as Adenovirus 2), papilloma virus, hepatitis-B virus, polyoma virus, and Simian Virus 40 (SV40), and various retroviral promoters. Mammalian promoters include, among many others, the actin promoter, immunoglobulin promoters, heat-shock promoters, and the like.
[0098] Various embodiments of the current invention can comprise a number of different vector constructions. Such constructions are typically and preferably used in plasmid rescue systems to create viruses for use in vaccines (e.g., in live attenuated vaccines, in killed or inactivated vaccines, etc.). Thus, the invention includes recombinant DNA molecules having a transcription control element that binds a DNA-directed RNA
polymerase that is operatively linked to a DNA sequence that encodes an RNA
molecule, wherein the RNA molecule comprises a binding site specific for an RNA-directed RNA
polymerase of a negative strand RNA virus, operatively linked to an RNA
sequence comprising the reverse complement of a mRNA coding sequence of a negative strand RNA
virus. Also, the invention includes a recombinant DNA molecule that, upon transcription yields an RNA template that contains an RNA sequence comprising the reverse complement of an mRNA coding sequence of a negative strand RNA virus, and vRNA terminal sequences. The invention also includes a recombinant DNA molecule that upon transcription yields a replicable RNA template comprising the reverse complement of an mRNA coding sequence of a negative strand RNA virus. Such above recombinant DNA
molecules typically involve wherein the negative strand RNA virus is influenza (e.g., influenza A or B, etc.). Also, the RNA molecule in such embodiments is typically an influenza genome segment and the RNA template is typically an influenza genome segment.
The recombinant DNA molecules typically comprise wherein the RNA template is replicable, wherein the negative strand RNA virus is influenza;and wherein the RNA
template is an influenza genome segment. Thus, the nucleic acids influenza segments typically comprise HA and/or NA genes (the corresponding nucleic acid of which is, e.g., in Figure 1, or within similar strains of the strains having the nucleic acids in, e.g., Figure 1.
[0099] The invention also includes methods of preparing an RNA
molecule comprising transcribing a recombinant DNA molecule with a DNA-directed RNA
polymerase, wherein the DNA molecule comprises a transcription control element that binds a DNA-directed RNA polymerase that is operatively linked to a DNA
sequence that encodes an RNA molecule, wherein the RNA molecule comprises a binding site specific for an RNA-directed RNA polymerase of a negative strand RNA virus, operatively linked to an RNA sequence comprising the reverse complement of an mRNA coding sequence of a negative strand RNA virus. The invention also includes a method of preparing an RNA
molecule comprising transcribing a recombinant DNA molecule with a DNA-directed RNA
polymerase, wherein the recombinant DNA molecule yields upon transcription an RNA
molecule that contains an RNA sequence comprising the reverse complement of an mRNA
coding sequence of a negative strand RNA virus, and vRNA terminal sequences.
Furthermore, the invention includes a method of preparing an RNA molecule comprising transcribing a recombinant DNA molecule with a DNA-directed RNA polymerase, wherein the recombinant DNA molecule yields upon transcription a replicable RNA
molecule comprising the reverse complement of an mRNA coding sequence of a negative strand , RNA virus. Such methods typically comprise wherein the negative strand RNA
virus is influenza, and wherein the RNA molecule is an influenza genome segment. Such methods preferably include wherein the DNA-directed RNA polymerase is poi I, pol II, polymerase, T3 polymerase, or Sp6 polymerase. Thus, again, the influenza nucleic acid segments typically comprise HA and/or NA genes as described throughout.
[0100] Other methods within the invention include methods of constructing a DNA
molecule comprising a transcription control element that binds a DNA-directed RNA
polymerase that is operatively linked to a DNA sequence that encodes an RNA
molecule, wherein the RNA molecule comprises a binding site specific for an RNA-directed RNA
polymerase of an influenza virus, operatively linked to an RNA sequence comprising the reverse complement of an mRNA coding sequence of an influenza virus, wherein the DNA
sequence comprises a nucleic acid corresponding to one or more of SEQ ID NO:1-34 or a fragment thereof or of one or more nucleic acid sequence of a similar strain (e.g., a strain similar to such strains having the sequences found in the sequences of Figure 1, etc.). Also, the invention includes a method of constructing a DNA molecule comprising a DNA
sequence that upon transcription yields an RNA template that contains an RNA
sequence comprising the reverse complement of an mRNA coding sequence of an influenza virus, and vRNA terminal sequences, wherein the DNA sequence comprises a nucleic acid corresponding to one or more of SEQ ID NO:1-34 or a fragment thereof, or of one or more nucleic acid of a similar strain (e.g., a strain similar to such strains that have the sequences found in Figure 1, etc.). Such methods also include wherein the RNA template is replicable.
Other methods of the invention include those of constructing a DNA molecule comprising a DNA sequence that upon transcription yields a replicable RNA template comprising the reverse complement of an mRNA coding sequence of an influenza virus: These methods of the invention typically include wherein the RNA molecule is an influenza genome segment, wherein the DNA-directed RNA polymerase is poi I, pol If, T7 polymerase, T3 polymerase, or 8p6 polymerase.
[0101] Transcription is optionally increased by including an enhancer sequence.
Enhancers are typically short, e.g., 10-500 bp, cis-acting DNA elements that act in concert with a promoter to increase transcription. Many enhancer sequences have been isolated from mammalian genes (hemoglobin, elastase, albumin, alpha-fetoprotein, and insulin), and eukaryotic cell viruses. The enhancer can be spliced into the vector at a position 5' or 3' to the heterologous coding sequence, but is typically inserted at a site 5' to the promoter.

Typically, the promoter, and if desired, additional transcription enhancing sequences are chosen to optimize expression in the host cell type into which the heterologous DNA is to be introduced (Scharf et al. (1994) Heat stress promoters and transcription factors Results Probl Cell Differ 20:125-62; Kriegler et al. (1990) Assembly of enhancers, promoters, and splice signals to control expression of transferred genes Methods in Enzymol 185: 512-27).
Optionally, the amplicon can also contain a ribosome binding site or an internal ribosome entry site (IRES) for translation initiation.
[01021 The vectors of the invention also favorably include sequences necessary for the termination of transcription and for stabilizing the mRNA, such as a polyadenylation site or a terminator sequence. Such sequences are commonly available from the 5' and, occasionally 3, untranslated regions of eukaryotic or viral DNAs or cDNAs. In one embodiment, the SV40 polyadenylation signal sequences can provide a bi-directional polyadenylation site that insulates transcription of (-1-) strand mRNA
molecules from the Poll promoter initiating replication of the (-) strand viral genome.
[0103] In addition, as described above, the expression vectors optionally include one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, in addition to genes previously listed, markers such as dihydrofolate reductase or neomycin resistance are suitable for selection in eukaryotic cell culture.
[0104] The vector containing the appropriate nucleic acid sequence as described above, as well as an appropriate promoter or control sequence, can be employed to transform a host cell permitting expression of the protein. While the vectors of the invention can be replicated in bacterial cells, most frequently it will be desirable to introduce them into mammalian cells, e.g., Vero cells, BIM cells, MDCK cell, 293 cells, COS cells, or the like, for the purpose of expression.
[01051 As described elsewhere, the HA and NA sequences herein, in various embodiments, can be comprised within plasmids involved in plasmid-rescue reassortment.
See, e.g., PCT publication WO 03/091401, US Published Patent Application No. US 2004/0029251, and PCT publication WO 2005/115448, all relating to "Multi-Plasmid System for the Production of Influenza Virus"; Hoffmann, E., 2000, PNAS, 97(11):6108-6113; U.S. Published Patent Application No.
20020164770 to Hoffwann; and U.S.P.N. 6,544,785 issued April 8, 2003 to Palese, et al. The reassortants produced can include the HA and NA genes arranged with the 6 other influenza genes from the A/Ann Arbor/6/60 donor strain, the B/Ann Arbor/1/66 donor strain (and/or derivatives and modifications thereof), the A/Puerto Rico/8/34 donor strain, etc.
Additional Expression Elements [0106] Most commonly, the genome segment encoding the influenza virus HA
and/or NA protein includes any additional sequences necessary for its expression, including translation into a functional viral protein. In other situations, a minigene, or other artificial construct encoding the viral proteins, e.g., an HA and/or NA protein, can be employed.
Again, in such case, it is often desirable to include specific initiation signals that aid in the efficient translation of the heterologous coding sequence. These signals can include, e.g., the ATG initiation codon and adjacent sequences. To insure translation of the entire insert, the initiation codon is inserted in the correct reading frame relative to the viral protein.
Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers appropriate to the cell system in use.
[0107] If desired, polynucleotide sequences encoding additional expressed elements, such as signal sequences, secretion or localization sequences, and the like can be incorporated into the vector, usually, in-frame with the polynucleotide sequence of interest, e.g., to target polypeptide expression to a desired cellular compartment, membrane, or organelle, or to direct polypeptide secretion to the periplasmic space or into the cell culture media. Such sequences are known to those of skill, and include secretion leader peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER
retention signals, mitochondrial transit sequences), membrane localization/anchor sequences (e.g., stop transfer sequences, GPI anchor sequences), and the like.
[0108] Where translation of a polypeptide encoded by a nucleic acid sequence of the invention is desired, additional translation specific initiation signals can improve the efficiency of translation. These signals can include, e.g., an ATG initiation codon and adjacent sequences, an 1RES region, etc. In some cases, for example, full-length cDNA
molecules or chromosomal segments including a coding sequence incorporating, e.g., a polynucleotide sequence of the invention (e.g., as in the sequences herein), a translation initiation codon and associated sequence elements are inserted into the appropriate expression vector simultaneously with the polynucleotide sequence of interest.
In such cases, additional translational control signals frequently are not required.
However, in cases where only a polypeptide coding sequence, or a portion thereof, is inserted, exogenous translational control signals, including, e.g., an ATG initiation codon is often provided for expression of the relevant sequence. The initiation codon is put in the correct reading frame to ensure transcription of the polynucleotide sequence of interest. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g., Scharf D. et al. (1994) Results Probl Cell Differ 20:125-62; Bittner et al. (1987) Methods in EnZymol 153:516-544).
Production of recombinant virus [0109] Negative strand RNA viruses can be genetically engineered and recovered using a recombinant reverse genetics approach (USPN 5,166,057 to Palese et al.). Such method was originally applied to engineer influenza viral genomes (Luytjes et al. (1989) Cell 59:1107-1113; Enami et al. (1990) Proc. Natl. Acad. Sci. USA 92:11563-11567), and has been successfully applied to a wide variety of segmented and nonsegmented negative strand RNA viruses, e.g., rabies (Schnell et al. (1994) EMBO J. 13: 4195-4203); VSV
(Lawson et al. (1995) Proc. Natl. Acad. Sci. USA 92: 4477-4481); measles virus (Radecke et al.(1995) EMBO J. 14:5773-5784); rinderpest virus (Baron & Barrett (1997) J. Virol. 71:
1265-1271); human paraMfluenza virus (Hoffman 8z. Banerjee (1997) J. Virol.
71: 3272-3277; Dubin et al. (1997) Virology 235:323-332); SV5 (He et al. (1997) Virology 237:249-260); canine distemper virus (Gassen et al. (2000) J. Virol. 74:10737-44); and Sendai virus (Park et al. (1991) Proc. Natl. Acad. Sci. USA 88: 5537-5541; Kato et al.
(1996) Genes to Cells 1:569-579). Those of skill in the art will be familiar with these and similar _techniques to produce influenza virus comprising the HA and NA sequences of the invention.
Recombinant influenza viruses produced according to such methods are also a feature of the invention, as are recombinant influenza virus comprising one or more nucleic acids and/or polypeptides of the invention. Of course, as will be appreciated by those of skill in the art, influenza virus in general (and those of the invention as well) are RNA
viruses. Thus, when the present invention describes influenza viruses as comprising, e.g., the sequences of Figure 1, etc., it is to be understood to mean the corresponding RNA version of the sequence. The nucleotide sequences in Figure 1 comprise DNA versions (e.g., coding plu sense, etc.) of the genes (along with some untranslated regions in the nucleotide sequences).

Those of skill in the art can easily convert between RNA and DNA sequences, between complementary nucleotide sequences, a corresponding RNA sequence, etc. Thus, for example, those of skill in the art can easily convert from a nucleotide sequence (e.g., one given in Figure 1 such as SEQ ID NO:1) to the corresponding amino acid sequence or a corresponding complementary sequence, etc. Also, as will be evident, when such HA
and/or NA sequences are described within DNA vectors, e.g., plasmids, etc.
then the corresponding DNA version of the sequences are to be used.
Cell Culture and Expression Hosts _ [0110] The present invention also relates to host cells that are introduced (transduced, transformed or transfected) with vectors of the invention, and the production of polypeptides of the invention by recombinant techniques. Host cells are genetically engineered (i.e., transduced, transformed or transfected) with a vector, such as an expression vector, of this invention. As described above, the vector can be in the form of a plasmid, a viral particle, a phage, etc. Examples of appropriate expression hosts include: bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium; fungal cells, such as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; or insect cells such as Drosophila and Spodoptera frugiperda.
[0111] Most commonly, mammalian cells are used to culture the HA and NA
molecules of the invention. Suitable host cells for the replication of influenza virus (e.g., with the HA and/or NA sequences herein) include, e.g., Vero cells, BHK cells, MDCK
cells, 293 cells and COS cells, including 293T cells, COS7 cells or the like.
Commonly, co-cultures including two of the above cell lines, e.g., MDCK cells and either 293T or COS
cells are employed at a ratio, e.g., of 1:1, to improve replication efficiency. Typically, cells are cultured in a standard commercial culture medium, such as Dulbecco's modified Eagle's medium supplemented with serum (e.g., 10% fetal bovine serum), or in serum free medium, under controlled humidity and CO2 concentration suitable for maintaining neutral buffered pH (e.g., at pH between 7.0 and 7.2). Optionally, the medium contains antibiotics to prevent bacterial growth, e.g., penicillin, streptomycin, etc., and/or additional nutrients, such as L-glutamine, sodium pyruvate, non-essential amino acids, additional supplements to promote favorable growth characteristics, e.g., trypsin,13-mercaptoethanol, and the like.
[0112] The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the inserted polynucleotide sequences, e.g., through production of viruses. The culture conditions, such as temperature, pH and the like, are typically those previously used with the particular host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, e.g., Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, 3rd edition, Wiley- Liss, New York and the references cited therein. Other helpful references include, e.g., Paul (1975) Cell and Tissue Culture, 5th ed., Livingston, Edinburgh; Adams (1980) Laboratory Techniques in Biochemistry and Molecular Biology-Cell Culture for Biochemists, Work and Burdon (eds.) Elsevier, Amsterdam. Additional details regarding tissue culture procedures of particular interest in the production of influenza virus in vitro include, e.g., Merten et al. (1996) Production of influenza virus in cell cultures for vaccine preparation. in Cohen and Shafferman (eds.) Novel Strategies in Design and Production of Vaccines, which is incorporated herein in its entirety for all purposes. Additionally, variations in such procedures adapted to the present invention are readily determined through routine experimentation and will be familiar to those skilled in the art.
[0113] Cells for production of influenza virus (e.g., having the HA
and/or NA
sequences of the invention) can be cultured in serum-containing or serum free medium. In some case, e.g., for the preparation of purified viruses, it is typically desirable to grow the host cells in serum free conditions. Cells can be cultured in small scale, e.g., less than 25 ml medium, culture tubes or flasks or in large flasks with agitation, in rotator bottles, or on microcarrier beads (e.g., DEAE-Dextran microcarrier beads, such as Dormacell, Pfeifer &
Langen; Superbead, Flow Laboratories; styrene copolymer-tri-methylamine beads, such as Hillex, SoloHill, Ann Arbor) in flasks, bottles or reactor cultures.
Microcarrier beads are small spheres (in the range of 100-200 microns in diameter) that provide a large surface area for adherent cell growth per volume of cell culture. For example a single liter of medium can include more than 20 million microcarrier beads providing greater than 8000 square centimeters of growth surface. For commercial production of viruses, e.g., for vaccine production, it is often desirable to culture the cells in a bioreactor or fermenter. Bioreactors are available in volumes from under 1 liter to in excess of 100 liters, e.g., Cyto3 Bioreactor (Osmonics, Minnetonka, MN); NBS bioreactors (New Brunswick Scientific, Edison, NJ);
laboratory and commercial scale bioreactors from B. Braun Biotech International (B. Braun Biotech, Melsungen, Germany).

[0114] Regardless of the culture volume, in many desired aspects of the current invention, it is important that the cultures be maintained at an appropriate temperature, to insure efficient recovery of recombinant and/or reassortant influenza virus using temperature dependent multi plasmid systems (see, e.g., PCT publication WO 03/091401, US Published Patent Application No. US 2004/0029251, and PCT publication WO 2005/115448, all relating to "Multi-Plasmid System for the Production of Influenza Virus"), heating of virus solutions for filtration, etc. Typically, a regulator, e.g., a thermostat, or other device for sensing and maintaining the temperature of the cell culture system and/or other solution, is employed to insure that the temperature is at the correct level during the appropriate period (e.g., virus replication, etc.).
[0115] In some embodiments herein (e.g., wherein reassorted viruses are to be produced from segments on vectors) vectors comprising influenza genome segments are introduced (e.g., transfected) into host cells according to methods well known in the art for introducing heterologous nucleic acids into eukaryotic cells, including, e.g., calcium phosphate co-precipitation, electroporation, microinjection, lipofection, and transfection employing polyamine transfection reagents. For example, vectors, e.g., plasmids, can be transfected into host cells, such as COS cells, 293T cells or combinations of COS or 293T
= cells and MDCK cells, using the polyamine transfection reagent TransIT-LT1 (Mirus) according to the manufacturer's instructions in order to produce reassorted viruses, etc.
Thus, in one example, approximately 1 g of each vector is introduced into a population of host cells with approximately 2 1.11 of TransIT-LT1 diluted in 160 I medium, preferably serum-free medium, in a total volume of 200 I. The DNA:tansfection reagent mixtures are incubated at room temperature for 45 minuets followed by addition of 800 I of medium. The transfection mixture is added to the host cells, and the cells are cultured as described via other methods well known to those skilled in the art.
Accordingly, for the production of recombinant or reassortant viruses in cell culture, vectors incorporating each of the 8 genome segments, (PB2, PB1, PA, NP, M, NS, HA and NA, e.g., of the invention) are mixed with approximately 20 I TransIT-LT1 and transfected into host cells.
Optionally, serum-containing medium is replaced prior to transfection with serum-free medium, e.g., Opti-MEM I, and incubated for 4-6 hours.
[0116] Alternatively, electroporation can be employed to introduce such vectors incorporating influenza genome segments into host cells. For example, plasmid vectors incorporating an influenza A or influenza B virus are favorably introduced into Vero cells using electroporation according to the following procedure. In brief, approximately 5 x 106 Vero cells, e.g., grown in Modified Eagle's Medium (MEM) supplemented with 10%
Fetal Bovine Serum (PBS) are resuspended in 0.4 ml OptiMEM and. placed in an electroporation cuvette. Twenty micrograms of DNA in a volume of up to 25 I is added to the cells in the cuvette, which is then mixed gently by tapping. Electroporation is performed according to the manufacturer's instructions (e.g., BioRad Gene Pulser II with Capacitance Extender Plus connected) at 300 volts, 950 microFarads with a time constant of between 28-33 msec.
The cells are remixed by gently tapping and approximately 1-2 minutes following electroporation 0.7 ml MEM with 10% FBS is added directly to the cuvette. The cells are then transferred to two wells of a standard 6 well tissue culture dish containing 2 ml MEM, 10% PBS. The cuvette is washed to recover any remaining cells and the wash suspension is divided between the two wells. Final volume is approximately 3.5 mL. The cells are then incubated under conditions permissive for viral growth, e.g., at approximately 33 C for cold adapted strains.
[0117] In mammalian host cells, a number of expression systems, such as viral-based systems, can be utilized. In cases where an adenovirus is used as an expression vector, a coding sequence is optionally ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence.
Insertion in a nonessential El or E3 region of the viral genom.e will result in a viable virus capable of expressing the polypeptides of interest in infected host cells (Logan and Shenk (1984) Proc Natl Acad Sci 81:3655-3659). In addition, transcription enhancers, such as the rous sarComa virus (RSV) enhancer, can be used to increase expression in mammalian host cells. -[0118] A host cell strain is optionally chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing, which cleaves a precursor form into a mature form, of the protein is sometimes important for correct insertion, folding and/or function. Additionally proper location within a host cell (e.g., on the cell surface) is also important. Different host cells such as COS, CHO, BHK, MDCK, 293, 293T, COS7, etc. have specific cellular machinery and characteristic mechanisms for such post=translational activities and can be chosen to ensure the correct modification and processing of the current introduced, foreign protein.
[0119] For long-term, high-yield production of recombinant proteins encoded by, or having subsequences encoded by, the polynucleotides of the invention, stable expression systems are optionally used. For example, cell lines, stably expressing a polypeptide of the invention, are transfected using expression vectors that contain viral origins of replication or endogenous expression elements and a selectable marker gene. For example, following the introduction of the vector, cells are allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Thus, resistant clumps of stably transformed cells, e.g., derived from single cell type, can be proliferated using tissue culture techniques appropriate to the cell type.
[0120] Host cells transformed with a nucleotide sequence encoding a polypeptide of the invention are optionally cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The cells expressing said protein can be sorted, isolated and/or purified. The protein or fragment thereof produced by a recombinant cell can be secreted, membrane-bound, or retained intracellularly, depending on the sequence (e.g., depending upon fusion proteins encoding a membrane retention signal or the like) and/or the vector used.
[0121] Expression products corresponding to the nucleic acids of the invention can also be produced in non-animal cells such as plants, yeast, fungi, bacteria and the like. In addition to Sambrook, Berger and Ausubel, all infra, details regarding cell culture can be found in Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &
Sons, Inc. New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.
[0122] In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the expressed product. For example, when large quantities of a polypeptide or fragments thereof are needed for the production of antibodies, vectors that direct high-level expression of fusion proteins that are readily purified are favorably employed. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUES CRIPT (Stratagene), in which the coding sequence of interest, e.g., sequences comprising those found herein, etc., can be ligated into the vector in-frame with sequences for the amino-terminal translation initiating methionine and the subsequent 7 residues of beta-galactosidase producing a catalytically active beta galactosidase fusion protein; ON vectors (Van Heeke & Schuster (1989) J Biol Chem 264:5503-5509); pET vectors (Novagen, Madison WI); and the like. Similarly, in the yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH can be used for production of the desired expression products. For reviews, see Ausubel, infra, and Grant et al., (1987);
Methods in Enzymology 153:516-544.
Nucleic Acid Hybridization [0123] Comparative hybridization can be used to identify nucleic acids of the invention, including conservative variations of nucleic acids of the invention. This comparative hybridization method is a preferred method of distinguishing nucleic acids of the invention. In addition, target nucleic acids which hybridize to the nucleic acids represented by, e.g., SEQ ID NO:1 through SEQ ID NO:34 under high, ultra-high and ultra-ultra-high stringency conditions are features of the invention. Examples of such nucleic acids include those with one or a few silent or conservative nucleic acid substitutions as compared to a given nucleic acid sequence.
[0124] A test target nucleic acid is said to specifically hybridize to a probe nucleic acid when it hybridizes at least one-half as well to the probe as to the perfectly matched complementary target, i.e., with a signal to noise ratio at least one-half as high as hybridization of the probe to the target under conditions in which the perfectly matched probe binds to the perfectly matched complementary target with a signal to noise ratio that is at least about 5x-10x as high as that observed for hybridization to any of the unmatched target nucleic acids.
[0125] Nucleic acids "hybridize" when they associate, typically in solution. Nucleic acids hybridize due to a variety of well-characterized physico-chernical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. Numerous protocols for nucleic acid hybridization are well known in the art. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes part I
chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," (Elsevier, New York), as well as in Ausubel, Sambrook, and Berger and Kimmel, all below. Hames and Higgins (1995) Gene Probes 1 IRL Press at Oxford University Press, Oxford, England, (Hames and Higgins 1) and Hames and Higgins (1995) Gene Probes 2 IRL Press at Oxford University Press, Oxford, England (Hames and Higgins 2) provide details on the synthesis, labeling, detection and quantification of DNA and RNA, including oligonucleotides.
[0126] An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of heparin at 42 C, with the hybridization being carried out overnight. An example of stringent wash conditions comprises a 0.2x SSC wash at 65 C for 15 minutes (see, Sambrook, infra for a description of SSC buffer). Often the high stringency wash is preceded by a low stringency wash to remove background probe signal. An example low stringency wash is 2x SSC at 40 C for 15 minutes. In general, a signal to noise ratio of 5x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
[0127] After hybridization, unhybridized nucleic acids can be removed by a series of washes, the stringency of which can be adjusted depending upon the desired results. Low stringency washing conditions (e.g., using higher salt and lower temperature) increase sensitivity, but can produce nonspecific hybridization signals and high background signals.
Higher stringency conditions (e.g., using lower salt and higher temperature that is closer to the Tm) lower the background signal, typically with primarily the specific signal remaining.
See, also, Rapley, R. and Walker, J.M. eds., Molecular Biomethods Handbook (Humana Press, Inc. 1998).
[0128] "Stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993), supra, and in Hames and Higgins, 1 and 2. Stringent hybridization and wash conditions can easily be determined empirically for any test nucleic acid. For example, in determining highly stringent hybridization and wash conditions, the hybridization and wash conditions are gradually increased (e.g., by increasing temperature, decreasing salt concentration, increasing detergent concentration and/or increasing the concentration of organic solvents such as formalin in the hybridization or wash), until a selected set of criteria is met. For example, the hybridization and wash conditions are gradually increased until a probe binds to a perfectly matched complementary target with a signal to noise ratio that is at least 5x as high as that observed for hybridization of the probe to-an unmatched target.
[0129] In general, a signal to noise ratio of at least 2x (or higher, e.g., at least 5x, 10x, 20x, 50x, 100x, or more) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Detection of at least stringent hybridization between two sequences in the context of the present invention indicates relatively strong structural similarity to, e.g., the nucleic acids of the present invention provided in the sequence listings herein.
[0130] "Very stringent" conditions are selected to be equal to the thermal melting point (T.) for a particular probe. The T. is the temperature (under defined ionic strength and pH) at which 50% of the test sequence hybridizes to a perfectly matched probe. For the purposes of the present invention, generally, "highly stringent" hybridization and wash conditions are selected to be about 5 C lower than the T. for the specific sequence at a defined ionic strength and pH (as noted below, highly stringent conditions can also be referred to in comparative terms). Target sequences that are closely related or identical to the nucleotide sequence of interest (e.g., "probe") can be identified under stringent or highly stringent conditions. Lower stringency conditions are appropriate for sequences that are less complementary.
[0131] "Ultra high-stringency" hybridization and wash conditions are those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least 10x as high as that observed for hybridization to any unmatched target nucleic acids.
A target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least one-half that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-high stringency conditions.

[0132] In determining stringent or highly stringent hybridization (or even more stringent hybridization) and wash conditions, the hybridization and wash conditions are gradually increased (e.g., by increasing temperature, decreasing salt concentration, increasing detergent concentration and/or increasing the concentration of organic solvents, such as formamide, in the hybridization or wash), until a selected set of criteria are met. For example, the hybridization and wash conditions are gradually increased until a probe comprising one or more polynucleotide sequences of the invention, e.g., sequences or unique subsequences selected from those given herein and/or complementary polynucleotide sequences, binds to a perfectly matched complementary target (again, a nucleic acid comprising one or more nucleic acid sequences or subsequences selected from those given herein and/or complementary polynucleotide sequences thereof), with a signal to noise ratio that is at least 2x (and optionally 5x, 10x, or 100x or more) as high as that observed for hybridization of the probe to an unmatched target (e.g., a polynucleotide sequence comprising one or more sequences or subsequences selected from known influenza sequences present in public databases such as GenBank at the time of filing, and/or complementary polynucleotide sequences thereof), as desired.
[0133] Using the polynucleotides of the invention, or subsequences thereof, novel target nucleic acids can be obtained; such target nucleic acids are also a feature of the invention. For example, such target nucleic acids include sequences that hybridize under stringent conditions to a unique oligonucleotide probe corresponding to any of the polynucleotides of the invention.
[0134] Similarly, even higher levels of stringency can be determined by gradually increasing the hybridization and/or wash conditions of the relevant hybridization assay. For example, those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least 10X, 20X, 50X, 100X, or 500X or more as high as that observed for hybridization to any unmatched target nucleic acids.
The particular signal will depend on the label used in the relevant assay, e.g., a fluorescent label, a colorimetric label, a radioactive label, or the like. A target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least one-half that of the perfectly matched complementary target nucleic acid, is said to bind to the probe under ultra-ultra-high stringency conditions.

[0135] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
Cloning, Mutagenesis and Expression of Biomolecules of Interest [0136] General texts which describe molecular biological techniques, which are applicable to the present invention, such as cloning, mutation, cell culture and the like, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2000 ("Sambrook") and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2002) ("Ausubel")). These texts describe mutagenesis, the use of vectors, promoters and many other relevant topics related to, e.g., the generation of HA and/or NA
molecules, etc.
[0137] Various types of mutagenesis are optionally used in the present invention, e.g., to produce and/or isolate, e.g., novel or newly isolated HA and/or NA
molecules and/or to further modify/mutate the polypeptides (e.g., HA and NA molecules) of the invention.
They include but are not limited to site-directed, random point mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA
mutagenesis, mutagenesis using gapped duplex DNA or the like. Additional suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and the like. Mutagenesis, e.g., involving chimeric constructs, is also included in the present invention. In one embodiment, mutagenesis can be guided by known information of the naturally occurring molecule or altered or mutated naturally occurring molecule, e.g., sequence, sequence comparisons, physical properties, crystal structure or the like.
[0138] The above texts and examples found herein describe these procedures as well as the following publications (and references cited within): Sieber, et al., Nature Biotechnology, 19:456-460 (2001); Ling et al., Approaches to DNA mutagenesis:
an overview, Anal Biochem 254(2): 157-178 (1997); Dale et al., Oligonucleotide-directed random mutagenesis using the phosphorothioate method, Methods Mol Biol 57:369-(1996); I. A. Lorimer, I. Pastan, Nucleic Acids Res 23, 3067-8 (1995); W. P.
C. Stemmer, Nature 370, 389-91 (1994); Arnold, Protein engineering for unusual environments, Current Opinion in Biotechnology 4:450-455 (1993); Bass et al., Mutant Tip repressors with new DNA-binding specificities, Science 242:240-245 (1988); Fritz et al., Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro, Nucl Acids Res 16: 6987-6999 (1988); Kramer et al., Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directed construction of mutations, Nucl Acids Res 16: 7207 (1988); Sakamar and Khorana, Total synthesis and expression of a gene for the a-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin), Nucl Acids Res 14: 6361-6372 (1988);
Sayers et al., Y-T Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, Nucl Acids Res 16:791-802 (1988); Sayers et al., Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide, (1988) Nucl Acids Res 16: 803-814; Carter, Improved oligonucleotide-directed mutagenesis using MI3 vectors, Methods in Enzymol 154: 382-403 (1987); Kramer & Fritz Oligonucleotide-directed construction of mutations via gapped duplex DNA, Methods in Enzymol 154:350-367 (1987); Kunkel, The efficiency of oligonucleotide directed mutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F.
and Lilley, D.M.J. eds., Springer Verlag, Berlin)) (1987); Kunkel et al., Rapid and efficient site-specific mutagenesis without phenotypic selection, Methods in Enzymol 154, 367-382 (1987); Zoller & Smith, Oligonucleotide-directed mutagenesis: a simple method using two olig6nucleotide primers and a single-stranded DNA template, Methods in Enzymol 154:329-350 (1987); Carter, Site-directed mutagenesis, Biochem J 237:1-7 (1986);
Eghtedarzadeh & Henikoff, Use of oligonucleotides to generate large deletions, Nucl Acids Res 14: 5115 (1986); Mandecki, Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis, Proc Nat! Acad Sci USA, 83:7177-7181 (1986); Nakamaye & Eckstein, Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis, Nucl Acids Res 14: 9679-9698 (1986); Wells et al., Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin, Phil Trans R
Soc Lond A 317:
415-423 (1986); Botstein & Shortie, Strategies and applications of in vitro mutagenesis, Science 229:1193-1201(1985); Carter et al., Improved oligonucleotide site-directed mutagenesis using M13 vectors, Nucl Acids Res 13: 4431-4443 (1985); Grundstrom et al., Oligonucleotide-directed mutagenesis by microscale 'shot-gun' gene synthesis, Nucl Acids Res 13: 3305-3316 (1985); Kunkel, Rapid and efficient site-specific mutagenesis without phenotypic selection, Proc Natl Acad Sci USA 82:488-492 (1985); Smith, In vitro rnutagenesis, Ann Rev Genet 19:423-462(1985); Taylor et al., The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA, Nucl Acids Res 13:
8749-8764 (1985); Taylor et at, The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modifiedD-NA, Nucl Acids Res 13: 8765-(1985); Wells et al., Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites, Gene 34:315-323 (1985); Kramer et al., The gapped duplex DNA
approach to oligonucleotide-directed mutation construction, NUcl Acids Res 12:

(1984); Kramer et al., Point Mismatch Repair, Cell 38:879-887 (1984); Nambiar et at, Total synthesis and cloning of a gene coding for the ribonuclease S protein, Science 223:
1299-1301 (1984); Zoller & Smith, Oligonucleotide-directed mutagenesis of DNA
fragments cloned into M13 vectors, Methods in Enzymol 100:468-500 (1983); and Zoller &
Smith, Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any DNA fragment, Nucl Acids Res 10:6487-6500 (1982). Additional details on many of the above methods can be found in Methods in Enzymol Volume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis, gene isolation, expression, and other methods.
[0139] Oligonucleotides, e.g., for use in mutagenesis of the present invention, e.g., mutating libraries of the HA and/or NA molecules of the invention, or altering such, are typically synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetrahedron Letts 22(20):1859-1862, (1981) e.g., using an automated synthesizer, as described in Needham-VanDevanter et at, Nucleic Acids Res, 12:6159-6168 (1984).
[0140] In addition, essentially any nucleic acid can be custom or standard ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company (Midland, TX, USA), The Great American Gene Company, ExpressGen Inc. (Chicago, IL, USA), Operon Technologies Inc. (Alameda, CA) and many others.
Similarly, peptides and antibodies can be custom ordered from any of a variety of sources, such as PeptidoGenic, I ITI Bio-products, Inc. (San Diego, CA, USA), BMA Biomedicals Ltd. (U.K.), Bio. Synthesis, Inc., and many others.
[0141] The present invention also relates to host cells and organisms comprising a HA and/or NA niolecule or other polypeptide and/or nucleic acid of the invention or such HA and/or NA or other sequences within various vectors such as 6:2 reassortant influenza viruses, plasmids in plasmid rescue systems, etc. Host cells are genetically engineered (e.g., transformed, transduced or transfected) with the vectors of this invention, which can be, for example, a cloning vector or an expression vector. The vector can be, for example, in the form of a plasrnid, a bacterium, a virus, a naked polynucleotide, or a conjugated polynucleotide. The vectors are introduced into cells and/or microorganisms by standard methods including electroporation (see, From et al., Proc Natl Acad Sci USA
82, 5824 (1985), infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327, 70-73 (1987)). Berger, Sambrook, and Ausubel provide a variety of appropriate transformation methods. See, above.
[0142] Several well-known methods of introducing target nucleic acids into bacterial cells are available, any of which can be used in the present invention. These include: fusion of the recipient cells with bacterial protoplasts containing the DNA, electroporation, projectile bombardment, and infection with viral vectors, etc. Bacterial cells can be used to amplify the number of plasmids containing DNA constructs of this invention. The bacteria are grown to log phase and the plasmids within the bacteria can be isolated by a variety of methods known in the art (see, for instance, Sambrook). In addition, a plethora of kits are commercially available for the purification of plasmids from bacteria, (see, e.g., EasyPrepTu, FlexiPrepTM, both from Pharmacia Biotech;
StrataCleanTm, from Stratagene; and, Q1Apreprm from Qiagen). The isolated and purified plasmids are then further manipulated to produce other plasmids, used to transfect cells or incorporated into related vectors to infect organisms. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. See, Giliman & Smith, Gene 8:81 (1979); Roberts, et al., Nature, 328:731 (1987); Schneider, B., et al., Protein Expr Purif 6435:10 (1995); Ausubel, Sambrook, Berger (all supra). A catalogue of Bacteria and Bacteriophages useful for cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria and Bactetiophage (1992) Gherna et al. (eds.) published by the ATCC. Additional basic procedures for sequencing, cloning and other aspects of molecular biology and underlying theoretical considerations are also found in Watson et al. (1992) Recombinant DNA Second Edition Scientific American Books, NY. See, above.
=
POLYPEPTIDE PRODUCTION AND RECOVERY
[0143] In some embodiments, following transduction of a suitable host cell line or strain and growth of the host cells to an appropriate cell density, a selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. In some embodiments, a secreted polypeptide product, e.g., a HA and/or NA polypeptide as in a secreted fusion protein form, etc., is then recovered from the culture medium. In other embodiments, a virus particle containing a HA
and/or a NA polypeptide of the invention is produced from the cell.
Alternatively, cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Eukaryotic or microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well know to those skilled in the art. Additionally, cells expressing a HA and/or a NA polypeptide product of the invention can be utilized without separating the polypeptide from the cell. In such situations, the polypeptide of the invention is optionally expressed on the cell surface and is examined thus (e.g., by having HA and/or NA molecules, or fragments thereof, e.g., comprising fusion proteins or the like) on the cell surface bind antibodies, etc. Such cells are also features of the invention.
[0144] Expressed polypeptides can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g., using any of the tagging systems known to those skilled in the art), hydroxylapatite chromatography, and lectin chromatography. Protein refolding steps can be used, as desired, in completing configuration of the mature protein. Also, high performance liquid chromatography (HPLC) can be employed in the final purification steps.
In addition to the references noted herein, a variety of purification methods are well known in the art, including, e.g., those set forth in Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; and Bollag et al. (1996) Protein Methods, 2n1 Edition Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ, Harris and Angal (1990) Protein Purification Applications: A Practical Approach IRL Press at Oxford, Oxford, England; Harris and Angal Protein Purification Methods: A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993) Protein Purification: Principles and Practice 3rd Edition Springer Verlag, NY; Janson and Ryden (1998) Protein Purification:
Principles, High Resolution Methods and Applications, Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ.
[0145] When the expressed polypeptides of the invention are produced in viruses, the viruses are typically recovered from the culture medium, in which infected (transfected) cells have been grown. Typically, crude medium is clarified prior to concentration of influenza viruses. Common methods include ultrafiltration, adsorption on barium sulfate and elution, and centrifugation. For example, crude medium from infected cultures can first be clarified by centrifugation at, e.g., 1000-2000 x g for a time sufficient to remove cell debris and other large particulate matter, e.g., between 10 and 30 minutes.
Optionally, the clarified medium supernatant is then centrifuged to pellet the influenza viruses, e.g., at 15,000 x g, for approximately 3-5 hours. Following resuspension of the virus pellet in an appropriate buffer, such as STE (0.01 M Tris-HC1; 0.15 M NaCl; 0.0001 M EDTA) or phosphate buffered saline (PBS) at pH 7.4, the virus is concentrated by density gradient centrifugation on sucrose (60%-12%) or potassium tartrate (50%-10%). Either continuous or step gradients, e.g., a sucrose gradient between 12% and 60% in four 12%
steps, are suitable. The gradients are centrifuged at a speed, and for a time, sufficient for the viruses to concentrate into a visible band for recovery. Alternatively, and for most large-scale commercial applications, virus is elutriated from density gradients using a zonal-centrifuge rotor operating in continuous mode. Additional details sufficient to guide one of skill through the preparation of influenza viruses from tissue culture are provided, e.g., in Funninger. Vaccine Production, in Nicholson et al. (eds.) Textbook of Influenza pp. 324-332; Merten et al. (1996) Production of influenza virus in cell cultures for vaccine preparation, in Cohen & Shafferman (eds.) Novel Strategies in Design and Production of Vaccines pp. 141-151, and United States Patent No. 5,690,937. If desired, the recovered viruses can be stored at -80 C in the presence of sucrose-phosphate-glutamate (SPG) as a stabilizer [0146] Alternatively, cell-free transcription/translation systems can be employed to produce polypeptides comprising an amino acid sequence or subsequence of, e.g., SEQ ID
NO:35 through SEQ ID NO:68, or encoded by the polynucleotide sequences of the invention. A number of suitable in vitro transcription and translation systems are commercially available. A general guide to in vitro transcription and translation protocols is found in Tymms (1995) In vitro Transcription and Translation Protocols:
Methods in Molecular Biology Volume 37, Garland Publishing, NY.
[0147] In addition, the polypeptides, or subsequences thereof, e.g., subsequences comprising antigenic peptides, can be produced manually or by using an automated system, by direct peptide synthesis using solid-phase techniques (see, Stewart et al.
(1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield J (1963) J
Am Chem Soc 85:2149-2154). Exemplary automated systems include the Applied Biosystems Peptide Synthesizer (Perkin Elmer, Foster City, CA). If desired, subsequences can be chemically synthesized separately, and combined using chemical methods to provide full-length polypeptides.
_ Modified Amino Acids [0148] Expressed polypeptides of the invention can contain one or more modified amino acids. The presence of modified amino acids can be advantageous in, for example, (a) increasing polypeptide serum half-life, (b) reducing/increasing polypeptide antigenicity, (c) increasing polypeptide storage stability, etc. Amino acid(s) are modified, for example, co-translationally or post-translationally during recombinant production (e.g.; N-linked glycosylation at N-X-SIT motifs during expression in mammalian cells) or modified by synthetic means (e.g., via PEGylation).

[0149] Non-limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenlyated (e.g., farnesylated, geranylgeranylated) amino acid, an acetylated amino acid, an acylated amino acid, a PEG-ylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like, as well as amino acids modified by conjugation to, e.g., lipid moieties or other organic derivatizing agents. References adequate to guide one of skill in the modification of amino acids are replete throughout the literature. Example protocols are found in Walker (1998) Protein Protocols on CD-ROM Human Press, Towata, NJ.
Fusion Proteins [0150] The present invention also provides fusion proteins comprising fusions of the sequences of the invention (e.g., encoding HA and/or NA polypeptides) or fragments thereof with, e.g., immunoglobulins (or portions thereof), sequences encoding, e.g., GFP
(green fluorescent protein), or other similar markers, etc. Nucleotide sequences encoding such fusion proteins are another aspect of the invention. Fusion proteins of the invention __ are optionally used for, e.g., similar applications (including, e.g., therapeutic, prophylactic, diagnostic, experimental, etc. applications as described herein) as the non-fusion proteins of the invention. In addition to fusion with inununoglobulin sequences and marker sequences, the proteins of the invention are also optionally fused with, e.g., sequences which allow sorting of the fusion proteins and/or targeting of the fusion proteins to specific cell types, __ regions, etc.
Antibodies [0151] The polypeptides of the invention can be used to produce antibodies specific for the polypeptides given herein and/or polypeptides encoded by the polynucleotides of the invention, e.g., those shown herein, and conservative variants thereof.
Antibodies specific __ for the above mentioned polypeptides are useful, e.g., for diagnostic and therapeutic purposes, e.g., related to the activity, distribution, and expression of target polypeptides.
For example, such antibodies can optionally be utilized to define other viruses within the same strain(s) as the HA/NA sequences herein.
[0152] Antibodies specific for the polypeptides of the invention can be generated by __ methods well known in the art. Such antibodies can include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by an Fab expression library.

-[0153] Polypeptides do not require biological activity for antibody production (e.g., full length functional hemagglutinin or neuraminidase is not required).
However, the polypeptide or oligopeptide must be antigenic. Peptides used to induce specific antibodies typically have an amino acid sequence of at least about 4 amino acids, and often at least 5 or 10 amino acids. Short stretches of a polypeptide can be fused with another protein, such as keyhole limpet hemocyanin, and antibody produced against the chimeric molecule.
[0154] Numerous methods for producing polyclonal and monoclonal antibodies are known to those of skill in the art, and can be adapted to produce antibodies specific for the polypeptides of the invention, and/or encoded by the polynucleotide sequences of the invention, etc. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; Paul (ed.) (1998) Fundamental Immunology, Fourth Edition, Lippincott-Raven, Lippincott Williams & Wilkins; Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited therein;
Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, NY;
and Kohler and Milstein (1975) Nature 256: 495-497. Other suitable techniques for antibody preparation include selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al. (1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546. Specific monoclonal and polyclonal antibodies and antisera will usually bind with a KD of, e.g., at least about 0.1 p.M, at least about 0.01 M
or better, and, typically and at least about 0.0011.tM or better.
[0155] For certain therapeutic applications, humanized antibodies are desirable.
Detailed methods for preparation of chimeric (humanized) antibodies can be found in U.S.
Patent 5,482,856. Additional details on humanization and other antibody production and engineering techniques can be found in Borrebaeck (ed.) (1995) Antibody Engineering, 2'd Edition Freeman and Company, NY (Borrebaeck); McCafferty et al. (1996) Antibody ,_M)racAL. Approach IRL at Oxford Press, Oxford, England (McCafferty), and Paul (1995) Antibody Engineering Protocols Humana Press, Towata, NJ
(Paul).
Additional details regarding specific procedures can be found, e.g., in Ostberg et al. (1983), Hybridoma 2: 361-367, Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al., U.S. Pat.
No. 4,634,666.

Defining Polypeptides by Immunoreactivity [0156] Because the polypeptides of the invention provide a variety of new polypeptide sequences (e.g., comprising HA and NA molecules), the polypeptides also provide new structural features which can be recognized, e.g., in immunological assays.
The generation of antisera which specifically bind the polypeptides of the invention, as well as the polypeptides which are bound by such antisera, are features of the invention.
[0157] For example, the invention includes polypeptides (e.g., HA and NA
molecules) that specifically bind to or that are specifically immunoreactive with an antibody or antisera generated against an immunogen comprising an amino acid sequence selected from one or more of the sequences given herein, etc. To eliminate cross-reactivity with other homologues, the antibody or antisera is subtracted with the HA and/or NA
molecules found in public databases at the time of filing, e.g., the "control"
polypeptide(s). Where the other control sequences correspond to a nucleic acid, a polypeptide encoded by the nucleic acid is generated and used for antibody/antisera subtraction purposes.
[0158] In one typical format, the immunoassay uses a polyclonal antiserum which was raised against one or more polypeptide comprising one or more of the sequences corresponding to the sequences herein, etc. or a substantial subsequence thereof (i.e., at least about 30% of the full length sequence provided). The set of potential polypeptide immunogens derived from the present sequences are collectively referred to below as "the immunogenic polypeptides." The resulting antisera is optionally selected to have low cross-reactivity against the control hemagglutinin and/or neuraminidase homologues and any such cross-reactivity is removed, e.g., by immunoabsorbtion, with one or more of the control hemagglutinin and neuraminidase homologues, prior to use of the polyclonal antiserum in the immunoassay.
[0159] In order to produce antisera for use in an immunoassay, one or more of the immunogenic polypeptides is produced and purified as described herein. For example, recombinant protein can be produced in a recombinant cell. An inbred strain of mice (used in this assay because results are more reproducible due to the virtual genetic identity of the mice) is immunized with the immunogenic protein(s) in combination with a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a standard description of antibody generation, immunoassay formats and conditions that can be used to determine specific immunoreactivity).
Additional references and discussion of antibodies is also found herein and can be applied here to defining polypeptides by immunoreactivity. Alternatively, one or more synthetic or recombinant polypeptide derived from the sequences disclosed herein is conjugated to a carrier protein and used as an immunogen.
[0160] Polyclonal sera are collected and titered against the immunogenic polypeptide in an immunoassay, for example, a solid phase immunoassay with one or more of the immunogenic proteins immobilized on a solid support. Polyclonal antisera with a titer of 106 or greater are selected, pooled and subtracted with the control hemagglutinin and/or neuraminidase polypeptide(s) to produce subtracted pooled titered polyclonal antisera.
[0161] The subtracted pooled titered polyclonal antisera are tested for cross reactivity against the control homologue(s) in a comparative immunoassay. In this comparative assay, discriminatory binding conditions are determined for the subtracted titered polyclonal antisera which result in at least about a 5-10 fold higher signal to noise ratio for binding of the titered polyclonal antisera to the immunogenic polypeptides as compared to binding to the control homologues. That is, the stringency of the binding reaction is adjusted by the addition of non-specific competitors such as albumin or non-fat dry milk, and/or by adjusting salt conditions, temperature, and/or the like.
These binding conditions are used in subsequent assays for determining whether a test polypeptide (a polypeptide being compared to the immunogenic polypeptides and/or the control polypeptides) is specifically bound by the pooled subtracted polyclonal antisera. In particular, test polypeptides which Show at least a 2-5x higher signal to noise ratio than the control receptor homologues under discriminatory binding conditions, and at least about al/2 signal to noise ratio as compared to the immunogenic polypeptide(s), shares substantial structural similarity with the immunogenic polypeptide as compared to the known receptor, etc., and is, therefore a polypeptide of the invention.
[0162] In another example, immunoassays in the competitive binding format are used for detection of a test polypeptide. For example, as noted, cross-reacting antibodies are removed from the pooled antisera mixture by immunoabsorbtion with the control polypeptides. The immunogenic polypeptide(s) are then immobilized to a solid support which is exposed to the subtracted pooled antisera. Test proteins are added to the assay to compete for binding to the pooled subtracted antisera. The ability of the test protein(s) to compete for binding to the pooled subtracted antisera as compared to the immobilized protein(s) is compared to the ability of the immunogenic polypeptide(s) added to the assay to compete for binding (the immunogenic polypeptides compete effectively with the immobilized immunogenic polypeptides for binding to the pooled antisera). The percent cross-reactivity for the test proteins is calculated, using standard calculations.
[0163] In a parallel assay, the ability of the control protein(s) to compete for binding to the pooled subtracted antisera is optionally determined as compared to the ability of the immunogenic polypeptide(s) to compete for binding to the antisera. Again, the percent cross-reactivity for the control polypeptide(s) is calculated, using standard calculations.
Where the percent cross-reactivity is at least 5-10x as high for the test polypeptides as compared to the control polypeptide(s) and or where the binding of the test polypeptides is approximately in the range of the binding of the immunogenic polypeptides, the test polypeptides are said to specifically bind the pooled subtracted antisera.
[0164] In general, the immunoabsorbed and pooled antisera can be used in a competitive binding immunoassay as described herein to compare any test polypeptide to the immunogenic and/or control polypeptide(s). In order to make this comparison, the immunogenic, test and control polypeptides are each assayed at a wide range of concentrations and the amount of each polypeptide required to inhibit 50% of the binding of the subtracted antisera to, e.g., an immobilized control, test or immunogenic protein is determined using standard techniques. If the amount of the test polypeptide required for binding in the competitive assay is less than twice the amount of the immunogenic polypeptide that is required, then the test polypeptide is said to specifically bind to an antibody generated to the immunogenic protein, provided the amount is at least about 5-10x as high as for the control polypeptide.
[0165] As an additional determination of specificity, the pooled antisera is optionally fully immunosorbed with the immunogenic polypeptide(s) (rather than the control polypeptide(s)) until little or no binding of the resulting immunogenic polypeptide subtracted pooled antisera to the immunogenic polypeptide(s) used in the immunosorbtion is detectable. This fully immunosorbed antisera is then tested for reactivity with the test polypeptide. If little or no reactivity is observed (i.e., no more than 2x the signal to noise =
ratio observed for binding of the fully immunosorbed antisera to the immunogenic polypeptide), then the test polypeptide is specifically bound by the antisera elicited by the immunogenic protein.
NUCLEIC ACID AND POLYPEPTIDE SEQUENCE VARIANTS
[0166] As described herein, the invention provides for nucleic acid polynucleotide sequences and polypeptide amino acid sequences, e.g., hemagglutinin and neuraminidase sequences, and, e.g., compositions and methods comprising said sequences.
Examples of said sequences are disclosed herein. However, one of skill in the art will appreciate that the invention is not necessarily limited to those sequences-disclosed herein and that the present invention also provides many related and unrelated sequences with the functions described herein, e.g., encoding a HA and/or a NA molecule.
[0167] One of skill will also appreciate that many variants of the disclosed sequences are included in the invention. For example, conservative variations of the disclosed sequences that yield a functionally identical sequence are included in the invention. Variants of the nucleic acid polynucleotide sequences, wherein the variants hybridize to at least one disclosed sequence, are considered to be included in the invention.
Unique subsequences of the sequences disclosed herein, as determined by, e.g., standard sequence comparison techniques, are also included in the invention.
Silent Variations [0168] Due to the degeneracy of the genetic code, any of a variety of nucleic acid sequences encoding polypeptides and/or viruses of the invention are optionally produced, some which can bear lower levels of sequence identity to the HA and NA nucleic acid and polypeptide sequences herein. The following provides a typical codon table specifying the genetic code, found in many biology and biochemistry texts.

Table 1 Codon Table Amino acids Codon Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU
Aspartic acid Asp D GAC GAU
Glutamic acid Glu E GAA GAG
Phenylalanine Phe F LTUC UUU
Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU
Isoleucine Ile I AUA AUC AUU
Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Glutamine Gin Q CAA CAG
Arginine Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU
Valine Val V GUA GUC GUG GLTU
Tryptophan Trp W UGG
Tyrosine Tyr Y UAC UAU
[0169] The codon table shows that many amino acids are encoded by more than one codon. For example, the codons AGA, AGO, CGA, CGC, COG, and CGU all encode the amino acid arginine. Thus, at every position in the nucleic acids of the invention where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that U in an RNA sequence corresponds to T in a DNA sequence.
[0170] Such "silent variations" are one species of "conservatively modified variations," discussed below. One of skill will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine, and TTG, which is ordinarily the only codon for tryptophan) can be modified by standard techniques to encode a functionally identical polypeptide. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in any described sequence. The invention, therefore, explicitly provides each and every possible variation of a nucleic acid sequence encoding a polypeptide of the invention that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standards triplet genetic code (e.g., as set forth in Table 1, or as is commonly available in the art) as applied to the nucleic acid sequence encoding a hemagglutinin or a neuraminidase polypeptide of the invention. All such variations of every nucleic acid herein are specifically provided and described by consideration of the sequence in combination with the genetic code. One of skill is fully able to make these silent, substitutions using the methods herein.
Conservative variations [0171] Owing to the degeneracy of the genetic code, "silent substitutions" (i.e., substitutions in a nucleic acid sequence which do not result in an alteration in an encoded polypeptide) are an implied feature of every nucleic acid sequence of the invention which encodes an amino acid. Similarly, "conservative amino acid substitutions," in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct such as those herein. Such conservative variations of each disclosed sequence are a feature of the present invention.
[0172] "Conservative variation" of a particular nucleic acid sequence refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences, see, Table 2 below. One of skill will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 4%, 3%, 2% or 1%) in an encoded sequence are "conservatively modified variations" where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid . .
with a chemically similar amino acid. Thus, "conservatiVe variTations" of a listed polypeptide sequence of the present invention include substitutions of a small percentage, typically less than 5%, more typically less than 4%, 3%, 2% or 1%, of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group. Finally, the addition of sequences which do not alter the encoded activity of a nucleic acid molecule, such as the addition of a non-functional sequence, is a conservative variation of the basic nucleic acid.
[0173] Table 2 -- Conservative Substitution Groups 1 Alanine (A) Serine (S) Threonine (T) 2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R) Lysine (K) Isoleucine (I) Leucine (L) Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan (W) Unique Polypeptide and Polynucleotide Subsequences [0174] In one aspect, the invention provides a nucleic acid which comprises a unique subsequence in a nucleic acid selected from the sequence of HA and NA
molecules 5 disclosed herein (e.g., SEQ ID NO:1-34). The unique subsequence is unique as compared to a nucleic acids corresponding to nucleic acids such as, e.g., those found in GenBank or other similar public databases at the time of filing (e.g., other known or characterized hemagglutinin and/or neuraminidase nucleic acid molecules). Alignment can be performed using, e.g., BLAST set to default parameters. Any unique subsequence is useful, e.g., as a probe to identify the nucleic acids of the invention. See, above.
[0175] Similarly, the invention includes a polypeptide (e.g., from SEQ lD NO:35 through 68) which comprises a unique subsequence in a polypeptide selected from the sequence of HA and NA molecules disclosed herein. Here, the unique subsequence is unique as compared to a polypeptide corresponding to, e.g., the amino acid corresponding to polynucleotide sequences found in, e.g., GenBank or other similar public databases at the time of filing.
[0176] The invention also provides for target nucleic acids which hybridize under stringent conditions to a unique coding oligonucleotide which encodes a unique subsequence in a polypeptide selected from the sequences of HA and NA
molecules of the invention wherein the unique subsequence is unique as compared to a polypeptide corresponding to any of the control polypeptides (sequences of, e.g., the nucleic acids corresponding to those found in, e.g., GenBank or other similar public databases at the time of filing). Unique sequences are determined as noted above.

Sequence comparison, identity, and homology [0177] The terms "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to persons of skill) or by visual inspection.
[0178] The phrase "substantially identical," in the context of two nucleic acids or polypeptides (e.g., DNAs encoding a HA or NA molecule, or the amino acid sequence of a HA or NA molecule) refers to two or more sequences or subsequences that have at least about 90%, preferably 91%, most preferably 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. Such "substantially identical" sequences are typically considered to be "homologous," without reference to actual ancestry. Preferably, "substantial identity" exists over a region of the amino acid sequences that is at least about 200 residues in length, more preferably over a region of at least about 250 residues, and most preferably the sequences are substantially identical over at least about 300 residues, 350 residues, 400 residues, 425 residues, 450 residues, 475 residues, 480 residues, 490 residues, 495 residues, 499 residues, 500 residues, 502 residues, 559 residues, 565 residues, or 566 residues, or over the full length of the two sequences to be compared when the amino acids are hemagglutinin or hemagglutinin fragments or which is substantially identical over at least about 350 amino acids; over at least about 400 amino acids; over at least about over at least about 436 amino acids, over at least about 450 amino acids; over at least about 451 amino acids; over at least about 465 amino acids; over at least about 466 amino acids; over at least about 469 amino acids; over at least about 470 amino acids; or over at least about 566 amino acids contiguous when the amino acid is neuraminidase.
[0179] For sequence comparison and homology determination, typically one sequence acts as a reference sequence to which test sequences are compared.
When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
[0180] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv Appl Math 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol Biol 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc Natl Acad Sci USA
85:2444 (1988), by computerized implementations of algorithms such as GAP, BESTIAT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by visual inspection (see generally, Ausubel et al., supra).
[0181] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in =
Altschul et al., J. Mol Biol 215:403-410 (1990). Software for performing BLAST
analyses is publicly available through the National Center for Biotechnology Information (NCBI, Nationl Library of Medicine, Bethesda, MD, USA). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T
when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (see, Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one =
or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both' strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff &
Henikoff (1989) Proc Natl Acad Sci USA 89:10915).
[0182] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc Natl Acad Sci USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
[0183] Another example of a useful sequence alignment algorithm is PT! FUR
PIT .FUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PTLEUP uses a simplification of the progressive alignment method of Feng & Doolittle (1987) J. Mol. Evol. 35:351-360. The method used is similar to the method described by Higgins & Sharp (1989) CABIOS5:151-153. The program can align, e.g., up to 300 sequences of a maximum length of 5,000 letters. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster can then be aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences can be aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments.
The program can also be used to plot a dendogram or tree representation of clustering relationships. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison.
[0184] An additional example of an algorithm that is suitable for multiple DNA, or amino acid, sequence alignments is the CLUSTALW program (Thompson, J. D. et al.
(1994) Nucl. Acids. Res. 22: 4673-4680). CLUSTALW performs multiple pairwise comparisons between groups of sequences and assembles them into a multiple alignment based on homology. Gap open and Gap extension penalties can be, e.g., 10 and 0.05 respectively. For amino acid alignments, the BLOSUM algorithm can be used as a protein weight matrix. See, e.g., Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci.
USA 89:
10915-10919.
DIGITAL SYSTEMS
[0185] The present invention provides digital systems, e.g., computers, computer readable media and integrated systems comprising character strings corresponding to the sequence information herein for the nucleic acids and isolated or recombinant polypeptides herein, including, e.g., the sequences shown herein, and the various silent substitutions and conservative substitutions thereof. Integrated systems-can further include, e.g., gene synthesis equipment for making genes corresponding to the character strings.
[0186] Various methods known in the art can be used to detect homology or = similarity between different character strings (see above), or cari be used to perform other desirable functions such as to control output files, provide the basis for making presentations of information including the sequences and the like. Examples include BLAST, discussed supra. Computer systems of the invention can include such programs, e.g., in conjunction with one or more data file or data base comprising a sequence as noted herein.
[0187] Thus, different types of homology and similarity of various stringency and length between various HA or NA sequences or fragments, etc. can be detected and recognized in the integrated systems herein. For example, many homology determination methods have been designed for comparative analysis of sequences of biopolymers, for spell-checking in word processing, and for data retrieval from various databases. With an understanding of double-helix pair-wise complement interactions among four principal nucleobases in natural polynucleotides, models that simulate annealing of complementary homologous polynucleotide strings can also be used as a foundation of sequence alignment or other operations typically performed on the character strings corresponding to the sequences herein (e.g., word-processing manipulations, construction of figures comprising sequence or subsequence character strings, output tables, etc.).
[0188] Thus, standard desktop applications such as word processing software (e.g., Microsoft WordTM or Corel WordPerfectTM) and database software (e.g., spreadsheet software such as Microsoft ExcelTM, Corel Quattro Pr0TM, or database programs such as Microsoft Access, ParadoxTM, GeneWorksTM, or MacyectorTM or other similar programs) can be adapted to the present invention by inputting a character string corresponding to one or more polynucleotides and polypeptides of the invention (either nucleic acids or proteins, or both). For example, a system of the invention can include the foregoing software having the appropriate character string information, e.g., used in conjunction with a user interface (e.g., a GUI in a standard operating system such as a Windows, Macintosh or LINUX
system) to manipulate strings of characters corresponding to the sequences herein. As noted, specialized alignment programs such as BLAST can also be incorporated into the systems of the invention for alignment of nucleic acids or proteins (or corresponding character strings).
[0189] Systems in the present invention typically include a digital computer with data sets entered into the software system comprising any of the sequences herein. The computer can be, e.g., a PC (Intel x86 or Pentium chip- compatible DOSTM, WINDOWSTM WINDOWSNTTm, WINDOWS95Tm, WINDOWS2000TM, WINDOWS98TM, LINUX based machine, a MACINTOSHTm, Power PC, or a UNIX based (e.g., SUNTM
work station) machine) or other commercially available computer that is known to one of skill. Software for aligning or otherwise manipulating sequences is available, or can easily be constructed by one of skill using a standard programming language such as Visualbasic, PERL, Fortran, Basic, Java, or the like.
[0190] Any controller or computer optionally includes a monitor which is often a cathode ray tube ("CRT") display, a flat panel display (e.g., active matrix liquid crystal display, liquid crystal display), or others. Computer circuitry is often placed in a box which includes numerous integrated circuit chips, such as a microprocessor, memory, interface circuits, and others. The box also optionally includes a hard disk drive, a floppy disk drive, a high capacity removable drive such as a writeable CD-ROM, and other common peripheral elements. Inputting devices such as a keyboard or mouse optionally provide for input from a user and for user selection of sequences to be compared or otherwise manipulated in the relevant computer system.
[0191] The computer typically includes appropriate software for receiving user instructions, either in the form of user input into a set parameter fields, e.g., in a GUI, or in the form of preprogrammed instructions, e.g., preprogrammed for a variety of different specific operations. The software then converts these instructions to appropriate language for instructing the operation, e.g., of appropriate mechanisms or transport controllers to carry out the desired operation. The software can also include output elements for controlling nucleic acid synthesis (e.g., based upon a sequence or an alignment of sequences herein), comparisons of samples for differential gene expression, or other operations.
KITS AND REAGENTS
[0192] The present invention is optionally provided to a user as a kit. For example, a kit of the invention contains one or more nucleic acid, polypeptide, antibody, or cell line described herein (e.g., comprising, or with, a HA and/or NA molecule of the invention).
The kit can contain a diagnostic nucleic acid or polypeptide, e.g., antibody, probe set, e.g., as a cDNA micro-array packaged in a suitable container, or other nucleic acid such as one or more expression vector. The kit typically further comprises, one or more additional reagents, e.g., substrates, labels, primers, for labeling expression products, tubes and/or other accessories, reagents for collecting samples, buffers, hybridization chambers, cover slips, etc. The kit optionally further comprises an instruction set or user manual detailing preferred methods of using the kit components for discovery or application of diagnostic sets, etc.
[0193] When used according to the instructions, the kit can be used, e.g., for evaluating a disease state or condition, for evaluating effects of a pharmaceutical agent or other treatment intervention on progression of a disease state or condition in a cell or organism, or for use as a vaccine, etc.
[0194] In an additional aspect, the present invention provides system kits embodying the methods, composition, systems and apparatus herein. System kits of the invention optionally comprise one or more of the following: (1) an apparatus, system, system component or apparatus component; (2) instructions for practicing methods described herein, and/or for operating the apparatus or apparatus components herein and/or for using the compositions herein. In a further aspect, the present invention provides for the use of any apparatus, apparatus component, composition or kit herein, for the practice of any method or assay herein, and/or for the use of any apparatus or kit to practice any assay or method herein.
[0195] Additionally, the kits can include one or more translation system as noted above (e.g., a cell) with appropriate packaging material, containers for holding the components of the kit, instructional materials for practicing the methods herein and/or the like. Similarly, products of the translation systems (e.g., proteins such as HA and/or NA
molecules) can be provided in kit form, e.g., with containers for holding the components of the kit, instructional materials for practicing the methods herein and/or the like.
Furthermore, the kits can comprise various vaccines (e.g., produced through plasmid rescue protocols) such as live attenuated vaccine (e.g., FluMistTm) comprising the HA
and/or NA
sequences herein.
[0196] To facilitate use of the methods and compositions of the invention, any of the vaccine components and/or compositions, e.g., reassorted virus in allantoic fluid, etc., and additional components, such as, buffer, cells, culture medium, useful for packaging and infection of influenza viruses for experimental or therapeutic vaccine purposes, can be packaged in the form of a kit. Typically, the kit contains, in addition to the above components, additional materials which can include, e.g., instructions for performing the methods of the invention, packaging material, and a container.
[0197] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above may be used in various combinations.
SEQUENCE LISTING
<110> MedImmune Vaccines, Inc.
Yang, Chin-Fen Kemble, George Liu, Chongguang <120> INFLUENZA HEMAGGLUTININ AND NEURAMINIDASE VARIANTS
<130> FL250PCT
<140> PCT/US2004/019372 <141> 2004-06-16 <150> 60/479,078 <151> 2003-06-16 <160> 68 <170> PatentIn version 3.2 <210> I
<211> 1745 <212> DNA
<213> Influenza A virus <400> 1 caggggataa ttctattaac catgaagact atcattgctt tgagctacat tttatgtctg 60 gttttcgctc aaaaacttcc cggaaatgac aacagcacag caacgctgtg cctgggacat 120 catgcagtgc caaacggaac gctagtgaaa acaatcacga atgatcaaat tgaagtgact 180 aatgctactg agttggttca gagttcctca acaggtagaa tatgcggcag tcctcaccga 240 atccttgatg gaaaaaactg cacactgata gatgctctat tgggagaccc tcattgtgat 300 ggcttccaaa ataaggaatg ggaccttttt gttgaacgca gcaaagctta cagcaactgt 360 tacccttatg atgtgccgga ttatgcctcc cttaggtcac tagttgcctc atcaggcacc 420 ctggagttta tcaatgaaga cttcaattgg actggagtcg ctcaggatgg gggaagctat 480 gcttgcaaaa gaggatctgt taacagtttc tttagtagat tgaattggtt gcacaaatta 540 gaatacaaat atccagcgct gaacgtgact atgccaaaca atggcaaatt tgacaaattg 600 tacatttggg gggttcacca cccgagcacg gacagtgacc aaaccagcct atatgttcga 660 gcatcaggga gagtcacagt ctctaccaaa agaagccaac aaactgtaac cccgaatatc 720 gggtctagac cctgggtaag gggtcagtcc agtagaataa gcatctattg gacaatagta 780 aaaccgggag acatactttt gattaatagc acagggaatc taattgctcc tcggggttac 840 ttcaaaatac gaaatgggaa aagctcaata atgaggtcag atgcacccat tggcaactgc 900 agttctgaat gcatcactcc aaatggaagc attcccaatg acaaaccttt tcaaaatgta 960 aacagaatca catatggggc ctgccccaga tatgttaagc aaaacactct gaaattggca 1020 acagggatgc ggaatgtacc agagaaacaa actagaggca tattcggcgc aatcgcaggt 1080 ttcatagaaa atggttggga gggaatggta gacggttggt acggtttcag gcatcaaaat 1140 tctgagggca caggacaagc agcagatctt aaaagcactc aagcagcaat cgaccaaatc 1200 aacgggaaac tgaataggtt aatcgagaaa acgaacgaga aattccatca aatcgaaaaa 1260 gaattctcag aagtagaagg gagaattcag gacctcgaga aatatgttga agacactaaa 1320 atagatctct ggtcttacaa cgcggagctt cttgttgccc tggagaacca acatacaatt 1380 gatctaactg actcagaaat gaacaaactg tttgaaaaaa caaggaagca actgagggaa 1440 aatgctgagg acatgggcaa tggttgcttc aaaatatacc acaaatgtga caatgcctgc 1500 atagggtcaa tcagaaatgg aacttatgac catgatgtat acagagacga agcattaaac 1560 aaccggttcc agatcaaagg tgttgagctg aagtcaggat acaaagattg gatcctatgg 1620 atttcctttg ccatatcatg ctttttgctt tgtgttgttt tgctggggtt catcatgtgg 1680 gcctgccaaa aaggcaacat taggtgcaac atttgcattt gagtgcatta attaaaaaca 1740 ccctg 1745 <210> 2 <211> 1429 <212> DNA
<213> Influenza A virus <400> 2 aaagataata acaattggct ctgtttctct cactattgcc acaatatgct tccttatgca 60 aattgccatc ctggtaacta ctgtaacatt gcacttcaag caatatgagt gcaactcccc 120 cccaaacaac caagtaatgc tgtgtgaacc aacaataata gaaagaaaca taacagagat 180 agtgtatctg accaacacca ccatagagaa agaaatatgc cccaaactag cagaatacag 240 aaattggtca aagccgcaat gtaaaattac aggatttgca cctttttcta aggacaattc 300 aattcggctt tcagctggtg gagacatttg ggtgacaaga gaaccttatg tgtcatgcga 360 tcctggcaag tgttatcaat ttgcccttgg acagggaaca acactaaaca acaggcactc 420 aaatgacaca gtacatgata ggacccctta tcgaacccta ttgatgaatg agttgggtgt 480 tccatttcat ttgggaacca agcaagtgtg catagcatgg tccagctcaa gttgtcacga 540 tggaaaagca tggctgcatg tttgtgtaac tgggcatgat gaaaatgcaa ctgctagctt 600 catttacgat gggaggcttg tagatagtat tggttcatgg tccaaaaata tcctcaggac 660 ccaggagtcg gaatgcgttt gtatcaatgg aacttgtaca gtagtaatga ctgatggaag 720 tgcttcagaa agagctgata ctaaaatact attcattgaa gaggggaaaa tcgttcatat 780 tagcccattg tcaggaagtg ctcagcatgt cgaggagtgc tcctgttatc ctcgatatcc 840 tggtgtcaga tgtgtctgca gagacaactg gaaaggctcc aataggccca tcgtagatat 900 aaatgtgaaa gattatagca ttgtttccag ttatgtgtgc tcaggacttg ttggagacac 960 acccagaaaa aacgacagct ccagcagtag ctattgccgg aatcctaaca atgagaaagg 1020 gagtcatgga gtgaaaggct gggcctttga tgatggaaat gacgtgtgga tgggaagaac 1080 gatcagcgag gagttacgct caggttatga aaccttcaaa gtcattggag gctggtccaa 1140 acctaactcc aaattgcaga taaataggca agtcatagtt gacagaggta ataggtccgg 1200 ttattctggt attttctctg ttgaaggcaa aagctgcatc aatcggtgct tttatgtgga 1260 gttgataagg ggaaggaaac aggaaactga agtctggtgg acctcaaaca gtattgttgt 1320 gttttgtggc acctcaggta catatggaac aggctcatgg ccctgatggg gcggacatca 1380 atctcatgcc tatataagct ttcgcaattt tagaaaaaaa ctccttgtt 1429 <210> 3 <211> 1755 <212> DNA
<213> Influenza A virus <400> 3 agcaaaagca ggggataatt ctattaacca tgaagactat cattgctttg agctacattt 60 tatgtctggt tttcgctcaa aaacttcccg gaaatgacaa cagcacagca acgctgtgcc 120 tgggacacca tgcagtgcca aacggaacgc tagtgaaaac aatcacgaat gatcaaattg 180 aagtgactaa tgctactgag ctggttcaga gttccccaac aggtagaata tgcgacagcc 240 ctcaccgaat ccttgatgga aagaactgca cactgataga tgctctattg ggagaccctc 300 attgtgatgg cttccaaaat aaggaatggg acctttttgt tgaacgcagc aaagcttaca 360 gcaactgtta cccttatgat gtgccggatt atgcctccct taggtcacta gttgcctcat 420 caggcaccct ggagtttatc aacgaaaact tcaattggac tggagtcgct caggatggga 480 aaagctatgc ttgcaaaagg ggatctgtta acagtttctt tagtagattg aattggttgc 540 acaaattaga atacaaatat ccagcgctga acgtgactat gccaaacaat ggcaaatttg 600 acaaattgta catttggggg gttcaccacc cgagcacgga cagtgtccaa accagcctat 660 atgtccgagc atcagggaga gtcacagtct ctaccaaaag aagccaacaa actgtaatcc 720 cggatatcgg gtatagacca tgggtaaggg gtcagtccag tagaataagc atctattgga 780 caatagtaaa accgggagac atacttttga ttaatagcac agggaatcta attgctcctc 840 ggggttactt caaaatacga aatgggaaaa gctcaataat gaggtcagat gcacccattg 900 gcaactgcag ttctgaatgc atcactccaa atggaagcat tcccaatgac aaaccttttc 960 aaaatgtaaa caggatcaca tatggggcct gccccagata tgttaagcaa aacactctga 1020 aattggcaac agggatgcgg aatgtaccag agaaacaaac tagaggcata ttcggcgcaa 1080 tcgcaggttt catagaaaat ggttgggagg gaatggtaga cggttggtac ggtttcaggc 1140 atcaaaattc tgagggcaca ggacaagctg cagatcttaa aagcactcaa gcagcaatcg 1200 accaaatcaa cgggaaactg aataggttag tcgagaaaac gaacgagaaa ttccatcaaa 1260 tcgaaaaaga attctcagaa gtagaaggga gaattcagga cctcgagaaa tatgttgaag 1320 acactaaaat agatctctgg tcttacaatg cggaacttct tgttgctctg gagaaccaac 1380 atacaattga tctaactgac tcagaaatga acaaactgtt tgaaagaaca aggaagcaac 1440 tgagggaaaa tgctgaggac atgggcaatg gttgtttcaa aatataccac aaatgtgaca 1500 atgcctgcat agggtcaatc agaaatggaa cttatgacca tgatgtatac agagacgaag 1560 cattaaacaa ccggttccag atcaaaggtg ttgagctgaa gtcaggatac aaagattgga 1620 ttctatggat ttcctttgcc atatcgtgct ttttgctttg tgttgttttg cttgggttca 1680 tcatgtgggc ctgccaaaaa ggcaacatta ggtgcaacat ttgcatttga gtgcattaat 1740 taaaaacacc cttgt 1755 <210> 4 <211> 1354 <212> DNA
<213> Influenza A virus <400> 4 gaaaatgaat ccaaatcaaa agataataac aattggctct gtttctctca ctattgccac 60 aatatgcttc cttatgcaaa ttgccatcct ggtaactact gtaacattgc atttcaagca 120 atatgagtgc aactcccccc caaacaacca agtaatgctg tgtgaaccaa caataataga 180 aagaaacata acagagatag tgtatctgac caacaccacc atagagaaag aaatatgccc 240 caaactagca gaatacagaa attggtcaaa gccgcaatgt aaaattacag gatttgcacc 300 tttttctaag gacaattcaa ttcggctttc cgctggtgga gacatttggg tgacaagaga 360 accttatgtg tcatgcgatc ctggcaagtg ttatcaattt gccctcggac agggaacaac 420 actaaacaac aggcattcaa atgacacagt acatgatagg accccttatc gaaccctatt 480 gatgaatgag ttgggtgttc catttcattt gggaaccaag caagtgtgca tagcatggtc 540 cagctcaagt tgtcacgatg gaaaagcatg gctgcatgtt tgtgtaactg ggcatgatga 600 aaatgcaact gctagcttca tttacgatgg gaggcttgta gatagtattg gttcatggtc 660 caaaaatatc ctcaggaccc aggagtcgga atgcgtttgt atcaatggaa cttgtacagt 720 agtaatgact gatggaagtg cttcagaaag agctgatact aaaatactat tcattgaaga 780 ggggaaaatc gttcatatta gcccattgtc aggaagtgct cagcatgtcg aggagtgctc 840 ctgttatcct cgatatcctg gtgtcagatg tgtctgcaga gacaactgga aaggctccaa 900 taggcccatc gtagatataa atgtgaaaga ttatagcatt gtttccagtt atgtgtgctc 960 aggacttgtt ggagacacac ccagaaaaaa cgacagctcc agcagtagct attgctggaa 1020 tcctaacaat gagaaagggg gtcatggagt gaaaggctgg gcctttgatg atggaaatga 1080 cgtgtggatg ggaagaacga tcagcgagga gttacgctca ggttatgaaa ccttcaaagt 1140 cattggaggc tggtccaaac ctaactccaa attgcagata aataggcaag tcatagttga 1200 cagaggtaat aggtccggtt attctggtat tttctctgtt gaaggcaaaa gctgcatcaa 1260 tcggtgcttt tatgtggagt tgataagggg aaggaaacag gaaactgaag tctggtggac 1320 ctcaaacagt attgttgtgt tttgtggcac ttca 1354 <210> 5 <211> 1762 <212> DNA
<213> Influenza A virus <400> 5 agcaaaagca ggggataatt ctattaacca tgaagactat cattgctttg agctacattt 60 tatgtctggt tttcgctcaa aaacttcccg gaaatgacaa cagcacggca acgctgtgcc 120 tgggacacca tgcagtgcca aacggaacgc tagtgaaaac aatcacgaat gaccaaattg 180 aagtgactaa tgctactgag ctggttcaga gttcctcaac aggtagaata tgcgacagtc 240 ctcaccgaat ccttgatgga aaaaactgca cactgataga tgctctattg ggagaccctc 300 attgtgatgg cttccaaaat aaggaatggg acctttttgt tgaacgcagc aaagcttaca 360 gcaactgtta cccttatgat gtgccggatt atgcttccct taggtcacta gttgcctcat 420 ccggcaccct ggagtttacc aatgaaggct tcaattggac tggagtcgct caggatggaa 480 caagctatgc ttgcaaaagg ggatctgtta aaagtttctt tagtagattg aattggttgc 540 acaaattaga atacaaatat ccagcactga acgtgactat gccaaacaat gacaaatttg 600 acaaattgta catttggggg gttcaccacc cgagtacgga cagtgaccaa accagcatat 660 atgttcaagc atcagggaga gtcacagtct ctaccaaaag aagccaacaa actgtaatcc 720 cgaatatcgg gtctagaccc tgggtaaggg ggatctccag cagaataagc atctattgga 780 caatagtaaa accgggagac atacttttga ttaacagcac agggaatcta attgctcctc 840 ggggttactt caaaatacga agtgggaaaa gctcaataat gaggtcagat gcacccattg 900 gcaactgcaa ttctgaatgc atcactccaa atggaagcat tcccaatgac aaaccttttc 960 aaaatgtaaa caggatcaca tatggggcct gtcccagata tgttaagcaa aacactctga 1020 aattggcaac agggatgcgg aatgtaccag agaaacaaac tagaggcata ttcggcgcaa 1080 tcgcaggttt catagaaaat ggttgggagg gaatggtaga cggttggtac ggtttcaggc 1140 atcaaaattc tgagggcaca ggacaagcag cagatcttaa aagcactcaa gcagcaatca 1200 accaaatcaa cgggaaactg aataggttaa tcgagaaaac gaacgagaaa ttccatcaaa 1260 tcgaaaaaga attctcagaa gtagaaggga gaattcagga cctcgagaaa tatgttgaag 1320 acactaaaat agatctctgg tcttacaacg cggagcttct tgttgccctg gagaaccaac 1380 atacaattga tctaactgac tcagaaatga acaaactgtt tgaaagaaca aggaagcaac 1440 tgagggaaaa tgctgaggac atgggcaatg gttgcttcaa aatataccac aaatgtgaca 1500 atgcctgcat agggtcaatc agaaatggaa cttatgacca tgatgtatac agagacgaag 1560 cattaaacaa ccggttccag atcaaaggtg ttgagctgaa gtcaggatac aaagattgga 1620 tcctatggat ttcctttgcc atatcatgct ttttgctttg tgttgttctg ctggggttca 1680 tcatgtgggc ctgccaaaaa ggcaacatta ggtgcaacat ttgcatttga gtgcattaat 1740 taaaaacacc cttgtttcta ct 1762 <210> 6 <211> 1451 <212> DNA
<213> Influenza A virus <400> 6 agcaaaagca ggagtgaaaa tgaatccaaa tcaaaagata ataactattg gctctgtttc 60 tctcactatt gccacaatat gcttccttat gcaaattgcc atcctggtaa ctactgtaac 120 attacatttc aagcaatatg aatgcaactc ccccccaaac aaccaagtaa tgctgtgtga 180 accaacaata atagaaagaa acataacaga gatagtgtat ctgaccaaca ccaccataga 240 gaaggaaata tgccccaaac tagcagaata cagaaattgg tcaaagccgc aatgtaaaat 300 tacaggattt gcaccttttt ctaaggacaa ttcaattcgg ctttccgctg gtggggacat 360 ttgggtgaca agagaacctt atgtgtcatg cgatcctgac aagtgttatc aatttgccct 420 tggacaggga acaacactaa acaacaggca ttcaaatgac acagtacatg ataggacccc 480 ttatcgaacc ctattgatga atgagttggg tgttccattt catttgggaa ccaagcaagt 540 gtgcatagca tggtccagct caagttgtca cgatggaaaa gcatggctgc atgtttgtgt 600 aactgggcat gatgaaaatg caactgctag cttcatttac gatgggaggc ttgtagatag 660 tattggttca tggtccaaaa aaatcctcag gacccaggag tcggaatgcg tttgtatcaa 720 tggaacttgt acagtagtaa tgactgatgg aagtgcttca ggaagagctg atactaaaat 780 actattcatt gaagagggga aaatcgttca tattagccca ttgtcaggaa gtgctcagca 840 tgtcgaggag tgctcctgtt atcctcgata ttctggtgtc agatgtgtct gcagagacaa 900 ctggaaaggc tccaataggc ccatcgtaga tataaatgtg aaagattata gcattgtttc 960 cagttatgtg tgctcaggac ttgttggaga cacacccaga aaaaacgaca gctccagcag 1020 tagccattgc ctgaatccta acaatgagga agggggtcat ggagtgaaag gctgggcctt 1080 tgatgatgga aatgacgtgt ggatgggaag aacgatcagc gagaagttac gctcaggtta 1140 tgaaaccttc aaagtcattg gaggctggtc caaacctaac tccaaattgc agataaatag 1200 acaagtcata gttgacagag gtaataggtc cggttattct ggtattttct ctgttgaagg 1260 caaaagctgc atcaatcggt gcttttatgt ggagttgata aggggaagga aacaggaaac 1320 tgaagtctgg tggacctcaa acagtattgt tgtgttttgt ggcacctcag gtacatatgg 1380 aacaggctca tggcctgatg gggcggacat caatctcatg cctatataag ctttcgcaat 1440 tttagaaaaa a 1451 <210> 7 <211> 1762 <212> DNA
<213> Influenza A virus <400> 7 agcaaaagca ggggataatt ctattaacca tgaagactat cattgctttg agctacattt 60 tatgtctggt tttcgctcaa aaaattcccg gaaatgacaa cagcacggca acgctgtgcc 120 tgggacacca tgcagtgcca aacggaacgc tagtgaaaac aatcacgaat gaccaaattg 180 aagtgactaa tgctactgag ctggttcaga gttcctcaac aggtagaata tgcgacagtc 240 ctcaccgaat ccttgatgga gaaaactgca cactgataga tgctctattg ggagaccctc 300 attgtgatgg cttccaaaat aaggaatggg acctttttgt tgaacgcagc aaagcctaca 360 gcaactgtta cccttatgat gtgccggatt atgcctccct taggtcacta gttgcctcat 420 ccggcaccct ggagtttaac aatgaaagct tcaattggac tggagtcgct cagaatggaa 480 caagctatgc ttgcaaaagg agttctatta aaagtttctt tagtagattg aattggttgc 540 accaattaaa atacaaatat ccagcactga acgtgactat gccaaacaat gacaaatttg 600 acaaattgta catttggggg gttcaccacc cgagtacgga cagtgaccaa accagcatat 660 atgctcaagc atcagggaga gtcacagtct ccaccaaaag aagccaacaa actgtaatcc 720 cgaatatcgg atctagaccc tgggtaaggg gtatctccag cagaataagc atccattgga 780 caatagtaaa accgggagac atacttttga ttaacagcac agggaatcta attgctcctc 840 ggggttactt caaaatacga agtgggaaaa gctcaataat gaggtcagat gcacccattg 900 gcaaatgcaa ttctgaatgc atcactccaa atggaagcat tcccaatgac aaaccatttc 960 aaaatgtaaa caggatcaca tatggggcct gtcccagata tgttaagcaa aacactctga 1020 aattggcaac agggatgcgg aatgtaccag agaaacaaac tagaggcata ttcggcgcaa 1080 tcgcaggttt catagaaaat ggttgggagg gaatggtaga cggttggtac ggtttcaggc 1140 atcaaaattc tgagggcaca ggacaagcag cagatcttaa aagcactcaa gcagcaatca 1200 accaaatcaa cgggaaactg aataggttaa tcgagaaaac gaacgagaaa ttccatcaaa 1260 ttgaaaaaga attctcagaa gtagaaggga gaattcagga cctcgagaaa tatgttgagg 1320 acactaaaat agatctctgg tcgtacaacg cggagcttct tgttgccctg gagaaccaac 1380 atacaattga tctaactgac tcagaaatga acaaactgtt tgaaagaaca aggaagcaac 1440 tgagggaaaa tgctgaggat atgggcaatg gttgtttcaa aatataccac aaatgtgaca 1500 atgcctgcat agggtcaatc agaaatggaa cttatgacca tgatgtatac agagacgaag 1560 cattaaacaa ccggttccag atcaaaggtg ttgagctgaa gtcaggatac aaagattgga 1620 tcctatggat ttcctttgcc atatcatgtt ttttgctttg tgttgttttg ctggggttca 1680 tcatgtgggc ctgccaaaaa ggcaacatta ggtgcaacat ttgcatttga gtgcattaat 1740 taaaaacacc cttgtttcta ct 1762 <210> 8 <211> 1467 <212> DNA
<213> Influenza A virus <400> 8 agcaaaagca ggagtaaaga tgaatccaaa tcaaaagata ataacgattg gctctgtttc 60 tctcactatt gccacaatat gcttccttat gcaaattgcc atcctggtaa ctactgtaac 120 attgcatttc aagcaatatg aatgcagctc tcccccaaac aaccaagtaa tgctgtgtga 180 accaacaata atagaaagaa acataacaga gatagtgtat ctgaccaaca ccaccataga 240 gaaggaaata tgccccaaac tagcagaata cagaaattgg tcaaagccac aatgtaaaat 300 tacaggattt gcaccttttt ctaaggacaa ttcaattcgg ctttccgctg gtggggacat 360 ttgggtgaca agggaacctt atgtgtcgtg cgatcctgac aagtgttatc aatttgccct 420 tggacaggga acaacactaa acaacaggca ttcaaatgac acagtacatg ataggacccc 480 ttatcgaacc ctattgatga atgagttggg tgttccattt catttgggaa ccaagcaagt 540 gtgcatagca tggtccagct caagttgtca cgatggaaaa gcatggctgc atgtttgtgt 600 aactgggcat gatgaaaatg caactgctag cttcatttac gatgggaggc ttgtagatag 660 tattggttca tggtccaaaa aaatcctcag gacccaggag tcggaatgcg tttgtatcaa 720 tggaacttgt acagtagtaa tgactgatgg gagtgcttca ggaagagctg atactaaaat 780 actattcatt gaggagggga aaatcgttca tatcagccca ctgtcaggaa gtgctcagca 840 tgtcgaggag tgctcctgtt atcctcgata tcctggtgtc agatgtgtct gcagagacaa 900 ctggaaaggc tccaataggc ccatcgtaga tataaatgta aaggattata gcattgtttc 960 cagttatgtg tgctcaggac ttgttggaga cacacccaga aaaaacgaca gctccagcag 1020 tagtcattgc ctgaatccta acaatgagga agggggtcat ggagtgaaag gctgggcctt 1080 tgatgatgga aatgacgtgt ggatgggaag aacgatcagc gagaagttcc gctcaggtta 1140 tgaaaccttc aaagtcattg aaggctggtc caaacctaac tccaaattgc agataaatag 1200 gcaagtcata gttgacagag gtaataggtc cggttattct ggtattttct ctgttgaagg 1260 caaaagctgc atcaatcggt gcttttatgt ggagttgata aggggaagga aacaggaaac 1320 tgaagtctgg tggacctcaa acagtattgt tgtgttttgt ggcacctcag gtacatatgg 1380 aacaggctca tggcctgatg gggcggacat caatctcatg cctatataag ctttcgcaat 1440 tttagaaaaa aactccttgt ttctact 1467 <210> 9 <211> 1762 <212> DNA
<213> Influenza A virus <400> 9 agcaaaagca ggggataatt ctattaacca tgaagactat cattgctttg agctacattt 60 tatgtctggt tttcgctcaa aaacttcccg gaaatgacaa cagcacggca acgctgtgcc 120 tggggcacca tgcagtgtca aacggaacgc tagtgaaaac aatcacgaat gaccaaattg 180 aagtgactaa tgctactgag ctggttcaga gttcctcaac aggtagaata tgcgacagtc 240 ctcaccaaat ccttgatgga gaaaactgca cactaataga tgctctattg ggagaccctc 300 attgtgatgg cttccaaaat aaggaatggg acctttttgt tgaacgcagc aaagcctaca 360 gcaactgtta cccttatgat gtgccggatt atgcctccct taggtcacta gttgcctcat 420 ccggcacact ggagtttaac aatgaaagct tcaattggac tggagtcgct cagaatggaa 480 caagctctgc ttgcaaaagg ggatctaata aaagtttctt tagtagattg aattggttgc 540 accaattaaa atacaaatat ccagcactga acgtgactat gccaaacaat gaaaaatttg 600 acaaattgta catttggggg gttctccacc cgagtacgga cagtgaccaa atcagcctat 660 atgctcaagc atcagggaga gtcacagtct ctaccaaaag aagccaacaa actgtaatcc 720 cgaatatcgg atctagaccc tgggtaaggg gtgtctccag cagaataagc atctattgga 780 caatagtaaa accgggagac atacttttga ttaacagcac agggaatcta attgctcctc 840 ggggttactt caaaatacga agtgggaaaa gctcaataat gaggtcagat gcacccattg 900 gcaaatgcaa ttctgaatgc atcactccaa atggaagcat tcccaatgac aaaccatttc 960 aaaatgtaaa caggatcaca tatggggcct gtcccagata tgttaagcaa aacactctga 1020 aattggcaac agggatgcgg aatgtaccag agaaacaaac tagaggcata ttcggcgcaa 1080 tcgcgggttt catagaaaat ggttgggagg gaatggtgga cggttggtac ggtttcaggc 1140 atcaaaattc tgagggcaca ggacaagcag cagatcttaa aagcactcaa gcagcaatca 1200 accaaatcaa cgggaaactg aataggttaa tcgagaaaac gaacgagaaa ttccatcaaa 1260 ttgaaaaaga attctcagaa gtagaaggga gaattcagga cctcgagaaa tatgttgagg 1320 acactaaaat agatctctgg tcgtacaacg cggagcttct tgttgccctg gagaaccaac 1380 atacaattga tctaactgac tcagaaatga acaaactgtt tgaaagaaca aagaagcaac 1440 tgagggaaaa tgctgaggat atgggcaatg gttgtttcaa aatataccac aaatgtgaca 1500 atgcctgcat agggtcaatc agaaatggaa cttatgacca tgatgtatac agagacgaag 1560 cattaaacaa ccggttccag atcaaaggtg ttgagctgaa gtcaggatac aaagattgga 1620 tcctatggat ttcctttgcc atatcatgct ttttgctttg tgttgttttg ctggggttca 1680 tcatgtgggc ctgccaaaaa ggcaacatta ggtgcaacat ttgcatttga gtgcattaat 1740 taaaaacacc cttgtttcta ct 1762 <210> 10 <211> 1466 <212> DNA
<213> Influenza A virus <400> 10 agcaaaagca ggagtaaaga tgaatccaaa tcaaaagata ataacgattg gctctgtttc 60 tctcactatt gccacaatat gcttccttat gcaaatagcc atcctggtaa ctactgtaac 120 attgcatttc aagcaatatg aatgcaactc ccccccaaac aaccaagtaa tgctgtgtga 180 accaacaata atagaaagaa acataacaga gatagtgtat ctgaccaaca ccaccataga 240 gaaggaaata tgccccaaac tagcagaata cagaaattgg tcaaagccgc aatgtaaaat 300 tacaggattt gcaccttttt ctaaggataa ttcaattcgg ctttccgctg gtggggacat 360 ttgggtgaca agagaacctt atgtgtcatg cgatcctgac aagtgttatc aatttgccct 420 tggacaggga acaacactaa acaacaggca ttcaaatgac acagtacatg ataggacccc 480 ttatcgaacc ctattgatga atgagttggg tgttccattt catttgggaa ccaagcaagt 540 gtgtatagca tggtccagct caagttgtca cgatggaaaa gcatggctgc atgtttgtgt 600 aactgggcat gatgaaaatg caactgctag cttcatttac gatgggagac ttgtagatag 660 tattggttca tggtccaaaa aaatcctcag gacccaggag tcggaatgcg tttgtatcaa 720 tggaacttgt acagtagtaa tgactgatgg gagtgcttca ggaagagctg atactaaaat 780 acttttcatt gaggagggga aaatcgttca tactagcaaa ttgtcaggaa gtgctcagca 840 tgtcgaggag tgctcctgtt atcctcgata tcctggtgtc agatgtgtct gcagagacaa 900 ctggaaaggc tccaataggc ccatcgtaga tataaatgta aaggattata gcattgtttc 960 cagttatgtg tgctcaggac ttgttggaga cacacccaga aaaaacgaca gctccagcag 1020 tagccattgc ctggatccta acaatgaaga agggggtcat ggagtgaaag gctgggcctt 1080 tgatgatgga aatgacgtgt ggatgggaag aacgatcagc gagaagtcac gctcaggtta 1140 tgaaaccttc aaggtcattg aaggctggtc caaacctaac tccaaattgc agataaatag 1200 gcaagtcata gttgaaagag gtaatatgtc cggttattct ggtattttct ctgttgaagg 1260 caaaagctgc atcaatcggt gcttttatgt ggagttgata aggggaagga aacaggaaac 1320 tgaagtctgg tggacctcaa acagtattgt tgtgttttgt ggcacctcag gtacatatgg 1380 aacaggctca tggcctgatg gggcggacat caatctcatg cctatataag ctttcgcaat 1440 tttagaaaaa actccttgtt tctact 1466 <210> 11 <211> 1762 <212> DNA
<213> Influenza A virus <400> 11 agcaaaagca ggggataatt ctattaacca tgaagactat cattgcttta agctacattc 60 tatgtctggt tttctctcaa aagcttcccg gaaatgacaa cagcacggca acgctgtgcc 120 ttgggcacca tgcagtacca aacggaacga tagtgaaaac aatcacgaat gaccaaattg 180 aagttactaa tgctactgag ctggttcaga gttcctcaac aggtggaata tgcgacagtc 240 ctcatcagat ccttgatgga gaaaactgca cactaataga tgctctattg ggagaccctc 300 agtgtgatgg cttccaaaat aagaaatggg acctttttgt tgaacgcagc aaagcctaca 360 gcaactgtta cccttatgat gtgccggatt atgcctccct taggtcacta gttgcctcat 420 ccggcacact ggagtttaac aatgaaagct tcaattgggc tggagtcact cagaatggaa 480 caagctctgc ttgcaaaagg agatctaata aaagtttctt tagtagattg aattggttga 540 cccacttaaa atacaaatac ccagcattga acgtgactat gccaaacaat gaaaaatttg 600 acaaattgta catttggggg gttcaccacc cggttacgga cagtgaccaa atcagcctat 660 atgctcaagc atcaggaaga atcacagtct ctaccaaaag aagccaacaa actgtaatcc 720 cgaatatcgg atatagaccc agggtaaggg atatctccag cagaataagc atctattgga 780 caatagtaaa accgggagac atacttttga ttaacagcac aggaaatcta attgctcctc 840 ggggttactt caaaatacga agtgggaaaa gctcaataat gagatcagat gcacccattg 900 gcaaatgcaa ttctgaatgc atcactccaa atggaagcat tcccaatgac aaaccatttc 960 aaaatgtaaa caggatcaca tatggggcct gtcccagata tgttaagcaa aacactctga 1020 aattggcaac agggatgcga aatgtaccag agaaacaaac tagaggcata tttggcgcaa 1080 tcgcgggttt catagaaaat ggttgggagg gaatggtgga cggttggtac ggtttcaggc 1140 atcaaaattc tgagggcaca ggacaagcag cagatctcaa aagcactcaa gcagcaatca 1200 accaaatcaa tgggaaactg aataggttaa tcgggaaaac aaacgagaaa ttccatcaga 1260 ttgaaaaaga attctcagaa gtagaaggga gaattcagga cctcgagaaa tatgttgagg 1320 acactaaaat agatctctgg tcatacaacg cggagcttct tgttgccctg gaaaaccaac 1380 atacaattga tctaactgac tcagaaatga acaaactgtt tgaaagaaca aagaagcaac 1440 tgagggaaaa tgctgaggat atgggcaatg gttgtttcaa aatataccac aaatgtgaca 1500 atgcctgcat agagtcaatc agaaatggaa cttatgacca tgatgtatac agagatgaag 1560 cattaaacaa ccggttccag atcaaaggtg ttgagctgaa gtcaggatac aaagattgga 1620 tcctatggat ttcctttgcc atatcatgtt ttttgctttg tgttgctttg ttggggttca 1680 tcatgtgggc ctgccaaaaa ggcaacatta ggtgcaacat ttgcatttga gtgcattaat 1740 taaaaacacc cttgtttcta ct 1762 <210> 12 <211> 1467 <212> DNA
<213> Influenza A virus <400> 12 agcaaaagca ggagtaaaga tgaatccaaa tcaaaagata ataacgattg gctctgtttc 60 cctcaccatt tccacaatat gcttcttcat gcaaattgcc atcctgataa ctactgtaac 120 attgcatttc aagcaatatg aattcaactc ccccccaaac aaccaagtga tgctgtgtga 180 accaacaata atagaaagaa acataacaga gatagtgtat ctgaccaaca ccaccataga 240 gaaggaaata tgccccaaac tagcagaata cagaaattgg tcaaagccgc aatgtaacat 300 tacaggattt gcaccttttt ctaaggacaa ttcgattcgg ctttccgctg gtggggacat 360 ctgggtgaca agagaacctt atgtgtcatg cgatcctgac aagtgttatc aatttgccct 420 tggacaggga acaacactaa acaacgtgca ttcaaatgac acagtacatg ataggacccc 480 ttatcggacc ctattgatga atgagttggg tgttccattt catctgggga ccaagcaagt 540 gtgcatagca tggtccagct caagttgtca cgatggaaaa gcatggctgc atgtttgtgt 600 aacgggggat gatgaaaatg caactgctag cttcatttac aatgggaggc ttgtagatag 660 tattgtttca tggtccaaaa aaatcctcag gacccaggag tcagaatgcg tttgtatcaa 720 tggaacttgt acagtagtaa tgactgatgg gagtgcttca ggaaaagctg atactaaaat 780 actattcatt gaggagggga aaattgttca tactagcaca ttatcaggaa gtgctcagca 840 tgtcgaggag tgctcctgtt atcctcgata tcctggtgtc agatgtgtct gcagagacaa 900 ctggaaaggc tccaataggc ccatcgtaga tataaacata aaggattata gcattgtttc 960 cagttatgtg tgctcaggac ttgttggaga cacacccaga aaaaacgaca gctccagcag 1020 tagccattgc ttggatccaa acaatgagga aggtggtcat ggagtgaaag gctgggcatt 1080 tgatgatgga aatgacgtgt ggatgggaag aacgatcagc gagaagttac gctcaggata 1140 tgaaaccttc aaagtcattg aaggctggtc caaccctaac tccaaattgc agataaatag 1200 gcaagtcata gttgacagag gtaacaggtc cggttattct ggtattttct ctgttgaagg 1260 caaaagctgc atcaatcggt gcttttatgt ggagttgata aggggaagaa aacaggaaac 1320 tgaagtcttg tggacctcaa acagtattgt tgtgttttgt ggcacctcag gtacatatgg 1380 aacaggctca tggcctgatg gggcggacat caatctcatg cctatataag ctttcgcaat 1440 tttagaaaaa aactccttgt ttctact 1467 <210> 13 <211> 1778 <212> DNA
<213> Influenza A virus <400> 13 agcaaaagca ggggaaaata aaaacaacca aaatgaaagc aaaactacta gtcctgttat 60 gtgcatttac agctacatat gcagacacaa tatgtatagg ctaccatgcg aacaactcaa 120 ccgacactgt tgacacagta cttgagaaga acgtgacagt gacacactct gtcaacctac 180 ttgaggacag tcacaacgga aaactatgtc gactaaaggg aatagcccca ctacaattgg 240 gtaattgcag cgttgccgga tggatcttag gaaacccaaa atgcgaatca ctgttttcta 300 aggaatcatg gtcctacatt gcagaaacac caaaccctga gaatggaaca tgttacccag 360 ggtatttcgc cgactatgag gaactgaggg agcaattgag ttcagtatca tcattcgaga 420 gattcgaaat attccccaaa gaaagctcat ggcccaacca caccgtaacc aaaggagtaa 480 cgacatcatg ctcccataat gggaaaagca gtttttacag aaatttgcta tggctgacga 540 agaagaatgg cttgtaccca aatgtgagca agtcctatgt aaacaacaaa gagaaagaag 600 tccttgtact atggggtgtt catcacccgt ctaacatagg ggaccaaagg gccatctatc 660 atacagaaaa tgcttatgtc tctgtagtgt cttcacatta tagcagaaga ttcaccccag 720 aaatagcaaa aagacccaaa gtaagagatc aagaaggaag aattaactac tactggactc 780 tgctggaacc cggggacaca ataatatttg aggcaaatgg aaatctaata gcgccatggt 840 atgctttcgc actgagtaga ggctttgggt caggaatcat cacctcaaac gcatcaatgg 900 atgaatgtga cgcgaagtgt caaacacccc agggagctat aaacagtagt cttcctttcc 960 agaatgtaca cccagtcaca ataggagagt gtccaaagta tgtcaggagt acaaaattaa 1020 ggatggttac aggactaagg aacatcccat ccattcaatc cagaggtttg tttggagcca 1080 ttgccggttt cattgaaggg gggtggactg gaatgataga tggatggtat ggttatcatc 1140 atcagaatga acaaggatct ggctatgctg cggaccaaaa aagcacacaa aatgccatta 1200 acgggattac aaacaaggtg aattctgtaa tcgagaaaat gaacactcaa ttcacagctg 1260 tgggcaaaga attcaacaaa ttagaaagaa ggatggaaaa cttaaataaa aaagttgatg 1320 atggatttct ggacatttgg acatataatg cagaattgtt ggttctactg gaaaatggaa 1380 ggactttgga ttttcatgac tcaaatgtga agaatctgta tgagaaagta aaaagccaat 1440 tgaagaataa tgccaaagaa atagggaacg ggtgttttga attctatcac aagtgtaaca 1500 atgaatgcat ggaaagtgtg aaaaatggaa cttatgacta tccaaaatat tccgaagaat 1560 caaagttaaa caggggaaaa attgatggag tgaaattgga atcaatggga gtctatcaga 1620 ttctggcgat ctactcaact gtcgccagtt cactggtgct tttggtctcc ctgggggcaa 1680 tcagcttctg gatgtgttct aatgggtctt tgcagtgtag aatatgcatc tgagaccaga 1740 atttcagaaa tataagaaaa aacacccttg tttctact 1778 <210> 14 <211> 1463 <212> DNA
<213> Influenza A virus <400> 14 agcaaaagca ggagtttaaa atgaatccaa atcaaaaaat aataatcata ggatcaatca 60 gtatggcaat cggaataatt agtctaatat tgcaaatagg aaatattatt tcaatatggg 120 ctagccactc aatccaaact ggaagtcaaa accacactgg aatatgcaac caaagaatca 180 ttacatatga aaatagcacc tgggtgaatc aaacatatgt taatattaac aacactaatg 240 ttgttgctgg aaaggacaaa acttcagtga cattggccgg caattcatct ctttgcccta 300 tccgtgggtg ggctatatac acaaaagaca acagcataag aattggttcc aaaggagatg 360 tttttgtcat aagagagcct tttatatcat gttctcactt ggaatgcaga accttttttc 420 tgacccaagg tgctctatta aatgacaagc attcaaatgg gaccgttaag gacagaagcc 480 cttatagggc cttaatgagc tgtcctctag gtgaagctcc gtctccatac aattcaagat 540 ttgaatcagt tgcttggtca gcaagcgcat gccatgatgg catgggctgg ctaacaatcg 600 gaatttctgg tccagataat ggagcagtgg ctgtactaaa atacaacggc ataataactg 660 aaaccataaa aagttggaag aagcgaatat taagaacaca agagtctgaa tgtgtctgtg 720 tgaacggttc atgttttacc ataatgaccg atggcccgag taatggggcc gcctcgtaca 780 gaatcttcaa aatcgagaag gggaaggtta ctaaatcaat agagttggat gcacccaatt 840 atcattacga ggaatgttcc tgttacccag acaccggcac agtgatgtgt gtgtgcaggg 900 acaattggca cggttcaaat cgaccttggg tgtcttttaa tcaaaacctg gattatcaaa 960 taggatacat ctgcagtggg gtgttcggtg acaatccgcg tcccaaagat ggagaaggca 1020 gctgtaatcc agtgactgtt gatggagcag acggagtaaa ggggttttca tacagatatg 1080 gtaatggtgt ttggatagga aggactaaaa gtaacagact cagaaaggga tttgagatga 1140 tttgggatcc taatggatgg acagataccg acagtgattt ctctgtgaaa caggatgtcg 1200 tggcaatgac tgattggtca gggtacagcg gaagtttcgt tcaacatcct gagctaacag 1260 gattggactg tatgagacct tgcttctggg ttgaattaat cagagggcga cctagagaaa 1320 atacaacaat ctggactagt gggagcagca tttctttttg tggcgtaaat agcgatactg 1380 caaactggtc ttggccagac ggtgccgagt tgccattcac cattgacaag tagtccgttg 1440 aaaaaaaact ccttgtttct act 1463 <210> 15 <211> 1689 <212> DNA
<213> Influenza A virus <400> 15 aaatgaaagc aaaactacta gtcctgttgt gtgcatttac agctacatat gcagacacaa 60 tatgtatagg ctaccatgcg aacaactcaa ccgacactgt tgacacagta cttgagaaga 120 acgtgacagt gacacactct gtcaacctac ttgaggacag tcacaacgga aaactatgcc 180 gactaaaagg aacagcccca ctacaattgg gtaattgcag cgttgccgga tggatcttag 240 gaaacccaga atgcgaatca ctgttttcta aggaatcatg gtcctacatt gcagaaacac 300 caaaccctga gaatggaaca tgttacccag ggtatttcgc cgactatgag gaactgaggg 360 agcaattgag ctcagtatca tcattcgaga gattcgaaat attccccaag gaaagctcat 420 ggcccaaaca caccgtaacc aaaggagtga cggcatcatg ctcccataat gggaaaagca 480 gtttttacaa aaatttgcta tggctgacgg aaaagaatgg cttgtaccca aatctgagca 540 agtcctatgt aaacaacaag gagaaagaag tccttgtact atggggtgtt catcacccgt 600 ctaacatagg ggaccaaagg gccatctatc atacagaaaa tgcttatgtc tctgtagtgt 660 cttcacatta tagcagaaga ttcaccccag aaatagcaaa aagacccaaa gtaagaggtc 720 aagaagggag aattaactac tactggactc tgctggaacc cggggacaca ataatatttg 780 aggcaaatgg aaatctaata gcgccatggt acgctttcgc actgagtaga ggctttgggt 840 caggaatcat cacctcaacc gcatcaatgg gtgaatgtga cgctaagtgt caaacacccc 900 aaggagctat aaacagtagt cttcctttcc agaatgtaca cccagtcaca ataggagagt 960 gtcccaagta tgtcaggagt acaaaattaa ggatggttac aggactaaga aacatcccat 1020 ccattcaatc tagaggtttg tttggagcca ttgccggttt cattgaaggg gggtggactg 1080 gaatgataga tggatggtat ggttatcatc atcagaatga acaaggatct ggctatgctg 1140 cagaccaaaa aagcacacaa aatgccattg atgggattac aaacaaggtg aattctgtaa 1200 tcgagaaaat gaacactcaa ttcacagctg taggcaaaga attcaacaaa ttagagagaa 1260 ggatggaaaa cttaaataag aaagttgatg atggatttct ggacatttgg acatataatg 1320 cagagttgtt ggttctcctg gaaaatggaa ggactttggg ttttcatgac tcaaatgtga 1380 agaatctgta tgagaaagta aaaaaccaat tgaagaataa tgccaaagaa atcgggaacg 1440 ggtgttttga attctatcac aagtgtaaca atgaatgcat ggaaagtgtg aaaaatggaa 1500 cttatgacta tccaaaatat tccgaagaat caaagttaaa cagggaaaaa attgatggag 1560 tgaaattgga atcaatggga gtctatcaga ttctggcgat ctactcaact gtcgccagtt 1620 cactggtgct tttggtctcc ctgggggcaa tcagtttctg gatgtgttct aatgggtctt 1680 tgcagtgta 1689 <210> 16 <211> 1447 <212> DNA
<213> Influenza A virus <400> 16 agcaaaagca ggagtttaaa atgaatccaa atcaaaaaat aataaccatt ggatcaatca 60 gtattgcaat tggaataatt agtctgatat tgcaaatagg aaatattatt tcaatatggg 120 ctagccactc aatccaaact ggaagtcaaa accacactgg aatatgcaac caaagaatca 180 ttacatatga aaatagcacc tgggtaaatc aaacatatgt taatattaac aacactaatg 240 ttgttgctgg aaaggacaaa acctcaatga cattggccgg caattcatct ctttgcccta 300 tccgtggatg ggctatatac acaaaagaca acagcataag aattggttcc aaaggagatg 360 tttttgtcat aagagagcct tttatatcat gttctcactt ggaatgcaga accttttttc 420 tgacccaagg tgctctatta aatgacaagc attcaaatgg gaccgttaag gacagaagcc 480 cttatagggc cttaatgagc tgtcctctag gtgaagctcc gtctccatac aattcaagat 540 ttgaatcagt tgcttggtca gcaagcgcat gccatgatgg cttgggctgg ctaacaatcg 600 gaatttctgg tccagataat ggggcagtgg ctgtactaaa atacaacggc ataataactg 660 aaaccattaa aagttggaag aagcgaatat taagaacaca agagtctgaa tgtgtctgta 720 tgaacggttc atgttttacc ataatgaccg atggcccgag taatggggcc gcatcgtaca 780 gaatcttcaa aatcgagaag gggagagtta ctaaatcaat agagttggat gcacccaatt 840 atcattacga ggaatgttca tgttacccag acaccggcac agtgatgtgt gtgtgcaggg 900 acaattggca cggttcaaat cgaccttggg tgtcttttaa tcaaaacctg gattatcaaa 960 taggatacat ctgcagtggg gtgttcggtg acaatccgcg tcccaaagat ggagaaggca 1020 gctgtaatcc agtgactgtt gatggagcag acggagtaaa ggggttttca tacagatatg 1080 gtaatggtgt ttggatagga aggactaaaa gtaacagact cagaaaggga tttgagatga 1140 tttgggatcc taatggatgg acagataccg acagtgattt ctcaatgaaa caggatatcg 1200 tggcaatgac tgattggtca gggtacagcg gaagttttgt tcaacatcct gagctaacag 1260 gattggactg tatgagacct tgcttttggg ttgaattagt cagagggcta cctagagaaa 1320 atacaacaat ctggactagt gggagcagca tttctttttg tggcgtaaat agcgatactg 1380 caaactggtc ttggccagac ggtgccgagt tgccattcac cattgacaag tagtccgttg 1440 aaaaaaa 1447 <210> 17 <211> 1775 <212> DNA
<213> Influenza A virus <400> 17 agcaaaagca ggggaaaata aaaacaacca aaatgaaagc aaaactacta gtcctgttat 60 gtacatttac agctacatat gcagacacaa tatgtatagg ctaccatgcc aacaactcaa 120 ccgacactgt tgacacagta cttgagaaga atgtgacagt gacacactct gtcaacctac 180 ttgaggacag tcacaatgga aaactatgtc tactaaaagg aatagcccca ctacaattgg 240 gtaattgcag cgttgccgga tggatcttag gaaacccaga atgcgaatca ctgatttcta 300 aggaatcatg gtcctacatt gtagagacac caaaccctga gaatggaaca tgttacccag 360 ggtatttcgc cgactatgag gaactgaggg agcaattgag ttcagtatca tcatttgaga 420 gattcgaaat attccccaaa gaaagctcat ggcccaaaca caccgtaaca ggagtaacgg 480 catcatgctc ccataatggg aaaagcagtt tttacagaaa tttgctatgg ctgacggaga 540 agaatggctt gtacccaaat ctgagcaatt cctatgtgaa caacaaagag aaagaagtcc 600 ttgtactatg gggtgttcat cacccatcta acatagggga ccaaagggcc atctatcata 660 cagaaaacgc ttatgtctct gtagtgtctt cacattatag cagaagattc accccagaaa 720 tagcaaaaag acccaaagta agaggtcagg aaggaagaat caactactac tggactctgc 780 tggaacccgg ggacacaata atatttgagg caaatggaaa tctaatagcg ccatggtatg 840 ctttcgcact gagtagaggc tttgggtcag gaatcatcac ctcaaatgca ccaatgaatg 900 aatgtgatgc gaagtgtcaa acacctcagg gagctataaa cagtagtctt cctttccaga 960 atgtacaccc agtcacaata ggagagtgtc caaagtatgt caggagtaca aaattaagga 1020 tggttacagg actaaggaat atcccatcca ttcaatccag aggtttgttt ggagccattg 1080 ccggtttcat tgaagggggg tggactggaa tgatggatgg gtggtatggt tatcatcatc 1140 agaatgagca aggatctggc tatgctgcag atcaaaaaag cacacaaaat gccattaacg 1200 ggattacaaa taaggtgaat tctgtaattg agaaaatgaa cactcaattc acagctgtgg 1260 gcaaagaatt caacaaatta gaaagaagga tggaaaactt aaataaaaaa gttgatgatg 1320 gatttctaga catttggaca tataatgcag aattgttggt tctactggaa aatgaaagga 1380 ctttggattt ccatgactca aatgtgaaga atctgtatga gaaagtgaaa agccaattaa 1440 agaataatgc caaagaaata gggaacgggt gttttgaatt ctatcacaag tgtaacaatg 1500 aatgcatgga aagtgtgaaa aatggaactt atgactatcc aaaatattcc gaagaatcaa 1560 agttaaacag ggagaaaatt gatggagtga aattggaatc aatgggagtc tatcagattc 1620 tggcgatcta ctcaactgtc gccagttcac tggttctttt ggtctccctg ggggcaatca 1680 gcttctggat gtgttccaat gggtctttgc agtgtagaat atgcatctga gaccagaatt 1740 tcagaaatat aagaaaaaac acccttgttt ctact 1775 <210> 18 <211> 1463 <212> DNA
<213> Influenza A virus <400> 18 agcaaaagca ggagtttaaa atgaatccaa atcaaaaaat aataaccatt ggatcaatca 60 gtatagtaat cgggataatt agtctaatgt tgcaaatagg aaatattatt tcaatatggg 120 ctagtcactc aatccaaact ggaagtcaaa accacactgg aatatgcaac caaagaatca 180 tcacatatga aaatagcacc tgggtgaatc acacatatgt taatattaac aacactaatg 240 ttgttgctgg aaaggacaaa acttcagtga cattggccgg caattcatca ctttgttcta 300 tcagtggatg ggctatatac acaaaagaca acagcataag aattggttcc aaaggagatg 360 tttttgtcat aagagagcct tttatatcat gttctcactt ggaatgcaga accttttttc 420 tgacccaagg tgctctatta aatgacaaac attcaaatgg gaccgttaag gacagaagtc 480 cttatagggc cttaatgagc tgtcctctag gcgaagctcc gtctccatat aattcaaagt 540 ttgaatcagt tgcttggtca gcaagcgcat gtcatgatgg catgggctgg ttaacaatcg 600 gaatttctgg tccagataat ggagcagtgg ctgtactaaa atacaacggc ataataactg 660 aaaccataaa aagttggaaa aagcgaatat taagaacaca agagtctgaa tgtgtctgtg 720 tgaacgggtc atgttttacc ataatgaccg atggcccgag taatggggcc gcctcgtaca 780 aaatcttcaa gattgagaag gggaaggtta ctaaatcaat agagttgaat gcacccaatt 840 ctcattatga ggaatgttcc tgttacccag acactggcac agtgatgtgt gtatgcaggg 900 acaattggca cggttcaaat cgaccttggg tgtcttttaa tcaaaacctg gattatcaaa 960 taggatacat ctgcagtggg gtgttcggtg acaatccgcg tcccaaagat ggagagggca 1020 gctgtaatcc agtgactgtt gatggagcag acggagtaaa ggggttttca tacagatatg 1080 gtaatggtgt ttggatagga aggactaaaa gtaacagact cagaaaggga tttgagatga 1140 tttgggatcc taatggatgg acagataccg acagtgattt ctcagtgaaa caggatgttg 1200 tggcaatgac tgattggtca gggtacagcg gaagtttcgt tcaacatcct gagctaacag 1260 gattggactg tataagacct tgcttctggg ttgaattagt cagaggacgg cctagagaaa 1320 atacaacaat ctggactagt gggagcagca tttctttttg tggcgtaaat agtgatactg 1380 caaactggtc ttggccagac ggtgctgagt tgccattcac cattgacaag tagtccgttg 1440 aaaaaaaact ccttgtttct act 1463 <210> 19 <211> 1775 <212> DNA
<213> Influenza A virus <400> 19 agcaaaagca ggggaaaata aaaacaacca aaatgaaagc aaaactactg gtcctgttat 60 gtacatttac agctacatat gcagacacaa tatgtatagg ctaccatgcc aacaactcaa 120 ccgacactgt tgacacagta cttgagaaga atgtgacagt gacacactct gtcaacctac 180 ttgaggacag tcacaatgga aaactatgtc tactaaaagg aatagcccca ctacaattgg 240 gtaattgcag cgttgccgga tggatcttag gaaacccaga atgcgaatta ctgatttcca 300 aggaatcatg gtcctacatt gtagaaacac caaatcctga gaatggaaca tgttacccag 360 ggtatttcgc cgactatgag gaactgaggg agcaattgag ttcagtatct tcatttgaga 420 gattcgaaat attccccaaa gaaagctcat ggcccaaaca caccgtaacc ggagtatcag 480 catcatgctc ccataatggg aaaaacagtt tttacagaaa tttgctatgg ctgacgggga 540 agaatggttt gtacccaaac ctgagcaagt cctatgtaaa caacaaagag aaagaagtcc 600 ttgtactatg gggtgttcat cacccgccta acatagggga ccaaagggcc ctctatcata 660 cagaaaatgc ttatgtctct gtagtgtctt cacattatag cagaagattc accccagaaa 720 tagccaaaag acccaaagta agagatcagg aaggaagaat caactactac tggactctgc 780 tggaacctgg ggatacaata atatttgagg caaatggaaa tctaatagcg ccatggtatg 840 cttttgcact gagtagaggc tttggatcag gaatcatcac ctcaaatgca ccaatggatg 900 aatgtgatgc gaagtgtcaa acacctcagg gagctataaa cagcagtctt cctttccaga 960 atgtacaccc agtcacaata ggagagtgtc caaagtatgt caggagtgca aaattgagga 1020 tggttacagg actaaggaac atcccatcca ttcaatccag aggtttgttt ggagccattg 1080 ccggtttcat tgaagggggg tggactggaa tggtagatgg gtggtatggt tatcatcatc 1140 agaatgagca aggatctggc tatgctgcag atcaaaaaag tacacaaaat gccattaacg 1200 ggattacaaa caaggtgaat tctgtaattg agaaaatgaa cactcaattc acagctgtgg 1260 gcaaagaatt caacaaattg gaaagaagga tggaaaactt aaataaaaaa gttgatgatg 1320 ggtttctaga catttggaca tataatgcag aattgttggt tctactggaa aatgaaagga 1380 ctttggattt ccatgactcc aatgtgaaga atctgtatga gaaagtaaaa agccaattaa 1440 agaataatgc caaagaaata ggaaacgggt gttttgaatt ctatcacaag tgtaacaatg 1500 aatgcatgga gagtgtgaaa aatggaactt atgactatcc aaaatattcc gaagaatcaa 1560 agttaaacag ggagaaaatt gatggagtga aattggaatc aatgggagtc tatcagattc 1620 tggcgatcta ctcaactgtc gccagttccc tggttctttt ggtctccctg ggggcaatca 1680 gcttctggat gtgttccaat gggtctttgc agtgtagaat atgcatctga gaccagaatt 1740 tcagaagtat aagaaaaaac acccttgttt ctact 1775 <210> 20 <211> 1463 <212> DNA
<213> Influenza A virus <400> 20 agcaaaagca ggagtttaaa atgaatccaa atcaaaaaat aataaccatt ggatcaatca 60 gtatagcaat cggaataatt agtctaatgt tgcaaatagg aaatattatt tcaatatggg 120 ctagtcactc aatccaaact ggaagtcaaa accacactgg agtatgcaac caaagaatca 180 tcacatatga aaacagcacc tgggtgaatc acacatatgt taatattaac aacactaatg 240 ttgttgctgg aaaggacaaa acttcagtga cattggccgg caattcatct ctttgttcta 300 tcagtggatg ggctatatac acaaaagaca acagcataag aattggctcc aaaggagatg 360 tttttgtcat aagagaacct ttcatatcat gttctcactt ggaatgcaga accttttttc 420 tgacccaagg tgctctatta aatgacaaac attcaaatgg gaccgttaag gacagaagtc 480 cttatagggc cttaatgagc tgtcctctag gtgaagctcc gtccccatac aattcaaagt 540 ttgaatcagt tgcatggtca gcaagcgcat gccatgatgg catgggctgg ttaacaatcg 600 gaatttctgg tccagacaat ggagctgtgg ctgtactaaa atacaacggc ataataactg 660 aaaccataaa aagttggaaa aagcgaatat taagaacaca agagtctgaa tgtgtctgtg 720 tgaacgggtc atgtttcacc ataatgaccg atggcccgag taatggggcc gcctcgtaca 780 aaatcttcaa gatcgaaaag gggaaggtta ctaaatcaat agagttgaat gcacccaatt 840 ttcattatga ggaatgttcc tgttacccag acactggcac agtgatgtgt gtatgcaggg 900 acaactggca tggttcaaat cgaccttggg tgtcttttaa tcaaaacctg gattatcaaa 960 taggatacat ctgcagtggg gtgttcggtg acaatccgcg tcccaaagat ggagagggca 1020 gctgtaatcc agtgactgtt gatggagcag acggagtaaa ggggttttca tacaaatatg 1080 gtaatggtgt ttggatagga aggactaaaa gtaacagact tagaaagggg tttgagatga 1140 tttgggatcc taatggatgg acagataccg acagtgattt ctcagtgaaa caggatgttg 1200 tggcaataac tgattggtca gggtacagcg gaagtttcgt tcaacatcct gagttaacag 1260 gattggactg tataagacct tgcttctggg ttgagttagt cagaggactg cctagagaaa 1320 atacaacaat ctggactagt gggagcagca tttctttttg tggcgtaaat agtgatactg 1380 caaactggtc ttggccagac ggtgctgagt tgccgttcac cattgacaag tagttcgttg 1440 aaaaaaaact ccttgtttct act 1463 <210> 21 <211> 1879 <212> DNA
<213> Influenza B virus <400> 21 agcagaagca gagcattttc taatatccac aaaatgaagg caataattgt actactcatg 60 gtagtaacat ccaacgcaga tcgaatctgc actgggataa catcttcaaa ctcacctcat 120 gtggtcaaaa cagctactca aggggaagtc aatgtgactg gtgtgatacc actgacaaca 180 acaccaacaa aatctcattt tgcaaatctc aaaggaacaa agaccagagg gaaactatgc 240 ccaaactgtc tcaactgcac agatctggat gtggccttgg gcagaccaat gtgtataggg 300 atcacacctt cggcaaaagc ttcaatactc cacgaagtca gacctgttac atccgggtgc 360 tttcctataa tgcacgacag aacaaaaatc agacagctac ccaatcttct cagaggatat 420 gaacatatca gattatcaac ccataacgtt atcaacgcag aaagggcacc aggaggaccc 480 tacagacttg gaacctcagg atcttgccct aacgttacca gtagaagcgg attcttcgca 540 acaatggctt gggctgtccc aagggacaac aaaacagcaa cgaacccact aacagtagaa 600 gtaccataca tttgtacaaa aggagaagac caaattactg tttgggggtt ccattctgat 660 aacaaaatcc aaatgaaaaa cctctatgga gactcaaatc ctcaaaagtt cacctcatct 720 gccaatggaa taaccacaca ttatgtttct cagattggtg gcttcccaaa tcaaacagaa 780 gacggagggc taccacaaag cggcagaatt gttgttgatt acatggtgca aaaacctggg 840 aaaacaggaa caattgtcta tcaaagaggt gttttgttgc ctcaaaaggt gtggtgtgca 900 agtggcagga gcaaggtaat aaaagggtcc ttgcctttaa ttggtgaagc agattgcctt 960 cacgaaaaat acggtggatt aaacaaaagc aagccttact acacaggaga acatgcaaaa 1020 gccataggaa attgcccaat atgggtgaaa acacctttaa agcttgccaa tggaaccaaa 1080 tatagacctc ccgcaaaact attaaaggaa aagggtttct tcggagctat tgctggtttc 1140 ttagaaggag gatgggaagg aatgattgca ggttggcacg gatacacatc tcatggagca 1200 catggggtgg cagtggcagc agaccttaag agtacgcaag aagccataaa caagataaca 1260 aaaaatctca attctttgag tgagctagaa gtaaagaatc ttcaaagact aagtggtgcc 1320 atggatgaac tccacaacga aatactcgag ctggatgaga aagtggatga tctcagagct 1380 gacacaataa gctcgcaaat agagcttgca gtcttgcttt ccaatgaagg aataataaac 1440 agtgaagatg agcatctatt ggcacttgag agaaaactaa agaaaatgct gggtccctct 1500 gctgtagaca tagggaatgg atgcttcgaa accaaacaca agtgcaacca gacctgctta 1560 gacaggatag ctgctggcac ctttaatgca ggagaatttt ctcttcccac ttttgattca 1620 ctgaatatta ctgctgcatc tttaaatgat gatggattgg ataatcatac tatactgctc 1680 tactactcaa ctgcggcttc tagtttggct gtaacattga tgatagctat ttttattgtt 1740 tatatggtct ccagagacaa tgtttcttgc tccatctgtc tatagggaaa attgagccct 1800 gtattttcct ttattgtggt gcttgtttgc ttgttgccat tacagagaaa cgttattgaa 1860 aaatgctctt gttactact 1879 <210> 22 <211> 1554 <212> DNA
<213> Influenza B virus <400> 22 agcagaagca gagcatcttc tcaaaactga agtaaagagg ccaaaaatga acaatgctac 60 cttcaactat acaaacgtta accctatttc tcacatcagg gggagtgtta ttatcactat 120 atgtgtcagc cttactgtca tacttattgt attcggatat attgctaaaa ttttcaccaa 180 aaataattgc accaacaacg tcgttggact gcgcgaacgc atcaaatgtt caggctgtga 240 accattctgc aacaaaagag atgaaattcc ttcccccaga accggagtgg acataccccc 300 gtttatcttg ccagggttca accttccaga aagcactctt aattagccct catagatttg 360 gagaagccaa aggaaactca gctcccttga taataaggga accttttatt gcttgtggac 420 caaaggagtg caaacacttt gctctaaccc attatgcagc tcaaccaggg ggatactaca 480 atggaacaag agaggacaga aacaagctga ggcatctgat ttcagtcaac ttaggcaaaa 540 tcccaactgt agaaaactcc attttccata tggcagcttg gagtggatcc gcatgccatg 600 atggtagaga atggacatat atcggagttg atggtcctga cagtaatgca ttgatcaaaa 660 taaaatatgg agaagcatac actgacacat accattccta tgcaaacaac atcctaagaa 720 cacaagaaag tgcctgcaat tgcatcgggg gagattgtta tcttatgata actgatggct 780 cagcttcagg aattagtaaa tgcagattcc ttaagatccg agagggtcga ataataaaag 840 aaatatttcc aacaggaagg gtagagcaca ctgaagaatg cacatgcgga tttgccagca 900 acaaaaccat agaatgtgcc tgtagagata acagttacac agcaaaaaga ccctttgtca 960 aattaaatgt ggagactgat acagctgaaa taagattgat gtgcacagag acttatttgg 1020 acacccccag accagatgat ggaagcataa cagggccttg cgaatctaat ggggacaaag 1080 ggagtggagg tgtcaaggga ggatttgttc atcaaagaat ggcatccaag attggaagat 1140 ggtactcccg aacgatgtct aaaactaaaa gaatggggat ggaactgtat gtcaagtatg 1200 atggagaccc atggactgac agtgacgccc ttgctcctag tggagtaatg gtctcaatgg 1260 aagaacctgg ttggtactct ttcggcttcg aaataaaaga taagaaatgt gatgtcccct 1320 gtattgggat agagatggta catgatggtg gaaaaaggac ttggcactca gcagcaacag 1380 ccatttactg tttaatgggc tcaggacagt tgctatggga cactgtcaca ggtgttaata 1440 tggctctgta atggaggaat ggttgaatct gttctaaacc ctttgttcct attttatttg 1500 aacaattgtc cttactggac ttaattgttt ctgaaaaatg ctcttgttac tact 1554 <210> 23 <211> 1881 <212> DNA
<213> Influenza B virus <400> 23 agcagaagca gagcattttc taatatccac aaaatgaagg caataattgt actactcatg 60 gtagtaacat ccaatgcaga tcgaatctgc actgggataa catcgtcaaa ctcacctcat 120 gtggtcaaaa cagctactca aggggaggtc aatgtgactg gtgtgatacc actgacaaca 180 acaccaacaa aatctcattt tgcaaatctc aaaggaacaa agaccagagg gaaactatgc 240 ccaacctgtc tcaactgcac agatctggat gtggccttag gcagaccaat gtgtgtgggg 300 gtcacacctt cggcaaaagc ttcaatactc cacgaagtca ggcctgttac atccggatgc 360 tttcctataa tgcacgacag aacaaaaatc agacagctac ccaatcttct cagaggatat 420 gaaaaaatca gattatcaac ccaaatcgtt atcaacgcag aaaaggcacc aggaggaccc 480 tacagacttg gaacctcagg atcttgccct aacgctacca gtagaagcgg atttttcgca 540 acaatggctt gggctgtccc aaaggacaac aacaaaacag caacgaatcc actaacagta 600 gaagtaccac acatctgtac aaaagaagaa gaccaaatta ctgtttgggg gttccattct 660 gatgacaaaa cccaaatgaa aaacctctat ggagactcaa atcctcaaaa gttcacctca 720 tctgctaatg gagtaaccac acattatgtt tctcagattg gcggcttccc ggatcaaaca 780 gaagacggag ggctaccaca aagcggcaga attgttgttg attacatggt gcaaaaacct 840 gggaaaacag gaacaattgt ctatcaaaga ggtattttgt tgcctcaaaa ggtgtggtgc 900 gcgagtggca ggagcaaagt aataaaaggg tccttgcctt taattggtga agcagattgc 960 cttcacgaaa aatacggtgg attaaacaaa agcaagcctt actacacagg agaacatgca 1020 aaagccatag gaaattgccc aatatgggtg aaaacacctt tgaagcttgc caatggaacc 1080 aaatatagac ctcctgcaaa actattaaag gaaaggggtt tcttcggagc tattgctggt 1140 ttcttagaag gaggatggga aggaatgatt gcaggttggc acggatacac atctcacgga 1200 gcacatggag tggcagtggc agcagacctt aagagtacgc aagaagccat aaacaagata 1260 acaaaaaatc tcaattcttt gagtgagcta gaagtaaaga atcttcaaag actaagtggt 1320 gccatggatg aactccacaa cgaaatactc gagctggatg agaaagtgga tgatctcaga 1380 gctgacacaa taagctcaca aatagaactt gcagtcttgc tttccaacga aggaataata 1440 aacagtgaag atgagcatct attggcactt gagagaaaac taaagaaaat gctgggtccc 1500 tctgctgtag acatagggaa tggatgcttc gaaaccaaac acaagtgcaa ccagacctgc 1560 ttagacagga tagctgctgg cacctttaat gcaggagaat tttctcttcc cacttttgat 1620 tcactgaata ttactgctgc atctttaaat gatgatggat tggataacca tactatactg 1680 ctctactact caactgctgc ttctagtttg gctgtaacat tgatgatagc tatttttatt 1740 gtttatatga tctccagaga caatgtttct tgctccatct gtctataggg aaattaagcc 1800 ctgtattttc ctttattgta gtgcttgttt gcttgttatc attacaaaga aacgttattg 1860 aaaaatgctc ttgttactac t 1881 <210> 24 <211> 1557 <212> DNA
<213> Influenza B virus <400> 24 agcagaagca gagcatcttc tcaaaactga ggcaaatagg ccaaaaatga acaatgctac 60 cttcaactat acaaacgtta accctatttc tcacatcagg gggagtgtta ttatcactat 120 atgtgtcagc ttcactgtca tacttactat attcggatat attgctaaaa ttttcaccaa 180 cagaaataac tgcaccaaca atgccattga attgtgcaaa cgcatcaaat gttcaggctg 240 tgaaccgttc tgcaacaaaa ggggtgacac ttcctctccc agaaccggag tggacatacc 300 ctcgtttatc ttgcccgggc tcaacctttc agaaagcact cctaattagc cctcatagat 360 tcggagaaac caaaggaaac tcagctccct tgataataag ggaacctttt attgcttgtg 420 gaccaaagga atgcagacac tttgctctaa cccattatgc agcccaacca gggggatact 480 acaatggaac aagagaagac agaaacaagc tgaggcatct aatttcagtc aaattgggca 540 aaatcccaac agtagaaaac tccattttcc acatggcagc ttggagcggg tccgcatgcc 600 atgatggtag agaatggaca tatatcggag ttgatggccc tgacagtaat gcattgctca 660 aaataaaata tggagaagca tatactgaca cataccattc ctatgcaaac aacatcctaa 720 gaacacaaga aagtgcctgc aattgcatcg ggggagattg ttatcttatg ataactgatg 780 gctcagcttc agggattagt gaatgcagat ttcttaagat tcgagagggc cgaataataa 840 aagaaatatt tccaacagga agagtagaac atactgaaga atgcacatgc ggatttgcca 900 gcaataaaac catagaatgt gcctgtagag ataacagtta cacagcaaaa agaccctttg 960 tcaaattaaa tgtggagact gatacagcag aaataagatt gatgtgcaca gagacttact 1020 tggacacccc cagaccagat gatggaagca taacagggcc ttgtgaatct aatggggata 1080 aagggagtgg aggcatcaag ggaggatttg ttcatcaaag aatggcatcc aagattggaa 1140 ggtggtactc tcgaacgatg tctaaaacta aaaggatggg gatgggactg tatgtcaagt 1200 atgatggaga cccatggatt gacagtgatg cccttactct tagcggagta atggtttcaa 1260 tggaagaacc tggttggtat tcctttggct tcgaaataaa agataagaaa tgtgatgtcc 1320 cctgtattgg gatagagatg gtacatgatg gtggaaagaa gacttggcac tcagcagcaa 1380 cagccattta ctgtttaatg ggctcaggac aactgctatg ggacactgtc acaggcgttg 1440 atatggctct gtaatggagg aatggttgag tctgttctaa accctttgtt cctattttgt 1500 ttgaacaatt gtccttactg aacttaattg tttctgaaaa atgctcttgt tactact 1557 <210> 25 <211> 1882 <212> DNA
<213> Influenza B virus <400> 25 agcagaagca gagcattttc taatatccac aaaatgaagg caataattgt actactcatg 60 gtagtaacat ccaatgcaga tcgaatctgc actgggataa catcgtcaaa ctcacctcat 120 gtggtcaaaa cagctactca aggggaggtc aatgtgactg gtgcgatacc attgacaaca 180 acaccaacaa aatctcattt tgcaaatctc aaaggaacaa agaccagagg gaaactatgc 240 ccaacctgtc tcaactgcac agatctggat gtggccttgg gcagaccaat gtgtgtgggg 300 atcacacctt cggcaaaagc ttcaatactc cacgaagtca gacctgttac atccggatgc 360 tttcctataa tgcacgacag aacaaaaatc agacagctac ccaatcttct cagaggatat 420 gaaaaaatca gattatcaac ccaaaacgtt atcaacgcag aaaaggcacc aggaggaccc 480 tacagacttg gaacttcagg atcttgccct aacgctacca gtaaaagcgg atttttcgca 540 acaatggctt gggctgtccc aagggacaac aacaaaacag caacgaatcc actaacagta 600 gaagtaccac acatctgtac aaaagaagaa gaccaaatta ctgtttgggg gttccattct 660 gatgacaaaa cccaaatgaa aaacctctat ggagactcaa atcctcaaaa gttcacctca 720 tctgctaatg gaataaccac acattatgtt tctcagattg gcggcttccc ggaccaaaca 780 gaagacggag ggctaccaca aagcggcaga attgttgttg attacatggt gcaaaaacct 840 gggaaaacag gaacaattgt ctatcaaaga gggatcttgt tgcctcaaaa ggtgtggtgc 900 gcgagtggca ggagcaaagt aataaaaggg tccttgcctt taattggtga agcagattgc 960 cttcacgaaa aatacggtgg attaaacaaa agcaagcctt actacacagg agaacatgca 1020 aaagccatag gaaattgccc aatatgggtg aaaacacctt tgaagcttgc caatggaacc 1080 aagtatagac ctcctgcaaa actattaaag gaaaggggtt tcttcggagc tattgctggt 1140 ttcttagaag gaggatggga aggaatgatt gcaggttggc acggatacac atctcacgga 1200 gcacacggag tggcagtggc agcagacctt aagagtacgc aagaagccat aaacaagata 1260 acaaaaaatc tcaattcttt gagtgagtta gaagtaaaga accttcaaag actaagtggt 1320 gccatggatg aactccataa cgaaatactc gagctggatg agaaagtgga tgatctcaga 1380 gctgacacaa taagctcaca aatagaactt gcagtcttgc tttccaacga aggaataata 1440 aacagtgaag atgagcatct attggcactt gagagaaaac taaagaagat gctgggtccc 1500 tctgctatag acatagggaa tggatgcttc gaaaccaaac acaagtgcaa ccagacctgc 1560 ttagacagga tagctgctgg cacctttaat gcaggagaat tttctcttcc cacttttgat 1620 tcactgaaca ttactgctgc atctttaaat gatgatggat tggataacca tactatactg 1680 ctctactact caactgctgc ttctagtttg gctgtaacat tgatgatagc tatttttatt 1740 gtttatatga tctccagaga caatgtttct tgctccatct gtctataagg aaaattaagc 1800 cctgtatttt cctttattgt agtgcttgtt tgcttgttat cattacaaag aaacgttatt 1860 gaaaaatgct cttgttacta ct 1882 <210> 26 <211> 1557 <212> DNA
<213> Influenza B virus <400> 26 agcagaagca gagcatcttc tcaaaactga ggcaaatagg ccaaaaatga acaatgctac 60 cctcaactat acaaacgtta accctattcc tcacatcagg gggagtgtta ttatcactat 120 atgtgtcagc ttcactgtca tacttactat attcggatat attgctaaaa ttttcaccaa 180 cagaaataac tgcaccagca atgcccttgg attgtgcaaa cgcatcaaat gttcaggctg 240 tgaaccgttc tgcaacaaaa ggggtgacac ttcttctccc agaaccggag tggacatacc 300 cgcgtttatc ttgcccgggc tcaacctttc agaaagcact cctaattagc cctcatagat 360 tcggagaaac caaaggaaac tcagctccct tgataataag ggaacctttt attgcttgtg 420 gaccaaagga atgcaaacac tttgctctaa cccattatgc agcccaacca gggggatact 480 acaatggaac aagagaagac agaaacaagc taaggcatct aatttcagtc aaatttggta 540 aaatcccaac agtagaaaac tccattttcc acatggcagc atggagcggg tccgcatgcc 600 atgatggtaa agaatggaca tatatcggag ttgatggccc tgacagtaat gcattgctca 660 aaataaaata tggagaagca tatactgaca cataccattc ctatgcaaac aacatcctaa 720 gaacacaaga aagtgcctgc aattgcatcg ggggaaattg ttatcttatg ataactgatg 780 gctcagcttc aggtattagt gagtgcagat ttcttaagat tcgagagggc cgaataataa 840 aagaaatatt tccaacagga agagtaaaac atactgaaga atgcacatgc ggatttgcca 900 gcaataaaac catagaatgt gcctgtagag ataacagtta cacagcaaaa agaccctttg 960 tcaaattaaa tgtggagact gatacagcag aaataagatt gatgtgcaca gagacttatt 1020 tggacacccc cagaccagat gatggaagca taacagggcc ttgtgaatct aatggggata 1080 aagggagtgg aggcatcaag ggaggatttg ttcatcaaag aatggcatcc aagattggaa 1140 ggtggtactc tcgaacaatg tctaaaacta aaaggatggg gatgggactg tatgtcaagt 1200 atgatggaga cccatggact gacagtgatg cccttgctct tagtggagta atggtttcaa 1260 tggaagaacc tggttggtac tcctttggct tcgaaataaa agataagaaa tgtgatgtcc 1320 cctgtattgg gatagagatg gtacatgatg gtggaaagga gacttggcac tcagcagcaa 1380 cagccattta ctgtttaatg ggctcaggac aactgctatg ggacactgtc acaggtgttg 1440 atatggctct gtaatggagg aatggttgag tctgttctaa accctttgtt cctattttgt 1500 ttgaacaatt gtccttactg aacttaattg tttctgaaaa atgctcttgt tactact 1557 <210> 27 <211> 1879 <212> DNA
<213> Influenza B virus <400> 27 agcagaagca gagcattttc taatatccac aaaatgaagg caataattgt actactcatg 60 gtagtaacat ccaacgcaga tcgaatctgc actgggataa catcttcaaa ctcacctcat 120 gtggtcaaaa cagctactca aggggaagtc aatgtgactg gtgtgatacc actgacaaca 180 acaccaacaa aatctcattt tgcaaatctc aaaggaacaa agaccagagg gaaactatgc 240 ccaaactgtc tcaactgcac agatctggat gtggccttgg gcagaccaat gtgtataggg 300 atcacacctt cggcaaaagc ttcaatactc cacgaagtca gacctgttac atccgggtgc 360 tttcctataa tgcacgacag aacaaaaatc agacagctac ccaatcttct cagaggatat 420 gaacatatca gattatcaac ccataacgtt atcaacgcag aaagggcacc aggaggaccc 480 tacagacttg gaacctcagg atcttgccct aacgttacca gtagaagcgg attcttcgca 540 acaatggctt gggctgtccc aagggacaac aaaacagcaa cgaacccact aacagtagaa 600 gtaccataca tttgtacaaa aggagaagac caaattactg tttgggggtt ccattctgat 660 aacaaaatcc aaatgaaaaa cctctatgga gactcaaatc ctcaaaagtt cacctcatct 720 gccaatggaa taaccacaca ttatgtttct cagattggtg gcttcccaaa tcaaacagaa 780 gacggagggc taccacaaag cggcagaatt gttgttgatt acatggtgca aaaacctggg 840 aaaacaggaa caattgtcta tcaaagaggt gttttgttgc ctcaaaaggt gtggtgtgca 900 agtggcagga gcaaggtaat aaaagggtcc ttgcctttaa ttggtgaagc agattgcctt 960 cacgaaaaat acggtggatt aaacaaaagc aagccttact acacaggaga acatgcaaaa 1020 gccataggaa attgcccaat atgggtgaaa acacctttaa agcttgccaa tggaaccaaa 1080 tatagacctc ccgcaaaact attaaaggaa aagggtttct tcggagctat tgctggtttc 1140 ttagaaggag gatgggaagg aatgattgca ggttggcacg gatacacatc tcatggagca 1200 catggggtgg cagtggcagc agaccttaag agtacgcaag aagccataaa caagataaca 1260 aaaaatctca attctttgag tgagctagaa gtaaagaatc ttcaaagact aagtggtgcc 1320 atggatgaac tccacaacga aatactcgag ctggatgaga aagtggatga tctcagagct 1380 gacacaataa gctcgcaaat agagcttgca gtcttgcttt ccaatgaagg aataataaac 1440 agtgaagatg agcatctatt ggcacttgag agaaaactaa agaaaatgct gggtccctct 1500 gctgtagaca tagggaatgg atgcttcgaa accaaacaca agtgcaacca gacctgctta 1560 gacaggatag ctgctggcac ctttaatgca ggagaatttt ctcttcccac ttttgattca 1620 ctgaatatta ctgctgcatc tttaaatgat gatggattgg ataatcatac tatactgctc 1680 tactactcaa ctgcggcttc tagtttggct gtaacattga tgatagctat ttttattgtt 1740 tatatggtct ccagagacaa tgtttcttgc tccatctgtc tatagggaaa attgagccct 1800 gtattttcct ttattgtggt gcttgtttgc ttgttgccat tacagagaaa cgttattgaa 1860 aaatgctctt gttactact 1879 <210> 28 <211> 1554 <212> DNA
<213> Influenza B virus <400> 28 agcagaagca gagcatcttc tcaaaactga agtaaagagg ccaaaaatga acaatgctac 60 cttcaactat acaaacgtta accctatttc tcacatcagg gggagtgtta ttatcactat 120 atgtgtcagc cttactgtca tacttattgt attcggatat attgctaaaa ttttcaccaa 180 aaataattgc accaacaacg tcgttggact gcgcgaacgc atcaaatgtt caggctgtga 240 accattctgc aacaaaagag atgaaattcc ttcccccaga accggagtgg acataccccc 300 gtttatcttg ccagggttca accttccaga aagcactctt aattagccct catagatttg 360 gagaagccaa aggaaactca gctcccttga taataaggga accttttatt gcttgtggac 420 caaaggagtg caaacacttt gctctaaccc attatgcagc tcaaccaggg ggatactaca 480 atggaacaag agaggacaga aacaagctga ggcatctgat ttcagtcaac ttaggcaaaa 540 tcccaactgt agaaaactcc attttccata tggcagcttg gagtggatcc gcatgccatg 600 atggtagaga atggacatat atcggagttg atggtcctga cagtaatgca ttgatcaaaa 660 taaaatatgg agaagcatac actgacacat accattccta tgcaaacaac atcctaagaa 720 cacaagaaag tgcctgcaat tgcatcgggg gagattgtta tcttatgata actgatggct 780 cagcttcagg aattagtaaa tgcagattcc ttaagatccg agagggtcga ataataaaag 840 aaatatttcc aacaggaagg gtagagcaca ctgaagaatg cacatgcgga tttgccagca 900 acaaaaccat agaatgtgcc tgtagagata acagttacac agcaaaaaga ccctttgtca 960 aattaaatgt ggagactgat acagctgaaa taagattgat gtgcacagag acttatttgg 1020 acacccccag accagatgat ggaagcataa cagggccttg cgaatctaat ggggacaaag 1080 ggagtggagg tgtcaaggga ggatttgttc atcaaagaat ggcatccaag attggaagat 1140 ggtactcccg aacgatgtct aaaactaaaa gaatggggat ggaactgtat gtcaagtatg 1200 atggagaccc atggactgac agtgacgccc ttgctcctag tggagtaatg gtctcaatgg 1260 aagaacctgg ttggtactct ttcggcttcg aaataaaaga taagaaatgt gatgtcccct 1320 gtattgggat agagatggta catgatggtg gaaaaaggac ttggcactca gcagcaacag 1380 ccatttactg tttaatgggc tcaggacagt tgctatggga cactgtcaca ggtgttaata 1440 tggctctgta atggaggaat ggttgaatct gttctaaacc ctttgttcct attttatttg 1500 aacaattgtc cttactggac ttaattgttt ctgaaaaatg ctcttgttac tact 1554 <210> 29 <211> 1885 <212> DNA
<213> Influenza B virus <400> 29 agcagaagca gagcattttc taatatccac aaaatgaagg caataattgt actactcatg 60 gtagtaacat ccaatgcaga tcgaatctgc actggaataa catcgtcaaa ctcaccccat 120 gtggtcaaaa ctgctactca aggggaagtc aatgtgactg gtgtgatacc actgacaaca 180 acacccacca aatctcattt tgcaaatctc aaaggaacaa aaaccagagg gaaactatgc 240 ccaaaatgtc tcaactgcac agatctggac gtggccttgg gcagaccaaa atgcacgggg 300 aacatacctt cggcaaaagt ttcaatactc catgaagtaa gacctgttac atctgggtgc 360 tttcctataa tgcacgacag aacaaaaatt agacagctgc ccaatcttct cagaggatac 420 gaacgtatca ggttatcaaa ccataacgtt atcaatgcag aaaaagcacc aggaggaccc 480 tacaaaattg gaacctcagg gtcttgccct aacgttacca atggaaacgg attcttcgca 540 acaatggctt gggctgtccc aaaaaacgaa aacaacaaaa cagcaacaaa ttcattaaca 600 atagaagtac catacatttg tacagaagga gaagaccaaa ttaccgtttg ggggttccac 660 tctgatagcg aaacccaaat ggcaaaactc tatggagact caaagcctca gaagttcact 720 tcatctgcta acggagtgac cacacattac gtttcacaga ttggtggctt cccaaatcaa 780 acagaagacg gaggactacc acaaagtggt agaattgttg ttgattacat ggtgcaaaaa 840 tctggaaaaa caggaacaat tacctatcaa agaggtattt tattgcctca aaaagtgtgg 900 tgcgcaagtg gcaggagcaa ggtaataaaa ggatccttgc ctttaattgg agaagcagat 960 tgcctccacg aaaaatacgg tggattaaac aaaagcaagc cttactatac aggggaacat 1020 gcaaaagcca taggaaattg cccaatatgg gtgaaaacac ccttgaagct ggccaatgga 1080 accaaatata gacctcctgc aaaactatta aaggaaaggg gtttcttcgg agctattgct 1140 ggtttcttag aaggaggatg ggaaggaatg attgcaggtt ggcacggata cacatcccat 1200 ggagcacatg gagtagcagt ggcagcagac cttaagagta ctcaagaagc cataaacaag 1260 atcacaaaaa atctcaactc tttgagtgag ctggaagtaa agaatcttca aagactaagc 1320 ggagccatgg atgaactcca caacgaaata ctagaactag atgagaaagt ggatgatctc 1380 agagctgata caataagctc gcaaatagaa ctcgcagtct tgctttccaa tgaaggaata 1440 ataaacagtg aagatgagca tctcttggcg cttgaaagaa aactgaagaa aatgctgggc 1500 ccctctgctg tagagatagg gaatggatgc ttcgaaacca aacacaagtg caaccagacc 1560 tgcctcgata gaatagctgc tggcaccttt aatgcaggag aattttctct ccccaccttt 1620 gattcactaa atattactgc tgcatcttta aatgacgatg gattggataa tcatactata 1680 ctgctttact actcaactgc tgcttccagt ttggctgtaa cattgatgat agctatcttt 1740 gttgtttata tggtctccag agacaatgtt tcttgttcca tctgtctata aggaaagtta 1800 agccccgtat tttcctttat tgtagtactt gtttgcttgt tatcattaca aaaaaacgtt 1860 attgaaaaat gctcttgtta ctact 1885 <210> 30 <211> 1544 <212> DNA
<213> Influenza B virus <400> 30 agcagagcat cttctcaaaa ctgaagcaaa taggccaaaa tgaacaatgc taccctcaac 60 tatacaaaca ttaaccctat ttctcacatc agggggagtg ttattatcac tatatgtgtc 120 agccttactg tcatacttac tgtattcgga tatattgcta aaattttcac caacaaaaat 180 aattgcacca acaacgtcgt tggactccgc gaacgcatca aattttcagg ccgtgaacca 240 ttctgcaaca aaagagatga catttcttct cccagaaccg gagtggacat accctcgttt 300 atcttgccag ggttcaacct ttcaaaaagc actcctaatt agccctcata gattcggaga 360 agccaaagga aactcagctc ccttgataat aagggaacct tttattgctt gtggaccaaa 420 ggagtgtaaa cactttgctc taacccatta tgcagctcaa ccagggggat actacaatgg 480 aacaagagag gacagaaaca agctgaggca tctgatttca gtcaacttag gcaaaatacc 540 aactgtagaa aactccattt tccacatggc agcttggagt gggtccgcat gccatgatgg 600 tagagagtgg acttatatcg gagttgatgg ccctgacagt aatgcattga tcaaaataaa 660 atatggagaa gcatacactg acacatacca ttcctatgca aacaacatcc taagaacaca 720 agaaagtgcc tgcaactgca tcgggggaga ttgttatctt atgataactg atggctcagc 780 ttcaggaatt agtaaatgca gattccttaa gattcgagag ggtcgaatag taaaagaaat 840 atttccaaca ggaagagtag agcatactga agaatgcaca tgcggatttg ccagcaataa 900 aaccatagaa tgtgcctgta gagataacag ttacacagca aaaagaccct ttgtcaaatt 960 aaatgtggaa actgatacag cagaaataag attgatgtgc acagagactt atttggacac 1020 ccccagacca gatgatggaa gcataacagg gccttgcgaa tctaatgggg acaaagggag 1080 tggaggtatc aagggaggat ttgtccatca aagaatggca tccaagattg gaagatggta 1140 ctctcgaacg atgtctaaaa ctaaaagaat ggggatggaa ctgtatgtca agtatgatgg 1200 agacccatgg actgacagtg atgcccttgc tcctagtgga gtaatggtct caatagaaga 1260 acctggttgg tattctttcg gcttcgaaat aaaagataag aaatgcgatg tcccctgtat 1320 tgggatagag atggtacacg atggtggaaa aacaacttgg cactcagcag caacagccat 1380 ttactgttta atgggctcag gacagttgct atgggacact atcacaggtg ttgatatggc 1440 tctgtaatgg aggaatggtt gaatctgttc taaacccttt gttcctattt tgtttgaaca 1500 attgtcctta ctggacttaa ttgtttctga aaaatgctct tgtt 1544 <210> 31 <211> 1885 <212> DNA
<213> Influenza B virus <400> 31 agcagaagca gagcattttc taatatccac aaaatgaagg caataattgt actactcatg 60 gtagtaacat ccaatgcaga tcgaatctgc actgggataa catcgtcaaa ctcaccccat 120 gtggtcaaaa ctgctactca aggggaggtc aatgtgactg gtgtgatacc actgacaaca 180 acacccacca aatctcattt tgcaaatctc aaaggaacaa aaaccagagg gaaactatgc 240 ccaaaatgcc tcaactgcac agatctggac gtggccttgg gcagaccaaa atgcacgggg 300 aacataccct cggcaaaagt ttcaatactc catgaagtca gacctgttac atctgggtgc 360 tttcctataa tgcacgacag aacaaaaatt agacagctgc ccaatcttct cagaggatac 420 gaacatatca ggttatcaac tcataacgtt atcaatgcag aaaaggcacc aggaggaccc 480 tacaaaattg gaacctcagg gtcttgccct aacgttacca atggaaacgg atttttcgca 540 acaatggctt gggccgtccc aaaaaacgac aacaacaaaa cagcaacaaa ttcattaaca 600 atagaagtac catacatttg tacagaagga gaagaccaaa ttaccgtttg ggggttccac 660 tctgataacg aagcccaaat ggcaaaactc tatggggact caaagcccca gaagttcacc 720 tcatctgcca acggagtgac cacacattac gtttcacaga ttggtggctt cccaaatcaa 780 acagaagacg gaggactacc acaaagtggt agaattgttg ttgattacat ggtgcaaaaa 840 tctgggaaaa caggaacaat tacctatcaa agaggtattt tattgcctca aaaagtgtgg 900 tgcgcaagtg gcaggagcaa ggtaataaaa ggatccttgc ctttaattgg agaagcagat 960 tgcctccacg aaaaatacgg tggattaaac aaaagcaagc cttactacac aggggaacat 1020 gcaaaggcca taggaaattg cccaatatgg gtgaaaacac ccttgaagct ggccaatgga 1080 accaaatata gacctcctgc aaaactatta aaggaaagag gtttcttcgg agctattgct 1140 ggtttcttag aaggaggatg ggaaggaatg attgcaggtt ggcacggata cacatcccat 1200 ggggcacatg gagtagcagt ggcagcagac cttaagagta ctcaagaagc cataaacaag 1260 ataacaaaaa atctcaactc tttgagtgag ctggaagtaa agaatcttca aagactaagc 1320 ggtgccatgg atgaactcca caacgaaata ctagaactag acgagaaagt ggatgatctc 1380 agagctgata caataagctc acaaatagaa ctcgcagtct tgctttccaa tgaaggaata 1440 ataaacagtg aagatgagca tctcttggcg cttgaaagaa agctgaagaa aatgctgggc 1500 ccctctgctg tagagatagg gaatggatgc ttcgaaacca aacacaagtg caaccagacc 1560 tgtctcgaca gaatagctgc tggtaccttt gatgcaggag aattttctct ccccactttt 1620 gattcactga atattactgc tgcatcttta aatgacgatg gattggataa tcatactata 1680 ctgctttact actcaactgc tgcctccagt ttggctgtaa cattgatgat agctatcttt 1740 gttgtttata tggtctccag agacaatgtt tcttgctcca tctgtctata aggaaagtta 1800 agccctgtat tttcctttat tgtagtgctt gtttgcttgt taccattaca aaaaaacgtt 1860 attgaaaaat gctcttgtta ctact 1885 <210> 32 <211> 1557 <212> DNA
<213> Influenza B virus <400> 32 agcagaagca gagcatcttc tcaaaactga ggcaaatagg ccaaaaatga acaatgctac 60 cttcaactat acaaacgtta accctatttc tcacatcagg gggagtatta ttatcactat 120 atgtgtcagc ttcattgtca tacttactat attcggatat attgctaaaa ttctcaccaa 180 cagaaataac tgcaccaaca atgccattgg attgtgcaaa cgcatcaaat gttcaggctg 240 tgaaccgttc tgcaacaaaa ggggtgacac ttcttctccc agaaccagag tggacatacc 300 cgcgtttatc ttgcccgggc tcaacctttc agaaagcact cctaattagc cctcatagat 360 tcggagaaac caaaggaaac tcagctccct tgataataag ggaacctttt attgcttgtg 420 gaccaaagga atgcaaacac tttgctctaa cccattatgc agcccaacca gggggatact 480 acaatggaac aagaggagac agaaacaagc tgaggcatct aatttcagtc aaattgggca 540 aaatcccaac agtagaaaac tccattttcc acatggcagc atggagcggg tccgcatgcc 600 atgatggtaa agaatggaca tatatcggag ttgatggccc tgacaataat gcattgctca 660 aaataaaata tggagaagca tatactgaca cataccattc ctatgcaaac aacatcctaa 720 gaacacaaga aagtgcctgc aattgcatcg ggggaaattg ttatcttatg ataactgatg 780 gctcagcttc aggtattagt gaatgcagat ttcttaaaat tcgagagggc cgaataataa 840 aagaaatatt tccaacagga agagtaaaac atactgaaga atgcacatgc ggatttgcca 900 gcaataagac catagaatgt gcctgtagag ataacagtta cacagcaaaa agaccctttg 960 tcaaattaaa cgtggagact gatacagcag aaataagatt gatgtgcaca gagacttatt 1020 tggacacccc cagaccagat gatggaagca taacagggcc ttgtgaatct aatggggaca 1080 aagggagtgg aggcatcaag ggaggatttg ttcatcaaag aatggcatcc aagattggaa 1140 ggtggtactc tcgaacgatg tctaaaacta aaaggatggg gatgggactg tatgtcaagt 1200 atgatggaga cccatgggct gacagtgatg cccttgctct tagtggagta atggtttcaa 1260 tggaagaacc tggttggtac tcctttggct tcgaaataaa agataagaaa tgtgatgtcc 1320 cctgtattgg aatagagatg gtacatgatg gtggaaaaga gacttggcac tcagcagcaa 1380 cagccattta ctgtttaatg ggctcaggac agctgctgtg ggacactgtc acaggtgttg 1440 atatggctct gtaatggagg aatggttgag tctgttctaa accctttgtt cctattttgt 1500 ttgaacaatt gtccttactg aacttaattg tttctgaaaa atgctcttgt tactact 1557 <210> 33 <211> 1853 <212> DNA
<213> Influenza B virus <400> 33 tctaatatcc acaaaatgaa ggcaataatt gtactactca tggtagtaac atccaatgca 60 gatcgaatct gcactgggat aacatcttca aactcacctc atgtggtcaa aacagctact 120 caaggggagg tcaatgtgac tggtgtaata ccactgacaa caacaccaac aaaatcttat 180 tttgcaaatc tcaaaggaac aaggaccaga gggaaactat gtccagactg tctcaactgt 240 acagatctgg atgtggcctt gggcagacca atgtgtgtgg ggaccacacc ttcggcaaaa 300 gcttcaatac tccacgaagt cagacctgtt acatccgggt gctttcctat aatgcacgac 360 agaacaaaaa tcagacaact acccaatctt ctcagaggat atgaaaatat cagattatca 420 acccaaaacg ttatcgatgc agaaaatgca ccaggaggac cctacagact tggaacctca 480 ggatcttgcc ctaacgctac cagtaaaagc ggatttttcg caacaatggc ttgggctgtc 540 ccaaaggaca acaacaaaaa tgcaacgaac ccactaacag tagaagtacc atacgtttgt 600 acagaagggg aagaccaaat tactgtttgg gggttccatt cagataacaa aaccccaatg 660 aagaacctct atggagactc aaatcctcaa aagttcacct catctgctaa tggagtaacc 720 acacattatg tttctcagat tggcggcttc ccagctcaaa cagaagacga aggactacca 780 caaagcggca gaattgttgt tgattacatg gtgcaaaaac ctaggaaaac aggaacaatt 840 gtctatcaaa gaggtgtttt gttgcctcaa aaggtgtggt gcgcgagtgg caggagcaaa 900 gtaataaaag ggtccttgcc tttaattggt gaagcagatt gccttcatga aaaatacggt 960 ggattaaaca aaagcaagcc ttactacaca ggagaacatg caaaagccat aggaaattgc 1020 ccaatatggg tgaaaacacc tttgaagctt gccaatggaa ccaaatatag acctcctgca 1080 aaactattaa aggaaagggg tttcttcgga gctattgctg gtttcctaga aggaggatgg 1140 gaaggaatga ttgcaggttg gcacggatac acatctcacg gagcacatgg agtggcagtg 1200 gcggcagacc ttaagagtac gcaagaagct ataaacaaga taacaaaaaa tctcaattct 1260 ttgagtgagc tagaagtaaa gaatcttcaa agactaagtg gtgccatgga tgaactccac 1320 aacgaaatac tcgagctgga tgagaaagtg gatgatctca gagctgacac tataagctcg 1380 caaatagaac ttgcagtctt gctttccaat gaaggaataa taaacagtga agatgagcat 1440 ctattggcac ttgagagaaa actaaagaaa atgctgggtc cctctgctgt agacatagga 1500 aatggatgct tcgaaaccaa acacaagtgc aaccagacct gcttagacag gatagctgct 1560 ggcaccttta atgcaggaga attttctctc cccacttttg attcactgaa cattactgct 1620 gcatctttaa atgatgatgg attggataac catactatac tgctctatta ctcaactgct 1680 gcttctagtt tggctgtaac attgatgcta gctattttta ttgtttatat ggtctccaga 1740 gacaacgttt catgctccat ctgtctataa ggaagattaa gccttgtatt ttcctttatt 1800 gtagtgcttg tttgcttgtc atcattacaa agaaacgtta ttgaaaaatg ctc 1853 <210> 34 <211> 1529 <212> DNA
<213> Influenza B virus <400> 34 tctcaaaact gaggcaaata ggccaaaaat gaacaatgct accctcaact atacaaacgt 60 taaccctatt cctcacatca gggggagtgt tattatcact atatgtgtca gcttcactgt 120 catacttact atattcggat atattgctaa aattttcaac aacagaaata actgcaccaa 180 caatgccatt ggattgtgca aacgcatcaa atgttcaggc tgtgaaccgt tctgcaacaa 240 aaggggtgac acttcttctc ccagaaccgg agtggacata cccgcgttta tcttgcccgg 300 gctcaacctt tcagaaagca ctcctaatta gccctcatag attcggagaa accaaaggaa 360 actcagctcc cttgataata agggaacctt ttattgcttg tggaccaaag gaatgcaaac 420 actttgctct aacccattat gcagcccaac cagggggata ctacaatgga acaaaagaag 480 acagaaacaa gctgaggcat ctaatttcag tcaaattggg caaaatccca acagtagaaa 540 actccatttt ccacatggca gcatggagcg ggtccgcatg ccatgatggt aaagaatgga 600 catatatcgg agttgatggc cctgacagta atgcattgct caaaataaaa tatggagaag 660 catatactga cacataccat tcctatgcaa acaacatcct aagaacacaa gaaagtgcct 720 gcaattgcat cgggggaaat tgttatctta tgataactga tggctcagct tcaggtatta 780 gtgagtgcag atttcttaag attcgagagg gccgaataat aaaagaaata tttccaacag 840 gaagagtaaa acatactgaa gaatgcacat gcggatttgc cagcaataaa accatagaat 900 gtgcctgtag agataacagt tacacagcaa aaagaccctt tgtcaaatta aatgtggaga 960 ctgatacagc agaaataaga ttgatgtgca cagagactta tttggacacc cccagaccag 1020 atgatggaag cataacaggg ccttgtgaat ctaatgggaa taaagggagt ggaggcatca 1080 agggaggatt tgttcatcaa agaatggcat ccaaaattgg aaggtggtac tctcgaacaa 1140 tgtctaaaac caaaaggatg ggaatgggac tgtatgtcaa gtatgatgga gacccatgga 1200 ctgacagtga tgcccttgct cttagtggag taatggtttc aatggaagaa cctggttggt 1260 actcatttgg cttcgaaata aaagataaga aatgtgatgt cccctgtatt gggatagaga 1320 tggtacatga tggtggaaag gagacttggc actcagcagc aacagccatt tactgtttaa 1380 tgggctcagg acaactgttg tgggacactg tcacaggtgt tgatatggct ctgtaatggg 1440 ggaatggttg agtctgttct aaaccctttg ttcctatttt gtttgaacaa ttgtccttgc 1500 tgaacttaat tgtttctgaa aaatgctct 1529 <210> 35 <211> 566 <212> PRT
<213> Influenza A virus <400> 35 Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Ala Gin Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile Thr Asn Asp Gin Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gin Ser Ser Ser Thr Gly Arg Ile Cys Gly Ser Pro His Arg Ile Leu Asp Gly Lys Asn Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Gly Phe Gin Asn Lys Glu Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Tyr Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe Ile Asn Glu Asp Phe Asn Trp Thr Gly Val Ala Gin Asp Gly Gly Ser Tyr Ala Cys Lys Arg Gly Ser Val Asn Ser Phe Phe Ser Arg Leu Asn Trp Leu His Lys Leu Glu Tyr Lys Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Gly Lys Phe Asp Lys Leu Tyr Ile Trp Gly Val His His Pro Ser Thr Asp Ser Asp Gin Thr Ser Leu Tyr Val Arg Ala Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser Gin Gin Thr Val Thr Pro Asn Ile Gly Ser Arg Pro Trp Val Arg Gly Gin Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile Ala Pro Arg Gly Tyr Phe Lys Ile Arg Asn Gly Lys Ser Ser Ile Met Arg Ser Asp Ala Pro Ile Gly Asn Cys Ser Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gin Asn Val Asn Arg Ile Thr Tyr Gly Ala Cys Pro Arg Tyr Val Lys Gin Asn Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gin Thr Arg Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe Arg His Gin Asn Ser Glu Gly Thr Gly Gin Ala Ala Asp Leu Lys Ser Thr Gin Ala Ala Ile Asp Gin Ile Asn Gly Lys Leu Asn Arg Leu Ile Glu Lys Thr Asn Glu Lys Phe His Gin Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gin Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gin His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Lys Gin Leu Arg Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gin Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gin Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile <210> 36 <211> 436 <212> PRT
<213> Influenza A virus <400> 36 Met Gin Ile Ala Ile Leu Val Thr Thr Val Thr Leu His Phe Lys Gin Tyr Glu Cys Asn Ser Pro Pro Asn Asn Gin Val Met Leu Cys Glu Pro Thr Ile Ile Glu Arg Asn Ile Thr Glu Ile Val Tyr Leu Thr Asn Thr Thr Ile Glu Lys Glu Ile Cys Pro Lys Leu Ala Glu Tyr Arg Asn Trp Ser Lys Pro Gin Cys Lys Ile Thr Gly Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Gly Lys Cys Tyr Gin Phe Ala Leu Gly Gin Gly Thr Thr Leu Asn Asn Arg His Ser Asn Asp Thr Val His Asp Arg Thr Pro Tyr Arg Thr Leu Leu Met Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Lys Gin Val Cys Ile Ala Trp Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val Cys Val Thr Gly His Asp Glu Asn Ala Thr Ala Ser Phe Ile Tyr Asp Gly Arg Leu Val Asp Ser Ile Gly Ser Trp Ser Lys Asn Ile Leu Arg Thr Gin Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val Met Thr Asp Gly Ser Ala Ser Glu Arg Ala Asp Thr Lys Ile Leu Phe Ile Glu Glu Gly Lys Ile Val His Ile Ser Pro Leu Ser Gly Ser Ala Gin His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg Cys Val Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Ile Val Asp Ile Asn Val Lys Asp Tyr Ser Ile Val Ser Ser Tyr Val Cys Ser Gly Leu Val Gly Asp Thr Pro Arg Lys Asn Asp Ser Ser Ser Ser Ser Tyr Cys Arg Asn Pro Asn Asn Glu Lys Gly Ser His Gly Val Lys Gly Trp Ala Phe Asp Asp Gly Asn Asp Val Trp Met Gly Arg Thr Ile Ser Glu Glu Leu Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile Gly Gly Trp Ser Lys Pro Asn Ser Lys Leu Gin Ile Asn Arg Gin Val Ile Val Asp Arg Gly Asn Arg Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Lys Gin Glu Thr Glu Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro <210> 37 <211> 566 <212> PRT
<213> Influenza A virus <400> 37 Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Ala Gin Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile Thr Asn Asp Gin Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gin Ser Ser Pro Thr Gly Arg Ile Cys Asp Ser Pro His Arg Ile Leu Asp Gly Lys Asn Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Gly Phe Gin Asn Lys Glu Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Tyr Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe Ile Asn Glu Asn Phe Asn Trp Thr Gly Val Ala Gin Asp Gly Lys Ser Tyr Ala Cys Lys Arg Gly Ser Val Asn Ser Phe Phe Ser Arg Leu Asn Trp Leu His Lys Leu Glu Tyr Lys Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Gly Lys Phe Asp Lys Leu Tyr Ile Trp Gly Val His His Pro Ser Thr Asp Ser Val Gin Thr Ser Leu Tyr Val Arg Ala Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser Gin Gin Thr Val Ile Pro Asp Ile Gly Tyr Arg Pro Trp Val Arg Gly Gin Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile Ala Pro Arg Gly Tyr Phe Lys Ile Arg Asn Gly Lys Ser Ser Ile Met Arg Ser Asp Ala Pro Ile Gly Asn Cys Ser Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gin Asn Val Asn Arg Ile Thr Tyr Gly Ala Cys Pro Arg Tyr Val Lys Gin Asn Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gin Thr Arg Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe Arg His Gin Asn Ser Glu Gly Thr Gly Gin Ala Ala Asp Leu Lys Ser Thr Gin Ala Ala Ile Asp Gin Ile Asn Gly Lys Leu Asn Arg Leu Val Glu Lys Thr Asn Glu Lys Phe His Gin Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gin Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gin His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Arg Thr Arg Lys Gin Leu Arg Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gin Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gin Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile <210> 38 <211> 451 <212> PRT
<213> Influenza A virus <400> 38 Lys Met Asn Pro Asn Gin Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Thr Ile Ala Thr Ile Cys Phe Leu Met Gin Ile Ala Ile Leu Val Thr Thr Val Thr Leu His Phe Lys Gin Tyr Glu Cys Asn Ser Pro Pro Asn Asn Gin Val Met Leu Cys Glu Pro Thr Ile Ile Glu Arg Asn Ile Thr Glu Ile Val Tyr Leu Thr Asn Thr Thr Ile Glu Lys Glu Ile Cys Pro Lys Leu Ala Glu Tyr Arg Asn Trp Ser Lys Pro Gin Cys Lys Ile Thr Gly Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Gly Lys Cys Tyr Gin Phe Ala Leu Gly Gin Gly Thr Thr Leu Asn Asn Arg His Ser Asn Asp Thr Val His Asp Arg Thr Pro Tyr Arg Thr Leu Leu Met Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Lys Gin Val Cys Ile Ala Trp Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val Cys Val Thr Gly His Asp Glu Asn Ala Thr Ala Ser Phe Ile Tyr Asp Gly Arg Leu Val Asp Ser Ile Gly Ser Trp Ser Lys Asn Ile Leu Arg Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val Met Thr Asp Gly Ser Ala Ser Glu Arg Ala Asp Thr Lys Ile Leu Phe Ile Glu Glu Gly Lys Ile Val His Ile Ser Pro Leu Ser Gly Ser Ala Gln His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg Cys Val Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Ile Val Asp Ile Asn Val Lys Asp Tyr Ser Ile Val Ser Ser Tyr Val Cys Ser Gly Leu Val Gly Asp Thr Pro Arg Lys Asn Asp Ser Ser Ser Ser Ser Tyr Cys Trp Asn Pro Asn Asn Glu Lys Gly Gly His Gly Val Lys Gly Trp Ala Phe Asp Asp Gly Asn Asp Val Trp Met Gly Arg Thr Ile Ser Glu Glu Leu Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile Gly Gly Trp Ser Lys Pro Asn Ser Lys Leu Gln Ile Asn Arg Gln Val Ile Val Asp Arg Gly Asn Arg Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Lys Gln Glu Thr Glu Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly Thr Ser <210> 39 <211> 566 <212> PRT
<213> Influenza A virus <400> 39 Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Ala Gln Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile Thr Asn Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr Gly Arg Ile Cys Asp Ser Pro His Arg Ile Leu Asp Gly Lys Asn Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Gly Phe Gln Asn Lys Glu Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Tyr Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe Thr Asn Glu Gly Phe Asn Trp Thr Gly Val Ala Gln Asp Gly Thr Ser Tyr Ala Cys Lys Arg Gly Ser Val Lys Ser Phe Phe Ser Arg Leu Asn Trp Leu His Lys Leu Glu Tyr Lys Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Asp Lys Phe Asp Lys Leu Tyr Ile Trp Gly Val His His Pro Ser Thr Asp Ser Asp Gln Thr Ser Ile Tyr Val Gln Ala Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser Gln Gln Thr Val Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg Gly Ile Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile Ala Pro Arg Gly Tyr Phe Lys Ile Arg Ser Gly Lys Ser Ser Ile Met Arg Ser Asp Ala Pro Ile Gly Asn Cys Asn Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg Ile Thr Tyr Gly Ala Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe Arg His Gin Asn Ser Glu Gly Thr Gly Gin Ala Ala Asp Leu Lys Ser Thr Gin Ala Ala Ile Asn Gin Ile Asn Gly Lys Leu Asn Arg Leu Ile Glu Lys Thr Asn Glu Lys Phe His Gin Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gin Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gin His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Arg Thr Arg Lys Gin Leu Arg Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gin Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gin Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile <210> 40 <211> 469 <212> PRT
<213> Influenza A virus <400> 40 Met Asn Pro Asn Gin Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Thr Ile Ala Thr Ile Cys Phe Leu Met Gin Ile Ala Ile Leu Val Thr Thr Val Thr Leu His Phe Lys Gin Tyr Glu Cys Asn Ser Pro Pro Asn Asn Gin Val Met Leu Cys Glu Pro Thr Ile Ile Glu Arg Asn Ile Thr Glu Ile Val Tyr Leu Thr Asn Thr Thr Ile Glu Lys Glu Ile Cys Pro Lys Leu Ala Glu Tyr Arg Asn Trp Ser Lys Pro Gin Cys Lys Ile Thr Gly Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Asp Lys Cys Tyr Gin Phe Ala Leu Gly Gin Gly Thr Thr Leu Asn Asn Arg His Ser Asn Asp Thr Val His Asp Arg Thr Pro Tyr Arg Thr Leu Leu Met Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Lys Gin Val Cys Ile Ala Trp Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val Cys Val Thr Gly His Asp Glu Asn Ala Thr Ala Ser Phe Ile Tyr Asp Gly Arg Leu Val Asp Ser Ile Gly Ser Trp Ser Lys Lys Ile Leu Arg Thr Gin Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val Met Thr Asp Gly Ser Ala Ser Gly Arg Ala Asp Thr Lys Ile Leu Phe Ile Glu Glu Gly Lys Ile Val His Ile Ser Pro Leu Ser Gly Ser Ala Gin His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Ser Gly Val Arg Cys Val Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Ile Val Asp Ile Asn Val Lys Asp Tyr Ser Ile Val Ser Ser Tyr Val Cys Ser Gly Leu Val Gly Asp Thr Pro Arg Lys Asn Asp Ser Ser Ser Ser Ser His Cys Leu Asn Pro Asn Asn Glu Glu Gly Gly His Gly Val Lys Gly Trp Ala Phe Asp Asp Gly Asn Asp Val Trp Met Gly Arg Thr Ile Ser Glu Lys Leu Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile Gly Gly Trp Ser Lys Pro Asn Ser Lys Leu Gin Ile Asn Arg Gin Val Ile Val Asp Arg Gly Asn Arg Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Lys Gin Glu Thr Glu Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asp Ile Asn Leu Met Pro Ile <210> 41 <211> 566 <212> PRT
<213> Influenza A virus <400> 41 Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Ala Gin Lys Ile Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile Thr Asn Asp Gin Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gin Ser Ser Ser Thr Gly Arg Ile Cys Asp Ser Pro His Arg Ile Leu Asp Gly Glu Asn Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Gly Phe Gin Asn Lys Glu Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Tyr Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe Asn Asn Glu Ser Phe Asn Trp Thr Gly Val Ala Gin Asn Gly Thr Ser Tyr Ala Cys Lys Arg Ser Ser Ile Lys Ser Phe Phe Ser Arg Leu Asn Trp Leu His Gin Leu Lys Tyr Lys Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Asp Lys Phe Asp Lys Leu Tyr Ile Trp Gly Val His His Pro Ser Thr Asp Ser Asp Gin Thr Ser Ile Tyr Ala Gin Ala Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser Gin Gin Thr Val Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg Gly Ile Ser Ser Arg Ile Ser Ile His Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile Ala Pro Arg Gly Tyr Phe Lys Ile Arg Ser Gly Lys Ser Ser Ile Met Arg Ser Asp Ala Pro Ile Gly Lys Cys Asn Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gin Asn Val Asn Arg Ile Thr Tyr Gly Ala Cys Pro Arg Tyr Val Lys Gin Asn Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gin Thr Arg Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe Arg His Gin Asn Ser Glu Gly Thr Gly Gin Ala Ala Asp Leu Lys Ser Thr Gin Ala Ala Ile Asn Gin Ile Asn Gly Lys Leu Asn Arg Leu Ile Glu Lys Thr Asn Glu Lys Phe His Gin Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gin Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gin His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Arg Thr Arg Lys Gin Leu Arg Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gin Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gin Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile <210> 42 <211> 469 <212> PRT
<213> Influenza A virus <400> 42 Met Asn Pro Asn Gin Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Thr Ile Ala Thr Ile Cys Phe Leu Met Gin Ile Ala Ile Leu Val Thr Thr Val Thr Leu His Phe Lys Gin Tyr Glu Cys Ser Ser Pro Pro Asn Asn Gin Val Met Leu Cys Glu Pro Thr Ile Ile Glu Arg Asn Ile Thr Glu Ile Val Tyr Leu Thr Asn Thr Thr Ile Glu Lys Glu Ile Cys Pro Lys Leu Ala Glu Tyr Arg Asn Trp Ser Lys Pro Gin Cys Lys Ile Thr Gly Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Asp Lys Cys Tyr Gin Phe Ala Leu Gly Gin Gly Thr Thr Leu Asn Asn Arg His Ser Asn Asp Thr Val His Asp Arg Thr Pro Tyr Arg Thr Leu Leu Met Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Lys Gin Val Cys Ile Ala Trp Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val Cys Val Thr Gly His Asp Glu Asn Ala Thr Ala Ser Phe Ile Tyr Asp Gly Arg Leu Val Asp Ser Ile Gly Ser Trp Ser Lys Lys Ile Leu Arg Thr Gin Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val Net Thr Asp Gly Ser Ala Ser Gly Arg Ala Asp Thr Lys Ile Leu Phe Ile Glu Glu Gly Lys Ile Val His Ile Ser Pro Leu Ser Gly Ser Ala Gin His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg Cys Val Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Ile Val Asp Ile Asn Val Lys Asp Tyr Ser Ile Val Ser Ser Tyr Val Cys Ser Gly Leu Val Gly Asp Thr Pro Arg Lys Asn Asp Ser Ser Ser Ser Ser His Cys Leu Asn Pro Asn Asn Glu Glu Gly Gly His Gly Val Lys Gly Trp Ala Phe Asp Asp Gly Asn Asp Val Trp Met Gly Arg Thr Ile Ser Glu Lys Phe Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile Glu Gly Trp Ser Lys Pro Asn Ser Lys Leu Gin Ile Asn Arg Gin Val Ile Val Asp Arg Gly Asn Arg Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Lys Gin Glu Thr Glu Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asp Ile Asn Leu Met Pro Ile <210> 43 <211> 566 <212> PRT
<213> Influenza A virus <400> 43 Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Ala Gin Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly His His Ala Val Ser Asn Gly Thr Leu Val Lys Thr Ile Thr Asn Asp Gin Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gin Ser Ser Ser Thr Gly Arg Ile Cys Asp Ser Pro His Gin Ile Leu Asp Gly Glu Asn Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Gly Phe Gin Asn Lys Glu Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Tyr Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe Asn Asn Glu Ser Phe Asn Trp Thr Gly Val Ala Gln Asn Gly Thr Ser Ser Ala Cys Lys Arg Gly Ser Asn Lys Ser Phe Phe Ser Arg Leu Asn Trp Leu His Gln Leu Lys Tyr Lys Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Glu Lys Phe Asp Lys Leu Tyr Ile Trp Gly Val Leu His Pro Ser Thr Asp Ser Asp Gln Ile Ser Leu Tyr Ala Gln Ala Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser Gln Gln Thr Val Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg Gly Val Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile Ala Pro Arg Gly Tyr Phe Lys Ile Arg Ser Gly Lys Ser Ser Ile Met Arg Ser Asp Ala Pro Ile Gly Lys Cys Asn Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg Ile Thr Tyr Gly Ala Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asn Gln Ile Asn Gly Lys Leu Asn Arg Leu Ile Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Arg Thr Lys Lys Gin Leu Arg Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gin Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gin Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile <210> 44 <211> 469 <212> PRT
<213> Influenza A virus <400> 44 Met Asn Pro Asn Gin Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Thr Ile Ala Thr Ile Cys Phe Leu Met Gin Ile Ala Ile Leu Val Thr Thr Val Thr Leu His Phe Lys Gin Tyr Glu Cys Asn Ser Pro Pro Asn Asn Gin Val Met Leu Cys Glu Pro Thr Ile Ile Glu Arg Asn Ile Thr Glu Ile Val Tyr Leu Thr Asn Thr Thr Ile Glu Lys Glu Ile Cys Pro Lys Leu Ala Glu Tyr Arg Asn Trp Ser Lys Pro Gin Cys Lys Ile Thr Gly Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Asp Lys Cys Tyr Gin Phe Ala Leu Gly Gin Gly Thr Thr Leu Asn Asn Arg His Ser Asn Asp Thr Val His Asp Arg Thr Pro Tyr Arg Thr Leu Leu Met Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Lys Gin Val Cys Ile Ala Trp Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val Cys Val Thr Gly His Asp Glu Asn Ala Thr Ala Ser Phe Ile Tyr Asp Gly Arg Leu Val Asp Ser Ile Gly Ser Trp Ser Lys Lys Ile Leu Arg Thr Gin Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val Met Thr Asp Gly Ser Ala Ser Gly Arg Ala Asp Thr Lys Ile Leu Phe Ile Glu Glu Gly Lys Ile Val His Thr Ser Lys Leu Ser Gly Ser Ala Gln His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg Cys Val Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Ile Val Asp Ile Asn Val Lys Asp Tyr Ser Ile Val Ser Ser Tyr Val Cys Ser Gly Leu Val Gly Asp Thr Pro Arg Lys Asn Asp Ser Ser Ser Ser Ser His Cys Leu Asp Pro Asn Asn Glu Glu Gly Gly His Gly Val Lys Gly Trp Ala Phe Asp Asp Gly Asn Asp Val Trp Met Gly Arg Thr Ile Ser Glu Lys Ser Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile Glu Gly Trp Ser Lys Pro Asn Ser Lys Leu Gin Ile Asn Arg Gin Val Ile Val Glu Arg Gly Asn Met Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Lys Gin Glu Thr Glu Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asp Ile Asn Leu Met Pro Ile <210> 45 <211> 566 <212> PRT
<213> Influenza A virus <400> 45 Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Ser Gln Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly His His Ala Val Pro Asn Gly Thr Ile Val Lys Thr Ile Thr Asn Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr Gly Gly Ile Cys Asp Ser Pro His Gln Ile Leu Asp Gly Glu Asn Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro Gln Cys Asp Gly Phe Gln Asn Lys Lys Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Tyr Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe Asn Asn Glu Ser Phe Asn Trp Ala Gly Val Thr Gln Asn Gly Thr Ser Ser Ala Cys Lys Arg Arg Ser Asn Lys Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr His Leu Lys Tyr Lys Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Glu Lys Phe Asp Lys Leu Tyr Ile Trp Gly Val His His Pro Val Thr Asp Ser Asp Gln Ile Ser Leu Tyr Ala Gln Ala Ser Gly Arg Ile Thr Val Ser Thr Lys Arg Ser Gln Gln Thr Val Ile Pro Asn Ile Gly Tyr Arg Pro Arg Val Arg Asp Ile Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile Ala Pro Arg Gly Tyr Phe Lys Ile Arg Ser Gly Lys Ser Ser Ile Met Arg Ser Asp Ala Pro Ile Gly Lys Cys Asn Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg Ile Thr Tyr Gly Ala Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe Arg His Gin Asn Ser Glu Gly Thr Gly Gin Ala Ala Asp Leu Lys Ser Thr Gin Ala Ala Ile Asn Gin Ile Asn Gly Lys Leu Asn Arg Leu Ile Gly Lys Thr Asn Glu Lys Phe His Gin Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gin Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gin His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Arg Thr Lys Lys Gin Leu Arg Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Glu Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gin Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val Ala Leu Leu Gly Phe Ile Met Trp Ala Cys Gin Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile <210> 46 <211> 469 <212> PRT
<213> Influenza A virus <400> 46 Met Asn Pro Asn Gin Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Thr Ile Ser Thr Ile Cys Phe Phe Met Gin Ile Ala Ile Leu Ile Thr Thr Val Thr Leu His Phe Lys Gin Tyr Glu Phe Asn Ser Pro Pro Asn Asn Gin Val Met Leu Cys Glu Pro Thr Ile Ile Glu Arg Asn Ile Thr Glu Ile Val Tyr Leu Thr Asn Thr Thr Ile Glu Lys Glu Ile Cys Pro Lys Leu Ala Glu Tyr Arg Asn Trp Ser Lys Pro Gin Cys Asn Ile Thr Gly Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Asp Lys Cys Tyr Gin Phe Ala Leu Gly Gin Gly Thr Thr Leu Asn Asn Val His Ser Asn Asp Thr Val His Asp Arg Thr Pro Tyr Arg Thr Leu Leu Met Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Lys Gin Val Cys Ile Ala Trp Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val Cys Val Thr Gly Asp Asp Glu Asn Ala Thr Ala Ser Phe Ile Tyr Asn Gly Arg Leu Val Asp Ser Ile Val Ser Trp Ser Lys Lys Ile Leu Arg Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val Met Thr Asp Gly Ser Ala Ser Gly Lys Ala Asp Thr Lys Ile Leu Phe Ile Glu Glu Gly Lys Ile Val His Thr Ser Thr Leu Ser Gly Ser Ala Gin His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg Cys Val Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Ile Val Asp Ile Asn Ile Lys Asp Tyr Ser Ile Val Ser Ser Tyr Val Cys Ser Gly Leu Val Gly Asp Thr Pro Arg Lys Asn Asp Ser Ser Ser Ser Ser His Cys Leu Asp Pro Asn Asn Glu Glu Gly Gly His Gly Val Lys Gly Trp Ala Phe Asp Asp Gly Asn Asp Val Trp Met Gly Arg Thr Ile Ser Glu Lys Leu Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile Glu Gly Trp Ser Asn Pro Asn Ser Lys Leu Gin Ile Asn Arg Gin Val Ile Val Asp Arg Gly Asn Arg Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Lys Gin Glu Thr Glu Val Leu Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asp Ile Asn Leu Met Pro Ile <210> 47 <211> 566 <212> PRT
<213> Influenza A virus <400> 47 Met Lys Ala Lys Leu Leu Val Leu Leu Cys Ala Phe Thr Ala Thr Tyr Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile Ala Pro Leu Gin Leu Gly Asn Cys Ser Val Ala Gly Trp Ile Leu Gly Asn Pro Lys Cys Glu Ser Leu Phe Ser Lys Glu Ser Trp Ser Tyr Ile Ala Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gin Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Thr Val Thr Lys Gly Val Thr Thr Ser Cys Ser His Asn Gly Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Lys Lys Asn Gly Leu Tyr Pro Asn Val Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu Val Leu Val Leu Trp Gly Val His His Pro Ser Asn Ile Gly Asp Gin Arg Ala Ile Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Asp Gin Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Ser Met Asp Glu Cys Asp Ala Lys Cys Gin Thr Pro Gin Gly Ala Ile Asn Ser Ser Leu Pro Phe Gin Asn Val His Pro Val Thr Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser Thr Lys Leu Arg Met Val Thr Gly Leu Arg Asn Ile Pro Ser Ile Gin Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gin Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Gin Lys Ser Thr Gin Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gin Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Gly Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gin Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Gly Lys Ile Asp Gly Val Lys Leu Glu Ser Met Gly Val Tyr Gin Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gin Cys Arg Ile Cys Ile <210> 48 <211> 470 <212> PRT
<213> Influenza A virus <400> 48 Met Asn Pro Asn Gin Lys Ile Ile Ile Ile Gly Ser Ile Ser Met Ala Ile Gly Ile Ile Ser Leu Ile Leu Gin Ile Gly Asn Ile Ile Ser Ile Trp Ala Ser His Ser Ile Gin Thr Gly Ser Gin Asn His Thr Gly Ile Cys Asn Gin Arg Ile Ile Thr Tyr Glu Asn Ser Thr Trp Val Asn Gin Thr Tyr Val Asn Ile Asn Asn Thr Asn Val Val Ala Gly Lys Asp Lys Thr Ser Val Thr Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly Trp Ala Ile Tyr Thr Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr Phe Phe Leu Thr Gin Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Ala Leu Met Ser Cys Pro Leu Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Ile Thr Glu Thr Ile Lys Ser Trp Lys Lys Arg Ile Leu Arg Thr Gin Glu Ser Glu Cys Val Cys Val Asn Gly Ser Cys Phe Thr Ile Met Thr Asp Gly Pro Ser Asn Gly Ala Ala Ser Tyr Arg Ile Phe Lys Ile Glu Lys Gly Lys Val Thr Lys Ser Ile Glu Leu Asp Ala Pro Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Thr Val Met Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gin Asn Leu Asp Tyr Gin Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly Asp Asn Pro Arg Pro Lys Asp Gly Glu Gly Ser Cys Asn Pro Val Thr Val Asp Gly Ala Asp Gly Val Lys Gly Phe Ser Tyr Arg Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Asn Arg Leu Arg Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Asp Thr Asp Ser Asp Phe Ser Val Lys Gin Asp Val Val Ala Met Thr Asp Trp Ser Gly Tyr Ser Gly Ser Phe Val Gin His Pro Glu Leu Thr Gly Leu Asp Cys Met Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Arg Glu Asn Thr Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr Ala Asn Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe Thr Ile Asp Lys <210> 49 <211> 562 <212> PRT
<213> Influenza A virus <400> 49 Net Lys Ala Lys Leu Leu Val Leu Leu Cys Ala Phe Thr Ala Thr Tyr Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Thr Ala Pro Leu Gin Leu Gly Asn Cys Ser Val Ala Gly Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Phe Ser Lys Glu Ser Trp Ser Tyr Ile Ala Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gin Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Lys His Thr Val Thr Lys Gly Val Thr Ala Ser Cys Ser His Asn Gly Lys Ser Ser Phe Tyr Lys Asn Leu Leu Trp Leu Thr Glu Lys Asn Gly Leu Tyr Pro Asn Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu Val Leu Val Leu Trp Gly Val His His Pro Ser Asn Ile Gly Asp Gin Arg Ala Ile Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Gly Gin Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Thr Ala Ser Met Gly Glu Cys Asp Ala Lys Cys Gin Thr Pro Gin Gly Ala Ile Asn Ser Ser Leu Pro Phe Gin Asn Val His Pro Val Thr Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser Thr Lys Leu Arg Met Val Thr Gly Leu Arg Asn Ile Pro Ser Ile Gin Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gin Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Gin Lys Ser Thr Gin Asn Ala Ile Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gin Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Gly Arg Thr Leu Gly Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Asn Gin Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met Gly Val Tyr Gin Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gin Cys <210> 50 <211> 470 <212> PRT
<213> Influenza A virus <400> 50 Met Asn Pro Asn Gin Lys Ile Ile Thr Ile Gly Ser Ile Ser Ile Ala Ile Gly Ile Ile Ser Leu Ile Leu Gin Ile Gly Asn Ile Ile Ser Ile Trp Ala Ser His Ser Ile Gin Thr Gly Ser Gin Asn His Thr Gly Ile Cys Asn Gin Arg Ile Ile Thr Tyr Glu Asn Ser Thr Trp Val Asn Gin Thr Tyr Val Asn Ile Asn Asn Thr Asn Val Val Ala Gly Lys Asp Lys Thr Ser Met Thr Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly Trp Ala Ile Tyr Thr Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr Phe Phe Leu Thr Gin Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Ala Leu Met Ser Cys Pro Leu Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Leu Gly Trp Leu Thr Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Ile Thr Glu Thr Ile Lys Ser Trp Lys Lys Arg Ile Leu Arg Thr Gin Glu Ser Glu Cys Val Cys Met Asn Gly Ser Cys Phe Thr Ile Net Thr Asp Gly Pro Ser Asn Gly Ala Ala Ser Tyr Arg Ile Phe Lys Ile Glu Lys Gly Arg Val Thr Lys Ser Ile Glu Leu Asp Ala Pro Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Thr Val Met Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gin Asn Leu Asp Tyr Gin Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly Asp Asn Pro Arg Pro Lys Asp Gly Glu Gly Ser Cys Asn Pro Val Thr Val Asp Gly Ala Asp Gly Val Lys Gly Phe Ser Tyr Arg Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Asn Arg Leu Arg Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Asp Thr Asp Ser Asp Phe Ser Met Lys Gin Asp Ile Val Ala Met Thr Asp Trp Ser Gly Tyr Ser Gly Ser Phe Val Gin His Pro Glu Leu Thr Gly Leu Asp Cys Met Arg Pro Cys Phe Trp Val Glu Leu Val Arg Gly Leu Pro Arg Glu Asn Thr Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr Ala Asn Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe Thr Ile Asp Lys <210> 51 <211> 565 <212> PRT
<213> Influenza A virus <400> 51 Net Lys Ala Lys Leu Leu Val Leu Leu Cys Thr Phe Thr Ala Thr Tyr Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu Leu Lys Gly Ile Ala Pro Leu Gin Leu Gly Asn Cys Ser Val Ala Gly Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ile Ser Lys Glu Ser Trp Ser Tyr Ile Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gin Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Lys His Thr Val Thr Gly Val Thr Ala Ser Cys Ser His Asn Gly Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Asn Gly Leu Tyr Pro Asn Leu Ser Asn Ser Tyr Val Asn Asn Lys Glu Lys Glu Val Leu Val Leu Trp Gly Val His His Pro Ser Asn Ile Gly Asp Gin Arg Ala Ile Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Gly Gin Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Pro Met Asn Glu Cys Asp Ala Lys Cys Gin Thr Pro Gin Gly Ala Ile Asn Ser Ser Leu Pro Phe Gin Asn Val His Pro Val Thr Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser Thr Lys Leu Arg Met Val Thr Gly Leu Arg Asn Ile Pro Ser Ile Gin Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Met Asp Gly Trp Tyr Gly Tyr His His Gin Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Gin Lys Ser Thr Gin Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gin Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gin Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met Gly Val Tyr Gin Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gin Cys Arg Ile Cys Ile <210> 52 <211> 470 <212> PRT
<213> Influenza A virus <400> 52 Met Asn Pro Asn Gin Lys Ile Ile Thr Ile Gly Ser Ile Ser Ile Val Ile Gly Ile Ile Ser Leu Met Leu Gin Ile Gly Asn Ile Ile Ser Ile Trp Ala Ser His Ser Ile Gin Thr Gly Ser Gin Asn His Thr Gly Ile Cys Asn Gin Arg Ile Ile Thr Tyr Glu Asn Ser Thr Trp Val Asn His Thr Tyr Val Asn Ile Asn Asn Thr Asn Val Val Ala Gly Lys Asp Lys Thr Ser Val Thr Leu Ala Gly Asn Ser Ser Leu Cys Ser Ile Ser Gly Trp Ala Ile Tyr Thr Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Ala Leu Met Ser Cys Pro Leu Gly Glu Ala Pro Ser Pro Tyr Asn Ser Lys Phe Glu Ser Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Ile Thr Glu Thr Ile Lys Ser Trp Lys Lys Arg Ile Leu Arg Thr Gln Glu Ser Glu Cys Val Cys Val Asn Gly Ser Cys Phe Thr Ile Met Thr Asp Gly Pro Ser Asn Gly Ala Ala Ser Tyr Lys Ile Phe Lys Ile Glu Lys Gly Lys Val Thr Lys Ser Ile Glu Leu Asn Ala Pro Asn Ser His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Thr Val Met Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly Asp Asn Pro Arg Pro Lys Asp Gly Glu Gly Ser Cys Asn Pro Val Thr Val Asp Gly Ala Asp Gly Val Lys Gly Phe Ser Tyr Arg Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Asn Arg Leu Arg Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Asp Thr Asp Ser Asp Phe Ser Val Lys Gln Asp Val Val Ala Met Thr Asp Trp Ser Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Val Arg Gly Arg Pro Arg Glu Asn Thr Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr Ala Asn Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe Thr Ile Asp Lys <210> 53 <211> 565 <212> PRT
<213> Influenza A virus <400> 53 Met Lys Ala Lys Leu Leu Val Leu Leu Cys Thr Phe Thr Ala Thr Tyr Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu Leu Lys Gly Ile Ala Pro Leu Gin Leu Gly Asn Cys Ser Val Ala Gly Trp Ile Leu Gly Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser Trp Ser Tyr Ile Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gin Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Lys His Thr Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly Lys Asn Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly Leu Tyr Pro Asn Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu Val Leu Val Leu Trp Gly Val His His Pro Pro Asn Ile Gly Asp Gin Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Asp Gin Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Pro Met Asp Glu Cys Asp Ala Lys Cys Gin Thr Pro Gin Gly Ala Ile Asn Ser Ser Leu Pro Phe Gin Asn Val His Pro Val Thr Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn Ile Pro Ser Ile Gin Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Gin Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Gin Lys Ser Thr Gin Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gin Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gin Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met Gly Val Tyr Gin Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gin Cys Arg Ile Cys Ile <210> 54 <211> 470 <212> PRT
<213> Influenza A virus <400> 54 Met Asn Pro Asn Gin Lys Ile Ile Thr Ile Gly Ser Ile Ser Ile Ala Ile Gly Ile Ile Ser Leu Met Leu Gin Ile Gly Asn Ile Ile Ser Ile Trp Ala Ser His Ser Ile Gin Thr Gly Ser Gin Asn His Thr Gly Val Cys Asn Gin Arg Ile Ile Thr Tyr Glu Asn Ser Thr Trp Val Asn His Thr Tyr Val Asn Ile Asn Asn Thr Asn Val Val Ala Gly Lys Asp Lys Thr Ser Val Thr Leu Ala Gly Asn Ser Ser Leu Cys Ser Ile Ser Gly Trp Ala Ile Tyr Thr Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr Phe Phe Leu Thr Gin Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Ala Leu Met Ser Cys Pro Leu Gly Glu Ala Pro Ser Pro Tyr Asn Ser Lys Phe Glu Ser Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Ile Thr Glu Thr Ile Lys Ser Trp Lys Lys Arg Ile Leu Arg Thr Gin Glu Ser Glu Cys Val Cys Val Asn Gly Ser Cys Phe Thr Ile Met Thr Asp Gly Pro Ser Asn Gly Ala Ala Ser Tyr Lys Ile Phe Lys Ile Glu Lys Gly Lys Val Thr Lys Ser Ile Glu Leu Asn Ala Pro Asn Phe His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Thr Val Met Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gin Asn Leu Asp Tyr Gin Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly Asp Asn Pro Arg Pro Lys Asp Gly Glu Gly Ser Cys Asn Pro Val Thr Val Asp Gly Ala Asp Gly Val Lys Gly Phe Ser Tyr Lys Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Asn Arg Leu Arg Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Asp Thr Asp Ser Asp Phe Ser Val Lys Gin Asp Val Val Ala Ile Thr Asp Trp Ser Gly Tyr Ser Gly Ser Phe Val Gin His Pro Glu Leu Thr Gly Leu Asp Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Val Arg Gly Leu Pro Arg Glu Asn Thr Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr Ala Asn Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe Thr Ile Asp Lys <210> 55 <211> 583 <212> PRT
<213> Influenza B virus <400> 55 Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys Thr Ala Thr Gin Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Lys Thr Arg Gly Lys Leu Cys Pro Asn Cys Leu Asn Cys Thr Asp Leu Asp Val Ala Leu Gly Arg Pro Met Cys Ile Gly Ile Thr Pro Ser Ala Lys Ala Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro Ile Met His Asp Arg Thr Lys Ile Arg Gin Leu Pro Asn Leu Leu Arg Gly Tyr Glu His Ile Arg Leu Ser Thr His Asn Val Ile Asn Ala Glu Arg Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys Pro Asn Val Thr Ser Arg Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro Arg Asp Asn Lys Thr Ala Thr Asn Pro Leu Thr Val Glu Val Pro Tyr Ile Cys Thr Lys Gly Glu Asp Gin Ile Thr Val Trp Gly Phe His Ser Asp Asn Lys Ile Gin Met Lys Asn Leu Tyr Gly Asp Ser Asn Pro Gin Lys Phe Thr Ser Ser Ala Asn Gly Ile Thr Thr His Tyr Val Ser Gin Ile Gly Gly Phe Pro Asn Gin Thr Glu Asp Gly Gly Leu Pro Gin Ser Gly Arg Ile Val Val Asp Tyr Met Val Gin Lys Pro Gly Lys Thr Gly Thr Ile Val Tyr Gin Arg Gly Val Leu Leu Pro Gin Lys Val Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg Pro Pro Ala Lys Leu Leu Lys Glu Lys Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys Ser Thr Gin Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu Ser Glu Leu Glu Val Lys Asn Leu Gin Arg Leu Ser Gly Ala Met Asp Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu Arg Ala Asp Thr Ile Ser Ser Gin Ile Glu Leu Ala Val Leu Leu Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Asp Ile Gly Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gin Thr Cys Leu Asp Arg Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr Phe Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala Val Thr Leu Met Ile Ala Ile Phe Ile Val Tyr Met Val Ser Arg Asp Asn Val Ser Cys Ser Ile Cys Leu <210> 56 <211> 465 <212> PRT
<213> Influenza B virus <400> 56 Met Leu Pro Ser Thr Ile Gin Thr Leu Thr Leu Phe Leu Thr Ser Gly Gly Val Leu Leu Ser Leu Tyr Val Ser Ala Leu Leu Ser Tyr Leu Leu Tyr Ser Asp Ile Leu Leu Lys Phe Ser Pro Lys Ile Ile Ala Pro Thr Thr Ser Leu Asp Cys Ala Asn Ala Ser Asn Val Gin Ala Val Asn His Ser Ala Thr Lys Glu Met Lys Phe Leu Pro Pro Glu Pro Glu Trp Thr Tyr Pro Arg Leu Ser Cys Gin Gly Ser Thr Phe Gin Lys Ala Leu Leu Ile Ser Pro His Arg Phe Gly Glu Ala Lys Gly Asn Ser Ala Pro Leu Ile Ile Arg Glu Pro Phe Ile Ala Cys Gly Pro Lys Glu Cys Lys His Phe Ala Leu Thr His Tyr Ala Ala Gin Pro Gly Gly Tyr Tyr Asn Gly Thr Arg Glu Asp Arg Asn Lys Leu Arg His Leu Ile Ser Val Asn Leu Gly Lys Ile Pro Thr Val Glu Asn Ser Ile Phe His Met Ala Ala Trp Ser Gly Ser Ala Cys His Asp Gly Arg Glu Trp Thr Tyr Ile Gly Val Asp Gly Pro Asp Ser Asn Ala Leu Ile Lys Ile Lys Tyr Gly Glu Ala Tyr Thr Asp Thr Tyr His Ser Tyr Ala Asn Asn Ile Leu Arg Thr Gln Glu Ser Ala Cys Asn Cys Ile Gly Gly Asp Cys Tyr Leu Met Ile Thr Asp Gly Ser Ala Ser Gly Ile Ser Lys Cys Arg Phe Leu Lys Ile Arg Glu Gly Arg Ile Ile Lys Glu Ile Phe Pro Thr Gly Arg Val Glu His Thr Glu Glu Cys Thr Cys Gly Phe Ala Ser Asn Lys Thr Ile Glu Cys Ala Cys Arg Asp Asn Ser Tyr Thr Ala Lys Arg Pro Phe Val Lys Leu Asn Val Glu Thr Asp Thr Ala Glu Ile Arg Leu Met Cys Thr Glu Thr Tyr Leu Asp Thr Pro Arg Pro Asp Asp Gly Ser Ile Thr Gly Pro Cys Glu Ser Asn Gly Asp Lys Gly Ser Gly Gly Val Lys Gly Gly Phe Val His Gln Arg Met Ala Ser Lys Ile Gly Arg Trp Tyr Ser Arg Thr Met Ser Lys Thr Lys Arg Met Gly Met Glu Leu Tyr Val Lys Tyr Asp Gly Asp Pro Trp Thr Asp Ser Asp Ala Leu Ala Pro Ser Gly Val Met Val Ser Met Glu Glu Pro Gly Trp Tyr Ser Phe Gly Phe Glu Ile Lys Asp Lys Lys Cys Asp Val Pro Cys Ile Gly Ile Glu Met Val His Asp Gly Gly Lys Arg Thr Trp His Ser Ala Ala Thr Ala Ile Tyr Cys Leu Met Gly Ser Gly Gln Leu Leu Trp Asp Thr Val Thr Gly Val Asn Met Ala Leu <210> 57 <211> 584 <212> PRT
<213> Influenza B virus <400> 57 Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Lys Thr Arg Gly Lys Leu Cys Pro Thr Cys Leu Asn Cys Thr Asp Leu Asp Val Ala Leu Gly Arg Pro Met Cys Val Gly Val Thr Pro Ser Ala Lys Ala Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro Ile Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg Gly Tyr Glu Lys Ile Arg Leu Ser Thr Gln Ile Val Ile Asn Ala Glu Lys Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys Pro Asn Ala Thr Ser Arg Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro Lys Asp Asn Asn Lys Thr Ala Thr Asn Pro Leu Thr Val Glu Val Pro His Ile Cys Thr Lys Glu Glu Asp Gln Ile Thr Val Trp Gly Phe His Ser Asp Asp Lys Thr Gln Met Lys Asn Leu Tyr Gly Asp Ser Asn Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val Ser Gln Ile Gly Gly Phe Pro Asp Gln Thr Glu Asp Gly Gly Leu Pro Gln Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Pro Gly Lys Thr Gly Thr Ile Val Tyr Gln Arg Gly Ile Leu Leu Pro Gln Lys Val Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys Ser Thr Gin Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu Ser Glu Leu Glu Val Lys Asn Leu Gin Arg Leu Ser Gly Ala Met Asp Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu Arg Ala Asp Thr Ile Ser Ser Gin Ile Glu Leu Ala Val Leu Leu Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Asp Ile Gly Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gin Thr Cys Leu Asp Arg Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr Phe Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala Val Thr Leu Met Ile Ala Ile Phe Ile Val Tyr Met Ile Ser Arg Asp Asn Val Ser Cys Ser Ile Cys Leu <210> 58 <211> 466 <212> PRT
<213> Influenza B virus <400> 58 Met Leu Pro Ser Thr Ile Gin Thr Leu Thr Leu Phe Leu Thr Ser Gly Gly Val Leu Leu Ser Leu Tyr Val Ser Ala Ser Leu Ser Tyr Leu Leu Tyr Ser Asp Ile Leu Leu Lys Phe Ser Pro Thr Glu Ile Thr Ala Pro Thr Met Pro Leu Asn Cys Ala Asn Ala Ser Asn Val Gln Ala Val Asn Arg Ser Ala Thr Lys Gly Val Thr Leu Pro Leu Pro Glu Pro Glu Trp Thr Tyr Pro Arg Leu Ser Cys Pro Gly Ser Thr Phe Gln Lys Ala Leu Leu Ile Ser Pro His Arg Phe Gly Glu Thr Lys Gly Asn Ser Ala Pro Leu Ile Ile Arg Glu Pro Phe Ile Ala Cys Gly Pro Lys Glu Cys Arg His Phe Ala Leu Thr His Tyr Ala Ala Gln Pro Gly Gly Tyr Tyr Asn Gly Thr Arg Glu Asp Arg Asn Lys Leu Arg His Leu Ile Ser Val Lys Leu Gly Lys Ile Pro Thr Val Glu Asn Ser Ile Phe His Met Ala Ala Trp Ser Gly Ser Ala Cys His Asp Gly Arg Glu Trp Thr Tyr Ile Gly Val Asp Gly Pro Asp Ser Asn Ala Leu Leu Lys Ile Lys Tyr Gly Glu Ala Tyr Thr Asp Thr Tyr His Ser Tyr Ala Asn Asn Ile Leu Arg Thr Gln Glu Ser Ala Cys Asn Cys Ile Gly Gly Asp Cys Tyr Leu Met Ile Thr Asp Gly Ser Ala Ser Gly Ile Ser Glu Cys Arg Phe Leu Lys Ile Arg Glu Gly Arg Ile Ile Lys Glu Ile Phe Pro Thr Gly Arg Val Glu His Thr Glu Glu Cys Thr Cys Gly Phe Ala Ser Asn Lys Thr Ile Glu Cys Ala Cys Arg Asp Asn Ser Tyr Thr Ala Lys Arg Pro Phe Val Lys Leu Asn Val Glu Thr Asp Thr Ala Glu Ile Arg Leu Met Cys Thr Glu Thr Tyr Leu Asp Thr Pro Arg Pro Asp Asp Gly Ser Ile Thr Gly Pro Cys Glu Ser Asn Gly Asp Lys Gly Ser Gly Gly Ile Lys Gly Gly Phe Val His Gln Arg Met Ala Ser Lys Ile Gly Arg Trp Tyr Ser Arg Thr Met Ser Lys Thr Lys Arg Met Gly Met Gly Leu Tyr Val Lys Tyr Asp Gly Asp Pro Trp Ile Asp Ser Asp Ala Leu Thr Leu Ser Gly Val Met Val Ser Met Glu Glu Pro Gly Trp Tyr Ser Phe Gly Phe Glu Ile Lys Asp Lys Lys Cys Asp Val Pro Cys Ile Gly Ile Glu Met Val His Asp Gly Gly Lys Lys Thr Trp His Ser Ala Ala Thr Ala Ile Tyr Cys Leu Met Gly Ser Gly Gin Leu Leu Trp Asp Thr Val Thr Gly Val Asp Met Ala Leu <210> 59 <211> 584 <212> PRT
<213> Influenza B virus <400> 59 Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys Thr Ala Thr Gin Gly Glu Val Asn Val Thr Gly Ala Ile Pro Leu Thr Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Lys Thr Arg Gly Lys Leu Cys Pro Thr Cys Leu Asn Cys Thr Asp Leu Asp Val Ala Leu Gly Arg Pro Met Cys Val Gly Ile Thr Pro Ser Ala Lys Ala Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro Ile Met His Asp Arg Thr Lys Ile Arg Gin Leu Pro Asn Leu Leu Arg Gly Tyr Glu Lys Ile Arg Leu Ser Thr Gin Asn Val Ile Asn Ala Glu Lys Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys Pro Asn Ala Thr Ser Lys Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro Arg Asp Asn Asn Lys Thr Ala Thr Asn Pro Leu Thr Val Glu Val Pro His Ile Cys Thr Lys Glu Glu Asp Gin Ile Thr Val Trp Gly Phe His Ser Asp Asp Lys Thr Gin Met Lys Asn Leu Tyr Gly Asp Ser Asn Pro Gin Lys Phe Thr Ser Ser Ala Asn Gly Ile Thr Thr His Tyr Val Ser Gin Ile Gly Gly Phe Pro Asp Gin Thr Glu Asp Gly Gly Leu Pro Gin Ser Gly Arg Ile Val Val Asp Tyr Met Val Gin Lys Pro Gly Lys Thr Gly Thr Ile Val Tyr Gin Arg Gly Ile Leu Leu Pro Gin Lys Val Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys Ser Thr Gin Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu Ser Glu Leu Glu Val Lys Asn Leu Gin Arg Leu Ser Gly Ala Met Asp Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu Arg Ala Asp Thr Ile Ser Ser Gin Ile Glu Leu Ala Val Leu Leu Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Ile Asp Ile Gly Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gin Thr Cys Leu Asp Arg Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr Phe Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala Val Thr Leu Met Ile Ala Ile Phe Ile Val Tyr Met Ile Ser Arg Asp Asn Val Ser Cys Ser Ile Cys Leu <210> 60 <211> 466 <212> PRT
<213> Influenza B virus <400> 60 Met Leu Pro Ser Thr Ile Gln Thr Leu Thr Leu Phe Leu Thr Ser Gly Gly Val Leu Leu Ser Leu Tyr Val Ser Ala Ser Leu Ser Tyr Leu Leu Tyr Ser Asp Ile Leu Leu Lys Phe Ser Pro Thr Glu Ile Thr Ala Pro Ala Met Pro Leu Asp Cys Ala Asn Ala Ser Asn Val Gln Ala Val Asn Arg Ser Ala Thr Lys Gly Val Thr Leu Leu Leu Pro Glu Pro Glu Trp Thr Tyr Pro Arg Leu Ser Cys Pro Gly Ser Thr Phe Gln Lys Ala Leu Leu Ile Ser Pro His Arg Phe Gly Glu Thr Lys Gly Asn Ser Ala Pro Leu Ile Ile Arg Glu Pro Phe Ile Ala Cys Gly Pro Lys Glu Cys Lys His Phe Ala Leu Thr His Tyr Ala Ala Gln Pro Gly Gly Tyr Tyr Asn Gly Thr Arg Glu Asp Arg Asn Lys Leu Arg His Leu Ile Ser Val Lys Phe Gly Lys Ile Pro Thr Val Glu Asn Ser Ile Phe His Met Ala Ala Trp Ser Gly Ser Ala Cys His Asp Gly Lys Glu Trp Thr Tyr Ile Gly Val Asp Gly Pro Asp Ser Asn Ala Leu Leu Lys Ile Lys Tyr Gly Glu Ala Tyr Thr Asp Thr Tyr His Ser Tyr Ala Asn Asn Ile Leu Arg Thr Gln Glu Ser Ala Cys Asn Cys Ile Gly Gly Asn Cys Tyr Leu Met Ile Thr Asp Gly Ser Ala Ser Gly Ile Ser Glu Cys Arg Phe Leu Lys Ile Arg Glu Gly Arg Ile Ile Lys Glu Ile Phe Pro Thr Gly Arg Val Lys His Thr Glu Glu Cys Thr Cys Gly Phe Ala Ser Asn Lys Thr Ile Glu Cys Ala Cys Arg Asp Asn Ser Tyr Thr Ala Lys Arg Pro Phe Val Lys Leu Asn Val Glu Thr Asp Thr Ala Glu Ile Arg Leu Met Cys Thr Glu Thr Tyr Leu Asp Thr Pro Arg Pro Asp Asp Gly Ser Ile Thr Gly Pro Cys Glu Ser Asn Gly Asp Lys Gly Ser Gly Gly Ile Lys Gly Gly Phe Val His Gin Arg Met Ala Ser Lys Ile Gly Arg Trp Tyr Ser Arg Thr Met Ser Lys Thr Lys Arg Met Gly Met Gly Leu Tyr Val Lys Tyr Asp Gly Asp Pro Trp Thr Asp Ser Asp Ala Leu Ala Leu Ser Gly Val Met Val Ser Met Glu Glu Pro Gly Trp Tyr Ser Phe Gly Phe Glu Ile Lys Asp Lys Lys Cys Asp Val Pro Cys Ile Gly Ile Glu Met Val His Asp Gly Gly Lys Glu Thr Trp His Ser Ala Ala Thr Ala Ile Tyr Cys Leu Met Gly Ser Gly Gin Leu Leu Trp Asp Thr Val Thr Gly Val Asp Met Ala Leu <210> 61 <211> 583 <212> PRT
<213> Influenza B virus <400> 61 Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Lys Thr Arg Gly Lys Leu Cys Pro Asn Cys Leu Asn Cys Thr Asp Leu Asp Val Ala Leu Gly Arg Pro Met Cys Ile Gly Ile Thr Pro Ser Ala Lys Ala Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro Ile Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg Gly Tyr Glu His Ile Arg Leu Ser Thr His Asn Val Ile Asn Ala Glu Arg Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys Pro Asn Val Thr Ser Arg Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro Arg Asp Asn Lys Thr Ala Thr Asn Pro Leu Thr Val Glu Val Pro Tyr Ile Cys Thr Lys Gly Glu Asp Gln Ile Thr Val Trp Gly Phe His Ser Asp Asn Lys Ile Gln Met Lys Asn Leu Tyr Gly Asp Ser Asn Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Ile Thr Thr His Tyr Val Ser Gln Ile Gly Gly Phe Pro Asn Gln Thr Glu Asp Gly Gly Leu Pro Gln Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Pro Gly Lys Thr Gly Thr Ile Val Tyr Gln Arg Gly Val Leu Leu Pro Gln Lys Val Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg Pro Pro Ala Lys Leu Leu Lys Glu Lys Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys Ser Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu Ser Glu Leu Glu Val Lys Asn Leu Gin Arg Leu Ser Gly Ala Met Asp Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu Arg Ala Asp Thr Ile Ser Ser Gin Ile Glu Leu Ala Val Leu Leu Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Asp Ile Gly Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gin Thr Cys Leu Asp Arg Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr Phe Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala Val Thr Leu Met Ile Ala Ile Phe Ile Val Tyr Met Val Ser Arg Asp Asn Val Ser Cys Ser Ile Cys Leu <210> 62 <211> 465 <212> PRT
<213> Influenza B virus <400> 62 Met Leu Pro Ser Thr Ile Gin Thr Leu Thr Leu Phe Leu Thr Ser Gly Gly Val Leu Leu Ser Leu Tyr Val Ser Ala Leu Leu Ser Tyr Leu Leu Tyr Ser Asp Ile Leu Leu Lys Phe Ser Pro Lys Ile Ile Ala Pro Thr Thr Ser Leu Asp Cys Ala Asn Ala Ser Asn Val Gin Ala Val Asn His Ser Ala Thr Lys Glu Met Lys Phe Leu Pro Pro Glu Pro Glu Trp Thr Tyr Pro Arg Leu Ser Cys Gin Gly Ser Thr Phe Gin Lys Ala Leu Leu Ile Ser Pro His Arg Phe Gly Glu Ala Lys Gly Asn Ser Ala Pro Leu Ile Ile Arg Glu Pro Phe Ile Ala Cys Gly Pro Lys Glu Cys Lys His Phe Ala Leu Thr His Tyr Ala Ala Gln Pro Gly Gly Tyr Tyr Asn Gly Thr Arg Glu Asp Arg Asn Lys Leu Arg His Leu Ile Ser Val Asn Leu Gly Lys Ile Pro Thr Val Glu Asn Ser Ile Phe His Met Ala Ala Trp Ser Gly Ser Ala Cys His Asp Gly Arg Glu Trp Thr Tyr Ile Gly Val Asp Gly Pro Asp Ser Asn Ala Leu Ile Lys Ile Lys Tyr Gly Glu Ala Tyr Thr Asp Thr Tyr His Ser Tyr Ala Asn Asn Ile Leu Arg Thr Gln Glu Ser Ala Cys Asn Cys Ile Gly Gly Asp Cys Tyr Leu Met Ile Thr Asp Gly Ser Ala Ser Gly Ile Ser Lys Cys Arg Phe Leu Lys Ile Arg Glu Gly Arg Ile Ile Lys Glu Ile Phe Pro Thr Gly Arg Val Glu His Thr Glu Glu Cys Thr Cys Gly Phe Ala Ser Asn Lys Thr Ile Glu Cys Ala Cys Arg Asp Asn Ser Tyr Thr Ala Lys Arg Pro Phe Val Lys Leu Asn Val Glu Thr Asp Thr Ala Glu Ile Arg Leu Met Cys Thr Glu Thr Tyr Leu Asp Thr Pro Arg Pro Asp Asp Gly Ser Ile Thr Gly Pro Cys Glu Ser Asn Gly Asp Lys Gly Ser Gly Gly Val Lys Gly Gly Phe Val His Gln Arg Met Ala Ser Lys Ile Gly Arg Trp Tyr Ser Arg Thr Met Ser Lys Thr Lys Arg Met Gly Met Glu Leu Tyr Val Lys Tyr Asp Gly Asp Pro Trp Thr Asp Ser Asp Ala Leu Ala Pro Ser Gly Val Met Val Ser Met Glu Glu Pro Gly Trp Tyr Ser Phe Gly Phe Glu Ile Lys Asp Lys Lys Cys Asp Val Pro Cys Ile Gly Ile Glu Met Val His Asp Gly Gly Lys Arg Thr Trp His Ser Ala Ala Thr Ala Ile Tyr Cys Leu Met Gly Ser Gly Gln Leu Leu Trp Asp Thr Val Thr Gly Val Asn Met Ala Leu <210> 63 <211> 585 <212> PRT
<213> Influenza B virus <400> 63 Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Lys Thr Arg Gly Lys Leu Cys Pro Lys Cys Leu Asn Cys Thr Asp Leu Asp Val Ala Leu Gly Arg Pro Lys Cys Thr Gly Asn Ile Pro Ser Ala Lys Val Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro Ile Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg Gly Tyr Glu Arg Ile Arg Leu Ser Asn His Asn Val Ile Asn Ala Glu Lys Ala Pro Gly Gly Pro Tyr Lys Ile Gly Thr Ser Gly Ser Cys Pro Asn Val Thr Asn Gly Asn Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro Lys Asn Glu Asn Asn Lys Thr Ala Thr Asn Ser Leu Thr Ile Glu Val Pro Tyr Ile Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe His Ser Asp Ser Glu Thr Gln Met Ala Lys Leu Tyr Gly Asp Ser Lys Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val Ser Gln Ile Gly Gly Phe Pro Asn Gln Thr Glu Asp Gly Gly Leu Pro Gln Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Ser Gly Lys Thr Gly Thr Ile Thr Tyr Gln Arg Gly Ile Leu Leu Pro Gln Lys Val Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys Ser Thr Gin Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu Ser Glu Leu Glu Val Lys Asn Leu Gin Arg Leu Ser Gly Ala Met Asp Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gin Thr Cys Leu Asp Arg Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr Phe Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala Val Thr Leu Met Ile Ala Ile Phe Val Val Tyr Met Val Ser Arg Asp Asn Val Ser Cys Ser Ile Cys Leu <210> 64 <211> 466 <212> PRT
<213> Influenza B virus <400> 64 Met Leu Pro Ser Thr Ile Gin Thr Leu Thr Leu Phe Leu Thr Ser Gly Gly Val Leu Leu Ser Leu Tyr Val Ser Ala Leu Leu Ser Tyr Leu Leu Tyr Ser Asp Ile Leu Leu Lys Phe Ser Pro Thr Lys Ile Ile Ala Pro Thr Thr Ser Leu Asp Ser Ala Asn Ala Ser Asn Phe Gin Ala Val Asn His Ser Ala Thr Lys Glu Met Thr Phe Leu Leu Pro Glu Pro Glu Trp Thr Tyr Pro Arg Leu Ser Cys Gin Gly Ser Thr Phe Gin Lys Ala Leu Leu Ile Ser Pro His Arg Phe Gly Glu Ala Lys Gly Asn Ser Ala Pro Leu Ile Ile Arg Glu Pro Phe Ile Ala Cys Gly Pro Lys Glu Cys Lys His Phe Ala Leu Thr His Tyr Ala Ala Gin Pro Gly Gly Tyr Tyr Asn Gly Thr Arg Glu Asp Arg Asn Lys Leu Arg His Leu Ile Ser Val Asn Leu Gly Lys Ile Pro Thr Val Glu Asn Ser Ile Phe His Met Ala Ala Trp Ser Gly Ser Ala Cys His Asp Gly Arg Glu Trp Thr Tyr Ile Gly Val Asp Gly Pro Asp Ser Asn Ala Leu Ile Lys Ile Lys Tyr Gly Glu Ala Tyr Thr Asp Thr Tyr His Ser Tyr Ala Asn Asn Ile Leu Arg Thr Gin Glu Ser Ala Cys Asn Cys Ile Gly Gly Asp Cys Tyr Leu Met Ile Thr Asp Gly Ser Ala Ser Gly Ile Ser Lys Cys Arg Phe Leu Lys Ile Arg Glu Gly Arg Ile Val Lys Glu Ile Phe Pro Thr Gly Arg Val Glu His Thr Glu Glu Cys Thr Cys Gly Phe Ala Ser Asn Lys Thr Ile Glu Cys Ala Cys Arg Asp Asn Ser Tyr Thr Ala Lys Arg Pro Phe Val Lys Leu Asn Val Glu Thr Asp Thr Ala Glu Ile Arg Leu Met Cys Thr Glu Thr Tyr Leu Asp Thr Pro Arg Pro Asp Asp Gly Ser Ile Thr Gly Pro Cys Glu Ser Asn Gly Asp Lys Gly Ser Gly Gly Ile Lys Gly Gly Phe Val His Gin Arg Met Ala Ser Lys Ile Gly Arg Trp Tyr Ser Arg Thr Met Ser Lys Thr Lys Arg Met Gly Met Glu Leu Tyr Val Lys Tyr Asp Gly Asp Pro Trp Thr Asp Ser Asp Ala Leu Ala Pro Ser Gly Val Met Val Ser Ile Glu Glu Pro Gly Trp Tyr Ser Phe Gly Phe Glu Ile Lys Asp Lys Lys Cys Asp Val Pro Cys Ile Gly Ile Glu Met Val His Asp Gly Gly Lys Thr Thr Trp His Ser Ala Ala Thr Ala Ile Tyr Cys Leu Met Gly Ser Gly Gin Leu Leu Trp Asp Thr Ile Thr Gly Val Asp Met Ala Leu <210> 65 <211> 585 <212> PRT
<213> Influenza B virus <400> 65 Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys Thr Ala Thr Gin Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Lys Thr Arg Gly Lys Leu Cys Pro Lys Cys Leu Asn Cys Thr Asp Leu Asp Val Ala Leu Gly Arg Pro Lys Cys Thr Gly Asn Ile Pro Ser Ala Lys Val Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro Ile Met His Asp Arg Thr Lys Ile Arg Gin Leu Pro Asn Leu Leu Arg Gly Tyr Glu His Ile Arg Leu Ser Thr His Asn Val Ile Asn Ala Glu Lys Ala Pro Gly Gly Pro Tyr Lys Ile Gly Thr Ser Gly Ser Cys Pro Asn Val Thr Asn Gly Asn Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro Lys Asn Asp Asn Asn Lys Thr Ala Thr Asn Ser Leu Thr Ile Glu Val Pro Tyr Ile Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe His Ser Asp Asn Glu Ala Gin Met Ala Lys Leu Tyr Gly Asp Ser Lys Pro Gin Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val Ser Gin Ile Gly Gly Phe Pro Asn Gin Thr Glu Asp Gly Gly Leu Pro Gin Ser Gly Arg Ile Val Val Asp Tyr Met Val Gin Lys Ser Gly Lys Thr Gly Thr Ile Thr Tyr Gin Arg Gly Ile Leu Leu Pro Gin Lys Val Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys Ser Thr Gin Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu Ser Glu Leu Glu Val Lys Asn Leu Gin Arg Leu Ser Gly Ala Met Asp Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu Arg Ala Asp Thr Ile Ser Ser Gin Ile Glu Leu Ala Val Leu Leu Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gin Thr Cys Leu Asp Arg Ile Ala Ala Gly Thr Phe Asp Ala Gly Glu Phe Ser Leu Pro Thr Phe Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala Val Thr Leu Met Ile Ala Ile Phe Val Val Tyr Met Val Ser Arg Asp Asn Val Ser Cys Ser Ile Cys Leu <210> 66 <211> 466 <212> PRT
<213> Influenza B virus <400> 66 Met Leu Pro Ser Thr Ile Gin Thr Leu Thr Leu Phe Leu Thr Ser Gly Gly Val Leu Leu Ser Leu Tyr Val Ser Ala Ser Leu Ser Tyr Leu Leu Tyr Ser Asp Ile Leu Leu Lys Phe Ser Pro Thr Glu Ile Thr Ala Pro Thr Met Pro Leu Asp Cys Ala Asn Ala Ser Asn Val Gin Ala Val Asn Arg Ser Ala Thr Lys Gly Val Thr Leu Leu Leu Pro Glu Pro Glu Trp Thr Tyr Pro Arg Leu Ser Cys Pro Gly Ser Thr Phe Gin Lys Ala Leu Leu Ile Ser Pro His Arg Phe Gly Glu Thr Lys Gly Asn Ser Ala Pro Leu Ile Ile Arg Glu Pro Phe Ile Ala Cys Gly Pro Lys Glu Cys Lys His Phe Ala Leu Thr His Tyr Ala Ala Gin Pro Gly Gly Tyr Tyr Asn Gly Thr Arg Gly Asp Arg Asn Lys Leu Arg His Leu Ile Ser Val Lys Leu Gly Lys Ile Pro Thr Val Glu Asn Ser Ile Phe His Met Ala Ala Trp Ser Gly Ser Ala Cys His Asp Gly Lys Glu Trp Thr Tyr Ile Gly Val Asp Gly Pro Asp Asn Asn Ala Leu Leu Lys Ile Lys Tyr Gly Glu Ala Tyr Thr Asp Thr Tyr His Ser Tyr Ala Asn Asn Ile Leu Arg Thr Gln Glu Ser Ala Cys Asn Cys Ile Gly Gly Asn Cys Tyr Leu Met Ile Thr Asp Gly Ser Ala Ser Gly Ile Ser Glu Cys Arg Phe Leu Lys Ile Arg Glu Gly Arg Ile Ile Lys Glu Ile Phe Pro Thr Gly Arg Val Lys His Thr Glu Glu Cys Thr Cys Gly Phe Ala Ser Asn Lys Thr Ile Glu Cys Ala Cys Arg Asp Asn Ser Tyr Thr Ala Lys Arg Pro Phe Val Lys Leu Asn Val Glu Thr Asp Thr Ala Glu Ile Arg Leu Met Cys Thr Glu Thr Tyr Leu Asp Thr Pro Arg Pro Asp Asp Gly Ser Ile Thr Gly Pro Cys Glu Ser Asn Gly Asp Lys Gly Ser Gly Gly Ile Lys Gly Gly Phe Val His Gin Arg Met Ala Ser Lys Ile Gly Arg Trp Tyr Ser Arg Thr Met Ser Lys Thr Lys Arg Met Gly Met Gly Leu Tyr Val Lys Tyr Asp Gly Asp Pro Trp Ala Asp Ser Asp Ala Leu Ala Leu Ser Gly Val Met Val Ser Met Glu Glu Pro Gly Trp Tyr Ser Phe Gly Phe Glu Ile Lys Asp Lys Lys Cys Asp Val Pro Cys Ile Gly Ile Glu Met Val His Asp Gly Gly Lys Glu Thr Trp His Ser Ala Ala Thr Ala Ile Tyr Cys Leu Met Gly Ser Gly Gin Leu Leu Trp Asp Thr Val Thr Gly Val Asp Met Ala Leu <210> 67 <211> 584 <212> PRT
<213> Influenza B virus <400> 67 Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr Thr Thr Pro Thr Lys Ser Tyr Phe Ala Asn Leu Lys Gly Thr Arg Thr Arg Gly Lys Leu Cys Pro Asp Cys Leu Asn Cys Thr Asp Leu Asp Val Ala Leu Gly Arg Pro Met Cys Val Gly Thr Thr Pro Ser Ala Lys Ala Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro Ile Met His Asp Arg Thr Lys Ile Arg Gin Leu Pro Asn Leu Leu Arg Gly Tyr Glu Asn Ile Arg Leu Ser Thr Gin Asn Val Ile Asp Ala Glu Asn Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys Pro Asn Ala Thr Ser Lys Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro Lys Asp Asn Asn Lys Asn Ala Thr Asn Pro Leu Thr Val Glu Val Pro Tyr Val Cys Thr Glu Gly Glu Asp Gin Ile Thr Val Trp Gly Phe His Ser Asp Asn Lys Thr Pro Met Lys Asn Leu Tyr Gly Asp Ser Asn Pro Gin Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val Ser Gin Ile Gly Gly Phe Pro Ala Gin Thr Glu Asp Glu Gly Leu Pro Gin Ser Gly Arg Ile Val Val Asp Tyr Met Val Gin Lys Pro Arg Lys Thr Gly Thr Ile Val Tyr Gin Arg Gly Val Leu Leu Pro Cln Lys Val Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys Ser Thr Gin Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu Ser Glu Leu Glu Val Lys Asn Leu Gin Arg Leu Ser Gly Ala Met Asp Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu Arg Ala Asp Thr Ile Ser Ser Gin Ile Glu Leu Ala Val Leu Leu Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Asp Ile Gly Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gin Thr Cys Leu Asp Arg Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr Phe Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala Val Thr Leu Met Leu Ala Ile Phe Ile Val Tyr Met Val Ser Arg Asp Asn Val Ser Cys Ser Ile Cys Leu <210> 68 <211> 466 <212> PRT
<213> Influenza B virus <400> 68 Met Leu Pro Ser Thr Ile Gin Thr Leu Thr Leu Phe Leu Thr Ser Gly Gly Val Leu Leu Ser Leu Tyr Val Ser Ala Ser Leu Ser Tyr Leu Leu Tyr Ser Asp Ile Leu Leu Lys Phe Ser Thr Thr Glu Ile Thr Ala Pro Thr Met Pro Leu Asp Cys Ala Asn Ala Ser Asn Val Gin Ala Val Asn Arg Ser Ala Thr Lys Gly Val Thr Leu Leu Leu Pro Glu Pro Glu Trp Thr Tyr Pro Arg Leu Ser Cys Pro Gly Ser Thr Phe Gin Lys Ala Leu Leu Ile Ser Pro His Arg Phe Gly Glu Thr Lys Gly Asn Ser Ala Pro Leu Ile Ile Arg Glu Pro Phe Ile Ala Cys Gly Pro Lys Glu Cys Lys His Phe Ala Leu Thr His Tyr Ala Ala Gin Pro Gly Gly Tyr Tyr Asn Gly Thr Lys Glu Asp Arg Asn Lys Leu Arg His Leu Ile Ser Val Lys Leu Gly Lys Ile Pro Thr Val Glu Asn Ser Ile Phe His Met Ala Ala Trp Ser Gly Ser Ala Cys His Asp Gly Lys Glu Trp Thr Tyr Ile Gly Val Asp Gly Pro Asp Ser Asn Ala Leu Leu Lys Ile Lys Tyr Gly Glu Ala Tyr Thr Asp Thr Tyr His Ser Tyr Ala Asn Asn Ile Leu Arg Thr Gin Glu Ser Ala Cys Asn Cys Ile Gly Gly Asn Cys Tyr Leu Met Ile Thr Asp Gly Ser Ala Ser Gly Ile Ser Glu Cys Arg Phe Leu Lys Ile Arg Glu Gly Arg Ile Ile Lys Glu Ile Phe Pro Thr Gly Arg Val Lys His Thr Glu Glu Cys Thr Cys Gly Phe Ala Ser Asn Lys Thr Ile Glu Cys Ala Cys Arg Asp Asn Ser Tyr Thr Ala Lys Arg Pro Phe Val Lys Leu Asn Val Glu Thr Asp Thr Ala Glu Ile Arg Leu Met Cys Thr Glu Thr Tyr Leu Asp Thr Pro Arg Pro Asp Asp Gly Ser Ile Thr Gly Pro Cys Glu Ser Asn Gly Asn Lys Gly Ser Gly Gly Ile Lys Gly Gly Phe Val His Gin Arg Met Ala Ser Lys Ile Gly Arg Trp Tyr Ser Arg Thr Met Ser Lys Thr Lys Arg Met Gly Met Gly Leu Tyr Val Lys Tyr Asp Gly Asp Pro Trp Thr Asp Ser Asp Ala Leu Ala Leu Ser Gly Val Met Val Ser Met Glu Glu Pro Gly Trp Tyr Ser Phe Gly Phe Glu Ile Lys Asp Lys Lys Cys Asp Val Pro Cys Ile Gly Ile Glu Met Val His Asp Gly Gly Lys Glu Thr Trp His Ser Ala Ala Thr Ala Ile Tyr Cys Leu Met Gly Ser Gly Gln Leu Leu Trp Asp Thr Val Thr Gly Val Asp Met Ala Leu

Claims (17)

1. A reassortant influenza virus comprising a genome segment encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 53.
2. The virus of claim 1, wherein the genome segment comprises a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 19.
3. The virus of claims 1 or 2, wherein the virus is a 6:2 reassortant virus comprising 6 internal genome segments from one or more donor viruses and 2 genome segments encoding surface antigens of an influenza virus.
4. The virus of claim 3, wherein the donor virus is A/Ann Arbor/6/60, B/Ann Arbor/1/66, or A/Puerto Rico/8/34.
5. The virus of claim 3, wherein the donor viruses comprises one or more phenotypic attributes selected from the group consisting of: attenuated, cold adapted and temperature sensitive.
6. An immunogenic composition comprising an immunologically effective amount of the reassortant influenza virus of any one of claims 1-5 and a physiologically acceptable carrier.
7. A live attenuated influenza vaccine comprising the immunogenic composition of claim 6.
8. A method for producing reassortant influenza viruses in cell culture, the method comprising:
i) introducing a plurality of vectors comprising polynucleotides corresponding to an influenza virus genome into a population of host cells capable of supporting replication of influenza virus, which plurality of vectors comprises polynucleotides corresponding to at least 6 internal genome segments of a first influenza strain, and at least one genome segment of a second influenza strain, wherein the at least one genome segment of the second influenza strain comprises a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 53;
ii) culturing the population of host cells; and iii) recovering a plurality of influenza viruses.
9. The method of claim 8, wherein the at least one genome segment of a second influenza strain comprises a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 19.
10. The method of claim 8 or 9, wherein the first influenza strain is an attenuated influenza virus strain, a cold-adapted influenza virus strain, or a temperature-sensitive influenza virus strain.
11. The method of any one of claims 8-10, wherein the plurality of vectors is eight vectors.
12. Use of a reassortant virus for prophylactic or therapeutic treatment of an influenza virus infection in a subject, the reassortant virus comprising:
a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO: 19; or b) a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 53, or a complementary polynucleotide sequence thereof;
in an amount effective to produce an immunogenic response against the viral infection.
13. An isolated or recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 53.
14. An isolated or recombinant polynucleotide, which polynucleotide is selected from the group consisting of:
a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO: 19, or a complementary sequence thereof; and b) a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 53, or a complementary polynucleotide sequence thereof.
15. An immunogenic composition comprising an immunologically effective amount of the polynucleotide of claim 14 or the polypeptide of claim 13 and a physiologically acceptable carrier.
16. A vector comprising the polynucleotide of claim 14.
17. A cell comprising the vector of claim 16.
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