US20080069821A1 - Influenza Hemagglutinin And Neuraminidase Variants - Google Patents

Abstract

Polypeptides, polynucleotides, methods, compositions, and vaccines comprising (avian pandemic) influenza hemagglutinin and neuraminidase variants are provided.

Description

CROSS REFERENCE TO RELATED APPLICATIONS
• This application claims the benefit under 35 U.S.C. § 119 (e) of U.S. Provisional Application Nos. 60/821,832 filed Aug. 9, 2006 and 60/942,804, filed Jun. 8, 2007, the disclosures of each of which are incorporated herein in their entirety for all purposes.
BACKGROUND OF THE INVENTION
• Vaccines against various and evolving strains of influenza are important from a community health stand point, 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 immuno-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 amongst various species, thereby necessitating the continuous production of new vaccines.
• Numerous vaccines capable of producing a protective immune response specific for such different and 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., U.S. Application Nos. 60/420,708, filed Oct. 23, 2002; 60/574,117, filed May 24, 2004; Ser. No. 10/423,828, filed Apr. 25, 2003; 60/578,962, filed Jun. 12, 2004; and Ser. No. 10/870,690 filed Jun. 16, 2004, the disclosure of which is incorporated by reference herein.
• Because of the continual emergence (or re-emergence) of different influenza strains, new influenza vaccines are continually desired. Such vaccines typically are created using antigenic moieties of the newly emergent virus strains, thus, polypeptides and polynucleotides of novel, newly emergent, or newly re-emergent virus strains (especially sequences of antigenic genes) are highly desirable.
• The present invention provides new and/or newly isolated influenza hemagglutinin and neuraminidase variants 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
• In some aspects herein, the invention comprises an isolated or recombinant polypeptide that is selected from: any one of the polypeptides encoded by SEQ ID NO:1 through SEQ ID NO:10 or SEQ ID NO:21 through SEQ ID NO:26 or SEQ ID NO:33 through SEQ ID NO:38; any one of the polypeptides of SEQ ID NO:11 through SEQ ID NO:20 or SEQ ID NO:27 through SEQ ID NO:32 or SEQ ID NO:39 through SEQ ID NO:44; only the open reading frame encoding the polypeptides of SEQ ID NO:11 through SEQ ID NO:20 or SEQ ID NO:27 through SEQ ID NO:32 or SEQ ID NO:39 through SEQ ID NO:44; any alternative (e.g., the mature form without the signal peptide, or without the 5′ and 3′ sequences outside of the open reading frame, or the sequences as expressed on the surface of a virus (e.g., influenza)) form of the polypeptides of SEQ ID NO:11-20 or SEQ ID NO:27-32 or SEQ ID NO:39-44; 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 SEQ ID NO:1 through SEQ ID NO:10 or SEQ ID NO:21 through SEQ ID NO:26, SEQ ID NO:33-38, or SEQ ID NO:45; any polypeptide that is encoded by a polynucleotide sequence which hybridizes under highly stringent conditions to a polynucleotide sequence of SEQ ID NO:1 through SEQ ID NO:10 or SEQ ID NO:21 through SEQ ID NO:26 or SEQ ID NO:33 through SEQ ID NO:38, or SEQ ID NO:45; and, a fragment of any of the above wherein the sequence comprises a hemagglutinin or neuraminidase polypeptide, or a fragment of a hemagglutinin or neuraminidase polypeptide, preferably where the fragments generate an antibody that specifically binds a full length polypeptide of the invention. 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, 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 520 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 above polypeptides. In some embodiments, the polypeptide sequence (e.g., as listed in “SEQUENCES” herein) 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. In still other embodiments, the invention comprises a polypeptide comprising a sequence having at least 85%, at least 90%, at least 93%, at least 95%, 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. The hemagglutinin sequences of the invention can comprise both those sequences with unmodified and modified polybasic cleavage sites (thereby allowing growth of the viruses in eggs). The hemagglutinin polypeptide sequences of SEQ ID NOS:11, 13, 15, 17, 19, 27, 29, 31, 39, 41, or 43 comprise the endogenous amino terminal signal peptide sequences, however, the hemagglutinin polypeptide sequences of the invention also include the mature (amino terminal signal peptide cleaved) form of the hemagglutinin polypeptides. The cleavage sites of any hemagglutinin polypeptide sequence of any influenza strain can be routinely measured or predicted using any number of methods in the art.
• 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.
• 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.
• Additionally, the invention includes recombinant influenza virus that comprises one or more of the polypeptides or polynucleotides 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.
• In other aspects, the invention comprises an isolated or recombinant nucleic acid that is selected from: any one of the polynucleotide sequences SEQ ID NO:1 through SEQ ID NO:10 or SEQ ID NO:21 through SEQ ID NO:26 or SEQ ID NO:33 through SEQ ID NO:38, or SEQ ID NO:45 (or complementary sequences thereof), any one of the polynucleotide sequences encoding a polypeptide of SEQ ID NO:11 through SEQ ID NO:20 or SEQ ID NO:27 through SEQ ID NO:32 or SEQ ID NO:39 through SEQ ID NO:44 (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 such polynucleotide sequences wherein the sequence preferably encodes a hemagglutinin or neuraminidase polypeptide or a fragment of a hemagglutinin or neuraminidase polypeptide. The invention also includes an isolated or recombinant nucleic acid that encodes an amino acid sequence which is substantially identical over at least about 300 amino acids of any polypeptide encoded by 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 polypeptide encoded by the above nucleic acids. Again, in situations wherein the amino acid is less than, e.g., 566, 565, 559, etc. in length (e.g., see, “SEQUENCES”) 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 polynucleotide encoding a hemagglutinin or neuraminidase polypeptide, or one or more fragments of one or more hemagglutinin or neuraminidase polypeptide. 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 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 polynucleotides. 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 sequences. 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).
• In yet other embodiments, the invention comprises a composition of matter having two or more above described nucleic acids (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.
• The invention also encompasses cells comprising at least one of the above described nucleic acids, 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 acids. Such vectors can optionally comprise an expression vector. Preferred expression vectors of the invention include, but are not limited to, vectors comprising pol I promoter and terminator sequences or vectors using both the pol I and pol II promoters “the polI/polII promoter system” (e.g., Zobel et al., Nucl. Acids Res. 1993, 21:3607; US20020164770; Neumann et al., Proc. Natl. Acad. Sci. USA 1999, 96:9345; Fodor et al., J. Virol. 1999, 73:9679; and US20030035814). Cells transduced by such vectors are also within the current invention.
• In some embodiments, the invention encompasses a virus (e.g., an influenza virus) comprising one or more above described nucleic acids (e.g., encoding hemagglutinin and/or neuraminidase), or one or more fragments thereof. Immunogenic compositions comprising such virus are also part of the current invention. Such viruses can comprises a reassortment virus such as a 6:2 reassortment virus (e.g., comprising 6 genes encoding regions from one or more donor virus and 2 genes encoding regions from one or more above described nucleotide sequence (or one or more fragment thereof) which can optionally comprise hemagglutinin and/or neuraminidase). Reassortment viruses (optionally live viruses) of the invention can include donor viruses that are one or more of, e.g., cold-sensitive, cold-adapted, or an attenuated. For example, reassortment viruses can comprise e.g., A/Ann Arbor/6/60, PR8, etc. Reassortment viruses of the invention may alternatively exclude A/Ann Arbor/6/60. One preferred embodiment of the invention is a reassortant influenza virus, wherein the virus is a 6:2 reassortment influenza virus and comprises 6 gene encoding regions from A/Ann Arbor/6/60 and 2 gene encoding regions that encode a polypeptide selected from the group consisting of: the polypeptides of SEQ ID NOS:11-20, SEQ ID NOS:27-32, and SEQ ID NOS:39-44. In an alternative embodiment, a reassortant influenza virus of the invention includes a 6:2 reassortment influenza virus, wherein said virus comprises 6 gene encoding regions from one or more donor viruses other than A/Ann Arbor/6/60 and 2 gene encoding regions that encode a polypeptide selected from the group consisting of: the polypeptides of SEQ ID NOS:11-20, SEQ ID NOS:27-32, and SEQ ID NOS:39-44. In another alternative embodiment, a reassortant influenza virus of the invention includes a 6:2 reassortment influenza virus, wherein said virus comprises 6 gene encoding regions from one or more donor viruses other than A/Ann Arbor/6/60 and 2 gene encoding regions, wherein the 2 gene encoding regions are HA or NA polypeptides from any pandemic influenza strain. Methods of producing 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 the recombinant influenza virus from one or more of the host cell or the medium are also part of the invention.
• In other embodiments herein, the invention comprises immunogenic compositions having an immunologically effective amount of any of the above described recombinant influenza 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 any of the recombinant influenza virus described above (optionally in a physiologically effective carrier).
• 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 cells or the medium in which is the cells are grown.
• Immunogenic compositions are also features of the invention. For example, immunogenic compositions comprising one or more of any of the polypeptides and/or nucleic acids described above 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 any one or more above described virus as well (e.g., along with one or more pharmaceutically acceptable administration component).
• Methods of producing immunogenic responses in a subject through administration of an effective amount of any of the above viruses (or immunogenic compositions) 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 any one or more above described virus (or immunogenic compositions) 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 are administered to the subject in an amount effect to prophylactically or therapeutically treat the viral infection.
• In other aspects the invention includes compositions of matter comprising nucleic acid sequences encoding hemagglutinin and/or neuraminidase polypeptides of one or more pandemic influenza strain and nucleic acid sequences encoding one or more polypeptide of A/Ann Arbor/6/60. Additionally, the invention includes compositions of matter comprising nucleic acid sequences encoding hemagglutinin and/or neuraminidase polypeptides of one or more pandemic influenza strain and nucleic acid sequences encoding one or more polypeptide of PR8 or A/Ann Arbor/6/60. Such sequences can include those listed in the “SEQUENCES” herein. Additionally, preferred embodiments of the invention include compositions of matter comprising sequences encoding hemagglutinin and/or neuraminidase of one or more pandemic influenza strain and nucleic acid sequences encoding a selected backbone strain in a 6:2 reassortment. Such compositions preferably include sequences encoding the hemagglutinin and neuraminidase selected from the “SEQUENCES” herein and a backbone strain, wherein the backbone strain is PR8 or A/Ann Arbor/6/60. The invention also includes such compositions as described above wherein the hemagglutinin comprises a modified polybasic cleavage site. The invention also includes live attenuated influenza vaccine comprising such above compositions.
• 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 and appendix.
BRIEF DESCRIPTION OF THE DRAWINGS
• FIG. 1: Shows modifications engineered into the HA gene of VN/1203/2004 to remove the polybasic cleavage site.
• FIG. 2: Displays results showing that intranasally administered H5N1 ca reassortant viruses do not replicate in chickens.
• FIG. 3: Illustrates that the H5N1/AA ca vaccine candidates are not lethal to mice.
• FIG. 4: Illustrates that the 1997 and 2004 H5N1 ca reassortant viruses are restricted in replication in mice.
• FIG. 5: Illustrates that the reassortant H5N1/AA ca influenza viruses are restricted in replication in lungs of mice.
• FIG. 6: Shows the serum HAI Ab titers elicited in mice following a single i.n. dose of vaccine.
• FIG. 7: Shows serum neutralizing Ab titers elicited in mice following a single i.n. dose of vaccine.
• FIG. 8: Illustrates that H5N1 ca reassortant viruses protect mice from lethal challenges with 50, 500 or 5000 LD₅₀of wild-type H5N1 viruses.
• FIG. 9: Illustrates the efficacy of protection from pulmonary replication of homologous and heterologous H5N1 challenge viruses in mice.
• FIG. 10: Illustrates the efficacy of protection from replication of homologous and heterologous H5N1 challenge viruses in the upper respiratory tract of mice.
• FIG. 11: Illustrates the efficacy of protection conferred by 2004 H5N1 ca vaccine against high dose (10₅TCID₅₀) challenge with homologous or heterologous H5N1 wt viruses in mice.
• FIG. 12: Illustrates the efficacy of protection conferred by 1997 and 2003 H5N1 ca vaccines against high dose (10₅TCID₅₀) challenges with homologous or heterologous H5N1 wild-type viruses in mice.
• FIG. 13: Illustrates the efficacy of protection conferred by 2004 H5N1 ca vaccine against low or high doses of homologous H5N1 wild-type virus challenges in mice.
• FIG. 14: Shows modifications that can be engineered into the HA gene of A/Netherland/219/03 HA to remove the polybasic cleavage site.
• FIG. 15: H5N1 ca vaccines elicit serum neutralizing antibody titers in mice. Sera were collected before (prebleed) and 28 days following each dose of vaccine; an undetectable titer is assigned a value of 10.
• FIG. 16: H6 ca viruses are attenuated in ferrets. *EID₅₀/g for lungs; PFU/g for nasal turbinates. 10⁷TCID₅₀inoculated intranasally, tissues were harvested onday 3 post-infection.
• FIG. 17: Immunogenicity of H6 ca vaccines in ferrets.
• FIGS.18(a) and18(b): Efficacy of H6 ca vaccines in ferrets. Virus titer was measured in (a) lungs and (b) nasal turbinates. Vaccine: 1 dose of 7 log₁₀PFU. Challenge: 7 log₁₀PFU; 3 days post-challenge.
• FIG. 19:H7N3 BC 04 ca is attenuated in ferrets. Inoculum: 10⁷TCID₅₀in 0.5 mL intranasally. Tissues were harvested onday 3 post-infection.
• FIG. 20:H7N3 BC 2004 ca is immunogenic in mice. a: Reciprocal geometric mean of serum neutralizing antibody titers against ck/BC/CN-6/04 wt. b: p<0.05 (Mann-Whitney U-test) compared to neutralization titers at 28 days post-infection.
• FIG. 21(a)-21(i):H7N3 BC 04 ca is efficacious against lethal challenge with H7 viruses in mice. Efficacy against a lethal challenge of 50 LD₅₀A/ck/BC/CN-7/04: four weeks following immunization with a single dose (a), eight weeks following immunization with a single dose (b), or eight weeks following immunization with 2 doses (2 doses administered at 4 weeks apart) (c). Efficacy against a lethal challenge of 50 LD₅₀A/NL/219/03: four weeks following immunization with a single dose (d), eight weeks following immunization with a single dose (e), or eight weeks following immunization with 2 doses (2 doses administered at 4 weeks apart) (f). Efficacy against a lethal challenge of 50 LD₅₀A/tk/Eng/63: four weeks following immunization with a single dose (g), eight weeks following immunization with a single dose (h), or eight weeks following immunization with 2 doses (2 doses administered at 4 weeks apart) (i).
• FIGS.22(a) and (b):H7N3 BC 04 ca vaccine is efficacious in mice. Virus titer was measured at 8 weeks in (a) nasal turbinates and (b) lungs.
• FIGS.23(a) and (b): H9N2 G9/AA ca is attenuated in ferrets. Virus titer was measured in (a) nasal turbinates and (b) lungs.
• FIGS.24(a) and (b): Efficacy of the H9N2 ca vaccine in mice.
• FIG. 25: Replication of H9N2 G9/AA ca is highly restricted in healthy adults.
• FIG. 26: HI antibody responses to 10^7.0TCID₅₀of H9N2 G9/AA ca in healthy adults.
• FIG. 27: Replication of H5N1 VN2004 A/AA ca is highly restricted in healthy adults.
• FIG. 28: HI antibody responses to 10^6.7TCID₅₀of VN2004 A/AA ca in healthy adults.
DETAILED DESCRIPTION
• The present invention includes polypeptide and polynucleotide sequences of influenza hemagglutinin and neuraminidase as well as vectors, compositions and the like comprising such sequences and methods of their use. Additional features of the invention are described in more detail herein.
Definitions
• 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.
• 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.
• 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.
• 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.
• 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′-O-methylated oligonucleotides), and the like. A nucleic acid can be e.g., single-stranded or double-stranded.
• 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 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.
• 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.
• 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.
• “Expression of a gene” or “expression of a nucleic acid” means transcription of DNA into RNA (optionally including modification of the RNA, e.g., splicing), 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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).
• 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.
• 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.
• The term “reassortant,” 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. 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 of the invention. 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 a different parental virus.
• 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 mitochondrial 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.
• The term “host cell” means a cell that contains a heterologous nucleic acid, such as a vector, and supports the replication and/or expression of the nucleic acid. Host cells can be prokaryotic cells such asE. 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., COS1, COS7 cells), etc.
• 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.
• 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 such influenza virus. In some instances, the influenza virus (e.g., naturally circulating) 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 vivo.
• 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 kD) and one “heavy” chain (about 50-70 kD). 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 (VH) 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
• The polypeptides and polynucleotides of the invention, e.g., SEQ ID NO:1-45, are variants of influenza HA and NA sequences. 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).
• Influenza is commonly grouped into influenza A and influenza B categories. 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 bysegment 2 andsegment 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.
• Influenza Virus Vaccines
• 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 β-propiolactone (or alternatively used in live attenuated vaccines). Thus, it will be appreciated that HA and NA sequences (e.g., SEQ ID NO:1-45) are quite useful in constructing influenza vaccines. The current invention includes viruses/vaccines comprising HA and/or NA sequences of pandemic influenza strains (including wherein the HA sequences comprise modified polybasic cleavage sites such as the modifications described herein); and including wherein the viruses/vaccines comprise a ca backbone such as A/AA/6/60 or the backbone of PR8.
• 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/or NA sequences herein. See, Multi-Plasmid System for the production of Influenza virus, U.S. Application No. 60/420,708, filed Oct. 23, 2002, U.S. application Ser. No. 10/423,828, filed Apr. 25, 2003 andU.S. Application 60/574,117 filed May 24, 2004. 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/or 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 Ser. No. 10/423,828, etc., various embodiments of the invention utilize A/Ann Arbor (AA)/6/60 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 PR8, etc., containing the other influenza genes present in a reassortant, namely, the non-HA and non-NA genes
• Various embodiments herein can comprise live attenuated vaccines, having the HA and/or NA sequences herein, for pandemic influenza. Such vaccines typically comprise, e.g., the HA and/or NA sequences of SEQ ID NO:11-20, 27-32, or 39-44, or their corresponding encoding nucleotides of SEQ ID NO:1-10, 21-26, 33-38, or 45. One problem arising from growth of vaccine virus strains (e.g., reassortants) in eggs is that avian strains (which can be involved in pandemics) can kill the eggs in which the vaccines are to be produced and are, thus, hard to manipulate, produce, etc. through use of traditional (non-plasmid rescue) reassortant production. Such avian strains are of interest since evidence indicates they can result in influenza in humans and possible pandemics. Thus, use of plasmid-rescue systems to create/manipulate influenza reassortants with pandemic strains such as various avian sequences (e.g., the HA and NA sequences herein) are quite desirable and are features of the invention. It will be appreciated, however, that the current sequences are also capable of use with non-plasmid or traditional systems.
• Aquatic birds (among others) can be infected by influenza A viruses of 15 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes. Such birds can serve as a reservoir from which novel influenza subtypes can be introduced into human populations and cause pandemics. The observation that avian H7N7 influenza A viruses infected humans in The Netherlands in 2003 and avian H5N1 and H9N2 viruses infected humans in Hong Kong and China earlier, raise concerns that these (and other) subtypes have the potential to cause pandemics. Thus, vaccines are needed to prevent human infections with avian influenza A viruses. 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 H3N2 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). Classical genetic reassortment and plasmid-based reverse genetics techniques have been applied to generate reassortant viruses that contain the hemagglutinin and neuraminidase genes from avian influenza A viruses (H4-H14 subtypes) and six internal gene segments from the AA ca virus. Such reassortant viruses are features of the invention. See the HA and NA gene sequences below. These viruses bear biological properties that are desirable in candidate vaccines because the phenotypes associated with the AA ca virus are present in the reassortant viruses. The generation and evaluation of these reassortant viruses as seed viruses for vaccines are important steps in pandemic preparedness. It is contemplated that clinical trials can establish the safety, infectivity and immunogenicity of such live attenuated pandemic vaccines. Other embodiments of the invention include reassortant viruses (e.g., those used in vaccines) comprising pandemic antigenic genes HA and/or NA from, e.g., avian, porcine, etc., pandemic virus strains in addition to those listed herein, to produce pandemic vaccines which are created through plasmid-rescue reassortment (e.g., reassortment with A/Ann Arbor 6/60 (i.e., A/AA/6/60), PR8, etc. Methods of construction and use of such viruses and vaccines are also included. “Pandemic virus strains” as used herein is defined as an influenza strain A virus subtype that it is not circulating in the human population, that is declared to be a pandemic strain by the Centers for Disease Control or the World Health Organization or generally acknowledged as such within the scientific community.
• In various embodiments herein, the antigenic sequences (e.g., the HA sequences) as well as viruses and vaccines from such viruses comprise modified polybasic cleavage sites. Some highly pathogenic avian pandemic influenza strains comprise multiple basic amino acid cleavage sites within hemagglutinin sequences. See, e.g., Li et al.,J. of Infectious Diseases,179:1132-8, 1999. Such cleavage sites, in typical embodiments herein, are, e.g., modified or altered in their sequences in comparison to the wild-type sequences from which the current sequences are derived (e.g., to disable the cleavage or reduce the cleavage there, etc.). Such modifications/alterations can be different in different strains due to the various sequences of the cleavage sites in the wild-type sequences. For example, 4 polybasic residues (RRKK) at 326-329 of mature H5 are typically removed in sequences herein (as compared to wt). See “SEQUENCES.” In various embodiments, the polybasic cleavage sites can be modified in a number of ways (all of which are contained within the invention). For example, the polybasic cleavage site can be removed one amino acid at a time (e.g., one R removed, two Rs removed, RRK removed, or RRKK removed). Additionally, the amino acid residue directly upstream of the cleavage site can also be removed or altered (e.g., from an R to a T, etc.); also, the nucleotides encoding the amino acid residue directly after the cleavage site can also be modified. See, e.g.,FIG. 1 for an illustration of cleavage site modification. In addition, hemagglutinin polypeptide sequences of influenza virus comprise amino terminal signal peptide sequences, thus, the hemagglutinin polypeptide sequences of the invention include both the mature (amino terminal signal peptide cleaved) form of the hemagglutinin polypeptides and the pre-cleaved form of hemagglutinin. The cleavage sites of any hemagglutinin polypeptide sequence of any influenza strain can be routinely measured or predicted using any number of methods in the art.
• 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 B 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” (att) 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.
• Again, the HA and NA sequences of the current invention are optionally utilized in the production of or in reassortment vaccines (and/or in other ts, cs, ca, 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 utilizes strain specific HA and NA antigens (e.g., any of SEQ ID NO:11-20, 27-32, or 39-44 or the corresponding nucleotides encoding the specific HA and NA antigens, e.g., SEQ ID NO:1-10, 21-26, 33-38, or 45).
• FLUMIST™
• As mentioned previously, numerous examples and types of influenza vaccine exist. An exemplary influenza vaccine is FluMist™ 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 Med338:1405-12; Nichol et al. (1999)Effectiveness of live, attenuated intranasal influenza virus vaccine in healthy, working adults: a randomized controlled trial JAMA282:137-44). In typical, and preferred, embodiments, the methods and compositions of the current invention are preferably adapted to/used with production of FluMist™ 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.
• FluMist™ vaccine strains contain, e.g., HA and NA gene segments derived from the strains (e.g., wild-type strains) to which the vaccine is addressed along with six gene segments, PB1, PB2, PA, NP, M and NS, from a common master donor virus (MDV). The HA sequences herein, thus, are part of various FluMist™ formulations. The MDV for influenza A strains of FluMist™ (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 at25degrees C. Nature213:612-4). MDV-A replicates efficiently at 25° C. (ca, cold adapted), but its growth is restricted at 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 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 Immunol15:295-323).
• 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 Med338:1405-12; Boyce et al. (2000)Safety and immunogenicity of adjuvanted and unadjuvanted subunit influenza vaccines administered intranasally to healthy adults Vaccine19:217-26; Edwards et al. (1994)A randomized controlled trial of cold adapted and inactivated vaccines for the prevention of influenza A disease J Infect Dis169:68-76; Nichol et al. (1999)Effectiveness of live, attenuated intranasal influenza virus vaccine in healthy, working adults: a randomized controlled trial JAMA282: 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).
• Production of such reassorted virus using B strains of influenza is more difficult, however, recent work (see, e.g., Multi-Plasmid System for the Production of Influenza Virus, U.S. Application No. 60/420,708, filed Oct. 23, 2002, U.S. application Ser. No. 10/423,828, filed Apr. 25, 2003, and U.S. Application No. 60/574,117, filed May 24, 2004) 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.
• 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 (e.g., nucleotide sequences SEQ ID NO:1-10, 21-26, 33-38, or 45 and the corresponding amino acids encoded by the nucleotide sequences in SEQ ID NO:11-20, 27-32, or 39-44), methods, etc. of the current invention, are optionally used in conjunction with, or in combination with, such previous work involving, e.g., reasserted influenza viruses for vaccine production to produce viruses for vaccines.
• Methods and Compositions for Prophylactic Administration of Vaccines
• As stated above, alternatively, or in addition to, use in production of FluMist™ 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:1-10, 21, 23-26, 33-38, or 45 or polypeptides of SEQ ID NO:11-20, 27-32, or 39-44, or fragments thereof) can be administered prophylactically in an immunologically effective amount and in an appropriate carrier or excipient to stimulate an immune 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, 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.
• 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., comprising an HA and/or 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.
• 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., U.S. Pat. No. 5,922,326; Wright et al.,Infect. Immun.37:397-400 (1982); Kim et al.,Pediatrics52:56-63 (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 10⁵-10⁸pfu (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.
• 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.
• 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.
• 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.
• 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 adjuvant QS-21.
• 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 cytokines, lymphokines and chemokines with immunostimulatory, immunopotentiating, and pro-inflammatory activities, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-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, e.g., any of SEQ ID NO:11-20, 27-32, or 39-44) or an expression vector comprising a nucleic acid (e.g., any of SEQ ID NO:1-10, 21-26, 33-38, or 45) encoding the protein can be administered to produce an immunostimulatory effect.
• 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 as described above in the sections entitled “Expression Vectors” and “Additional Expression Elements.” 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, cytokines, antibodies, etc., and/or markers, and the like.
• 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 (e.g., those against pandemic influenza strains such as various avian strains, see, e.g., the sequences herein, or other pandemic strains) 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.
• Polynucleotides of the Invention
• It is well known in the art that the HA and NA polynucleotide segments of influenza viruses comprise both a coding region (encoding the ORF) and noncoding regions (NCRs), both 5′ and 3′ of the HA and NA coding sequence. An example of these NCRs are shown in SEQ ID NOS:1-9 (outside of the ORFs). It is also known that primers can be made to these NCRs to facilitate amplification of the entire HA and NA segments of influenza virus. (see, e.g., Hoffmann et al. Arch Virol. 2001 December; 146(12):2275-89). Further, it is known that the NCRs of the HA and NA of influenza may increase the efficiency of achieving reassortants. Therefore, the polynucleotide sequences of these NCRs (including fragments and variants (e.g., at least about 60%, or at least 70%, or at least 80%, or at least 90%, or at least about 91% or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 98.5%, or at least about 98.7%, or at least about 99%, or at least about 99.1%, or at least about 99.2%, or at least about 99.3%, or at least about 99.4%, or at least about 99.5%, or at least about 99.6% or at least about 99.7%, or at least about 99.8%, or at least about 99.9% identity) thereof) are within the scope of this invention. When amplifying the HA and NA segments of any pandemic strain, one could make and use polynucleotide primers to bind conserved (e.g., among related strains) regions of the HA and NA NCRs for amplification (e.g., by RT-PCR). In one embodiment, HA and NA polynucleotides of the invention include both the NCR and ORF of the HA and NA sequences (including fragments and variants (e.g., at least about 60%, or at least 70%, or at least 80%, or at least 90%, or at least about 91% or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 98.5%, or at least about 98.7%, or at least about 99%, or at least about 99.1%, or at least about 99.2%, or at least about 99.3%, or at least about 99.4%, or at least about 99.5%, or at least about 99.6% or at least about 99.7%, or at least about 99.8%, or at least about 99.9%) thereof) of pandemic virus strains. In alternative embodiments, the HA and NA polynucleotides of the invention exclude the NCR, but include the ORF (including fragments and variants (e.g., at least about 60%, or at least 70%, or at least 80%, or at least 90%, or at least about 91% or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 98.5%, or at least about 98.7%, or at least about 99%, or at least about 99.1%, or at least about 99.2%, or at least about 99.3%, or at least about 99.4%, or at least about 99.5%, or at least about 99.6% or at least about 99.7%, or at least about 99.8%, or at least about 99.9% thereof)) of the HA and NA sequences of pandemic virus strains (e.g., SEQ ID NOS:1-9).
• The HA and NA polynucleotides of the invention, e.g., SEQ ID NO:1 through SEQ ID NO:10, SEQ ID NO:21 through SEQ ID NO:26, SEQ ID NO:33 through SEQ ID NO:38, SEQ ID NO:45, 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. Different methods of construction, purification, and characterization of the nucleotide sequences of the invention are also described herein. 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 the 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 through SEQ ID NO:10, SEQ ID NO:21 through SEQ ID NO:26, SEQ ID NO:33 through SEQ ID NO:38, SEQ ID NO:45, 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 HA 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.
• 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 the sequences herein, 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.
• 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 Letts22(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 Chrom255:137-149. 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 Enzymology65:499-560. Custom oligos can also easily be ordered from a variety of commercial sources known to persons of skill.
• 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 (e.g., SEQ ID NO:11-20, SEQ ID NO: 27-32, SEQ ID NO:39-44) and/or encoded by polynucleotide sequences of the invention, e.g., selected from SEQ ID NO:1 through SEQ ID NO:10, SEQ ID NO: 21 through SEQ ID NO: 26, SEQ ID NO:33 through SEQ ID NO:38, and SEQ ID NO:45 are favorably used to identify and isolate antibodies, e.g., from phage display libraries, combinatorial libraries, polyclonal sera, and the like. Polypeptide fragments of the inventions include a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, or at least 10 contiguous amino acid residues, or at least 15 contiguous amino acid residues, or at least 20 contiguous amino acid residues, or at least 25 contiguous amino acid residues, or at least 40 contiguous amino acid residues, or at least 50 contiguous amino acid residues, or at least 60 contiguous amino residues, or at least 70 contiguous amino acid residues, or at least contiguous 80 amino acid residues, or at least contiguous 90 amino acid residues, or at least contiguous 100 amino acid residues, or at least contiguous 125 amino acid residues, or at least 150 contiguous amino acid residues, or at least contiguous 175 amino acid residues, or at least contiguous 200 amino acid residues, or at least contiguous 250 amino acid residues, or at least contiguous 350, or at least contiguous 400, or at least contiguous 450, or at least contiguous 500, or at least contiguous 550 amino acid residues of the amino acid sequence an HA or NA polypeptide of the invention (e.g., SEQ ID NOS:11-20, SEQ ID NOS: 27-32, and SEQ ID NOS: 39-44). Polynucleotides encoding said polypeptide fragments and antibodies that specifically bind said polypeptides are also preferred embodiments of the invention.
• Antibodies specific for any polypeptide sequence or subsequence, e.g., of SEQ ID NO:11 through SEQ ID NO: 20, SEQ ID NO: 27 through SEQ ID NO: 32, and/or SEQ ID NO: 39 through SEQ ID NO: 44, and/or encoded by polynucleotide sequences of the invention, e.g., selected from SEQ ID NO:1 through SEQ ID NO:10, SEQ ID NO: 21 through SEQ ID NO: 26, SEQ ID NO: 33 through SEQ ID NO: 38, and SEQ ID NO:45 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. Additional details regarding production of specific antibodies are provided below.
• Diagnostic Assays
• 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.
• The probes of the invention, e.g., as exemplified by unique subsequences selected from those given herein, 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.
• 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.
• 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 T_mwhere 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
• 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:10, SEQ ID NO:21 through SEQ ID NO:26, SEQ ID NO:33 through SEQ ID NO:38, SEQ ID NO:45 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.
• 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.
• 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. colilac or trp promoter, phage lambda P_Lpromoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
• A variety of promoters are suitable for use in expression vectors for regulating transcription of influenza virus genome segments. In certain embodiments, the cytomegalovirus (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.
• 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 aposition 5′ or 3′ to the heterologous coding sequence, but is typically inserted at asite 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 Differ20:125-62; Kriegler et al. (1990)Assembly of enhancers, promoters, and splice signals to control expression of transferred genes Methods in Enzymol185: 512-27). Optionally, the amplicon can also contain a ribosome binding site or an internal ribosome entry site (IRES) for translation initiation.
• 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 (+) strand mRNA molecules from the Poll promoter initiating replication of the (−) strand viral genome.
• 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.
• 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, BHK cells, MDCK cell, 293 cells, COS cells, or the like, for the purpose of expression.
• 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., U.S. Application Nos. 60/420,708, filed Oct. 23, 2002; 60/574,117, filed May 24, 2004; Ser. No. 10/423,828, filed Apr. 25, 2003; 60/578,962, filed Jun. 12, 2004; and Ser. No. 10/870,690 filed Jun. 16, 2004; and US20020164770, which are incorporated by reference herein. For example, preferred expression vectors of the invention include, but are not limited to, vectors comprising pol I promoter and terminator sequences or vectors using both the pol I and pol II promoters “the polI/polII promoter system” (e.g., Zobel et al., Nucl. Acids Res. 1993, 21:3607; US20020164770; Neumann et al., Proc. Natl. Acad. Sci. USA 1999, 96:9345; Fodor et al., J. Virol. 1999, 73:9679; and US20030035814). 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 (and/or derivatives and modifications thereof), the PR8 donor strain backbone, the A/Leningrad/17 donor strain backbone, etc. Other backbone strains are described, for example, in 20040137013 and 20030147916, which are incorporated by reference herein.
• Additional Expression Elements
• 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.
• 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.
• 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 IRES 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 Differ20:125-62; Bittner et al. (1987)Methods in Enzymol153:516-544).
• Production of Recombinant Virus
• Negative strand RNA viruses can be genetically engineered and recovered using a recombinant reverse genetics approach (see, e.g., U.S. Pat. No. 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. USA92:11563-11567), 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. USA92: 4477-4481); measles virus (Radecke et al. (1995)EMBO J.14:5773-5784); rinderpest virus (Baron & Barrett (1997)J. Virol.71: 1265-1271); human parainfluenza virus (Hoffman & Banerjee (1997)J. Virol.71: 3272-3277; Dubin et al. (1997)Virology235:323-332); SV5 (He et al. (1997)Virology237:249-260); canine distemper virus (Gassen et al. (2000)J. Virol.74:10737-44); and Sendai virus (Park et al. (1991)Proc. Natl. Acad. Sci. USA88: 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.
• Cell Culture and Expression Hosts
• 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 asE. coli, Streptomyces, andSalmonella typhimurium; fungal cells, such asSaccharomyces cerevisiae, Pichia pastoris, andNeurospora crassa; or insect cells such asDrosophilaandSpodoptera frugiperda.
• 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 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 CO₂concentration 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, β-mercaptoethanol, and the like.
• 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. 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,3^rdedition, Wiley-Liss, New York and the references cited therein. Other helpful references include, e.g., Paul (1975)Cell and Tissue Culture,5^thed., 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.
• 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 cases, 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, Minn.); NBS bioreactors (New Brunswick Scientific, Edison, N.J.); laboratory and commercial scale bioreactors from B. Braun Biotech International (B. Braun Biotech, Melsungen, Germany).
• 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., Multi-Plasmid System for the Production of Influenza Virus, U.S. Application No. 60/420,708, filed Oct. 23, 2002, U.S. application Ser. No. 10/423,828, filed Apr. 25, 2003, and U.S. Application No. 60/574,117, filed May 24, 2004), 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.).
• 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 μg of each vector is introduced into a population of host cells with approximately 2 μl of TransIT-LT1 diluted in 160 μl medium, preferably serum-free medium, in a total volume of 200 μl. The DNA:transfection reagent mixtures are incubated at room temperature for 45 minutes followed by addition of 800 μl 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 μl 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.
• 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 5×10⁶Vero cells, e.g., grown in Modified Eagle's Medium (MEM) supplemented with 10% Fetal Bovine Serum (FBS) are resuspended in 0.4 ml OptiMEM and placed in an electroporation cuvette. Twenty micrograms of DNA in a volume of up to 25 μl 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% FBS. 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.
• 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 E1 or E3 region of the viral genome will result in a viable virus capable of expressing the polypeptides of interest in infected host cells (Logan and Shenk (1984)Proc Natl Acad Sci81:3655-3659). In addition, transcription enhancers, such as the rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
• 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.
• 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.
• 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.
• 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 SystemsJohn Wiley & Sons, Inc. New York, N.Y.; 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, Fla.
• 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, multifunctionalE. colicloning and expression vectors such as BLUESCRIPT (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; pIN vectors (Van Heeke & Schuster (1989)J Biol Chem264:5503-5509); pET vectors (Novagen, Madison Wis.); and the like. Similarly, in the yeastSaccharomyces cerevisiaea 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 Enzymology153:516-544.
• Nucleic Acid Hybridization
• Comparative hybridization can be used to identify nucleic acids (e.g., SEQ ID NO:1-10, SEQ ID NO: 21-26, SEQ ID NO:33-38, SEQ ID NO:45) 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., those shown herein 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.
• 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 and target under conditions in which a perfectly matched probe binds to a perfectly matched complementary target with a signal to noise ratio that is at least about 5×-10× as high as that observed for hybridization to any of the unmatched target nucleic acids.
• Nucleic acids “hybridize” when they associate, typically in solution. Nucleic acids hybridize due to a variety of well-characterized physico-chemical 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 Probespart 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 Probes1 IRL Press at Oxford University Press, Oxford, England, (Hames and Higgins 1) and Hames and Higgins (1995)Gene Probes2 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.
• 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 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook, infra for a description of SSC buffer and other nucleic acid hybridization parameters). Often the high stringency wash is preceded by a low stringency wash to remove background probe signal. An example low stringency wash is 2×SSC at 40° C. for 15 minutes. In general, a signal to noise ratio of 5× (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
• 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 T_m) 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).
• “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 5× as high as that observed for hybridization of the probe to an unmatched target.
• In general, a signal to noise ratio of at least 2× (or higher, e.g., at least 5×, 10×, 20×, 50×, 100×, 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.
• “Very stringent” conditions are selected to be equal to the thermal melting point (T_m) for a particular probe. The T_mis 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_mfor 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.
• “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 a probe to a perfectly matched complementary target nucleic acid is at least 10× 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.
• 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 (e.g., SEQ ID NO:1-10, 21-26, 33-38, SEQ ID NO:45) 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 2× (and optionally 5×, 10×, or 100× 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.
• 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, e.g., SEQ ID NO:1-10, 21-26, 33-38, 45).
• 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 10×, 20×, 50×, 100×, or 500× 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 calorimetric 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 and are also features of the invention.
• 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
• 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 Enzymologyvolume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al.,Molecular Cloning—A Laboratory Manual(3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2000 (“Sambrook”) andCurrent 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.
• 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 as in SEQ ID NO: 11-20 or 27-32 or 39-44) 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.
• 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 Biochem254(2): 157-178 (1997); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioate method, Methods Mol Biol57:369-374 (1996); I. A. Lorimer, I. Pastan,Nucleic Acids Res23, 3067-8 (1995); W. P. C. Stemmer,Nature370, 389-91 (1994); Arnold,Protein engineering for unusual environments, Current Opinion in Biotechnology4:450-455 (1993); Bass et al.,Mutant Trp repressors with new DNA-binding specificities, Science242:240-245 (1988); Fritz et al.,Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro, Nucl Acids Res16: 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 Res16: 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 Res14: 6361-6372 (1988); Sayers et al.,Y-T Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, Nucl Acids Res16:791-802 (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 Res16: 803-814; Carter,Improved oligonucleotide-directed mutagenesis using M13vectors, Methods in Enzymol154: 382-403 (1987); Kramer & FritzOligonucleotide-directed construction of mutations via gapped duplex DNA, Methods in Enzymol154:350-367 (1987); Kunkel,The efficiency of oligonucleotide directed mutagenesis, inNucleic 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 Enzymol154, 367-382 (1987); Zoller & Smith,Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template, Methods in Enzymol154:329-350 (1987); Carter,Site-directed mutagenesis, Biochem J237:1-7 (1986); Eghtedarzadeh & Henikoff,Use of oligonucleotides to generate large deletions, Nucl Acids Res14: 5115 (1986); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis, Proc Natl 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 Res14: 9679-9698 (1986); Wells et al.,Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin, Phil Trans R Soc Lond A317: 415-423 (1986); Botstein & Shortle,Strategies and applications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter et al.,Improved oligonucleotide site-directed mutagenesis using M13vectors, Nucl Acids Res13: 4431-4443 (1985); Grundström et al.,Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’ gene synthesis, Nucl Acids Res13:3305-3316 (1985); Kunkel,Rapid and efficient site-specific mutagenesis without phenotypic selection, Proc Natl Acad Sci USA82:488-492 (1985); Smith,In vitro mutagenesis, 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 Res13: 8749-8764 (1985); Taylor et al.,The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA, Nucl Acids Res13: 8765-8787 (1985); Wells et al.,Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites, Gene34:315-323 (1985); Kramer et al.,The gapped duplex DNA approach to oligonucleotide-directed mutation construction, Nucl Acids Res12: 9441-9456 (1984); Kramer et al.,Point Mismatch Repair, Cell38:879-887 (1984); Nambiar et al.,Total synthesis and cloning of a gene coding for the ribonuclease S protein, Science223: 1299-1301 (1984); Zoller & Smith,Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors, Methods in Enzymol100:468-500 (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 Res10:6487-6500 (1982). Additional details on many of the above methods can be found inMethods inEnzymolVolume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis, gene isolation, expression, and other methods.
• 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 Letts22(20):1859-1862, (1981) e.g., using an automated synthesizer, as described in Needham-Van Devanter et al.,Nucleic Acids Res,12:6159-6168 (1984).
• 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 (mcrc@oligos.com), The Great American Gene Company (www.genco.com), ExpressGen Inc. (www.expressgen.com), Operon Technologies Inc. (Alameda, Calif.) and many others. Similarly, peptides and antibodies can be custom ordered from any of a variety of sources, such as PeptidoGenic (available at pkim@ccnet.com), HTI Bio-products, Inc. (www.htibio.com), BMA Biomedicals Ltd. (U.K.), Bio.Synthesis, Inc., and many others.
• The present invention also relates to host cells and organisms comprising a HA and/or NA molecule or other polypeptide and/or nucleic acid of the invention, e.g., SEQ ID NOS:1-45. 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 plasmid, 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 USA82, 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.,Nature327, 70-73 (1987)). Berger, Sambrook, and Ausubel provide a variety of appropriate transformation methods. See, above.
• 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., EasyPrep™, FlexiPrep™, both from Pharmacia Biotech; StrataClean™, from Stratagene; and, QIAprep™ 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 optionally both. See, Giliman & Smith,Gene8:81 (1979); Roberts, et al.,Nature,328:731 (1987); Schneider, B., et al.,Protein Expr Purif6435:10 (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 Bacteriophage(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 EditionScientific American Books, NY. See, above. Further vectors useful with the sequences herein are illustrated above in the section concerning production of influenza virus for vaccines and the references cited therein.
• Polypeptide Production and Recovery
• Following transduction of a suitable host cell line or strain and growth of the host cells to an appropriate cell density, the 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.
• 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,2^ndEditionWiley-Liss, NY; Walker (1996)The Protein Protocols HandbookHumana Press, NJ, Harris and Angal (1990)Protein Purification Applications: A Practical ApproachIRL Press at Oxford, Oxford, England; Harris and AngalProtein Purification Methods: A Practical ApproachIRL Press at Oxford, Oxford, England; Scopes (1993)Protein Purification: Principles andPractice3^rdEditionSpringer Verlag, NY; Janson and Ryden (1998)Protein Purification: Principles, High Resolution Methods and Applications, Second EditionWiley-VCH, NY; and Walker (1998)Protein Protocols on CD-ROMHumana Press, NJ.
• 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×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×g, for approximately 3-5 hours. Following resuspension of the virus pellet in an appropriate buffer, such as STE (0.01 M Tris-HCl; 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 Furminger.Vaccine Production, in Nicholson et al. (eds.)Textbook of Influenzapp. 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 Vaccinespp. 141-151, and U.S. Pat. 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
• Alternatively, cell-free transcription/translation systems can be employed to produce polypeptides comprising an amino acid sequence or subsequence of, e.g., the sequences given herein such as SEQ ID NOS:11-20 or 27-32 or 39-44, or encoded by the polynucleotide sequences of the invention, e.g., SEQ ID NOS:1-10 or 21-26 or 33-38 or 45. 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 inMolecular BiologyVolume 37, Garland Publishing, NY.
• 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 Soc85:2149-2154). Exemplary automated systems include the Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.). If desired, subsequences can be chemically synthesized separately, and combined using chemical methods to provide full-length polypeptides.
• Modified Amino Acids
• 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-S/T motifs during expression in mammalian cells) or modified by synthetic means (e.g., via PEGylation).
• 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-ROMHuman Press, Towata, N.J.
• Fusion Proteins
• The present invention also provides fusion proteins comprising fusions of the sequences of the invention (e.g., encoding HA and/or NA polypeptides as exampled by SEQ ID NOS:11-20, 27-32, and 39-44) 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 immunoglobulin 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
• 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.
• 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.
• 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.
• 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 ImmunologyWiley/Greene, NY; Paul (ed.) (1998)Fundamental Immunology Fourth Edition, Lippincott-Raven, Lippincott Williams & Wilkins; Harlow and Lane (1989)Antibodies: A Laboratory ManualCold Spring Harbor Press, NY; Stites et al. (eds.)Basic and Clinical Immunology(4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therein; Goding (1986)Monoclonal Antibodies: Principles and Practice(2d ed.) Academic Press, New York, N.Y.; 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)Science246: 1275-1281; and Ward, et al. (1989)Nature341: 544-546. Specific monoclonal and polyclonal antibodies and antisera will usually bind with a K_Dof, e.g., at least about 0.1 μM, at least about 0.01 μM or better, and, typically and at least about 0.001 μM or better.
• For certain therapeutic applications, humanized antibodies are desirable. Detailed methods for preparation of chimeric (humanized) antibodies can be found in U.S. Pat. No. 5,482,856. Additional details on humanization and other antibody production and engineering techniques can be found in Borrebaeck (ed.) (1995)Antibody Engineering,2^ndEditionFreeman and Company, NY (Borrebaeck); McCafferty et al. (1996)Antibody Engineering, A Practical ApproachIRL at Oxford Press, Oxford, England (McCafferty), and Paul (1995)Antibody Engineering ProtocolsHumana Press, Towata, N.J. (Paul). Additional details regarding specific procedures can be found, e.g., in Ostberg et al. (1983),Hybridoma2: 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
• 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.
• 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 (e.g., SEQ ID NOS:11-20, 27-32, and 39-44), 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.
• 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 (e.g., SEQ ID NOS:11-20, 27-32, and 39-44), 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.
• 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.
• 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 10⁶or greater are selected, pooled and subtracted with the control hemagglutinin and/or neuraminidase polypeptide(s) to produce subtracted pooled titered polyclonal antisera.
• 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 2-5× higher signal to noise ratio than the control receptor homologues under discriminatory binding conditions, and at least about a ½ 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.
• 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.
• 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 5-10× 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.
• 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 5-10× as high as for the control polypeptide.
• 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 2× 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
• 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 (e.g., SEQ ID NOS:1-45). 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.
• 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
• 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	UUC 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	Gln	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 GUU
  	Tryptophan	Trp	W	UGG
  	Tyrosine	Tyr	Y	UAC UAU

• The codon table shows that many amino acids are encoded by more than one codon. For example, the codons AGA, AGG, CGA, CGC, CGG, 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.
• 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 standard 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
• 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.
• “Conservative variations” 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 variations” 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.

  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)
  5	Isoleucine (I)	Leucine (L)	Methionine (M)	Valine (V)
  6	Phenylalanine (F)	Tyrosine (Y)	Tryptophan (W)

• Unique Polypeptide and Polynucleotide Subsequences
• 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 disclosed herein, e.g., SEQ ID NOS:1-10, 21-26, 33-38, and 45. 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. 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.
• Similarly, the invention includes a polypeptide which comprises a unique subsequence in a polypeptide selected from the sequence of HA and NA molecules disclosed herein, e.g., SEQ ID NOS:11-20, 27-32, and 39-44. 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.
• 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
• 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.
• 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, at least about 250 residues, 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.
• 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.
• Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv Appl Math2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch,J Mol Biol48:443 (1970), by the search for similarity method of Pearson & Lipman,Proc Natl Acad Sci USA85:2444 (1988), by computerized implementations of algorithms such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis., or by visual inspection (see generally, Ausubel et al., supra).
• 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 Biol215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). 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 USA89:10915).
• 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 USA90:5873-5787 (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.
• Another example of a useful sequence alignment algorithm is PILEUP. PILEUP 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. PILEUP 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.
• 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. USA89: 10915-10919.
• Digital Systems
• 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.
• Various methods known in the art can be used to detect homology or similarity between different character strings (see, above), or can 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.
• 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 4 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.).
• Thus, standard desktop applications such as word processing software (e.g., Microsoft Word™ or Corel WordPerfect™) and database software (e.g., spreadsheet software such as Microsoft Excel™, Corel Quattro Pro™, or database programs such as Microsoft Access™, Paradox™, GeneWorks™, or MacVector™ 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).
• 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 DOS™, OS2™ WINDOWS™ WINDOWSNT™, WINDOWS95™, WINDOWS2000™, WINDOWS98™, LINUX based machine, a MACINTOSH™, Power PC, or a UNIX based (e.g., SUN™ 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.
• 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.
• 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
• 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 can also further comprise, 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.
• 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.
• 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.
• 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.
• To facilitate use of the methods and compositions of the invention, any of the vaccine components and/or compositions, e.g., reasserted 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.
EXAMPLESExample 1Construction and Analysis of H5N1 ca Viruses and Vaccines
• Various sequences herein comprising H5N1 HA/NA sequences were used to create influenza viruses and vaccines. The HA sequences in such vaccines were altered from wild-type by removal of the polybasic cleavage site within the HA. The HA/NA sequences were reasserted (in a 6:2 reassortment) with ca A/AA/6/60 (a ts, att, ca virus, see above).
• Three strains of H5N1 influenza were used in this example: A/VN/1203/2004, A/HK/491/1997, and A/HK/213/2003. Such strains are also referred to within this example as the '97, '03, and '04 strains based on their year designations. The HA sequence homology of these three strains is 95-96%.FIG. 1 illustrates modification of the polybasic cleavage site of an exemplary HA sequence, the '04 HA sequences, used to construct the viruses/vaccines. As stated previously, various embodiments of the invention comprise sequences which have differing regions of the polybasic cleavage site removed. See above.
• As stated, the modified H5N1 sequences (i.e., the modified '97, '03, and '04 genes) were used to construct 6:2 reassortant viruses with ca A/AA/6/60. It will be appreciated, and is pointed out elsewhere herein, that other desirable backbones could also have been used (e.g., PR8, etc.).
• In the 6:2 reassortants of this example, the HA and NA gene sequences were derived from one or more wild type parent virus, i.e., the HA and NA gene sequences of the '03 virus were derived from A/HK/213/2003, the HA and NA gene sequences of the '04 virus were derived from A % VN/1203/2004, and the HA gene sequence of the '97 virus was derived from A/HK/491/1997 while the NA gene sequence was derived from A/HK/486/1997. The remaining genes of the 6:2 reassortants were characterized by sequence analysis as derived from the A/AA/6/60 ca parent virus. The reasserted viruses replicated to 8.0-8.5 log₁₀TCID₅₀in eggs. However, it will be appreciated that other embodiments wherein the log₁₀TCID₅₀comprises from about 7.0 to about 9.0, from about 7.5-8.5, or from about 8.0-8.5 are also within the scope of the invention. The cleavability of the modified HA in the constructed viruses by endogenous proteases was restricted in vitro and the viruses were dependent on trypsin (e.g., from about 0.1 ug/ml to about 1.0 ug/ml) for growth. The constructed viruses were temperature sensitive as assayed by an in vitro assay.
• The H5N1 ca reassortant viruses (having the modified '97, '03, or '04 HA genes) were not lethal for chickens. For example, when 4-week-old SPF white Plymouth Rock chickens were inoculated intravenously with a 1:10 dilution of stock virus (10^8-8.75TCID₅₀/ml) and observed for 10 days, it was observed that 8 out of 8 chickens died within 1-2 days when wild-type '97, '03, and '04 H5N1 were used, while 0 of 8 chickens died when the H5N1 ca reassortant viruses were used. As can be seen inFIG. 2, the intranasally administered H5N1 ca reassortant viruses did not replicate in chickens.
• The H5N1/AA ca reassortants were also not lethal for mice. SeeFIG. 3, which also shows the TCID₅₀for the H5N1 wild-type strains.FIG. 4 shows that the 1997 and 2004 H5N1 ca reassortant viruses were restricted in replication in mice.FIG. 5, shows that the H5N1 ca reassorted viruses are restricted in replication in lungs of mice.
• A comparison of the serum HAI antibody titers elicited in mice following a single intranasal dose of vaccine (2003 ca as compared against 2003 wild-type), is shown inFIG. 6.FIGS. 7 and 15 show similar measurements, but using serum neutralizing antibody titers.
• FIG. 8 displays that the H5N1 ca reassortant viruses protect mice from lethal challenge with 50, 500, or 5,000 LD₅₀of wild-type H5N1 virus.FIG. 9 shows the efficacy of protection from pulmonary replication of homologous and heterologous H5N1 challenge viruses in mice. As can be seen, the ca reassortants replicated less well than the wild-type viruses did.FIG. 10 shows related data using upper respiratory tracts of mice. Those of skill in the art will be familiar with homologous and heterologous challenges (e.g., testing whether 2003 vaccine protects against a 2003 wild-type challenge (homologous) or whether a 2003 vaccine protects against a 1997 wild-type challenge (heterologous), etc.).
• FIG. 11 shows efficacy of protection conferred by 2004 H5N1 ca vaccine against high dose (10⁵TCID₅₀) challenge with homologous or heterologous H5N1 wild-type viruses in mice.FIG. 12 shows efficacy of protection conferred by 1997 and 2003 H5N1 ca vaccines against high dose (10⁵TCID₅₀) challenge with homologous or heterologous H5N1 wild-type viruses in mice.FIG. 13 shows efficacy of protection conferred by 2004 H5N1 ca vaccine against low or high doses of homologous H5N1 wild-type virus challenge in mice.FIGS. 11-13 demonstrate that the tested vaccines could protect against other related viruses.
• In healthy human adults nasal spray administration the '04 vaccine was well tolerated and its replication was highly restricted. SeeFIG. 27 for replication restriction of the vaccine in healthy adults. HI antibody responses to 10^6.7TCID₅₀of the '04 vaccine were also observed in some of the healthy adults. SeeFIG. 28.
• The current example demonstrates several points concerning exemplary H5N1 ca reassortant viruses/vaccines of the invention. The modified ca reassortant '97, '03, and '04 viruses were shown to have in vitro ts phenotype, loss of pathogenicity in chickens and attenuation in mice. It is expected that attenuation is also present in ferrets. Efficacy of protection and cross-protection against lethal challenge and systemic spread with wild-type viruses in mice was also shown. Efficacy of protection and cross-protections against replication of wild-type challenge viruses in the respiratory tract of mice is also expected.
• It is contemplated to use these (and similar) viruses/vaccines to determine whether immunogenicity and efficacy is improved following 2 doses of vaccine; to assess immunogenicity in non-human primates; to assess attenuation and vaccine efficacy in ferrets; to determine the contribution of humoral and cellular immunity to observed efficacy of the produced vaccines in mice; to determine which residues of the 2003 HA contribute to enhanced immunogenicity and introduce them into 1997 and 2004 HAs; and to determine the effects of deleting the multibasic amino acid cleavage site and of the gene constellation.
Example 2Construction and Analysis of H6 ca Viruses and Vaccines
• A set of three recombinant influenza viruses and vaccines comprising H6 HA sequences were prepared: (a) A/Duck, which comprised the H6 HA and N9 NA of A/Duck77; (b) A/Teal, which comprised the H6 HA and N1 NA of A/Teal97; and (c) A/Mallard, which comprised the H6 HA and N2 NA of A/Mallard85. The six internal genome segments of each recombinant virus were those of ca A/AA/6/60.
• Each of the A/Duck, A/Teal, and A/Mallard recombinant viruses was attenuated in nasal turbinates and lungs of ferrets. Ferrets were intranasally inoculated with 10⁷TCID₅₀recombinant (ca; see paragraph immediately above) or wild-type (wt) H6 influenza virus. Nasal turbinate and lung tissue was harvested from the ferrets three days post-infection for examination.FIG. 16 shows that the nasal turbinate and lung tissue of ferrets inoculated with recombinant virus (ca) exhibited lower virus titers than did the nasal turbinate and lung tissue of ferrets inoculated with the respective counterpart wt virus.
• Each of the A/Duck, A/Teal, and A/Mallard recombinant (ca) viruses was also immunogenic in the ferrets. SeeFIG. 17.
• FIG. 18 shows the efficacy of protection conferred by the A/Duck, A/Teal, and A/Mallard vaccines. Ferrets were vaccinated with a single dose of 7 log₁₀PFU recombinant A/Duck, A/Teal, or A/Mallard vaccine. The ferrets were then challenged with 7 log₁₀PFU wt A/Duck, A/Teal, or A/Mallard virus. Three days post challenge lungs and nasal turbinates of the ferrets were harvested and virus titer in the tissues was determined.
• FIG. 18 shows efficacy of protection conferred by the recombinant (ca) H6 vaccines against homologous and heterologous wild-type H6 viruses in ferrets.
Example 3Construction and Analysis of anH7N3 BC 04 ca, Virus and Vaccine
• A further recombinant influenza virus and vaccine was prepared using the HA H7 and NA N3 sequences of A/ck/BC/CN-6/04 (BC 04 ca). These HA and NA sequences were combined with the six internal genome segments of ca A/AA/6/60.
• TheBC 04 ca vaccine was attenuated in the ferrets. Ferrets were intranasally inoculated with 10⁷TCID₅₀vaccine in 0.5 mL. Three days following inoculation, ferret nasal turbinates, lungs, brain, and olfactory bulb were harvested. Virus titer in each of these tissues was diminished in ferrets inoculated with the vaccine virus relative to ferrets inoculated with wt viruses A/BC/CN-6/04 or A/BC/CN-7/04. SeeFIG. 19.
• TheBC 04 ca vaccine was immunogenic in mice. In mice receiving theBC 04 ca vaccine, neutralizing antibodies were detected at 4 weeks and these titers rose over 8 weeks. A second dose of vaccine boosted antibody titer but final titer achieved was similar to that following a single dose. SeeFIG. 20.
• FIGS. 21 and 22 show the efficacy of protection conferred by theBC 04 ca vaccine against both homologous and heterologous H7 wt viruses. ForFIG. 21, mice were intranasally inoculated with 1 dose vaccine four weeks before challenge, 1dose vaccine 8 weeks before challenge, or 2 doses vaccine (administered 4 weeks apart) before lethal challenge with 50 LD₅₀homologous (A/ck/BC/CN-7/04) and heterologous (A/NL/219/03 or A/tk/Eng/63) H7 wt viruses. Weight change of the mice following lethal challenged was monitored each day for fourteen days, to monitor morbidity associated with the wt influenza virus challenge.
• For each of the mice lethally challenged with the homologous A/ck/BC/CN-7/04 virus little or no weight change was observed regardless of whether 1 dose of vaccine was administered 4 weeks prior to challenge (a), 1 dose of vaccine was administered 8 weeks prior to challenge (b) or 2 doses of vaccine were administered prior to challenge (c). Likewise, little to no weight loss occurred following challenge of the mice with either heterologous influenza virus, A/NL/2109/03 (d, e, f) or A/tk/Eng/63 (g, h, i). Again, the lack of weight loss was observed regardless of whether 1 dose of vaccine was administered 4 weeks prior to challenge (d or g), 1 dose of vaccine was administered 8 weeks prior to challenge (e, or h), or 2 doses of vaccine were administered prior to challenge (f or i).
• FIG. 22 provides further evidence of the efficacy of theH7N3 BC 04 ca vaccine. In both nasal turbinates (a) and lungs (b) of mice receiving theH7N3 BC 04 ca vaccine, protection was observed against challenge using ck/BC/CN-6/04 (H7N3), ck/BC/CN-7/04 (H7N3), NL/219/03 (H7N7), tk/Eng/63 (H7N3), tk/UT/95 (H7N3), and tk/VA/02 (H7N2) viruses.
Example 4Construction and Analysis of an H9N2 G9/AA ca, Virus and Vaccine
• A further recombinant influenza virus and vaccine was prepared using the HA H9 and NA N2 sequences of A/ck/Hong Kong/G9/97 (G9/AA ca). These HA and NA sequences were combined with the six internal genome segments of ca A/AA/6/60.
• The H9N2 G9/AA ca vaccine was attenuated in the ferrets. SeeFIG. 23, which shows reduced virus titers in nasal turbinates (a) and lungs (b) of ferrets following administration of the H9N2 G9/AA ca virus relative to the H9N2 G9 wt virus.
• FIG. 24 provides evidence of the efficacy of the H9N2 G9 ca vaccine in mice. In the mice receiving the H9N2 G9 ca vaccine, protection was observed against challenge using H9N2 G9 wt and A/HK/1073/99 viruses.
• The H9N2 G9/AA ca vaccine was also well tolerated in healthy adults in a clinical trial setting. Healthy adults were administered the H9N2 G9/AA ca vaccine by nose drop. In the healthy adults, the H9N2 G9/AA ca vaccine was highly restricted in replication. SeeFIG. 25. Furthermore, administration of 10^7.0TCID₅₀of H9N2 G9/AA ca vaccine induced ≧4-fold increases in HI titer in 92% of healthy volunteers. SeeFIG. 26.
• 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. All publications, patents, patent applications, or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes. In particular, U.S. provisional application Nos. 60/821,832 filed Aug. 9, 2006 and 60/942,804, filed Jun. 8, 2007, are incorporated herein in their entirety for all purposes.
SPECIFIC EMBODIMENTS
• Additional embodiments of the present invention are presented in Table 3 and 4.

  TABLE 3
  Specific embodiments

  1	An isolated polypeptide, wherein said polypeptide is selected from the group consisting
  	of:
  	a) a polypeptide encoded by a polynucleotide sequence as shown in any one of SEQ ID
  	NOS: 21-26 or 33-38 or 45;
  	b) a polypeptide as shown in any one of SEQ ID NOS: 27-32 or 39-44;
  	c) the mature form of the polypeptide as shown in any one of SEQ ID NOS: 27-32 or 39-44;
  	d) a polypeptide encoded by a polynucleotide sequence which hybridizes under highly
  	stringent conditions to a polynucleotide sequence encoding (a) (b) or (c); and
  	e) a polypeptide having at least 90% sequence identity to the polypeptide of (b).
  2	An immunogenic composition comprising an immunologically effective amount of at
  	least one polypeptide ofembodiment 1.
  3	An isolated antibody that specifically binds the polypeptide ofembodiment 1.
  4	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to the
  	individual an immunologically effective amount of the polypeptide ofembodiment 1 in a
  	physiologically acceptable carrier.
  5	A recombinant influenza virus comprising the polypeptide ofembodiment 1.
  6	An immunogenic composition comprising an immunologically effective amount of the
  	recombinant influenza virus ofembodiment 5.
  7	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to the
  	individual an immunologically effective amount of the recombinant influenza virus of
  	embodiment 5 in a physiologically acceptable carrier.
  8	An isolated nucleic acid, wherein said nucleic acid is selected from the group consisting
  	of:
  	a) a polynucleotide sequence as shown in any one of SEQ ID NOS: 21-26 or 33-38 or 45,
  	or a complementary sequence thereof;
  	b) a polynucleotide sequence encoding a polypeptide as shown in any one of SEQ ID
  	NOS: 27-32 or 39-44, or a complementary polynucleotide sequence thereof;
  	c) a polynucleotide sequence which hybridizes under highly stringent conditions over
  	substantially the entire length of polynucleotide sequence (a); and
  	d) a polynucleotide sequence having at least 98% sequence identity to the polynucleotide
  	sequence of (a).
  9	An immunogenic composition comprising at least one of the nucleic acids of
  	embodiment 8.
  10	A cell comprising at least one nucleic acid ofembodiment 8.
  11	A vector comprising the nucleic acid ofembodiment 8.
  12	The vector ofembodiment 12, wherein the vector is a plasmid, a cosmid, a phage, a
  	virus, or a fragment of a virus.
  13	The vector ofembodiment 12, wherein the vector is an expression vector.
  14	A cell comprising the vector ofembodiment 13.
  15	An influenza virus comprising one or more nucleic acids ofembodiment 8.
  16	The virus ofembodiment 15, wherein the virus is a reassortment virus.
  17	A 6:2 reassortment influenza virus, wherein said virus comprises 6 gene encoding
  	regions from A/Ann Arbor/6/60 and 2 gene encoding regions that encode polypeptides
  	selected from the group consisting of: the polypeptides of SEQ ID NOS: 27-32, and 39-44.
  18	A method of producing a recombinant influenza virus, the method comprising: culturing
  	the cell ofembodiment 14 in a suitable culture medium under conditions permitting
  	expression of nucleic acid; and, isolating the recombinant influenza virus from a cell
  	population comprising said cell or the medium.
  19	An immunogenic composition comprising an immunologically effective amount of the
  	recombinant influenza virus of embodiment 17.
  20	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to the
  	individual an immunologically effective amount of the recombinant influenza virus of
  	embodiment 17 in a physiologically effective carrier.
  21	A method of producing an isolated or recombinant polypeptide, the method comprising:
  	culturing the host cell ofembodiment 10 in a suitable culture medium under conditions
  	permitting expression of said nucleic acid; and, isolating the polypeptide from one or
  	more of the host cells or the medium.
  22	A method of prophylactic or therapeutic treatment of a viral infection in a subject, the
  	method comprising: administering to the subject, a virus of embodiment 17 in an
  	amount effective to produce an immunogenic response against the viral infection.
  23	The method ofembodiment 22, wherein the subject is a human.
  24	The immunogenic composition ofembodiment 19, wherein the hemagglutinin comprises
  	a modified polybasic cleavage site.
  25	A live attenuated influenza vaccine comprising the composition ofembodiment 19.
  26	A split virus or killed virus vaccine comprising the composition ofembodiment 19.
  27	A live attenuated influenza vaccine comprising the composition ofembodiment 24.
  28	A split virus or killed virus vaccine comprising the composition ofembodiment 24.
  29	A method for producing influenza viruses in cell culture, the method comprising:
  	i) introducing into a population of host cells, which population of host cells is capable of
  	supporting replication of influenza virus, a plurality of vectors comprising nucleic acid
  	encoding at least 6 internal genome segments of a first influenza strain, wherein the first
  	influenza strain is A/Ann Arbor/6/60; and, at least one genome segment encoding an
  	immunogenic influenza surface antigen of a second influenza strain, wherein said second
  	strain is a pandemic influenza strain,
  	ii) culturing the population of host cells at a temperature less than or equal to 35° C.; and,
  	iii) recovering a plurality of influenza viruses.
  30	The method of embodiment 29, wherein the plurality of vectors comprise at least one
  	isolated nucleic acid, wherein said nucleic acid is selected from the group consisting of:
  	a) a polynucleotide sequence of one of SEQ ID NOS: 21-26 or 33-38, or 45, or a
  	complementary sequence thereof;
  	b) a polynucleotide sequence encoding a polypeptide of one of SEQ ID NOS: 27-32 or
  	39-44, or a complementary polynucleotide sequence thereof;
  	c) a polynucleotide sequence which hybridizes under highly stringent conditions over
  	substantially the entire length of polynucleotide sequence (a); and
  	d) a polynucleotide sequence having at least 98% sequence identity to the polynucleotide
  	sequence of (a).
  31	An immunogenic composition comprising an immunologically effective amount of the
  	influenza virus of embodiment 29.
  32	An immunogenic composition comprising an immunologically effective amount of the
  	influenza virus ofembodiment 30.
  33	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to the
  	individual an immunologically effective amount of the influenza virus of embodiment 29
  	in a physiologically effective carrier.
  34	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to the
  	individual an immunologically effective amount of the influenza virus ofembodiment 30
  	in a physiologically effective carrier.
  35	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to the
  	individual the immunogenic composition ofembodiment 31.
  36	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to the
  	individual the immunogenic composition ofembodiment 32.
  37	A live attenuated influenza vaccine comprising the immunogenic composition of
  	embodiment 31.
  38	A split virus or killed virus vaccine comprising the immunogenic composition of
  	embodiment 32.

• TABLE 4
  Specific embodiments.

  1	An isolated polypeptide, wherein said polypeptide is selected from the group
  	consisting of:
  	a) a polypeptide comprising the amino acid sequence encoded by the nucleotide
  	sequence as shown in any one of SEQ ID NOS: 21-26 or 33-38 or 45;
  	b) a polypeptide comprising the amino acid sequence as shown in any one of SEQ ID
  	NOS: 27-32 or 39-44;
  	c) the mature form of a polypeptide comprising the amino acid sequence as shown in
  	any one of SEQ ID NOS: 27-32 or 39-44;
  	d) a polypeptide comprising an amino acid sequence encoded by a polynucleotide
  	which hybridizes under highly stringent conditions to a polynucleotide comprising a
  	nucleotide sequence encoding (a) (b) or (c); and
  	e) a polypeptide comprising an amino acid sequence having at least 90% sequence
  	identity to the polypeptide of (b).
  2	An immunogenic composition comprising an immunologically effective amount of at
  	least one polypeptide ofembodiment 1.
  3	An isolated antibody that specifically binds the polypeptide ofembodiment 1.
  4	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to
  	the individual an immunologically effective amount of the polypeptide of
  	embodiment 1 in a physiologically acceptable carrier.
  5	A recombinant influenza virus comprising the polypeptide ofembodiment 1.
  6	An immunogenic composition comprising an immunologically effective amount of
  	the recombinant influenza virus ofembodiment 5.
  7	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to
  	the individual an immunologically effective amount of the recombinant influenza
  	virus ofembodiment 5 in a physiologically acceptable carrier.
  8	An isolated polynucleotide, wherein said polynucleotide is selected from the group
  	consisting of:
  	a) a polynucleotide comprising the nucleotide sequence as shown in any one of SEQ
  	ID NOS: 21-26 or 33-38 or 45, or a complementary sequence thereof;
  	b) a polynucleotide comprising a nucleotide sequence encoding a polypeptide
  	comprising the amino acid sequence as shown in any one of SEQ ID NOS: 27-32 or
  	39-44, or a complementary nucleotide sequence thereof;
  	c) a polynucleotide which hybridizes under highly stringent conditions over
  	substantially the entire length of the polynucleotide of (a); and
  	d) a polynucleotide comprising a nucleotide sequence having at least 98% sequence
  	identity to the polynucleotide of (a).
  9	An immunogenic composition comprising at least one polynucleotide ofembodiment
  	8.
  10	A cell comprising at least one polynucleotide ofembodiment 8.
  11	A vector comprising the polynucleotide ofembodiment 8.
  12	The vector ofembodiment 11, wherein the vector is a plasmid, a cosmid, a phage, a
  	virus, or a fragment of a virus.
  13	The vector ofembodiment 12, wherein the vector is an expression vector.
  14	A cell comprising the vector ofembodiment 13.
  15	An influenza virus comprising one or more polynucleotides ofembodiment 8.
  16	The virus ofembodiment 15, wherein the virus is a reassortant virus.
  17	A 6:2 reassortant influenza virus, wherein said virus comprises 6 internal genome
  	segments from A/Ann Arbor/6/60 and 2 genome segments that encode an HA and/or
  	a NA polypeptide selected from the group consisting of: the polypeptides of SEQ ID
  	NOS: 27-32, and 39-44.
  18	A method of producing a reassortant influenza virus, the method comprising:
  	culturing the cell ofembodiment 14 in a suitable culture medium under conditions
  	permitting expression of said polynucleotide; and, isolating the reassortant influenza
  	virus from a cell population comprising said cell or the medium.
  19	An immunogenic composition comprising an immunologically effective amount of
  	the reassortant influenza virus of embodiment 17.
  20	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to
  	the individual an immunologically effective amount of the reassortant influenza virus
  	of embodiment 17 in a physiologically effective carrier.
  21	A method of producing an isolated or recombinant polypeptide, the method
  	comprising: culturing the cell ofembodiment 10 in a suitable culture medium under
  	conditions permitting expression of said polynucleotide; and, isolating the
  	polypeptide from the cell or the medium.
  22	A method of prophylactic or therapeutic treatment of a viral infection in a subject,
  	the method comprising: administering to the subject, the virus of embodiment 17 in
  	an amount effective to produce an immunogenic response against the viral infection.
  23	The method ofembodiment 22, wherein the subject is a human.
  24	The immunogenic composition ofembodiment 19, wherein the hemagglutinin
  	comprises a modified polybasic cleavage site.
  25	A live attenuated influenza vaccine comprising the composition ofembodiment 19.
  26	A split virus or killed virus vaccine comprising the composition ofembodiment 19.
  27	A live attenuated influenza vaccine comprising the composition ofembodiment 24.
  28	A split virus or killed virus vaccine comprising the composition ofembodiment 24.
  29	A method for producing an influenza virus in cell culture, the method comprising:
  	i) introducing into a population of host cells, which population of host cells is
  	capable of supporting replication of influenza virus, a plurality of vectors comprising
  	nucleotide sequences corresponding to at least 6 internal genome segments of A/Ann
  	Arbor/6/60; and, at least one genome segment comprising a polynucleotide encoding
  	an HA and/or a NA polypeptide selected from the group consisting of: the
  	polypeptides of SEQ ID NOS: 27-32, and 39-44,
  	ii) culturing the population of host cells at a temperature less than or equal to 35° C.;
  	and,
  	iii) recovering an influenza virus.
  30	The method of embodiment 29, wherein the polynucleotide encoding the HA and/or
  	NA polypeptide is selected from the group consisting of:
  	a) a polynucleotide comprising the nucleotide sequence of any one of SEQ ID
  	NOS: 21, 23-26 or 33-38, or 45, or a complementary nucleotide sequence thereof;
  	b) a polynucleotide comprising a nucleotide sequence encoding a polypeptide
  	comprising the amino acid sequence as shown in any one of SEQ ID NOS: 27-32 or
  	39-44, or a complementary nucleotide sequence thereof;
  	c) a polynucleotide which hybridizes under highly stringent conditions over
  	substantially the entire length of the polynucleotide of (a); and
  	d) a polynucleotide comprising a nucleotide sequence having at least 98%
  	sequence identity to the polynucleotide of (a).
  31	An immunogenic composition comprising an immunologically effective amount of
  	the influenza virus produced by the method of embodiment 29.
  32	An immunogenic composition comprising an immunologically effective amount of
  	the influenza virus produced by the method ofembodiment 30.
  33	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to
  	the individual an immunologically effective amount of the influenza virus produced
  	by the method of embodiment 29 in a physiologically effective carrier.
  34	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to
  	the individual an immunologically effective amount of the influenza virus produced
  	by the method ofembodiment 30 in a physiologically effective carrier.
  35	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to
  	the individual the immunogenic composition ofembodiment 31.
  36	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to
  	the individual the immunogenic composition ofembodiment 32.
  37	A live attenuated influenza vaccine comprising the immunogenic composition of
  	embodiment 31.
  38	A split virus or killed virus vaccine comprising the immunogenic composition of
  	embodiment 32.
  39	A 6:2 reassortant influenza virus, wherein said virus comprises 6 internal genome
  	segments from one or more donor viruses other than A/Ann Arbor/6/60 and 2
  	genome segments that encode an HA and/or a NA polypeptide selected from the
  	group consisting of: the polypeptides of SEQ ID NOS: 27-32, and 39-44.
  40	The 6:2 reassortant influenza virus of embodiment 39, wherein said donor virus
  	comprises one or more of the following phenotypes: temperature-sensitive, cold-
  	adaped, or attenuated.
  41	The 6:2 reassortant influenza virus of embodiment 39, wherein said donor virus is
  	PR8.
  42	The 6:2 reassortant influenza virus of embodiment 39, wherein said donor virus is
  	A/Leningrad/17.
  43	An immunogenic composition comprising an immunologically effective amount of
  	the reassortant influenza virus of embodiment 39.
  44	An immunogenic composition comprising an immunologically effective amount of
  	the reassortant influenza virus ofembodiment 40.
  45	An immunogenic composition comprising an immunologically effective amount of
  	the reassortant influenza virus of embodiment 41.
  46	An immunogenic composition comprising an immunologically effective amount of
  	the reassortant influenza virus of embodiment 42.
  47	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to
  	the individual an immunologically effective amount of the reassortant influenza virus
  	of embodiment 39 in a physiologically effective carrier.
  48	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to
  	the individual an immunologically effective amount of the reassortant influenza virus
  	ofembodiment 40 in a physiologically effective carrier.
  49	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to
  	the individual an immunologically effective amount of the reassortant influenza virus
  	of embodiment 41 in a physiologically effective carrier.
  50	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to
  	the individual an immunologically effective amount of the reassortant influenza virus
  	of embodiment 42 in a physiologically effective carrier.
  51	A method of prophylactic or therapeutic treatment of a viral infection in a subject,
  	the method comprising: administering to the subject, the virus of embodiment 39 in
  	an amount effective to produce an immunogenic response against the viral infection.
  52	A method of prophylactic or therapeutic treatment of a viral infection in a subject,
  	the method comprising: administering to the subject, the virus of embodiment 41 in
  	an amount effective to produce an immunogenic response against the viral infection.
  53	A method of prophylactic or therapeutic treatment of a viral infection in a subject,
  	the method comprising: administering to the subject, the virus of embodiment 42 in
  	an amount effective to produce an immunogenic response against the viral infection.
  54	The method ofembodiment 51, wherein said virus is killed or inactivated.
  55	The method of embodiment 52, wherein said virus is killed or inactivated.
  56	The method of embodiment 53, wherein said virus is killed or inactivated.
  57	The immunogenic composition of embodiment 43, wherein the hemagglutinin
  	comprises a modified polybasic cleavage site.
  58	The immunogenic composition of embodiment 44, wherein the hemagglutinin
  	comprises a modified polybasic cleavage site.
  59	The immunogenic composition ofembodiment 45, wherein the hemagglutinin
  	comprises a modifiedpolybasic cleavage site
  60	The immunogenic composition of embodiment 46, wherein the hemagglutinin
  	comprises a modified polybasic cleavage site.
  61	The method of embodiment 47, wherein the subject is a human.
  62	The method of embodiment 48, wherein the subject is a human.
  63	The method of embodiment 49, wherein the subject is a human.
  64	A live attenuated influenza vaccine comprising the composition ofembodiment 45.
  65	A live attenuated influenza vaccine comprising the composition of embodiment 46.
  66	A method for producing an influenza virus in cell culture, the method comprising:
  	i) introducing into a population of host cells, which population of host cells is
  	capable of supporting replication of influenza virus, a plurality of vectors comprising
  	nucleotide sequences corresponding to:
  	(a) at least 6 internal genome segments of a first influenza strain, wherein the
  	first influenza strain is not A/Ann Arbor/6/60; and, at least one genome segment
  	encoding an HA or an NA polypeptide selected from the group consisting of: the
  	polypeptides of SEQ ID NOS: 27-32, and 39-44; or
  	(b) at least 6 internal genome segments of a first influenza strain, wherein the
  	first influenza strain is not A/Ann Arbor/6/60 and which influenza strain comprises
  	one or more phenotypic attributes selected from the group consisting of: attenuated,
  	cold adapted and temperature sensitive; and, at least one genome segment encoding
  	an HA or an NA polypeptide selected from the group consisting of: the polypeptides
  	of SEQ ID NOS: 27-32, and 39-44,
  	ii) culturing the population of host cells at a temperature less than or equal to 35° C.;
  	and,
  	iii) recovering an influenza virus.
  67	An immunogenic composition comprising an immunologically effective amount of
  	the influenza virus produced by the method of embodiment 66.
  68	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to
  	the individual an immunologically effective amount of the influenza virus produced
  	by the method of embodiment 66 in a physiologically effective carrier.
  69	A method for stimulating the immune system of an individual to produce a protective
  	immune response against influenza virus, the method comprising administering to
  	the individual the immunogenic composition of embodiment 67.
  70	A live attenuated influenza vaccine comprising the immunogenic composition of
  	embodiment 67.
  71	A split virus or killed virus vaccine comprising the immunogenic composition of
  	embodiment 67.

SEQUENCES
• ca A/Vietnam/1203/04
• Nucleotide Sequence of ca A/Vietnam/1203/04H5 (SEQ ID NO:1)
• Entire molecule length: 1767 nt

  1	agcaaaagca ggggttcaat ctgtcaaaat ggagaaaata gtgcttcttt
  51	ttgcaatagt cagtcttgtt aaaagtgatc agatttgcat tggttaccat
  101	gcaaacaact cgacagagca ggttgacaca ataatggaaa agaacgttac
  151	tgttacacat gcccaagaca tactggaaaa gaaacacaac gggaagctct
  201	gcgatctaga tggagtgaag cctctaattt tgagagattg tagcgtagct
  251	ggatggctcc tcggaaaccc aatgtgtgac gaattcatca atgtgccgga
  301	atggtcttac atagtggaga aggccaatcc agtcaatgac ctctgttacc
  351	caggggattt caatgactat gaagaattga aacacctatt gagcagaata
  401	aaccattttg agaaaattca gatcatcccc aaaagttctt ggtccagtca
  451	tgaagcctca ttaggggtga gctcagcatg tccataccag ggaaagtcct
  501	cctttttcag aaatgtggta tggcttatca aaaagaacag tacataccca
  551	acaataaaga ggagctacaa taataccaac caagaagatc ttttggtact
  601	gtgggggatt caccatccta atgatgcggc agagcagaca aagctctatc
  651	aaaacccaac cacctatatt tccgttggga catcaacact aaaccagaga
  701	ttggtaccaa gaatagctac tagatccaaa gtaaacgggc aaagtggaag
  751	gatggagttc ttctggacaa ttttaaagcc gaatgatgca atcaacttcg
  801	agagtaatgg aaatttcatt gctccagaat atgcatacaa aattgtcaag
  851	aaaggggact caacaattat gaaaagtgaa ttggaatatg gtaactgcaa
  901	caccaagtgt caaactccaa tgggggcgat aaactctagc atgccattcc
  951	acaatataca ccctctcacc attggggaat gccccaaata tgtgaaatca
  1001	aacagattag tccttgcgac tgggctcaga aatagccctc aaagagagac
  1051	tcgaggatta tttggagcta tagcaggttt tatagaggga ggatggcagg
  1101	gaatggtaga tggttggtat gggtaccacc atagcaatga gcaggggagt
  1151	gggtacgctg cagacaaaga atccactcaa aaggcaatag atggagtcac
  1201	caataaggtc aactcgatca ttgacaaaat gaacactcag tttgaggccg
  1251	ttggaaggga atttaacaac ttagaaagga gaatagagaa tttaaacaag
  1301	aagatggaag acgggttcct agatgtctgg acttataatg ctgaacttct
  1351	ggttctcatg gaaaatgaga gaactctaga ctttcatgac tcaaatgtca
  1401	agaaccttta cgacaaggtc cgactacagc ttagggataa tgcaaaggag
  1451	ctgggtaacg gttgtttcga gttctatcat aaatgtgata atgaatgtat
  1501	ggaaagtgta agaaatggaa cgtatgacta cccgcagtat tcagaagaag
  1551	cgagactaaa aagagaggaa ataagtggag taaaattgga atcaatagga
  1601	atttaccaaa tactgtcaat ttattctaca gtggcgagtt ccctagcact
  1651	ggcaatcatg gtagctggtc tatccttatg gatgtgctcc aatgggtcgt
  1701	tacaatgcag aatttgcatt taaatttgtg agttcagatt gtagttaaaa
  1751	acacccttgt ttctact

• Amino acid sequence of ca A/Vietnam/1203/04H5 (SEQ ID NO:11)
• Entire molecule length: 564aa

  1	mekivllfai vslvksdqic igyhannste qvdtimeknv tvthaqdile
  51	kkhngklcdl dgvkplilrd csvagwllgn pmcdefinvp ewsyivekan
  101	pvndlcypgd fndyeelkhl lsrinhfeki qiipksswss heaslgvssa
  151	cpyqgkssff rnvvwlikkn styptikrsy nntnqedllv lwgihhpnda
  201	aeqtklyqnp ttyisvgtst lnqrlvpria trskvngqsg rmeffwtilk
  251	pndainfesn gnfiapeyay kivkkgdsti mkseleygnc ntkcqtpmga
  301	inssmpfhni hpltigecpk yvksnrlvla tglrnspqre trglfgaiag
  351	fieggwqgmv dgwygyhhsn eqgsgyaadk estqkaidgv tnkvnsiidk
  401	mntqfeavgr efnnlerrie nlnkkmedgf ldvwtynael lvlmenertl
  451	dfhdsnvknl ydkvrlqlrd nakelgngcf efyhkcdnec mesvrngtyd
  501	ypqyseearl kreeisgvkl esigiyqils iystvassla laimvaglsl
  551	wmcsngslqc rici

• Nucleotide Sequence of ca A/Vietnam/1203/04 N1 (SEQ ID NO: 2)
• Entire molecule length: 1398 nt

  1	agcaaaagca ggagttcaaa atgaatccaa atcagaagat aataaccatc
  51	gggtcaatct gtatggtaac tggaatagtt agcttaatgt tacaaattgg
  101	gaacatgatc tcaatatggg tcagtcattc aattcacaca gggaatcaac
  151	accaatctga accaatcagc aatactaatt ttcttactga gaaagctgtg
  201	gcttcagtaa aattagcggg caattcatct ctttgcccca ttaacggatg
  251	ggctgtatac agtaaggaca acagtataag gatcggttcc aagggggatg
  301	tgtttgttat aagagagccg ttcatctcat gctcccactt ggaatgcaga
  351	actttctttt tgactcaggg agccttgctg aatgacaagc actccaatgg
  401	gactgtcaaa gacagaagcc ctcacagaac attaatgagt tgtcctgtgg
  451	gtgaggctcc ctccccatat aactcaaggt ttgagtctgt tgcttggtca
  501	gcaagtgctt gccatgatgg caccagttgg ttgacgattg gaatttctgg
  551	cccagacaat ggggctgtgg ctgtattgaa atacaatggc ataataacag
  601	acactatcaa gagttggagg aacaacatac tgagaactca agagtctgaa
  651	tgtgcatgtg taaatggctc ttgctttact gtaatgactg acggaccaag
  701	taatggtcag gcatcacata agatcttcaa aatggaaaaa gggaaagtgg
  751	ttaaatcagt cgaattggat gctcctaatt atcactatga ggaatgctcc
  801	tgttatccta atgccggaga aatcacatgt gtgtgcaggg ataattggca
  851	tggctcaaat cggccatggg tatctttcaa tcaaaatttg gagtatcaaa
  901	taggatatat atgcagtgga gttttcggag acaatccacg ccccaatgat
  951	ggaacaggta gttgtggtcc ggtgtcctct aacggggcat atggggtaaa
  1001	agggttttca tttaaatacg gcaatggtgt ctggatcggg agaaccaaaa
  1051	gcactaattc caggagcggc tttgaaatga tttgggatcc aaatgggtgg
  1101	actgaaacgg acagtagctt ttcagtgaaa caagatatcg tagcaataac
  1151	tgattggtca ggatatagcg ggagttttgt ccagcatcca gaactgacag
  1201	gactagattg cataagacct tgtttctggg ttgagttgat cagagggcgg
  1251	cccaaagaga gcacaatttg gactagtggg agcagcatat ctttttgtgg
  1301	tgtaaatagt gacactgtgg gttggtcttg gccagacggt gctgagttgc
  1351	cattcaccat tgacaagtag tttgttcaaa aaactccttg tttctact

• Amino acid sequence of ca A/Vietnam/1203/04 N1 (SEQ ID NO:12)
• Entire molecule length: 449aa

  1	mnpnqkiiti gsicmvtgiv slmlqignmi siwvshsiht gnqhqsepis
  51	ntnfltekav asvklagnss lcpingwavy skdnsirigs kgdvfvirep
  101	fiscshlecr tffltqgall ndkhsngtvk drsphrtlms cpvgeapspy
  151	nsrfesvaws asachdgtsw ltigisgpdn gavavlkyng iitdtikswr
  201	nnilrtqese cacvngscft vmtdgpsngq ashkifkmek gkvvksveld
  251	apnyhyeecs cypnageitc vcrdnwhgsn rpwvsfnqnl eyqigyicsg
  301	vfgdnprpnd gtgscgpvss ngaygvkgfs fkygngvwig rtkstnsrsg
  351	femiwdpngw tetdssfsvk qdivaitdws gysgsfvqhp eltgldcirp
  401	cfwvelirgr pkestiwtsg ssisfcgvns dtvgwswpdg aelpftidk

  
  ca A/Hong Kong/213/03
• Nucleotide Sequence of ca A/Hong Kong/213/03H5 (SEQ ID NO: 3)
• Entire molecule length: 1767 nt

  1	agcaaaagca ggggttcaat ctgtcaaaat ggagaaaata gtgcttcttt
  51	ttgcaatagt cagtcttgtt aaaagtgatc agatttgcat tggttaccat
  101	gcaaacaact cgacagagca ggttgacaca ataatggaaa agaacgttac
  151	tgttacacat gcccaagaca tactggaaaa gacacacaac gggaagctct
  201	gcgatctaga tggagtgaag cctctaattt tgagagattg tagtgtagct
  251	ggatggctcc tcggaaaccc aatgtgtgac gaattcatca atgtgccgga
  301	atggtcttac atagtggaga aggccaatcc agccaatgac ctctgttacc
  351	caggggattt caacgactat gaagaattga aacacctatt gagcagaata
  401	aaccattttg agaaaattca gatcatcccc aaaaattctt ggtccagtca
  451	tgaagcctca ttaggggtga gctcagcatg tccataccaa ggaaagtcct
  501	cctttttcag gaatgtggta tggcttatca aaaagaacaa tgcataccca
  551	acaataaaga ggagctacaa taataccaac caagaagatc ttttggtatt
  601	gtgggggatt caccatccta atgatgcggc agagcagact aggctctatc
  651	aaaacccaac cacctacatt tccgttggga catcaacact aaaccagaga
  701	ttggtaccaa aaatagctac tagatccaaa gtaaacgggc aaaatggaag
  751	gatggagttc ttctggacaa ttttaaaacc gaatgatgca atcaacttcg
  801	agagcaatgg aaatttcatt gctccagaat atgcatacaa aattgtcaag
  851	aaaggggact cagcaattat gaaaagtgaa ttggaatatg gtaactgcaa
  901	caccaagtgt caaactccaa tgggggcgat aaactctagt atgccattcc
  951	acaatataca ccctctcacc atcggggaat gccccaaata tgtgaaatca
  1001	aacagattag tccttgcgac tgggctcaga aatagccctc aaagagagac
  1051	tcgaggatta tttggagcta tagcaggttt tatagaggga ggatggcagg
  1101	gaatggtaga tggttggtat gggtaccacc atagcaatga gcaggggagt
  1151	gggtacgctg cagacaaaga atccactcaa aaggcaatag atggagtcac
  1201	caataaggtc aactcgatca ttgacaaaat gaacactcag tttgaggccg
  1251	ttggaaggga atttaataac ttagaaagga gaatagagaa tttaaacaag
  1301	aagatggaag acggattcct agatgtctgg acttataatg ctgaacttct
  1351	ggttctcatg gaaaatgaga gaactctaga ctttcatgac tcaaatgtca
  1401	agaaccttta cgacaaggtc cgactacagc ttagggataa tgcaaaggag
  1451	ctgggtaacg gttgtttcga gttctatcac aaatgtgata atgaatgtat
  1501	ggaaagtgta agaaacggaa cgtatgacta cccgcagtat tcagaagaag
  1551	caagactaaa aagagaggaa ataagtggag taaaattgga gtcaatagga
  1601	acttaccaaa tactgtcaat ttattctaca gtggcgagtt ccctagcact
  1651	ggcaatcatg gtagctggtc tatctttatg gatgtgctcc aatgggtcgt
  1701	tacaatgcag aatttgcatt taaatttgtg agttcagatt gtagttaaaa
  1751	acacccttqt ttctact

• Amino acid sequence of ca A/Hong Kong/213/03H5 (SEQ ID NO:13)
• Entire molecule length: 564aa

  1	mekivllfai vslvksdqic igyhannste qvdtimeknv tvthaqdile
  51	kthngklcdl dgvkplilrd csvagwllgn pmcdefinvp ewsyivekan
  101	pandlcypgd fndyeelkhl lsrinhfeki qiipknswss heaslgvssa
  151	cpyqgkssff rnvvwlikkn nayptikrsy nntnqedllv lwgihhpnda
  201	aeqtrlyqnp ttyisvgtst lnqrlvpkia trskvngqng rmeffwtilk
  251	pndainfesn gnfiapeyay kivkkgdsai mkseleygnc ntkcqtpmga
  301	inssmpfhni hpltigecpk yvksnrlvla tglrnspqre trglfgaiag
  351	fieggwqgmv dgwygyhhsn eqgsgyaadk estqkaidgv tnkvnsiidk
  401	mntqfeavgr efnnlerrie nlnkkmedgf ldvwtynael lvlmenertl
  451	dfhdsnvknl ydkvrlqlrd nakelgngcf efyhkcdnec mesvrngtyd
  501	ypqyseearl kreeisgvkl esigtyqils iystvassla laimvaglsl
  551	wmcsngslqc rici

• Nucleotide Sequence of ca A/Hong Kong/213/03 N1 (SEQ ID NO: 4)
• Entire molecule length: 1458 nt

  1	agcaaaagca ggagttcaaa atgaatccaa atcagaagat aacaaccatt
  51	ggatcaatct gtatggtaat tggaatagtt agcttgatgt tacaaattgg
  101	gaacataatc tcaatatggg ttagtcattc aattcaaaca gggaatcaac
  151	accaggctga accatgcaat caaagcatta ttacttatga aaacaacacc
  201	tgggtaaacc agacatatgt caacatcagc aataccaatt ttcttactga
  251	gaaagctgtg gcttcagtaa cattagcggg caattcatct ctttgcccca
  301	ttagtggatg ggctgtatac agtaaggaca acggtataag aatcggttcc
  351	aagggggatg tgtttgttat aagagagccg ttcatctcat gctcccactt
  401	ggaatgcaga actttctttt tgactcaggg agccttgctg aatgacaagc
  451	attctaatgg gaccgtcaaa gacagaagcc ctcacagaac attaatgagt
  501	tgtcccgtgg gtgaggctcc ttccccatac aactcgaggt ttgagtctgt
  551	tgcttggtcg gcaagtgctt gtcatgatgg cactagttgg ttgacaattg
  601	gaatttctgg cccagacaat ggggctgtgg ctgtattgaa atacaatggc
  651	ataataacag acactatcaa gagttggagg aacaacataa tgagaactca
  701	agagtctgaa tgtgcatgtg taaatggctc ttgctttact gttatgactg
  751	atggaccaag taatgggcag gcttcataca aaatcttcag aatagaaaaa
  801	gggaaagtag ttaaatcagc cgaattaaat gcccctaatt atcactatga
  851	ggagtgctcc tgttatcctg atgctggaga aatcacatgt gtgtgcaggg
  901	ataactggca tggctcaaat cggccatggg tatctttcaa tcaaaatttg
  951	gagtatcgaa taggatatat atgcagtgga gttttcggag acaatccacg
  1001	ccccaatgat gggacaggca gttgtggtcc ggtgtcccct aaaggggcat
  1051	atggaataaa agggttctca tttaaatacg gcaatggtgt ttggatcggg
  1101	agaaccaaaa gcactaattc caggagcggc tttgaaatga tttgggatcc
  1151	aaatggatgg actggtacgg acagtaattt ttcagtaaag caagatattg
  1201	tagctataac cgattggtca ggatatagcg ggagttttgt ccagcatcca
  1251	gaactgacag gattagattg cataagacct tgtttctggg ttgagctaat
  1301	cagagggcgg cccaaagaga gcacaatttg gactagtggg agcagcatat
  1351	ccttttgtgg tgtaaatagt gacactgtgg gttggtcttg gccagacggt
  1401	gctgagttgc cattcaccat tgacaagtag tttgttcaaa aaactccttg
  1451	tttctact

• Amino acid sequence of ca A/Hong Kong/213/03 N1 (SEQ ID NO:14)
• Entire molecule length: 469aa

  1	mnpnqkitti gsicmvigiv slmlqignii siwvshsiqt gnqhqaepcn
  51	qsiityennt wvnqtyvnis ntnfltekav asvtlagnss lcpisgwavy
  101	skdngirigs kgdvfvirep fiscshlecr tffltqgall ndkhsngtvk
  151	drsphrtlms cpvgeapspy nsrfesvaws asachdgtsw ltigisgpdn
  201	gavavlkyng iitdtikswr nnimrtqese cacvngscft vmtdgpsngq
  251	asykifriek gkvvksaeln apnyhyeecs cypdageitc vcrdnwhgsn
  301	rpwvsfnqnl eyrigyicsg vfgdnprpnd gtgscgpvsp kgaygikgfs
  351	fkygngvwig rtkstnsrsg femiwdpngw tgtdsnfsvk qdivaitdws
  401	gysgsfvqhp eltgldcirp cfwvelirgr pkestiwtsg ssisfcgvns
  451	dtvgwswpdg aelpftidk

  
  ca A/Hong Kong/491/97 (HA)+A/Hong Kong/486/97 (NA)
• Nucleotide Sequence of ca A/Hong Kong/491/97H5 (SEQ ID NO: 5)
• Entire molecule length: 1767 nt

  1	agcaaaagca ggggtataat ctgtcaaaat ggagaaaata gtgcttcttc
  51	ttgcaacagt cagccttgtt aaaagtgacc agatttgcat tggttaccat
  101	gcaaacaact cgacagagca agttgacaca ataatggaaa agaatgttac
  151	tgttacacat gcccaagaca tactggaaag gacacacaac gggaagctct
  201	gcgatctaaa tggagtgaag cctctgattt tgagggattg tagtgtagct
  251	ggatggctcc tcggaaaccc tatgtgtgac gaattcatca atgtgccgga
  301	atggtcttac atagtggaga aggccagtcc agccaatgac ctctgttatc
  351	cagggaattt caacgactat gaagaactga aacacctatt gagcagaata
  401	aaccattttg agaaaattca gataatcccc aaaagttctt ggtccaatca
  451	tgatgcctca tcaggggtga gctcagcatg tccatacctt gggaggtcct
  501	cctttttcag aaatgtggta tggcttatca aaaagaacag tagctaccca
  551	acaataaaga ggagctacaa taataccaac caagaagatc ttttggtact
  601	gtgggggatt caccatccta atgatgcggc agagcagaca aggctctatc
  651	aaaacccaac cacctacatt tccgttggaa catcaacact gaaccagaga
  701	ttggttccag aaatagctac tagacccaaa gtaaacgggc aaagtggaag
  751	aatggagttc ttctggacaa ttttaaagcc gaatgatgcc atcaatttcg
  801	agagtaatgg aaatttcatt gctccagaat atgcatacaa aattgtcaag
  851	aaaggggact caacaattat gaaaagtgaa ttggaatatg gtaactgcaa
  901	caccaagtgt caaactccaa tgggggcaat aaactctagt atgccattcc
  951	acaacataca ccccctcacc atcggggaat gccccaaata tgtgaaatca
  1001	aacagattag tccttgcaac tggactcaga aatacccctc aacgagagac
  1051	gcgaggacta tttggagcta tagcaggttt tatagaggga ggatggcagg
  1101	gaatggtaga tggttggtat gggtaccacc atagcaatga gcaggggagt
  1151	ggatacgctg cagaccaaga atccacacaa aaggcaatag atggagtcac
  1201	caataaggtc aactcgatca ttaacaaaat gaacactcag tttgaggccg
  1251	ttggaaggga atttaataac ttggaaagga ggatagagaa tttaaacaag
  1301	aaaatggaag acggattcct agatgtctgg acttacaatg ccgaacttct
  1351	ggttctcatg gaaaatgaga gaactctaga ctttcatgac tcaaatgtca
  1401	agaaccttta cgacaaggtc cgactacagc ttagggataa tgcaaaggag
  1451	ctgggtaatg gttgtttcga attctatcac aaatgtgata acgaatgtat
  1501	ggaaagtgta aaaaacggaa cgtatgacta cccgcagtat tcagaagaag
  1551	caagactaaa cagagaggaa ataagtggag taaaattgga atcaatggga
  1601	acttaccaaa tactgtcaat ttattcaaca gtggcgagtt ccctagcact
  1651	ggcaatcatg gtagctggtc tatctttatg gatgtgctcc aatggatcgt
  1701	tacaatgcag aatttgcatt taaatttgtg agttcagatt gtagttaaaa
  1751	acacccttgt ttctact

• Amino acid sequence of ca A/Hong Kong/491/97H5 (SEQ ID NO:15)
• Entire molecule length: 564aa

  1	mekivlllat vslvksdqic igyhannste qvdtimeknv tvthaqdile
  51	rthngklcdl ngvkplilrd csvagwllgn pmcdefinvp ewsyivekas
  101	pandlcypgn fndyeelkhl lsrinhfeki qiipksswsn hdassgvssa
  151	cpylgrssff rnvvwlikkn ssyptikrsy nntnqedllv lwgihhpnda
  201	aeqtrlyqnp ttyisvgtst lnqrlvpeia trpkvngqsg rmeffwtilk
  251	pndainfesn gnfiapeyay kivkkgdsti mkseleygnc ntkcqtpmga
  301	inssmpfhni hpltigecpk yvksnrlvla tglrntpqre trglfgaiag
  351	fieggwqgmv dgwygyhhsn eqgsgyaadq estqkaidgv tnkvnsiink
  401	mntqfeavgr efnnlerrie nlnkkmedgf ldvwtynael lvlmenertl
  451	dfhdsnvknl ydkvrlqlrd nakelgngcf efyhkcdnec mesvkngtyd
  501	ypqyseearl nreeisgvkl esmgtyqils iystvassla laimvaglsl
  551	wmcsngslqc rici

• Nucleotide Sequence of ca A/Hong Kong/486/97 N1 (SEQ ID NO: 6)
• Entire molecule length: 1401 nt

  1	agcaaaagca ggagtttaaa atgaatccaa atcagaagat aataaccatt
  51	ggatcaatct gcatggtagt tgggataatc agcttgatgt tacaaattgg
  101	aaacacaata tcagtatggg tcagccacat aattaaaact tggcacccaa
  151	accagcctga accatgcaac caaagcatca atttttacac tgagcaggct
  201	gcagcttcag tgacattagc gggcaattcc tctctctgcc ctattagtgg
  251	atgggctata tacagcaagg acaatagtat aagaattggt tccaaagggg
  301	atgtgtttgt tataagagaa ccattcatct catgctccca tttggaatgc
  351	agaacctttt tcttgaccca aggagcccta ttgaatgaca agcattctaa
  401	tgggaccgtc aaagacagga gcccctatag aactttaatg agctgtcctg
  451	ttggtgaggc cccttcccca tacaactcaa ggtttgagtc tgttgcttgg
  501	tcagcaagtg cttgccatga tggcattagt tggctaacaa ttggaatttc
  551	cggtccggat aatggggctg tggctgtgtt gaaatacaat ggcataataa
  601	cagacaccat caagagttgg aggaacaaca cactgaggac gcaagagtct
  651	gaatgtgcat gtgtgaatgg ttcttgtttt actgtaatga cagatggacc
  701	gagtaatgaa caggcctcat acaagatttt caagatagaa aaggggaggg
  751	tagtcaaatc agttgagttg aacgccccta attatcatta cgaggaatgc
  801	tcctgttatc ctgatgctgg cgaaatcaca tgtgtgtgca gggataattg
  851	gcatggctcg aaccgaccat gggtgtcttt caatcagaat ctggagtatc
  901	aaataggata tatatgcagt ggggttttcg gagacagtcc acgccccaat
  951	gatgggacag gcagttgtgg tccagtgtct cttaacggag cgtatggagt
  1001	aaaagggttt tcatttaaat acggcaatgg tgtttggatc gggagaacca
  1051	aaagcactag ttccaggagc ggttttgaaa tgatttggga tccaaatggg
  1101	tggaccgaaa cagacagtag cttctcgttg aagcaagaca tcatagcgat
  1151	aactgattgg tcaggataca gcgggagttt tattcaacat ccagaactga
  1201	caggattaaa ttgcatgaga ccttgcttct gggttgaact aatcagaggg
  1251	aggcccaaag agaaaacaat ctggactagt gggagcagta tatctttctg
  1301	tggtgtaaat agtgacactg tgggttggtc ttggccagac ggtgctgagt
  1351	tgccatacac cattgacaag tagtttgttc aaaaaactcc ttgtttctac
  1401	t

• Amino acid sequence of ca A/Hong Kong/486/97 N1 (SEQ ID NO:16)
• Entire molecule length: 450aa

  1	mnpnqkiiti gsicmvvgii slmlqignti svwvshiikt whpnqpepcn
  51	qsinfyteqa aasvtlagns slcpisgwai yskdnsirig skgdvfvire
  101	pfiscshlec rtffltqgal lndkhsngtv kdrspyrtlm scpvgeapsp
  151	ynsrfesvaw sasachdgis wltigisgpd ngavavlkyn giitdtiksw
  201	rnntlrtqes ecacvngscf tvmtdgpsne qasykifkie kgrvvksvel
  251	napnyhyeec scypdageit cvcrdnwhgs nrpwvsfnqn leyqigyics
  301	gvfgdsprpn dgtgscgpvs lngaygvkgf sfkygngvwi grtkstssrs
  351	gfemiwdpng wtetdssfsl kqdiiaitdw sgysgsfiqh peltglncmr
  401	pcfwvelirg rpkektiwts gssisfcgvn sdtvgwswpd gaelpytidk

  
  ca A/Hong Kong/491/97 (Ser211) (HA)+ca A/Hong Kong/486/97 (NA)
• Nucleotide Sequence of ca A/Hong Kong/491/97 (Ser211) H5 (SEQ ID NO: 7)
• Entire molecule length: 1767 nt

  1	agcaaaagca ggggtataat ctgtcaaaat ggagaaaata gtgcttcttc
  51	ttgcaacagt cagccttgtt aaaagtgacc agatttgcat tggttaccat
  101	gcaaacaact cgacagagca agttgacaca ataatggaaa agaatgttac
  151	tgttacacat gcccaagaca tactggaaag gacacacaac gggaagctct
  201	gcgatctaaa tggagtgaag cctctgattt tgagggattg tagtgtagct
  251	ggatggctcc tcggaaaccc tatgtgtgac gaattcatca atgtgccgga
  301	atggtcttac atagtggaga aggccagtcc agccaatgac ctctgttatc
  351	cagggaattt caacgactat gaagaactga aacacctatt gagcagaata
  401	aaccattttg agaaaattca gataatcccc aaaagttctt ggtccaatca
  451	tgatgcctca tcaggggtga gctcagcatg tccatacctt gggaggtcct
  501	cctttttcag aaatgtggta tggcttatca aaaagaacag tagctaccca
  551	acaataaaga ggagctacaa taataccaac caagaagatc ttttggtact
  601	gtgggggatt caccatccta atgatgcggc agagcagaca aggctctatc
  651	aaaacccaac cacctacatt tccgttggaa catcaacact gaaccagaga
  701	ttggtttcag aaatagctac tagacccaaa gtaaacgggc aaagtggaag
  751	aatggagttc ttctggacaa ttttaaagcc gaatgatgcc atcaatttcg
  801	agagtaatgg aaatttcatt gctccagaat atgcatacaa aattgtcaag
  851	aaaggggact caacaattat gaaaagtgaa ttggaatatg gtaactgcaa
  901	caccaagtgt caaactccaa tgggggcaat aaactctagt atgccattcc
  951	acaacataca ccccctcacc atcggggaat gccccaaata tgtgaaatca
  1001	aacagattag tccttgcaac tggactcaga aatacccctc aacgagagac
  1051	gcgaggacta tttggagcta tagcaggttt tatagaggga ggatggcagg
  1101	gaatggtaga tggttggtat gggtaccacc atagcaatga gcaggggagt
  1151	ggatacgctg cagaccaaga atccacacaa aaggcaatag atggagtcac
  1201	caataaggtc aactcgatca ttaacaaaat gaacactcag tttgaggccg
  1251	ttggaaggga atttaataac ttggaaagga ggatagagaa tttaaacaag
  1301	aaaatggaag acggattcct agatgtctgg acttacaatg ccgaacttct
  1351	ggttctcatg gaaaatgaga gaactctaga ctttcatgac tcaaatgtca
  1401	agaaccttta cgacaaggtc cgactacagc ttagggataa tgcaaaggag
  1451	ctgggtaatg gttgtttcga attctatcac aaatgtgata acgaatgtat
  1501	ggaaagtgta aaaaacggaa cgtatgacta cccgcagtat tcagaagaag
  1551	caagactaaa cagagaggaa ataagtggag taaaattgga atcaatggga
  1601	acttaccaaa tactgtcaat ttattcaaca gtggcgagtt ccctagcact
  1651	ggcaatcatg gtagctggtc tatctttatg gatgtgctcc aatggatcgt
  1701	tacaatgcag aatttgcatt taaatttgtg agttcagatt gtagttaaaa
  1751	acacccttgt ttctact

• Amino acid sequence of ca A/Hong Kong/491/97 (Ser211) H5 (SEQ ID NO:17)
• Entire molecule length: 564aa

  1	mekivlllat vslvksdqic igyhannste qvdtimeknv tvthaqdile
  51	rthngklcdl ngvkplilrd csvagwllgn pmcdefinvp ewsyivekas
  101	pandlcypgn fndyeelkhl lsrinhfeki qiipksswsn hdassgvssa
  151	cpylgrssff rnvvwlikkn ssyptikrsy nntnqedllv lwgihhpnda
  201	aeqtrlyqnp ttyisvgtst lnqrlvseia trpkvngqsg rmeffwtilk
  251	pndainfesn gnfiapeyay kivkkgdsti mkseleygnc ntkcqtpmga
  301	inssmpfhni hpltigecpk yvksnrlvla tglrntpqre trglfgaiag
  351	fieggwqgmv dgwygyhhsn eqgsgyaadq estqkaidgv tnkvnsiink
  401	mntqfeavgr efnnlerrie nlnkkmedgf ldvwtynael lvlmenertl
  451	dfhdsnvknl ydkvrlqlrd nakelgngcf efyhkcdnec mesvkngtyd
  501	ypqyseearl nreeisgvkl esmgtyqils iystvassla laimvaglsl
  551	wmcsngslqc rici

• Nucleotide Sequence of ca A/Hong Kong/486/97 N1 (SEQ ID NO: 8)
• Entire molecule length: 1401 nt

  1	agcaaaagca ggagtttaaa atgaatccaa atcagaagat aataaccatt
  51	ggatcaatct gcatggtagt tgggataatc agcttgatgt tacaaattgg
  101	aaacacaata tcagtatggg tcagccacat aattaaaact tggcacccaa
  151	accagcctga accatgcaac caaagcatca atttttacac tgagcaggct
  201	gcagcttcag tgacattagc gggcaattcc tctctctgcc ctattagtgg
  251	atgggctata tacagcaagg acaatagtat aagaattggt tccaaagggg
  301	atgtgtttgt tataagagaa ccattcatct catgctccca tttggaatgc
  351	agaacctttt tcttgaccca aggagcccta ttgaatgaca agcattctaa
  401	tgggaccgtc aaagacagga gcccctatag aactttaatg agctgtcctg
  451	ttggtgaggc cccttcccca tacaactcaa ggtttgagtc tgttgcttgg
  501	tcagcaagtg cttgccatga tggcattagt tggctaacaa ttggaatttc
  551	cggtccggat aatggggctg tggctgtgtt gaaatacaat ggcataataa
  601	cagacaccat caagagttgg aggaacaaca cactgaggac gcaagagtct
  651	gaatgtgcat gtgtgaatgg ttcttgtttt actgtaatga cagatggacc
  701	gagtaatgaa caggcctcat acaagatttt caagatagaa aaggggaggg
  751	tagtcaaatc agttgagttg aacgccccta attatcatta cgaggaatgc
  801	tcctgttatc ctgatgctgg cgaaatcaca tgtgtgtgca gggataattg
  851	gcatggctcg aaccgaccat gggtgtcttt caatcagaat ctggagtatc
  901	aaataggata tatatgcagt ggggttttcg gagacagtcc acgccccaat
  951	gatgggacag gcagttgtgg tccagtgtct cttaacggag cgtatggagt
  1001	aaaagggttt tcatttaaat acggcaatgg tgtttggatc gggagaacca
  1051	aaagcactag ttccaggagc ggttttgaaa tgatttggga tccaaatggg
  1101	tggaccgaaa cagacagtag cttctcgttg aagcaagaca tcatagcgat
  1151	aactgattgg tcaggataca gcgggagttt tattcaacat ccagaactga
  1201	caggattaaa ttgcatgaga ccttgcttct gggttgaact aatcagaggg
  1251	aggcccaaag agaaaacaat ctggactagt gggagcagta tatctttctg
  1301	tggtgtaaat agtgacactg tgggttggtc ttggccagac ggtgctgagt
  1351	tgccatacac cattgacaag tagtttgttc aaaaaactcc ttgtttctac
  1401	t

• Amino acid sequence of ca A/Hong Kong/486/97 N1 (SEQ ID NO:18)
• Entire molecule length: 450aa

  1	mnpnqkiiti gsicmvvgii slmlqignti svwvshiikt whpnqpepcn
  51	qsinfyteqa aasvtlagns slcpisgwai yskdnsirig skgdvfvire
  101	pfiscshlec rtffltqgal lndkhsngtv kdrspyrtlm scpvgeapsp
  151	ynsrfesvaw sasachdgis wltigisgpd ngavavlkyn giitdtiksw
  201	rnntlrtqes ecacvngscf tvmtdgpsne qasykifkie kgrvvksvel
  251	napnyhyeec scypdageit cvcrdnwhgs nrpwvsfnqn leyqigyics
  301	gvfgdsprpn dgtgscgpvs lngaygvkgf sfkygngvwi grtkstssrs
  351	gfemiwdpng wtetdssfsl kqdiiaitdw sgysgsfiqh peltglncmr
  401	pcfwvelirg rpkektiwts gssisfcgvn sdtvgwswpd gaelpytidk

  
  ca A/ck/Hong Kong/G9/97
• Nucleotide sequence of ca A/ck/Hong Kong/G9/97 (SEQ ID NO: 9)
• Entire molecule length: 1690bp

  1	ttaaccactc aagatggaag caataccact aataactata ctactagtag
  51	taacagcaag caatgcagac aaaatctgca tcggctacca atcaacaaac
  101	tccacagaaa ccgtagacac gctaacagaa aacaatgttc ctgtgacaca
  151	tgccaaagaa ttgctccaca cagagcacaa tgggatgctg tgtgcaacaa
  201	atctgggacg tcctcttatt ctagacactt gcaccattga aggactgatc
  251	tatggcaacc cttcttgtga tctactgttg ggaggaagag aatggtccta
  301	catcgtcgaa agaccatcgg ctgttaatgg aatgtgttac cccgggaatg
  351	tagaaaacct agaggaacta aggtcatttt ttagttctgc tagttcctac
  401	caaagaatcc agatctttcc agacacaatc tggaatgtgt cttacagtgg
  451	aacaagcaaa gcatgttcag attcattcta caggagcatg agatggttga
  501	ctcaaaagaa caacgcttac cctattcaag acgcccaata cacaaataat
  551	agaggaaaga gcattctttt catgtggggc ataaatcacc cacctaccga
  601	tactgcacag acaaatctgt acacaaggac tgacacaaca acaagtgtgg
  651	caacagaaga tataaatagg accttcaaac cagtgatagg gccaaggccc
  701	cttgtcaatg gtctgcaggg aagaattgat tattattggt cggtattgaa
  751	accaggtcag acattgcgag taagatccaa tgggaatcta atcgctccat
  801	ggtatgggca cattctttca ggagagagcc acggaagaat cctgaagact
  851	gatttaaaca gtggtagctg tgtagtgcaa tgtcaaacag aaagaggtgg
  901	cttaaatact actttgccat tccacaatgt cagtaaatat gcatttggaa
  951	actgcccaaa atatgttgga gtaaagagtc tcaaactggc agttggtctg
  1001	aggaatgtgc ctgctagatc aagtagagga ctatttgggg ccatagctgg
  1051	attcatagag ggaggttggt cagggctggt cgctggttgg tatgggttcc
  1101	agcattcaaa tgatcaaggg gttggtatag ctgcagatag agactcaact
  1151	caaagggcaa ttgacaaaat aacgtccaaa gtgaataata tagtcgataa
  1201	aatgaacaag caatatgaaa ttattgatca tgaattcagc gaggttgaaa
  1251	atagactcaa tatgatcaat aataagattg atgaccagat acaagacata
  1301	tgggcatata acgctgaatt gctagtgctg cttgaaaacc agaaaacact
  1351	cgatgagcat gatgcgaatg taaacaatct atataacaaa gtgaagaggg
  1401	cactgggttc caatgcaatg gaagatggga aaggatgttt cgagctatac
  1451	cataaatgtg atgatcagtg catggagaca attcggaacg ggacctataa
  1501	caggaggaag tataaagagg aatcaagact agaaagacag aaaatagaag
  1551	gggtcaagct ggaatctgaa ggaacttaca aaatcctcac catttattcg
  1601	actgtcgcct catctcttgt gattgcaatg gggtttgctg ccttcttgtt
  1651	ctgggccatg tccaatggat cttgcagatg caacatttga

• Amino acid sequence of ca A/ck/Hong Kong/G9/97H9 (SEQ ID NO:19)
• Entire molecule length: 558aa

  1	meaiplitil lvvtasnadk icigyqstns tetvdtlten nvpvthakel
  51	lhtehngmlc atnlgrplil dtctiegliy gnpscdlllg grewsyiver
  101	psavngmcyp gnvenleelr sffssassyq riqifpdtiw nvsysgtska
  151	csdsfyrsmr wltqknnayp iqdaqytnnr gksilfmwgi nhpptdtaqt
  201	nlytrtdttt svatedinrt fkpvigprpl vnglqgridy ywsvlkpgqt
  251	lrvrsngnli apwyghilsg eshgrilktd lnsgscvvqc qtergglntt
  301	lpfhnvskya fgncpkyvgv kslklavglr nvparssrgl fgaiagfieg
  351	gwsglvagwy gfqhsndqgv giaadrdstq raidkitskv nnivdkmnkq
  401	yeiidhefse venrlnminn kiddqiqdiw aynaellvll enqktldehd
  451	anvnnlynkv kralgsname dgkgcfelyh kcddqcmeti rngtynrrky
  501	keesrlerqk iegvkleseg tykiltiyst vasslviamg faaflfwams
  551	ngscrcni

• Nucleotide sequence of ca A/ck/Hong Kong/G9/97 N2 (SEQ ID NO:10)
• Entire molecule length: 1428bp

  1	aaatgaatcc aaatcagaag ataatagcaa ttggctctgt ttctctaact
  51	attgcgacaa tatgcctcct catgcagatt gctatcttag caacgactat
  101	gacactacat ttcaagcaga atgaatgcat caactcctcg aataatcaag
  151	tagtgccatg tgaaccaatc ataatagaaa ggaacataac agagatagtg
  201	catttgaata gtactacctt agagaaggaa atttgtccta aagtagcaga
  251	ctacaggaat tggtcaaaac cacaatgtca aatcacaggg ttcgctcctt
  301	tctccaagga caattcaatt aggctctccg caggtggaga tatttgggtg
  351	acaagagaac cttatgtatc gtgcggtctt ggtaaatgtt atcaatttgc
  401	acttgggcag ggaaccactt tggagaacaa acactcaaac ggcacagcac
  451	atgatagaac tcctcataga acccttttaa tgaatgagtt gggtgttccg
  501	tttcatttgg caaccaaaca agtgtgcata gcatggtcca gctcaagctg
  551	ccatgatggg aaagcatggt tacatgtttg tgtcactggg gatgatagaa
  601	atgcaacggc tagcatcatt tatgatggga tacttgttga cagtattggt
  651	tcatggtcta aaaacatcct cagaactcag gagtcagaat gcgtttgcat
  701	caatggaacc tgtgcagtag taatgactga tggaagtgca tcaggaaggg
  751	ctgacactag aatactattt attagagagg ggaaaattgc acacattagc
  801	ccattgtcag gaagtgctca gcatgtggag gaatgctcct gttacccccg
  851	atatccagaa gttagatgtg tttgcagaga caattggaag ggatccaata
  901	ggcccgttct atatataaat atggcaaatt atagtattga ttccagttat
  951	gtgtgctcag gacttgttgg cgacacacca agaaatgatg ataggtctag
  1001	cagcagcaac tgcagagatc ctaataacga gagaggggcc ccaggagtaa
  1051	aagggtgggc ctttgacaat ggaaatgaca tttggatggg aagaacaatc
  1101	aaaaaggatt cgcgctcagg ttatgagact ttcagggtca ttggtggttg
  1151	gaccactgct aattccaagt cacagataaa tagacaagtc atagttgaca
  1201	gtgataactc gtctgggtat tctggtatct tctctgttga aggcaaaagc
  1251	tgcatcaaca ggtgttttta cgtggagttg ataagaggaa gaccaaagga
  1301	gactagggtg tggtggactt caaatagcat cattgtattt tgtggaactt
  1351	caggtaccta tggaacaggc tcatggcctg atggggcgaa tatcaatttc
  1401	atgcctatat aagctttcgc aattttag

• Amino acid sequence of ca A/ck/Hong Kong/G9/97 N2 (SEQ ID NO: 20)
• Entire molecule length: 469aa

  1	mnpnqkiiai gsvsltiati cllmqiaila ttmtlhfkqn ecinssnnqv
  51	vpcepiiier niteivhlns ttlekeicpk vadyrnwskp qcqitgfapf
  101	skdnsirlsa ggdiwvtrep yvscglgkcy qfalgqgttl enkhsngtah
  151	drtphrtllm nelgvpfhla tkqvciawss sschdgkawl hvcvtgddrn
  201	atasilydgi lvdsigswsk nhlrtqesec vcingtcavv mtdgsasgra
  251	dtrilfireg kiahisplsg saqhveecsc yprypevrcv crdnwkgsnr
  301	pvlyinmany sidssyvcsg lvgdtprndd rssssncrdp nnergapgvk
  351	gwafdngndi wmgrtikkds rsgyetfrvi ggwttansks qinrqvivds
  401	dnssgysgif svegkscinr cfyvelirgr pketrvwwts nsiivfcgts
  451	gtygtgswpd ganinfmpi

  
  A/Netherlands/219/03
• Nucleotide sequence of A/Netherlands/219/03H7 (SEQ ID NO: 21)
• Entire molecule length: 1737bp

  1	agcaaaagca ggggatacaa aatgaacact caaatcctgg tattcgctct
  51	ggtggcgagc attccgacaa atgcagacaa gatctgcctt gggcatcatg
  101	ccgtgtcaaa cgggactaaa gtaaacacat taactgagag aggagtggaa
  151	gtcgttaatg caactgaaac ggtggaacga acaaacgttc ccaggatctg
  201	ctcaaaaggg aaaaggacag ttgacctcgg tcaatgtgga cttctgggaa
  251	caatcactgg gccaccccaa tgtgaccaat tcctagaatt ttcggccgac
  301	ttaattattg agaggcgaga aggaagtgat gtctgttatc ctgggaaatt
  351	cgtgaatgaa gaagctctga ggcaaattct cagagagtca ggcggaattg
  401	acaaggagac aatgggattc acctacagcg gaataagaac taatggaaca
  451	accagtgcat gtaggagatc aggatcttca ttctatgcag agatgaaatg
  501	gctcctgtca aacacagaca atgctgcttt cccgcaaatg actaagtcat
  551	acaagaacac aaggaaagac ccagctctga taatatgggg gatccaccat
  601	tccggatcaa ctacagaaca gaccaagcta tatgggagtg gaaacaaact
  651	gataacagtt gggagttcta attaccaaca gtcctttgta ccgagtccag
  701	gagcgagacc acaagtgaat ggccaatctg gaagaattga ctttcattgg
  751	ctgatactaa accctaatga cacggtcact ttcagtttca atggggcctt
  801	catagctcca gaccgtgcaa gctttctgag agggaagtcc atgggaattc
  851	agagtgaagt acaggttgat gccaattgtg aaggagattg ctatcatagt
  901	ggagggacaa taataagtaa tttgcccttt cagaacataa atagcagggc
  951	agtaggaaaa tgtccgagat atgttaagca agagagtctg ctgttggcaa
  1001	caggaatgaa gaatgttccc gaaatcccaa agaggaggag gagaggccta
  1051	tttggtgcta tagcgggttt cattgaaaat ggatgggaag gtttgattga
  1101	tgggtggtat ggcttcaggc atcaaaatgc acaaggggag ggaactgctg
  1151	cagattacaa aagcacccaa tcagcaattg atcaaataac agggaaatta
  1201	aatcggctta tagaaaaaac taaccaacag tttgagttaa tagacaacga
  1251	attcactgag gttgaaaggc aaattggcaa tgtgataaac tggaccagag
  1301	attccatgac agaagtgtgg tcctataacg ctgaactctt agtagcaatg
  1351	gagaatcagc acacaattga tctggccgac tcagaaatga acaaactgta
  1401	cgaacgagtg aagagacaac tgagagagaa tgccgaagaa gatggcactg
  1451	gttgcttcga aatatttcac aagtgtgatg acgactgcat ggccagtatt
  1501	agaaacaaca cctatgatca cagcaagtac agggaagaag caatacaaaa
  1551	tagaatacag attgacccag tcaaactaag cagcggctac aaagatgtga
  1601	tactttggtt tagcttcggg gcatcatgtt tcatacttct ggccattgca
  1651	atgggccttg tcttcatatg tgtgaagaat ggaaacatgc ggtgcactat
  1701	ttgtatataa gtttggaaaa acacccttgt ttctact

• Amino acid sequence of A/Netherlands/219/03H7 (SEQ ID NO: 27)
• Entire molecule length: 562aa

  1	mntqilvfal vasiptnadk iclghhavsn gtkvntlter gvevvnatet
  51	vertnvpric skgkrtvdlg qcgllgtitg ppqcdqflef sadliierre
  101	gsdvcypgkf vneealrqil resggidket mgftysgirt ngttsacrrs
  151	gssfyaemkw llsntdnaaf pqmtksyknt rkdpaliiwg ihhsgstteq
  201	tklygsgnkl itvgssnyqq sfvpspgarp qvngqsgrid fhwlilnpnd
  251	tvtfsfngaf iapdrasflr gksmgiqsev qvdancegdc yhsggtiisn
  301	lpfqninsra vgkcpryvkq eslllatgmk nvpeipkrrr rglfgaiagf
  351	iengweglid gwygfrhqna qgegtaadyk stqsaidqit gklnrliekt
  401	nqqfelidne fteverqign vinwtrdsmt evwsynaell vamenqhtid
  451	ladsemnkly ervkrqlren aeedgtgcfe ifhkcdddcm asirnntydh
  501	skyreeaiqn riqidpvkls sgykdvilwf sfgascfill aiamglvfic
  551	vkngnmrcti ci

• Nucleotide Sequence of A/Netherlands/219/03 N7 (SEQ ID NO: 22)
• Entire molecule length: 1465 nt

  1	agcaaaagca gggtgatcga gaatgaatcc aaatcagaaa ctatttgcat
  51	tatctggagt ggcaatagca cttagtgtac tgaacttatt gataggaatc
  101	tcaaacgtcg gattgaacgt atctctacat ctaaaggaaa aaggacccaa
  151	acaggaggag aatttaacat gcacgaccat taatcaaaac aacactactg
  201	tagtagaaaa cacatatgta aataatacaa caataattac caagggaact
  251	gatttgaaaa caccaagcta tctgctgttg aacaagagcc tgtgcaatgt
  301	tgaagggtgg gtcgtgatag caaaagacaa tgcagtaaga tttggggaaa
  351	gtgaacaaat cattgttacc agggagccat atgtatcatg cgacccaaca
  401	ggatgcaaaa tgtatgcctt gcaccaaggg actaccatta ggaacaaaca
  451	ttcaaatgga acgattcatg acagaacagc tttcagaggt ctcatctcca
  501	ctccattggg cactccacca accgtaagta acagtgactt tatgtgtgtt
  551	ggatggtcaa gcacaacttg ccatgatggg attgctagga tgactatctg
  601	tatacaagga aataatgaca atgctacagc aacggtttat tacaacagaa
  651	ggctgaccac taccattaag acctgggcca gaaacattct gaggactcaa
  701	gaatcagaat gtgtgtgcca caatggcaca tgtgcagttg taatgaccga
  751	cggatcggct agtagtcaag cctatacaaa agtaatgtat ttccacaagg
  801	gattagtagt taaggaggag gagttaaggg gatcagccag acatattgag
  851	gaatgctcct gttatggaca caatcaaaag gtgacctgtg tgtgcagaga
  901	taactggcag ggagcaaaca ggcctattat agaaattgat atgagcacat
  951	tggagcacac aagtagatac gtgtgcactg gaattctcac agacaccagc
  1001	agacctgggg acaaatctag tggtgattgt tccaatccaa taactgggag
  1051	tcccggcgtt ccgggagtga agggattcgg gtttctaaat ggggataaca
  1101	catggcttgg taggaccatc agccccagat caagaagtgg attcgaaatg
  1151	ttgaaaatac ctaatgcagg tactgatccc aattctagaa tagcagaacg
  1201	acaggaaatt gtcgacaata acaattggtc aggctattcc ggaagcttta
  1251	ttgactattg gaatgataac agtgaatgct acaatccatg cttttacgta
  1301	gagttaatta gaggaagacc cgaagaggct aaatacgtat ggtgggcaag
  1351	taacagtcta attgccctat gtggaagccc attcccagtt gggtctggtt
  1401	ccttccccga tggggcacaa atccaatact tttcgtaaaa tgcaaaaaaa
  1451	ctccttgttt ctact

• Nucleotide Sequence of A/Netherlands/219/03 N7 (SEQ ID NO: 45)
• Entire molecule length: 1464 nt

  1	agcaaaagca gggtgatcga gaatgaatcc aaatcagaaa ctatttgcat tatctggagt
  61	ggcaatagca cttagtgtac tgaacttatt gataggaatc tcaaacgtcg gattgaacgt
  121	atctctacat ctaaaggaaa aaggacccaa acaggaggag aatttaacat gcacgaccat
  181	taatcaaaac aacactactg tagtagaaaa cacatatgta aataatacaa caataattac
  241	caagggaact gatttgaaaa caccaagcta tctgctgttg aacaagagcc tgtgcaatgt
  301	tgaagggtgg gtcgtgatag caaaagacaa tgcagtaaga tttggggaaa gtgaacaaat
  361	cattgttacc agggagccat atgtatcatg cgacccaaca ggatgcaaaa tgtatgcctt
  421	gcaccaaggg actaccatta ggaacaaaca ttcaaatgga acgattcatg acagaacagc
  481	tttcagaggt ctcatctcca ctccattggg cactccacca accgtaagta acagtgactt
  541	tatgtgtgtt ggatggtcaa gcacaacttg ccatgatggg attgctagga tgactatctg
  601	tatacaagga aataatgaca atgctacagc aacggtttat tacaacagaa ggctgaccac
  661	taccattaag acctgggcca gaaacattct gaggactcaa gaatcagaat gtgtgtgcca
  721	caatggcaca tgtgcagttg taatgaccga cggatcggct agtagtcaag cctatacaaa
  781	agtaatgtat ttccacaagg gattagtagt taaggaggag gagttaaggg gatcagccag
  841	acatattgag gaatgctcct gttatggaca caatcaaaag gtgacctgtg tgtgcagaga
  901	taactggcag ggagcaaaca ggcctattat agaaattgat atgagcacat tggagcacac
  961	aagtagatac gtgtgcactg gaattctcac agacaccagc agacctgggg acaaatctag
  1021	tggtgattgt tccaatccaa taactgggag tcccggcgtt ccgggagtga agggattcgg
  1081	gtttctaaat ggggataaca catggcttgg taggaccatc agccccagat caagaagtgg
  1141	attcgaaatg ttgaaaatac ctaatgcagg tactgatccc aattctagaa tagcagaacg
  1201	acaggaaatt gtcgacaata acaattggtc aggctattcc ggaagcttta ttgactattg
  1261	gaatgataac agtgaatgct acaatccatg cttttacgta gagttaatta gaggaagacc
  1321	cgaagaggct aaatacgtat ggtgggcaag taacagtcta attgccctat gtggaagccc
  1381	attcccagtt gggtctggtt ccttccccga tggggcacaa atccaatact tttcgtaaaa
  1441	tgcaaaaaca cccttgtttc tact

• Amino acid sequence of A/Netherlands/219/03 N7 (SEQ ID NO: 28)
• Entire molecule length: 471aa

  1	mnpnqklfal sgvaialsvl nlligisnvg lnvslhlkek gpkqeenltc
  51	ttinqnnttv ventyvnntt iitkgtdlkt psylllnksl cnvegwvvia
  101	kdnavrfges eqiivtrepy vscdptgckm yalhqgttir nkhsngtihd
  151	rtafrglist plgtpptvsn sdfmcvgwss ttchdgiarm ticiqgnndn
  201	atatvyynrr ltttiktwar nilrtqesec vchngtcavv mtdgsassqa
  251	ytkvmyfhkg lvvkeeelrg sarhieecsc yghnqkvtcv crdnwqganr
  301	piieidmstl ehtsryvctg iltdtsrpgd kssgdcsnpi tgspgvpgvk
  351	gfgflngdnt wlgrtisprs rsgfemlkip nagtdpnsri aerqeivdnn
  401	nwsgysgsfi dywndnsecy npcfyvelir grpeeakyvw wasnslialc
  451	gspfpvgsgs fpdgaqiqyf s

  
  A/ck/BC/CN-7/04
• Nucleotide sequence of A/ck/BC/CN-7/04H7 (SEQ ID NO: 23)
• Entire molecule length: 1754bp

  1	agcaaaagca ggggatacaa aatgaatact caaattttgg cattcattgc
  51	ttgtatgctg attggaacta aaggagacaa aatatgtctt gggcaccatg
  101	ctgtggcaaa tgggacaaaa gtgaacacac taacagagag gggaattgaa
  151	gtagtcaatg ccacggagac ggtggaaact gtaaatatta aaaaaatatg
  201	cactcaagga aaaaggccaa cagatctggg acaatgtgga cttctaggaa
  251	ccctaatagg acctccccaa tgcgatcaat ttctggagtt tgacgctaat
  301	ttgataattg aacgaagaga aggaaccgat gtgtgctatc ccgggaagtt
  351	cacaaatgaa gaatcactga ggcagatcct tcgagggtca ggaggaattg
  401	ataaagagtc aatgggtttc acctatagtg gaataagaac caatggggcg
  451	acgagtgcct gcagaagatc aggttcttct ttctatgcgg agatgaagtg
  501	gttactgtcg aattcagaca atgcggcatt tccccaaatg actaagtcgt
  551	ataggaatcc caggaacaaa ccagctctga taatctgggg agtgcatcac
  601	tctggatcag ctactgagca gaccaaactc tatggaagtg gaaacaagtt
  651	gataacagta ggaagctcga aataccagca atcattcact ccaagtccgg
  701	gagcacggcc acaagtgaat ggacaatcag gaaggattga ttttcattgg
  751	ctactccttg accccaatga cacagtgacc ttcactttca atggggcatt
  801	catagcccct gacagggcaa gtttctttag aggagaatcg ctaggagtcc
  851	agagtgatgt tcctttggat tctggttgtg aaggggattg cttccacagt
  901	gggggtacga tagtcagttc cctgccattc caaaacatca accctagaac
  951	agtggggaaa tgccctcgat atgtcaaaca gacaagcctc cttttggcta
  1001	caggaatgag aaacgtccca gagaacccca agcaggccta ccggaaacgg
  1051	atgaccagag gcctttttgg agcgattgct ggattcatag agaatggatg
  1101	ggaaggtctc atcgatggat ggtatggttt cagacatcaa aatgcacaag
  1151	gagaaggaac tgcagctgac tacaaaagca cccaatctgc aatagatcag
  1201	atcacaggca aattgaatcg tctgattgac aaaacaaacc agcagtttga
  1251	actgatagac aatgaattca gtgagataga acaacaaatc gggaatgtca
  1301	ttaactggac acgagactca atgactgagg tatggtcgta taatgctgag
  1351	ctgttggtgg caatggagaa tcagcataca atagatcttg cagactcaga
  1401	aatgaacaaa ctttacgaac gcgtcagaaa acaactaagg gaaaatgctg
  1451	aagaagatgg aactggatgc tttgagatat tccataagtg tgatgatcag
  1501	tgtatggaga gcataaggaa caacacttat gaccataccc aatacaggac
  1551	agagtcattg cagaatagaa tacagataga cccagtgaaa ttgagtagtg
  1601	gatacaaaga cataatctta tggtttagct tcggggcatc atgttttctt
  1651	cttctagcca ttgcaatggg attggttttc atttgcataa agaatggaaa
  1701	catgcggtgc actatttgta tatagtttga gaaaaaaaca cccttgtttc
  1751	tact

• Amino acid sequence of A/ck/BC/CN-7/04H7 (SEQ ID NO: 29)
• Entire molecule length: 567aa

  1	mntqilafia cmligtkgdk iclghhavan gtkvntlter gievvnatet
  51	vetvnikkic tqgkrptdlg qcgllgtlig ppqcdqflef danlilerre
  101	gtdvcypgkf tneeslrqil rgsggidkes mgftysgirt ngatsacrrs
  151	gssfyaemkw llsnsdnaaf pqmtksyrnp rnkpaliiwg vhhsgsateq
  201	tklygsgnkl itvgsskyqq sftpspgarp qvngqsgrid fhwllldpnd
  251	tvtftfngaf iapdrasffr geslgvqsdv pldsgcegdc fhsggtivss
  301	lpfqninprt vgkcpryvkq tslllatgmr nvpenpkqay rkrmtrglfg
  351	aiagfiengw eglidgwygf rhqnaqgegt aadykstqsa idqitgklnr
  401	lidktnqqfe lidnefseie qqignvinwt rdsmtevwsy naellvamen
  451	qhtidladse mnklyervrk qlrenaeedg tgcfeifhkc ddqcmesirn
  501	ntydhtqyrt eslqnriqid pvklssgykd iilwfsfgas cflllaiamg
  551	lvficikngn mrctici

• Nucleotide Sequence of A/ck/BC/CN-7/04 N3 (SEQ ID NO: 24)
• Entire molecule length: 1453 nt

  1	agcaaaagca ggtgcgagat gaatccgaat cagaagataa taacaatcgg
  51	ggtagtgaat accactctgt caacaatagc ccttctcatt ggagtgggaa
  101	acttagtttt caacacagtc atacatgaga aaataggaga ccatcaaata
  151	gtgacccatc caacaataat gacccctgaa gtaccgaact gcagtgacac
  201	tataataaca tacaataaca ctgttataaa caacataaca acaacaataa
  251	taactgaagc agaaaggcct ttcaagtctc cactaccgct gtgccccttc
  301	agaggattct tcccttttca caaggacaat gcaatacgac tgggtgaaaa
  351	caaagacgtc atagtcacaa gggagcctta tgttagctgc gataatgaca
  401	actgctggtc ctttgctctc gcacaaggag cattgctagg gactaaacat
  451	agcaatggga ccattaaaga cagaacacca tataggtctc taattcgttt
  501	cccaatagga acagctccag tactaggaaa ttacaaagag atatgcattg
  551	cttggtcgag cagcagttgc tttgacggga aagagtggat gcatgtgtgc
  601	atgacaggga atgataatga tgcaagtgcc cagataatat atggaggaag
  651	aatgacagac tccattaaat catggaggaa ggacatacta agaacccagg
  701	agtctgaatg tcaatgcatt gacgggactt gtgttgttgc tgtcacagat
  751	ggccctgctg ctaatagtgc agatcacagg gtttactgga tacgggaggg
  801	aagaataata aagtatgaaa atgttcccaa aacaaagata caacacttag
  851	aagaatgttc ctgctatgtg gacattgatg tttactgtat atgtagggac
  901	aattggaagg gctctaacag accttggatg agaatcaaca acgagactat
  951	actggaaaca ggatatgtat gtagtaaatt tcactcagac acccccaggc
  1001	cagctgaccc ttcaataatg tcatgtgact ccccaagcaa tgtcaatgga
  1051	ggacccggag tgaaggggtt tggtttcaaa gctggcaatg atgtatggtt
  1101	aggtagaaca gtgtcaacta gtggtagatc gggctttgaa attatcaaag
  1151	ttacagaagg gtggatcaac tctcctaacc atgtcaaatc aattacacaa
  1201	acactagtgt ccaacaatga ctggtcaggc tattcaggta gcttcattgt
  1251	caaagccaag gactgttttc agccctgttt ttatgttgag cttatacgag
  1301	ggaggcccaa caagaatgat gacgtctctt ggacaagtaa tagtatagtt
  1351	actttctgtg gactagacaa tgaacctgga tcgggaaatt ggccagatgg
  1401	ttctaacatt gggtttatgc ccaagtaata gaaaaaagca ccttgtttct
  1451	act

• Amino acid sequence of A/ck/BC/CN-7/04 N3 (SEQ ID NO: 30)
• Entire molecule length: 469aa

  1	mnpnqkiiti gvvnttlsti alligvgnlv fntvihekig dhqivthpti
  51	mtpevpncsd tiitynntvi nnitttiite aerpfksplp lcpfrgffpf
  101	hkdnairlge nkdvivtrep yvscdndncw sfalaqgall gtkhsngtik
  151	drtpyrslir fpigtapvlg nykeiciaws ssscfdgkew mhvcmtgndn
  201	dasaqiiygg rmtdsikswr kdilrtqese cqcidgtcvv avtdgpaans
  251	adhrvywire griikyenvp ktkiqhleec scyvdidvyc icrdnwkgsn
  301	rpwmrinnet iletgyvcsk fhsdtprpad psimscdsps nvnggpgvkg
  351	fgfkagndvw lgrtvstsgr sgfeiikvte gwinspnhvk sitqtlvsnn
  401	dwsgysgsfi vkakdcfqpc fyvelirgrp nknddvswts nsivtfcgld
  451	nepgsgnwpd gsnigfmpk

  
  ca A/ck/BC/CN-6/04
• Nucleotide sequence of ca A/ck/BC/CN-6/04H7 (SEQ ID NO: 25)
• Entire molecule length: 1734bp

  1	agcgaaagca ggggatacaa aatgaatact caaattttgg cattcattgc
  51	ttgtatgctg attggaacta aaggagacaa aatatgtctt gggcaccatg
  101	ctgtggcaaa tgggacaaaa gtgaacacac taacagagag gggaattgaa
  151	gtagtcaatg ccacggagac ggtggaaact gtaaatatta agaaaatatg
  201	cactcaagga aaaaggccaa cagatctggg acaatgtgga cttctaggaa
  251	ccctaatagg acctccccaa tgcgatcaat ttctggagtt tgacgctaat
  301	ttgataattg aacgaagaga aggaaccgat gtgtgctatc ccgggaagtt
  351	cacaaatgaa gaatcactga ggcagatcct tcgagggtca ggaggaattg
  401	ataaagagtc aatgggtttc acctatagtg gaataagaac caatggggcg
  451	acgagtgcct gcagaagatc aggttcttct ttctatgcgg agatgaagtg
  501	gttactgtcg aattcagaca atgcggcatt tccccaaatg actaagtcgt
  551	ataggaatcc caggaacaaa ccagctctga taatctgggg agtgcatcac
  601	tctggatcag ctactgagca gaccaaactc tatggaagtg gaaacaagtt
  651	gataacagta ggaagctcga aataccagca atcattcact ccaagtccgg
  701	gagcacggcc acaagtgaat ggacaatcag gaaggattga ttttcattgg
  751	ctactccttg accccaatga cacagtgacc ttcactttca atggggcatt
  801	catagcccct gacagggcaa gtttctttag aggagaatcg ctaggagtcc
  851	agagtgatgt tcctttggat tctggttgtg aaggggattg cttccacagt
  901	gggggtacga tagtcagttc cctgccattc caaaacatca accctagaac
  951	agtggggaaa tgccctcgat atgtcaaaca gacaagcctc cttttggcta
  1001	caggaatgag aaacgtccca gagaacccca agaccagagg cctttttgga
  1051	gcgattgctg gattcataga gaatggatgg gaaggtctca tcgatggatg
  1101	gtatggtttc agacatcaaa atgcacaagg agaaggaact gcagctgact
  1151	acaaaagcac ccaatctgca atagatcaga tcacaggcaa attgaatcgt
  1201	ctgattgaca aaacaaacca gcagtttgaa ctgatagaca atgaattcag
  1251	tgagatagaa caacaaatcg ggaatgtcat taactggaca cgagactcaa
  1301	tgactgaggt atggtcgtat aatgctgagc tgttggtggc aatggagaat
  1351	cagcatacaa tagatcttgc agactcagaa atgaacaaac tttacgaacg
  1401	cgtcagaaaa caactaaggg aaaatgctga agaagatgga actggatgct
  1451	ttgagatatt ccataagtgt gatgatcagt gtatggagag cataaggaac
  1501	aacacttatg accataccca atacaggaca gagtcattgc agaatagaat
  1551	acagatagac ccagtgaaat tgagtagtgg atacaaagac ataatcttat
  1601	ggtttagctt cggggcatca tgttttcttc ttctagccat tgcaatggga
  1651	ttggttttca tttgcataaa gaatggaaac atgcggtgca ctatttgtat
  1701	atagtttgag aaaaaaacac ccttgtttct act

• Amino acid sequence of ca A/ck/BC/CN-6/04H7 (SEQ ID NO: 31)
• Entire molecule length: 560aa

  1	mntqilafia cmligtkgdk iclghhavan gtkvntlter gievvnatet
  51	vetvnikkic tqgkrptdlg qcgllgtlig ppqcdqflef danliierre
  101	gtdvcypgkf tneeslrqil rgsggidkes mgftysgirt ngatsacrrs
  151	gssfyaemkw llsnsdnaaf pqmtksyrnp rnkpaliiwg vhhsgsateq
  201	tklygsgnkl itvgsskyqq sftpspgarp qvngqsgrid fhwllldpnd
  251	tvtftfngaf iapdrasffr geslgvqsdv pldsgcegdc fhsggtivss
  301	lpfqninprt vgkcpryvkq tslllatgmr nvpenpktrg lfgaiagfie
  351	ngweglidgw ygfrhqnaqg egtaadykst qsaidqitgk lnrlidktnq
  401	qfelidnefs eieqqignvi nwtrdsmtev wsynaellva menqhtidla
  451	dsemnklyer vrkqlrenae edgtgcfeif hkcddqcmes irnntydhtq
  501	yrteslqnri qidpvklssg ykdiilwfsf gascflllai amglvficik
  551	ngnmrctici

• Nucleotide sequence of ca A/ck/BC/CN-6/04 N3 (SEQ ID NO: 26)
• Entire molecule length: 1453 nt

  1	agcaaaagca ggtgcgagat gaatccgaat cagaagataa taacaatcgg
  51	ggtagtgaat accactctgt caacaatagc ccttctcatt ggagtgggaa
  101	acttagtttt caacacagtc atacatgaga aaataggaga ccatcaaata
  151	gtgacccatc caacaataat gacccctgaa gtaccgaact gcagtgacac
  201	tataataaca tacaataaca ctgttataaa caacataaca acaacaataa
  251	taactgaagc agaaaggcct ttcaagtctc cactaccgct gtgccccttc
  301	agaggattct tcccttttca caaggacaat gcaatacgac tgggtgaaaa
  351	caaagacgtc atagtcacaa gggagcctta tgttagctgc gataatgaca
  401	actgctggtc ctttgctctc gcacaaggag cattgctagg gactaaacat
  451	agcaatggga ccattaaaga cagaacacca tataggtctc taattcgttt
  501	cccaatagga acagctccag tactaggaaa ttacaaagag atatgcattg
  551	cttggtcgag cagcagttgc tttgacggga aagagtggat gcatgtgtgc
  601	atgacaggga atgataatga tgcaagtgcc cagataatat atggaggaag
  651	aatgacagac tccattaaat catggaggaa ggacatacta agaacccagg
  701	agtctgaatg tcaatgcatt gacgggactt gtgttgttgc tgtcacagat
  751	ggccctgctg ctaatagtgc agatcacagg gtttactgga tacgggaggg
  801	aagaataata aagtatgaaa atgttcccaa aacaaagata caacacttag
  851	aagaatgttc ctgctatgtg gacattgatg tttactgtat atgtagggac
  901	aattggaagg gctctaacag accttggatg agaatcaaca acgagactat
  951	actggaaaca ggatatgtat gtagtaaatt tcactcagac acccccaggc
  1001	cagctgaccc ttcaataatg tcatgtgact ccccaagcaa tgtcaatgga
  1051	ggacccggag tgaaggggtt tggtttcaaa gctggcaatg atgtatggtt
  1101	aggtagaaca gtgtcaacta gtggtagatc gggctttgaa attatcaaag
  1151	ttacagaagg gtggatcaac tctcctaacc atgtcaaatc aattacacaa
  1201	acactagtgt ccaacaatga ctggtcaggc tattcaggta gcttcattgt
  1251	caaagccaag gactgttttc agccctgttt ttatgttgag cttatacgag
  1301	ggaggcccaa caagaatgat gacgtctctt ggacaagtaa tagtatagtt
  1351	actttctgtg gactagacaa tgaacctgga tcgggaaatt ggccagatgg
  1401	ttctaacatt gggtttatgc ccaagtaata gaaaaaagca ccttgtttct
  1451	act

• Amino acid sequence of ca A/ck/BC/CN-6/04 N3 (SEQ ID NO: 32)
• Entire molecule length: 469aa

  1	mnpnqkiiti gvvnttlsti alligvgnlv fntvihekig dhqivthpti
  51	mtpevpncsd tiitynntvi nnitttiite aerpfksplp lcpfrgffpf
  101	hkdnairlge nkdvivtrep yvscdndncw sfalaqgall gtkhsngtik
  151	drtpyrslir fpigtapvlg nykeiciaws ssscfdgkew mhvcmtgndn
  201	dasaqiiygg rmtdsikswr kdilrtqese cqcidgtcvv avtdgpaans
  251	adhrvywire griikyenvp ktkiqhleec scyvdidvyc icrdnwkgsn
  301	rpwmrinnet iletgyvcsk fhsdtprpad psimscdsps nvnggpgvkg
  351	fgfkagndvw lgrtvstsgr sgfeiikvte gwinspnhvk sitqtlvsnn
  401	dwsgysgsfi vkakdcfqpc fyvelirgrp nknddvswts nsivtfcgld
  451	nepgsgnwpd gsnigfmpk

  
  ca A/Duck
• Nucleotide sequence of ca A/Duck H6 (SEQ ID NO: 33)
• Entire molecule length: 1743bp

  1	agcaaaagca ggggaaaatg attgcagtca ttataatagc ggtactggca acggccggaa
  61	aatcagacaa gatctgcatt gggtatcatg ccaacaattc aacaacacaa gtggatacga
  121	tacttgagaa gaatgtaacc gtcacacact cagttgaatt gctggagaac caaaaagaag
  181	aaagattctg caagatcttg aacaaggccc ctctcgattt aagaggatgt accatagagg
  241	gttggatctt ggggaatccc caatgcgacc tattgcttgg tgatcaaagc tggtcatata
  301	tagtggaaag acctacagct caaaatggga tctgctaccc aggaattttg aatgaagtag
  361	aagaactgaa ggcacttatt ggatcaggag aaagagtgga gagatttgaa atgtttccca
  421	aaagtacatg ggcaggagta gacaccagca gtggggtaac aaaggcttgc ccttatacta
  481	gtggttcgtc tttctacaga aacctcctat ggataataaa aaccaagtcc gcagcatatc
  541	cagtaattaa gggaacctac aataacactg gaagccagcc aatcctctat ttctggggtg
  601	tgcaccatcc tcctgacacc aatgagcaaa acactttgta tggctctggt gatcgatatg
  661	tcaggatggg aactgaaagc atgaattttg ccaagagccc agaaattgcg gcaaggcctg
  721	ctgtgaatgg tcaaagaggc agaattgatt attactggtc tgttttaaag ccgggggaaa
  781	ccttgaatgt ggaatctaat ggaaatctaa tcgccccttg gtatgcatac aaatttgtca
  841	gcaccaatag taaaggagcc gtcttcaagt caaatttacc aatcgagaac tgtgatgcca
  901	catgccagac tattgcagga gtcttaagaa ccaataaaac atttcagaat gtaagccctc
  961	tgtggatagg agaatgcccc aaatatgtga aaagtgaaag tttgaggctt gcaactggac
  1021	taagaaatat tccacagatt gagactagag gacttttcgg agctatcgca gggtttattg
  1081	aaggaggatg gactggaatg atagatgggt ggtatggcta tcaccatgaa aattctcaag
  1141	gctcagggta tgcggcagac agagaaagca ctcaaagggc tatagacgga attacaaata
  1201	aggtcaattc cattatagac aaaatgaaca cacaattcga agctatagac cacgaattct
  1261	caaatttgga gagaagaatt gacagtctga acaaaagaat ggaagatgga tttctggacg
  1321	tttggacata caatgctgaa ctgttggttc ttcttgaaaa cgaaaggaca ctagacctac
  1381	atgacgcgaa tgtgaagaac ctgtatgaaa aggtcaaatc acaactacgg gacaatgcta
  1441	atgatctagg aaatggatgc tttgaatttt ggcataagtg tgacaatgaa tgcatagagt
  1501	ctgtcaaaaa tggtacctat gactatccca aatatcagga tgaaagcaaa ttgaacaggc
  1561	aggaaataga atcggtgaag ctggagaacc ttggtgtgta tcaaatcctc gccatttata
  1621	gtacggtatc gagcagtcta gtcttggtag ggctgattat agcaatgggt ctttggatgt
  1681	gttcaaatgg ttcaatgcaa tgcaggatat gtatataatt aagaaaaaca cccttgttct
  1741	act

• Amino acid sequence of ca A/Duck H6 (SEQ ID NO: 39)
• Entire molecule length: 566aa

  1	miaviiiavl atagksdkic igyhannstt qvdtileknv tvthsvelle nqkeerfcki
  61	lnkapldlrg ctiegwilgn pqcdlllgdq swsyiverpt aqngicypgi lneveelkal
  121	igsgerverf emfpkstwag vdtssgvtka cpytsgssfy rnllwiiktk saaypvikgt
  181	ynntgsqpil yfwgvhhppd tneqntlygs gdryvrmgte smnfakspei aarpavngqr
  241	gridyywsvl kpgetlnves ngnliapwya ykfvstnskg avfksnlpie ncdatcqtia
  301	gvlrtnktfq nvsplwigec pkyvkseslr latglrnipq ietrglfgai agfieggwtg
  361	midgwygyhh ensqgsgyaa drestqraid gitnkvnsii dkmntqfeai dhefsnlerr
  421	idslnkrmed gfldvwtyna ellvllener tldlhdanvk nlyekvksql rdnandlgng
  481	cfefwhkcdn eciesvkngt ydypkyqdes klnrqeiesv klenlgvyqi laiystvsss
  541	lvlvgliiam glwmcsngsm qcrici

• Nucleotide sequence of ca A/Duck N9 (SEQ ID NO: 34)
• Entire molecule length: 1460 nt

  1	agcaaaagca gggtcaagat gaatccaaat cagaagattc tatgcacatc tgctactgcc
  61	attgcaatag gcacaattgc tgtattaata ggaatagcaa acctgggttt gaacatagga
  121	ctacacctga aaccgagctg caactgctcc aaccctcctc ctgaaacaac aaatgtaagc
  181	caaacaataa taaacaatta ctacaatgaa acaaatgtta cccaaataag taacacaaac
  241	attcaacata tggggggaac cgaaaaggac ttcaacaatc tgactaaagg gctctgcaca
  301	ataaattcat ggcatatatt cggaaaggac aatgctataa gaatagggga gaactctgat
  361	gttttagtca caagagagcc atatgtttct tgtgatccag atgaatgcag attctatgct
  421	ctcagccaag gaacaacaat acggggaaag cactcaaatg gaacaataca cgatagatcc
  481	caataccgtg ctttagtgag ctggccttta tcatcaccac ccactgtgta caataccaga
  541	gtagaatgca ttggatggtc cagtacaagc tgccatgatg ggaaagcacg aatgtctata
  601	tgtgtctcag gtcccaacaa caatgcatca gcagtgattt ggtacaaagg gcggcctatc
  661	acggaaatca atacgtgggc ccgaaacata ttgagaaccc aagaatctga gtgtgtatgc
  721	cacaatggaa tatgtccagt agtgttcact gacggttctg ccaccggtcc agcagaaact
  781	aggatatact atttcaaaga ggggaaaatc ctcaaatggg agccactaac tggaaccgcc
  841	aagcacattg aagaatgctc ttgctatggg aaagactcag aaataacgtg cacatgtaga
  901	gacaattggc aaggctcgaa tagaccagta atacaaataa accccacaat gatgactcac
  961	actagtcaat acatatgcag ccctgtcctc acagacaatc cacgccccaa tgaccccacg
  1021	gtaggcaagt gtaatgatcc ttatccagga aacaacaata atggagtcaa aggattctca
  1081	tatttagatg gtgacaatac atggctagga agaacgataa gcacagcctc taggtctggg
  1141	tatgaaatgc tgaaagtgcc taatgcattg acagatgata gatcaaaacc tactcaaggt
  1201	cagacaattg tattaaacac agactggagt ggttacagtg ggtctttcat tgattactgg
  1261	gcaaaagggg agtgctatag agcatgcttc tacgttgagc tgatccgtgg aaggccaaaa
  1321	gaggacaaag tgtggtggac cagtaatagt atagtgtcga tgtgttccag cacagagttc
  1381	cttggacaat ggaactggcc agatggggct aaaatagagt acttcctcta agatgtagaa
  1441	aaaagaccct tgtttctact

• Amino acid sequence of ca A/Duck N9 (SEQ ID NO: 40)
• Entire molecule length: 470aa

  1	mnpnqkilct sataiaigti avilgianlg lniglhlkps cncsnpppet tnvsqtiinn
  61	yynetnvtqi sntniqhmgg tekdfnnltk glctinswhi fgkdnairig ensdvlvtre
  121	pyvscdpdec rfyalsqgtt irgkhsngti hdrsqyralv swplsspptv yntrvecigw
  181	sstschdgka rmsicvsgpn nnasaviwyk grpiteintw arnilrtqes ecvchngicp
  241	vvftdgsatg paetriyyfk egkilkwepl tgtakhieec scygkdseit ctcrdnwqgs
  301	nrpviqinpt mmthtsqyic spvltdnprp ndptvgkcnd pypgnnnngv kgfsyldgdn
  361	twlgrtista srsgyemlkv pnaltddrsk ptqgqtivln tdwsgysgsf idywakgecy
  421	racfyvelir grpkedkvww tsnsivsmcs steflgqwnw pdgakieyfl

  
  ca A/Teal
• Nucleotide sequence of ca A/Teal H6 (SEQ ID NO: 35)
• Entire molecule length: 1747bp

  1	agcaaaagca ggggaaaatg attgcaatca ttgtaatagc aatactggca gcagccggaa
  61	aatcagacaa gatctgcatt gggtatcatg ccaacaattc aacaacacag gtagatacga
  121	tacttgagaa gaatgtgact gtcacacact caattgaatt gctggaaaat cagaaggaag
  181	aaagattctg caagatattg aacaaggccc ctctcgactt aagggaatgt accatagagg
  241	gttggatctt ggggaatccc caatgcgacc tattgcttgg tgatcaaagc tggtcataca
  301	ttgtggaaag acctactgct caaaacggga tctgctaccc aggaacctta aatgaggtag
  361	aagaactgag ggcacttatt ggatcaggag aaagggtaga gagatttgag atgtttcccc
  421	aaagcacctg gcaaggagtt gacaccaaca gtggaacaac aagatcctgc ccttattcta
  481	ctggtgatcc gtctttctac agaaacctcc tatggataat aaaaaccaag acagcagaat
  541	atccagtaat taagggaatt tacaacaaca ctggaaccca gccaatcctc tatttctggg
  601	gtgtgcatca tcctcctaac accgacgagc aagatactct gtatggctct ggtgatcgat
  661	acgttagaat gggaactgaa agcatgaatt ttgccaagag tccggaaatt gcggcaaggc
  721	ctgctgtgaa tggacaaaga ggcagaattg attattattg gtcggtttta aaaccagggg
  781	aaaccttgaa tgtggaatct aatggaaatc taatcgcccc ttggtatgca tacaaatttg
  841	tcaacacaaa tagtaaagga gccgtcttca ggtcagattt accaatcgag aactgcgatg
  901	ccacatgcca gactattgca ggggttctaa ggaccaataa aacatttcag aatgtgagtc
  961	ccctgtggat aggagaatgt cccaaatacg tgaaaagtga aagtctgagg cttgcaactg
  1021	gactaagaaa tgttccacag attgaaacta gaggactctt cggagctatt gcagggttta
  1081	ttgaaggagg atggactggg atgatagatg ggtggtatgg ctatcaccat gaaaattctc
  1141	aagggtcagg atatgcagcg gacagagaaa gcactcaaaa ggctgtaaac agaattacaa
  1201	ataaggtcaa ttccatcatc aacaaaatga acacacaatt tgaagctgtc gatcacgaat
  1261	tttcaaatct ggagaggaga atcgacaatc tgaacaaaag aatgcaagat ggatttctgg
  1321	atgtttggac atacaatgct gaactgttgg ttcttcttga aaacgaaaga acactagaca
  1381	tgcatgacgc aaatgtgaag aacctacatg aaaaggtcaa atcacaacta agggacaatg
  1441	ctaacgatct agggaatggt tgctttgaat tttggcataa gtgtgacaat gaatgcatag
  1501	agtctgtcaa aaatggtaca tatgactatc ccaaatacca gactgaaagc aaattaaaca
  1561	ggctaaaaat agaatcagta aagctagaga accttggtgt gtatcaaatt cttgccattt
  1621	atagtacggt atcgagcagc ctagtgttgg tagggctgat catggcaatg ggtctttgga
  1681	tgtgttcaaa tggttcaatg cagtgcaatg tgtgtatatg attaagaaaa acacccttgt
  1741	ttctact

• Amino acid sequence of ca A/Teal H6 (SEQ ID NO: 41)
• Entire molecule length: 567aa

  1	miaiiviail aaagksdkic igyhannstt qvdtileknv tvthsielle nqkeerfcki
  61	lnkapldlre ctiegwilgn pqcdlllgdq swsyiverpt aqngicypgt lneveelral
  121	igsgerverf emfpqstwqg vdtnsgttrs cpystgdpsf yrnllwiikt ktaeypvikg
  181	iynntgtqpi lyfwgvhhpp ntdeqdtlyg sgdryvrmgt esmnfakspe iaarpavngq
  241	rgridyywsv lkpgetlnve sngnliapwy aykfvntnsk gavfrsdlpi encdatcqti
  301	agvlrtnktf qnvsplwige cpkyvksesl rlatglrnvp qietrglfga iagfieggwt
  361	gmidgwygyh hensqgsgya adrestqkav nritnkvnsi inkmntqfea vdhefsnler
  421	ridnlnkrmq dgfldvwtyn aellvllene rtldmhdanv knlhekvksq lrdnandlgn
  481	gcfefwhkcd neciesvkng tydypkyqte sklnrlkies vklenlgvyq ilaiystvss
  541	slvlvglima mglwmcsngs mqcnvci

• Nucleotide sequence of ca A/Teal N1 (SEQ ID NO: 36)
• Entire molecule length: 1401 nt

  1	agcaaaagca ggagtttaac atgaatccaa atcagaagat aataaccatt gggtcaatct
  61	gtatggtagt tggaataatc agcttgatgt tacaaattgg aaacataata tcaatatggg
  121	ttagccacat aattcagact gggcatccaa accagcctgg gccatgcaat caaagcatca
  181	atttttacac tgagcaggct gcagcttcag tgacattagc gggtaattcc tctctctgcc
  241	ctattagtgg atgggctata tacagtaaag acaatagtat aagaattggt tccaaagggg
  301	atgtgtttgt tatgagagaa ccattcgttt catgctccca tttggaatgc agaacctttt
  361	tcttgactca aggagcccta ttgaatgaca agcattctaa tgggaccgtt aaagacagaa
  421	gcccctatag aactttaatg agctgtcctg ttggtgaggc tccttcccca tacaactcaa
  481	ggtttgagtc tgttgcttgg tcagcaagtg cttgccatga tggcattagt tggctaacaa
  541	ttggaatttc cggtccggat aatggggctg tggctgtgtt gaaatacaat ggcataataa
  601	cagacaccat caagagttgg aggaacaaca tactgaggac acaagagtct gaatgtgcat
  661	gtgtgaatgg ttcttgtttt actgtaatga cagatggacc gagtaatgaa caggcctcat
  721	acaagatttt caagatagag aaggggaaag tagtcaaatc agttgagttg aacgccccta
  781	attatcatta cgaggaatgc tcctgttatc ctgatgctgg cgaaatcaca tgtgtgtgca
  841	gggataattg gcatggctcg aaccgaccgt gggtgtcttt caatcagaat ctggagtatc
  901	aaataggata tatatgcagt ggggttttcg gagacagtcc acgccccaat gatggaacag
  961	gcagttgcgg tccagtgtct cttaacggag agtatggagt aaaagggttt tcatttaagt
  1021	acggtgatgg tgtttggatc gggagaacca aaagcactag ttccaggagc gggtttgaaa
  1081	tgatttggga tccaaatggg tggaccgaaa cagatagtaa cttctcattg aagcaagaca
  1141	tcatagcaat aactgattgg tcaggataca gcgggagttt tgtccaacat ccagaactga
  1201	caggattaaa ttgcatgagg ccttgcttct gggttgaact aatcagaggg aggcccaaag
  1261	agaaaacaat ctggactagt gggagcagta tatctttctg tggtgtaaat agtgacactg
  1321	tgggttggtc ttggccagac ggtgctgagg tgccattcac cattgacaag tagtttgttc
  1381	aaaaaactcc ttgtttctac t

• Amino acid sequence of ca A/Teal N1 (SEQ ID NO: 42)
• Entire molecule length: 450aa

  1	mnpnqkiiti gsicmvvgii slmlqignii siwvshiiqt ghpnqpgpcn qsinfyteqa
  61	aasvtlagns slcpisgwai yskdnsirig skgdvfvmre pfvscshlec rtffltqgal
  121	lndkhsngtv kdrspyrtlm scpvgeapsp ynsrfesvaw sasachdgis wltigisgpd
  181	ngavavlkyn giitdtiksw rnnilrtqes ecacvngscf tvmtdgpsne qasykifkie
  241	kgkvvksvel napnyhyeec scypdageit cvcrdnwhgs nrpwvsfnqn leyqigyics
  301	gvfgdsprpn dgtgscgpvs lngeygvkgf sfkygdgvwi grtkstssrs gfemiwdpng
  361	wtetdsnfsl kqdiiaitdw sgysgsfvqh peltglncmr pcfwvelirg rpkektiwts
  421	gssisfcgvn sdtvgwswpd gaevpftidk

  
  ca A/Mallard
• Nucleotide sequence of ca A/Mallard H6 (SEQ ID NO: 37)
• Entire molecule length: 1745bp

  1	agcaaaagca ggggaaaatg attgcaatca taatacttgc aatagtggtc tctaccagca
  61	agtcagacag gatctgcatt ggttaccatg caaacaactc gacaacacaa gtggacacaa
  121	tattagagaa gaatgtgaca gtgacacact cagtggagct cctagaaaac cagaaggaga
  181	atagattctg cagagtcttg aataaagcgc cactggatct aatggactgc accactgagg
  241	gttggatcct tggaaacccc cgatgtgata acttactcgg tgatcaaagt tggtcataca
  301	tagtagagag gcctgatgcc caaaatggga tatgttaccc aggggtattg aaggagacgg
  361	aagagctgaa agcactcatt gggtctatag atagcataca aagatttgaa atgtttccca
  421	agagcacgtg gaccggggta gatactaata gcggagttac gagcgcttgc ccctacaatg
  481	gtgaatcttc cttttacagg aatctgttgt ggataataaa aataagatct gatccgtact
  541	cattgatcaa ggggacatat accaatacag gctctcagcc aatcttatat ttctggggtg
  601	tgcaccatcc tccagatgaa gttgagcaag ctaacttgta tggaattggt acccggtatg
  661	ttaggatggg aactgaaagt atgaattttg ccaaaggtcc tgaaatagca ggcagaccac
  721	ctgcgaatgg gcaacgagga agaattgatt attattggtc tgtgttgaag ccaggagaaa
  781	ccttgaatgt ggaatccaat ggaaatttaa tagctccttg gtatgcttac aagttcacta
  841	gttccagaaa caagggagct attttcaaat cagaccttcc aattgagaat tgtgatgctg
  901	tctgtcaaac tttagctgga gcaataaata caaacaaaac cttccaaaat attagtccag
  961	tctggattgg agaatgcccc aaatatgtta aaagtaagag cctaaaacta gcaactggtc
  1021	tgagaaatgt tccacaggca gaaacaagag gattgtttgg agcaatagct gggtttatag
  1081	aaggaggatg gacaggtatg gtagacggat ggtacggata ccaccatgaa aattcacagg
  1141	ggtctggtta tgcagcagat aaagaaagca ctcagaaagc aatagacggg atcaccaata
  1201	aagtcaattc aatcattgac aaaatgaaca cacaatttga ggcagtagag catgagttct
  1261	caagtctcga aaggagaata ggcaatctga acaaaagaat ggaagatgga tttttagacg
  1321	tgtggacata caatgctgaa cttctggttc tactggaaaa tgagaggact ttggacatgc
  1381	atgatgctaa tgtaaagaat ctacatgaaa aggtgaaatc acaattaagg gataatgcaa
  1441	aggatttggg taatgggtgt tttgaatttt ggcacaaatg cgacaatgaa tgcatcaact
  1501	cagttaaaaa tggcacatat gactacccaa agtaccagga agagagcaga cttaataggc
  1561	aggaaataaa atcagtgatg ctggaaaatc tgggagtata ccaaatcctt gctatttata
  1621	gtacggtatc gagcagtctg gttttggtgg gactgatcat tgccatgggt ctttggatgt
  1681	gctcaaatgg ctcaatgcaa tgcaagatat gtatataatt agaaaaaaac acccttgttt
  1741	ctact

• Amino acid sequence of ca A/Mallard H6 (SEQ ID NO: 43)
• Entire molecule length: 566aa

  1	miaiiilaiv vstsksdric igyhannstt qvdtileknv tvthsvelle nqkenrfcrv
  61	lnkapldlmd cttegwilgn prcdnllgdq swsyiverpd aqngicypgv lketeelkal
  121	igsidsiqrf emfpkstwtg vdtnsgvtsa cpyngessfy rnllwiikir sdpyslikgt
  181	ytntgsqpil yfwgvhhppd eveqanlygi gtryvrmgte smnfakgpei agrppangqr
  241	gridyywsvl kpgetlnves ngnliapwya ykftssrnkg aifksdlpie ncdavcqtla
  301	gaintnktfq nispvwigec pkyvkskslk latglrnvpq aetrglfgai agfieggwtg
  361	mvdgwygyhh ensqgsgyaa dkestqkaid gitnkvnsii dkmntqfeav ehefsslerr
  421	ignlnkrmed gfldvwtyna ellvllener tldmhdanvk nlhekvksql rdnakdlgng
  481	cfefwhkcdn ecinsvkngt ydypkyqees rlnrqeiksv mlenlgvyqi laiystvsss
  541	lvlvgliiam glwmcsngsm qckici

• Nucleotide sequence of ca A/Mallard N2 (SEQ ID NO: 38)
• Entire molecule length: 1467 nt

  1	agcaaaagca ggagtgaaaa tgaatccaaa tcagaggata ataacaattg gatccgtctc
  61	tctaactatt gcaacagtgt gtttcctcat gcagattgcc atcctagcaa cgactgtgac
  121	actgcatttc aaacaaaatg aatgcagcat tcccgcaaac aaccaagtaa cgccatgtga
  181	accaatagta atagagagga acataacaga gatagtgtat ttgaataata ctaccataga
  241	aaaagagatt tgtcctgaag tagtagaata caggaattgg tcaaaaccgc aatgtcaaat
  301	tacagggttt gctcctttct ccaaggacaa ctcaattcgg ctttctgctg gtggggacat
  361	ttggataaca agagaacctt atgtgtcatg cgaccccagt aaatgttatc aatttgcact
  421	cgggcagggg accacgctgg acaacaaaca ctcaaatggc acaatacatg atagaatccc
  481	tcatcggacc cttttgatga atgaattggg tgttccgttt catttgggaa ccaaacaagt
  541	gtgcatagca tggtccagct caagctgtca tgatgggaaa gcatggttgc acgtttgtgt
  601	cactggggat gatagaaatg caactgctag tttcatttat gatgggatgc ttattgacag
  661	tattggttcc tggtctcaaa atatcctcag gactcaggag tcagaatgcg tttgtatcag
  721	tggaacttgt acagtagtaa tgactgatgg aagtgcatca ggaagggcag acactagaat
  781	actattcatt agagagggga aaattgtcca cattagtcca ttgtcaggaa gtgctcagca
  841	tgtagaggaa tgttcttgtt atccccggta cccaaacgtc agatgtgtct gcagagacaa
  901	ctggaagggc tctaataggc ccgttataga tataaatatg gcagattata gcattgactc
  961	aagttatgtg tgctcaggac ttgttggaga cacaccaagg aacgatgata gctctagcag
  1021	cagcaactgc agggatccta ataatgagag agggaaccca ggagtgaaag ggtgggcctt
  1081	tgataatgga aatgatgtgt ggatgggaag aacaatcagt aaagattcgc gctcaggcta
  1141	tgagaccttc aaggtcattg gtggttgggc cattgctaat tccaagtcac agaccaatag
  1201	acaagtcata gttgataata acaactggtc tggttattct ggtattttct ctgttgaaag
  1261	caaaggctgc atcaataggt gtttttatgt ggagttgata agaggaaggc cacaggagac
  1321	tagagtatgg tggacctcaa acagtattgt cgtattttgt ggcacttcag ggacatatgg
  1381	aacaggctca tggcctgatg gggcgaatat cgatttcatg cctatataag ctttcgcaat
  1441	tttagaaaaa aactccttgt ttctact

• Amino acid sequence of ca A/Mallard N2 (SEQ ID NO: 44)
• Entire molecule length: 469aa

  1	mnpnqriiti gsvsltiatv cflmqiaila ttvtlhfkqn ecsipannqv tpcepivier
  61	niteivylnn ttiekeicpe vveyrnwskp qcqitgfapf skdnsirlsa ggdiwitrep
  121	yvscdpskcy qfalgqgttl dnkhsngtih driphrtllm nelgvpfhlg tkqvciawss
  181	sschdgkawl hvcvtgddrn atasfiydgm lidsigswsq nilrtqesec vcisgtctvv
  241	mtdgsasgra dtrilfireg kivhisplsg saqhveecsc yprypnvrcv crdnwkgsnr
  301	pvidinmady sidssyvcsg lvgdtprndd sssssncrdp nnergnpgvk gwafdngndv
  361	wmgrtiskds rsgyetfkvi ggwaiansks qtnrqvivdn nnwsgysgif sveskgcinr
  421	cfyvelirgr pqetrvwwts nsivvfcgts gtygtgswpd ganidfmpi

• Summary of SEQ ID NO Designations

  			Amino Acid or
  SEQ ID NO	HA or NA	STRAIN NAME	Nucleotide
  SEQ ID NO: 1	HA (H5)	ca A/Vietnam/1203/04	Nucleotide
  SEQ ID NO: 2	NA (N1)	ca A/Vietnam/1203/04	Nucleotide
  SEQ ID NO: 3	HA (H5)	ca A/Hong	Nucleotide
  		Kong/213/03
  SEQ ID NO: 4	NA (N1)	ca A/Hong	Nucleotide
  		Kong/213/03
  SEQ ID NO: 5	HA (H5)	ca A/Hong	Nucleotide
  		Kong/491/97
  SEQ ID NO: 6	NA (N1)	ca A/Hong	Nucleotide
  		Kong/486/97
  SEQ ID NO: 7	HA (H5)	ca A/Hong	Nucleotide
  		Kong/491/97 (Ser211)
  SEQ ID NO: 8	NA (N1)	ca A/Hong	Nucleotide
  		Kong/486/97
  SEQ ID NO: 9	HA (H9)	ca A/ck/Hong	Nucleotide
  		Kong/G9/97
  SEQ ID NO: 10	NA (N2)	ca A/ck/Hong	Nucleotide
  		Kong/G9/97
  SEQ ID NO: 21	HA (H7)	A/Netherlands/219/03	Nucleotide
  SEQ ID NOs: 22	NA (N7)	A/Netherlands/219/03	Nucleotide
  & 45
  SEQ ID NO: 23	HA (H7)	A/ck/BC/CN-7/04	Nucleotide
  SEQ ID NO: 24	NA (N3)	A/ck/BC/CN-7/04	Nucleotide
  SEQ ID NO: 25	HA (H7)	ca A/ck/BC/CN-6/04	Nucleotide
  SEQ ID NO: 26	NA (N3)	ca A/ck/BC/CN-6/04	Nucleotide
  SEQ ID NO: 33	HA (H6)	ca A/Duck	Nucleotide
  SEQ ID NO: 34	NA (N9)	ca A/Duck	Nucleotide
  SEQ ID NO: 35	HA (H6)	ca A/Teal	Nucleotide
  SEQ ID NO: 36	NA (N1)	ca A/Teal	Nucleotide
  SEQ ID NO: 37	HA (H6)	ca A/Mallard	Nucleotide
  SEQ ID NO: 38	NA (N2)	ca A/Mallard	Nucleotide
  SEQ ID NO: 11	HA (H5)	ca A/Vietnam/1203/04	Amino Acid
  SEQ ID NO: 12	NA (N1)	ca A/Vietnam/1203/04	Amino Acid
  SEQ ID NO: 13	HA (H5)	ca A/Hong	Amino Acid
  		Kong/213/03
  SEQ ID NO: 14	NA (N1)	ca A/Hong	Amino Acid
  		Kong/213/03
  SEQ ID NO: 15	HA (H5)	ca A/Hong	Amino Acid
  		Kong/491/97
  SEQ ID NO: 16	NA (N1)	ca A/Hong	Amino Acid
  		Kong/486/97
  SEQ ID NO: 17	HA (H5)	ca A/Hong	Amino Acid
  		Kong/491/97 (Ser211)
  SEQ ID NO: 18	NA (N1)	ca A/Hong	Amino Acid
  		Kong/486/97
  SEQ ID NO: 19	HA (H9)	ca A/ck/Hong	Amino Acid
  		Kong/G9/97
  SEQ ID NO: 20	NA (N2)	ca A/ck/Hong	Amino Acid
  		Kong/G9/97
  SEQ ID NO: 27	HA (H7)	A/Netherlands/219/03	Amino Acid
  SEQ ID NO: 28	NA (N7)	A/Netherlands/219/03	Amino Acid
  SEQ ID NO: 29	HA (H7)	A/ck/BC/CN-7/04	Amino Acid
  SEQ ID NO: 30	NA (N3)	A/ck/BC/CN-7/04	Amino Acid
  SEQ ID NO: 31	HA (H7)	ca A/ck/BC/CN-6/04	Amino Acid
  SEQ ID NO: 32	NA (N3)	ca A/ck/BC/CN-6/04	Amino Acid
  SEQ ID NO: 39	HA (H6)	ca A/Duck	Amino Acid
  SEQ ID NO: 40	NA (N9)	ca A/Duck	Amino Acid
  SEQ ID NO: 41	HA (H6)	ca A/Teal	Amino Acid
  SEQ ID NO: 42	NA (N1)	ca A/Teal	Amino Acid
  SEQ ID NO: 43	HA (H6)	ca A/Mallard	Amino Acid
  SEQ ID NO: 44	NA (N2)	ca A/Mallard	Amino Acid

Claims (38)

1. An isolated polypeptide, wherein said polypeptide is selected from the group consisting of:

a) a polypeptide comprising the amino acid sequence encoded by the nucleotide sequence as shown in any one of SEQ ID NOS:21-26 or 33-38 or 45;

b) a polypeptide comprising the amino acid sequence as shown in any one of SEQ ID NOS:27-32 or 39-44;

c) the mature form of a polypeptide comprising the amino acid sequence as shown in any one of SEQ ID NOS: 27-32 or 39-44;

d) a polypeptide comprising an amino acid sequence encoded by a polynucleotide which hybridizes under highly stringent conditions to a polynucleotide comprising a nucleotide sequence encoding (a) (b) or (c); and

e) a polypeptide comprising an amino acid sequence having at least 90% sequence identity to the polypeptide of (b).

2. An immunogenic composition comprising an immunologically effective amount of at least one polypeptide ofclaim 1.

3. An isolated antibody that specifically binds the polypeptide ofclaim 1.

4. A method for stimulating the immune system of an individual to produce a protective immune response against influenza virus, the method comprising administering to the individual an immunologically effective amount of the polypeptide ofclaim 1 in a physiologically acceptable carrier.

5. A recombinant influenza virus comprising the polypeptide ofclaim 1.

6. An immunogenic composition comprising an immunologically effective amount of the recombinant influenza virus ofclaim 5.

7. A method for stimulating the immune system of an individual to produce a protective immune response against influenza virus, the method comprising administering to the individual an immunologically effective amount of the recombinant influenza virus ofclaim 5 in a physiologically acceptable carrier.

8. An isolated polynucleotide, wherein said polynucleotide is selected from the group consisting of:

a) a polynucleotide comprising the nucleotide sequence as shown in any one of SEQ ID NOS: 21-26 or 33-38 or 45, or a complementary sequence thereof;

b) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence as shown in any one of SEQ ID NOS: 27-32 or 39-44, or a complementary nucleotide sequence thereof;

c) a polynucleotide which hybridizes under highly stringent conditions over substantially the entire length of the polynucleotide of (a); and

d) a polynucleotide comprising a nucleotide sequence having at least 98% sequence identity to the polynucleotide of (a).

9. An immunogenic composition comprising at least one polynucleotide ofclaim 8.

10. A cell comprising at least one polynucleotide ofclaim 8.

11. A vector comprising the polynucleotide ofclaim 8.

12. The vector ofclaim 11, wherein the vector is a plasmid, a cosmid, a phage, a virus, or a fragment of a virus.

13. The vector ofclaim 12, wherein the vector is an expression vector.

14. A cell comprising the vector ofclaim 13.

15. An influenza virus comprising one or more polynucleotides ofclaim 8.

16. The virus ofclaim 15, wherein the virus is a reassortant virus.

17. A 6:2 reassortant influenza virus, wherein said virus comprises 6 internal genome segments from A/Ann Arbor/6/60 and 2 genome segments that encode an HA and/or a NA polypeptide selected from the group consisting of: the polypeptides of SEQ ID NOS:27-32, and 39-44.

18. A method of producing a reassortant influenza virus, the method comprising: culturing the cell ofclaim 14 in a suitable culture medium under conditions permitting expression of said polynucleotide; and, isolating the reassortant influenza virus from a cell population comprising said cell or the medium.

19. An immunogenic composition comprising an immunologically effective amount of the reassortant influenza virus ofclaim 17.

20. A method for stimulating the immune system of an individual to produce a protective immune response against influenza virus, the method comprising administering to the individual an immunologically effective amount of the reassortant influenza virus ofclaim 17 in a physiologically effective carrier.

21. A method of producing an isolated or recombinant polypeptide, the method comprising: culturing the cell ofclaim 10 in a suitable culture medium under conditions permitting expression of said polynucleotide; and, isolating the polypeptide from the cell or the medium.

22. A method of prophylactic or therapeutic treatment of a viral infection in a subject, the method comprising: administering to the subject, the virus ofclaim 17 in an amount effective to produce an immunogenic response against the viral infection.

23. The method ofclaim 22, wherein the subject is a human.

24. The immunogenic composition ofclaim 19, wherein the hemagglutinin comprises a modified polybasic cleavage site.

25. A live attenuated influenza vaccine comprising the composition ofclaim 19.

26. A split virus or killed virus vaccine comprising the composition ofclaim 19.

27. A live attenuated influenza vaccine comprising the composition ofclaim 24.

28. A split virus or killed virus vaccine comprising the composition ofclaim 24.

29. A method for producing an influenza virus in cell culture, the method comprising:

i) introducing into a population of host cells, which population of host cells is capable of supporting replication of influenza virus, a plurality of vectors comprising nucleotide sequences corresponding to at least 6 internal genome segments of A/Ann Arbor/6/60; and, at least one genome segment comprising a polynucleotide encoding an HA and/or a NA polypeptide selected from the group consisting of: the polypeptides of SEQ ID NOS:27-32, and 39-44,

ii) culturing the population of host cells at a temperature less than or equal to 35° C.; and,

iii) recovering an influenza virus.

30. The method ofclaim 29, wherein the polynucleotide encoding the HA and/or NA polypeptide is selected from the group consisting of:

a) a polynucleotide comprising the nucleotide sequence of any one of SEQ ID NOS:21, 23-26 or 33-38, or 45, or a complementary nucleotide sequence thereof;

b) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence as shown in any one of SEQ ID NOS: 27-32 or 39-44, or a complementary nucleotide sequence thereof;

c) a polynucleotide which hybridizes under highly stringent conditions over substantially the entire length of the polynucleotide of (a); and

d) a polynucleotide comprising a nucleotide sequence having at least 98% sequence identity to the polynucleotide of (a).

31. An immunogenic composition comprising an immunologically effective amount of the influenza virus produced by the method ofclaim 29.

32. An immunogenic composition comprising an immunologically effective amount of the influenza virus produced by the method ofclaim 30.

33. A method for stimulating the immune system of an individual to produce a protective immune response against influenza virus, the method comprising administering to the individual an immunologically effective amount of the influenza virus produced by the method ofclaim 29 in a physiologically effective carrier.

34. A method for stimulating the immune system of an individual to produce a protective immune response against influenza virus, the method comprising administering to the individual an immunologically effective amount of the influenza virus produced by the method ofclaim 30 in a physiologically effective carrier.

35. A method for stimulating the immune system of an individual to produce a protective immune response against influenza virus, the method comprising administering to the individual the immunogenic composition ofclaim 31.

36. A method for stimulating the immune system of an individual to produce a protective immune response against influenza virus, the method comprising administering to the individual the immunogenic composition ofclaim 32.

37. A live attenuated influenza vaccine comprising the immunogenic composition ofclaim 31.

38. A split virus or killed virus vaccine comprising the immunogenic composition ofclaim 32.

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