U.S. patent application number 11/836413 was filed with the patent office on 2008-03-20 for influenza hemagglutinin and neuraminidase variants.
This patent application is currently assigned to MedImmune Vaccines, Inc.. Invention is credited to George Kemble, Brian Murphy, Kanta Subbarao, Chin-Fen Yang.
Application Number | 20080069821 11/836413 |
Document ID | / |
Family ID | 39082962 |
Filed Date | 2008-03-20 |
United States Patent
Application |
20080069821 |
Kind Code |
A1 |
Yang; Chin-Fen ; et
al. |
March 20, 2008 |
Influenza Hemagglutinin And Neuraminidase Variants
Abstract
Polypeptides, polynucleotides, methods, compositions, and
vaccines comprising (avian pandemic) influenza hemagglutinin and
neuraminidase variants are provided.
Inventors: |
Yang; Chin-Fen; (San Jose,
CA) ; Kemble; George; (Saratoga, CA) ;
Subbarao; Kanta; (Washington, DC) ; Murphy;
Brian; (Bethesda, MD) |
Correspondence
Address: |
JOHNATHAN KLEIN-EVANS
ONE MEDIMMUNE WAY
GAITHERSBURG
MD
20878
US
|
Assignee: |
MedImmune Vaccines, Inc.
Gaithersburg
MD
The Government of the United States of America National
Institutes of Health
Rockville
MD
|
Family ID: |
39082962 |
Appl. No.: |
11/836413 |
Filed: |
August 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60821832 |
Aug 9, 2006 |
|
|
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60942804 |
Jun 8, 2007 |
|
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Current U.S.
Class: |
424/139.1 ;
424/186.1; 424/206.1; 424/93.2; 435/235.1; 435/252.33; 435/254.2;
435/325; 435/349; 435/350; 435/352; 435/364; 435/365; 435/69.3;
530/350 |
Current CPC
Class: |
A61P 31/16 20180101;
C12N 2760/16134 20130101; C07K 14/005 20130101; A61K 2039/5252
20130101; A61K 39/145 20130101; A61K 39/12 20130101; A61K 2039/5254
20130101; A61K 2039/543 20130101; A61K 39/00 20130101; A61P 43/00
20180101; A61P 37/04 20180101; C12N 7/00 20130101; C12N 2760/16122
20130101; C12N 2760/16164 20130101 |
Class at
Publication: |
424/139.1 ;
424/186.1; 424/206.1; 424/093.2; 435/235.1; 435/252.33; 435/254.2;
435/325; 435/349; 435/350; 435/352; 435/364; 435/365; 435/069.3;
530/350 |
International
Class: |
A61K 39/145 20060101
A61K039/145; A61K 35/76 20060101 A61K035/76; A61K 39/00 20060101
A61K039/00; A61P 31/16 20060101 A61P031/16; C12N 1/19 20060101
C12N001/19; C12N 15/63 20060101 C12N015/63; C12N 7/01 20060101
C12N007/01; C12P 21/00 20060101 C12P021/00; C12N 5/10 20060101
C12N005/10; C12N 1/21 20060101 C12N001/21; C07K 14/00 20060101
C07K014/00; A61K 39/395 20060101 A61K039/395; A61K 39/12 20060101
A61K039/12 |
Claims
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 of claim 1.
3. An isolated antibody that specifically binds the polypeptide of
claim 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 claim 1 in a
physiologically acceptable carrier.
5. A recombinant influenza virus comprising the polypeptide of
claim 1.
6. An immunogenic composition comprising an immunologically
effective amount of the recombinant influenza virus of claim 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 claim 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 of claim 8.
10. A cell comprising at least one polynucleotide of claim 8.
11. A vector comprising the polynucleotide of claim 8.
12. The vector of claim 11, wherein the vector is a plasmid, a
cosmid, a phage, a virus, or a fragment of a virus.
13. The vector of claim 12, wherein the vector is an expression
vector.
14. A cell comprising the vector of claim 13.
15. An influenza virus comprising one or more polynucleotides of
claim 8.
16. The virus of claim 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 of claim 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 claim
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 claim 17 in a physiologically effective carrier.
21. A method of producing an isolated or recombinant polypeptide,
the method comprising: culturing the cell of claim 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 claim 17 in an amount effective to produce an
immunogenic response against the viral infection.
23. The method of claim 22, wherein the subject is a human.
24. The immunogenic composition of claim 19, wherein the
hemagglutinin comprises a modified polybasic cleavage site.
25. A live attenuated influenza vaccine comprising the composition
of claim 19.
26. A split virus or killed virus vaccine comprising the
composition of claim 19.
27. A live attenuated influenza vaccine comprising the composition
of claim 24.
28. A split virus or killed virus vaccine comprising the
composition of claim 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.degree. C.; and, iii) recovering an
influenza virus.
30. The method of claim 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
claim 29.
32. An immunogenic composition comprising an immunologically
effective amount of the influenza virus produced by the method of
claim 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 claim 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 of claim 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 of claim 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 of claim 32.
37. A live attenuated influenza vaccine comprising the immunogenic
composition of claim 31.
38. A split virus or killed virus vaccine comprising the
immunogenic composition of claim 32.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn.
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
[0002] 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.
[0003] 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.
[0004] 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.
[0005] 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
[0006] 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.
[0007] In other aspects, the invention comprises a composition with
one or more polypeptide listed above, or fragments thereof. The
invention also includes polypeptides that are specifically bound by
a polyclonal antisera raised against at least 1 antigen that
comprises at least one amino acid sequence described above, or a
fragment thereof. Such antibodies specific for the polypeptides
described above are also features of the invention. The
polypeptides of the invention are optionally immunogenic.
[0008] The invention also encompasses immunogenic compositions
comprising an immunologically effective amount of one or more of
any of the polypeptides described above as well as methods for
stimulating the immune system of an individual to produce a
protective immune response against influenza virus by administering
to the individual an immunologically effective amount of any of the
above polypeptides in a physiologically acceptable carrier.
[0009] Additionally, the invention 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.
[0010] 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).
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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).
[0015] 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.
[0016] 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).
[0017] 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.
[0018] 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.
[0019] 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
[0020] FIG. 1: Shows modifications engineered into the HA gene of
VN/1203/2004 to remove the polybasic cleavage site.
[0021] FIG. 2: Displays results showing that intranasally
administered H5N1 ca reassortant viruses do not replicate in
chickens.
[0022] FIG. 3: Illustrates that the H5N1/AA ca vaccine candidates
are not lethal to mice.
[0023] FIG. 4: Illustrates that the 1997 and 2004 H5N1 ca
reassortant viruses are restricted in replication in mice.
[0024] FIG. 5: Illustrates that the reassortant H5N1/AA ca
influenza viruses are restricted in replication in lungs of
mice.
[0025] FIG. 6: Shows the serum HAI Ab titers elicited in mice
following a single i.n. dose of vaccine.
[0026] FIG. 7: Shows serum neutralizing Ab titers elicited in mice
following a single i.n. dose of vaccine.
[0027] FIG. 8: Illustrates that H5N1 ca reassortant viruses protect
mice from lethal challenges with 50, 500 or 5000 LD.sub.50 of
wild-type H5N1 viruses.
[0028] FIG. 9: Illustrates the efficacy of protection from
pulmonary replication of homologous and heterologous H5N1 challenge
viruses in mice.
[0029] FIG. 10: Illustrates the efficacy of protection from
replication of homologous and heterologous H5N1 challenge viruses
in the upper respiratory tract of mice.
[0030] FIG. 11: Illustrates the efficacy of protection conferred by
2004 H5N1 ca vaccine against high dose (10.sub.5TCID.sub.50)
challenge with homologous or heterologous H5N1 wt viruses in
mice.
[0031] FIG. 12: Illustrates the efficacy of protection conferred by
1997 and 2003 H5N1 ca vaccines against high dose
(10.sub.5TCID.sub.50) challenges with homologous or heterologous
H5N1 wild-type viruses in mice.
[0032] 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.
[0033] FIG. 14: Shows modifications that can be engineered into the
HA gene of A/Netherland/219/03 HA to remove the polybasic cleavage
site.
[0034] 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.
[0035] FIG. 16: H6 ca viruses are attenuated in ferrets.
*EID.sub.50/g for lungs; PFU/g for nasal turbinates. 10.sup.7
TCID.sub.50 inoculated intranasally, tissues were harvested on day
3 post-infection.
[0036] FIG. 17: Immunogenicity of H6 ca vaccines in ferrets.
[0037] FIGS. 18(a) and 18(b): Efficacy of H6 ca vaccines in
ferrets. Virus titer was measured in (a) lungs and (b) nasal
turbinates. Vaccine: 1 dose of 7 log.sub.10 PFU. Challenge: 7
log.sub.10 PFU; 3 days post-challenge.
[0038] FIG. 19: H7N3 BC 04 ca is attenuated in ferrets. Inoculum:
10.sup.7 TCID.sub.50 in 0.5 mL intranasally. Tissues were harvested
on day 3 post-infection.
[0039] 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.
[0040] 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 LD.sub.50 A/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 LD.sub.50
A/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 LD.sub.50 A/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).
[0041] 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.
[0042] FIGS. 23(a) and (b): H9N2 G9/AA ca is attenuated in ferrets.
Virus titer was measured in (a) nasal turbinates and (b) lungs.
[0043] FIGS. 24(a) and (b): Efficacy of the H9N2 ca vaccine in
mice.
[0044] FIG. 25: Replication of H9N2 G9/AA ca is highly restricted
in healthy adults.
[0045] FIG. 26: HI antibody responses to 10.sup.7.0 TCID.sub.50 of
H9N2 G9/AA ca in healthy adults.
[0046] FIG. 27: Replication of H5N1 VN2004 A/AA ca is highly
restricted in healthy adults.
[0047] FIG. 28: HI antibody responses to 10.sup.6.7 TCID.sub.50 of
VN2004 A/AA ca in healthy adults.
DETAILED DESCRIPTION
[0048] 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
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] "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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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).
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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 as E. coli, or eukaryotic cells such as
yeast, insect, amphibian, avian or mammalian cells, including human
cells. Exemplary host cells can include, e.g., Vero (African green
monkey kidney) cells, BHK (baby hamster kidney) cells, primary
chick kidney (PCK) cells, Madin-Darby Canine Kidney (MDCK) cells,
Madin-Darby Bovine Kidney (MDBK) cells, 293 cells (e.g., 293T
cells), and COS cells (e.g., COS1, COS7 cells), etc.
[0070] 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.
[0071] 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.
[0072] 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
[0073] 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).
[0074] 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 by segment 2 and segment
3, respectively. In addition, segment 1 of some influenza strains
encodes a small protein, PB1-F2, produced from an alternative
reading frame within the PB1 coding region. Segment 4 encodes the
hemagglutinin (HA) surface glycoprotein involved in cell attachment
and entry during infection. Segment 5 encodes the nucleocapsid
nucleoprotein (NP) polypeptide, the major structural component
associated with viral RNA. Segment 6 encodes a neuraminidase (NA)
envelope glycoprotein. Segment 7 encodes two matrix proteins,
designated M1 and M2, which are translated from differentially
spliced mRNAs. Segment 8 encodes NS1 and NS2, two nonstructural
proteins, which are translated from alternatively spliced mRNA
variants. The eight genome segments of influenza B encode 11
proteins. The three largest genes code for components of the RNA
polymerase, PB1, PB2 and PA. Segment 4 encodes the HA protein.
Segment 5 encodes NP. Segment 6 encodes the NA protein and the NB
protein. Both proteins, NB and NA, are translated from overlapping
reading frames of a bicistronic mRNA. Segment 7 of influenza B also
encodes two proteins: M1 and BM2. The smallest segment encodes two
products: NS1 is translated from the full length RNA, while NS2 is
translated from a spliced mRNA variant.
Influenza Virus Vaccines
[0075] 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 .beta.-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.
[0076] 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 and U.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 CO.sub.2, at
constant temperature using a temperature regulator, such as a
thermostat to insure that the temperature does not exceed
35.degree. 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
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.degree. C.
relative to 33.degree. C. for influenza A strains, or that the
virus exhibits a 100 fold or greater reduction in titer at
37.degree. C. relative to 33.degree. C. for influenza B strains.
The term "cold adapted" (ca) indicates that the virus exhibits
growth at 25.degree. C. within 100 fold of its growth at 33.degree.
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.degree. C. (for A strain viruses) or 37.degree. C. (for
B strain viruses), or exhibiting growth at 25.degree. C. that is
more than 100 fold than its growth at 33.degree. 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.
[0081] 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.TM.
[0082] As mentioned previously, numerous examples and types of
influenza vaccine exist. An exemplary influenza vaccine is
FluMist.TM. which is a live, attenuated vaccine that protects
children and adults from influenza illness (Belshe et al. (1998)
The efficacy of live attenuated, cold-adapted, trivalent,
intranasal influenza virus vaccine in children N Engl J Med
338:1405-12; Nichol et al. (1999) Effectiveness of live, attenuated
intranasal influenza virus vaccine in healthy, working adults: a
randomized controlled trial JAMA 282:137-44). In typical, and
preferred, embodiments, the methods and compositions of the current
invention are preferably adapted to/used with production of
FluMist.TM. 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.
[0083] FluMist.TM. 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.TM.
formulations. The MDV for influenza A strains of FluMist.TM.
(MDV-A), was created by serial passage of the wild-type A/Ann
Arbor/6/60 (A/AA/6/60) strain in primary chicken kidney tissue
culture at successively lower temperatures (Maassab (1967)
Adaptation and growth characteristics of influenza virus at 25
degrees C. Nature 213:612-4). MDV-A replicates efficiently at
25.degree. C. (ca, cold adapted), but its growth is restricted at
38 and 39.degree. 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 Immunol 15:295-323).
[0084] Clinical studies in over 20,000 adults and children
involving 12 separate 6:2 reassortant strains have shown that these
vaccines are attenuated, safe and efficacious (Belshe et al. (1998)
The efficacy of live attenuated, cold-adapted, trivalent,
intranasal influenza virus vaccine in children N Engl J Med
338:1405-12; Boyce et al. (2000) Safety and immunogenicity of
adjuvanted and unadjuvanted subunit influenza vaccines administered
intranasally to healthy adults Vaccine 19:217-26; Edwards et al.
(1994) A randomized controlled trial of cold adapted and
inactivated vaccines for the prevention of influenza A disease J
Infect Dis 169:68-76; Nichol et al. (1999) Effectiveness of live,
attenuated intranasal influenza virus vaccine in healthy, working
adults: a randomized controlled trial JAMA 282: 137-44).
Reassortants carrying the six internal genes of MDV-A and the two
HA and NA gene segments of a wild-type virus (i.e., a 6:2
reassortant) consistently maintain ca, ts and att phenotypes
(Maassab et al. (1982) Evaluation of a cold-recombinant influenza
virus vaccine in ferrets J. Infect. Dis. 146:780-900).
[0085] 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.
[0086] 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
[0087] As stated above, alternatively, or in addition to, use in
production of FluMist.TM. 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.
[0088] 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.
[0089] 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., Pediatrics 52:56-63 (1973); and Wright et al., J. Pediatr.
88:931-936 (1976). For example, influenza viruses are provided in
the range of about 1-1000 HID.sub.50 (human infectious dose), i.e.,
about 10.sup.5-10.sup.8 pfu (plaque forming units) per dose
administered. Typically, the dose will be adjusted within this
range based on, e.g., age, physical condition, body weight, sex,
diet, time of administration, and other clinical factors. The
prophylactic vaccine formulation is systemically administered,
e.g., by subcutaneous or intramuscular injection using a needle and
syringe, or a needle-less injection device. Alternatively, the
vaccine formulation is administered intranasally, either by drops,
large particle aerosol (greater than about 10 microns), or spray
into the upper respiratory tract. While any of the above routes of
delivery results in a protective systemic immune response,
intranasal administration confers the added benefit of eliciting
mucosal immunity at the site of entry of the influenza virus. For
intranasal administration, attenuated live virus vaccines are often
preferred, e.g., an attenuated, cold adapted and/or temperature
sensitive recombinant or reassortant influenza virus. See above.
While stimulation of a protective immune response with a single
dose is preferred, additional dosages can be administered, by the
same or different route, to achieve the desired prophylactic
effect.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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
[0097] 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).
[0098] 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.
[0099] 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.
[0100] Oligonucleotide probes are optionally produced via a variety
of methods well known to those skilled in the art. Most typically,
they are produced by well known synthetic methods, such as the
solid phase phosphoramidite triester method described by Beaucage
and Caruthers (1981) Tetrahedron Letts 22(20):1859-1862, e.g.,
using an automated synthesizer, or as described in Needham-Van
Devanter et al. (1984) Nucl Acids Res, 12:6159-6168.
Oligonucleotides can also be custom made and ordered from a variety
of commercial sources known to persons of skill. Purification of
oligonucleotides, where necessary, is typically performed by either
native acrylamide gel electrophoresis or by anion-exchange HPLC as
described in Pearson and Regnier (1983) J Chrom 255:137-149. The
sequence of the synthetic oligonucleotides can be verified using
the chemical degradation method of Maxam and Gilbert (1980) in
Grossman and Moldave (eds.) Academic Press, New York, Methods in
Enzymology 65:499-560. Custom oligos can also easily be ordered
from a variety of commercial sources known to persons of skill.
[0101] 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.
[0102] 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.
[0103] Diagnostic Assays
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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.sub.m where different
probes have different GC contents). Although melting temperature is
a primary consideration in probe design, other factors are
optionally used to further adjust probe construction, such as
selecting against primer self-complementarity and the like.
[0108] Vectors, Promoters and Expression Systems
[0109] 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.
[0110] 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.
[0111] In an expression vector, the HA and/or NA polynucleotide
sequence of interest is physically arranged in proximity and
orientation to an appropriate transcription control sequence (e.g.,
promoter, and optionally, one or more enhancers) to direct mRNA
synthesis. That is, the polynucleotide sequence of interest is
operably linked to an appropriate transcription control sequence.
Examples of such promoters include: LTR or SV40 promoter, E. coli
lac or trp promoter, phage lambda P.sub.L promoter, and other
promoters known to control expression of genes in prokaryotic or
eukaryotic cells or their viruses.
[0112] 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.
[0113] Transcription is optionally increased by including an
enhancer sequence. Enhancers are typically short, e.g., 10-500 bp,
cis-acting DNA elements that act in concert with a promoter to
increase transcription. Many enhancer sequences have been isolated
from mammalian genes (hemoglobin, elastase, albumin,
alpha-fetoprotein, and insulin), and eukaryotic cell viruses. The
enhancer can be spliced into the vector at a position 5' or 3' to
the heterologous coding sequence, but is typically inserted at a
site 5' to the promoter. Typically, the promoter, and if desired,
additional transcription enhancing sequences are chosen to optimize
expression in the host cell type into which the heterologous DNA is
to be introduced (Scharf et al. (1994) Heat stress promoters and
transcription factors Results Probl Cell Differ 20:125-62; Kriegler
et al. (1990) Assembly of enhancers, promoters, and splice signals
to control expression of transferred genes Methods in Enzymol 185:
512-27). Optionally, the amplicon can also contain a ribosome
binding site or an internal ribosome entry site (IRES) for
translation initiation.
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] Additional Expression Elements
[0119] 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.
[0120] 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.
[0121] 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 Differ
20:125-62; Bittner et al. (1987) Methods in Enzymol
153:516-544).
[0122] Production of Recombinant Virus
[0123] 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. USA 92:11563-11567), and has been successfully applied to a
wide variety of segmented and nonsegmented negative strand RNA
viruses, e.g., rabies (Schnell et al. (1994) EMBO J. 13:
4195-4203); VSV (Lawson et al. (1995) Proc. Natl. Acad. Sci. USA
92: 4477-4481); measles virus (Radecke et al. (1995) EMBO J.
14:5773-5784); rinderpest virus (Baron & Barrett (1997) J.
Virol. 71: 1265-1271); human parainfluenza virus (Hoffman &
Banerjee (1997) J. Virol. 71: 3272-3277; Dubin et al. (1997)
Virology 235:323-332); SV5 (He et al. (1997) Virology 237:249-260);
canine distemper virus (Gassen et al. (2000) J. Virol.
74:10737-44); and Sendai virus (Park et al. (1991) Proc. Natl.
Acad. Sci. USA 88: 5537-5541; Kato et al. (1996) Genes to Cells
1:569-579). Those of skill in the art will be familiar with these
and similar techniques to produce influenza virus comprising the HA
and NA sequences of the invention. Recombinant influenza viruses
produced according to such methods are also a feature of the
invention, as are recombinant influenza virus comprising one or
more nucleic acids and/or polypeptides of the invention.
[0124] Cell Culture and Expression Hosts
[0125] The present invention also relates to host cells that are
introduced (transduced, transformed or transfected) with vectors of
the invention, and the production of polypeptides of the invention
by recombinant techniques. Host cells are genetically engineered
(i.e., transduced, transformed or transfected) with a vector, such
as an expression vector, of this invention. As described above, the
vector can be in the form of a plasmid, a viral particle, a phage,
etc. Examples of appropriate expression hosts include: bacterial
cells, such as E. coli, Streptomyces, and Salmonella typhimurium;
fungal cells, such as Saccharomyces cerevisiae, Pichia pastoris,
and Neurospora crassa; or insect cells such as Drosophila and
Spodoptera frugiperda.
[0126] 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 CO.sub.2 concentration
suitable for maintaining neutral buffered pH (e.g., at pH between
7.0 and 7.2). Optionally, the medium contains antibiotics to
prevent bacterial growth, e.g., penicillin, streptomycin, etc.,
and/or additional nutrients, such as L-glutamine, sodium pyruvate,
non-essential amino acids, additional supplements to promote
favorable growth characteristics, e.g., trypsin,
.beta.-mercaptoethanol, and the like.
[0127] 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.sup.rd edition, Wiley-Liss, New York and the
references cited therein. Other helpful references include, e.g.,
Paul (1975) Cell and Tissue Culture, 5.sup.th ed., Livingston,
Edinburgh; Adams (1980) Laboratory Techniques in Biochemistry and
Molecular Biology-Cell Culture for Biochemists, Work and Burdon
(eds.) Elsevier, Amsterdam. Additional details regarding tissue
culture procedures of particular interest in the production of
influenza virus in vitro include, e.g., Merten et al. (1996)
Production of influenza virus in cell cultures for vaccine
preparation. in Cohen and Shafferman (eds.) Novel Strategies in
Design and Production of Vaccines, which is incorporated herein in
its entirety for all purposes. Additionally, variations in such
procedures adapted to the present invention are readily determined
through routine experimentation and will be familiar to those
skilled in the art.
[0128] 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).
[0129] 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.).
[0130] 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
.mu.g of each vector is introduced into a population of host cells
with approximately 2 .mu.l of TransIT-LT1 diluted in 160 .mu.l
medium, preferably serum-free medium, in a total volume of 200
.mu.l. The DNA:transfection reagent mixtures are incubated at room
temperature for 45 minutes followed by addition of 800 .mu.l 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 .mu.l 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.
[0131] 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.times.10.sup.6 Vero 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 .mu.l 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.degree. C.
for cold adapted strains.
[0132] 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 Sci 81:3655-3659). In addition, transcription
enhancers, such as the rous sarcoma virus (RSV) enhancer, can be
used to increase expression in mammalian host cells.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] Expression products corresponding to the nucleic acids of
the invention can also be produced in non-animal cells such as
plants, yeast, fungi, bacteria and the like. In addition to
Sambrook, Berger and Ausubel, all infra, details regarding cell
culture can be found in Payne et al. (1992) Plant Cell and Tissue
Culture in Liquid Systems John Wiley & Sons, Inc. New York,
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.
[0137] In bacterial systems, a number of expression vectors can be
selected depending upon the use intended for the expressed product.
For example, when large quantities of a polypeptide or fragments
thereof are needed for the production of antibodies, vectors that
direct high-level expression of fusion proteins that are readily
purified are favorably employed. Such vectors include, but are not
limited to, multifunctional E. coli cloning and expression vectors
such as 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 Chem 264:5503-5509); pET vectors (Novagen,
Madison Wis.); and the like. Similarly, in the yeast Saccharomyces
cerevisiae a number of vectors containing constitutive or inducible
promoters such as alpha factor, alcohol oxidase and PGH can be used
for production of the desired expression products. For reviews, see
Ausubel, infra, and Grant et al., (1987); Methods in Enzymology
153:516-544.
[0138] Nucleic Acid Hybridization
[0139] 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.
[0140] 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.times.-10.times. as high as that observed for hybridization to
any of the unmatched target nucleic acids.
[0141] 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 Probes part I
chapter 2, "Overview of principles of hybridization and the
strategy of nucleic acid probe assays," (Elsevier, New York), as
well as in Ausubel, Sambrook, and Berger and Kimmel, all below.
Hames and Higgins (1995) Gene Probes 1 IRL Press at Oxford
University Press, Oxford, England, (Hames and Higgins 1) and Hames
and Higgins (1995) Gene Probes 2 IRL Press at Oxford University
Press, Oxford, England (Hames and Higgins 2) provide details on the
synthesis, labeling, detection and quantification of DNA and RNA,
including oligonucleotides.
[0142] 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.degree. C., with
the hybridization being carried out overnight. An example of
stringent wash conditions comprises a 0.2.times.SSC wash at
65.degree. 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.times.SSC at 40.degree. C. for 15 minutes. In
general, a signal to noise ratio of 5.times. (or higher) than that
observed for an unrelated probe in the particular hybridization
assay indicates detection of a specific hybridization.
[0143] 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.sub.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).
[0144] "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.times. as high as that observed for
hybridization of the probe to an unmatched target.
[0145] In general, a signal to noise ratio of at least 2.times. (or
higher, e.g., at least 5.times., 10.times., 20.times., 50.times.,
100.times., 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.
[0146] "Very stringent" conditions are selected to be equal to the
thermal melting point (T.sub.m) for a particular probe. The T.sub.m
is the temperature (under defined ionic strength and pH) at which
50% of the test sequence hybridizes to a perfectly matched probe.
For the purposes of the present invention, generally, "highly
stringent" hybridization and wash conditions are selected to be
about 5.degree. C. lower than the T.sub.m for the specific sequence
at a defined ionic strength and pH (as noted below, highly
stringent conditions can also be referred to in comparative terms).
Target sequences that are closely related or identical to the
nucleotide sequence of interest (e.g., "probe") can be identified
under stringent or highly stringent conditions. Lower stringency
conditions are appropriate for sequences that are less
complementary.
[0147] "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.times. 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.
[0148] 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.times. (and optionally 5.times., 10.times., or
100.times. 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.
[0149] 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).
[0150] 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.times., 20.times., 50.times., 100.times., or 500.times. 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.
[0151] 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.
[0152] Cloning, Mutagenesis and Expression of Biomolecules of
Interest
[0153] General texts which describe molecular biological
techniques, which are applicable to the present invention, such as
cloning, mutation, cell culture and the like, include Berger and
Kimmel, Guide to Molecular Cloning Techniques, Methods in
Enzymology volume 152 Academic Press, Inc., San Diego, 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") and Current Protocols in Molecular
Biology, F. M. Ausubel et al., eds., Current Protocols, a joint
venture between Greene Publishing Associates, Inc. and John Wiley
& Sons, Inc., (supplemented through 2002) ("Ausubel")). These
texts describe mutagenesis, the use of vectors, promoters and many
other relevant topics related to, e.g., the generation of HA and/or
NA molecules, etc.
[0154] 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.
[0155] The above texts and examples found herein describe these
procedures as well as the following publications (and references
cited within): Sieber, et al., Nature Biotechnology, 19:456-460
(2001); Ling et al., Approaches to DNA mutagenesis: an overview,
Anal Biochem 254(2): 157-178 (1997); Dale et al.,
Oligonucleotide-directed random mutagenesis using the
phosphorothioate method, Methods Mol Biol 57:369-374 (1996); I. A.
Lorimer, I. Pastan, Nucleic Acids Res 23, 3067-8 (1995); W. P. C.
Stemmer, Nature 370, 389-91 (1994); Arnold, Protein engineering for
unusual environments, Current Opinion in Biotechnology 4:450-455
(1993); Bass et al., Mutant Trp repressors with new DNA-binding
specificities, Science 242:240-245 (1988); Fritz et al.,
Oligonucleotide-directed construction of mutations: a gapped duplex
DNA procedure without enzymatic reactions in vitro, Nucl Acids Res
16: 6987-6999 (1988); Kramer et al., Improved enzymatic in vitro
reactions in the gapped duplex DNA approach to
oligonucleotide-directed construction of mutations, Nucl Acids Res
16: 7207 (1988); Sakamar and Khorana, Total synthesis and
expression of a gene for the a-subunit of bovine rod outer segment
guanine nucleotide-binding protein (transducin), Nucl Acids Res 14:
6361-6372 (1988); Sayers et al., Y-T Exonucleases in
phosphorothioate-based oligonucleotide-directed mutagenesis, Nucl
Acids Res 16:791-802 (1988); Sayers et al., Strand specific
cleavage of phosphorothioate-containing DNA by reaction with
restriction endonucleases in the presence of ethidium bromide,
(1988) Nucl Acids Res 16: 803-814; Carter, Improved
oligonucleotide-directed mutagenesis using M13 vectors, Methods in
Enzymol 154: 382-403 (1987); Kramer & Fritz
Oligonucleotide-directed construction of mutations via gapped
duplex DNA, Methods in Enzymol 154:350-367 (1987); Kunkel, The
efficiency of oligonucleotide directed mutagenesis, in Nucleic
Acids & Molecular Biology (Eckstein, F. and Lilley, D. M. J.
eds., Springer Verlag, Berlin)) (1987); Kunkel et al., Rapid and
efficient site-specific mutagenesis without phenotypic selection,
Methods in Enzymol 154, 367-382 (1987); Zoller & Smith,
Oligonucleotide-directed mutagenesis: a simple method using two
oligonucleotide primers and a single-stranded DNA template, Methods
in Enzymol 154:329-350 (1987); Carter, Site-directed mutagenesis,
Biochem J 237:1-7 (1986); Eghtedarzadeh & Henikoff, Use of
oligonucleotides to generate large deletions, Nucl Acids Res 14:
5115 (1986); Mandecki, Oligonucleotide-directed double-strand break
repair in plasmids of Escherichia coli: a method for site-specific
mutagenesis, Proc 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 Res 14: 9679-9698
(1986); Wells et al., Importance of hydrogen-bond formation in
stabilizing the transition state of subtilisin, Phil Trans R Soc
Lond A 317: 415-423 (1986); Botstein & Shortle, Strategies and
applications of in vitro mutagenesis, Science 229:1193-1201 (1985);
Carter et al., Improved oligonucleotide site-directed mutagenesis
using M13 vectors, Nucl Acids Res 13: 4431-4443 (1985); Grundstrom
et al., Oligonucleotide-directed mutagenesis by microscale
`shot-gun` gene synthesis, Nucl Acids Res 13:3305-3316 (1985);
Kunkel, Rapid and efficient site-specific mutagenesis without
phenotypic selection, Proc Natl Acad Sci USA 82:488-492 (1985);
Smith, In vitro 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 Res
13: 8749-8764 (1985); Taylor et al., The rapid generation of
oligonucleotide-directed mutations at high frequency using
phosphorothioate-modified DNA, Nucl Acids Res 13: 8765-8787 (1985);
Wells et al., Cassette mutagenesis: an efficient method for
generation of multiple mutations at defined sites, Gene 34:315-323
(1985); Kramer et al., The gapped duplex DNA approach to
oligonucleotide-directed mutation construction, Nucl Acids Res 12:
9441-9456 (1984); Kramer et al., Point Mismatch Repair, Cell
38:879-887 (1984); Nambiar et al., Total synthesis and cloning of a
gene coding for the ribonuclease S protein, Science 223: 1299-1301
(1984); Zoller & Smith, Oligonucleotide-directed mutagenesis of
DNA fragments cloned into M13 vectors, Methods in Enzymol
100:468-500 (1983); and Zoller & Smith,
Oligonucleotide-directed mutagenesis using M13-derived vectors: an
efficient and general procedure for the production of point
mutations in any DNA fragment, Nucl Acids Res 10:6487-6500 (1982).
Additional details on many of the above methods can be found in
Methods in Enzymol Volume 154, which also describes useful controls
for trouble-shooting problems with various mutagenesis, gene
isolation, expression, and other methods.
[0156] Oligonucleotides, e.g., for use in mutagenesis of the
present invention, e.g., mutating libraries of the HA and/or NA
molecules of the invention, or altering such, are typically
synthesized chemically according to the solid phase phosphoramidite
triester method described by Beaucage and Caruthers, Tetrahedron
Letts 22(20):1859-1862, (1981) e.g., using an automated
synthesizer, as described in Needham-Van Devanter et al., Nucleic
Acids Res, 12:6159-6168 (1984).
[0157] 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.
[0158] 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 USA
82, 5824 (1985), infection by viral vectors, high velocity
ballistic penetration by small particles with the nucleic acid
either within the matrix of small beads or particles, or on the
surface (Klein et al., Nature 327, 70-73 (1987)). Berger, Sambrook,
and Ausubel provide a variety of appropriate transformation
methods. See, above.
[0159] 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.TM., FlexiPrep.TM., both from
Pharmacia Biotech; StrataClean.TM., from Stratagene; and,
QIAprep.TM. 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, Gene 8:81 (1979); Roberts, et al., Nature, 328:731 (1987);
Schneider, B., et al., Protein Expr Purif 6435:10 (1995); Ausubel,
Sambrook, Berger (all supra). A catalogue of Bacteria and
Bacteriophages useful for cloning is provided, e.g., by the ATCC,
e.g., The ATCC Catalogue of Bacteria and 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 Edition Scientific
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
[0160] 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.
[0161] 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.sup.nd Edition Wiley-Liss, NY;
Walker (1996) The Protein Protocols Handbook Humana Press, NJ,
Harris and Angal (1990) Protein Purification Applications: A
Practical Approach IRL Press at Oxford, Oxford, England; Harris and
Angal Protein Purification Methods: A Practical Approach IRL Press
at Oxford, Oxford, England; Scopes (1993) Protein Purification:
Principles and Practice 3.sup.rd Edition Springer Verlag, NY;
Janson and Ryden (1998) Protein Purification: Principles, High
Resolution Methods and Applications, Second Edition Wiley-VCH, NY;
and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ.
[0162] 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.times.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.times.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 Influenza pp. 324-332; Merten et al. (1996) Production of
influenza virus in cell cultures for vaccine preparation, in Cohen
& Shafferman (eds.) Novel Strategies in Design and Production
of Vaccines pp. 141-151, and U.S. Pat. No. 5,690,937. If desired,
the recovered viruses can be stored at -80.degree. C. in the
presence of sucrose-phosphate-glutamate (SPG) as a stabilizer
[0163] 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 in Molecular Biology Volume 37, Garland Publishing, NY.
[0164] In addition, the polypeptides, or subsequences thereof,
e.g., subsequences comprising antigenic peptides, can be produced
manually or by using an automated system, by direct peptide
synthesis using solid-phase techniques (see, Stewart et al. (1969)
Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco;
Merrifield J (1963) J Am Chem Soc 85:2149-2154). Exemplary
automated systems include the Applied Biosystems 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.
[0165] Modified Amino Acids
[0166] 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).
[0167] Non-limiting examples of a modified amino acid include a
glycosylated amino acid, a sulfated amino acid, a prenlyated (e.g.,
farnesylated, geranylgeranylated) amino acid, an acetylated amino
acid, an acylated amino acid, a PEG-ylated amino acid, a
biotinylated amino acid, a carboxylated amino acid, a
phosphorylated amino acid, and the like, as well as amino acids
modified by conjugation to, e.g., lipid moieties or other organic
derivatizing agents. References adequate to guide one of skill in
the modification of amino acids are replete throughout the
literature. Example protocols are found in Walker (1998) Protein
Protocols on CD-ROM Human Press, Towata, N.J.
[0168] Fusion Proteins
[0169] 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.
[0170] Antibodies
[0171] 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.
[0172] 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.
[0173] 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.
[0174] Numerous methods for producing polyclonal and monoclonal
antibodies are known to those of skill in the art, and can be
adapted to produce antibodies specific for the polypeptides of the
invention, and/or encoded by the polynucleotide sequences of the
invention, etc. See, e.g., Coligan (1991) Current Protocols in
Immunology Wiley/Greene, NY; Paul (ed.) (1998) Fundamental
Immunology Fourth Edition, Lippincott-Raven, Lippincott Williams
& Wilkins; Harlow and Lane (1989) Antibodies: A Laboratory
Manual Cold Spring Harbor Press, NY; Stites et al. (eds.) Basic and
Clinical Immunology (4th ed.) Lange Medical Publications, Los
Altos, 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) Science 246: 1275-1281;
and Ward, et al. (1989) Nature 341: 544-546. Specific monoclonal
and polyclonal antibodies and antisera will usually bind with a
K.sub.D of, e.g., at least about 0.1 .mu.M, at least about 0.01
.mu.M or better, and, typically and at least about 0.001 .mu.M or
better.
[0175] 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.sup.nd Edition Freeman and Company, NY
(Borrebaeck); McCafferty et al. (1996) Antibody Engineering, A
Practical Approach IRL at Oxford Press, Oxford, England
(McCafferty), and Paul (1995) Antibody Engineering Protocols Humana
Press, Towata, N.J. (Paul). Additional details regarding specific
procedures can be found, e.g., in Ostberg et al. (1983), Hybridoma
2: 361-367, Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al.,
U.S. Pat. No. 4,634,666.
[0176] Defining Polypeptides by Immunoreactivity
[0177] 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.
[0178] 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.
[0179] 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.
[0180] 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.
[0181] 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
10.sup.6 or greater are selected, pooled and subtracted with the
control hemagglutinin and/or neuraminidase polypeptide(s) to
produce subtracted pooled titered polyclonal antisera.
[0182] 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.times. higher signal to
noise ratio than the control receptor homologues under
discriminatory binding conditions, and at least about a 1/2 signal
to noise ratio as compared to the immunogenic polypeptide(s),
shares substantial structural similarity with the immunogenic
polypeptide as compared to the known receptor, etc., and is,
therefore a polypeptide of the invention.
[0183] 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.
[0184] 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.times. 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.
[0185] 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.times. as high as for the control polypeptide.
[0186] 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.times.
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
[0187] 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.
[0188] 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.
[0189] Silent Variations
[0190] 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-US-00001 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
[0191] 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.
[0192] 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.
[0193] Conservative Variations
[0194] 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.
[0195] "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-US-00002 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)
[0196] Unique Polypeptide and Polynucleotide Subsequences
[0197] 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.
[0198] 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.
[0199] 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.
[0200] Sequence Comparison, Identity, and Homology
[0201] 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.
[0202] 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.
[0203] 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.
[0204] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv Appl Math 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J Mol Biol 48:443 (1970), by
the search for similarity method of Pearson & Lipman, Proc Natl
Acad Sci USA 85:2444 (1988), by computerized implementations of
algorithms such as GAP, 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).
[0205] One example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity is the BLAST
algorithm, which is described in Altschul et al., J Mol Biol
215:403-410 (1990). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (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 USA 89:10915).
[0206] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul,
Proc Natl Acad Sci USA 90:5873-5787 (1993)). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0207] 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) CABIOS 5:151-153. 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.
[0208] An additional example of an algorithm that is suitable for
multiple DNA, or amino acid, sequence alignments is the CLUSTALW
program (Thompson, J. D. et al. (1994) Nucl. Acids. Res. 22:
4673-4680). CLUSTALW performs multiple pairwise comparisons between
groups of sequences and assembles them into a multiple alignment
based on homology. Gap open and Gap extension penalties can be,
e.g., 10 and 0.05 respectively. For amino acid alignments, the
BLOSUM algorithm can be used as a protein weight matrix. See, e.g.,
Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:
10915-10919.
Digital Systems
[0209] 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.
[0210] 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.
[0211] 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.).
[0212] Thus, standard desktop applications such as word processing
software (e.g., Microsoft Word.TM. or Corel WordPerfect.TM.) and
database software (e.g., spreadsheet software such as Microsoft
Excel.TM., Corel Quattro Pro.TM., or database programs such as
Microsoft Access.TM., Paradox.TM., GeneWorks.TM., or MacVector.TM.
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).
[0213] 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.TM., OS2.TM. WINDOWS.TM.
WINDOWSNT.TM., WINDOWS95.TM., WINDOWS2000.TM., WINDOWS98.TM., LINUX
based machine, a MACINTOSH.TM., Power PC, or a UNIX based (e.g.,
SUN.TM. 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.
[0214] 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.
[0215] 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
[0216] 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.
[0217] 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.
[0218] 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.
[0219] 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.
[0220] 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.
EXAMPLES
Example 1
Construction and Analysis of H5N1 ca Viruses and Vaccines
[0221] 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).
[0222] 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.
[0223] 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.).
[0224] 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 log.sub.10TCID.sub.50 in eggs. However, it
will be appreciated that other embodiments wherein the
log.sub.10TCID.sub.50 comprises 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.
[0225] 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.sup.8-8.75
TCID.sub.50/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 in FIG. 2, the
intranasally administered H5N1 ca reassortant viruses did not
replicate in chickens.
[0226] The H5N1/AA ca reassortants were also not lethal for mice.
See FIG. 3, which also shows the TCID.sub.50 for 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.
[0227] 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 in FIG. 6. FIGS. 7 and
15 show similar measurements, but using serum neutralizing antibody
titers.
[0228] FIG. 8 displays that the H5N1 ca reassortant viruses protect
mice from lethal challenge with 50, 500, or 5,000 LD.sub.50 of
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.).
[0229] FIG. 11 shows efficacy of protection conferred by 2004 H5N1
ca vaccine against high dose (10.sup.5TCID.sub.50) 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 (10.sup.5TCID.sub.50) 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.
[0230] In healthy human adults nasal spray administration the '04
vaccine was well tolerated and its replication was highly
restricted. See FIG. 27 for replication restriction of the vaccine
in healthy adults. HI antibody responses to 10.sup.6.7 TCID.sub.50
of the '04 vaccine were also observed in some of the healthy
adults. See FIG. 28.
[0231] 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.
[0232] 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 2
Construction and Analysis of H6 ca Viruses and Vaccines
[0233] 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.
[0234] 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 10.sup.7 TCID.sub.50
recombinant (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.
[0235] Each of the A/Duck, A/Teal, and A/Mallard recombinant (ca)
viruses was also immunogenic in the ferrets. See FIG. 17.
[0236] 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 log.sub.10 PFU recombinant A/Duck, A/Teal,
or A/Mallard vaccine. The ferrets were then challenged with 7
log.sub.10 PFU 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.
[0237] FIG. 18 shows efficacy of protection conferred by the
recombinant (ca) H6 vaccines against homologous and heterologous
wild-type H6 viruses in ferrets.
Example 3
Construction and Analysis of an H7N3 BC 04 ca, Virus and
Vaccine
[0238] 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.
[0239] The BC 04 ca vaccine was attenuated in the ferrets. Ferrets
were intranasally inoculated with 10.sup.7 TCID.sub.50 vaccine 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. See FIG. 19.
[0240] The BC 04 ca vaccine was immunogenic in mice. In mice
receiving the BC 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. See FIG. 20.
[0241] FIGS. 21 and 22 show the efficacy of protection conferred by
the BC 04 ca vaccine against both homologous and heterologous H7 wt
viruses. For FIG. 21, mice were intranasally inoculated with 1 dose
vaccine four weeks before challenge, 1 dose vaccine 8 weeks before
challenge, or 2 doses vaccine (administered 4 weeks apart) before
lethal challenge with 50 LD.sub.50 homologous (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.
[0242] 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).
[0243] FIG. 22 provides further evidence of the efficacy of the
H7N3 BC 04 ca vaccine. In both nasal turbinates (a) and lungs (b)
of mice receiving the H7N3 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 4
Construction and Analysis of an H9N2 G9/AA ca, Virus and
Vaccine
[0244] 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.
[0245] The H9N2 G9/AA ca vaccine was attenuated in the ferrets. See
FIG. 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.
[0246] 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.
[0247] 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. See FIG. 25. Furthermore, administration of 10.sup.7.0
TCID.sub.50 of H9N2 G9/AA ca vaccine induced .gtoreq.4-fold
increases in HI titer in 92% of healthy volunteers. See FIG.
26.
[0248] 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
[0249] Additional embodiments of the present invention are
presented in Table 3 and 4. TABLE-US-00003 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 of
embodiment 1. 3 An isolated antibody that specifically binds the
polypeptide of embodiment 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 of
embodiment 1. 6 An immunogenic composition comprising an
immunologically effective amount of the recombinant influenza virus
of embodiment 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 of embodiment 8. 11 A vector comprising the nucleic acid of
embodiment 8. 12 The vector of embodiment 12, wherein the vector is
a plasmid, a cosmid, a phage, a virus, or a fragment of a virus. 13
The vector of embodiment 12, wherein the vector is an expression
vector. 14 A cell comprising the vector of embodiment 13. 15 An
influenza virus comprising one or more nucleic acids of embodiment
8. 16 The virus of embodiment 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 of embodiment 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 of embodiment 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 of embodiment 22, wherein the
subject is a human. 24 The immunogenic composition of embodiment
19, wherein the hemagglutinin comprises a modified polybasic
cleavage site. 25 A live attenuated influenza vaccine comprising
the composition of embodiment 19. 26 A split virus or killed virus
vaccine comprising the composition of embodiment 19. 27 A live
attenuated influenza vaccine comprising the composition of
embodiment 24. 28 A split virus or killed virus vaccine comprising
the composition of embodiment 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.degree. 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 of
embodiment 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 of
embodiment 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 of embodiment 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 of
embodiment 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.
[0250] TABLE-US-00004 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 of embodiment 1. 3 An isolated antibody that
specifically binds the polypeptide of embodiment 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 of embodiment 1. 6 An immunogenic
composition comprising an immunologically effective amount of the
recombinant influenza virus of embodiment 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
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 of
embodiment 8. 10 A cell comprising at least one polynucleotide of
embodiment 8. 11 A vector comprising the polynucleotide of
embodiment 8. 12 The vector of embodiment 11, wherein the vector is
a plasmid, a cosmid, a phage, a virus, or a fragment of a virus. 13
The vector of embodiment 12, wherein the vector is an expression
vector. 14 A cell comprising the vector of embodiment 13. 15 An
influenza virus comprising one or more polynucleotides of
embodiment 8. 16 The virus of embodiment 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 of embodiment 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 of
embodiment 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 of
embodiment 22, wherein the subject is a human. 24 The immunogenic
composition of embodiment 19, wherein the hemagglutinin comprises a
modified polybasic cleavage site. 25 A live attenuated influenza
vaccine comprising the composition of embodiment 19. 26 A split
virus or killed virus vaccine comprising the composition of
embodiment 19. 27 A live attenuated influenza vaccine comprising
the composition of embodiment 24. 28 A split virus or killed virus
vaccine comprising the composition of embodiment 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.degree. 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 of embodiment 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 of
embodiment 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 of embodiment 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 of
embodiment 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 of embodiment 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 of embodiment
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 of
embodiment 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 of embodiment 45, wherein the
hemagglutinin comprises a modified polybasic 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 of embodiment 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.degree. 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
[0251] Nucleotide Sequence of ca A/Vietnam/1203/04H5 (SEQ ID
NO:1)
[0252] Entire molecule length: 1767 nt TABLE-US-00005 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
[0253] Amino acid sequence of ca A/Vietnam/1203/04H5 (SEQ ID
NO:11)
[0254] Entire molecule length: 564 aa TABLE-US-00006 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
[0255] Nucleotide Sequence of ca A/Vietnam/1203/04 N1 (SEQ ID NO:
2)
[0256] Entire molecule length: 1398 nt TABLE-US-00007 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
[0257] Amino acid sequence of ca A/Vietnam/1203/04 N1 (SEQ ID
NO:12)
[0258] Entire molecule length: 449 aa TABLE-US-00008 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
[0259] Nucleotide Sequence of ca A/Hong Kong/213/03H5 (SEQ ID NO:
3)
[0260] Entire molecule length: 1767 nt TABLE-US-00009 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
[0261] Amino acid sequence of ca A/Hong Kong/213/03H5 (SEQ ID
NO:13)
[0262] Entire molecule length: 564 aa TABLE-US-00010 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
[0263] Nucleotide Sequence of ca A/Hong Kong/213/03 N1 (SEQ ID NO:
4)
[0264] Entire molecule length: 1458 nt TABLE-US-00011 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
[0265] Amino acid sequence of ca A/Hong Kong/213/03 N1 (SEQ ID
NO:14)
[0266] Entire molecule length: 469 aa TABLE-US-00012 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)
[0267] Nucleotide Sequence of ca A/Hong Kong/491/97H5 (SEQ ID NO:
5)
[0268] Entire molecule length: 1767 nt TABLE-US-00013 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
[0269] Amino acid sequence of ca A/Hong Kong/491/97H5 (SEQ ID
NO:15)
[0270] Entire molecule length: 564 aa TABLE-US-00014 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
[0271] Nucleotide Sequence of ca A/Hong Kong/486/97 N1 (SEQ ID NO:
6)
[0272] Entire molecule length: 1401 nt TABLE-US-00015 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
[0273] Amino acid sequence of ca A/Hong Kong/486/97 N1 (SEQ ID
NO:16)
[0274] Entire molecule length: 450 aa TABLE-US-00016 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)
[0275] Nucleotide Sequence of ca A/Hong Kong/491/97 (Ser211) H5
(SEQ ID NO: 7)
[0276] Entire molecule length: 1767 nt TABLE-US-00017 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
[0277] Amino acid sequence of ca A/Hong Kong/491/97 (Ser211) H5
(SEQ ID NO:17)
[0278] Entire molecule length: 564 aa TABLE-US-00018 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
[0279] Nucleotide Sequence of ca A/Hong Kong/486/97 N1 (SEQ ID NO:
8)
[0280] Entire molecule length: 1401 nt TABLE-US-00019 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
[0281] Amino acid sequence of ca A/Hong Kong/486/97 N1 (SEQ ID
NO:18)
[0282] Entire molecule length: 450 aa TABLE-US-00020 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
[0283] Nucleotide sequence of ca A/ck/Hong Kong/G9/97 (SEQ ID NO:
9)
[0284] Entire molecule length: 1690 bp TABLE-US-00021 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
[0285] Amino acid sequence of ca A/ck/Hong Kong/G9/97H9 (SEQ ID
NO:19)
[0286] Entire molecule length: 558 aa TABLE-US-00022 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
[0287] Nucleotide sequence of ca A/ck/Hong Kong/G9/97 N2 (SEQ ID
NO:10)
[0288] Entire molecule length: 1428 bp TABLE-US-00023 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
[0289] Amino acid sequence of ca A/ck/Hong Kong/G9/97 N2 (SEQ ID
NO: 20)
[0290] Entire molecule length: 469 aa TABLE-US-00024 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
[0291] Nucleotide sequence of A/Netherlands/219/03H7 (SEQ ID NO:
21)
[0292] Entire molecule length: 1737 bp TABLE-US-00025 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
[0293] Amino acid sequence of A/Netherlands/219/03H7 (SEQ ID NO:
27)
[0294] Entire molecule length: 562 aa TABLE-US-00026 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
[0295] Nucleotide Sequence of A/Netherlands/219/03 N7 (SEQ ID NO:
22)
[0296] Entire molecule length: 1465 nt TABLE-US-00027 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
[0297] Nucleotide Sequence of A/Netherlands/219/03 N7 (SEQ ID NO:
45)
[0298] Entire molecule length: 1464 nt TABLE-US-00028 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
[0299] Amino acid sequence of A/Netherlands/219/03 N7 (SEQ ID NO:
28)
[0300] Entire molecule length: 471 aa TABLE-US-00029 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
[0301] Nucleotide sequence of A/ck/BC/CN-7/04H7 (SEQ ID NO: 23)
[0302] Entire molecule length: 1754 bp TABLE-US-00030 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
[0303] Amino acid sequence of A/ck/BC/CN-7/04H7 (SEQ ID NO: 29)
[0304] Entire molecule length: 567 aa TABLE-US-00031 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
[0305] Nucleotide Sequence of A/ck/BC/CN-7/04 N3 (SEQ ID NO:
24)
[0306] Entire molecule length: 1453 nt TABLE-US-00032 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
[0307] Amino acid sequence of A/ck/BC/CN-7/04 N3 (SEQ ID NO:
30)
[0308] Entire molecule length: 469 aa TABLE-US-00033 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
[0309] Nucleotide sequence of ca A/ck/BC/CN-6/04H7 (SEQ ID NO:
25)
[0310] Entire molecule length: 1734 bp TABLE-US-00034 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
[0311] Amino acid sequence of ca A/ck/BC/CN-6/04H7 (SEQ ID NO:
31)
[0312] Entire molecule length: 560 aa TABLE-US-00035 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
[0313] Nucleotide sequence of ca A/ck/BC/CN-6/04 N3 (SEQ ID NO:
26)
[0314] Entire molecule length: 1453 nt TABLE-US-00036 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
[0315] Amino acid sequence of ca A/ck/BC/CN-6/04 N3 (SEQ ID NO:
32)
[0316] Entire molecule length: 469 aa TABLE-US-00037 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
[0317] Nucleotide sequence of ca A/Duck H6 (SEQ ID NO: 33)
[0318] Entire molecule length: 1743 bp TABLE-US-00038 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
[0319] Amino acid sequence of ca A/Duck H6 (SEQ ID NO: 39)
[0320] Entire molecule length: 566 aa TABLE-US-00039 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
[0321] Nucleotide sequence of ca A/Duck N9 (SEQ ID NO: 34)
[0322] Entire molecule length: 1460 nt TABLE-US-00040 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
[0323] Amino acid sequence of ca A/Duck N9 (SEQ ID NO: 40)
[0324] Entire molecule length: 470 aa TABLE-US-00041 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
[0325] Nucleotide sequence of ca A/Teal H6 (SEQ ID NO: 35)
[0326] Entire molecule length: 1747 bp TABLE-US-00042 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
[0327] Amino acid sequence of ca A/Teal H6 (SEQ ID NO: 41)
[0328] Entire molecule length: 567 aa TABLE-US-00043 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
[0329] Nucleotide sequence of ca A/Teal N1 (SEQ ID NO: 36)
[0330] Entire molecule length: 1401 nt TABLE-US-00044 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
[0331] Amino acid sequence of ca A/Teal N1 (SEQ ID NO: 42)
[0332] Entire molecule length: 450 aa TABLE-US-00045 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
[0333] Nucleotide sequence of ca A/Mallard H6 (SEQ ID NO: 37)
[0334] Entire molecule length: 1745 bp TABLE-US-00046 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
[0335] Amino acid sequence of ca A/Mallard H6 (SEQ ID NO: 43)
[0336] Entire molecule length: 566 aa TABLE-US-00047 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
[0337] Nucleotide sequence of ca A/Mallard N2 (SEQ ID NO: 38)
[0338] Entire molecule length: 1467 nt TABLE-US-00048 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
[0339] Amino acid sequence of ca A/Mallard N2 (SEQ ID NO: 44)
[0340] Entire molecule length: 469 aa TABLE-US-00049 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
[0341] Summary of SEQ ID NO Designations TABLE-US-00050 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
[0342]
Sequence CWU 1
1
45 1 1767 DNA Influenza A virus 1 agcaaaagca ggggttcaat ctgtcaaaat
ggagaaaata gtgcttcttt ttgcaatagt 60 cagtcttgtt aaaagtgatc
agatttgcat tggttaccat gcaaacaact cgacagagca 120 ggttgacaca
ataatggaaa agaacgttac tgttacacat gcccaagaca tactggaaaa 180
gaaacacaac gggaagctct gcgatctaga tggagtgaag cctctaattt tgagagattg
240 tagcgtagct ggatggctcc tcggaaaccc aatgtgtgac gaattcatca
atgtgccgga 300 atggtcttac atagtggaga aggccaatcc agtcaatgac
ctctgttacc caggggattt 360 caatgactat gaagaattga aacacctatt
gagcagaata aaccattttg agaaaattca 420 gatcatcccc aaaagttctt
ggtccagtca tgaagcctca ttaggggtga gctcagcatg 480 tccataccag
ggaaagtcct cctttttcag aaatgtggta tggcttatca aaaagaacag 540
tacataccca acaataaaga ggagctacaa taataccaac caagaagatc ttttggtact
600 gtgggggatt caccatccta atgatgcggc agagcagaca aagctctatc
aaaacccaac 660 cacctatatt tccgttggga catcaacact aaaccagaga
ttggtaccaa gaatagctac 720 tagatccaaa gtaaacgggc aaagtggaag
gatggagttc ttctggacaa ttttaaagcc 780 gaatgatgca atcaacttcg
agagtaatgg aaatttcatt gctccagaat atgcatacaa 840 aattgtcaag
aaaggggact caacaattat gaaaagtgaa ttggaatatg gtaactgcaa 900
caccaagtgt caaactccaa tgggggcgat aaactctagc atgccattcc acaatataca
960 ccctctcacc attggggaat gccccaaata tgtgaaatca aacagattag
tccttgcgac 1020 tgggctcaga aatagccctc aaagagagac tcgaggatta
tttggagcta tagcaggttt 1080 tatagaggga ggatggcagg gaatggtaga
tggttggtat gggtaccacc atagcaatga 1140 gcaggggagt gggtacgctg
cagacaaaga atccactcaa aaggcaatag atggagtcac 1200 caataaggtc
aactcgatca ttgacaaaat gaacactcag tttgaggccg ttggaaggga 1260
atttaacaac ttagaaagga gaatagagaa tttaaacaag aagatggaag acgggttcct
1320 agatgtctgg acttataatg ctgaacttct ggttctcatg gaaaatgaga
gaactctaga 1380 ctttcatgac tcaaatgtca agaaccttta cgacaaggtc
cgactacagc ttagggataa 1440 tgcaaaggag ctgggtaacg gttgtttcga
gttctatcat aaatgtgata atgaatgtat 1500 ggaaagtgta agaaatggaa
cgtatgacta cccgcagtat tcagaagaag cgagactaaa 1560 aagagaggaa
ataagtggag taaaattgga atcaatagga atttaccaaa tactgtcaat 1620
ttattctaca gtggcgagtt ccctagcact ggcaatcatg gtagctggtc tatccttatg
1680 gatgtgctcc aatgggtcgt tacaatgcag aatttgcatt taaatttgtg
agttcagatt 1740 gtagttaaaa acacccttgt ttctact 1767 2 1398 DNA
Influenza A virus 2 agcaaaagca ggagttcaaa atgaatccaa atcagaagat
aataaccatc gggtcaatct 60 gtatggtaac tggaatagtt agcttaatgt
tacaaattgg gaacatgatc tcaatatggg 120 tcagtcattc aattcacaca
gggaatcaac accaatctga accaatcagc aatactaatt 180 ttcttactga
gaaagctgtg gcttcagtaa aattagcggg caattcatct ctttgcccca 240
ttaacggatg ggctgtatac agtaaggaca acagtataag gatcggttcc aagggggatg
300 tgtttgttat aagagagccg ttcatctcat gctcccactt ggaatgcaga
actttctttt 360 tgactcaggg agccttgctg aatgacaagc actccaatgg
gactgtcaaa gacagaagcc 420 ctcacagaac attaatgagt tgtcctgtgg
gtgaggctcc ctccccatat aactcaaggt 480 ttgagtctgt tgcttggtca
gcaagtgctt gccatgatgg caccagttgg ttgacgattg 540 gaatttctgg
cccagacaat ggggctgtgg ctgtattgaa atacaatggc ataataacag 600
acactatcaa gagttggagg aacaacatac tgagaactca agagtctgaa tgtgcatgtg
660 taaatggctc ttgctttact gtaatgactg acggaccaag taatggtcag
gcatcacata 720 agatcttcaa aatggaaaaa gggaaagtgg ttaaatcagt
cgaattggat gctcctaatt 780 atcactatga ggaatgctcc tgttatccta
atgccggaga aatcacatgt gtgtgcaggg 840 ataattggca tggctcaaat
cggccatggg tatctttcaa tcaaaatttg gagtatcaaa 900 taggatatat
atgcagtgga gttttcggag acaatccacg ccccaatgat ggaacaggta 960
gttgtggtcc ggtgtcctct aacggggcat atggggtaaa agggttttca tttaaatacg
1020 gcaatggtgt ctggatcggg agaaccaaaa gcactaattc caggagcggc
tttgaaatga 1080 tttgggatcc aaatgggtgg actgaaacgg acagtagctt
ttcagtgaaa caagatatcg 1140 tagcaataac tgattggtca ggatatagcg
ggagttttgt ccagcatcca gaactgacag 1200 gactagattg cataagacct
tgtttctggg ttgagttgat cagagggcgg cccaaagaga 1260 gcacaatttg
gactagtggg agcagcatat ctttttgtgg tgtaaatagt gacactgtgg 1320
gttggtcttg gccagacggt gctgagttgc cattcaccat tgacaagtag tttgttcaaa
1380 aaactccttg tttctact 1398 3 1767 DNA Influenza A virus 3
agcaaaagca ggggttcaat ctgtcaaaat ggagaaaata gtgcttcttt ttgcaatagt
60 cagtcttgtt aaaagtgatc agatttgcat tggttaccat gcaaacaact
cgacagagca 120 ggttgacaca ataatggaaa agaacgttac tgttacacat
gcccaagaca tactggaaaa 180 gacacacaac gggaagctct gcgatctaga
tggagtgaag cctctaattt tgagagattg 240 tagtgtagct ggatggctcc
tcggaaaccc aatgtgtgac gaattcatca atgtgccgga 300 atggtcttac
atagtggaga aggccaatcc agccaatgac ctctgttacc caggggattt 360
caacgactat gaagaattga aacacctatt gagcagaata aaccattttg agaaaattca
420 gatcatcccc aaaaattctt ggtccagtca tgaagcctca ttaggggtga
gctcagcatg 480 tccataccaa ggaaagtcct cctttttcag gaatgtggta
tggcttatca aaaagaacaa 540 tgcataccca acaataaaga ggagctacaa
taataccaac caagaagatc ttttggtatt 600 gtgggggatt caccatccta
atgatgcggc agagcagact aggctctatc aaaacccaac 660 cacctacatt
tccgttggga catcaacact aaaccagaga ttggtaccaa aaatagctac 720
tagatccaaa gtaaacgggc aaaatggaag gatggagttc ttctggacaa ttttaaaacc
780 gaatgatgca atcaacttcg agagcaatgg aaatttcatt gctccagaat
atgcatacaa 840 aattgtcaag aaaggggact cagcaattat gaaaagtgaa
ttggaatatg gtaactgcaa 900 caccaagtgt caaactccaa tgggggcgat
aaactctagt atgccattcc acaatataca 960 ccctctcacc atcggggaat
gccccaaata tgtgaaatca aacagattag tccttgcgac 1020 tgggctcaga
aatagccctc aaagagagac tcgaggatta tttggagcta tagcaggttt 1080
tatagaggga ggatggcagg gaatggtaga tggttggtat gggtaccacc atagcaatga
1140 gcaggggagt gggtacgctg cagacaaaga atccactcaa aaggcaatag
atggagtcac 1200 caataaggtc aactcgatca ttgacaaaat gaacactcag
tttgaggccg ttggaaggga 1260 atttaataac ttagaaagga gaatagagaa
tttaaacaag aagatggaag acggattcct 1320 agatgtctgg acttataatg
ctgaacttct ggttctcatg gaaaatgaga gaactctaga 1380 ctttcatgac
tcaaatgtca agaaccttta cgacaaggtc cgactacagc ttagggataa 1440
tgcaaaggag ctgggtaacg gttgtttcga gttctatcac aaatgtgata atgaatgtat
1500 ggaaagtgta agaaacggaa cgtatgacta cccgcagtat tcagaagaag
caagactaaa 1560 aagagaggaa ataagtggag taaaattgga gtcaatagga
acttaccaaa tactgtcaat 1620 ttattctaca gtggcgagtt ccctagcact
ggcaatcatg gtagctggtc tatctttatg 1680 gatgtgctcc aatgggtcgt
tacaatgcag aatttgcatt taaatttgtg agttcagatt 1740 gtagttaaaa
acacccttgt ttctact 1767 4 1458 DNA Influenza A virus 4 agcaaaagca
ggagttcaaa atgaatccaa atcagaagat aacaaccatt ggatcaatct 60
gtatggtaat tggaatagtt agcttgatgt tacaaattgg gaacataatc tcaatatggg
120 ttagtcattc aattcaaaca gggaatcaac accaggctga accatgcaat
caaagcatta 180 ttacttatga aaacaacacc tgggtaaacc agacatatgt
caacatcagc aataccaatt 240 ttcttactga gaaagctgtg gcttcagtaa
cattagcggg caattcatct ctttgcccca 300 ttagtggatg ggctgtatac
agtaaggaca acggtataag aatcggttcc aagggggatg 360 tgtttgttat
aagagagccg ttcatctcat gctcccactt ggaatgcaga actttctttt 420
tgactcaggg agccttgctg aatgacaagc attctaatgg gaccgtcaaa gacagaagcc
480 ctcacagaac attaatgagt tgtcccgtgg gtgaggctcc ttccccatac
aactcgaggt 540 ttgagtctgt tgcttggtcg gcaagtgctt gtcatgatgg
cactagttgg ttgacaattg 600 gaatttctgg cccagacaat ggggctgtgg
ctgtattgaa atacaatggc ataataacag 660 acactatcaa gagttggagg
aacaacataa tgagaactca agagtctgaa tgtgcatgtg 720 taaatggctc
ttgctttact gttatgactg atggaccaag taatgggcag gcttcataca 780
aaatcttcag aatagaaaaa gggaaagtag ttaaatcagc cgaattaaat gcccctaatt
840 atcactatga ggagtgctcc tgttatcctg atgctggaga aatcacatgt
gtgtgcaggg 900 ataactggca tggctcaaat cggccatggg tatctttcaa
tcaaaatttg gagtatcgaa 960 taggatatat atgcagtgga gttttcggag
acaatccacg ccccaatgat gggacaggca 1020 gttgtggtcc ggtgtcccct
aaaggggcat atggaataaa agggttctca tttaaatacg 1080 gcaatggtgt
ttggatcggg agaaccaaaa gcactaattc caggagcggc tttgaaatga 1140
tttgggatcc aaatggatgg actggtacgg acagtaattt ttcagtaaag caagatattg
1200 tagctataac cgattggtca ggatatagcg ggagttttgt ccagcatcca
gaactgacag 1260 gattagattg cataagacct tgtttctggg ttgagctaat
cagagggcgg cccaaagaga 1320 gcacaatttg gactagtggg agcagcatat
ccttttgtgg tgtaaatagt gacactgtgg 1380 gttggtcttg gccagacggt
gctgagttgc cattcaccat tgacaagtag tttgttcaaa 1440 aaactccttg
tttctact 1458 5 1767 DNA Influenza A virus 5 agcaaaagca ggggtataat
ctgtcaaaat ggagaaaata gtgcttcttc ttgcaacagt 60 cagccttgtt
aaaagtgacc agatttgcat tggttaccat gcaaacaact cgacagagca 120
agttgacaca ataatggaaa agaatgttac tgttacacat gcccaagaca tactggaaag
180 gacacacaac gggaagctct gcgatctaaa tggagtgaag cctctgattt
tgagggattg 240 tagtgtagct ggatggctcc tcggaaaccc tatgtgtgac
gaattcatca atgtgccgga 300 atggtcttac atagtggaga aggccagtcc
agccaatgac ctctgttatc cagggaattt 360 caacgactat gaagaactga
aacacctatt gagcagaata aaccattttg agaaaattca 420 gataatcccc
aaaagttctt ggtccaatca tgatgcctca tcaggggtga gctcagcatg 480
tccatacctt gggaggtcct cctttttcag aaatgtggta tggcttatca aaaagaacag
540 tagctaccca acaataaaga ggagctacaa taataccaac caagaagatc
ttttggtact 600 gtgggggatt caccatccta atgatgcggc agagcagaca
aggctctatc aaaacccaac 660 cacctacatt tccgttggaa catcaacact
gaaccagaga ttggttccag aaatagctac 720 tagacccaaa gtaaacgggc
aaagtggaag aatggagttc ttctggacaa ttttaaagcc 780 gaatgatgcc
atcaatttcg agagtaatgg aaatttcatt gctccagaat atgcatacaa 840
aattgtcaag aaaggggact caacaattat gaaaagtgaa ttggaatatg gtaactgcaa
900 caccaagtgt caaactccaa tgggggcaat aaactctagt atgccattcc
acaacataca 960 ccccctcacc atcggggaat gccccaaata tgtgaaatca
aacagattag tccttgcaac 1020 tggactcaga aatacccctc aacgagagac
gcgaggacta tttggagcta tagcaggttt 1080 tatagaggga ggatggcagg
gaatggtaga tggttggtat gggtaccacc atagcaatga 1140 gcaggggagt
ggatacgctg cagaccaaga atccacacaa aaggcaatag atggagtcac 1200
caataaggtc aactcgatca ttaacaaaat gaacactcag tttgaggccg ttggaaggga
1260 atttaataac ttggaaagga ggatagagaa tttaaacaag aaaatggaag
acggattcct 1320 agatgtctgg acttacaatg ccgaacttct ggttctcatg
gaaaatgaga gaactctaga 1380 ctttcatgac tcaaatgtca agaaccttta
cgacaaggtc cgactacagc ttagggataa 1440 tgcaaaggag ctgggtaatg
gttgtttcga attctatcac aaatgtgata acgaatgtat 1500 ggaaagtgta
aaaaacggaa cgtatgacta cccgcagtat tcagaagaag caagactaaa 1560
cagagaggaa ataagtggag taaaattgga atcaatggga acttaccaaa tactgtcaat
1620 ttattcaaca gtggcgagtt ccctagcact ggcaatcatg gtagctggtc
tatctttatg 1680 gatgtgctcc aatggatcgt tacaatgcag aatttgcatt
taaatttgtg agttcagatt 1740 gtagttaaaa acacccttgt ttctact 1767 6
1401 DNA Influenza A virus 6 agcaaaagca ggagtttaaa atgaatccaa
atcagaagat aataaccatt ggatcaatct 60 gcatggtagt tgggataatc
agcttgatgt tacaaattgg aaacacaata tcagtatggg 120 tcagccacat
aattaaaact tggcacccaa accagcctga accatgcaac caaagcatca 180
atttttacac tgagcaggct gcagcttcag tgacattagc gggcaattcc tctctctgcc
240 ctattagtgg atgggctata tacagcaagg acaatagtat aagaattggt
tccaaagggg 300 atgtgtttgt tataagagaa ccattcatct catgctccca
tttggaatgc agaacctttt 360 tcttgaccca aggagcccta ttgaatgaca
agcattctaa tgggaccgtc aaagacagga 420 gcccctatag aactttaatg
agctgtcctg ttggtgaggc cccttcccca tacaactcaa 480 ggtttgagtc
tgttgcttgg tcagcaagtg cttgccatga tggcattagt tggctaacaa 540
ttggaatttc cggtccggat aatggggctg tggctgtgtt gaaatacaat ggcataataa
600 cagacaccat caagagttgg aggaacaaca cactgaggac gcaagagtct
gaatgtgcat 660 gtgtgaatgg ttcttgtttt actgtaatga cagatggacc
gagtaatgaa caggcctcat 720 acaagatttt caagatagaa aaggggaggg
tagtcaaatc agttgagttg aacgccccta 780 attatcatta cgaggaatgc
tcctgttatc ctgatgctgg cgaaatcaca tgtgtgtgca 840 gggataattg
gcatggctcg aaccgaccat gggtgtcttt caatcagaat ctggagtatc 900
aaataggata tatatgcagt ggggttttcg gagacagtcc acgccccaat gatgggacag
960 gcagttgtgg tccagtgtct cttaacggag cgtatggagt aaaagggttt
tcatttaaat 1020 acggcaatgg tgtttggatc gggagaacca aaagcactag
ttccaggagc ggttttgaaa 1080 tgatttggga tccaaatggg tggaccgaaa
cagacagtag cttctcgttg aagcaagaca 1140 tcatagcgat aactgattgg
tcaggataca gcgggagttt tattcaacat ccagaactga 1200 caggattaaa
ttgcatgaga ccttgcttct gggttgaact aatcagaggg aggcccaaag 1260
agaaaacaat ctggactagt gggagcagta tatctttctg tggtgtaaat agtgacactg
1320 tgggttggtc ttggccagac ggtgctgagt tgccatacac cattgacaag
tagtttgttc 1380 aaaaaactcc ttgtttctac t 1401 7 1767 DNA Influenza A
virus 7 agcaaaagca ggggtataat ctgtcaaaat ggagaaaata gtgcttcttc
ttgcaacagt 60 cagccttgtt aaaagtgacc agatttgcat tggttaccat
gcaaacaact cgacagagca 120 agttgacaca ataatggaaa agaatgttac
tgttacacat gcccaagaca tactggaaag 180 gacacacaac gggaagctct
gcgatctaaa tggagtgaag cctctgattt tgagggattg 240 tagtgtagct
ggatggctcc tcggaaaccc tatgtgtgac gaattcatca atgtgccgga 300
atggtcttac atagtggaga aggccagtcc agccaatgac ctctgttatc cagggaattt
360 caacgactat gaagaactga aacacctatt gagcagaata aaccattttg
agaaaattca 420 gataatcccc aaaagttctt ggtccaatca tgatgcctca
tcaggggtga gctcagcatg 480 tccatacctt gggaggtcct cctttttcag
aaatgtggta tggcttatca aaaagaacag 540 tagctaccca acaataaaga
ggagctacaa taataccaac caagaagatc ttttggtact 600 gtgggggatt
caccatccta atgatgcggc agagcagaca aggctctatc aaaacccaac 660
cacctacatt tccgttggaa catcaacact gaaccagaga ttggtttcag aaatagctac
720 tagacccaaa gtaaacgggc aaagtggaag aatggagttc ttctggacaa
ttttaaagcc 780 gaatgatgcc atcaatttcg agagtaatgg aaatttcatt
gctccagaat atgcatacaa 840 aattgtcaag aaaggggact caacaattat
gaaaagtgaa ttggaatatg gtaactgcaa 900 caccaagtgt caaactccaa
tgggggcaat aaactctagt atgccattcc acaacataca 960 ccccctcacc
atcggggaat gccccaaata tgtgaaatca aacagattag tccttgcaac 1020
tggactcaga aatacccctc aacgagagac gcgaggacta tttggagcta tagcaggttt
1080 tatagaggga ggatggcagg gaatggtaga tggttggtat gggtaccacc
atagcaatga 1140 gcaggggagt ggatacgctg cagaccaaga atccacacaa
aaggcaatag atggagtcac 1200 caataaggtc aactcgatca ttaacaaaat
gaacactcag tttgaggccg ttggaaggga 1260 atttaataac ttggaaagga
ggatagagaa tttaaacaag aaaatggaag acggattcct 1320 agatgtctgg
acttacaatg ccgaacttct ggttctcatg gaaaatgaga gaactctaga 1380
ctttcatgac tcaaatgtca agaaccttta cgacaaggtc cgactacagc ttagggataa
1440 tgcaaaggag ctgggtaatg gttgtttcga attctatcac aaatgtgata
acgaatgtat 1500 ggaaagtgta aaaaacggaa cgtatgacta cccgcagtat
tcagaagaag caagactaaa 1560 cagagaggaa ataagtggag taaaattgga
atcaatggga acttaccaaa tactgtcaat 1620 ttattcaaca gtggcgagtt
ccctagcact ggcaatcatg gtagctggtc tatctttatg 1680 gatgtgctcc
aatggatcgt tacaatgcag aatttgcatt taaatttgtg agttcagatt 1740
gtagttaaaa acacccttgt ttctact 1767 8 1401 DNA Influenza A virus 8
agcaaaagca ggagtttaaa atgaatccaa atcagaagat aataaccatt ggatcaatct
60 gcatggtagt tgggataatc agcttgatgt tacaaattgg aaacacaata
tcagtatggg 120 tcagccacat aattaaaact tggcacccaa accagcctga
accatgcaac caaagcatca 180 atttttacac tgagcaggct gcagcttcag
tgacattagc gggcaattcc tctctctgcc 240 ctattagtgg atgggctata
tacagcaagg acaatagtat aagaattggt tccaaagggg 300 atgtgtttgt
tataagagaa ccattcatct catgctccca tttggaatgc agaacctttt 360
tcttgaccca aggagcccta ttgaatgaca agcattctaa tgggaccgtc aaagacagga
420 gcccctatag aactttaatg agctgtcctg ttggtgaggc cccttcccca
tacaactcaa 480 ggtttgagtc tgttgcttgg tcagcaagtg cttgccatga
tggcattagt tggctaacaa 540 ttggaatttc cggtccggat aatggggctg
tggctgtgtt gaaatacaat ggcataataa 600 cagacaccat caagagttgg
aggaacaaca cactgaggac gcaagagtct gaatgtgcat 660 gtgtgaatgg
ttcttgtttt actgtaatga cagatggacc gagtaatgaa caggcctcat 720
acaagatttt caagatagaa aaggggaggg tagtcaaatc agttgagttg aacgccccta
780 attatcatta cgaggaatgc tcctgttatc ctgatgctgg cgaaatcaca
tgtgtgtgca 840 gggataattg gcatggctcg aaccgaccat gggtgtcttt
caatcagaat ctggagtatc 900 aaataggata tatatgcagt ggggttttcg
gagacagtcc acgccccaat gatgggacag 960 gcagttgtgg tccagtgtct
cttaacggag cgtatggagt aaaagggttt tcatttaaat 1020 acggcaatgg
tgtttggatc gggagaacca aaagcactag ttccaggagc ggttttgaaa 1080
tgatttggga tccaaatggg tggaccgaaa cagacagtag cttctcgttg aagcaagaca
1140 tcatagcgat aactgattgg tcaggataca gcgggagttt tattcaacat
ccagaactga 1200 caggattaaa ttgcatgaga ccttgcttct gggttgaact
aatcagaggg aggcccaaag 1260 agaaaacaat ctggactagt gggagcagta
tatctttctg tggtgtaaat agtgacactg 1320 tgggttggtc ttggccagac
ggtgctgagt tgccatacac cattgacaag tagtttgttc 1380 aaaaaactcc
ttgtttctac t 1401 9 1690 DNA Influenza A virus 9 ttaaccactc
aagatggaag caataccact aataactata ctactagtag taacagcaag 60
caatgcagac aaaatctgca tcggctacca atcaacaaac tccacagaaa ccgtagacac
120 gctaacagaa aacaatgttc ctgtgacaca tgccaaagaa ttgctccaca
cagagcacaa 180 tgggatgctg tgtgcaacaa atctgggacg tcctcttatt
ctagacactt gcaccattga 240 aggactgatc tatggcaacc cttcttgtga
tctactgttg ggaggaagag aatggtccta 300 catcgtcgaa agaccatcgg
ctgttaatgg aatgtgttac cccgggaatg tagaaaacct 360 agaggaacta
aggtcatttt ttagttctgc tagttcctac caaagaatcc agatctttcc 420
agacacaatc tggaatgtgt cttacagtgg aacaagcaaa gcatgttcag attcattcta
480 caggagcatg agatggttga ctcaaaagaa caacgcttac cctattcaag
acgcccaata 540 cacaaataat agaggaaaga gcattctttt catgtggggc
ataaatcacc cacctaccga 600 tactgcacag acaaatctgt acacaaggac
tgacacaaca acaagtgtgg caacagaaga 660 tataaatagg accttcaaac
cagtgatagg gccaaggccc cttgtcaatg gtctgcaggg 720 aagaattgat
tattattggt cggtattgaa accaggtcag acattgcgag taagatccaa 780
tgggaatcta atcgctccat ggtatgggca cattctttca ggagagagcc acggaagaat
840 cctgaagact gatttaaaca gtggtagctg tgtagtgcaa tgtcaaacag
aaagaggtgg 900 cttaaatact actttgccat tccacaatgt cagtaaatat
gcatttggaa actgcccaaa 960 atatgttgga gtaaagagtc tcaaactggc
agttggtctg aggaatgtgc ctgctagatc 1020 aagtagagga ctatttgggg
ccatagctgg attcatagag ggaggttggt cagggctggt 1080 cgctggttgg
tatgggttcc agcattcaaa tgatcaaggg gttggtatag ctgcagatag 1140
agactcaact caaagggcaa ttgacaaaat aacgtccaaa gtgaataata tagtcgataa
1200 aatgaacaag caatatgaaa ttattgatca tgaattcagc gaggttgaaa
atagactcaa 1260 tatgatcaat aataagattg atgaccagat acaagacata
tgggcatata acgctgaatt 1320 gctagtgctg cttgaaaacc agaaaacact
cgatgagcat gatgcgaatg taaacaatct 1380 atataacaaa gtgaagaggg
cactgggttc caatgcaatg gaagatggga aaggatgttt 1440 cgagctatac
cataaatgtg atgatcagtg catggagaca attcggaacg ggacctataa 1500
caggaggaag tataaagagg aatcaagact agaaagacag aaaatagaag gggtcaagct
1560 ggaatctgaa ggaacttaca aaatcctcac catttattcg actgtcgcct
catctcttgt 1620 gattgcaatg gggtttgctg ccttcttgtt ctgggccatg
tccaatggat cttgcagatg 1680 caacatttga 1690 10 1428 DNA Influenza A
virus 10
aaatgaatcc aaatcagaag ataatagcaa ttggctctgt ttctctaact attgcgacaa
60 tatgcctcct catgcagatt gctatcttag caacgactat gacactacat
ttcaagcaga 120 atgaatgcat caactcctcg aataatcaag tagtgccatg
tgaaccaatc ataatagaaa 180 ggaacataac agagatagtg catttgaata
gtactacctt agagaaggaa atttgtccta 240 aagtagcaga ctacaggaat
tggtcaaaac cacaatgtca aatcacaggg ttcgctcctt 300 tctccaagga
caattcaatt aggctctccg caggtggaga tatttgggtg acaagagaac 360
cttatgtatc gtgcggtctt ggtaaatgtt atcaatttgc acttgggcag ggaaccactt
420 tggagaacaa acactcaaac ggcacagcac atgatagaac tcctcataga
acccttttaa 480 tgaatgagtt gggtgttccg tttcatttgg caaccaaaca
agtgtgcata gcatggtcca 540 gctcaagctg ccatgatggg aaagcatggt
tacatgtttg tgtcactggg gatgatagaa 600 atgcaacggc tagcatcatt
tatgatggga tacttgttga cagtattggt tcatggtcta 660 aaaacatcct
cagaactcag gagtcagaat gcgtttgcat caatggaacc tgtgcagtag 720
taatgactga tggaagtgca tcaggaaggg ctgacactag aatactattt attagagagg
780 ggaaaattgc acacattagc ccattgtcag gaagtgctca gcatgtggag
gaatgctcct 840 gttacccccg atatccagaa gttagatgtg tttgcagaga
caattggaag ggatccaata 900 ggcccgttct atatataaat atggcaaatt
atagtattga ttccagttat gtgtgctcag 960 gacttgttgg cgacacacca
agaaatgatg ataggtctag cagcagcaac tgcagagatc 1020 ctaataacga
gagaggggcc ccaggagtaa aagggtgggc ctttgacaat ggaaatgaca 1080
tttggatggg aagaacaatc aaaaaggatt cgcgctcagg ttatgagact ttcagggtca
1140 ttggtggttg gaccactgct aattccaagt cacagataaa tagacaagtc
atagttgaca 1200 gtgataactc gtctgggtat tctggtatct tctctgttga
aggcaaaagc tgcatcaaca 1260 ggtgttttta cgtggagttg ataagaggaa
gaccaaagga gactagggtg tggtggactt 1320 caaatagcat cattgtattt
tgtggaactt caggtaccta tggaacaggc tcatggcctg 1380 atggggcgaa
tatcaatttc atgcctatat aagctttcgc aattttag 1428 11 564 PRT Influenza
A virus 11 Met Glu Lys Ile Val Leu Leu Phe Ala Ile Val Ser Leu Val
Lys Ser 1 5 10 15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser
Thr Glu Gln Val 20 25 30 Asp Thr Ile Met Glu Lys Asn Val Thr Val
Thr His Ala Gln Asp Ile 35 40 45 Leu Glu Lys Lys His Asn Gly Lys
Leu Cys Asp Leu Asp Gly Val Lys 50 55 60 Pro Leu Ile Leu Arg Asp
Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 65 70 75 80 Pro Met Cys Asp
Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val 85 90 95 Glu Lys
Ala Asn Pro Val Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn 100 105 110
Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu 115
120 125 Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Ser His Glu Ala
Ser 130 135 140 Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Lys Ser
Ser Phe Phe 145 150 155 160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn
Ser Thr Tyr Pro Thr Ile 165 170 175 Lys Arg Ser Tyr Asn Asn Thr Asn
Gln Glu Asp Leu Leu Val Leu Trp 180 185 190 Gly Ile His His Pro Asn
Asp Ala Ala Glu Gln Thr Lys Leu Tyr Gln 195 200 205 Asn Pro Thr Thr
Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg 210 215 220 Leu Val
Pro Arg Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 225 230 235
240 Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn
245 250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr
Lys Ile 260 265 270 Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu
Leu Glu Tyr Gly 275 280 285 Asn Cys Asn Thr Lys Cys Gln Thr Pro Met
Gly Ala Ile Asn Ser Ser 290 295 300 Met Pro Phe His Asn Ile His Pro
Leu Thr Ile Gly Glu Cys Pro Lys 305 310 315 320 Tyr Val Lys Ser Asn
Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser 325 330 335 Pro Gln Arg
Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile 340 345 350 Glu
Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His 355 360
365 Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln
370 375 380 Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile
Asp Lys 385 390 395 400 Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu
Phe Asn Asn Leu Glu 405 410 415 Arg Arg Ile Glu Asn Leu Asn Lys Lys
Met Glu Asp Gly Phe Leu Asp 420 425 430 Val Trp Thr Tyr Asn Ala Glu
Leu Leu Val Leu Met Glu Asn Glu Arg 435 440 445 Thr Leu Asp Phe His
Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val 450 455 460 Arg Leu Gln
Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe 465 470 475 480
Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn 485
490 495 Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys
Arg 500 505 510 Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Ile
Tyr Gln Ile 515 520 525 Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu
Ala Leu Ala Ile Met 530 535 540 Val Ala Gly Leu Ser Leu Trp Met Cys
Ser Asn Gly Ser Leu Gln Cys 545 550 555 560 Arg Ile Cys Ile 12 449
PRT Influenza A virus 12 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile
Gly Ser Ile Cys Met Val 1 5 10 15 Thr Gly Ile Val Ser Leu Met Leu
Gln Ile Gly Asn Met Ile Ser Ile 20 25 30 Trp Val Ser His Ser Ile
His Thr Gly Asn Gln His Gln Ser Glu Pro 35 40 45 Ile Ser Asn Thr
Asn Phe Leu Thr Glu Lys Ala Val Ala Ser Val Lys 50 55 60 Leu Ala
Gly Asn Ser Ser Leu Cys Pro Ile Asn Gly Trp Ala Val Tyr 65 70 75 80
Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val Phe Val 85
90 95 Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr
Phe 100 105 110 Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His Ser
Asn Gly Thr 115 120 125 Val Lys Asp Arg Ser Pro His Arg Thr Leu Met
Ser Cys Pro Val Gly 130 135 140 Glu Ala Pro Ser Pro Tyr Asn Ser Arg
Phe Glu Ser Val Ala Trp Ser 145 150 155 160 Ala Ser Ala Cys His Asp
Gly Thr Ser Trp Leu Thr Ile Gly Ile Ser 165 170 175 Gly Pro Asp Asn
Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Ile 180 185 190 Thr Asp
Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu Arg Thr Gln Glu 195 200 205
Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr Val Met Thr Asp 210
215 220 Gly Pro Ser Asn Gly Gln Ala Ser His Lys Ile Phe Lys Met Glu
Lys 225 230 235 240 Gly Lys Val Val Lys Ser Val Glu Leu Asp Ala Pro
Asn Tyr His Tyr 245 250 255 Glu Glu Cys Ser Cys Tyr Pro Asn Ala Gly
Glu Ile Thr Cys Val Cys 260 265 270 Arg Asp Asn Trp His Gly Ser Asn
Arg Pro Trp Val Ser Phe Asn Gln 275 280 285 Asn Leu Glu Tyr Gln Ile
Gly Tyr Ile Cys Ser Gly Val Phe Gly Asp 290 295 300 Asn Pro Arg Pro
Asn Asp Gly Thr Gly Ser Cys Gly Pro Val Ser Ser 305 310 315 320 Asn
Gly Ala Tyr Gly Val Lys Gly Phe Ser Phe Lys Tyr Gly Asn Gly 325 330
335 Val Trp Ile Gly Arg Thr Lys Ser Thr Asn Ser Arg Ser Gly Phe Glu
340 345 350 Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp Ser Ser
Phe Ser 355 360 365 Val Lys Gln Asp Ile Val Ala Ile Thr Asp Trp Ser
Gly Tyr Ser Gly 370 375 380 Ser Phe Val Gln His Pro Glu Leu Thr Gly
Leu Asp Cys Ile Arg Pro 385 390 395 400 Cys Phe Trp Val Glu Leu Ile
Arg Gly Arg Pro Lys Glu Ser Thr Ile 405 410 415 Trp Thr Ser Gly Ser
Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr 420 425 430 Val Gly Trp
Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe Thr Ile Asp 435 440 445 Lys
13 564 PRT Influenza A virus 13 Met Glu Lys Ile Val Leu Leu Phe Ala
Ile Val Ser Leu Val Lys Ser 1 5 10 15 Asp Gln Ile Cys Ile Gly Tyr
His Ala Asn Asn Ser Thr Glu Gln Val 20 25 30 Asp Thr Ile Met Glu
Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 35 40 45 Leu Glu Lys
Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50 55 60 Pro
Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 65 70
75 80 Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile
Val 85 90 95 Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly
Asp Phe Asn 100 105 110 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg
Ile Asn His Phe Glu 115 120 125 Lys Ile Gln Ile Ile Pro Lys Asn Ser
Trp Ser Ser His Glu Ala Ser 130 135 140 Leu Gly Val Ser Ser Ala Cys
Pro Tyr Gln Gly Lys Ser Ser Phe Phe 145 150 155 160 Arg Asn Val Val
Trp Leu Ile Lys Lys Asn Asn Ala Tyr Pro Thr Ile 165 170 175 Lys Arg
Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180 185 190
Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Arg Leu Tyr Gln 195
200 205 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln
Arg 210 215 220 Leu Val Pro Lys Ile Ala Thr Arg Ser Lys Val Asn Gly
Gln Asn Gly 225 230 235 240 Arg Met Glu Phe Phe Trp Thr Ile Leu Lys
Pro Asn Asp Ala Ile Asn 245 250 255 Phe Glu Ser Asn Gly Asn Phe Ile
Ala Pro Glu Tyr Ala Tyr Lys Ile 260 265 270 Val Lys Lys Gly Asp Ser
Ala Ile Met Lys Ser Glu Leu Glu Tyr Gly 275 280 285 Asn Cys Asn Thr
Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser 290 295 300 Met Pro
Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310 315
320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser
325 330 335 Pro Gln Arg Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly
Phe Ile 340 345 350 Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr
Gly Tyr His His 355 360 365 Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala
Asp Lys Glu Ser Thr Gln 370 375 380 Lys Ala Ile Asp Gly Val Thr Asn
Lys Val Asn Ser Ile Ile Asp Lys 385 390 395 400 Met Asn Thr Gln Phe
Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu 405 410 415 Arg Arg Ile
Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp 420 425 430 Val
Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg 435 440
445 Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val
450 455 460 Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly
Cys Phe 465 470 475 480 Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met
Glu Ser Val Arg Asn 485 490 495 Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser
Glu Glu Ala Arg Leu Lys Arg 500 505 510 Glu Glu Ile Ser Gly Val Lys
Leu Glu Ser Ile Gly Thr Tyr Gln Ile 515 520 525 Leu Ser Ile Tyr Ser
Thr Val Ala Ser Ser Leu Ala Leu Ala Ile Met 530 535 540 Val Ala Gly
Leu Ser Leu Trp Met Cys Ser Asn Gly Ser Leu Gln Cys 545 550 555 560
Arg Ile Cys Ile 14 469 PRT Influenza A virus 14 Met Asn Pro Asn Gln
Lys Ile Thr Thr Ile Gly Ser Ile Cys Met Val 1 5 10 15 Ile Gly Ile
Val Ser Leu Met Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30 Trp
Val Ser His Ser Ile Gln Thr Gly Asn Gln His Gln Ala Glu Pro 35 40
45 Cys Asn Gln Ser Ile Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gln
50 55 60 Thr Tyr Val Asn Ile Ser Asn Thr Asn Phe Leu Thr Glu Lys
Ala Val 65 70 75 80 Ala Ser Val Thr Leu Ala Gly Asn Ser Ser Leu Cys
Pro Ile Ser Gly 85 90 95 Trp Ala Val Tyr Ser Lys Asp Asn Gly Ile
Arg Ile Gly Ser Lys Gly 100 105 110 Asp Val Phe Val Ile Arg Glu Pro
Phe Ile Ser Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe Phe Leu
Thr Gln Gly Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn Gly Thr
Val Lys Asp Arg Ser Pro His Arg Thr Leu Met Ser 145 150 155 160 Cys
Pro Val Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170
175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Thr Ser Trp Leu Thr
180 185 190 Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu
Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg
Asn Asn Ile Met 210 215 220 Arg Thr Gln Glu Ser Glu Cys Ala Cys Val
Asn Gly Ser Cys Phe Thr 225 230 235 240 Val Met Thr Asp Gly Pro Ser
Asn Gly Gln Ala Ser Tyr Lys Ile Phe 245 250 255 Arg Ile Glu Lys Gly
Lys Val Val Lys Ser Ala Glu Leu Asn Ala Pro 260 265 270 Asn Tyr His
Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ala Gly Glu Ile 275 280 285 Thr
Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val 290 295
300 Ser Phe Asn Gln Asn Leu Glu Tyr Arg Ile Gly Tyr Ile Cys Ser Gly
305 310 315 320 Val Phe Gly Asp Asn Pro Arg Pro Asn Asp Gly Thr Gly
Ser Cys Gly 325 330 335 Pro Val Ser Pro Lys Gly Ala Tyr Gly Ile Lys
Gly Phe Ser Phe Lys 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg
Thr Lys Ser Thr Asn Ser Arg 355 360 365 Ser Gly Phe Glu Met Ile Trp
Asp Pro Asn Gly Trp Thr Gly Thr Asp 370 375 380 Ser Asn Phe Ser Val
Lys Gln Asp Ile Val Ala Ile Thr Asp Trp Ser 385 390 395 400 Gly Tyr
Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp 405 410 415
Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys 420
425 430 Glu Ser Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly
Val 435 440 445 Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala
Glu Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465 15 564 PRT
Influenza A virus 15 Met Glu Lys Ile Val Leu Leu Leu Ala Thr Val
Ser Leu Val Lys Ser 1 5 10 15 Asp Gln Ile Cys Ile Gly Tyr His Ala
Asn Asn Ser Thr Glu Gln Val 20 25 30 Asp Thr Ile Met Glu Lys Asn
Val Thr Val Thr His Ala Gln Asp Ile 35 40 45 Leu Glu Arg Thr His
Asn Gly Lys Leu Cys Asp Leu Asn Gly Val Lys 50 55 60 Pro Leu Ile
Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 65 70 75 80 Pro
Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val 85 90
95 Glu Lys Ala Ser Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn
100 105 110 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His
Phe Glu 115 120 125 Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asn
His Asp Ala Ser 130 135 140 Ser Gly Val Ser Ser Ala Cys Pro Tyr Leu
Gly Arg Ser Ser Phe Phe 145 150 155
160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Ser Tyr Pro Thr Ile
165 170 175 Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val
Leu Trp 180 185 190 Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr
Arg Leu Tyr Gln 195 200 205 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr
Ser Thr Leu Asn Gln Arg 210 215 220 Leu Val Pro Glu Ile Ala Thr Arg
Pro Lys Val Asn Gly Gln Ser Gly 225 230 235 240 Arg Met Glu Phe Phe
Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn 245 250 255 Phe Glu Ser
Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile 260 265 270 Val
Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly 275 280
285 Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser
290 295 300 Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys
Pro Lys 305 310 315 320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr
Gly Leu Arg Asn Thr 325 330 335 Pro Gln Arg Glu Thr Arg Gly Leu Phe
Gly Ala Ile Ala Gly Phe Ile 340 345 350 Glu Gly Gly Trp Gln Gly Met
Val Asp Gly Trp Tyr Gly Tyr His His 355 360 365 Ser Asn Glu Gln Gly
Ser Gly Tyr Ala Ala Asp Gln Glu Ser Thr Gln 370 375 380 Lys Ala Ile
Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asn Lys 385 390 395 400
Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu 405
410 415 Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu
Asp 420 425 430 Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu
Asn Glu Arg 435 440 445 Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn
Leu Tyr Asp Lys Val 450 455 460 Arg Leu Gln Leu Arg Asp Asn Ala Lys
Glu Leu Gly Asn Gly Cys Phe 465 470 475 480 Glu Phe Tyr His Lys Cys
Asp Asn Glu Cys Met Glu Ser Val Lys Asn 485 490 495 Gly Thr Tyr Asp
Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Asn Arg 500 505 510 Glu Glu
Ile Ser Gly Val Lys Leu Glu Ser Met Gly Thr Tyr Gln Ile 515 520 525
Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu Ala Ile Met 530
535 540 Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser Leu Gln
Cys 545 550 555 560 Arg Ile Cys Ile 16 450 PRT Influenza A virus 16
Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Val 1 5
10 15 Val Gly Ile Ile Ser Leu Met Leu Gln Ile Gly Asn Thr Ile Ser
Val 20 25 30 Trp Val Ser His Ile Ile Lys Thr Trp His Pro Asn Gln
Pro Glu Pro 35 40 45 Cys Asn Gln Ser Ile Asn Phe Tyr Thr Glu Gln
Ala Ala Ala Ser Val 50 55 60 Thr Leu Ala Gly Asn Ser Ser Leu Cys
Pro Ile Ser Gly Trp Ala Ile 65 70 75 80 Tyr Ser Lys Asp Asn Ser Ile
Arg Ile Gly Ser Lys Gly Asp Val Phe 85 90 95 Val Ile Arg Glu Pro
Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr 100 105 110 Phe Phe Leu
Thr Gln Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly 115 120 125 Thr
Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser Cys Pro Val 130 135
140 Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser Val Ala Trp
145 150 155 160 Ser Ala Ser Ala Cys His Asp Gly Ile Ser Trp Leu Thr
Ile Gly Ile 165 170 175 Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu
Lys Tyr Asn Gly Ile 180 185 190 Ile Thr Asp Thr Ile Lys Ser Trp Arg
Asn Asn Thr Leu Arg Thr Gln 195 200 205 Glu Ser Glu Cys Ala Cys Val
Asn Gly Ser Cys Phe Thr Val Met Thr 210 215 220 Asp Gly Pro Ser Asn
Glu Gln Ala Ser Tyr Lys Ile Phe Lys Ile Glu 225 230 235 240 Lys Gly
Arg Val Val Lys Ser Val Glu Leu Asn Ala Pro Asn Tyr His 245 250 255
Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ala Gly Glu Ile Thr Cys Val 260
265 270 Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe
Asn 275 280 285 Gln Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly
Val Phe Gly 290 295 300 Asp Ser Pro Arg Pro Asn Asp Gly Thr Gly Ser
Cys Gly Pro Val Ser 305 310 315 320 Leu Asn Gly Ala Tyr Gly Val Lys
Gly Phe Ser Phe Lys Tyr Gly Asn 325 330 335 Gly Val Trp Ile Gly Arg
Thr Lys Ser Thr Ser Ser Arg Ser Gly Phe 340 345 350 Glu Met Ile Trp
Asp Pro Asn Gly Trp Thr Glu Thr Asp Ser Ser Phe 355 360 365 Ser Leu
Lys Gln Asp Ile Ile Ala Ile Thr Asp Trp Ser Gly Tyr Ser 370 375 380
Gly Ser Phe Ile Gln His Pro Glu Leu Thr Gly Leu Asn Cys Met Arg 385
390 395 400 Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys Glu
Lys Thr 405 410 415 Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly
Val Asn Ser Asp 420 425 430 Thr Val Gly Trp Ser Trp Pro Asp Gly Ala
Glu Leu Pro Tyr Thr Ile 435 440 445 Asp Lys 450 17 564 PRT
Influenza A virus 17 Met Glu Lys Ile Val Leu Leu Leu Ala Thr Val
Ser Leu Val Lys Ser 1 5 10 15 Asp Gln Ile Cys Ile Gly Tyr His Ala
Asn Asn Ser Thr Glu Gln Val 20 25 30 Asp Thr Ile Met Glu Lys Asn
Val Thr Val Thr His Ala Gln Asp Ile 35 40 45 Leu Glu Arg Thr His
Asn Gly Lys Leu Cys Asp Leu Asn Gly Val Lys 50 55 60 Pro Leu Ile
Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 65 70 75 80 Pro
Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val 85 90
95 Glu Lys Ala Ser Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Phe Asn
100 105 110 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His
Phe Glu 115 120 125 Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asn
His Asp Ala Ser 130 135 140 Ser Gly Val Ser Ser Ala Cys Pro Tyr Leu
Gly Arg Ser Ser Phe Phe 145 150 155 160 Arg Asn Val Val Trp Leu Ile
Lys Lys Asn Ser Ser Tyr Pro Thr Ile 165 170 175 Lys Arg Ser Tyr Asn
Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180 185 190 Gly Ile His
His Pro Asn Asp Ala Ala Glu Gln Thr Arg Leu Tyr Gln 195 200 205 Asn
Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg 210 215
220 Leu Val Ser Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Gln Ser Gly
225 230 235 240 Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp
Ala Ile Asn 245 250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu
Tyr Ala Tyr Lys Ile 260 265 270 Val Lys Lys Gly Asp Ser Thr Ile Met
Lys Ser Glu Leu Glu Tyr Gly 275 280 285 Asn Cys Asn Thr Lys Cys Gln
Thr Pro Met Gly Ala Ile Asn Ser Ser 290 295 300 Met Pro Phe His Asn
Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310 315 320 Tyr Val
Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Thr 325 330 335
Pro Gln Arg Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile 340
345 350 Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His
His 355 360 365 Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Glu
Ser Thr Gln 370 375 380 Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn
Ser Ile Ile Asn Lys 385 390 395 400 Met Asn Thr Gln Phe Glu Ala Val
Gly Arg Glu Phe Asn Asn Leu Glu 405 410 415 Arg Arg Ile Glu Asn Leu
Asn Lys Lys Met Glu Asp Gly Phe Leu Asp 420 425 430 Val Trp Thr Tyr
Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg 435 440 445 Thr Leu
Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val 450 455 460
Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys Phe 465
470 475 480 Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val
Lys Asn 485 490 495 Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala
Arg Leu Asn Arg 500 505 510 Glu Glu Ile Ser Gly Val Lys Leu Glu Ser
Met Gly Thr Tyr Gln Ile 515 520 525 Leu Ser Ile Tyr Ser Thr Val Ala
Ser Ser Leu Ala Leu Ala Ile Met 530 535 540 Val Ala Gly Leu Ser Leu
Trp Met Cys Ser Asn Gly Ser Leu Gln Cys 545 550 555 560 Arg Ile Cys
Ile 18 450 PRT Influenza A virus 18 Met Asn Pro Asn Gln Lys Ile Ile
Thr Ile Gly Ser Ile Cys Met Val 1 5 10 15 Val Gly Ile Ile Ser Leu
Met Leu Gln Ile Gly Asn Thr Ile Ser Val 20 25 30 Trp Val Ser His
Ile Ile Lys Thr Trp His Pro Asn Gln Pro Glu Pro 35 40 45 Cys Asn
Gln Ser Ile Asn Phe Tyr Thr Glu Gln Ala Ala Ala Ser Val 50 55 60
Thr Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Ser Gly Trp Ala Ile 65
70 75 80 Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp
Val Phe 85 90 95 Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu
Glu Cys Arg Thr 100 105 110 Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn
Asp Lys His Ser Asn Gly 115 120 125 Thr Val Lys Asp Arg Ser Pro Tyr
Arg Thr Leu Met Ser Cys Pro Val 130 135 140 Gly Glu Ala Pro Ser Pro
Tyr Asn Ser Arg Phe Glu Ser Val Ala Trp 145 150 155 160 Ser Ala Ser
Ala Cys His Asp Gly Ile Ser Trp Leu Thr Ile Gly Ile 165 170 175 Ser
Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile 180 185
190 Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Thr Leu Arg Thr Gln
195 200 205 Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr Val
Met Thr 210 215 220 Asp Gly Pro Ser Asn Glu Gln Ala Ser Tyr Lys Ile
Phe Lys Ile Glu 225 230 235 240 Lys Gly Arg Val Val Lys Ser Val Glu
Leu Asn Ala Pro Asn Tyr His 245 250 255 Tyr Glu Glu Cys Ser Cys Tyr
Pro Asp Ala Gly Glu Ile Thr Cys Val 260 265 270 Cys Arg Asp Asn Trp
His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn 275 280 285 Gln Asn Leu
Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly 290 295 300 Asp
Ser Pro Arg Pro Asn Asp Gly Thr Gly Ser Cys Gly Pro Val Ser 305 310
315 320 Leu Asn Gly Ala Tyr Gly Val Lys Gly Phe Ser Phe Lys Tyr Gly
Asn 325 330 335 Gly Val Trp Ile Gly Arg Thr Lys Ser Thr Ser Ser Arg
Ser Gly Phe 340 345 350 Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu
Thr Asp Ser Ser Phe 355 360 365 Ser Leu Lys Gln Asp Ile Ile Ala Ile
Thr Asp Trp Ser Gly Tyr Ser 370 375 380 Gly Ser Phe Ile Gln His Pro
Glu Leu Thr Gly Leu Asn Cys Met Arg 385 390 395 400 Pro Cys Phe Trp
Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Lys Thr 405 410 415 Ile Trp
Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp 420 425 430
Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Tyr Thr Ile 435
440 445 Asp Lys 450 19 558 PRT Influenza A virus 19 Met Glu Ala Ile
Pro Leu Ile Thr Ile Leu Leu Val Val Thr Ala Ser 1 5 10 15 Asn Ala
Asp Lys Ile Cys Ile Gly Tyr Gln Ser Thr Asn Ser Thr Glu 20 25 30
Thr Val Asp Thr Leu Thr Glu Asn Asn Val Pro Val Thr His Ala Lys 35
40 45 Glu Leu Leu His Thr Glu His Asn Gly Met Leu Cys Ala Thr Asn
Leu 50 55 60 Gly Arg Pro Leu Ile Leu Asp Thr Cys Thr Ile Glu Gly
Leu Ile Tyr 65 70 75 80 Gly Asn Pro Ser Cys Asp Leu Leu Leu Gly Gly
Arg Glu Trp Ser Tyr 85 90 95 Ile Val Glu Arg Pro Ser Ala Val Asn
Gly Met Cys Tyr Pro Gly Asn 100 105 110 Val Glu Asn Leu Glu Glu Leu
Arg Ser Phe Phe Ser Ser Ala Ser Ser 115 120 125 Tyr Gln Arg Ile Gln
Ile Phe Pro Asp Thr Ile Trp Asn Val Ser Tyr 130 135 140 Ser Gly Thr
Ser Lys Ala Cys Ser Asp Ser Phe Tyr Arg Ser Met Arg 145 150 155 160
Trp Leu Thr Gln Lys Asn Asn Ala Tyr Pro Ile Gln Asp Ala Gln Tyr 165
170 175 Thr Asn Asn Arg Gly Lys Ser Ile Leu Phe Met Trp Gly Ile Asn
His 180 185 190 Pro Pro Thr Asp Thr Ala Gln Thr Asn Leu Tyr Thr Arg
Thr Asp Thr 195 200 205 Thr Thr Ser Val Ala Thr Glu Asp Ile Asn Arg
Thr Phe Lys Pro Val 210 215 220 Ile Gly Pro Arg Pro Leu Val Asn Gly
Leu Gln Gly Arg Ile Asp Tyr 225 230 235 240 Tyr Trp Ser Val Leu Lys
Pro Gly Gln Thr Leu Arg Val Arg Ser Asn 245 250 255 Gly Asn Leu Ile
Ala Pro Trp Tyr Gly His Ile Leu Ser Gly Glu Ser 260 265 270 His Gly
Arg Ile Leu Lys Thr Asp Leu Asn Ser Gly Ser Cys Val Val 275 280 285
Gln Cys Gln Thr Glu Arg Gly Gly Leu Asn Thr Thr Leu Pro Phe His 290
295 300 Asn Val Ser Lys Tyr Ala Phe Gly Asn Cys Pro Lys Tyr Val Gly
Val 305 310 315 320 Lys Ser Leu Lys Leu Ala Val Gly Leu Arg Asn Val
Pro Ala Arg Ser 325 330 335 Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly
Phe Ile Glu Gly Gly Trp 340 345 350 Ser Gly Leu Val Ala Gly Trp Tyr
Gly Phe Gln His Ser Asn Asp Gln 355 360 365 Gly Val Gly Ile Ala Ala
Asp Arg Asp Ser Thr Gln Arg Ala Ile Asp 370 375 380 Lys Ile Thr Ser
Lys Val Asn Asn Ile Val Asp Lys Met Asn Lys Gln 385 390 395 400 Tyr
Glu Ile Ile Asp His Glu Phe Ser Glu Val Glu Asn Arg Leu Asn 405 410
415 Met Ile Asn Asn Lys Ile Asp Asp Gln Ile Gln Asp Ile Trp Ala Tyr
420 425 430 Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Gln Lys Thr Leu
Asp Glu 435 440 445 His Asp Ala Asn Val Asn Asn Leu Tyr Asn Lys Val
Lys Arg Ala Leu 450 455 460 Gly Ser Asn Ala Met Glu Asp Gly Lys Gly
Cys Phe Glu Leu Tyr His 465 470 475 480 Lys Cys Asp Asp Gln Cys Met
Glu Thr Ile Arg Asn Gly Thr Tyr Asn 485 490 495 Arg Arg Lys Tyr Lys
Glu Glu Ser Arg Leu Glu Arg Gln Lys Ile Glu 500 505 510 Gly Val Lys
Leu Glu Ser Glu Gly Thr Tyr Lys Ile Leu Thr Ile Tyr 515 520 525 Ser
Thr Val Ala Ser Ser Leu Val Ile Ala Met Gly Phe Ala Ala Phe 530 535
540 Leu Phe Trp Ala Met Ser Asn Gly Ser Cys Arg Cys Asn Ile 545 550
555 20 469 PRT Influenza A virus 20 Met Asn Pro Asn Gln Lys Ile Ile
Ala Ile Gly Ser Val Ser Leu
Thr 1 5 10 15 Ile Ala Thr Ile Cys Leu Leu Met Gln Ile Ala Ile Leu
Ala Thr Thr 20 25 30 Met Thr Leu His Phe Lys Gln Asn Glu Cys Ile
Asn Ser Ser Asn Asn 35 40 45 Gln Val Val Pro Cys Glu Pro Ile Ile
Ile Glu Arg Asn Ile Thr Glu 50 55 60 Ile Val His Leu Asn Ser Thr
Thr Leu Glu Lys Glu Ile Cys Pro Lys 65 70 75 80 Val Ala Asp Tyr Arg
Asn Trp Ser Lys Pro Gln Cys Gln Ile Thr Gly 85 90 95 Phe Ala Pro
Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly 100 105 110 Asp
Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Gly Leu Gly Lys 115 120
125 Cys Tyr Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Glu Asn Lys His
130 135 140 Ser Asn Gly Thr Ala His Asp Arg Thr Pro His Arg Thr Leu
Leu Met 145 150 155 160 Asn Glu Leu Gly Val Pro Phe His Leu Ala Thr
Lys Gln Val Cys Ile 165 170 175 Ala Trp Ser Ser Ser Ser Cys His Asp
Gly Lys Ala Trp Leu His Val 180 185 190 Cys Val Thr Gly Asp Asp Arg
Asn Ala Thr Ala Ser Ile Ile Tyr Asp 195 200 205 Gly Ile Leu Val Asp
Ser Ile Gly Ser Trp Ser Lys Asn Ile Leu Arg 210 215 220 Thr Gln Glu
Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Ala Val Val 225 230 235 240
Met Thr Asp Gly Ser Ala Ser Gly Arg Ala Asp Thr Arg Ile Leu Phe 245
250 255 Ile Arg Glu Gly Lys Ile Ala His Ile Ser Pro Leu Ser Gly Ser
Ala 260 265 270 Gln His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro
Glu Val Arg 275 280 285 Cys Val Cys Arg Asp Asn Trp Lys Gly Ser Asn
Arg Pro Val Leu Tyr 290 295 300 Ile Asn Met Ala Asn Tyr Ser Ile Asp
Ser Ser Tyr Val Cys Ser Gly 305 310 315 320 Leu Val Gly Asp Thr Pro
Arg Asn Asp Asp Arg Ser Ser Ser Ser Asn 325 330 335 Cys Arg Asp Pro
Asn Asn Glu Arg Gly Ala Pro Gly Val Lys Gly Trp 340 345 350 Ala Phe
Asp Asn Gly Asn Asp Ile Trp Met Gly Arg Thr Ile Lys Lys 355 360 365
Asp Ser Arg Ser Gly Tyr Glu Thr Phe Arg Val Ile Gly Gly Trp Thr 370
375 380 Thr Ala Asn Ser Lys Ser Gln Ile Asn Arg Gln Val Ile Val Asp
Ser 385 390 395 400 Asp Asn Ser Ser Gly Tyr Ser Gly Ile Phe Ser Val
Glu Gly Lys Ser 405 410 415 Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu
Ile Arg Gly Arg Pro Lys 420 425 430 Glu Thr Arg Val Trp Trp Thr Ser
Asn Ser Ile Ile Val Phe Cys Gly 435 440 445 Thr Ser Gly Thr Tyr Gly
Thr Gly Ser Trp Pro Asp Gly Ala Asn Ile 450 455 460 Asn Phe Met Pro
Ile 465 21 1737 DNA Influenza A virus 21 agcaaaagca ggggatacaa
aatgaacact caaatcctgg tattcgctct ggtggcgagc 60 attccgacaa
atgcagacaa gatctgcctt gggcatcatg ccgtgtcaaa cgggactaaa 120
gtaaacacat taactgagag aggagtggaa gtcgttaatg caactgaaac ggtggaacga
180 acaaacgttc ccaggatctg ctcaaaaggg aaaaggacag ttgacctcgg
tcaatgtgga 240 cttctgggaa caatcactgg gccaccccaa tgtgaccaat
tcctagaatt ttcggccgac 300 ttaattattg agaggcgaga aggaagtgat
gtctgttatc ctgggaaatt cgtgaatgaa 360 gaagctctga ggcaaattct
cagagagtca ggcggaattg acaaggagac aatgggattc 420 acctacagcg
gaataagaac taatggaaca accagtgcat gtaggagatc aggatcttca 480
ttctatgcag agatgaaatg gctcctgtca aacacagaca atgctgcttt cccgcaaatg
540 actaagtcat acaagaacac aaggaaagac ccagctctga taatatgggg
gatccaccat 600 tccggatcaa ctacagaaca gaccaagcta tatgggagtg
gaaacaaact gataacagtt 660 gggagttcta attaccaaca gtcctttgta
ccgagtccag gagcgagacc acaagtgaat 720 ggccaatctg gaagaattga
ctttcattgg ctgatactaa accctaatga cacggtcact 780 ttcagtttca
atggggcctt catagctcca gaccgtgcaa gctttctgag agggaagtcc 840
atgggaattc agagtgaagt acaggttgat gccaattgtg aaggagattg ctatcatagt
900 ggagggacaa taataagtaa tttgcccttt cagaacataa atagcagggc
agtaggaaaa 960 tgtccgagat atgttaagca agagagtctg ctgttggcaa
caggaatgaa gaatgttccc 1020 gaaatcccaa agaggaggag gagaggccta
tttggtgcta tagcgggttt cattgaaaat 1080 ggatgggaag gtttgattga
tgggtggtat ggcttcaggc atcaaaatgc acaaggggag 1140 ggaactgctg
cagattacaa aagcacccaa tcagcaattg atcaaataac agggaaatta 1200
aatcggctta tagaaaaaac taaccaacag tttgagttaa tagacaacga attcactgag
1260 gttgaaaggc aaattggcaa tgtgataaac tggaccagag attccatgac
agaagtgtgg 1320 tcctataacg ctgaactctt agtagcaatg gagaatcagc
acacaattga tctggccgac 1380 tcagaaatga acaaactgta cgaacgagtg
aagagacaac tgagagagaa tgccgaagaa 1440 gatggcactg gttgcttcga
aatatttcac aagtgtgatg acgactgcat ggccagtatt 1500 agaaacaaca
cctatgatca cagcaagtac agggaagaag caatacaaaa tagaatacag 1560
attgacccag tcaaactaag cagcggctac aaagatgtga tactttggtt tagcttcggg
1620 gcatcatgtt tcatacttct ggccattgca atgggccttg tcttcatatg
tgtgaagaat 1680 ggaaacatgc ggtgcactat ttgtatataa gtttggaaaa
acacccttgt ttctact 1737 22 1465 DNA Influenza A virus 22 agcaaaagca
gggtgatcga gaatgaatcc aaatcagaaa ctatttgcat tatctggagt 60
ggcaatagca cttagtgtac tgaacttatt gataggaatc tcaaacgtcg gattgaacgt
120 atctctacat ctaaaggaaa aaggacccaa acaggaggag aatttaacat
gcacgaccat 180 taatcaaaac aacactactg tagtagaaaa cacatatgta
aataatacaa caataattac 240 caagggaact gatttgaaaa caccaagcta
tctgctgttg aacaagagcc tgtgcaatgt 300 tgaagggtgg gtcgtgatag
caaaagacaa tgcagtaaga tttggggaaa gtgaacaaat 360 cattgttacc
agggagccat atgtatcatg cgacccaaca ggatgcaaaa tgtatgcctt 420
gcaccaaggg actaccatta ggaacaaaca ttcaaatgga acgattcatg acagaacagc
480 tttcagaggt ctcatctcca ctccattggg cactccacca accgtaagta
acagtgactt 540 tatgtgtgtt ggatggtcaa gcacaacttg ccatgatggg
attgctagga tgactatctg 600 tatacaagga aataatgaca atgctacagc
aacggtttat tacaacagaa ggctgaccac 660 taccattaag acctgggcca
gaaacattct gaggactcaa gaatcagaat gtgtgtgcca 720 caatggcaca
tgtgcagttg taatgaccga cggatcggct agtagtcaag cctatacaaa 780
agtaatgtat ttccacaagg gattagtagt taaggaggag gagttaaggg gatcagccag
840 acatattgag gaatgctcct gttatggaca caatcaaaag gtgacctgtg
tgtgcagaga 900 taactggcag ggagcaaaca ggcctattat agaaattgat
atgagcacat tggagcacac 960 aagtagatac gtgtgcactg gaattctcac
agacaccagc agacctgggg acaaatctag 1020 tggtgattgt tccaatccaa
taactgggag tcccggcgtt ccgggagtga agggattcgg 1080 gtttctaaat
ggggataaca catggcttgg taggaccatc agccccagat caagaagtgg 1140
attcgaaatg ttgaaaatac ctaatgcagg tactgatccc aattctagaa tagcagaacg
1200 acaggaaatt gtcgacaata acaattggtc aggctattcc ggaagcttta
ttgactattg 1260 gaatgataac agtgaatgct acaatccatg cttttacgta
gagttaatta gaggaagacc 1320 cgaagaggct aaatacgtat ggtgggcaag
taacagtcta attgccctat gtggaagccc 1380 attcccagtt gggtctggtt
ccttccccga tggggcacaa atccaatact tttcgtaaaa 1440 tgcaaaaaaa
ctccttgttt ctact 1465 23 1754 DNA Influenza A virus 23 agcaaaagca
ggggatacaa aatgaatact caaattttgg cattcattgc ttgtatgctg 60
attggaacta aaggagacaa aatatgtctt gggcaccatg ctgtggcaaa tgggacaaaa
120 gtgaacacac taacagagag gggaattgaa gtagtcaatg ccacggagac
ggtggaaact 180 gtaaatatta aaaaaatatg cactcaagga aaaaggccaa
cagatctggg acaatgtgga 240 cttctaggaa ccctaatagg acctccccaa
tgcgatcaat ttctggagtt tgacgctaat 300 ttgataattg aacgaagaga
aggaaccgat gtgtgctatc ccgggaagtt cacaaatgaa 360 gaatcactga
ggcagatcct tcgagggtca ggaggaattg ataaagagtc aatgggtttc 420
acctatagtg gaataagaac caatggggcg acgagtgcct gcagaagatc aggttcttct
480 ttctatgcgg agatgaagtg gttactgtcg aattcagaca atgcggcatt
tccccaaatg 540 actaagtcgt ataggaatcc caggaacaaa ccagctctga
taatctgggg agtgcatcac 600 tctggatcag ctactgagca gaccaaactc
tatggaagtg gaaacaagtt gataacagta 660 ggaagctcga aataccagca
atcattcact ccaagtccgg gagcacggcc acaagtgaat 720 ggacaatcag
gaaggattga ttttcattgg ctactccttg accccaatga cacagtgacc 780
ttcactttca atggggcatt catagcccct gacagggcaa gtttctttag aggagaatcg
840 ctaggagtcc agagtgatgt tcctttggat tctggttgtg aaggggattg
cttccacagt 900 gggggtacga tagtcagttc cctgccattc caaaacatca
accctagaac agtggggaaa 960 tgccctcgat atgtcaaaca gacaagcctc
cttttggcta caggaatgag aaacgtccca 1020 gagaacccca agcaggccta
ccggaaacgg atgaccagag gcctttttgg agcgattgct 1080 ggattcatag
agaatggatg ggaaggtctc atcgatggat ggtatggttt cagacatcaa 1140
aatgcacaag gagaaggaac tgcagctgac tacaaaagca cccaatctgc aatagatcag
1200 atcacaggca aattgaatcg tctgattgac aaaacaaacc agcagtttga
actgatagac 1260 aatgaattca gtgagataga acaacaaatc gggaatgtca
ttaactggac acgagactca 1320 atgactgagg tatggtcgta taatgctgag
ctgttggtgg caatggagaa tcagcataca 1380 atagatcttg cagactcaga
aatgaacaaa ctttacgaac gcgtcagaaa acaactaagg 1440 gaaaatgctg
aagaagatgg aactggatgc tttgagatat tccataagtg tgatgatcag 1500
tgtatggaga gcataaggaa caacacttat gaccataccc aatacaggac agagtcattg
1560 cagaatagaa tacagataga cccagtgaaa ttgagtagtg gatacaaaga
cataatctta 1620 tggtttagct tcggggcatc atgttttctt cttctagcca
ttgcaatggg attggttttc 1680 atttgcataa agaatggaaa catgcggtgc
actatttgta tatagtttga gaaaaaaaca 1740 cccttgtttc tact 1754 24 1453
DNA Influenza A virus 24 agcaaaagca ggtgcgagat gaatccgaat
cagaagataa taacaatcgg ggtagtgaat 60 accactctgt caacaatagc
ccttctcatt ggagtgggaa acttagtttt caacacagtc 120 atacatgaga
aaataggaga ccatcaaata gtgacccatc caacaataat gacccctgaa 180
gtaccgaact gcagtgacac tataataaca tacaataaca ctgttataaa caacataaca
240 acaacaataa taactgaagc agaaaggcct ttcaagtctc cactaccgct
gtgccccttc 300 agaggattct tcccttttca caaggacaat gcaatacgac
tgggtgaaaa caaagacgtc 360 atagtcacaa gggagcctta tgttagctgc
gataatgaca actgctggtc ctttgctctc 420 gcacaaggag cattgctagg
gactaaacat agcaatggga ccattaaaga cagaacacca 480 tataggtctc
taattcgttt cccaatagga acagctccag tactaggaaa ttacaaagag 540
atatgcattg cttggtcgag cagcagttgc tttgacggga aagagtggat gcatgtgtgc
600 atgacaggga atgataatga tgcaagtgcc cagataatat atggaggaag
aatgacagac 660 tccattaaat catggaggaa ggacatacta agaacccagg
agtctgaatg tcaatgcatt 720 gacgggactt gtgttgttgc tgtcacagat
ggccctgctg ctaatagtgc agatcacagg 780 gtttactgga tacgggaggg
aagaataata aagtatgaaa atgttcccaa aacaaagata 840 caacacttag
aagaatgttc ctgctatgtg gacattgatg tttactgtat atgtagggac 900
aattggaagg gctctaacag accttggatg agaatcaaca acgagactat actggaaaca
960 ggatatgtat gtagtaaatt tcactcagac acccccaggc cagctgaccc
ttcaataatg 1020 tcatgtgact ccccaagcaa tgtcaatgga ggacccggag
tgaaggggtt tggtttcaaa 1080 gctggcaatg atgtatggtt aggtagaaca
gtgtcaacta gtggtagatc gggctttgaa 1140 attatcaaag ttacagaagg
gtggatcaac tctcctaacc atgtcaaatc aattacacaa 1200 acactagtgt
ccaacaatga ctggtcaggc tattcaggta gcttcattgt caaagccaag 1260
gactgttttc agccctgttt ttatgttgag cttatacgag ggaggcccaa caagaatgat
1320 gacgtctctt ggacaagtaa tagtatagtt actttctgtg gactagacaa
tgaacctgga 1380 tcgggaaatt ggccagatgg ttctaacatt gggtttatgc
ccaagtaata gaaaaaagca 1440 ccttgtttct act 1453 25 1733 DNA
Influenza A virus 25 agcgaaagca ggggatacaa aatgaatact caaattttgg
cattcattgc ttgtatgctg 60 attggaacta aaggagacaa aatatgtctt
gggcaccatg ctgtggcaaa tgggacaaaa 120 gtgaacacac taacagagag
gggaattgaa gtagtcaatg ccacggagac ggtggaaact 180 gtaaatatta
agaaaatatg cactcaagga aaaaggccaa cagatctggg acaatgtgga 240
cttctaggaa ccctaatagg acctccccaa tgcgatcaat ttctggagtt tgacgctaat
300 ttgataattg aacgaagaga aggaaccgat gtgtgctatc ccgggaagtt
cacaaatgaa 360 gaatcactga ggcagatcct tcgagggtca ggaggaattg
ataaagagtc aatgggtttc 420 acctatagtg gaataagaac caatggggcg
acgagtgcct gcagaagatc aggttcttct 480 ttctatgcgg agatgaagtg
gttactgtcg aattcagaca atgcggcatt tccccaaatg 540 actaagtcgt
ataggaatcc caggaacaaa ccagctctga taatctgggg agtgcatcac 600
tctggatcag ctactgagca gaccaaactc tatggaagtg gaaacaagtt gataacagta
660 ggaagctcga aataccagca atcattcact ccaagtccgg gagcacggcc
acaagtgaat 720 ggacaatcag gaaggattga ttttcattgg ctactccttg
accccaatga cacagtgacc 780 ttcactttca atggggcatt catagcccct
gacagggcaa gtttctttag aggagaatcg 840 ctaggagtcc agagtgatgt
tcctttggat tctggttgtg aaggggattg cttccacagt 900 gggggtacga
tagtcagttc cctgccattc caaaacatca accctagaac agtggggaaa 960
tgccctcgat atgtcaaaca gacaagcctc cttttggcta caggaatgag aaacgtccca
1020 gagaacccca agaccagagg cctttttgga gcgattgctg gattcataga
gaatggatgg 1080 gaaggtctca tcgatggatg gtatggtttc agacatcaaa
atgcacaagg agaaggaact 1140 gcagctgact acaaaagcac ccaatctgca
atagatcaga tcacaggcaa attgaatcgt 1200 ctgattgaca aaacaaacca
gcagtttgaa ctgatagaca atgaattcag tgagatagaa 1260 caacaaatcg
ggaatgtcat taactggaca cgagactcaa tgactgaggt atggtcgtat 1320
aatgctgagc tgttggtggc aatggagaat cagcatacaa tagatcttgc agactcagaa
1380 atgaacaaac tttacgaacg cgtcagaaaa caactaaggg aaaatgctga
agaagatgga 1440 actggatgct ttgagatatt ccataagtgt gatgatcagt
gtatggagag cataaggaac 1500 aacacttatg accataccca atacaggaca
gagtcattgc agaatagaat acagatagac 1560 ccagtgaaat tgagtagtgg
atacaaagac ataatcttat ggtttagctt cggggcatca 1620 tgttttcttc
ttctagccat tgcaatggga ttggttttca tttgcataaa gaatggaaac 1680
atgcggtgca ctatttgtat atagtttgag aaaaaaacac ccttgtttct act 1733 26
1453 DNA Influenza A virus 26 agcaaaagca ggtgcgagat gaatccgaat
cagaagataa taacaatcgg ggtagtgaat 60 accactctgt caacaatagc
ccttctcatt ggagtgggaa acttagtttt caacacagtc 120 atacatgaga
aaataggaga ccatcaaata gtgacccatc caacaataat gacccctgaa 180
gtaccgaact gcagtgacac tataataaca tacaataaca ctgttataaa caacataaca
240 acaacaataa taactgaagc agaaaggcct ttcaagtctc cactaccgct
gtgccccttc 300 agaggattct tcccttttca caaggacaat gcaatacgac
tgggtgaaaa caaagacgtc 360 atagtcacaa gggagcctta tgttagctgc
gataatgaca actgctggtc ctttgctctc 420 gcacaaggag cattgctagg
gactaaacat agcaatggga ccattaaaga cagaacacca 480 tataggtctc
taattcgttt cccaatagga acagctccag tactaggaaa ttacaaagag 540
atatgcattg cttggtcgag cagcagttgc tttgacggga aagagtggat gcatgtgtgc
600 atgacaggga atgataatga tgcaagtgcc cagataatat atggaggaag
aatgacagac 660 tccattaaat catggaggaa ggacatacta agaacccagg
agtctgaatg tcaatgcatt 720 gacgggactt gtgttgttgc tgtcacagat
ggccctgctg ctaatagtgc agatcacagg 780 gtttactgga tacgggaggg
aagaataata aagtatgaaa atgttcccaa aacaaagata 840 caacacttag
aagaatgttc ctgctatgtg gacattgatg tttactgtat atgtagggac 900
aattggaagg gctctaacag accttggatg agaatcaaca acgagactat actggaaaca
960 ggatatgtat gtagtaaatt tcactcagac acccccaggc cagctgaccc
ttcaataatg 1020 tcatgtgact ccccaagcaa tgtcaatgga ggacccggag
tgaaggggtt tggtttcaaa 1080 gctggcaatg atgtatggtt aggtagaaca
gtgtcaacta gtggtagatc gggctttgaa 1140 attatcaaag ttacagaagg
gtggatcaac tctcctaacc atgtcaaatc aattacacaa 1200 acactagtgt
ccaacaatga ctggtcaggc tattcaggta gcttcattgt caaagccaag 1260
gactgttttc agccctgttt ttatgttgag cttatacgag ggaggcccaa caagaatgat
1320 gacgtctctt ggacaagtaa tagtatagtt actttctgtg gactagacaa
tgaacctgga 1380 tcgggaaatt ggccagatgg ttctaacatt gggtttatgc
ccaagtaata gaaaaaagca 1440 ccttgtttct act 1453 27 562 PRT Influenza
A virus 27 Met Asn Thr Gln Ile Leu Val Phe Ala Leu Val Ala Ser Ile
Pro Thr 1 5 10 15 Asn Ala Asp Lys Ile Cys Leu Gly His His Ala Val
Ser Asn Gly Thr 20 25 30 Lys Val Asn Thr Leu Thr Glu Arg Gly Val
Glu Val Val Asn Ala Thr 35 40 45 Glu Thr Val Glu Arg Thr Asn Val
Pro Arg Ile Cys Ser Lys Gly Lys 50 55 60 Arg Thr Val Asp Leu Gly
Gln Cys Gly Leu Leu Gly Thr Ile Thr Gly 65 70 75 80 Pro Pro Gln Cys
Asp Gln Phe Leu Glu Phe Ser Ala Asp Leu Ile Ile 85 90 95 Glu Arg
Arg Glu Gly Ser Asp Val Cys Tyr Pro Gly Lys Phe Val Asn 100 105 110
Glu Glu Ala Leu Arg Gln Ile Leu Arg Glu Ser Gly Gly Ile Asp Lys 115
120 125 Glu Thr Met Gly Phe Thr Tyr Ser Gly Ile Arg Thr Asn Gly Thr
Thr 130 135 140 Ser Ala Cys Arg Arg Ser Gly Ser Ser Phe Tyr Ala Glu
Met Lys Trp 145 150 155 160 Leu Leu Ser Asn Thr Asp Asn Ala Ala Phe
Pro Gln Met Thr Lys Ser 165 170 175 Tyr Lys Asn Thr Arg Lys Asp Pro
Ala Leu Ile Ile Trp Gly Ile His 180 185 190 His Ser Gly Ser Thr Thr
Glu Gln Thr Lys Leu Tyr Gly Ser Gly Asn 195 200 205 Lys Leu Ile Thr
Val Gly Ser Ser Asn Tyr Gln Gln Ser Phe Val Pro 210 215 220 Ser Pro
Gly Ala Arg Pro Gln Val Asn Gly Gln Ser Gly Arg Ile Asp 225 230 235
240 Phe His Trp Leu Ile Leu Asn Pro Asn Asp Thr Val Thr Phe Ser Phe
245 250 255 Asn Gly Ala Phe Ile Ala Pro Asp Arg Ala Ser Phe Leu Arg
Gly Lys 260 265 270 Ser Met Gly Ile Gln Ser Glu Val Gln Val Asp Ala
Asn Cys Glu Gly 275 280 285 Asp Cys Tyr His Ser Gly Gly Thr Ile Ile
Ser Asn Leu Pro Phe Gln 290 295 300 Asn Ile Asn Ser Arg Ala Val Gly
Lys Cys Pro Arg Tyr Val Lys Gln 305 310 315 320 Glu Ser Leu Leu Leu
Ala Thr Gly Met Lys Asn Val Pro Glu Ile Pro 325 330 335 Lys Arg Arg
Arg Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu 340 345 350 Asn
Gly Trp Glu Gly Leu Ile Asp Gly Trp Tyr Gly Phe Arg His Gln 355 360
365
Asn Ala Gln Gly Glu Gly Thr Ala Ala Asp Tyr Lys Ser Thr Gln Ser 370
375 380 Ala Ile Asp Gln Ile Thr Gly Lys Leu Asn Arg Leu Ile Glu Lys
Thr 385 390 395 400 Asn Gln Gln Phe Glu Leu Ile Asp Asn Glu Phe Thr
Glu Val Glu Arg 405 410 415 Gln Ile Gly Asn Val Ile Asn Trp Thr Arg
Asp Ser Met Thr Glu Val 420 425 430 Trp Ser Tyr Asn Ala Glu Leu Leu
Val Ala Met Glu Asn Gln His Thr 435 440 445 Ile Asp Leu Ala Asp Ser
Glu Met Asn Lys Leu Tyr Glu Arg Val Lys 450 455 460 Arg Gln Leu Arg
Glu Asn Ala Glu Glu Asp Gly Thr Gly Cys Phe Glu 465 470 475 480 Ile
Phe His Lys Cys Asp Asp Asp Cys Met Ala Ser Ile Arg Asn Asn 485 490
495 Thr Tyr Asp His Ser Lys Tyr Arg Glu Glu Ala Ile Gln Asn Arg Ile
500 505 510 Gln Ile Asp Pro Val Lys Leu Ser Ser Gly Tyr Lys Asp Val
Ile Leu 515 520 525 Trp Phe Ser Phe Gly Ala Ser Cys Phe Ile Leu Leu
Ala Ile Ala Met 530 535 540 Gly Leu Val Phe Ile Cys Val Lys Asn Gly
Asn Met Arg Cys Thr Ile 545 550 555 560 Cys Ile 28 471 PRT
Influenza A virus 28 Met Asn Pro Asn Gln Lys Leu Phe Ala Leu Ser
Gly Val Ala Ile Ala 1 5 10 15 Leu Ser Val Leu Asn Leu Leu Ile Gly
Ile Ser Asn Val Gly Leu Asn 20 25 30 Val Ser Leu His Leu Lys Glu
Lys Gly Pro Lys Gln Glu Glu Asn Leu 35 40 45 Thr Cys Thr Thr Ile
Asn Gln Asn Asn Thr Thr Val Val Glu Asn Thr 50 55 60 Tyr Val Asn
Asn Thr Thr Ile Ile Thr Lys Gly Thr Asp Leu Lys Thr 65 70 75 80 Pro
Ser Tyr Leu Leu Leu Asn Lys Ser Leu Cys Asn Val Glu Gly Trp 85 90
95 Val Val Ile Ala Lys Asp Asn Ala Val Arg Phe Gly Glu Ser Glu Gln
100 105 110 Ile Ile Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Thr
Gly Cys 115 120 125 Lys Met Tyr Ala Leu His Gln Gly Thr Thr Ile Arg
Asn Lys His Ser 130 135 140 Asn Gly Thr Ile His Asp Arg Thr Ala Phe
Arg Gly Leu Ile Ser Thr 145 150 155 160 Pro Leu Gly Thr Pro Pro Thr
Val Ser Asn Ser Asp Phe Met Cys Val 165 170 175 Gly Trp Ser Ser Thr
Thr Cys His Asp Gly Ile Ala Arg Met Thr Ile 180 185 190 Cys Ile Gln
Gly Asn Asn Asp Asn Ala Thr Ala Thr Val Tyr Tyr Asn 195 200 205 Arg
Arg Leu Thr Thr Thr Ile Lys Thr Trp Ala Arg Asn Ile Leu Arg 210 215
220 Thr Gln Glu Ser Glu Cys Val Cys His Asn Gly Thr Cys Ala Val Val
225 230 235 240 Met Thr Asp Gly Ser Ala Ser Ser Gln Ala Tyr Thr Lys
Val Met Tyr 245 250 255 Phe His Lys Gly Leu Val Val Lys Glu Glu Glu
Leu Arg Gly Ser Ala 260 265 270 Arg His Ile Glu Glu Cys Ser Cys Tyr
Gly His Asn Gln Lys Val Thr 275 280 285 Cys Val Cys Arg Asp Asn Trp
Gln Gly Ala Asn Arg Pro Ile Ile Glu 290 295 300 Ile Asp Met Ser Thr
Leu Glu His Thr Ser Arg Tyr Val Cys Thr Gly 305 310 315 320 Ile Leu
Thr Asp Thr Ser Arg Pro Gly Asp Lys Ser Ser Gly Asp Cys 325 330 335
Ser Asn Pro Ile Thr Gly Ser Pro Gly Val Pro Gly Val Lys Gly Phe 340
345 350 Gly Phe Leu Asn Gly Asp Asn Thr Trp Leu Gly Arg Thr Ile Ser
Pro 355 360 365 Arg Ser Arg Ser Gly Phe Glu Met Leu Lys Ile Pro Asn
Ala Gly Thr 370 375 380 Asp Pro Asn Ser Arg Ile Ala Glu Arg Gln Glu
Ile Val Asp Asn Asn 385 390 395 400 Asn Trp Ser Gly Tyr Ser Gly Ser
Phe Ile Asp Tyr Trp Asn Asp Asn 405 410 415 Ser Glu Cys Tyr Asn Pro
Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg 420 425 430 Pro Glu Glu Ala
Lys Tyr Val Trp Trp Ala Ser Asn Ser Leu Ile Ala 435 440 445 Leu Cys
Gly Ser Pro Phe Pro Val Gly Ser Gly Ser Phe Pro Asp Gly 450 455 460
Ala Gln Ile Gln Tyr Phe Ser 465 470 29 567 PRT Influenza A virus 29
Met Asn Thr Gln Ile Leu Ala Phe Ile Ala Cys Met Leu Ile Gly Thr 1 5
10 15 Lys Gly Asp Lys Ile Cys Leu Gly His His Ala Val Ala Asn Gly
Thr 20 25 30 Lys Val Asn Thr Leu Thr Glu Arg Gly Ile Glu Val Val
Asn Ala Thr 35 40 45 Glu Thr Val Glu Thr Val Asn Ile Lys Lys Ile
Cys Thr Gln Gly Lys 50 55 60 Arg Pro Thr Asp Leu Gly Gln Cys Gly
Leu Leu Gly Thr Leu Ile Gly 65 70 75 80 Pro Pro Gln Cys Asp Gln Phe
Leu Glu Phe Asp Ala Asn Leu Ile Ile 85 90 95 Glu Arg Arg Glu Gly
Thr Asp Val Cys Tyr Pro Gly Lys Phe Thr Asn 100 105 110 Glu Glu Ser
Leu Arg Gln Ile Leu Arg Gly Ser Gly Gly Ile Asp Lys 115 120 125 Glu
Ser Met Gly Phe Thr Tyr Ser Gly Ile Arg Thr Asn Gly Ala Thr 130 135
140 Ser Ala Cys Arg Arg Ser Gly Ser Ser Phe Tyr Ala Glu Met Lys Trp
145 150 155 160 Leu Leu Ser Asn Ser Asp Asn Ala Ala Phe Pro Gln Met
Thr Lys Ser 165 170 175 Tyr Arg Asn Pro Arg Asn Lys Pro Ala Leu Ile
Ile Trp Gly Val His 180 185 190 His Ser Gly Ser Ala Thr Glu Gln Thr
Lys Leu Tyr Gly Ser Gly Asn 195 200 205 Lys Leu Ile Thr Val Gly Ser
Ser Lys Tyr Gln Gln Ser Phe Thr Pro 210 215 220 Ser Pro Gly Ala Arg
Pro Gln Val Asn Gly Gln Ser Gly Arg Ile Asp 225 230 235 240 Phe His
Trp Leu Leu Leu Asp Pro Asn Asp Thr Val Thr Phe Thr Phe 245 250 255
Asn Gly Ala Phe Ile Ala Pro Asp Arg Ala Ser Phe Phe Arg Gly Glu 260
265 270 Ser Leu Gly Val Gln Ser Asp Val Pro Leu Asp Ser Gly Cys Glu
Gly 275 280 285 Asp Cys Phe His Ser Gly Gly Thr Ile Val Ser Ser Leu
Pro Phe Gln 290 295 300 Asn Ile Asn Pro Arg Thr Val Gly Lys Cys Pro
Arg Tyr Val Lys Gln 305 310 315 320 Thr Ser Leu Leu Leu Ala Thr Gly
Met Arg Asn Val Pro Glu Asn Pro 325 330 335 Lys Gln Ala Tyr Arg Lys
Arg Met Thr Arg Gly Leu Phe Gly Ala Ile 340 345 350 Ala Gly Phe Ile
Glu Asn Gly Trp Glu Gly Leu Ile Asp Gly Trp Tyr 355 360 365 Gly Phe
Arg His Gln Asn Ala Gln Gly Glu Gly Thr Ala Ala Asp Tyr 370 375 380
Lys Ser Thr Gln Ser Ala Ile Asp Gln Ile Thr Gly Lys Leu Asn Arg 385
390 395 400 Leu Ile Asp Lys Thr Asn Gln Gln Phe Glu Leu Ile Asp Asn
Glu Phe 405 410 415 Ser Glu Ile Glu Gln Gln Ile Gly Asn Val Ile Asn
Trp Thr Arg Asp 420 425 430 Ser Met Thr Glu Val Trp Ser Tyr Asn Ala
Glu Leu Leu Val Ala Met 435 440 445 Glu Asn Gln His Thr Ile Asp Leu
Ala Asp Ser Glu Met Asn Lys Leu 450 455 460 Tyr Glu Arg Val Arg Lys
Gln Leu Arg Glu Asn Ala Glu Glu Asp Gly 465 470 475 480 Thr Gly Cys
Phe Glu Ile Phe His Lys Cys Asp Asp Gln Cys Met Glu 485 490 495 Ser
Ile Arg Asn Asn Thr Tyr Asp His Thr Gln Tyr Arg Thr Glu Ser 500 505
510 Leu Gln Asn Arg Ile Gln Ile Asp Pro Val Lys Leu Ser Ser Gly Tyr
515 520 525 Lys Asp Ile Ile Leu Trp Phe Ser Phe Gly Ala Ser Cys Phe
Leu Leu 530 535 540 Leu Ala Ile Ala Met Gly Leu Val Phe Ile Cys Ile
Lys Asn Gly Asn 545 550 555 560 Met Arg Cys Thr Ile Cys Ile 565 30
469 PRT Influenza A virus 30 Met Asn Pro Asn Gln Lys Ile Ile Thr
Ile Gly Val Val Asn Thr Thr 1 5 10 15 Leu Ser Thr Ile Ala Leu Leu
Ile Gly Val Gly Asn Leu Val Phe Asn 20 25 30 Thr Val Ile His Glu
Lys Ile Gly Asp His Gln Ile Val Thr His Pro 35 40 45 Thr Ile Met
Thr Pro Glu Val Pro Asn Cys Ser Asp Thr Ile Ile Thr 50 55 60 Tyr
Asn Asn Thr Val Ile Asn Asn Ile Thr Thr Thr Ile Ile Thr Glu 65 70
75 80 Ala Glu Arg Pro Phe Lys Ser Pro Leu Pro Leu Cys Pro Phe Arg
Gly 85 90 95 Phe Phe Pro Phe His Lys Asp Asn Ala Ile Arg Leu Gly
Glu Asn Lys 100 105 110 Asp Val Ile Val Thr Arg Glu Pro Tyr Val Ser
Cys Asp Asn Asp Asn 115 120 125 Cys Trp Ser Phe Ala Leu Ala Gln Gly
Ala Leu Leu Gly Thr Lys His 130 135 140 Ser Asn Gly Thr Ile Lys Asp
Arg Thr Pro Tyr Arg Ser Leu Ile Arg 145 150 155 160 Phe Pro Ile Gly
Thr Ala Pro Val Leu Gly Asn Tyr Lys Glu Ile Cys 165 170 175 Ile Ala
Trp Ser Ser Ser Ser Cys Phe Asp Gly Lys Glu Trp Met His 180 185 190
Val Cys Met Thr Gly Asn Asp Asn Asp Ala Ser Ala Gln Ile Ile Tyr 195
200 205 Gly Gly Arg Met Thr Asp Ser Ile Lys Ser Trp Arg Lys Asp Ile
Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Gln Cys Ile Asp Gly Thr
Cys Val Val 225 230 235 240 Ala Val Thr Asp Gly Pro Ala Ala Asn Ser
Ala Asp His Arg Val Tyr 245 250 255 Trp Ile Arg Glu Gly Arg Ile Ile
Lys Tyr Glu Asn Val Pro Lys Thr 260 265 270 Lys Ile Gln His Leu Glu
Glu Cys Ser Cys Tyr Val Asp Ile Asp Val 275 280 285 Tyr Cys Ile Cys
Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Trp Met 290 295 300 Arg Ile
Asn Asn Glu Thr Ile Leu Glu Thr Gly Tyr Val Cys Ser Lys 305 310 315
320 Phe His Ser Asp Thr Pro Arg Pro Ala Asp Pro Ser Ile Met Ser Cys
325 330 335 Asp Ser Pro Ser Asn Val Asn Gly Gly Pro Gly Val Lys Gly
Phe Gly 340 345 350 Phe Lys Ala Gly Asn Asp Val Trp Leu Gly Arg Thr
Val Ser Thr Ser 355 360 365 Gly Arg Ser Gly Phe Glu Ile Ile Lys Val
Thr Glu Gly Trp Ile Asn 370 375 380 Ser Pro Asn His Val Lys Ser Ile
Thr Gln Thr Leu Val Ser Asn Asn 385 390 395 400 Asp Trp Ser Gly Tyr
Ser Gly Ser Phe Ile Val Lys Ala Lys Asp Cys 405 410 415 Phe Gln Pro
Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Asn Lys 420 425 430 Asn
Asp Asp Val Ser Trp Thr Ser Asn Ser Ile Val Thr Phe Cys Gly 435 440
445 Leu Asp Asn Glu Pro Gly Ser Gly Asn Trp Pro Asp Gly Ser Asn Ile
450 455 460 Gly Phe Met Pro Lys 465 31 560 PRT Influenza A virus 31
Met Asn Thr Gln Ile Leu Ala Phe Ile Ala Cys Met Leu Ile Gly Thr 1 5
10 15 Lys Gly Asp Lys Ile Cys Leu Gly His His Ala Val Ala Asn Gly
Thr 20 25 30 Lys Val Asn Thr Leu Thr Glu Arg Gly Ile Glu Val Val
Asn Ala Thr 35 40 45 Glu Thr Val Glu Thr Val Asn Ile Lys Lys Ile
Cys Thr Gln Gly Lys 50 55 60 Arg Pro Thr Asp Leu Gly Gln Cys Gly
Leu Leu Gly Thr Leu Ile Gly 65 70 75 80 Pro Pro Gln Cys Asp Gln Phe
Leu Glu Phe Asp Ala Asn Leu Ile Ile 85 90 95 Glu Arg Arg Glu Gly
Thr Asp Val Cys Tyr Pro Gly Lys Phe Thr Asn 100 105 110 Glu Glu Ser
Leu Arg Gln Ile Leu Arg Gly Ser Gly Gly Ile Asp Lys 115 120 125 Glu
Ser Met Gly Phe Thr Tyr Ser Gly Ile Arg Thr Asn Gly Ala Thr 130 135
140 Ser Ala Cys Arg Arg Ser Gly Ser Ser Phe Tyr Ala Glu Met Lys Trp
145 150 155 160 Leu Leu Ser Asn Ser Asp Asn Ala Ala Phe Pro Gln Met
Thr Lys Ser 165 170 175 Tyr Arg Asn Pro Arg Asn Lys Pro Ala Leu Ile
Ile Trp Gly Val His 180 185 190 His Ser Gly Ser Ala Thr Glu Gln Thr
Lys Leu Tyr Gly Ser Gly Asn 195 200 205 Lys Leu Ile Thr Val Gly Ser
Ser Lys Tyr Gln Gln Ser Phe Thr Pro 210 215 220 Ser Pro Gly Ala Arg
Pro Gln Val Asn Gly Gln Ser Gly Arg Ile Asp 225 230 235 240 Phe His
Trp Leu Leu Leu Asp Pro Asn Asp Thr Val Thr Phe Thr Phe 245 250 255
Asn Gly Ala Phe Ile Ala Pro Asp Arg Ala Ser Phe Phe Arg Gly Glu 260
265 270 Ser Leu Gly Val Gln Ser Asp Val Pro Leu Asp Ser Gly Cys Glu
Gly 275 280 285 Asp Cys Phe His Ser Gly Gly Thr Ile Val Ser Ser Leu
Pro Phe Gln 290 295 300 Asn Ile Asn Pro Arg Thr Val Gly Lys Cys Pro
Arg Tyr Val Lys Gln 305 310 315 320 Thr Ser Leu Leu Leu Ala Thr Gly
Met Arg Asn Val Pro Glu Asn Pro 325 330 335 Lys Thr Arg Gly Leu Phe
Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly 340 345 350 Trp Glu Gly Leu
Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ala 355 360 365 Gln Gly
Glu Gly Thr Ala Ala Asp Tyr Lys Ser Thr Gln Ser Ala Ile 370 375 380
Asp Gln Ile Thr Gly Lys Leu Asn Arg Leu Ile Asp Lys Thr Asn Gln 385
390 395 400 Gln Phe Glu Leu Ile Asp Asn Glu Phe Ser Glu Ile Glu Gln
Gln Ile 405 410 415 Gly Asn Val Ile Asn Trp Thr Arg Asp Ser Met Thr
Glu Val Trp Ser 420 425 430 Tyr Asn Ala Glu Leu Leu Val Ala Met Glu
Asn Gln His Thr Ile Asp 435 440 445 Leu Ala Asp Ser Glu Met Asn Lys
Leu Tyr Glu Arg Val Arg Lys Gln 450 455 460 Leu Arg Glu Asn Ala Glu
Glu Asp Gly Thr Gly Cys Phe Glu Ile Phe 465 470 475 480 His Lys Cys
Asp Asp Gln Cys Met Glu Ser Ile Arg Asn Asn Thr Tyr 485 490 495 Asp
His Thr Gln Tyr Arg Thr Glu Ser Leu Gln Asn Arg Ile Gln Ile 500 505
510 Asp Pro Val Lys Leu Ser Ser Gly Tyr Lys Asp Ile Ile Leu Trp Phe
515 520 525 Ser Phe Gly Ala Ser Cys Phe Leu Leu Leu Ala Ile Ala Met
Gly Leu 530 535 540 Val Phe Ile Cys Ile Lys Asn Gly Asn Met Arg Cys
Thr Ile Cys Ile 545 550 555 560 32 469 PRT Influenza A virus 32 Met
Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Val Val Asn Thr Thr 1 5 10
15 Leu Ser Thr Ile Ala Leu Leu Ile Gly Val Gly Asn Leu Val Phe Asn
20 25 30 Thr Val Ile His Glu Lys Ile Gly Asp His Gln Ile Val Thr
His Pro 35 40 45 Thr Ile Met Thr Pro Glu Val Pro Asn Cys Ser Asp
Thr Ile Ile Thr 50 55 60 Tyr Asn Asn Thr Val Ile Asn Asn Ile Thr
Thr Thr Ile Ile Thr Glu 65 70 75 80 Ala Glu Arg Pro Phe Lys Ser Pro
Leu Pro Leu Cys Pro Phe Arg Gly 85 90 95 Phe Phe Pro Phe His Lys
Asp Asn Ala Ile Arg Leu Gly Glu Asn Lys 100 105 110 Asp Val Ile Val
Thr Arg Glu Pro Tyr Val Ser Cys Asp Asn Asp Asn 115 120 125 Cys Trp
Ser Phe Ala Leu Ala Gln Gly Ala Leu Leu Gly Thr Lys His 130 135 140
Ser Asn Gly Thr Ile Lys Asp Arg Thr Pro Tyr Arg Ser Leu Ile Arg 145
150 155 160 Phe Pro Ile Gly Thr Ala Pro Val Leu Gly Asn Tyr Lys Glu
Ile Cys 165
170 175 Ile Ala Trp Ser Ser Ser Ser Cys Phe Asp Gly Lys Glu Trp Met
His 180 185 190 Val Cys Met Thr Gly Asn Asp Asn Asp Ala Ser Ala Gln
Ile Ile Tyr 195 200 205 Gly Gly Arg Met Thr Asp Ser Ile Lys Ser Trp
Arg Lys Asp Ile Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Gln Cys
Ile Asp Gly Thr Cys Val Val 225 230 235 240 Ala Val Thr Asp Gly Pro
Ala Ala Asn Ser Ala Asp His Arg Val Tyr 245 250 255 Trp Ile Arg Glu
Gly Arg Ile Ile Lys Tyr Glu Asn Val Pro Lys Thr 260 265 270 Lys Ile
Gln His Leu Glu Glu Cys Ser Cys Tyr Val Asp Ile Asp Val 275 280 285
Tyr Cys Ile Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Trp Met 290
295 300 Arg Ile Asn Asn Glu Thr Ile Leu Glu Thr Gly Tyr Val Cys Ser
Lys 305 310 315 320 Phe His Ser Asp Thr Pro Arg Pro Ala Asp Pro Ser
Ile Met Ser Cys 325 330 335 Asp Ser Pro Ser Asn Val Asn Gly Gly Pro
Gly Val Lys Gly Phe Gly 340 345 350 Phe Lys Ala Gly Asn Asp Val Trp
Leu Gly Arg Thr Val Ser Thr Ser 355 360 365 Gly Arg Ser Gly Phe Glu
Ile Ile Lys Val Thr Glu Gly Trp Ile Asn 370 375 380 Ser Pro Asn His
Val Lys Ser Ile Thr Gln Thr Leu Val Ser Asn Asn 385 390 395 400 Asp
Trp Ser Gly Tyr Ser Gly Ser Phe Ile Val Lys Ala Lys Asp Cys 405 410
415 Phe Gln Pro Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Asn Lys
420 425 430 Asn Asp Asp Val Ser Trp Thr Ser Asn Ser Ile Val Thr Phe
Cys Gly 435 440 445 Leu Asp Asn Glu Pro Gly Ser Gly Asn Trp Pro Asp
Gly Ser Asn Ile 450 455 460 Gly Phe Met Pro Lys 465 33 1743 DNA
Influenza A virus 33 agcaaaagca ggggaaaatg attgcagtca ttataatagc
ggtactggca acggccggaa 60 aatcagacaa gatctgcatt gggtatcatg
ccaacaattc aacaacacaa gtggatacga 120 tacttgagaa gaatgtaacc
gtcacacact cagttgaatt gctggagaac caaaaagaag 180 aaagattctg
caagatcttg aacaaggccc ctctcgattt aagaggatgt accatagagg 240
gttggatctt ggggaatccc caatgcgacc tattgcttgg tgatcaaagc tggtcatata
300 tagtggaaag acctacagct caaaatggga tctgctaccc aggaattttg
aatgaagtag 360 aagaactgaa ggcacttatt ggatcaggag aaagagtgga
gagatttgaa atgtttccca 420 aaagtacatg ggcaggagta gacaccagca
gtggggtaac aaaggcttgc ccttatacta 480 gtggttcgtc tttctacaga
aacctcctat ggataataaa aaccaagtcc gcagcatatc 540 cagtaattaa
gggaacctac aataacactg gaagccagcc aatcctctat ttctggggtg 600
tgcaccatcc tcctgacacc aatgagcaaa acactttgta tggctctggt gatcgatatg
660 tcaggatggg aactgaaagc atgaattttg ccaagagccc agaaattgcg
gcaaggcctg 720 ctgtgaatgg tcaaagaggc agaattgatt attactggtc
tgttttaaag ccgggggaaa 780 ccttgaatgt ggaatctaat ggaaatctaa
tcgccccttg gtatgcatac aaatttgtca 840 gcaccaatag taaaggagcc
gtcttcaagt caaatttacc aatcgagaac tgtgatgcca 900 catgccagac
tattgcagga gtcttaagaa ccaataaaac atttcagaat gtaagccctc 960
tgtggatagg agaatgcccc aaatatgtga aaagtgaaag tttgaggctt gcaactggac
1020 taagaaatat tccacagatt gagactagag gacttttcgg agctatcgca
gggtttattg 1080 aaggaggatg gactggaatg atagatgggt ggtatggcta
tcaccatgaa aattctcaag 1140 gctcagggta tgcggcagac agagaaagca
ctcaaagggc tatagacgga attacaaata 1200 aggtcaattc cattatagac
aaaatgaaca cacaattcga agctatagac cacgaattct 1260 caaatttgga
gagaagaatt gacagtctga acaaaagaat ggaagatgga tttctggacg 1320
tttggacata caatgctgaa ctgttggttc ttcttgaaaa cgaaaggaca ctagacctac
1380 atgacgcgaa tgtgaagaac ctgtatgaaa aggtcaaatc acaactacgg
gacaatgcta 1440 atgatctagg aaatggatgc tttgaatttt ggcataagtg
tgacaatgaa tgcatagagt 1500 ctgtcaaaaa tggtacctat gactatccca
aatatcagga tgaaagcaaa ttgaacaggc 1560 aggaaataga atcggtgaag
ctggagaacc ttggtgtgta tcaaatcctc gccatttata 1620 gtacggtatc
gagcagtcta gtcttggtag ggctgattat agcaatgggt ctttggatgt 1680
gttcaaatgg ttcaatgcaa tgcaggatat gtatataatt aagaaaaaca cccttgttct
1740 act 1743 34 1460 DNA Influenza A virus 34 agcaaaagca
gggtcaagat gaatccaaat cagaagattc tatgcacatc tgctactgcc 60
attgcaatag gcacaattgc tgtattaata ggaatagcaa acctgggttt gaacatagga
120 ctacacctga aaccgagctg caactgctcc aaccctcctc ctgaaacaac
aaatgtaagc 180 caaacaataa taaacaatta ctacaatgaa acaaatgtta
cccaaataag taacacaaac 240 attcaacata tggggggaac cgaaaaggac
ttcaacaatc tgactaaagg gctctgcaca 300 ataaattcat ggcatatatt
cggaaaggac aatgctataa gaatagggga gaactctgat 360 gttttagtca
caagagagcc atatgtttct tgtgatccag atgaatgcag attctatgct 420
ctcagccaag gaacaacaat acggggaaag cactcaaatg gaacaataca cgatagatcc
480 caataccgtg ctttagtgag ctggccttta tcatcaccac ccactgtgta
caataccaga 540 gtagaatgca ttggatggtc cagtacaagc tgccatgatg
ggaaagcacg aatgtctata 600 tgtgtctcag gtcccaacaa caatgcatca
gcagtgattt ggtacaaagg gcggcctatc 660 acggaaatca atacgtgggc
ccgaaacata ttgagaaccc aagaatctga gtgtgtatgc 720 cacaatggaa
tatgtccagt agtgttcact gacggttctg ccaccggtcc agcagaaact 780
aggatatact atttcaaaga ggggaaaatc ctcaaatggg agccactaac tggaaccgcc
840 aagcacattg aagaatgctc ttgctatggg aaagactcag aaataacgtg
cacatgtaga 900 gacaattggc aaggctcgaa tagaccagta atacaaataa
accccacaat gatgactcac 960 actagtcaat acatatgcag ccctgtcctc
acagacaatc cacgccccaa tgaccccacg 1020 gtaggcaagt gtaatgatcc
ttatccagga aacaacaata atggagtcaa aggattctca 1080 tatttagatg
gtgacaatac atggctagga agaacgataa gcacagcctc taggtctggg 1140
tatgaaatgc tgaaagtgcc taatgcattg acagatgata gatcaaaacc tactcaaggt
1200 cagacaattg tattaaacac agactggagt ggttacagtg ggtctttcat
tgattactgg 1260 gcaaaagggg agtgctatag agcatgcttc tacgttgagc
tgatccgtgg aaggccaaaa 1320 gaggacaaag tgtggtggac cagtaatagt
atagtgtcga tgtgttccag cacagagttc 1380 cttggacaat ggaactggcc
agatggggct aaaatagagt acttcctcta agatgtagaa 1440 aaaagaccct
tgtttctact 1460 35 1747 DNA Influenza A virus 35 agcaaaagca
ggggaaaatg attgcaatca ttgtaatagc aatactggca gcagccggaa 60
aatcagacaa gatctgcatt gggtatcatg ccaacaattc aacaacacag gtagatacga
120 tacttgagaa gaatgtgact gtcacacact caattgaatt gctggaaaat
cagaaggaag 180 aaagattctg caagatattg aacaaggccc ctctcgactt
aagggaatgt accatagagg 240 gttggatctt ggggaatccc caatgcgacc
tattgcttgg tgatcaaagc tggtcataca 300 ttgtggaaag acctactgct
caaaacggga tctgctaccc aggaacctta aatgaggtag 360 aagaactgag
ggcacttatt ggatcaggag aaagggtaga gagatttgag atgtttcccc 420
aaagcacctg gcaaggagtt gacaccaaca gtggaacaac aagatcctgc ccttattcta
480 ctggtgatcc gtctttctac agaaacctcc tatggataat aaaaaccaag
acagcagaat 540 atccagtaat taagggaatt tacaacaaca ctggaaccca
gccaatcctc tatttctggg 600 gtgtgcatca tcctcctaac accgacgagc
aagatactct gtatggctct ggtgatcgat 660 acgttagaat gggaactgaa
agcatgaatt ttgccaagag tccggaaatt gcggcaaggc 720 ctgctgtgaa
tggacaaaga ggcagaattg attattattg gtcggtttta aaaccagggg 780
aaaccttgaa tgtggaatct aatggaaatc taatcgcccc ttggtatgca tacaaatttg
840 tcaacacaaa tagtaaagga gccgtcttca ggtcagattt accaatcgag
aactgcgatg 900 ccacatgcca gactattgca ggggttctaa ggaccaataa
aacatttcag aatgtgagtc 960 ccctgtggat aggagaatgt cccaaatacg
tgaaaagtga aagtctgagg cttgcaactg 1020 gactaagaaa tgttccacag
attgaaacta gaggactctt cggagctatt gcagggttta 1080 ttgaaggagg
atggactggg atgatagatg ggtggtatgg ctatcaccat gaaaattctc 1140
aagggtcagg atatgcagcg gacagagaaa gcactcaaaa ggctgtaaac agaattacaa
1200 ataaggtcaa ttccatcatc aacaaaatga acacacaatt tgaagctgtc
gatcacgaat 1260 tttcaaatct ggagaggaga atcgacaatc tgaacaaaag
aatgcaagat ggatttctgg 1320 atgtttggac atacaatgct gaactgttgg
ttcttcttga aaacgaaaga acactagaca 1380 tgcatgacgc aaatgtgaag
aacctacatg aaaaggtcaa atcacaacta agggacaatg 1440 ctaacgatct
agggaatggt tgctttgaat tttggcataa gtgtgacaat gaatgcatag 1500
agtctgtcaa aaatggtaca tatgactatc ccaaatacca gactgaaagc aaattaaaca
1560 ggctaaaaat agaatcagta aagctagaga accttggtgt gtatcaaatt
cttgccattt 1620 atagtacggt atcgagcagc ctagtgttgg tagggctgat
catggcaatg ggtctttgga 1680 tgtgttcaaa tggttcaatg cagtgcaatg
tgtgtatatg attaagaaaa acacccttgt 1740 ttctact 1747 36 1401 DNA
Influenza A virus 36 agcaaaagca ggagtttaac atgaatccaa atcagaagat
aataaccatt gggtcaatct 60 gtatggtagt tggaataatc agcttgatgt
tacaaattgg aaacataata tcaatatggg 120 ttagccacat aattcagact
gggcatccaa accagcctgg gccatgcaat caaagcatca 180 atttttacac
tgagcaggct gcagcttcag tgacattagc gggtaattcc tctctctgcc 240
ctattagtgg atgggctata tacagtaaag acaatagtat aagaattggt tccaaagggg
300 atgtgtttgt tatgagagaa ccattcgttt catgctccca tttggaatgc
agaacctttt 360 tcttgactca aggagcccta ttgaatgaca agcattctaa
tgggaccgtt aaagacagaa 420 gcccctatag aactttaatg agctgtcctg
ttggtgaggc tccttcccca tacaactcaa 480 ggtttgagtc tgttgcttgg
tcagcaagtg cttgccatga tggcattagt tggctaacaa 540 ttggaatttc
cggtccggat aatggggctg tggctgtgtt gaaatacaat ggcataataa 600
cagacaccat caagagttgg aggaacaaca tactgaggac acaagagtct gaatgtgcat
660 gtgtgaatgg ttcttgtttt actgtaatga cagatggacc gagtaatgaa
caggcctcat 720 acaagatttt caagatagag aaggggaaag tagtcaaatc
agttgagttg aacgccccta 780 attatcatta cgaggaatgc tcctgttatc
ctgatgctgg cgaaatcaca tgtgtgtgca 840 gggataattg gcatggctcg
aaccgaccgt gggtgtcttt caatcagaat ctggagtatc 900 aaataggata
tatatgcagt ggggttttcg gagacagtcc acgccccaat gatggaacag 960
gcagttgcgg tccagtgtct cttaacggag agtatggagt aaaagggttt tcatttaagt
1020 acggtgatgg tgtttggatc gggagaacca aaagcactag ttccaggagc
gggtttgaaa 1080 tgatttggga tccaaatggg tggaccgaaa cagatagtaa
cttctcattg aagcaagaca 1140 tcatagcaat aactgattgg tcaggataca
gcgggagttt tgtccaacat ccagaactga 1200 caggattaaa ttgcatgagg
ccttgcttct gggttgaact aatcagaggg aggcccaaag 1260 agaaaacaat
ctggactagt gggagcagta tatctttctg tggtgtaaat agtgacactg 1320
tgggttggtc ttggccagac ggtgctgagg tgccattcac cattgacaag tagtttgttc
1380 aaaaaactcc ttgtttctac t 1401 37 1745 DNA Influenza A virus 37
agcaaaagca ggggaaaatg attgcaatca taatacttgc aatagtggtc tctaccagca
60 agtcagacag gatctgcatt ggttaccatg caaacaactc gacaacacaa
gtggacacaa 120 tattagagaa gaatgtgaca gtgacacact cagtggagct
cctagaaaac cagaaggaga 180 atagattctg cagagtcttg aataaagcgc
cactggatct aatggactgc accactgagg 240 gttggatcct tggaaacccc
cgatgtgata acttactcgg tgatcaaagt tggtcataca 300 tagtagagag
gcctgatgcc caaaatggga tatgttaccc aggggtattg aaggagacgg 360
aagagctgaa agcactcatt gggtctatag atagcataca aagatttgaa atgtttccca
420 agagcacgtg gaccggggta gatactaata gcggagttac gagcgcttgc
ccctacaatg 480 gtgaatcttc cttttacagg aatctgttgt ggataataaa
aataagatct gatccgtact 540 cattgatcaa ggggacatat accaatacag
gctctcagcc aatcttatat ttctggggtg 600 tgcaccatcc tccagatgaa
gttgagcaag ctaacttgta tggaattggt acccggtatg 660 ttaggatggg
aactgaaagt atgaattttg ccaaaggtcc tgaaatagca ggcagaccac 720
ctgcgaatgg gcaacgagga agaattgatt attattggtc tgtgttgaag ccaggagaaa
780 ccttgaatgt ggaatccaat ggaaatttaa tagctccttg gtatgcttac
aagttcacta 840 gttccagaaa caagggagct attttcaaat cagaccttcc
aattgagaat tgtgatgctg 900 tctgtcaaac tttagctgga gcaataaata
caaacaaaac cttccaaaat attagtccag 960 tctggattgg agaatgcccc
aaatatgtta aaagtaagag cctaaaacta gcaactggtc 1020 tgagaaatgt
tccacaggca gaaacaagag gattgtttgg agcaatagct gggtttatag 1080
aaggaggatg gacaggtatg gtagacggat ggtacggata ccaccatgaa aattcacagg
1140 ggtctggtta tgcagcagat aaagaaagca ctcagaaagc aatagacggg
atcaccaata 1200 aagtcaattc aatcattgac aaaatgaaca cacaatttga
ggcagtagag catgagttct 1260 caagtctcga aaggagaata ggcaatctga
acaaaagaat ggaagatgga tttttagacg 1320 tgtggacata caatgctgaa
cttctggttc tactggaaaa tgagaggact ttggacatgc 1380 atgatgctaa
tgtaaagaat ctacatgaaa aggtgaaatc acaattaagg gataatgcaa 1440
aggatttggg taatgggtgt tttgaatttt ggcacaaatg cgacaatgaa tgcatcaact
1500 cagttaaaaa tggcacatat gactacccaa agtaccagga agagagcaga
cttaataggc 1560 aggaaataaa atcagtgatg ctggaaaatc tgggagtata
ccaaatcctt gctatttata 1620 gtacggtatc gagcagtctg gttttggtgg
gactgatcat tgccatgggt ctttggatgt 1680 gctcaaatgg ctcaatgcaa
tgcaagatat gtatataatt agaaaaaaac acccttgttt 1740 ctact 1745 38 1467
DNA Influenza A virus 38 agcaaaagca ggagtgaaaa tgaatccaaa
tcagaggata ataacaattg gatccgtctc 60 tctaactatt gcaacagtgt
gtttcctcat gcagattgcc atcctagcaa cgactgtgac 120 actgcatttc
aaacaaaatg aatgcagcat tcccgcaaac aaccaagtaa cgccatgtga 180
accaatagta atagagagga acataacaga gatagtgtat ttgaataata ctaccataga
240 aaaagagatt tgtcctgaag tagtagaata caggaattgg tcaaaaccgc
aatgtcaaat 300 tacagggttt gctcctttct ccaaggacaa ctcaattcgg
ctttctgctg gtggggacat 360 ttggataaca agagaacctt atgtgtcatg
cgaccccagt aaatgttatc aatttgcact 420 cgggcagggg accacgctgg
acaacaaaca ctcaaatggc acaatacatg atagaatccc 480 tcatcggacc
cttttgatga atgaattggg tgttccgttt catttgggaa ccaaacaagt 540
gtgcatagca tggtccagct caagctgtca tgatgggaaa gcatggttgc acgtttgtgt
600 cactggggat gatagaaatg caactgctag tttcatttat gatgggatgc
ttattgacag 660 tattggttcc tggtctcaaa atatcctcag gactcaggag
tcagaatgcg tttgtatcag 720 tggaacttgt acagtagtaa tgactgatgg
aagtgcatca ggaagggcag acactagaat 780 actattcatt agagagggga
aaattgtcca cattagtcca ttgtcaggaa gtgctcagca 840 tgtagaggaa
tgttcttgtt atccccggta cccaaacgtc agatgtgtct gcagagacaa 900
ctggaagggc tctaataggc ccgttataga tataaatatg gcagattata gcattgactc
960 aagttatgtg tgctcaggac ttgttggaga cacaccaagg aacgatgata
gctctagcag 1020 cagcaactgc agggatccta ataatgagag agggaaccca
ggagtgaaag ggtgggcctt 1080 tgataatgga aatgatgtgt ggatgggaag
aacaatcagt aaagattcgc gctcaggcta 1140 tgagaccttc aaggtcattg
gtggttgggc cattgctaat tccaagtcac agaccaatag 1200 acaagtcata
gttgataata acaactggtc tggttattct ggtattttct ctgttgaaag 1260
caaaggctgc atcaataggt gtttttatgt ggagttgata agaggaaggc cacaggagac
1320 tagagtatgg tggacctcaa acagtattgt cgtattttgt ggcacttcag
ggacatatgg 1380 aacaggctca tggcctgatg gggcgaatat cgatttcatg
cctatataag ctttcgcaat 1440 tttagaaaaa aactccttgt ttctact 1467 39
566 PRT Influenza A virus 39 Met Ile Ala Val Ile Ile Ile Ala Val
Leu Ala Thr Ala Gly Lys Ser 1 5 10 15 Asp Lys Ile Cys Ile Gly Tyr
His Ala Asn Asn Ser Thr Thr Gln Val 20 25 30 Asp Thr Ile Leu Glu
Lys Asn Val Thr Val Thr His Ser Val Glu Leu 35 40 45 Leu Glu Asn
Gln Lys Glu Glu Arg Phe Cys Lys Ile Leu Asn Lys Ala 50 55 60 Pro
Leu Asp Leu Arg Gly Cys Thr Ile Glu Gly Trp Ile Leu Gly Asn 65 70
75 80 Pro Gln Cys Asp Leu Leu Leu Gly Asp Gln Ser Trp Ser Tyr Ile
Val 85 90 95 Glu Arg Pro Thr Ala Gln Asn Gly Ile Cys Tyr Pro Gly
Ile Leu Asn 100 105 110 Glu Val Glu Glu Leu Lys Ala Leu Ile Gly Ser
Gly Glu Arg Val Glu 115 120 125 Arg Phe Glu Met Phe Pro Lys Ser Thr
Trp Ala Gly Val Asp Thr Ser 130 135 140 Ser Gly Val Thr Lys Ala Cys
Pro Tyr Thr Ser Gly Ser Ser Phe Tyr 145 150 155 160 Arg Asn Leu Leu
Trp Ile Ile Lys Thr Lys Ser Ala Ala Tyr Pro Val 165 170 175 Ile Lys
Gly Thr Tyr Asn Asn Thr Gly Ser Gln Pro Ile Leu Tyr Phe 180 185 190
Trp Gly Val His His Pro Pro Asp Thr Asn Glu Gln Asn Thr Leu Tyr 195
200 205 Gly Ser Gly Asp Arg Tyr Val Arg Met Gly Thr Glu Ser Met Asn
Phe 210 215 220 Ala Lys Ser Pro Glu Ile Ala Ala Arg Pro Ala Val Asn
Gly Gln Arg 225 230 235 240 Gly Arg Ile Asp Tyr Tyr Trp Ser Val Leu
Lys Pro Gly Glu Thr Leu 245 250 255 Asn Val Glu Ser Asn Gly Asn Leu
Ile Ala Pro Trp Tyr Ala Tyr Lys 260 265 270 Phe Val Ser Thr Asn Ser
Lys Gly Ala Val Phe Lys Ser Asn Leu Pro 275 280 285 Ile Glu Asn Cys
Asp Ala Thr Cys Gln Thr Ile Ala Gly Val Leu Arg 290 295 300 Thr Asn
Lys Thr Phe Gln Asn Val Ser Pro Leu Trp Ile Gly Glu Cys 305 310 315
320 Pro Lys Tyr Val Lys Ser Glu Ser Leu Arg Leu Ala Thr Gly Leu Arg
325 330 335 Asn Ile Pro Gln Ile Glu Thr Arg Gly Leu Phe Gly Ala Ile
Ala Gly 340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly
Trp Tyr Gly Tyr 355 360 365 His His Glu Asn Ser Gln Gly Ser Gly Tyr
Ala Ala Asp Arg Glu Ser 370 375 380 Thr Gln Arg Ala Ile Asp Gly Ile
Thr Asn Lys Val Asn Ser Ile Ile 385 390 395 400 Asp Lys Met Asn Thr
Gln Phe Glu Ala Ile Asp His Glu Phe Ser Asn 405 410 415 Leu Glu Arg
Arg Ile Asp Ser Leu Asn Lys Arg Met Glu Asp Gly Phe 420 425 430 Leu
Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn 435 440
445 Glu Arg Thr Leu Asp Leu His Asp Ala Asn Val Lys Asn Leu Tyr Glu
450 455 460 Lys Val Lys Ser Gln Leu Arg Asp Asn Ala Asn Asp Leu Gly
Asn Gly 465 470 475 480 Cys Phe Glu Phe Trp His Lys Cys Asp Asn Glu
Cys Ile Glu Ser Val 485 490 495 Lys Asn Gly Thr Tyr Asp Tyr Pro Lys
Tyr Gln Asp Glu Ser Lys Leu 500 505 510 Asn Arg Gln Glu Ile Glu Ser
Val Lys Leu Glu Asn Leu Gly Val Tyr
515 520 525 Gln Ile Leu Ala Ile Tyr Ser Thr Val Ser Ser Ser Leu Val
Leu Val 530 535 540 Gly Leu Ile Ile Ala Met Gly Leu Trp Met Cys Ser
Asn Gly Ser Met 545 550 555 560 Gln Cys Arg Ile Cys Ile 565 40 470
PRT Influenza A virus 40 Met Asn Pro Asn Gln Lys Ile Leu Cys Thr
Ser Ala Thr Ala Ile Ala 1 5 10 15 Ile Gly Thr Ile Ala Val Leu Ile
Gly Ile Ala Asn Leu Gly Leu Asn 20 25 30 Ile Gly Leu His Leu Lys
Pro Ser Cys Asn Cys Ser Asn Pro Pro Pro 35 40 45 Glu Thr Thr Asn
Val Ser Gln Thr Ile Ile Asn Asn Tyr Tyr Asn Glu 50 55 60 Thr Asn
Val Thr Gln Ile Ser Asn Thr Asn Ile Gln His Met Gly Gly 65 70 75 80
Thr Glu Lys Asp Phe Asn Asn Leu Thr Lys Gly Leu Cys Thr Ile Asn 85
90 95 Ser Trp His Ile Phe Gly Lys Asp Asn Ala Ile Arg Ile Gly Glu
Asn 100 105 110 Ser Asp Val Leu Val Thr Arg Glu Pro Tyr Val Ser Cys
Asp Pro Asp 115 120 125 Glu Cys Arg Phe Tyr Ala Leu Ser Gln Gly Thr
Thr Ile Arg Gly Lys 130 135 140 His Ser Asn Gly Thr Ile His Asp Arg
Ser Gln Tyr Arg Ala Leu Val 145 150 155 160 Ser Trp Pro Leu Ser Ser
Pro Pro Thr Val Tyr Asn Thr Arg Val Glu 165 170 175 Cys Ile Gly Trp
Ser Ser Thr Ser Cys His Asp Gly Lys Ala Arg Met 180 185 190 Ser Ile
Cys Val Ser Gly Pro Asn Asn Asn Ala Ser Ala Val Ile Trp 195 200 205
Tyr Lys Gly Arg Pro Ile Thr Glu Ile Asn Thr Trp Ala Arg Asn Ile 210
215 220 Leu Arg Thr Gln Glu Ser Glu Cys Val Cys His Asn Gly Ile Cys
Pro 225 230 235 240 Val Val Phe Thr Asp Gly Ser Ala Thr Gly Pro Ala
Glu Thr Arg Ile 245 250 255 Tyr Tyr Phe Lys Glu Gly Lys Ile Leu Lys
Trp Glu Pro Leu Thr Gly 260 265 270 Thr Ala Lys His Ile Glu Glu Cys
Ser Cys Tyr Gly Lys Asp Ser Glu 275 280 285 Ile Thr Cys Thr Cys Arg
Asp Asn Trp Gln Gly Ser Asn Arg Pro Val 290 295 300 Ile Gln Ile Asn
Pro Thr Met Met Thr His Thr Ser Gln Tyr Ile Cys 305 310 315 320 Ser
Pro Val Leu Thr Asp Asn Pro Arg Pro Asn Asp Pro Thr Val Gly 325 330
335 Lys Cys Asn Asp Pro Tyr Pro Gly Asn Asn Asn Asn Gly Val Lys Gly
340 345 350 Phe Ser Tyr Leu Asp Gly Asp Asn Thr Trp Leu Gly Arg Thr
Ile Ser 355 360 365 Thr Ala Ser Arg Ser Gly Tyr Glu Met Leu Lys Val
Pro Asn Ala Leu 370 375 380 Thr Asp Asp Arg Ser Lys Pro Thr Gln Gly
Gln Thr Ile Val Leu Asn 385 390 395 400 Thr Asp Trp Ser Gly Tyr Ser
Gly Ser Phe Ile Asp Tyr Trp Ala Lys 405 410 415 Gly Glu Cys Tyr Arg
Ala Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg 420 425 430 Pro Lys Glu
Asp Lys Val Trp Trp Thr Ser Asn Ser Ile Val Ser Met 435 440 445 Cys
Ser Ser Thr Glu Phe Leu Gly Gln Trp Asn Trp Pro Asp Gly Ala 450 455
460 Lys Ile Glu Tyr Phe Leu 465 470 41 567 PRT Influenza A virus 41
Met Ile Ala Ile Ile Val Ile Ala Ile Leu Ala Ala Ala Gly Lys Ser 1 5
10 15 Asp Lys Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Thr Gln
Val 20 25 30 Asp Thr Ile Leu Glu Lys Asn Val Thr Val Thr His Ser
Ile Glu Leu 35 40 45 Leu Glu Asn Gln Lys Glu Glu Arg Phe Cys Lys
Ile Leu Asn Lys Ala 50 55 60 Pro Leu Asp Leu Arg Glu Cys Thr Ile
Glu Gly Trp Ile Leu Gly Asn 65 70 75 80 Pro Gln Cys Asp Leu Leu Leu
Gly Asp Gln Ser Trp Ser Tyr Ile Val 85 90 95 Glu Arg Pro Thr Ala
Gln Asn Gly Ile Cys Tyr Pro Gly Thr Leu Asn 100 105 110 Glu Val Glu
Glu Leu Arg Ala Leu Ile Gly Ser Gly Glu Arg Val Glu 115 120 125 Arg
Phe Glu Met Phe Pro Gln Ser Thr Trp Gln Gly Val Asp Thr Asn 130 135
140 Ser Gly Thr Thr Arg Ser Cys Pro Tyr Ser Thr Gly Asp Pro Ser Phe
145 150 155 160 Tyr Arg Asn Leu Leu Trp Ile Ile Lys Thr Lys Thr Ala
Glu Tyr Pro 165 170 175 Val Ile Lys Gly Ile Tyr Asn Asn Thr Gly Thr
Gln Pro Ile Leu Tyr 180 185 190 Phe Trp Gly Val His His Pro Pro Asn
Thr Asp Glu Gln Asp Thr Leu 195 200 205 Tyr Gly Ser Gly Asp Arg Tyr
Val Arg Met Gly Thr Glu Ser Met Asn 210 215 220 Phe Ala Lys Ser Pro
Glu Ile Ala Ala Arg Pro Ala Val Asn Gly Gln 225 230 235 240 Arg Gly
Arg Ile Asp Tyr Tyr Trp Ser Val Leu Lys Pro Gly Glu Thr 245 250 255
Leu Asn Val Glu Ser Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Tyr 260
265 270 Lys Phe Val Asn Thr Asn Ser Lys Gly Ala Val Phe Arg Ser Asp
Leu 275 280 285 Pro Ile Glu Asn Cys Asp Ala Thr Cys Gln Thr Ile Ala
Gly Val Leu 290 295 300 Arg Thr Asn Lys Thr Phe Gln Asn Val Ser Pro
Leu Trp Ile Gly Glu 305 310 315 320 Cys Pro Lys Tyr Val Lys Ser Glu
Ser Leu Arg Leu Ala Thr Gly Leu 325 330 335 Arg Asn Val Pro Gln Ile
Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala 340 345 350 Gly Phe Ile Glu
Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly 355 360 365 Tyr His
His Glu Asn Ser Gln Gly Ser Gly Tyr Ala Ala Asp Arg Glu 370 375 380
Ser Thr Gln Lys Ala Val Asn Arg Ile Thr Asn Lys Val Asn Ser Ile 385
390 395 400 Ile Asn Lys Met Asn Thr Gln Phe Glu Ala Val Asp His Glu
Phe Ser 405 410 415 Asn Leu Glu Arg Arg Ile Asp Asn Leu Asn Lys Arg
Met Gln Asp Gly 420 425 430 Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu
Leu Leu Val Leu Leu Glu 435 440 445 Asn Glu Arg Thr Leu Asp Met His
Asp Ala Asn Val Lys Asn Leu His 450 455 460 Glu Lys Val Lys Ser Gln
Leu Arg Asp Asn Ala Asn Asp Leu Gly Asn 465 470 475 480 Gly Cys Phe
Glu Phe Trp His Lys Cys Asp Asn Glu Cys Ile Glu Ser 485 490 495 Val
Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Gln Thr Glu Ser Lys 500 505
510 Leu Asn Arg Leu Lys Ile Glu Ser Val Lys Leu Glu Asn Leu Gly Val
515 520 525 Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ser Ser Ser Leu
Val Leu 530 535 540 Val Gly Leu Ile Met Ala Met Gly Leu Trp Met Cys
Ser Asn Gly Ser 545 550 555 560 Met Gln Cys Asn Val Cys Ile 565 42
450 PRT Influenza A virus 42 Met Asn Pro Asn Gln Lys Ile Ile Thr
Ile Gly Ser Ile Cys Met Val 1 5 10 15 Val Gly Ile Ile Ser Leu Met
Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30 Trp Val Ser His Ile
Ile Gln Thr Gly His Pro Asn Gln Pro Gly Pro 35 40 45 Cys Asn Gln
Ser Ile Asn Phe Tyr Thr Glu Gln Ala Ala Ala Ser Val 50 55 60 Thr
Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Ser Gly Trp Ala Ile 65 70
75 80 Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val
Phe 85 90 95 Val Met Arg Glu Pro Phe Val Ser Cys Ser His Leu Glu
Cys Arg Thr 100 105 110 Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp
Lys His Ser Asn Gly 115 120 125 Thr Val Lys Asp Arg Ser Pro Tyr Arg
Thr Leu Met Ser Cys Pro Val 130 135 140 Gly Glu Ala Pro Ser Pro Tyr
Asn Ser Arg Phe Glu Ser Val Ala Trp 145 150 155 160 Ser Ala Ser Ala
Cys His Asp Gly Ile Ser Trp Leu Thr Ile Gly Ile 165 170 175 Ser Gly
Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile 180 185 190
Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu Arg Thr Gln 195
200 205 Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr Val Met
Thr 210 215 220 Asp Gly Pro Ser Asn Glu Gln Ala Ser Tyr Lys Ile Phe
Lys Ile Glu 225 230 235 240 Lys Gly Lys Val Val Lys Ser Val Glu Leu
Asn Ala Pro Asn Tyr His 245 250 255 Tyr Glu Glu Cys Ser Cys Tyr Pro
Asp Ala Gly Glu Ile Thr Cys Val 260 265 270 Cys Arg Asp Asn Trp His
Gly Ser Asn Arg Pro Trp Val Ser Phe Asn 275 280 285 Gln Asn Leu Glu
Tyr Gln Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly 290 295 300 Asp Ser
Pro Arg Pro Asn Asp Gly Thr Gly Ser Cys Gly Pro Val Ser 305 310 315
320 Leu Asn Gly Glu Tyr Gly Val Lys Gly Phe Ser Phe Lys Tyr Gly Asp
325 330 335 Gly Val Trp Ile Gly Arg Thr Lys Ser Thr Ser Ser Arg Ser
Gly Phe 340 345 350 Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr
Asp Ser Asn Phe 355 360 365 Ser Leu Lys Gln Asp Ile Ile Ala Ile Thr
Asp Trp Ser Gly Tyr Ser 370 375 380 Gly Ser Phe Val Gln His Pro Glu
Leu Thr Gly Leu Asn Cys Met Arg 385 390 395 400 Pro Cys Phe Trp Val
Glu Leu Ile Arg Gly Arg Pro Lys Glu Lys Thr 405 410 415 Ile Trp Thr
Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp 420 425 430 Thr
Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Val Pro Phe Thr Ile 435 440
445 Asp Lys 450 43 566 PRT Influenza A virus 43 Met Ile Ala Ile Ile
Ile Leu Ala Ile Val Val Ser Thr Ser Lys Ser 1 5 10 15 Asp Arg Ile
Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Thr Gln Val 20 25 30 Asp
Thr Ile Leu Glu Lys Asn Val Thr Val Thr His Ser Val Glu Leu 35 40
45 Leu Glu Asn Gln Lys Glu Asn Arg Phe Cys Arg Val Leu Asn Lys Ala
50 55 60 Pro Leu Asp Leu Met Asp Cys Thr Thr Glu Gly Trp Ile Leu
Gly Asn 65 70 75 80 Pro Arg Cys Asp Asn Leu Leu Gly Asp Gln Ser Trp
Ser Tyr Ile Val 85 90 95 Glu Arg Pro Asp Ala Gln Asn Gly Ile Cys
Tyr Pro Gly Val Leu Lys 100 105 110 Glu Thr Glu Glu Leu Lys Ala Leu
Ile Gly Ser Ile Asp Ser Ile Gln 115 120 125 Arg Phe Glu Met Phe Pro
Lys Ser Thr Trp Thr Gly Val Asp Thr Asn 130 135 140 Ser Gly Val Thr
Ser Ala Cys Pro Tyr Asn Gly Glu Ser Ser Phe Tyr 145 150 155 160 Arg
Asn Leu Leu Trp Ile Ile Lys Ile Arg Ser Asp Pro Tyr Ser Leu 165 170
175 Ile Lys Gly Thr Tyr Thr Asn Thr Gly Ser Gln Pro Ile Leu Tyr Phe
180 185 190 Trp Gly Val His His Pro Pro Asp Glu Val Glu Gln Ala Asn
Leu Tyr 195 200 205 Gly Ile Gly Thr Arg Tyr Val Arg Met Gly Thr Glu
Ser Met Asn Phe 210 215 220 Ala Lys Gly Pro Glu Ile Ala Gly Arg Pro
Pro Ala Asn Gly Gln Arg 225 230 235 240 Gly Arg Ile Asp Tyr Tyr Trp
Ser Val Leu Lys Pro Gly Glu Thr Leu 245 250 255 Asn Val Glu Ser Asn
Gly Asn Leu Ile Ala Pro Trp Tyr Ala Tyr Lys 260 265 270 Phe Thr Ser
Ser Arg Asn Lys Gly Ala Ile Phe Lys Ser Asp Leu Pro 275 280 285 Ile
Glu Asn Cys Asp Ala Val Cys Gln Thr Leu Ala Gly Ala Ile Asn 290 295
300 Thr Asn Lys Thr Phe Gln Asn Ile Ser Pro Val Trp Ile Gly Glu Cys
305 310 315 320 Pro Lys Tyr Val Lys Ser Lys Ser Leu Lys Leu Ala Thr
Gly Leu Arg 325 330 335 Asn Val Pro Gln Ala Glu Thr Arg Gly Leu Phe
Gly Ala Ile Ala Gly 340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met
Val Asp Gly Trp Tyr Gly Tyr 355 360 365 His His Glu Asn Ser Gln Gly
Ser Gly Tyr Ala Ala Asp Lys Glu Ser 370 375 380 Thr Gln Lys Ala Ile
Asp Gly Ile Thr Asn Lys Val Asn Ser Ile Ile 385 390 395 400 Asp Lys
Met Asn Thr Gln Phe Glu Ala Val Glu His Glu Phe Ser Ser 405 410 415
Leu Glu Arg Arg Ile Gly Asn Leu Asn Lys Arg Met Glu Asp Gly Phe 420
425 430 Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu
Asn 435 440 445 Glu Arg Thr Leu Asp Met His Asp Ala Asn Val Lys Asn
Leu His Glu 450 455 460 Lys Val Lys Ser Gln Leu Arg Asp Asn Ala Lys
Asp Leu Gly Asn Gly 465 470 475 480 Cys Phe Glu Phe Trp His Lys Cys
Asp Asn Glu Cys Ile Asn Ser Val 485 490 495 Lys Asn Gly Thr Tyr Asp
Tyr Pro Lys Tyr Gln Glu Glu Ser Arg Leu 500 505 510 Asn Arg Gln Glu
Ile Lys Ser Val Met Leu Glu Asn Leu Gly Val Tyr 515 520 525 Gln Ile
Leu Ala Ile Tyr Ser Thr Val Ser Ser Ser Leu Val Leu Val 530 535 540
Gly Leu Ile Ile Ala Met Gly Leu Trp Met Cys Ser Asn Gly Ser Met 545
550 555 560 Gln Cys Lys Ile Cys Ile 565 44 469 PRT Influenza A
virus 44 Met Asn Pro Asn Gln Arg Ile Ile Thr Ile Gly Ser Val Ser
Leu Thr 1 5 10 15 Ile Ala Thr Val Cys Phe Leu Met Gln Ile Ala Ile
Leu Ala Thr Thr 20 25 30 Val Thr Leu His Phe Lys Gln Asn Glu Cys
Ser Ile Pro Ala Asn Asn 35 40 45 Gln Val Thr Pro Cys Glu Pro Ile
Val Ile Glu Arg Asn Ile Thr Glu 50 55 60 Ile Val Tyr Leu Asn Asn
Thr Thr Ile Glu Lys Glu Ile Cys Pro Glu 65 70 75 80 Val Val Glu Tyr
Arg Asn Trp Ser Lys Pro Gln Cys Gln Ile Thr Gly 85 90 95 Phe Ala
Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly 100 105 110
Asp Ile Trp Ile Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Ser Lys 115
120 125 Cys Tyr Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Asp Asn Lys
His 130 135 140 Ser Asn Gly Thr Ile His Asp Arg Ile Pro His Arg Thr
Leu Leu Met 145 150 155 160 Asn Glu Leu Gly Val Pro Phe His Leu Gly
Thr Lys Gln Val Cys Ile 165 170 175 Ala Trp Ser Ser Ser Ser Cys His
Asp Gly Lys Ala Trp Leu His Val 180 185 190 Cys Val Thr Gly Asp Asp
Arg Asn Ala Thr Ala Ser Phe Ile Tyr Asp 195 200 205 Gly Met Leu Ile
Asp Ser Ile Gly Ser Trp Ser Gln Asn Ile Leu Arg 210 215 220 Thr Gln
Glu Ser Glu Cys Val Cys Ile Ser Gly Thr Cys Thr Val Val 225 230 235
240 Met Thr Asp Gly Ser Ala Ser Gly Arg Ala Asp Thr Arg Ile Leu Phe
245 250 255 Ile Arg Glu Gly Lys Ile Val His Ile Ser Pro Leu Ser Gly
Ser Ala 260 265 270 Gln His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr
Pro Asn Val Arg 275 280 285 Cys Val Cys Arg Asp Asn Trp Lys Gly Ser
Asn Arg Pro Val Ile Asp 290 295 300 Ile Asn Met Ala Asp Tyr Ser Ile
Asp Ser Ser Tyr Val Cys Ser Gly 305 310 315 320 Leu Val Gly Asp Thr
Pro Arg Asn Asp Asp Ser Ser Ser Ser Ser Asn 325 330
335 Cys Arg Asp Pro Asn Asn Glu Arg Gly Asn Pro Gly Val Lys Gly Trp
340 345 350 Ala Phe Asp Asn Gly Asn Asp Val Trp Met Gly Arg Thr Ile
Ser Lys 355 360 365 Asp Ser Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile
Gly Gly Trp Ala 370 375 380 Ile Ala Asn Ser Lys Ser Gln Thr Asn Arg
Gln Val Ile Val Asp Asn 385 390 395 400 Asn Asn Trp Ser Gly Tyr Ser
Gly Ile Phe Ser Val Glu Ser Lys Gly 405 410 415 Cys Ile Asn Arg Cys
Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Gln 420 425 430 Glu Thr Arg
Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly 435 440 445 Thr
Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asn Ile 450 455
460 Asp Phe Met Pro Ile 465 45 1464 DNA Influenza A virus 45
agcaaaagca gggtgatcga gaatgaatcc aaatcagaaa ctatttgcat tatctggagt
60 ggcaatagca cttagtgtac tgaacttatt gataggaatc tcaaacgtcg
gattgaacgt 120 atctctacat ctaaaggaaa aaggacccaa acaggaggag
aatttaacat gcacgaccat 180 taatcaaaac aacactactg tagtagaaaa
cacatatgta aataatacaa caataattac 240 caagggaact gatttgaaaa
caccaagcta tctgctgttg aacaagagcc tgtgcaatgt 300 tgaagggtgg
gtcgtgatag caaaagacaa tgcagtaaga tttggggaaa gtgaacaaat 360
cattgttacc agggagccat atgtatcatg cgacccaaca ggatgcaaaa tgtatgcctt
420 gcaccaaggg actaccatta ggaacaaaca ttcaaatgga acgattcatg
acagaacagc 480 tttcagaggt ctcatctcca ctccattggg cactccacca
accgtaagta acagtgactt 540 tatgtgtgtt ggatggtcaa gcacaacttg
ccatgatggg attgctagga tgactatctg 600 tatacaagga aataatgaca
atgctacagc aacggtttat tacaacagaa ggctgaccac 660 taccattaag
acctgggcca gaaacattct gaggactcaa gaatcagaat gtgtgtgcca 720
caatggcaca tgtgcagttg taatgaccga cggatcggct agtagtcaag cctatacaaa
780 agtaatgtat ttccacaagg gattagtagt taaggaggag gagttaaggg
gatcagccag 840 acatattgag gaatgctcct gttatggaca caatcaaaag
gtgacctgtg tgtgcagaga 900 taactggcag ggagcaaaca ggcctattat
agaaattgat atgagcacat tggagcacac 960 aagtagatac gtgtgcactg
gaattctcac agacaccagc agacctgggg acaaatctag 1020 tggtgattgt
tccaatccaa taactgggag tcccggcgtt ccgggagtga agggattcgg 1080
gtttctaaat ggggataaca catggcttgg taggaccatc agccccagat caagaagtgg
1140 attcgaaatg ttgaaaatac ctaatgcagg tactgatccc aattctagaa
tagcagaacg 1200 acaggaaatt gtcgacaata acaattggtc aggctattcc
ggaagcttta ttgactattg 1260 gaatgataac agtgaatgct acaatccatg
cttttacgta gagttaatta gaggaagacc 1320 cgaagaggct aaatacgtat
ggtgggcaag taacagtcta attgccctat gtggaagccc 1380 attcccagtt
gggtctggtt ccttccccga tggggcacaa atccaatact tttcgtaaaa 1440
tgcaaaaaca cccttgtttc tact 1464
* * * * *