Note: Descriptions are shown in the official language in which they were submitted.
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NUCELOTIDE SEQUENCE OF
INFLUENZA A/Udorn/72 (H3N2) GENOME
Field Of The Invention
This invention relates to the nucleotide
sequence of each of the segments of the Influenza
virus strain A/Udorn/72 (H3N2).
Background Of The Invention
The influenza viruses consist of subtypes
designated A, B and C. Influenza A viruses possess a
single negative strand RNA genome of eight segments,
which encodes 10 polypeptides (proteins) that are
required for the life cycle of the virus. The order
of the proteins is as follows:
Segment: 1 2 3 4 5 6 7 8
Protein: PB2 PB1 PA HA NP NA M1 NS1
Spliced M2 NS2
Product:
Each of the eight RNA,segments of the
influenza subtype A complete genome is encapsidated
with multiple subunits of the nucleocapsid protein
(NP) and associated with a few molecules of the
trimeric polymerase (PB1, PB2 and PA subunits),
thereby forming the ribonucleoprotein complex (RNP)
(Lamb, R.A., pages 1-87 of The Influenza Viruses, R.M.
Krug, ed. (Plenum Press, 1989)). The NP protein is a
structural and transcription/replication regulatory
protein. Surrounding these structures is a.layer of
the matrix protein (M1), which is the major structural
component of the virion and appears to serve as a
nexus between the core and the viral envelope. This
host cell-derived envelope is studded with the two
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major virally encoded surface glycoproteins
hemagglutinin (HA) and neuraminidase (NA), which are
antigenic determinants, and a much smaller amount of a
nonglycosylated small protein M2 (Lamb 1989; Lamb,
R.A., et al., Cell, 40, 627-633 (1985)). The M2
protein is a spliced gene product from segment #7
(which also encodes the M1 protein) which has ion
channel activity; spliced genes are only present in
infected cells. Similarly, the nonstructural NS2
protein is a spliced gene product from segment #8
(which also encodes the nonstructural NS1 protein);
spliced genes are only present in infected cells. The
HA glycoprotein is cleaved by a protease to form HAl
and HA2.
Influenza viral infection is initiated by
the attachment of the surface hemagglutinin to a
sialic acid-containing cellular receptor. This first
virus-cell interaction induces the uptake of the viral
particle into the cell by receptor-mediated
endocytosis. Under the low pH conditions of the
endosome, the HA undergoes a conformational change
that facilitates the interaction of the hydrophobic NH2
terminal domain of HA2 and the endosomal membrane,
resulting in membrane fusion and subsequent release of
the core RNPs and matrix protein (M1) into the
cytosol. Disassociation of the RNPs and matrix
proteins occurs in the cytosol before the RNPs are
translocated to the nucleus where transcription and
replication of the complete genome take place (Martin,
K., and Helenius, A., Cell, 67, 117-130 (1991);
Shapiro, G.I., et al., J. Virology, 61, 764-773
(1987) ) .
Following primary transcription, newly
synthesized proteins initiate the replication of the
viral genome which in turn increases transcription and
protein synthesis. At this point of the virus life
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cycle, the surface glycoproteins HA and NA start to
accumulate at discrete areas of the plasma membrane
from where newly assembled virus will be released.
Virus assembly is assumed to begin via some sort of
interaction between the cytoplasmic and/or
transmembrane domains of the four virally encoded
structural proteins: the membrane anchored proteins
(HA, NA and M2), and the underlying matrix protein
(M1), which in turn maintains a close association with
the RNPs (Garoff, H., et al., Microbiology and
Molecular Biology Reviews, 62, 1171-1190 (1998);
Nayak, D.P., ASM News, 62, 411-414 (1996)). The
contacts between matrix protein M1 and the RNP
complexes, as well as the mechanism by which a
complete set of eight RNPs gets incorporated into the
mature virion particle, have not been well defined.
Specific molecular contacts among the structural
components are assumed to dictate how the process of
morphogenesis initiates and progresses to the point of
mature particle assembly and budding from the surface
of the host cell.
There are a large number of influenza A
virus subtypes and antigenic variants' that
continuously evolve by mutations introduced by the RNA
polymerase during the replication of the viral genome.
Mutations also occur by genetic reassortment between
the segments of two different subtypes when they
happen to infect the same cellular host. This
biological characteristic of influenza A virus gives
rise to new virus strains, which necessitates periodic
updating of the immunogenic compositions against
influenza. Therefore, the availability of a complete
nucleotide sequence and a full length clone of one
particular strain of influenza A virus is essential
for the development of new and innovative immunogenic
compositions against influenza. Thus, there a.s a need
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to determine the complete nucleotide sequence of an
influenza A virus and to construct a full length clone
containing that sequence.
In particular. the influenza A strain
designated Influenza A/Udorn/72 (H3N2) was first
isolated in 1972. The nucleotide sequences of the
polymerase genes PB2, PBl and PA have not been
sequenced. The nucleotide sequences of the segments
encoding HA, NP, M1, M2, NS1 and NS2 have been
sequenced. However, those sequences were obtained a
number of years ago using instrumentation which may
not have provided accurate sequences. Thus, there is
a need to repeat the sequencing of those segments in
order to provide updated, accurate sequences.
Summary Of The Invention
It is an object of this invention to
determine the complete nucleotide sequence of the
Influenza A/Udorn/72 (H3N2) viral strain and the
spliced products of the segment #7 and #8 mRNAs. It
is a~further object of this invention to generate a
clone of each of the eight genes and spliced messages.
It is yet another object of this invention
to determine the deduced amino acid sequences of all
ten of the products of these genes and spliced
messages.
These and other objects of this invention
are achieved by the elucidation of isolated nucleic
acid molecules constituting the nucleotide sequences
of all of the segments of the Influenza A/Udorn/72
(H3N2) strain which together comprise the complete
nucleotide sequence of that strain, as well as their
spliced messages. All of these nucleotide sequences
are presented in positive strand, antigenomic message
sense.
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In particular, the complete nucleotide
sequence consists of SEQ ID N0:1, SEQ ID N0:3, SEQ ID
N0:5, SEQ ID N0:7, SEQ ID N0:9, SEQ ID N0:11, SEQ ID
N0:13 and SEQ ID N0:17, and biological equivalents
thereof .
Alternatively, the complete nucleotide
sequence includes a variant of the HA sequence,
designated HA (P1) (SEQ ID N0:21), and consists of SEQ
ID N0:1, SEQ ID N0:3, SEQ ID N0:5, SEQ ID N0:9, SEQ ID
N0:11, SEQ ID N0:13, SEQ ID N0:17 and SEQ ID N0:21,
and biological equivalents thereof.
In another embodiment of this invention,
these isolated nucleic acid molecules are directed to
individual isolated influenza A virus nucleic acid
molecules which encode individual proteins and are
selected from the group consisting of SEQ ID N0:1, SEQ
ID N0:3, SEQ ID N0:5, SEQ ID N0:7, SEQ ID N0:11 and
SEQ ID N0:21 (the variant of SEQ ID N0:7), and
biological equivalents thereof.
In yet another embodiment of this invention,
these individual isolated nucleic acid molecules
encode proteins having amino acid sequences selected
from the group consisting of SEQ ID N0:2, SEQ ID N0:4
and SEQ ID N0:6, and biological equivalents thereof.
In a further embodiment of this invention,
there are provided individual isolated influenza A
virus amino acid sequences, where said sequences are
selected from the group consisting of SEQ ID N0:2, SEQ
ID N0:4 and SEQ ID N0:6, and biological equivalents
thereof.
In a still further embodiment of this
invention, there is provided an individual influenza A
virus amino acid sequence, where said sequence has the
sequence of SEQ ID N0:8 or SEQ ID N0:12.
In still another embodiment of this
invention, an isolated nucleic acid molecule having
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the nucleotide sequence of a segment of the Influenza
A/Udorn/72 (H3N2) strain is used: (1) to design
polymerase chain reaction (PCR) primers for use in a
PCR assay to detect. the presence of the corresponding
virus segment in a sample; or (2) to design and select
peptides for use in an ELISA to detect the presence of
the corresponding protein produced by that segment in
a sample.
Detailed Description of the Invention
The nucleotide sequences and deduced amino
acid sequences of several of the segments of the
Influenza A/Udorn/72 (H3N2) strain have been
published. Yuferov et al. reported the sequence of
the HA gene of segment 4 (Yuferov, V.P., et al.,
Proceedings of the Academy of Sciences of the USSR,
278, 738-742 (1984)). Buckler-White et al. reported
the sequence of the NP gene of segment 5 (Buckler-
White, A.J., and Murphy, B.R., Virology, 155, 345-355
(1986)). Markoff et al. reported the sequence of the
NA gene of segment 6 (Markoff, L., and Lai, C.J.,
Virology, 119, 288-297 (1982)). Lamb et al. in 1981
reported the sequence of the' M1 gene and the M2 gene
splice product of segment 7 (M1 in Lamb. R.A., and
Lai, C.J., Virology, 112, 746-751 (1981)("Lamb
Virology 1981"), and M2 in Lamb, R.A., et al., Proc.
Natl. Acad. Sci. USA, 78, 4170-4174 (1981)("Lamb PNAS
1981")). Lamb et al. in 1980 reported the sequence of
the NS1 gene and the NS2 gene splice product of
segment 8 (Lamb, R.A., and Lai, C.J., Cell, 21, 475-
485 (1980)
In order to resequence these segments, as
well as to sequence those segments not previously
elucidated, the Tnfluenza A/Udorn/72 (H3N2) strain was
grown in and purified from Madin-Darby canine kidney
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(MDCK) cells. The viral RNA was extracted from
purified virions and amplified by RT-PCR using
termini-specific primers. Gel purified genes were
cloned into pGemT vectors (Promega) and sequenced
using multiple sets of primers. The sequences of the
termini were determined by 3' and 5' ligation and
sequencing of the RT-PCR fragments crossing the.
junction (Galarza, J.M., et al., J. Virol., 70, 2360-
2368 (1996)). The M2 and NS2 genes are spliced
products of the mRNA of segments seven and eight,
respectively. These genes were recovered from mRNA
purified from MDCK-Influenza A/Udorn-infected cells by
RT-PCR using oligo-dT and gene-specific primers. The
RT-PCR products were gel purified and cloned into
pGemT vectors. All gene sequences were determined by
using a fluorescence dye terminator with AmpliTaq DNA
polymerase (Perkin-Elmer) and an Applied Biosystems
ABI 377 DNA sequencer. This procedure was repeated
with multiple clones for each fragment; the sequences
obtained were consistent.
The complete genome of the Influenza
A/Udorn/72 (H3N2) strain totaled 13628 nucleotides.
This is the first description of the complete
nucleotide sequence of the Influenza A/Udorn/72 (H3N2)
strain.
The eight segments were sequenced in their
coding regions, and their 5' and 3' non-coding
regions, including regulatory sequences such as
promoters, enhancers and polyadenylation signals. The
segments, their number of nucleotides, their isolated
nucleic acid molecule sequences (shown in positive
strand, antigenomic, message sense, that is, in 5' to
3' orientation), their coding regions and amino acid
translations are as follows:
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Segment #1: 2341 nucleotides, SEQ ID N0:1, coding
region nucleotides 28-2304, encodes PB2, 759 amino
acids (SEQ ID N0:2).
Segment #2: 2341 nucleotides, SEQ ID N0:3, coding
region nucleotides 25-2295, encodes PB1, 757 amino
acids (SEQ ID N0:4).
Segment #3: 2233 nucleotides, SEQ ID N0:5, coding
region nucleotides 25-2172, encodes PA, 716 amino
acids (SEQ ID N0:6).
Segment #4: 1765 nucleotides, SEQ ID N0:7, coding
region nucleotides 30-1727, encodes HA, 566 amino
acids (SEQ ID N0:8).
SEQ ID N0:7 and SEQ ID N0:8 differ from the
published Yuferov et al. nucleotide and amino acid
sequences for HA in several respects.
Yuferov SEQ ID NO: 7 or 8
Codon # Amino Acid # Codon # Amino Acid #
GAC Asp AAC Asn
81-83 18 81-83 18
GGT Gly GGG Gly
1101-1103 358 1101-1103 358
TAC Tyr TTC Phe
1485-1487 486 1485-1487 486
GAC Asp AAC Asn
1614-1616 529 1614-1616 529
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Segment #5: 1565 nucleotides, SEQ ID N0:9, coding
region nucleotides 46-1539, encodes NP, 498 amino
acids (SEQ ID N0:10).
SEQ ID N0:9 and SEQ ID NO:10 are identical
to the published Buckler-White et al. nucleotide and
amino acid sequences for NP.
Segment #6: 1466 nucleotides, SEQ ID N0:11, coding
region nucleotides 20-1426, encodes NA, 469 amino
acids (SEQ ID N0:12).
SEQ ID N0:11 and SEQ ID N0:12 differ from
the published Markoff et al. nucleotide and amino acid
sequences for NA in several respects.
Markoff SEQ ID NO: 11 or 12
Codon # Amino Acid # Codon # Amino Acid #
CAG Gln CTG Leu
104-106 29 104-106 29
GGA Gly GCA Ala
546-548 177 546-548 177
CAA Gln CAG Gln
695-697 226 695-697 226
ACA Thr GCA Ala
992-994 325 992-994 325
There are also several differences in the leader
sequence upstream of the methionine at amino acid 1.
Segment #7: 1027 nucleotides, SEQ ID N0:13, coding
region nucleotides 26-781, encodes M1, 252 amino acids
(SEQ ID N0:14).
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SEQ ID N0:13 and SEQ ID NO:14 are identical
to the published Lamb Virology 1981 nucleotide and
amino acid sequences for M1.
Segment #7, spliced product: 322 nucleotides, SEQ ID
N0:15, coding region nucleotides 26-316, encodes M2,
97 amino acids (SEQ ID N0:16) [first spliced amino
acid at residue 10, nucleotides 53-55].
SEQ ID N0:15 and SEQ ID N0:16 are identical
to the published Lamb PNAS 1981 nucleotide and amino
acid sequences for M2.
Segment #8: 890 nucleotides, SEQ ID N0:17, coding
region nucleotides 27-737, encodes NS1, 237 amino
acids (SEQ TD NO: 18).
SEQ ID N0:17 and SEQ ID N0:18 are identical ,
to the published Lamb et a1. 1980 nucleotide and amino
acid sequences for NS1.
Segment #8, spliced product: 402 nucleotides, SEQ ID
N0:19, coding region nucleotides 27-389, encodes NS2,
121 amino acids (SEQ ID N0:20) [first spliced amino
acid at residue 11, nucleotides 57-59].
SEQ ID N0:19 and SEQ ID N0:20 are identical
to the published Lamb et al. 1980 nucleotide and amino
acid sequences for NS2.
As mentioned above, the is also an alternate
HA sequence, designated HA (P1):
Segment #4: 1764 nucleotides, SEQ ID N0:21, coding
region nucleotides 30-1727, encodes HA, 566 amino
acids (SEQ ID N0:22).
Without being bound by theory, the HA (P1)
sequence may incorporate minor mutations from the
Influenza A/Udorn/72 (H3N2) strain which occurred over
time. The HA (P1) sequence (SEQ ID N0:21) has 1764
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nucleotides, which is one fewer than in the HA
sequence (SEQ ID N0:7), due to a one nucleotide
deletion in the non,-coding region of segment #4
(position 1756) .
The two HA sequences of this invention
differ a.n their nucleotide and amino acid sequences as
follows:
Nucleotide _HA HA (P1) Amino Acid
Position Change
35 G A Silent
81 A G Asn to Asp
1103 G T Silent
1486 T A Phe to Tyr
1614 A G Asn to Asp
1756 T deletion Outside coding
region
The HA (P1) sequence also differs slightly
from the Yuferov et al. HA nucleotide sequence as
follows:
Nucleotide HA (P1) Yuferov Amino Acid
PO S 3. tl.On Change
35 A G Silent
1756 deletion T Outside coding
region
The nucleic acid sequences of the mRNAs of
the segments, which are in genome, negative sense
(that is, in 3' to 5' orientation), are the
complements of the positive strand, antigenomic,
message sense sequences set forth above.
In addition to the nucleotide sequences
which encode the Influenza A/Udorn/72 (H3N2) viral
proteins set forth above, the present invention
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further comprises isolated nucleic acid molecules
containing individual influenza A virus nucleotide
sequences which, by virtue of"the redundancy of the
genetic code, are biologically equivalent to those
sequences which encode the viral proteins, that is,
these other nucleotide sequences are characterized by
nucleotide sequences which differ from those set forth
herein, but which encode a protein having the same
amino acid sequence as that encoded by any of the
nucleotide sequences in SEQ ID~N0:1, SEQ ID N0:3, SEQ
ID N0:5, SEQ ID N0:7, SEQ ID N0:11 and SEQ ID N0:21.
In particular, the invention contemplates
those nucleotide sequences which are sufficiently
duplicative of any of the sequences of SEQ ID N0:1,
SEQ ID N0:3, SEQ ID N0:5, SEQ ID N0:7, SEQ ID N0:11
and SEQ ID NO:21, so as to permit hybridization
therewith under standard high stringency Southern
hybridization conditions, such as those described in
Sambrook et al. (Sambrook, J., et al., Molecular
Cloning: A Laboratory Manual, 2nd ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989) ) .
This invention also comprises nucleotide
sequences which encode amino acid sequences which
differ from those of the Influenza A/Udorn/72 (H3N2)
viral proteins, but which are biologically equivalent
to those described for one of these viral proteins
(SEQ ID N0:2, SEQ ID N0:4 and SEQ ID N0:6). Such
amino acid sequences may be said to be biologically
equivalent to any of the viral proteins if their
sequences differ only by minor deletions from,
insertions into or substitutions to the viral protein
sequences, such that the tertiary configurations of
the sequences are essentially unchanged from those of
the viral proteins.
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For example, a codon for the amino acid
alanine, a hydrophobic amino acid, may be substituted
by a codon encoding another less hydrophobic residue,
such as glycine, or a more hydrophobic residue, such
as valine, leucine, or isoleucine. Similarly, changes
which result in substitution of one negatively charged
(acidic) residue for another, such as aspartic acid
for glutamic acid, or one positively charged (basic)
residue for another, such as lysine for arginine or
histidine, as well as changes based on similarities of
residues in their hydropathic index, can also be
expected to produce a biologically equivalent product.
Nucleotide changes which result in alteration of the
N-terminal or C-terminal portions of the protein
molecule would also not be expected to alter the
activity of the protein.
Each of the proposed modifications is well
within the routine skill in the art, as is determi-
nation of retention of structural and biological
activity of the encoded products.
This invention further comprises the
isolated amino acid sequences comprising the
individual influenza A virus amino acid sequences
selected from the group consisting of SEQ ID N0:2, SEQ
ID N0:4 and SEQ ID N0:6.
This invention still further comprises the ,
individual isolated amino acid sequences of,SEQ ID
N0:8 and SEQ ID N0:12.
The fundamental biological properties of the
Influenza A/Udorn/72 (H3N2) virus depend on its
genome, which consists of eight segments. The
segments of influenza virus consists of a single
strand of negative polarity RNA, and each viral strain
has its own specific nucleotide sequence.
Complete differential identification between
viral strains is, therefore, necessary to determine
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the nucleotide sequence of the virus. Since the
entire specific nucleotide sequence, consisting of
13,628 bases, has been clearly determined as described
herein, the virus can be identified at the genetic
level, and the identification technique can therefore
provide an absolute determination.
The sequences described herein are useful to
provide an identification method comprising detecting
a part of the nucleotide sequence of influenza viral
antigenomic DNA using the polymerase chain reaction
(PCR) method, as well as to generate peptides to
detect an antigen produced by that DNA using an ELISA.
Having a completely sequenced and cloned
genome of one particular influenza strain is
advantageous for the research and development of new
and innovative immunogenic compositions based on this
genetic blueprint.
Defined mutations are introduced where
desired into one or more of the segments whose
sequences are set forth herein. One or more mutations
are introduced by using site-directed mutagenesis.
However, a mutant influenza virus cannot be directly
generated from viral RNA, because neither the genomic
viral RNA nor the antigenomic cDNA can serve as a
direct template for synthesis. Instead, the viral
RNA, after its encapsidation by NP, must be
transcribed into positive-sense mRNA by the viral RNA
polymerase complex.
Palese et al. (U. S. Patent 5,166,057, which
is hereby incorporated by reference) described a
reverse genetics helper virus-dependent system for the
"rescue" of an influenza A virus segment. Briefly, a
ribonucleoprotein (RNP) complex is generated by in
vitro synthesis in the presence of the three
polymerase proteins and NP. The RNP complex is then
used to transfect eukaryotic cells. Subsequent
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infection with influenza A helper virus results in the
generation of viruses possessing a gene derived from
the cloned cDNA segment. A selection method is then
used to separate the desired transfectants from the
larger number of helper viruses.
A different system is used if an entire
mutant influenza A strain is to be rescued. Using the
system described by Neumann et al. (Neumann, G., et
al., Proc. Natl. Acad. Sci. USA, 96, 9345-9350 (1999),
which is hereby incorporated by reference), a modified
Influenza virus strain A/Udorn/72 (H3N2) is generated
entirely from cloned cDNAs. This plasmid-based system
does not require the use of helper virus infection.
In summary, a cell line, such as human embryonic
kidney cell line 293T, is transfected with eight
plasmids, each encoding a viral RNA segment of the
strain and flanked by a suitable RNA polymerase
promoter and terminator, together with another four
plasmids encoding the viral NP, PB2, PB1 and PA
proteins (which serve to synthesize RNPs
intracellularly in vivo). Yields are increased
substantially by the addition of five further plasmids
expressing the viral structural proteins HA, NA, M1,
M2 and NS2.
In a variation of this system, only eight
plasmids are required (Hoffmann, E., et al., Proc.
Natl. Acad. Sci. USA, 97, 6108-6113 (2000), which is
hereby incorporated by reference). Each plasmid
contains two promoters, a human RNA polymerase I (pol
I) promoter and a human RNA polymerase II (pol II)
promoter. After transfection of eukaryotic cells with
the eight expression plasmids, the human pol I and II
promoters each transcribe the plasmid template. This
results in the synthesis of both viral mRNAs and
vRNAs, ultimately leading to the generation of
infectious influenza A virus.
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Because helper virus is not required in any
of the 8, 12 or 17 plasmid transfection systems, the
transfectant viruses are recovered without plaque
purification. This system facilitates the production
of live attenuated influenza A viruses for use in
combating epidemics involving new HA or NA subtypes,
where the corresponding segment of the Influenza
A/Udorn/72 (H3N2) strain is mutated to match the
sequence of the new subtypes.
Predetermined mutations to the segment
encoding HA or NA in the Influenza A/Udorn/72 (H3N2)
strain which correspond to. the sequence of a new
circulating subtype are made by using site-directed
mutagenesis. Such mutations are introduced by
standard recombinant DNA methods into a DNA copy of
the viral genome.
If desired, reassortant viruses are
generated by substituting for the segment encoding the
HA or NA of the Influenza A/Udorn/72 (H3N2) strain (or
mutant thereof) the corresponding segment from a
different influenza virus strain.
In another embodiment of this invention, an
isolated nucleic acid molecule having the nucleotide
sequence of a segment of the Influenza A/Udorn/72
(H3N2) strain is used to generate oligonucleotide
probes (from either positive strand antigenomic
message sense or negative strand complementary genomic
sense) and to express peptides (from positive strand
antigenomic message sense only), which are used to
detect the presence of a segment of that influenza A
strain (or a mutant thereof) in samples of body fluids
and tissues. The nucleotide sequences are used to
design.highly specific and sensitive diagnostic tests
to detect the presence of the viral segment in a
sample.
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PCR primers are synthesized with sequences
based on the viral sequences described herein. The
test sample is subjected to reverse transcription of
RNA, followed by PCR amplification, of selected cDNA
regions corresponding to the nucleotide sequence
described herein which have nucleotides which are
distinct for a defined segment of that viral strain.
Amplified PCR products are identified on gels and
their specificity confirmed by hybridization with
specific nucleotide probes.
ELISA tests are~used to detect the presence
of a protein produced by a viral segment. Peptides
are designed and selected to contain one or more
distinct residues based on the viral sequences
described herein. These peptides are then coupled to
a hapten (e.g., keyhole limpet hemocyanin (KLH) and
used to immunize animals (e.g., rabbits) for the
production of monospecific polyclonal antibody. A
selection of these polyclonal antibodies, or a
combination of polyclonal and monoclonal antibodies
can then be used in a "capture ELISA" to detect a
protein produced by that viral segment.
All references cited herein are hereby
incorporated by reference.
In order that this invention may be better
understood, the following examples are set forth. The
examples are for the purpose of illustration only and
are not to be construed as limiting the scope of the
invention.
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Examples
Example 1
Sequencing of the Influenza A/Udorn/72 (H3N2) Strain
The sequencing of each of the eight segments
was carried out as follows:
RT-PCR Protocol
Influenza A/Udorn/72 (H3N2) was grown in
Madin-Darby canine kidney (MDCK) cells and
concentrated from clarified culture supernatant.
Genomic viral RNA (vRNA) was prepared from 100,u1 of
virus stock using a RNAeasy extraction kit (Qiagen).
The spliced products of mRNAs of segment numbers 7 and
8 (M2 and NS2 respectively) were. isolated from total
mRNA of influenza/Udorn-infected MDCK cells using an
Oligotex mRNA kit (Qiagen). Approximately l~,g of
either vRNA or mRNA was added to an eppendorf tube
containing 2pmol of gene-specific primer and diethyl
pyrocarbonate-treated water (to inhibit RNAses) to a
final volume of 12~C1. Oligo-dT primers were used to
reverse transcribe the M2 and NS2 spliced messages.
The RNA-primer mixture was heated at 70°C for 10
minutes and quickly chilled on ice. The following
reagents were added to this tube: 4~C1 of 5X first
strand buffer (250mM Tris-HC1 pH 8.3, 375mM KC1, lSmM
MgClz), 2~1 of 0.1M DTT, and 1~C1 of dNTP mix (10 mM
each dATP, dTTP, dGTP, dCTP) and incubated at 42°C for
two minutes. Subsequently, 1~,1 of Superscript II
(Gibco) reverse transcriptase was added and re-
incubated at 42°C for an additional 50 minutes. The
RT reaction was stopped by incubation at 70°C for 15
minutes. PCR reactions were set up by adding 2~C1 of
the previous RT to a mixture of the following
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reagents: 50mM .Tris-HC1 (pH 8.0), l.5mM MgCl2, 50mM
KCl, 1~,M each primer, 0.2mM each dNTP and 2 units of
Taq polymerase. DNA was amplified in a Perkin-Elmer
9600 PCR machine as follows: One cycle of denaturation
at 94°C for two minutes, followed by 30 cycles of
denaturation at 94°C for 30 seconds, annealing at 42°C
for 30 seconds, and. elongation at 72°C for one minute.
This was followed by one cycle of elongation at 72°C
for seven minutes. PCR-amplified DNA was analyzed on
a 1% agarose gel and purified using a Qiagen
purification kit.
Sequencing Protocol
Approximately 500ng of plasmid pGEM-T
containing the corresponding DNA segment generated by
RT-PCR was added to a 0.2m1 tube containing 3.2pmol of
sequencing primer and 8 ~Cl of Terminator ready
reaction mix (Perkin-Elmer) and placed into a Perkin-
Elmer 9600 PCR machine. The reaction was amplified by
cycles at 96°C for 10 seconds, 50°C for five
seconds, and 60°C for four minutes. The products were
then purified over a G50 Sephadex spin column,
lyophilized, and resuspended in 3~C1 of 25mM EDTA
25 (pH8.0) with 50 mg/ml Blue dextran loading dye. The
samples were run on an ABI 377 automated sequencer and
the sequence analyzed using Sequencher (sequence
analysis program from Gene Codes Corporation).
Determination of the nucleotide sequence of the 3' and
5' termini
Purified genomic vRNA (2.5~g) (see above)
was treated with 10U of tobacco acid pyrophosphatase
(Epicenter Technologies, Madison, Wis.) at 37°C for 30
minutes to remove phosphate groups from the 5'
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terminus. Treated RNA was phenol-chloroform extracted
and precipitated with ethanol. Subsequently, the RNA
5' and 3' termini were ligated with 50U of T4 RNA-
ligase (Pharmacia Biotech.) at 37°C for one hour,
extracted with phenol-chloroform and precipitated with
ethanol. Ligated RNA was reverse transcribed and PCR
in a single reaction with a GeneAmp Gold RNA PCR
Reagent Kit ( PE Biosystems) using a forward and
reverse pair of primers (see below). Sequencing
reactions were carried out as described above, using
the primers specific for each RNA segment (Galarza, J.
M., et al., 1996, J of Virol. 70:2360-2368).
Primers for sequencing influenza A/Udorn/72 (H3N2)
Primers in the forward direction are marked
"F" and are in antigenomic, plus sense. Primers in
the reverse direction are marked "R" and are in
genomic negative sense.
PB2 (Segment 1) Primers '
Primer F/R Primer sequence by spanned
~ RT Primer
1107 F 5'-TATGGAAAGAATAAA.AGAACTACGGAA-3' 27-53
(SEQ ID N0:23)
~ PCR Primers
1108 R 5'-TCGTTTTTAA.ACTATTCAACAT-3' 2307-2328
(SEQ ID N0:24)
1107 F 5'-TATGGAAAGAATAAA.AGAACTACGGAA-3' 27-53
(SEQ ID N0:23)
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Sequencing Primers
1101 F 5'-AGCAGGTCAATTATATTCAATATG-3' 1-24
(SEQ ID N0:25)
The primer sequence differs from the PB2
gene sequence as follows:
Position PB2 Gene 1101 F
5 A G
6 A G
7 ~ A T
8 G C
9 C A
11 G T
12 G T
13 T A
14 C T
16 A T
18 T . C
20 T A
21 A T
22 T A
24 C G
1102 R 5'-AACAAGGTCGTTTTTAAACTATTC-3' 2312-2335
(SEQ ID N0:26)
1103 F 5'-AGAACTCTATTCCAACAAATG-3' 1816-1836
(SEQ ID N0:27)
1104 R 5'-AATCGGATATTTCATTGCCAT-3' 178-198
(SEQ ID N0:28; the primer sequence has
a C at nucleotide 195, while the PB2
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gene sequence has a T)
1105 F 5'-AGAACTCTATTCCAACAAATGAGGGATGTAGTT-3'
1816-1848
(SEQ ID N0:29; the primer sequence has
a G at nucleotide 1846, while the PB2
gene sequence has a C)
1106 R 5'-AATCGGATATTTCATTGCCATCATCCATTTCAT-3'
166-198
(SEQ ID N0:30; the primer sequence has
a C at nucleotide 195, while the PB2
gene sequence has a T)
1107 F 5'-TATGGAAAGAATAA.A.AGAACTACGGAA-3' 27-53
(SEQ ID N0:23)
1108 R 5'-TCGTTTTTAAA,CTATTCAACAT-3' 2307-2328
(SEQ ID N0:24)
1109 F 5'-AGACGTGGTGTTGGTAATGAA-3' 2214-2234
(SEQ ID N0:31)
1110 F 5'-CGAAGAGTTGACATAAACCCT-3' 454-474
(SEQ ID N0:32)
1111 R 5'-TCATCCCTCATCCCCTCACAT-3' 1943-1963
(SEQ ID N0:33; the primer'sequence has
a C at nucleotide 1955, while the PB2
gene sequence has a G)
1112 R 5'-ATTTTCTGTTATCCTCTTGTCA-3' 204-224
(SEQ ID N0:34; this primer has an extra
nucleotide (a T) after nucleotide 220
compared to the actual PB2 gene
sequence)
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1113 F 5' -GTGGAGTCCGCTGTTTTGAG-3' 2083-2102
(SEQ ID N0:35)
1114 F 5' -AGGATGGTGGACATTCTTAGG-3' 904-924
(SEQ ID N0:36)
1115 R 5' -CCGATCAATGCTAACCACTAC-3' 1507-1527
(SEQ ID N0:37)
1116 F 5' -AGCAAAAGCAGGTCAATTATATTCA-3' 1-25
(SEQ ID N0:38)
1117 R 5' -AGTAGAAACAAGGTCGTTTTTAAAC-3' 2317-2341
(SEQ ID N0:39)
Primers used to determine terminal sequences
1104 R 5' -AATCGGATATTTCATTGCCAT-3' 17$-198'
(SEQ ID N0:28)
1109 F 5' -AGACGTGGTGTTGGTAATGAA-3' 2214-2234
(SEQ ID N0:31)
1112 R 5' -ATTTTCTGTTATCCTCTTGTCA-3' 204-224
(SEQ ID N0:34)
1113 F 5' -GTGGAGTCCGCTGTTTTGAG-3' 2083-2102
(SEQ ID N0:35)
PB1 (Segment 2) Primers
Primer F/R Primer sequence by spanned
~ RT primer
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1215 F 5'-AGCAAAAGCAGGCAAACCAT-3' 1-20
(SEQ ID N0:40; the primer sequence has
an A at nucleotide 4, while the PB1
gene sequence has a G)
~ PCR primers
1216 R 5' -AGTAGAA.ACAAGGCATTTTTT-3' 2321-2341
(SEQ ID N0:41)
1215 F 5' -AGCAAAAGCAGGCAAACCAT-3' 1-20
(SEQ ID N0:40)
Sequenc ing primers
1201 F 5' -TCGAGCTGAAGAAGCTATGG-3' 1745-1764
(SEQ ID N0:42; the primer sequence has
a G at nucleotide 1752, while the PB1
gene sequence has an A, and the primer
sequence has an A at nucleotide 1761,
while the PB1 gene sequence has a G)
1202 R 5' -GTTCTGTTGACTGTGTCCAT-3' 142-161
(SEQ ID N0:43)
1203 F 5' -TCGAGCTGAAGAAGCTATGGGAGCAGACCCGT-3'
1745-1776
(SEQ ID N0:44)
The primer sequence differs from the PB1
gene sequence as follows:
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Position
PB1 Gene
1203 F
1752 A G
1761 G A
1770 A G
1776 C T
1204 R 5' -GTTCTGTTGACTGTGTCCATGGTGTATCC-3' 133-161
(SEQ ID N0:45)
1205 F 5' -AGCGAAAGCAGGCAA.ACCATTTGAATGGATGTCAA-3'
1-35
(SEQ ID N0:46)
1206 R 5' -ATTAAAAACAAGGCATTTTTTCATGAAGGAC-3'
2341-2311
(SEQ ID N0:47; the primer sequence has
an A at nucleotide 2337, while the PB1
gene sequence has a G, and the primer
sequence has a T at nucleotide 2340,
while the PB1 gene sequence has a G)
1207 F 5' -AATTTCCAGCATGGTGGAGGCCATGGTG-3' 2154-2181
(SEQ ID N0:48)
1208 F 5' -GGGATCTTTGAAAACTCGTG-3' 325-344
(SEQ ID N0:49)
1209 R 5' -CAGCATTGTTTACAGACTC-3' 1936-1954
(SEQ ID N0:50)
1210 F 5' -ACCAAAGATGCAGAAAGAGG-3' 706-725
(SEQ ID N0:51)
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1211 R 5' -CTCATATTGATTCCGACTAA-3' 1438-1457
(SEQ ID N0:52)
1212 R 5' -TGTCCACTTCCCTTTTTCTGA-3' 172-192
(SEQ ID N0:53)
1213 F 5' -ATTTTTCCCCAGTAGTTCATAC-3' 2118-2139
(SEQ ID N0:54)
1214 F 5' -ACGCTGTTGCAACTACACACTCCTG-3' 1997-2021
(SEQ ID N0:55)
1215 F 5' -AGCAAAAGCAGGCAAACCAT-3' 1-20
(SEQ ID N0:40)
1216 R 5' -AGTAGAAACAAGGCATTTTTT-3' 2321-2341
(SEQ ID N0:41)
Primers used to determine terminal sequences
1204 R 5' -GTTCTGTTGACTGTGTCCATGGTGTATCC-3'133-161
(SEQ ID N0:45)
1212 R 5' -TGTCCACTTCCCTTTTTCTGA-3' 172-192
(SEQ ID N0:53)
1213 F 5' -ATTTTTCCCCAGTAGTTCATAC-3' 2118-2139
(SEQ ID N0:54)
1214 F 5' -ACGCTGTTGCAACTACACACTCCTG-3' 1997-2021
(SEQ ID N0:55)
PA (Segment 3) primers
Primer F/R Primer sequence by spanned
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t RT Primer
1315 F 5'-AGCAAAAGCAGGTACTGATTCGAGA-3' 1-25
(SEQ ID N0:56)
PCR Primers
1315 F 5'-AGCAAA.AGCAGGTACTGATTCGAGA-3' 1-25
(SEQ ID N0:56)
1316 R 5'-AGTAGAAACAAGGTACTTTTTTGGA-3' 2209-2233
(SEQ ID N0:57)
t Sequencing primers
1301 F 5'-GAACCTGGAACCTTTGATCTT-3' 2053-2073
(SEQ ID N0:58)
1302 R 5'-ATTCACCACTGTCCAGGCCAT-3' 280-300
(SEQ ID N0:59)
The primer sequence differs from the PA
gene sequence as follows:
Position PA Gene 1302 F
294 T C
297 T C
300 G A
1303 F 5'-GAACCTGGAACCTTTGATCTTGAGGGGCTA-3'
2053-2082
(SEQ ID N0:60; the primer sequence has
an A at nucleotide 2075, while the PA
gene sequence has a G)
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1304 R 5'-ATTCACCACTGTCCAGGCCATTGACGCGTC-3'
271-300
(SEQ ID N0:61)
The primer sequence differs from the PA
gene sequence as follows:
Position PA Gene 1304 F
274 G C
275 C G
276 G C
277 T A
294 T C
297 T C
300 G A
1305 F 5'-AGCAAAAGCAGGTAGTGATAG-3' 1-21
(SEQ ID N0:62)
The primer sequence differs from the PA
gene sequence as follows:
Position PA Gene 1302 F
C G
T A
21 C G
1306 R 5'-AGTAGAA.ACAAGGTA-3' 2219-2233
(SEQ ID N0:63)
1307 F 5'-AGCGAAAGCAGGTAGTGATTCGAGATGGA-3' 1-29
(SEQ ID N0:64; the primer sequence has
a G at nucleotide 4, while the PA
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gene sequence has an A)
1308 R 5' -AGTAGAA.ACAAGGTACTTTTTTGGACAG-3'2206-2233
(SEQ ID N0:65)
s
1309 R 5' -TCGATTTGTTGGAGTGACTGA-3' 1782-1802
(SEQ ID N0:66)
1310 F 5' -GCCGAACTTCTCCTGCCTTGA-3' 684-704
(SEQ ID N0:67)
1311 F 5' -GTATTCAATAGCCTGTATG-3' 1957-1975
(SEQ ID N0:68)
1312 R 5' -GACTTCTCTCCTTGTCACTC-3' 386-405
(SEQ ID N0:69)
1313 F 5' -ACGAGTCAGCTAAAGTGGGCA-3' 1111-1131
(SEQ ID N0:70)
1314 R 5' -GACACCTCTGCTGTGAAGTAA-3' 1356-1376
' (SEQ ID N0:71)
1315 F 5' -AGCGAAAGCAGGTAGTGATTCGAGA-3' 1-25
(SEQ ID NO:56)
1316 R 5' -AGTAGAAACAAGGTACTTTTTTGGA-3' 2209-2233
(SEQ ID N0:57)
1317 F 5' -GGAGCTGAGAAACCGAAGTTT-3' 319-339
(SEQ ID N0:72)
1318 R 5' -GACTTGGCCAATAAAGTCCTA-3' 1935-1955
(SEQ ID N0:73)
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1319 R 5' -GCCTGCGCATAGTTCCTGTGA-3' 644-664
(SEQ ID N0:74)
1320 R 5' -TCTACCACTATTGACTCGCC-3' 196-215
(SEQ ID N0:75)
Primers used to determine terminal sequences
1301 F 5' -GAACCTGGAACCTTTGATCTT-3' 2053-2073
(SEQ ID N0:58)
1303 F 5' -GAACCTGGAACCTTTGATCTTGAGGGGGCTA-3'
2053-2082
(SEQ ID N0:60)
1311 F 5' -GTATTCAATAGCCTGTATG-3' 1957-1975
(SEQ ID N0:68)
1312 R 5' -GACTTCTCTCCTTGTCACTC-3' 386-405
(SEQ ID N0:69)
1320 R 5' -TCTACCACTATTGACTCGCC-3' 196-215
(SEQ ID N0:75)
HA (Segment 4) Primers
Primer F/R Primer sequence by spanned
RTprimer
1401 F 5' -AGCAAAAGCAGGGGATAATTCTA-3' 1-23
(SEQ ID N0:76)
PCR
primers
1401 F 5' -AGCAAAAGCAGGGGATAATTCTA-3' 1-23
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(SEQ ID N0:76)
1402 R 5' -AGTAGAA.ACAAGGGTGTTTTTAA-3' 1743-1765
(SEQ ID N0:77)
Sequex~,cix~,g
Primers
1401 F 5' -AGCAAAAGCAGGGGATATTCTA-3' 1-23
(SEQ ID N0:76)
1402 R 5' -AGTAGAAACAAGGGTGTTTTTAA-3' 1743-1765
(SEQ ID N0:77)
1403 R 5' -ACAGTTTGTTCATTTCCGAG-3' 1400-1419
(SEQ ID N0:78)
1404 R 5' -ACTTCAGGGTGTTTTGCTTA-3'. 1004-1023
(SEQ ID N0:79)
1405 R 5' -GAACCCCCCAAATGTATAGT-3' 605-624
(SEQ ID N0:80)
1406 R 5' -TGAGGAACTCTGAACCAGCT-3' 199-218
(SEQ ID N0:81)
1407 F 5' -GTCACTAGTTGCCTCGTCAG-3' 404-423
(SEQ ID N0:82)
1408 F 5' -CCGGGAGACATACTGGTAAT-3' 792-811
(SEQ ID N0:83)
1409 F 5' -CAAATCAATGGGAAACTGAA-3' 1203-1222
(SEQ ID N0:84)
1410 F 5' -TGAACTGAAGTCAGGATACA-3' 1592-1611
(SEQ ID N0:85)
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Primersused to determine terminal sequences
1405 R 5' -GAACCCCCCAAATGTATAGT-3' 605-624
(SEQ ID N0:80)
1406 R 5' -TGAGGAACTCTGAACCAGCT-3' 199-218
(SEQ ID N0:81)
1409 F 5' -CAAATCAATGGGAAACTGAA-3' 1203-1222
(SEQ ID N0:84)
1410 F 5' -TGAACTGAAGTCAGGATACA-3' 1592-1611
(SEQ ID N0:85)
NP (Segment 5) Primers
Primer F/R Primer sequence by spanned
~ RT Primer
1509 F 5'-AGCAAAAGCAGGGTTAATAATCAC-3' 1-24
(SEQ ID N0:86)
~ PCR Primers
1509 F 5'-AGCA.AAAGCAGGGTTAATAATCAC-3' 1-24
(SEQ ID N0:86)
1510 R .5'-AGTAGAAACAAGGGTATTTTTCCT-3' 1542-1565
(SEQ ID N0:87)
~ Sequencing primers
1501 R 5'-TTGCACCTTCCATCATCCTT-3' 1380-1399
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(SEQ ID N0:88)
1502 R 5' -GTCTATTCCCACTAAAGAGT-3' 932-951
(SEQ ID N0:89)
1503 R 5' -TGCATCAGAGAGCACATCCT-3' 529-548
(SEQ ID N0:90)
1504 F 5' -GAACTCGTCCTTTATGACAA-3' 364-383
(SEQ ID N0:91)
1505 F 5' -AGAGCAATGGATCAAGT-3' 751-770
(SEQ ID N0:92; there is a
deletion of
the three bases ATG at nucleotides
761-
763)
1506 F 5' -TACTATGGAATCAAGTACTC-3' 1161-1180
(SEQ ID N0:93)
1507 R 5' -ATCAATCATCTTCCCGAC-3' 130-147
(SEQ ID N0:94)
1508 F 5' -AGTGTCCTTCCGTGGGCG-3' 1410-1427
(SEQ ID N0:95)
1509 F 5' -AGCAAAAGCAGGGTTAATAATCAC-3'1-24
(SEQ ID N0:86)
1510 R 5' -AGTAGAAACAAGGGTATTTTTCCT-3'1542-1565
(SEQ ID N0:87)
t Primers used to determine terminal sequences
1503 R 5' -TGCATCAGAGAGCACATCCT-3' 529-548
(SEQ ID N0:90)
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1506 F 5'-TACTATGGAATCAAGTACTC-3' 1161-1180
(SEQ ID N0:93)
NA (Segment 6) primers
Primer Primer sequence by spanned
F/R
t RT Primer
1601 F 5' -AGCAAAAGCAGGAGTGAAGATGA-3' 1-23
(SEQ ID N0:96)
t PCR mers
pri
1601 F 5' -AGCAAAAGCAGGAGTGAAGATGA-3' 1-23
(SEQ ID N0:96)
1602 R 5' -AGTAGAAACAAGGAGTTTTTTCTA-3' 1443-1466
(SEQ ID N0:97)
Sequencing primers
1601 F 5' -AGCAAAAGCAGGAGTGAAGATGA-3' 1-23
(SEQ ID N0:96)
1602 R 5' -AGTAGAAACAAGGAGTTTTTTCTA-3' 1443-1466
(SEQ ID N0:97)
1603 R 5' -TCGTTGTTTCTGGGTGTGTC-3' 989-1008
(SEQ ID N0:98; the primer sequence
has
a T at nucleotide 992, while the NA
gene sequence has an C)
1604 R 5' -TTATCATACCCAGTGACACA-3' 596-615
(SEQ ID N0:99)
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1605 R 5' -ATTATTATTGGTTCACACGG-3' 173-192
(SEQ ID N0:100)
1606 F 5' -TTATGTGTCATGCGATCCTG-3' 379-398
(SEQ ID N0:101)
1607 F 5' -ATTGTTCATATTAGCCCATT-3' 803-822
(SEQ ID N0:102)
1608 F 5' -ATAGGTCAGGTTATTCTGGT-3' 1224-1243
(SEQ ID N0:.103)
Primers used to determine terminal sequences
1608 F 5' -ATAGGTCAGGTTATTCTGGT-3' 1224-1243
(SEQ ID N0:103)
1605 R 5' -ATTATTATTGGTTCACACGG-3' 173-192
(SEQ ID N0:100)
M1 (Segment 7) Primers
Primer F/R Primer sequence by spanned
~ RT Primer
*1706 F 5'-AGCAAAAGCAGGTAG-3' 1-15
(SEQ ID N0:104)
~ PCR primers
1707 R 5'-AGTAGAA.ACAAGGTA-3' 1013-1027
(SEQ ID N0:105)
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1706 F 5' -AGCAAAAGCAGGTAG-3' 1-15
(SEQ ID N0:104)
*1701 R 5' -TTTACTCCAGCTCTATGCTGACAA-3' 985-1008
(SEQ ID N0:106)
* The se primers and M2 genes.
will
amplify
both
the
M1
t Sequenc ing primers
1701 R 5' -TTTACTCCAGCTCTATGCTGACAA-3' 985-1008
(SEQ ID N0:106)
1702 R 5' -GATCCAGCCATTTGCTCCAT-3' 590-609
(SEQ ID N0:107)
1703 R 5' -GAGGTGACAGGATTGGTCTT-3' 169-188
(SEQ ID N0:108)
1704 F 5' -CATGGACAGAGCAGTTAAAC-3' 301-320
(SEQ ID N0:109)
0
1705F 5'-GCGAGTATCATTGGGATCTT-3' 801-820
(SEQ ID N0:110)
0
Common to M1 and M2 sequence.
Primers used to determine terminal sequences
1705 F 5'-GCGAGTATCATTGGGATCTT-3' 801-820
' (SEQ ID N0:110)
1702 R 5'-GATCCAGCCATTTGCTCCAT-3' 590-609
(SEQ ID N0:107)
1703 R 5'-GAGGTGACAGGATTGGTCTT-3 169-188
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(SEQ ID N0:108)
Primers 1701, 1705 and 1706 are sufficient to sequence
M2.
NS1 (Segment 8) Primers
Primer F/R Primer sequence by spanned
RT Primer
*1801 F 5'-AGCAAAAGCAGGGTGACAAAGACA-3' 1-24
(SEQ ID N0:111)
PCR Primers
1801 F 5' -AGCAAAAGCAGGGTGACAAAGACA-3' 1-24
(SEQ ID N0:111)
1802 R 5' -AGTAGAAACAAGGGTGTTTTTTAT-3' 867-890
(SEQ ID N0:112)
*1803 R 5' -TTTTTTATCATTAAATAAGCTGAA-3' 857.-874
(SEQ ID~ N0:113)
* These primers and NS2
will
amplify
both
the
NS1
genes.
~ Sequenc ing Primers
0
1801 F 5' -AGCAAAAGCAGGGTGACAAAGACA-3' 1-24
(SEQ ID N0:111)
1802 R 5' -AGTAGAAACAAGGGTGTTTTTTAT-3' 867-890
(SEQ ID N0:112)
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0
1803 R 5'-TTTTTTATCATTAAATAAGCTGAA-3' 851-874
(SEQ ID N0:1I3)
1804 R 5'-AATTGCATTTTTGACATCCT-3' 541-560
(SEQ ID N0:114)
1805 R 5'-ATCTTCTCTACTATCTGCTT-3' 210-229
(SEQ ID N0:115)
1806 F 5'-CAAGCAATCATGGATAAGAA-3' 387-406
(SEQ ID N0:116)
0
1807 F 5'-GGCGAGAACAGCTAGGTCAA-3' 692-711
(SEQ ID N0:117)
0
Common to NS1 and NS2 sequence
t Pri mers used to determine terminal
sequences
1807 F 5'-GGCGAGAACAGCTAGGTCAA-3' 692-711
(SEQ ID N0:117)
1804 R 5'-AATTGCATTTTTGACATCCT-3' 541-560
(SEQ ID N0:114)
1806 F 5'-CAAGCAATCATGGATAAGAA-3' 387-406
(SEQ ID N0:116)
1805 R 5'-ATCTTCTCTACTATCTGCTT-3' 210-229
(SEQ ID N0:115)
Primers 1801, 1803 and 1807 are sufficient to sequence
NS2.
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Example 2
PCR Assay to Detect a Segment of
the Influenza A/Udorn/72 (H3N2) Strain
A PCR assay is used to detect the presence
of a segment of the Influenza A/Udorn/72 (H3N2)
strain. PCR primers are designed and selected based
on homologies to the viral sequences described herein.
The assay is conducted by subjecting the sample to
reverse transcription of RNA, followed by PCR
amplification of selected cDNA regions corresponding
to the specific nucleotide sequence described herein.
Amplified PCR products are identified on gels and
their specificity confirmed by hybridization with
specific nucleotide probes.
Example 3
ELISA to Detect an Antigen Produced by a Segment
of the Influenza A/Udorn/72 (H3N2) Strain
An ELISA test is used to detect the presence
of an antigen produced by a segment of the Influenza
A/Udorn/72 (H3N2) strain. Peptides are designed and
selected based on homologies to the viral sequences
described herein. These peptides are then coupled to
KLH and used to immunize rabbits for the production of
monospecific polyclonal antibody. A selection of
these polyclonal antibodies, or a combination of
polyclonal and monoclonal antibodies is then used in a
"capture ELISA" to detect the presence of a protein
produced by that viral segment.
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SEQUENCE LISTING
<110> American Cyanamid Company
<120> Nucleotide Sequence of Influenza A/Udorn/72 (H3N2)
Genome
<130> AM100289PCT
<140>
<141>
<150> 60/213,650
<151> 2000-06-23
<160> 117
<170> PatentIn Ver. 2.1
<210> 1
<211> 2341
<212> DNA
<213> Influenza A virus
<220>
<221> CDS
<222> (28) .. (2304)
<400> 1
agcaaaagca ggtcaattat attcaat atg gaa aga ata aaa gaa cta cgg aat 54
Met Glu Arg Ile Lys Glu Leu Arg Asn
1 5
ctg atg tcg cag tct cgc act cgc gag ata cta aca aaa acc aca gtg 102
Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu Thr Lys Thr Thr Val
15 20 25
gac cat atg gcc ata att aag aag tac aca tca ggg aga cag gaa aag 150
Asp His Met Ala Ile Ile Lys Lys Tyr Thr Ser Gly Arg Gln Glu Lys
30 35 40
aac ccg tca ctt agg atg aaa tgg atg atg gca atg aaa tat cca att 198
Asn Pro Ser Leu Arg Met Lys Trp Met Met Ala Met Lys Tyr Pro Ile
45 50 55
aca get gac aag agg ata aca gaa atg gtt cct gag aga aat gag caa 246
Thr Ala Asp Lys Arg Ile Thr Glu Met Val Pro Glu Arg Asn Glu Gln
60 65 70
gga caa acc cta tgg agt aaa atg agt gat gcc ggg tca gat cga gtg 294
Gly Gln Thr Leu Trp Ser Lys Met Ser Asp Ala Gly Ser Asp Arg Val
75 80 85
atg gta tca cct ttg gcg gtg aca tgg tgg aat aga aat gga cca gtg 342
Met Val Ser Pro Leu Ala Val Thr Trp Trp Asn Arg Asn Gly Pro Val
90 95 100 105
aca agt acg gtt cat tat cca aaa gtc tac aag act tat ttt gat aaa 390
Thr Ser Thr Va1 His Tyr Pro Lys Va1 Tyr Lys Thr Tyr Phe Asp Lys
110 115 120
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
gtc gaa agg tta aaa cat gga acc ttt ggc cct gtc cat ttt aga aac 438
Val Glu Arg Leu Lys His Gly Thr Phe Gly Pro Val His Phe Arg Asn
125 . 130 135
caa gtc aaa ata cgc cga aga gtt gac ata aac cct ggt cat gca gac 486
Gln Val Lys Ile Arg Arg Arg Val Asp Ile Asn Pro Gly His Ala Asp
140 145 150
ctc agt gcc aag gag gca caa gat gta atc atg gaa gtt gtt ttc ccc 534
Leu Ser Ala Lys Glu Ala Gln Asp Val Ile Met Glu Val Val Phe Pro
155 160 165
aat gaa gtg ggg gcc agg ata cta acg tcg gaa tca caa tta aca ata 582
Asn Glu Val Gly Ala Arg Ile Leu Thr Ser Glu Ser Gln Leu Thr Ile
170 175 180 185
acc aaa gag aaa aaa gaa gaa ctc caa gat tgc aaa att tct cct ttg 630
Thr Lys Glu Lys Lys Glu Glu Leu Gln Asp Cys Lys Ile Ser Pro Leu
190 195 200
atg gtt gca tac atg tta gag aga gaa ctt gtc cga aaa acg aga ttt 678
Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys Thr Arg Phe
205 210 215
ctc cca gtt get ggt gga aca agc agt gtg tac att gaa gtg tta cac 726
Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu Val Leu His
220 225 230
ttg act caa gga acg tgt tgg gaa cag atg tac act cca ggt gga gaa 774
Leu Thr Gln Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro Gly Gly Glu
235 240 245
gtg agg aat gac gat gtt gac caa agc cta att att gca gcc agg aac 822
Val Arg Asn Asp Asp Val Asp Gln Ser Leu Ile Ile Ala Ala Arg Asn
250 255 260 265
ata gtg aga aga gca gca gta tca gca gat cca cta gca tct tta ttg 870
Ile Val Arg Arg Ala Ala Val Ser Ala Asp Pro Leu Ala Ser Leu Leu
270 275 280
gag atg tgc cac agc aca ctg att ggc ggg aca agg atg gtg gac att 918
Glu Met Cys His Ser Thr Leu Ile Gly Gly Thr Arg Met Val Asp Ile
285 290 295
ctt agg cag aac ccg acg gaa gaa caa get gtg gat ata tgc aag get 966
Leu Arg Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys Ala
300 305 310
gca atg gga ctg agg atc agc tca tcc ttc agt ttt ggt ggg ttc aca 1014
Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr
315 320 325
ttt aag aga aca agc ggg tca tca atc aaa aga gag gaa gaa gtg ctt 2062
Phe Lys Arg Thr Ser Gly Ser Ser Ile Lys Arg Glu Glu Glu Val Leu
330 335 340 345
acg ggc aat ctc caa aca ttg aaa ata agg gtg cat gag ggg tac gag 1110
Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu
350 355 360
gag ttc aca atg gtg ggg aaa agg gca aca get ata ctc aga aaa gca 1158
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
Glu Phe Thr Met Val Gly Lys Arg Ala Thr Ala Ile Leu Arg Lys Ala
365 370 375
acc agg aga ttg gtt cag ctt ata gtg agt gga agg gac gag cag tca 1206
Thr Arg Arg Leu Val Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser
380 385 390
ata gcc gaa gcg ata att gta gcc atg gtg ttt tca caa gag gat tgc 1254
Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys
395 400 405
atg ata aaa gca gtt aga ggt gac ctg aat ttc gtt aac agg gca aat 1302
Met Ile Lys Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn
410 415 420 425
cag cgg ttg aat ccc atg cat caa ctt tta agg cat ttt cag aaa gat 1350
Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp
430 435 440
gcg aaa gtg ctt ttt cag aat tgg gga att gaa cat atc gac aat gtg 1398
Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu His Ile Asp Asn Val
445 450 455
atg gga atg gtt gga gta tta cca gac atg act cca agc aca gag atg ' 1446
Met Gly Met Val Gly Val Leu Pro Asp Met Thr Pro Ser Thr Glu Met
460 465 470
tca atg aga gga ata aga gtc agc aaa atg ggc gtg gat gaa tac tcc 1494
Ser Met Arg Gly Ile Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser
475 480 485
agc aca gag agg gta gtg gtt agc att gat cgg ttt ttg aga gtt cga 1542
Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg
490 495 500 505
gac caa cgt ggg aat gta tta cta tct cct gag gag gtc agt gaa aca 1590
Asp Gln Arg Gly Asn Val Leu Leu Ser Pro Glu Glu Val Ser Glu Thr
510 515 520
cag ggg aca gag aga ctg aca ata act tac tca tcg tca atg atg tgg 1638
Gln Gly Thr Glu Arg Leu Thr Ile Thr Tyr Ser Ser Ser Met Met Trp
525 530 535
gag att aat ggc cct gag tca gtg ttg gtc aat acc tat caa tgg atc 1686
Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile
540 545 550
atc aga aac tgg gaa act gtt aaa att caa tgg tct cag aat cct aca 1734
Ile Arg Asn Trp Glu Thr Val Lys Ile Gln Trp Ser Gln Asn Pro Thr
555 560 565
atg ttg tac aac aaa atg gaa ttt gag cca ttt cag tct tta gtt cct 1782
Met Leu Tyr Asn Lys Met Glu Phe Glu Pro Phe Gln Ser Leu Val Pro
570 575 580 585
aag gcc att aga ggc caa tac agt gga ttt gtc aga act cta ttc caa 1830
Lys Ala Ile Arg Gly Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe Gln
590 595 600
caa atg agg gat gta ctt ggg aca ttt gat acc acc cag ata ata aag 1878
Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Thr Gln Ile Ile Lys
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
605 610 615
ctt ctc ccc ttt gca gcc gcc cca cca aag caa agt aga atg cag ttc 1926
Leu Leu Pro Phe Ala Ala Ala Pro Pro Lys Gln Ser Arg Met Gln Phe
620 625 630
tct tca ttg act gtg aat gtg agg gga tca ggg atg aga ata ctt gta 1974
Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu Val
635 640 645
agg ggc aat tct cct gta ttc aac tac aac aag acc act aaa aga cta 2022
Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Thr Thr Lys Arg Leu
650 655 660 665
aca att ctc gga aaa gat get ggc act tta att gaa gac cca gat gaa 2070
Thr Ile Leu Gly Lys Asp Ala Gly Thr Leu'Ile Glu Asp Pro Asp Glu
670 675 680
agc aca tcc gga gtg gag tcc get gtt ttg aga gga ttt ctc att cta 2118
Ser Thr Ser Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu
685 1690 695
ggt aag gaa gat aga aga tac gga cca gca tta agc atc aat gaa ctg 2166
Gly Lys Glu Asp Arg Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu
700 705 710
agt aac ctt gca aaa gga gaa aag get aat gtg cta att ggg caa gga 2214
Ser Asn Leu Ala Lys Gly Glu Lys Ala Asn Va1 Leu Ile Gly Gln Gly
715 720 725
gac gtg gtg ttg gta atg aaa cga aaa cgg gac tct agc ata ctt act 2262
Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr
730 735 740 745
gac agc cag aca gcg acc aaa aga att cgg atg gcc atc aat 2304
Asp Ser Gln Thr Ala Thr Lys Arg Tle Arg Met Ala Ile Asn
750 755
taatgttgaa tagtttaaaa acgaccttgt ttctact 2341
<210> 2
<211> 759
<212> PRT
<213> Influenza A virus
<400> 2
Met Glu Arg Ile Lys Glu Leu Arg Asn Leu Met Ser Gln Ser Arg Thr
1 5 10 15
Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys
20 25 30
Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ser Leu Arg Met Lys
35 40 45
Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Thr
50 55 60
Glu Met Val Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys
65 70 75 80
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
Met Ser Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val
85 90 95
Thr Trp Trp Asn Arg Asn Gly Pro Val Thr Ser Thr Val His Tyr Pro
100 105 110
Lys Val Tyr Lys Thr Tyr Phe Asp Lys Val Glu Arg Leu Lys His Gly
115 120 125
Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg
130 135 140
Val Asp Ile Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln
145 150 155 160
Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile
165 170 175
Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu
180 185 190
Leu Gln Asp Cys Lys Ile Ser Pro Leu Met Val Ala Tyr Met Leu Glu
195 200 205
Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr
210 215 220
Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp
225 230 235 240
Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Val Asp
245 250 255
Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Ala Val
260 265 270
Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Leu
275 280 285
Ile Gly Gly Thr Arg Met Val Asp Ile Leu Arg Gln Asn Pro Thr Glu
290 295 300
Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser
305 310 315 320
Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser
325 330 335
Ser Ile Lys Arg Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu
340 345 350
Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Lys
355 360 365
Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Val Gln Leu
370 375 380
Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val
385 390 395 400
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly
405 410 415
Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His
420 425 430
Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn
435 440 445
Trp Gly Ile Glu His Ile Asp Asn Val Met Gly Met Val Gly Val Leu
450 455 ' 460
Pro Asp Met Thr Pro Ser Thr G1u Met Ser Met Arg Gly Ile Arg Val
465 470 ~ 475 480
Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val
485 490 495
Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Val Leu
500 505 510
Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Arg Leu Thr
515 520 525
Ile Thr Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser
530 535 540
Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Thr Val
545 550 555 560
Lys Ile Gln Trp Ser Gln Asn Pro Thr Met Leu Tyr Asn Lys Met Glu
565 570 575
Phe Glu Pro Phe Gln Ser Leu Val Pro Lys Ala Ile Arg Gly Gln Tyr
580 585 590
Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly
595 600 605
Thr Phe Asp Thr Thr Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala
610 615 620
Pro Pro Lys Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val
625 630 635 ' 640
Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe
645 650 655
Asn Tyr Asn Lys Thr Thr Lys Arg Leu Thr Ile Leu Gly Lys Asp Ala
660 665 670
Gly Thr Leu Ile Glu Asp Pro Asp Glu Ser Thr Ser Gly Val Glu Ser
675 680 685
Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asp Arg Arg Tyr
690 695 700
Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Asn Leu Ala Lys Gly Glu
705 710 715 720
Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
725 730 735
Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys
740 745 750
Arg Ile Arg Met Ala Ile Asn
755
<210> 3
<211> 2341
<212> DNA
<213> Influenza A virus
<220>
<221> CDS
<222> (25)..(2295)
<400> 3
agcgaaagca ggcaaaccat ttga atg gat gtc aac ccg act tta ctt ttc 51
Met Asp Val Asn Pro Thr Leu Leu Phe
1 5
ttg aaa gtt cca gcg caa aat gcc ata agc acc aca ttc cct tat act 99
Leu Lys Val Pro Ala Gln Asn Ala Ile Ser Thr Thr Phe Pro Tyr Thr
15 20 25
gga gat cct cca tac agc cat gga aca gga aca gga tac acc atg gac 147
Gly Asp Pro Pro Tyr Ser His Gly Thr Gly Thr Gly Tyr Thr Met Asp
30 35 40
aca gtc aac aga aca cat caa tat tca gaa aaa ggg aag tgg aca aca 195
Thr Val Asn Arg Thr His Gln Tyr Ser Glu Lys Gly Lys Trp Thr Thr
45 50 55
aac aca gaa act ggg gcg CCC Caa Ctt aac cca att gat gga cca cta 243
Asn Thr Glu Thr Gly Ala Pro Gln Leu Asn Pro Ile Asp Gly Pro Leu
60 65 70
cct gag gat aat gag cca agt gga tat gca caa aca gac tgt gtc ctg 291
Pro Glu Asp Asn Glu Pro Ser Gly Tyr Ala Gln Thr Asp Cys Val Leu
75 80 85
gaa gca atg get ttc ctt gaa gaa tcc cac cca ggg atc ttt gaa aac 339
Glu Ala Met Ala Phe Leu Glu Glu Ser His Pro Gly Ile Phe Glu Asn
90 95 100 105
tcg tgc ctt gaa acg atg gaa gtc gtt caa caa aca agg gtg gac aga 387
Ser Cys Leu Glu Thr Met Glu Val Val Gln Gln Thr Arg Val Asp Arg
110 115 120
ctg acc caa ggt cgt cag acc tat gat tgg aca tta aac aga aat caa 435
Leu Thr Gln Gly Arg Gln Thr Tyr Asp Trp Thr Leu Asn Arg Asn Gln
125 130 135
f
CCg gCC gca act gca tta gcc aac act ata gaa gtc ttc aga tcg aat 483
Pro Ala Ala Thr Ala Leu Ala Asn Thr Ile Glu Val Phe Arg Ser Asn
140 145 150
ggt cta aca get aat gag tcg gga agg cta ata gat ttc ctc aag gat 531
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
Gly Leu Thr Ala Asn Glu Ser Gly Arg Leu Ile Asp Phe Leu Lys Asp
155 160 165
gtg atg gaa tca atg gat aaa gag gaa atg gag ata aca aca cac ttc 579
Val Met Glu Ser Met Asp Lys Glu Glu Met Glu Ile Thr Thr His Phe
170 175 180 185
caa aga aaa aga aga gta aga gac aac atg acc aag aaa atg gtc aca 627
Gln Arg Lys Arg Arg Val Arg Asp Asn Met Thr Lys Lys Met Val Thr
190 195 200
caa aga aca ata gga aag aag aag cag aga gtg aac aag aga agc tat 675
Gln Arg Thr Ile Gly Lys Lys Lys Gln Arg Val Asn Lys Arg Ser Tyr
205 210 215
cta ata aga gca tta aca ttg aac aca atg acc aaa gat gca gaa aga 723
Leu Ile Arg Ala Leu Thr Leu Asn Thr Met Thr Lys Asp Ala Glu Arg
220 225 230
ggt aaa tta aag aga aga get att gca aca ccc ggg atg caa atc aga 771
Gly Lys Leu Lys Arg Arg Ala Ile Ala Thr Pro Gly Met Gln Ile Arg
235 240 245
ggg ttc gtg tac ttt gtt gaa act cta get agg agc att tgt gag aag 819
Gly Phe Val Tyr Phe Val Glu Thr Leu Ala Arg Ser Ile Cys Glu Lys
250 255 260 265
ctt gaa cag tct gga ctt cca gtt gga ggt aat gaa aag aag gcc aaa 867
Leu Glu Gln Ser Gly Leu Pro Val Gly Gly Asn Glu Lys Lys Ala Lys
270 275 280
ctg gca aat gtt gtg aga aag atg atg act aat tca caa gac aca gag 915
Leu Ala Asn Val Val Arg Lys Met Met Thr Asn Ser Gln Asp Thr Glu
285 290 295
ctt tct ttc aca att act gga gac aat act aag tgg aat gaa aat caa 963
Leu Ser Phe Thr Ile Thr Gly Asp Asn Thr Lys Trp Asn Glu Asn Gln
300 305 310
aat cct cga atg ttc ctg gcg atg att aca tat atc aca aaa aat.caa 1011
Asn Pro Arg Met Phe Leu Ala Met Ile Thr Tyr Ile Thr Lys Asn Gln
315 320 325
cct gaa tgg ttc aga aac att ctg agc ate gca ccc ata atg ttc tca 1059
Pro Glu Trp Phe Arg Asn Ile Leu Ser Ile Ala Pro I1e Met Phe Ser
330 335 340 345
aac aaa atg gcg aga cta ggg aaa gga tac atg ttc gaa agt aag aga 1107
Asn Lys Met Ala Arg Leu Gly Lys Gly Tyr Met Phe Glu Ser Lys Arg
350 355 360
atg aag ctc cga aca caa ata cca gca gaa atg cta gca agc att gac 1155
Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser Ile Asp
365 370 375
cta aag tat ttc aat gaa tca aca aga aag aaa att gag aaa ata aag 1203
Leu Lys Tyr Phe Asn Glu Ser Thr Arg Lys Lys Ile Glu Lys Ile Lys
380 385 390
cct ctt cta ata gat ggc aca gcg tca ttg agt cct gga atg atg atg 1251
Pro Leu Leu Ile Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
395 400 405
ggc atg ttc aac atg cta agt acg gtt tta gga gtc tca atc ctg aat 1299
Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn
410 415 420 425
ctt ggg caa aag aaa tac acc aaa aca aca tac tgg tgg gat gga ctc 1347
Leu Gly Gln Lys Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu
430 435 440
caa tcc tct gat gat ttt get ctc ata gtg aat gca cca aat cat gag 1395
Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu
445 450 455
gga ata caa gca gga gtg gat aga ttc tac agg acc tgc aag tta gtc 1443
Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val
460 465 470
gga atc aat atg agc aag aag aag tcc tat ata aat agg aca gga aca 1491
Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Thr
475 480 485
ttt gaa ttc aca agc ttt ttt tat cgc tat gga ttt gta gcc aat ttt 1539
Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe
490 495 500 505
agc atg gag ctg ccc agt ttt gga gtg tct ggg att aat gag tca get 1587
Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala
510 515 520
gat atg agc att gga gta aca gtg ata aag aac aac atg ata aac aat 1635
Asp Met Ser Ile Gly Val Thr Val Ile Lys Asn Asn Met Ile Asn Asn
525 530 535
gac ctt gga cca gca aca gcc cag atg get ctt caa ctg ttc ate aag 1683
Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys
540 545 550
gac tac aga tat aca tat cgg tgc cac aga gga gac aca caa att cag 1731
Asp Tyr Arg Tyr Thr Tyr Arg Cys His Arg Gly Asp Th'r Gln Ile Gln
555 560 565
acg agg aga tca ttc gag cta aag aag ctg tgg gag caa acc cgc tca 1779
Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser
570 575 580 585
aag gca gga cta ttg gtt tca gat gga gga cca aac tta tac aat atc 1827
Lys Ala Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile
590 595 600
cgg aat ctt cac atc cct gaa gtc tgc tta aag tgg gag cta atg gat 1875
Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp
605 610 615
gag gac tat cag gga aga ctt tgt aat ccc ctg aat cca ttt gtc agc 1923
Glu Asp Tyr Gln Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser
620 625 630
cat aag gag att gag tct gta aac aat get gtg gta atg cca get cat 1971
His Lys Glv. Ile Glu Ser Val Asn Asn Ala Val Val Met Pro Ala His
635 640 645
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
ggt cca gcc aag agc atg gaa tat gac get gtt gca act aca cac tcc 2019
Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser
650 655 660 ' 665
tgg att ccc aag agg aac cgc tct att ctc aac aca agc caa agg gga 2067
Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln Arg Gly
670 675 680
att ctt gag gat gaa cag atg tat cag aag tgc tgc aac ctg ttc gag 2115
Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu
685 690 695
aaa ttt ttc ccc agt agt tca tac agg aga ccg gtt gga att tcc agc 2163
Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly Ile Ser Ser
700 705 710
atg gtg gag gcc atg gtg tct agg gcc cgg att gat gcc aga att gac 2211
Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp
715 720 725
ttc gaa tct gga cgg att aag aaa gaa gag ttc gcc gag atc atg aag 2259
Phe Glu Ser Gly Arg Ile Lys Lys Glu Glu Phe Ala G1u Ile Met Lys
730 735 740 745
atc tgt tcc acc att gaa gag ctc aga cgg caa aaa taatgaattt 2305
Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys
750 755
agcttgtcct tcatgaaaaa atgccttgtt tctact 2341
<210> 4
<211> 757
<212> PRT
<213> Influenza A virus
<400> 4
Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn
1 5 10 15
Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His
20 25 30
Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln
35 40 ~ 45
Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Thr Gly Ala Pro
50 55 60
Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser
65 70 75 80
Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu
85 90 95
Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu
100 105 110
Val Val Gln Gln Thr Arg Val Asp Arg Leu Thr'Gln Gly Arg Gln Thr
115 120 125
CA 02410284 2002-11-20
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Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala
130 135 140
A5n Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr Ala Asn Glu Ser
145 150 155 160
Gly Arg Leu Ile Asp Phe Leu Lys Asp Val Met Glu Ser Met Asp Lys
165 170 175
Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg
180 185 190
Asp Asn Met Thr Lys Lys Met Val Thr Gln Arg Thr Ile Gly Lys Lys
195 200 205
Lys Gln Arg Val Asn Lys Arg Ser Tyr Leu Ile Arg Ala Leu Thr Leu
210 215 220
Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala
225 230 235 240
Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu
245 250 255
Thr Leu Ala Arg Ser Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro
260 265 270
Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys
275 280 285
Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly
290 295 300
Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala
305 310 315 320
Met Ile Thr Tyr Ile Thr Lys Asn Gln Pro Glu Trp Phe Arg Asn Ile
325 330 335
Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly
340 345 350
Lys Gly Tyr Met Phe Glu Ser Lys Arg Met Lys Leu Arg Thr Gln Ile
355 360 365
Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Glu Ser
370 375 380
Thr Arg Lys Lys Ile Glu Lys Ile Lys Pro Leu Leu Ile Asp Gly Thr
385 390 395 400
Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser
405 410 415
Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Lys Lys Tyr Thr
420 425 430
Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala
435 440 445
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Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp
450 455 460
Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys
465 470 475 480
Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe
485 490 495
Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe
500 505 510
Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr
515 520 525
Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala
530 535 540
Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg
545 550 555 560
Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu
565 570 575
Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Ala Gly Leu Leu Val Ser
580 585 590
Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu
595 600 605
Va1 Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Gln Gly Arg Leu
610' 615 620
Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val
625 630 635 640
Asn Asn Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu
645 650 655
Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg
660 665 670
Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met
675 680 685
Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser
690 695 700
Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser
705 710 715 720
Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys
725 730 735
Lys Glu Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu
740 745 750
Leu Arg Arg Gln Lys
755
CA 02410284 2002-11-20
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<210> 5
<211> 2233
<212> DNA
<213> Influenza A virus
<220>
<221> CDS
<222> (25)..(2172)
<400> 5
agcaaaagca ggtactgatt cgag atg gaa gat ttt gtg cga caa tgc ttc 51
Met Glu Asp Phe Val Arg Gln Cys Phe
1 5
aat ccg atg att gtc gag ctt gca gaa aag gca atg aaa gag tat gga 99
Asn Pro Met Ile Val Glu Leu Ala Glu Lys Ala Met Lys Glu Tyr Gly
15 20 25
gag gat ctg aaa atc gaa aca aac aaa ttt gca gca ata tgc act cac 147
Glu Asp Leu Lys Ile Glu Thr Asn Lys Phe Ala Ala Ile Cys Thr His
30 35 40
ttg gag ata tgt ttc atg tat tca gat ttt cat ttc atc aat gaa caa 195
Leu Glu Ile Cys Phe Met Tyr Ser Asp Phe His Phe Ile Asn Glu Gln
45 50 55
ggc gag tca ata gtg gta gag ctt gat gat cca aat gca ctg tta aag 243
Gly Glu Ser Ile Val Val Glu Leu Asp Asp Pro Asn Ala Leu Leu Lys
60 65 70
cac aga ttt gaa ata ata gag gga aga gac cgc aca atg gcc tgg aca 291
His Arg Phe Glu Ile Ile Glu Gly Arg Asp Arg Thr Met Ala Trp Thr
75 80 85
gta gta aac agt att tgc aac act act gga get gag aaa ccg aag ttt 339
Val Val Asn Ser Ile Cys Asn Thr Thr Gly Ala Glu Lys Pro Lys Phe
90~ 95 100 105
ctg cca gat ttg tat gat tac aag gag aat aga ttc atc gag att gga 387
Leu Pro Asp Leu Tyr Asp Tyr Lys Glu Asn Arg Phe Ile Glu Ile Gly
110 115 120
gtg aca agg aga gaa gtc cac ata tac tac ctt gaa aag gcc aat aaa 435
Val Thr Arg Arg Glu Val His Ile Tyr Tyr Leu Glu Lys Ala Asn Lys
125 130 135
att aaa tct gag aat aca cac atc cac att ttc tca ttc act ggg gag 483
Ile Lys Ser Glu Asn Thr His Ile His Ile Phe Ser Phe Thr Gly Glu
140 145 150
gaa atg gcc aca aag gcc gac tac act ctt gat gat gaa agc agg get 531
Glu Met Ala Thr Lys Ala Asp Tyr Thr Leu Asp Asp Glu Ser Arg Ala
155 160 165
agg atc aaa acc agg cta ttt acc ata aga caa gaa atg gcc aac aga 579
Arg Ile Lys Thr Arg Leu Phe Thr Ile Arg Gln Glu Met Ala Asn Arg
170 175 180 185
ggC CtC tgg gat tcc ttt cgt cag tcc gaa aga ggc gaa gaa aca att 627
Gly Leu Trp Asp Ser Phe Arg Gln Ser Glu Arg Gly Glu Glu Thr Ile
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190 195 200
gaa gaa aga ttt gaa atc aca gga act atg cgc agg ctt gcc gac caa 675
Glu Glu Arg Phe Glu Ile Thr Gly Thr Met Arg Arg Leu Ala Asp Gln
205 210 ' 215
agt ctc ccg ccg aac ttc tcc tgc ctt gag aat ttt aga gcc tat gtg 723
Ser Leu Pro Pro Asn Phe Ser Cys Leu Glu Asn Phe Arg Ala Tyr Val
220 225 230
gat gga ttc gaa ccg aac ggc tgc att gag ggc aag ctt tct caa atg 771
Asp Gly Phe Glu Pro Asn Gly Cys Ile Glu Gly Lys Leu Ser Gln Met
235 240 245
tcc aaa gaa gtg aat gca aga att gaa cct ttt ctg aag aca aca cca 819
Ser Lys Glu Val Asn Ala Arg Ile Glu Pro Phe Leu Lys Thr Thr Pro
250 255 260 265
aga cca atc aaa ctt ccg gat ggg cct cct tgt ttt cag cgg tcc aaa 867
Arg Pro Ile Lys Leu Pro Asp Gly Pro Pro Cys Phe Gln Arg Ser Lys
270 275 280
ttc ctt ctg atg gat get tta aaa tta agc att gaa gac cca agt cac 915
Phe Leu Leu Met Asp Ala Leu Lys Leu Ser Ile Glu Asp Pro Ser His
285 290 295
gaa gga gag gga ata cca cta tat gat gcg atc aag tgc atg aga aca 963
Glu Gly Glu Gly Ile Pro Leu Tyr Asp Ala Ile Lys Cys Met Arg Thr
300 305 310
ttc ttt gga tgg aaa gaa ccc tat atc gtc aaa cca cac gaa aag gga 1011
Phe Phe Gly Trp Lys Glu Pro Tyr Ile Val Lys Pro His Glu Lys Gly
315 320 325
ata aat cca aat tat ctg ctg tca tgg aag caa gta ctg gca gaa cta 1059
Ile Asn Pro Asn Tyr Leu Leu Ser Trp Lys Gln Val Leu Ala Glu Leu
330 335 340 345
cag gac att gaa aat gag gag aag att cca aga act aaa aac atg aag 1107
Gln Asp Ile Glu Asn Glu Glu Lys Ile Pro Arg Thr Lys Asn Met Lys
350 355 360
aaa acg agt cag cta aag tgg gca ctt ggt gag aac atg gca cct gag 1155
Lys Thr Ser Gln Leu Lys Trp Ala Leu Gly Glu Asn Met Al.a Pro Glu
365 370 375
aaa gta gac ttt gac aac tgt aga gac ata agc gat ttg aag caa tat 1203
Lys Val Asp Phe Asp Asn Cys Arg Asp Ile Ser Asp Leu Lys Gln Tyr
380 385 390
gat agt gac gaa cct gaa tta agg tca ctt tca agc tgg atc cag aat 1251
Asp Ser Asp Glu Pro Glu Leu Arg Ser Leu Ser Ser Trp Ile Gln Asn
395 400 405
gag ttc aac aag gca tgc gag ctg acc gat tca atc tgg ata gag ctc 1299
Glu Phe Asn Lys Ala Cys Glu Leu Thr Asp Ser Ile Trp Ile Glu Leu
410 415 420 425
gat gag att gga gaa gac gtg get cca att gaa tac att gca agc atg 1347
Asp Glu Ile Gly Glu Asp Val Ala Pro Ile Glu Tyr Ile Ala Ser Met
430 435 440
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agg agg aat tac ttc aca gca gag gtg tcc cat tgc aga gcc aca gaa 1395
Arg Arg Asn Tyr Phe Thr Ala Glu Val Ser His Cys Arg Ala ~'hr Glu
445 450 455
tac ata atg aag ggg gta tac att aat act gcc ttg ctt aat gca tcc 1443
Tyr Ile Met Lys Gly Val Tyr Ile Asn Thr Ala Leu Leu Asn Ala Ser
460 465 470
tgt gca gca atg gac gat ttc caa cta att ccc atg ata agc aag tgc 1491
Cys Ala Ala Met Asp Asp Phe Gln Leu Ile Pro Met Ile Ser Lys Cys
475 480 485
aga act aaa gag gga agg cga aaa acc aat tta tat gga ttc atc ata 1539
Arg Thr Lys Glu Gly Arg Arg Lys Thr Asn Leu Tyr Gly Phe Il'e Ile
490 ' 495 500 505
aaa gga aga tct cac tta agg aat gac acc gac gtg gta aac ttt gtg 1587
Lys Gly Arg Ser His Leu Arg Asn Asp Thr Asp Val Val Asn Phe Val
510 515 520
agc atg gag ttt tct ctc act gac ccg aga ctt gag cca cat aaa tgg 1635
Ser Met Glu Phe Ser Leu Thr Asp Pro Arg Leu Glu Pro His Lys Trp
525 530 535
gag aaa tac tgt gtc ctt gag ata gga gat atg cta cta aga agt gcc 1683
Glu Lys Tyr Cys Val Leu Glu Ile Gly Asp Met Leu Leu Arg Ser Ala
540 545 550
ata ggc cag atg tca agg cct atg ttc ttg tat gtg agg aca aat gga 1731
Ile Gly Gln Met Ser Arg Pro Met Phe Leu Tyr Val Arg Thr Asn Gly
555 560 565
aca tca aag att aaa atg aaa tgg gga atg gag atg aga cgt tgc ctc 1779
Thr Ser Lys Ile Lys Met Lys Trp Gly Met Glu Met Arg Arg Cys Leu
570 575 580 585
ctt cag tca ctc caa caa atc gag agc atg att gaa gcc gag tct tct 1827
Leu Gln Ser Leu Gln Gln Ile Glu Ser Met Ile Glu Ala Glu Ser Ser
590 595 600
gtc aaa gag aaa gac atg acc aaa gag ttt ttt gag aat aaa tca gaa 1875
Val Lys Glu Lys Asp Met Thr Lys Glu Phe Phe Glu Asn Lys Ser Glu
605 620 615
aca tgg ccc att ggg gag tcc ccc aag gga gtg gaa gaa ggt tcc att 1923
Thr Trp Pro Ile Gly Glu Ser Pro Lys Gly Val Glu Glu Gly Ser Ile
620 625 630
gga aag gtc tgt agg act tta ttg gcc aag tcg gta ttc aat agc ctg 1971
Gly Lys Val Cys Arg Thr Leu Leu Ala Lys Ser Val Phe Asn Ser Leu
635 640 645
tat gca tcc cca caa ttg gaa gga ttt tca gcg gag tca aga aaa ctg 2019
Tyr Ala Ser Pro Gln Leu Glu Gly Phe Ser Ala Glu Ser Arg Lys Leu
650 655 660 ~ 665
ctt ctt gtc gtt cag get ctt agg gac aac ctt gaa cct gga acc ttt 2067
Leu Leu Val Val Gln Ala Leu Arg Asp Asn Leu Glu Pro Gly Thr Phe
670 675 680
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gat ctt ggg ggg cta tat gaa gca att gag gag tgc ctg att aat gat 2115
Asp Leu Gly Gly Leu Tyr Glu Ala Ile Glu Glu Cys Leu Ile Asn Asp
685 690 695
ccc tgg gtt ttg ctt aat gcg tct tgg ttc aac tcc ttc cta aca cat 2163
Pro Trp Val Leu Leu Asn Ala Ser Trp Phe Asn Ser Phe Leu Thr His
700 705 710
gca tta aga tagttgtggc aatgctacta tttgctatcc atactgtcca 2212
Ala Leu Arg
715
aaaaagtacc ttgtttctac t 2233
<210> 6
<211> 716
<212> PRT
<213> Influenza A virus
<400> 6
Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu
1 5 10 15
Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asp Leu Lys Ile Glu Thr
20 25 30
Asn Lys Phe Ala Ala Ile Cys Thr His Leu Glu Ile Cys Phe Met Tyr
35 40 45
Ser Asp Phe His Phe Ile Asn Glu Gln Gly Glu Ser Ile Val Val Glu
50 55 60
Leu Asp Asp Pro Asn A1a Leu Leu Lys His Arg Phe Glu Ile Ile Glu
65 70 75 80
Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn
85 90 95
Thr Thr Gly Ala Glu Lys-Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr
100 105 110
Lys Glu Asn Arg Phe Ile Glu Ile Gly Val Thr Arg Arg Glu Val His
115 120 125
Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Asn Thr His
130 135 140
Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys Ala Asp
145 150 155 160
Tyr Thr Leu Asp Asp Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe
165 170 175
Thr Ile Arg Gln Glu Met Ala Asn Arg Gly Leu Trp Asp Ser Phe Arg
180 185 190
Gln Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr
195 200 205
Gly Thr Met Arg Arg Leu Ala Asp Gln Ser Leu Pro Pro Asn Phe Ser
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210 215 220
Cys Leu Glu Asn Phe Arg Ala Tyr Val Asp Gly Phe Glu Pro Asn Gly
225 230 235 240
Cys Ile Glu Gly Lys Leu Ser Gln Met Ser Lys.Glu Val Asn Ala Arg
245 250 255
Ile Glu Pro Phe Leu Lys Thr Thr Pro Arg Pro Ile Lys Leu Pro Asp
260 265 270
Gly Pro Pro Cys Phe Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu
275 280 285
Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu
290 295 300
Tyr Asp Ala Ile Lys Cys Met Arg Thr Phe Phe Gly Trp Lys Glu Pro
305 310 315 320
Tyr Ile Val Lys Pro His Glu Lys Gly Ile Asn Pro Asn Tyr Leu Leu
325 330 335
Ser Trp Lys Gln Val Leu Ala Glu Leu Gln Asp Ile Glu Asn Glu Glu
340 345 350
Lys Ile Pro Arg Thr Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp
355 360 365
Ala Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Asp Asn Cys
370 375 380
Arg Asp Ile Ser Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Leu
385 390 395 400
Arg Ser Leu Ser Ser Trp Ile Gln Asn Glu Phe Asn Lys Ala Cys Glu
405 410 415
Leu Thr Asp Ser Ile Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val
420 425 430
Ala Pro Ile Glu Tyr Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala
435 440 445
Glu Val Ser His Cys Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr
450 455 460
Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Asp Phe
465 470 475 480
Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg
485 490 495
Lys Thr, Asn Leu Tyr Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg
500 505 510
Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr
515 520 525
Asp Pro Arg Leu Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu
530 535 540
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Ile Gly Asp Met Leu Leu Arg Ser Ala Ile Gly Gln Met Ser Arg Pro
545 550 555 560
Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys
565 570 575
Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile
580 585 590
Glu Ser Met Ile Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr
595 600 605
Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser
610 615 620
Pro Lys Gly Val Glu Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu
625 630 635 640
Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu
645 650 655
Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu Val Val Gln Ala Leu
660 665 670
Arg Asp Asn Leu Glu Pro Gly Thr Phe Asp Leu Gly Gly Leu Tyr Glu
675 680 685
Ala Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala
690 695 700
Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Arg
705 710 715
<210> 7
<211> 1765
<212> DNA
<213> Influenza A virus
<220>
<221> CDS
<222> (30)..(1727)
<400> 7
agcaaaagca ggggataatt ctattaacc atg aag act atc att get ttg agc 53
Met Lys Thr Ile Ile Ala Leu Ser
1 5
tac att ttc tgt ctg gtt ctc ggc caa aac ttt cca gga aat gac aac 101
Tyr Ile Phe Cys Leu Val Leu Gly Gln Asn Phe Pro Gly Asn Asp Asn
15 20
agc aca gca acg ctg tgc ctg gga cat cat gcg gtg cca aac gga aca 149
Ser Thr Ala Thr Leu Cys Leu Gly His His Ala Val Pro Asn Gly Thr
25 30 35 40
cta gtg aaa aca atc aca aat gat cag att gaa gtg act aat get act 197
Leu Val Lys Thr Ile Thr Asn Asp Gln Ile Glu Val Thr Asn Ala Thr
45 50 55
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gag ctg gtt cag agt tcc tca acg ggg aaa ata tgc aac aat cct cat 245
Glu Leu Val Gln Ser Ser Ser Thr Gly Lys Ile Cys Asn Asn Pro His
60 65 70
cga atc ctt gat gga ata gac tgc aca ctg ata gat get cta ttg ggg 293
Arg Ile Leu Asp Gly Ile Asp Cys Thr Leu Ile Asp Ala Leu Leu Gly
75 80 85
gac cct cat tgt gat ggc ttt caa aat gag aca tgg gac ctt ttc gtt 341
Asp Pro His Cys Asp Gly Phe Gln Asn Glu Thr Trp Asp Leu Phe Val
90 95 100
gaa cgc agc aaa get ttc agc aac tgt tac cct tat gat gtg cca gat 389
Glu Arg Ser Lys Ala Phe Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp
105 110 115 120
tat gcc tcc ctt agg tca cta gtt gcc tcg tca ggc act ctg gag ttt 437
Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe
125 130 135
atc agt gaa ggc ttc act tgg act ggg gtc act cag aat ggg gga agc 485
Ile Ser Glu Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly Gly Ser
140 145 150
aat get tgc aaa agg gga cct gat agc ggt ttt ttc agt aga ctg aac 533
Asn Ala Cys Lys Arg Gly Pro Asp Ser Gly Phe Phe Ser Arg Leu Asn
155 160 165
tgg ttg tac aaa tca gga agc aca tat cca gtg ctg aac gtg act atg 581
Trp Leu Tyr Lys Ser Gly Ser Thr Tyr Pro Val Leu Asn Val Thr Met
170 175 180
cca aac aat gac aat ttt gac aaa cta tac att tgg ggg gtt cac cac 629
Pro Asn Asn Asp Asn Phe Asp Lys Leu Tyr Ile Trp Gly Val His His
185 190 195 200
ceg agc acg gac caa gaa caa acc agc cta tat gtt caa gca tca ggg 677
Pro Ser Thr Asp Gln Glu Gln Thr Ser Leu Tyr Val Gln Ala Ser Gly
205 210 215
aga gtc aca gtc tct acc aag aga agc cag caa act ata atc ccg aat 725
Arg Val Thr Val Ser Thr Lys Arg Ser Gln Gln Thr Ile Ile Pro Asn
220 225 230
atc ggg tct aga ccc tgg gta agg ggt ctg tct agt aga ata agc atc 773
Ile Gly Ser Arg Pro Trp Val Arg Gly Leu Ser Ser Arg Ile Ser Ile
235 240 245
tat tgg aca ata gtt aaa ccg gga gac ata ctg gta att aat agt aat 821
Tyr Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Val Ile Asn Ser Asn
250 255 260
ggg aac cta att get cct cgg ggt tat ttt aaa atg cgc act ggg aaa 869
Gly Asn Leu Ile Ala Pro Arg Gly Tyr Phe Lys Met Arg Thr Gly Lys
265 270 275 280
agc tca ata atg agg tca gat gca cct att ggc acc tgc att tct gaa 917
Ser Ser Ile Met Arg Ser Asp Ala Pro Ile Gly Thr Cys Ile Ser Glu
285 290 295
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tgc atc act cca aat gga agc att ccc aat gac aag ccc ttt caa aac 965
Cys Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn
300 305 310
gta aac aag atc aca tat ggg gca tgt ccc aag tat gtt aag caa aac 1013
Val Asn Lys Ile Thr Tyr Gly Ala Cys Pro Lys Tyr Val Lys Gln Asn
315 320 325
acc ctg aag ttg gca aca ggg atg cgg aat gta cca gag aaa caa act 1061
Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr
330 335 340
aga ggc cta ttc agc gca ata gca ggt ttc ata gaa aat ggg tgg gag 1109
Arg Gly Leu Phe Ser Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu
345 350 355 360
gga atg ata gac ggt tgg tac ggt ttc agg cat caa aat tct gag ggc 1157
Gly Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly
365 370 375
aca gga caa gca gca gat ctt aaa agc act caa gca gcc atc gac caa 1205
Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln
380 385 390
atc aat ggg aaa ctg aat agg gta atc gag aag acg aac gag aaa ttc 1253
Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe
395 400 405
cat caa atc gaa aag gaa ttc tca, gaa gta gaa ggg aga att cag gac 1301
His Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp
410 415 420
ctc gag aaa tac gtt gaa gac act aaa ata gat ctc tgg tct tac aat 1349
Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn
425 430 435 440
gcg gag ctt ctt gtc get ctg gag aac caa cat aca att gat ctg act 1397
Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr
445 450 455
gac tcg gaa atg aac aaa ctg ttt gaa aaa aca agg agg caa ctg agg 1445
Asp Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg
460 465 470
gaa aat get gag gac atg ggc aat ggt tgc ttc aaa ata ttc cac aaa 1493
Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Phe His Lys
475 480 485
tgt gac aat get tgc ata ggg tca atc aga aat ggg act tat gac cat 1541
Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His
490 495 500
gat gta tac aga gac gaa gca tta aac aac cgg ttt cag atc aaa ggt 1589
Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly
505 510 515 520
gtt gaa ctg aag tca gga tac aaa aac tgg atc ctg tgg att tcc ttt 1637
Val Glu Leu Lys Ser Gly Tyr Lys Asn Trp Ile Leu Trp Ile Ser Phe
525 530 535
gcc ata tca tgc ttt ttg ctt tgt gtt gtt ttg ctg ggg ttc atc atg 1685
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Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met
540 545 550
tgg gcc tgc cag aaa ggc aac att agg tgc aac att tgc att 1727
Trp Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile
555 560 565
tgagtgtatt agtaattaaa aacacccttg tttctact 1765
<210> 8
<211> 566
<212> PRT
<213> Influenza A virus
<400> 8
Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Leu Gly
1 5 10 15
Gln Asn Phe Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly
20 25 30
His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile Thr Asn Asp
35 40 45
Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr
50 55 60
Gly Lys Ile Cys Asn Asn Pro His Arg Ile Leu Asp Gly Ile Asp Cys
65 70 75 80
Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Gly Phe Gln
85 90 95
Asn Glu Thr Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Phe~Ser Asn
100 105 110
Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val
115 120 125
Ala Ser Ser Gly Thr Leu Glu Phe Ile Ser Glu Gly Phe Thr Trp Thr
130 135 140
Gly Val Thr Gln Asn Gly Gly Ser Asn Ala Cys Lys Arg Gly Pro Asp
145 150 155 160
Ser Gly Phe Phe Ser Arg Leu Asn Trp Leu Tyr Lys Ser Gly Ser Thr
165 170 175
Tyr Pro Val Leu Asn Val Thr Met Pro Asn Asn Asp Asn Phe Asp Lys
180 185 190
Leu Tyr Ile Trp Gly Val His His Pro Ser Thr Asp Gln Glu Gln Thr
195 200 205
Ser Leu Tyr Val Gln Ala Ser Gly Arg Val Thr Val Ser Thr Lys Arg
210 215 220
Ser Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg
225 230 235 240
CA 02410284 2002-11-20
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Gly Leu Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly
245 250 ~ 255
Asp Ile Leu Val Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro Arg Gly
260 265 270
Tyr Phe Lys Met Arg Thr Gly Lys Ser Ser Ile Met Arg Ser Asp Ala
275 280 285
Pro Ile Gly Thr Cys Ile Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile
290 295 300
Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Ile Thr Tyr Gly Ala
305 310 315 320
Cys Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met
325 330 335
Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Leu Phe Ser Ala Ile Ala
340 345 350
Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly
355 360 365
Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys
370 375 380
Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val
385 390 395 400
Ile Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser
405 4l0 415
Glu Val Glu Gly Arg I1e Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr
420 425 430
Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu
435 440 445
Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe
450 455 460
Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn
465 470 475 480
Gly Cys Phe Lys Ile Phe His Lys Cys Asp Asn Ala Cys Ile Gly Ser
485 490 495
Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu
500 505 510
Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys
515 520 525
Asn Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys
530 535 540
Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile
545 550 555 560
Arg Cys Asn Ile Cys Ile
CA 02410284 2002-11-20
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565
<210> 9
<211> 1565
<212> DNA
<213> Influenza A virus
<220>
<221> CDS
<222> (46)..(1539)
<400> 9
agcaaaagca gggttaataa tcactcactg agtgacatca aaatc atg gcg tcc caa 57
Met Ala Ser Gln
1
ggc acc aaa cgg tct tat gaa cag atg gaa act gat ggg gaa cgc cag 105
Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp Gly Glu Arg Gln
10 15 20
aat gca act gag atc agg gca tcc gtc ggg aag atg att gat gga att 153
Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Lys Met Ile Asp Gly Ile
25 30 35
gga cga ttc tac atc caa atg tgc act gaa ctt aaa ctc agt gat tat 201
Gly Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys Leu Ser Asp Tyr
40 45 50
gag ggg cga ttg atc cag aac agc tta aca ata gag aga atg gtg ctc 249
Glu Gly Arg Leu Ile Gln Asn Ser Leu Thr Ile Glu Arg Met Val Leu
55 60 0 65
tct get ttt gat gag aga agg aat aga tat ctg gaa gaa cat ccc agc 297
Ser Ala Phe Asp Glu Arg Arg Asn Arg Tyr Leu Glu Glu His Pro Ser
70 75 80
gcg ggg aaa gat cct aag aaa act gga ggg ccc ata tac aag aga gta 345
Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile Tyr Lys Arg Val
85 90 95 100
gat aga aag tgg atg agg gaa ctc gtc ctt tat gac aaa gaa gaa ata 393
Asp Arg Lys Trp Met Arg Glu Leu Val Leu Tyr Asp Lys Glu Glu Ile
105 110 115
agg cga atc tgg cgc caa gcc aat aat ggt gat gat gcg aca get ggt 441
Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Asp Asp Ala Thr Ala Gly
120 125 130
cta acc cac atg atg atc tgg cat tcc aat ttg aat gat aca aca tac 489
Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn Asp Thr Thr Tyr
135 140 145
cag agg aca aga get ctt gtt cgc acc gga atg gat ccc agg atg tgc 537
Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp Pro Arg Met Cys
150 155 160
tct ctg atg cag ggt tcg act ctc cct agg agg tct gga get gca ggc~ 585
Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser Gly Ala Ala Gly
165 170 175 180
CA 02410284 2002-11-20
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get gca gtc aaa gga gtc ggg aca atg gtg atg gag ctg atc aga atg 633
Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu Leu Ile Arg Met
185 190 195
atc aaa cgt ggg atc aac gat cgg aac ttc tgg aga ggt gag aat gga 681
Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg Gly Glu Asn Gly
200 205 210
cga aaa aca agg ggt get tat gag aga atg tgc aac att ctc aaa gga 729
Arg Lys Thr Arg Gly Ala Tyr Glu Arg Met Cys Asn Ile Leu Lys Gly
215 220 225
aaa ttt caa aca get gca caa aga gca atg atg gat caa gtg aga gaa 777
Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp Gln Val Arg Glu
230 ' 235 240
agc cgg aac cca gga aat get gag atc gaa gat ctc ata ttt ttg gca 825
Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu Ile Phe Leu Ala
245 250 255 260
cgg tct gca cta ata ttg aga ggg tca gtt get cac aaa tct tgt ctg 873
Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His Lys Ser Cys Leu
265 270 275
cct gcc tgt gtg tat gga cct gcc gta gcc agt ggg tac gac ttc gaa 921
Pro Ala Cys Val Tyr Gly Pro Ala Val Ala Ser Gly Tyr Asp Phe Glu
280 285 290
aaa gag gga tac tct tta gtg gga ata gac cct ttc aaa cta ctt caa 969
Lys Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe Lys Leu Leu Gln
295 300 305
aac agc caa gta tac agc cta atc aga ccg aac gag aat cca gca cac 1017
Asn Ser Gln Val Tyr Ser Leu Ile Arg Pro Asn Glu Asn Pro Ala His
310 315 320
aag agt cag ctg gtg tgg atg gca tgc aat tct get gca ttt gaa gat 1065
Lys Ser Gln Leu Val Trp Met Ala Cys Asn Ser Ala Ala Phe Glu Asp
325 330 335 340
cta aga tta tta agc ttc atc aga ggg acc aaa gta tct cca agg ggg 1113
Leu Arg Leu Leu Ser Phe Ile Arg Gly Thr Lys Val Ser Pro Arg Gly
345 350 355
aaa ctt tca act aga gga gta caa att get tca aat gaa aac atg gat 1161
Lys Leu Ser Thr Arg Gly Va1 Gln Ile Ala Ser Asn Glu Asn Met Asp
360 365 370
act atg gaa tca agt act ctt gaa ctg aga agc agg tac tgg gcc ata 1209
Thr Met Glu Ser Ser Thr Leu Glu Leu Arg Ser Arg Tyr Trp Ala Ile
375 380 385
agg acc aga agt gga gga aac act aat caa cag agg gcc tcc gca ggc 1257
Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Arg Ala Ser Ala Gly
390 395 400
caa atc agt gtg caa cct gca ttt tct gtg caa aga aac ctc cca ttt 1305
Gln Ile Ser Val Gln Pro Ala Phe Ser Val Gln Arg Asn Leu Pro Phe
405 410 415 420
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gac aaa tca acc atc atg gca gca ttc act ggg aat acg gag gga aga 1353
Asp Lys Ser Thr Ile Met Ala Ala Phe Thr Gly Asn Thr Glu Gly Arg
425 430 435
acc tca gac atg agg gca gaa atc ata agg atg atg gaa ggt gca aaa 1401
Thr Ser Asp Met Arg Ala Glu Ile Ile Arg Met Met Glu Gly Ala Lys
440 445 450
cca gaa gaa gtg tcc ttc cgt ggg cgg gga gtc ttc gag ctc tcg gac 1449
Pro Glu Glu Val Ser Phe Arg Gly Arg Gly Val Phe Glu Leu Ser Asp
455 460 465
gaa aag gca acg aac ccg atc gtg ccc tct ttt gac atg agt aat gaa 1497
Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp Met Ser Asn Glu
470 475 480
gga tct tat ttc ttc gga gac aat gca gag gag tac gac aat 1539
Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr Asp Asn
485 490 495
taaggaaaaa tacccttgtt tctact 1565
<210> 10
<211> 498
<212> PRT
<213> Influenza A virus
<400> 10
Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp
1 5 10 . 15
Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Lys Met
20 25 30
Ile Asp Gly Ile Gly Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys
35 40 45
Leu Ser Asp Tyr Glu Gly Arg Leu Ile Gln Asn Ser Leu Thr Ile Glu
50 55 60
Arg Met Va1 Leu Ser Ala Phe Asp Glu Arg Arg Asn Arg Tyr Leu Glu
65 70 75 80
Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile
85 90 95
Tyr Lys Arg Val Asp Arg Lys Trp Met Arg Glu Leu Val Leu Tyr Asp
100 105 110
Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Asp Asp
115 120 125
Ala Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn
130 135 140
Asp Thr Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp
145 150 155 160
Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser
165 170 175
CA 02410284 2002-11-20
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Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu
180 185 190
Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg
195 200 205
Gly Glu Asn Gly Arg Lys Thr Arg Gly Ala Tyr Glu Arg Met Cys Asn
210 215 220
Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp
225 230 235 240
Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Tle Glu Asp Leu
245 250 255
Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His
260 265 270
Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Pro Ala Val Ala Ser Gly
275 280 285
Tyr Asp Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe
290 295 300
Lys Leu Leu Gln Asn Ser Gln Val Tyr Ser Leu Ile Arg Pro Asn Glu
305 310 315 320
Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys Asn Ser Ala
325 330 335
Ala Phe Glu Asp Leu Arg Leu Leu Ser Phe Ile Arg Gly Thr Lys Val
340 345 350
Ser Pro Arg Gly Lys Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn
355 360 365
Glu Asn Met Asp Thr Met Glu Ser Ser Thr Leu Glu Leu Arg Ser Arg
370 375 380
Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Arg
385 390 395 400
Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Ala Phe Ser Val Gln Arg
405 410 415
Asn Leu Pro Phe Asp Lys Ser Thr Ile Met Ala Ala Phe Thr Gly Asn
420 425 430
Thr Glu Gly Arg Thr Ser Asp Met Arg Ala Glu Ile Ile Arg Met Met
435 440 445
Glu Gly Ala Lys Pro Glu Glu Val Ser Phe Arg Gly Arg Gly Val Phe
450 455 460
Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp
465 470 475 480
Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr
485 490 495
CA 02410284 2002-11-20
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Asp Asn
<210> 11
<211> 1466
<212> DNA
<213> Influenza A virus
<220>
<221> CDS
<222> (20)..(1426)
<400> 11
agcaaaagca ggagtgaag atg aat cca aat caa aag ata ata aca att ggc 52
Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly
1 5 10
tct gtc tct ctc acc att gca aca ata tgc ttc ctc atg cag att gcc 100
Ser Val Ser Leu Thr Ile Ala Thr Ile Cys Phe Leu Met Gln Ile Ala
15 20 25
atc ctg gta act act gta aca ttg cat ttc aag caa tat gag tgc gac 148
Ile Leu Val Thr Thr Val Thr Leu His Phe Lys Gln Tyr Glu Cys Asp
30 35 40
tcc ccc gcg aac aac caa gta atg ccg tgt gaa cca ata ata ata gaa 196
Ser Pro Ala Asn Asn Gln Val Met Pro Cys Glu Pro Ile Ile Ile Glu
45 50 55
aggaac ataacagag atagtgtat ttgactaac accaccata gagaaa 244
ArgAsn IleThrGlu IleValTyr LeuThrAsn ThrThrIle GluLys.
60 65 70 75
gagata tgccccaaa ttagtggaa tacaggaat tggtcaaag ccgcaa 292
GluIle CysProLys LeuValGlu TyrArgAsn TrpSerLys ProGln
80 85 90
tgtaaa attacagga tttgcacct ttttctaag gacaattca attcgg 340
CysLys IleThrGly PheAlaPro PheSerLys AspAsnSer IleArg
95 100 105
ctttct getggtggg gacatttgg gtgacgaga gaaccttat gtgtca 388
LeuSer AlaGlyG1y AspIleTrp ValThrArg GluProTyr ValSer
110 115 120
tgcgat cctggcaag tgttatcaa tttgcactc gggcagggg accaca 436
CysAsp ProGlyLys CysTyrGln PheAlaLeu GlyGlnGly ThrThr
125 130 135
ctagac aacaaacat tcaaatgac acaatacat gatagaacc cctcat 484
LeuAsp AsnLysHis SerAsnAsp ThrIleHis AspArgThr ProHis
140 145 150 155
cgaacc ctattgatg aatgagttg ggtgttcca tttcatttg ggaacc 532
ArgThr LeuLeuMet AsnGluLeu GlyValPro PheHisLeu GlyThr
160 165 170
aggcaa gtgtgtata gcatggtcc agctcaagt tgtcacgat ggaaaa 580
ArgGln ValCysIle AlaTrpSer SerSerSer CysHisAsp GlyLys
CA 02410284 2002-11-20
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175 180 185
gca tgg ctg cat gtt tgt gtc act ggg tat gat aaa aat gca act get 628
Ala Trp Leu His Val Cys Val Thr Gly Tyr Asp Lys Asn Ala Thr Ala
190 195 200
agc ttc att tac gat ggg agg ctt gta gac agt att ggt tca tgg tct 676
Ser Phe Ile Tyr Asp Gly Arg Leu Val Asp Ser Ile Gly Ser Trp Ser
205 210 215
caa aat atc ctc agg acc cag gag tcg gaa tgc gtt tgt atc aat ggg 724
Gln Asn Ile Leu Arg Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly
220 225 230 235
act tgt aca gta gta atg act gat gga agt get tca gga aga get gat 772
Thr Cys Thr Val Val Met Thr Asp Gly Ser Ala Ser Gly Arg Ala Asp
240 245 250
act aaa ata cta ttc att gaa gag ggg aaa att gtt cat att agc cca 820
Thr Lys Ile Leu Phe Ile Glu Glu Gly Lys Ile Val His Ile Ser Pro
255 260 265
ttg tca gga agt get cag cat gta gag gag tgt tcc tgt tat cct cga 868
Leu Ser Gly Ser Ala Gln His Val Glu Glu Cys Ser Cys Tyr Pro Arg
270 275 280
tat'cct ggt gtc aga tgt atc tgc aga gac aac tgg aaa ggc tct aat 916
Tyr Pro Gly Val Arg Cys Ile Cys Arg Asp Asn Trp Lys Gly Ser Asn
285 290 295
agg ccc gtc gta gat ata aat gtg aaa gat tat agc att gat tcc agt 964
Arg Pro Val Val Asp Ile Asn Val Lys Asp Tyr Ser Ile Asp Ser Ser
300 305 310 315
tat gtg tgc tca ggg ctt gtt ggc gac gca ccc aga aac aac gac aga 1012
Tyr Val Cys Ser Gly Leu Val Gly Asp Ala Pro Arg Asn Asn Asp Arg
320 325 330
tct agc aat agc tat tgc cgg aat cct aac aat gag aaa ggg aat cac 1060
Ser Ser Asn Ser Tyr Cys Arg Asn Pro Asn Asn Glu Lys Gly Asn His
335 340 345
gga gtg aaa ggc tgg gcc ttt gac gat gga aat gac gtg tgg atg gga 1108
Gly Val Lys Gly Trp Ala Phe Asp Asp Gly Asn Asp Val Trp Met Gly
350 355 360
aga acg atc agc gag ga.t tca cgc tca ggt tat gaa acc ttc aaa gtc 1156
Arg Thr Ile Ser Glu Asp Ser Arg Ser Gly Tyr Glu Thr Phe Lys Val
365 370 375
att ggt ggt tgg tcc aca cct aat tcc aaa ttg cag ata aat agg caa 1204
Ile Gly Gly Trp Ser Thr Pro Asn Ser Lys Leu Gln Ile Asn Arg Gln
380 385 390 395
gtc ata gtt gac agc gat aat agg tca ggt tat tct ggt att ttc tct 1252
Val Ile Val Asp Ser Asp Asn Arg Ser Gly Tyr Ser Gly Ile Phe Ser
400 405 410
gtt gag ggc aaa agc tgc atc aat agg tgc ttt tat gtg gag ttg ata 1300
Val Glu Gly Lys Ser Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile
415 420 425
CA 02410284 2002-11-20
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agg gga agg gaa cag gaa act aga gta tgg tgg acc tca aac agt att 1348
Arg Gly Arg Glu Gln Glu Thr Arg Val Trp Trp Thr Ser Asn Ser Ile
430 435 440
gtt gtg ttt tgt ggc act tca ggt acc tat ggg aca ggc tca tgg cct 1396
Val Val Phe Cys Gly Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro
445 450 455
gat ggg gcg gac atc aat ctc atg cct ata taagctttcg caattttaga 1446
Asp Gly Ala Asp Ile Asn Leu.Met Pro Ile
460 465
aaaaactcct tgtttctact 1466
<210> 12
<211> 469
<212> PRT
<213> Influenza A virus
<400> 12
Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Thr
1 5 10 15
Ile Ala Thr Ile Cys Phe Leu Met Gln Ile Ala Ile Leu Val Thr Thr
20 25 30
Val Thr Leu His Phe Lys Gln Tyr Glu Cys Asp Ser Pro Ala Asn Asn
35 40 45
Gln Va1 Met Pro Cys Glu Pro Ile Ile Ile Glu Arg Asn Ile Thr Glu
50 55 60
Ile Val Tyr Leu Thr Asn Thr Thr Ile Glu Lys Glu Ile Cys Pro Lys
65 70 75 80
Leu Val Glu Tyr Arg Asn Trp Ser Lys Pro Gln Cys Lys 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 Asp Pro Gly 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 Asp Thr Ile His Asp Arg Thr Pro His Arg Thr Leu Leu Met
145 150 155 160
Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Arg 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 Tyr Asp Lys Asn Ala Thr Ala Ser Phe Ile Tyr Asp
195 200 205
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Gly Arg Leu Val Asp Ser Ile Gly Ser Trp Ser Gln Asn Ile Leu Arg
210 215 220
Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val
225 230 235 240
Met Thr Asp Gly Ser Ala Ser Gly Arg Ala Asp Thr Lys Ile Leu Phe
245 250 255
Ile Glu 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 Gly Val Arg
275 280 285
Cys Ile Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Val Val Asp
290 295 300
Ile Asn Val Lys Asp Tyr Ser Ile Asp Ser Ser Tyr Val Cys Ser Gly
305 310 315 320
Leu Val Gly Asp Ala Pro Arg Asn Asn Asp Arg Ser Ser Asn Ser Tyr
325 330 335
Cys Arg Asn Pro Asn Asn Glu Lys Gly Asn His Gly Val Lys Gly Trp
340 345 350
Ala Phe Asp Asp Gly Asn Asp Val Trp Met Gly Arg Thr Ile Ser Glu
355 360 365
Asp Ser Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile Gly Gly Trp Ser
370 375 380
Thr Pro Asn Ser Lys Leu Gln Ile Asn Arg Gln Val Ile Val Asp Ser
385 390 395 400
Asp Asn Arg 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 Glu 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 Asp Ile
450 455 460
Asn Leu Met Pro Ile
465
<210> 13
<211> 1027
<212> DNA
<213> Influenza A virus
<220>
<221> CDS
<222> (26)..(781)
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<400> 13
agcaaaagca ggtagatatt gaaag atg agc ctt ctg acc gag gtc gaa acg 52
Met Ser Leu Leu Thr Glu Val Glu Thr
1 5
tat gtt ctc tct atc gtt ccg tca ggc ccc ctc aaa gcc gag atc gcg 100
Tyr Val Leu Ser Ile Val Pro Ser Gly Pro Leu Lys Ala Glu Ile Ala
15 20 25
cag aga ctt gaa gat gtc ttt get gga aag aac aca gat ctt gag get 148
Gln Arg Leu Glu Asp Val Phe Ala Gly Lys Asn Thr Asp Leu Glu Ala
30 35 40
ctc atg gaa tgg cta aag aca aga cca atc ctg tca cct ctg act aaa 196
Leu Met Glu Trp Leu Lys Thr Arg Pro Ile Leu Ser Pro Leu Th.r Lys
45 50 55
ggg att ttg gga ttt gta ttc acg ctc acc gtg ccc agt gag cga gga 244
Gly Ile Leu Gly Phe Val Phe Thr Leu Thr Val Pro Ser Glu Arg Gly
60 65 70
ctg cag cgt aga cgc ttt gtc caa aat gcc ctc aat ggg aat ggg gat 292
Leu Gln Arg Arg Arg Phe Val Gln Asn Ala Leu Asn Gly Asn Gly Asp
75 80 85
cca aat aac atg gac aga gca gtt aaa ctg tat aga aaa ctt aag agg 340
Pro Asn Asn Met Asp Arg Ala Val Lys Leu Tyr Arg Lys Leu Lys Arg
90 95 100 105
gag ata aca ttc cat ggg gcc aaa gaa ata gca ctc agt tat tct gct, 388
Glu Ile Thr Phe His Gly Ala Lys Glu Ile Ala Leu Ser Tyr Ser Ala
110 115 120
ggt gca ctt gcc agt tgc atg ggc ctc ata tac aac agg atg ggg get 436
Gly Ala Leu Ala Ser Cys Met Gly Leu Ile Tyr Asn Arg Met Gly Ala
125 130 135
gtg acc act gaa gtg gcc ttt ggc ctg gtt tgt gca acc tgt gaa cag 484
Val Thr Thr Glu Val Ala Phe Gly Leu Val Cys Ala Thr Cys Glu Gln
140 145 150
att get gac tcc cag cac agg tct cat agg caa atg gtg gca aca acc 532
Ile Ala Asp Ser Gln His Arg Ser His Arg Gln Met Val Ala Thr Thr
155 160 165
aat cca cta ata aga cat gag aac aga atg gtt ctg gcc agc act aca 580
Asn Pro Leu Ile Arg His Glu Asn Arg Met Val Leu Ala Ser Thr Thr
170 175 180 185
get aag get atg gag caa atg get gga tca agt gag cag gca gca gag 628
Ala Lys Ala Met Glu Gln Met Ala Gly Ser Ser Glu Gln Ala Ala Glu
190 195 200
gcc atg gag gtt get agt cag gcc agg caa atg gtg cag gca atg aga 676
Ala Met Glu Val Ala Ser Gln Ala Arg Gln Met Val Gln Ala Met Arg
205 210 215
gcc att ggg act cat cct agc tcc agt get ggt cta aaa gat gat ctt 724
Ala Ile Gly Thr His Pro Ser Ser Ser Ala Gly Leu Lys Asp Asp Leu
220 225 230
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ctt gaa aat ttg cag gcc tat cag aaa cga atg ggg gtg cag atg caa 772
Leu Glu Asn Leu Gln Ala Tyr Gln Lys Arg Met Gly Val Gln Met Gln
235 240 245
cga ttc aag tgaccctctt gttgttgctg cgagtatcat tgggatcttg 821
Arg Phe Lys
250
cacttgatat tgtggattct tgatcgtctt tttttcaaat gcatctatcg attctttgaa 881
cacggtctga aaagagggcc ttctacggaa ggagtacctg agtctatgag ggaagaatat 941
cgaaaggaac agcagagtgc tgtggatgct gacgacagtc attttgtcag catagagctg 1001
gagtaaaaaa ctaccttgtt tctact 1027
<210> 14
<211> 252
<212> PRT
<213> Influenza A virus
<400> 14
Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro
1 5 10 15
Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe
20 25 30
Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr
35 40 45
Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe
50 55 60
Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val
65 70 75 80
Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala
85 90 95
Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala
100 105 110
Lys Glu Ile Ala Leu Ser Tyr Ser Ala Gly Ala Leu Ala Ser Cys Met
115 120 125
Gly Leu Ile Tyr Asn Arg Met Gly Ala Val Thr Thr Glu Val Ala Phe
130 135 140
Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg
145 150 155 160
Ser His Arg Gln Met Val Ala Thr Thr Asn Pro Leu Ile Arg His Glu
165 170 175
Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met
180 185 190
Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln
CA 02410284 2002-11-20
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195 200 205
Ala Arg Gln Met Val Gln Ala Met Arg Ala Ile Gly Thr His Pro Ser
210 215 220
Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr
225 230 235 240
Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys
245 250
<210> 15
<211> 322
<212> DNA
<213> Influenza A virus
<220>
<221> CDS
<222> (26)..(316)
<400> 15
agcaaaagca ggtagatatt gaaag atg agc ctt ctg acc gag gtc gaa acg 52
Met Ser Leu Leu Thr Glu Val Glu Thr
1 5
cct atc aga aac gaa tgg ggg tgc aga tgc aac gat tca agt gac cct 100
Pro Ile Arg Asn Glu Trp Gly Cys Arg Cys Asn Asp Ser Ser Asp Pro
15 20 25
ctt gtt gtt get gcg agt atc att ggg atc ttg cac ttg ata ttg tgg 148
Leu Val Va1 Ala Ala Ser Ile Ile Gly Ile Leu His Leu Ile Leu Trp
30 35 40
att ctt gat cgt ctt ttt ttc aaa tgc atc tat cga ttc ttt gaa cac 196
Ile Leu Asp Arg Leu Phe Phe Lys Cys Ile Tyr Arg Phe Phe Glu His
45 50 55
ggt ctg aaa aga ggg cct tct acg gaa gga gta cct gag tct atg agg 244
Gly Leu Lys Arg Gly Pro Ser Thr Glu Gly Val Pro Glu Ser Met Arg
60 65 70
gaa gaa tat cga aag gaa cag cag agt get gtg gat get gac gac agt 292
Glu Glu Tyr Arg Lys Glu Gln Gln Ser Ala Val Asp Ala Asp Asp Ser
75 80 85
cat ttt gtc agc ata gag ctg gag taaaaa 322
His Phe Val Ser Ile Glu Leu Glu
90 95
<210> 16
<211> 97
<212> PRT
<213> Influenza A virus
<400> 16
Met Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly
1 5 10 15
CA 02410284 2002-11-20
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Cys Arg Cys Asn Asp Ser Ser Asp Pro Leu Val Val Ala Ala Ser Ile
20 ~ 25 30
Ile Gly Ile Leu His Leu Ile Leu Trp Ile Leu Asp Arg Leu Phe Phe
35 40 45
Lys Cys Ile Tyr Arg Phe Phe Glu His Gly Leu Lys Arg Gly Pro Ser
50 55 60
Thr Glu Gly Val Pro Glu Ser Met Arg Glu Glu Tyr Arg Lys Glu Gln
65 70 75 80
Gln Ser Ala Val Asp Ala Asp Asp Ser His Phe Val Ser Ile Glu Leu
85 90 95
Glu
<210> 17
<211> 890
<212> DNA
<213> Influenza A virus
<220>
<221> CDS
<222> (27)..(737)
<400> 17
agcaaaagca gggtgacaaa gacata atg gat tcc aac act gtg tca agt ttt 53
Met Asp Ser Asn Thr Val Ser Ser Phe
1 5
cag gta gac tgc ttc ctt tgg cat gtc cga aaa caa gtt gta gac caa 101
Gln Val Asp Cys Phe Leu Trp His Val Arg Lys Gln Val Val Asp Gln
15 20 25
gaa cta ggt gat gcc cca ttc ctt gat cgg ctt cgc cga gat cag aag 149
Glu Leu Gly Asp Ala Pro Phe Leu Asp Arg Leu Arg Arg Asp Gln Lys
30 35 40
tcc cta agg gga aga ggc agc act ctc ggt cta aac atc gaa gca gcc 197
Ser Leu Arg Gly Arg Gly Ser Thr Leu Gly Leu Asn Ile Glu Ala Ala
45 50 55
acc cat gtt gga aag cag ata gta gag aag att ctg aag gaa gaa tct 245
Thr His Val Gly Lys Gln Ile Val Glu Lys Ile Leu Lys Glu Glu Ser
60 65 70
gat gag gca ctt aaa atg acc atg gcc tcc aca cct get tcg cga tac 293
Asp Glu Ala Leu Lys Met Thr Met Ala Ser Thr Pro Ala Ser Arg Tyr
'75 80 85
ata act gac atg act att gag gaa ttg tca agg gac tgg ttc atg cta 341
Ile Thr Asp Met Thr Ile Glu Glu Leu Ser Arg Asp Trp Phe Met Leu
90 95 100 105
atg ccc aag cag aaa gtg gaa gga cct ctt tgc atc aga ata gac caa 389
Met Pro Lys Gln Lys Val Glu Gly Pro Leu Cys Ile Arg Ile Asp Gln
110 115 120
CA 02410284 2002-11-20
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gca atc atg gat aag aac atc atg ttg aaa gcg aat ttc agt gtg att 437
Ala Ile Met Asp Lys Asn Ile Met Leu Lys Ala Asn Phe Ser Val Ile
125 130 135
ttt gac cgg cta gag acc cta ata tta cta agg get ttc acc gaa gag 485
Phe Asp Arg Leu Glu Thr Leu Ile Leu Leu Arg Ala Phe Thr Glu Glu
140 145 150
gga gca att gtt ggc gaa atc tca cca ttg cct tct ttt cca gga cat 533
Gly Ala Ile Val Gly Glu Ile Ser Pro Leu Pro Ser Phe Pro Gly His
155 160 165
act att gag gat gtc aaa aat gca att ggg gtc ctc atc gga gga ctt 581
Thr Ile Glu Asp Val Lys Asn Ala Ile Gly Val Leu Ile Gly Gly Leu
170 175 180 185
gaa tgg aat gat aac aca gtt cga gtc tct aaa act cta cag aga ttc 629
Glu Trp Asn Asp Asn Thr Val Arg Val Ser Lys Thr Leu Gln Arg Phe
190 195 200
get tgg gga agc agt aat gag aat ggg aga cct cca ctt act cca aaa 677
Ala Trp Gly Ser Ser Asn Glu Asn Gly Arg Pro Pro Leu Thr Pro Lys
205 210 215
cag aaa cgg aaa atg gcg aga aca get agg tca aaa gtt cga aga gat 725
Gln Lys Arg Lys Met Ala Arg Thr Ala Arg Ser Lys Val Arg Arg Asp
220 225 230
aag atg get gat tgaagaagtg agacacagac tgaagacaac agagaatagt 777
Lys Met Ala Asp
235
tttgagcaaa taacattcat gcaagcctta cagctactat ttgaagtgga acaggagata 837
agaactttct cgtttcagct tatttaatga taaaaaacac ccttgtttct act 890
<210> 18
<211> 237
<212> PRT
<213> Influenza A virus
<400> 18
Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
His Val Arg Lys Gln Val Val Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
35 40 45
Thr Leu Gly Leu Asn Ile Glu Ala Ala Thr His Val Gly Lys Gln Ile
50 55 60
Val Glu Lys Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80
Met Ala Ser Thr Pro Ala Ser Arg Tyr Ile Thr Asp Met Thr Ile Glu
85 90 95
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Glu Leu Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Glu
100 105 110
G1y Pro Leu Cys Ile Arg Ile Asp Gln Ala Ile Met Asp Lys Asn Ile
115 120 125
Met Leu Lys Ala Asn Phe Ser Val Ile Phe Asp Arg Leu Glu Thr Leu
130 135 140
Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Ile Val Gly Glu Ile
145 150 155 160
Ser Pro Leu Pro Ser Phe Pro Gly His Thr Ile Glu Asp Val Lys Asn
165 170 175
Ala Ile Gly Val Leu Ile Gly Gly Leu Glu Trp Asn Asp Asn Thr Val
180 185 190
Arg Val Ser Lys Thr Leu Gln Arg Phe Ala Trp Gly Ser Ser Asn Glu
195 200 205
Asn Gly Arg Pro Pro Leu Thr Pro Lys Gln Lys Arg Lys Met Ala Arg
210 215 220
Thr Ala Arg Ser Lys Val Arg Arg Asp Lys Met Ala Asp
225 230 235
<210> 19
<211> 402
<212> DNA
<213> Influenza A virus
<220>
<221> CDS
<222> (27)..(389)
<400> 19
agcaaaagca gggtgacaaa gacata atg gat tcc aac act gtg tca agt ttt 53
Met Asp Ser Asn Thr Va1 Ser Ser Phe
1 5
cag gac ata cta ttg agg atg tca aaa atg caa ttg ggg tcc tca tcg 101
Gln Asp Ile Leu Leu Arg Met Ser Lys Met Gln Leu Gly Ser Ser Ser
15 20 25
gag gac ttg aat gga atg ata aca cag ttc gag tct cta aaa ctc tac 149
Glu Asp Leu Asn Gly Met Ile Thr Gln Phe Glu Ser Leu Lys Leu Tyr
30 35 40
aga gat tcg ctt ggg gaa gca gta atg aga atg gga gac ctc cac tta 197
Arg Asp Ser Leu Gly Glu Ala Val Met Arg Met Gly Asp Leu His Leu
45 50 55
ctc caa aac aga aac gga aaa tgg cga gaa cag cta ggt caa aag ttc 245
Leu Gln Asn Arg Asn Gly Lys Trp Arg Glu Gln Leu Gly Gln Lys Phe
60 65 70
gaa gag ata aga tgg ctg att gaa gaa gtg aga cac aga ctg aag aca 293
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Glu Glu Ile Arg Trp Leu Ile Glu Glu Val Arg His Arg Leu Lys Thr
75 80 85
aca gag aat agt ttt gag caa ata aca ttc atg caa gcc tta cag cta 341
Thr Glu Asn Ser Phe Glu Gln Ile Thr Phe Met Gln Ala Leu Gln Leu
90 95 100 105
cta ttt gaa gtg gaa cag gag ata aga act ttc tcg ttt cag ctt att 389
Leu Phe Glu Val Glu Gln Glu Ile Arg Thr Phe Ser Phe Gln Leu Ile
110 115 120
taatgataaa aaa 402
<210> 20
<211> 121
<212> PRT
<213> Influenza A virus
<400> 20
Met Asp Ser Asn Thr Val Ser Ser Phe Gln Asp Ile Leu Leu Arg Met
1 5 10 15
Ser Lys Met Gln Leu Gly Ser Ser Ser Glu Asp Leu Asn Gly Met Ile
20 25 30
Thr Gln Phe Glu Ser Leu Lys Leu Tyr Arg Asp Ser Leu Gly Glu Ala
35 40 45
Val Met Arg Met Gly Asp Leu His Leu Leu Gln Asn Arg Asn Gly Lys
50. 55 60
Trp Arg Glu Gln Leu Gly Gln Lys Phe Glu Glu Ile Arg Trp Leu Ile
65 70 75 80
Glu Glu Val Arg His Arg Leu Lys Thr Thr Glu Asn Ser Phe Glu Gln
85 90 95
Ile Thr Phe Met Gln Ala Leu Gln Leu Leu Phe Glu Val Glu Gln Glu
100 105 110
Ile Arg Thr Phe Ser Phe Gln Leu Ile
115 120
<210> 21
<211> 1764
<212> DNA
<213> Influenza A virus
<220>
<221> CDS
<222> (30)..(1727)
<400> 21
agcaaaagca ggggataatt ctattaacc atg aaa act atc att get ttg agc 53
Met Lys Thr Ile Ile Ala Leu Ser
1 5
tac att ttc tgt ctg gtt ctc ggc caa gac ttt cca gga aat gac aac 101
CA 02410284 2002-11-20
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Tyr Ile Phe Cys Leu Val Leu Gly Gln Asp Phe Pro Gly Asn Asp Asn
15 20
agc aca gca acg ctg tgc ctg gga cat cat gcg gtg cca aac gga aca 149
Ser Thr Ala Thr Leu Cys Leu Gly His His Ala Val Pro Asn Gly Thr
25 30 35 40
cta gtg aaa aca atc aca aat gat cag att gaa gtg act aat get act 197
Leu Val Lys Thr Ile Thr Asn Asp Gln Ile Glu Val Thr Asn Ala Thr
45 50 55
gag ctg gtt cag agt tcc tca acg ggg aaa ata tgc aac aat cct cat 245
Glu Leu Val Gln Ser Ser Ser Thr Gly Lys Ile Cys Asn Asn Pro His
60 65 70
cga atc ctt gat gga ata gac tgc aca ctg ata gat get cta ttg ggg 293
Arg Ile Leu Asp Gly Ile Asp Cys Thr Leu Ile Asp Ala Leu Leu Gly
75 80 85
gac cct cat tgt gat ggc ttt caa aat gag aca tgg gac ctt ttc gtt 341
Asp Pro His Cys Asp Gly Phe Gln Asn Glu Thr Trp Asp Leu Phe Val
90 95 100
gaa cgc agc aaa get ttc agc aac tgt tac cct tat gat gtg cca gat 389
Glu Arg Ser Lys Ala Phe Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp
105 110 115 120
tat gcc tCC Ctt agg tca cta gtt gcc tcg tca ggc act ctg gag ttt 437
Tyr Ala Ser Leu Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe
125 130 135
atc agt gaa ggc ttc act tgg act ggg gtc act cag aat ggg gga agc 485
Ile Ser Glu Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly Gly Ser
140 145 150
aat get tgc aaa agg gga cct gat agc ggt ttt ttc agt aga ctg aac 533
Asn Ala Cys Lys Arg Gly Pro Asp Ser Gly Phe Phe Ser Arg Leu Asn
155 160 165
tgg ttg tac aaa tca gga agc aca tat cca gtg ctg aac gtg act atg 581
Trp Leu Tyr Lys Ser Gly Ser Thr Tyr Pro Val Leu Asn Val Thr Met
170 175 180
cca aac aat gac aat ttt gac aaa cta tac att tgg ggg gtt cac cac 629
Pro Asn Asn Asp Asn Phe Asp Lys Leu Tyr Ile Trp Gly Val His His
185 190 195 200
ccg agc acg gac caa gaa caa acc agc cta tat gtt caa gca tca ggg 677
Pro Ser Thr Asp Gln Glu Gln Thr Ser Leu Tyr Val Gln Ala Ser Gly
205 210 215
aga gtc aca gtc tct acc aag aga agc cag caa act ata atc ccg aat 725
Arg Val Thr Val Ser Thr Lys Arg Ser Gln Gln Thr Ile Ile Pro Asn
220 225 230
atc ggg tct aga ccc tgg gta agg ggt ctg tct agt aga ata agc atc 773
Ile Gly Ser Arg Pro Trp Val Arg Gly Leu Ser Ser Arg Ile Ser Ile
235 240 245
tat tgg aca ata gtt aaa ccg gga gac ata ctg .gta att aat agt aat 821
Tyr Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Val Ile Asn Ser Asn
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
250 255 260
ggg aac cta att get cct cgg ggt tat ttt aaa atg cgc act ggg aaa 869
Gly Asn Leu Ile Ala Pro Arg Gly Tyr Phe Lys Met Arg Thr Gly Lys
265 270 275 280
agc tca ata atg agg tca gat gca cct att ggc acc tgc att tct gaa 917
Ser Ser Ile Met Arg Ser Asp Ala Pro Ile Gly Thr Cys Ile Ser Glu
285 290 295
tgc atc act cca aat gga agc att ccc aat gac aag ccc ttt caa aac 965
Cys Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn
300 305 310
gta aac aag atc aca tat ggg gca tgt ccc aag tat gtt aag caa aac 1013
Val Asn Lys Ile Thr Tyr Gly Ala Cys Pro Lys Tyr Val Lys Gln Asn
315 320 325
acc ctg aag ttg gca aca ggg atg cgg aat gta cca gag aaa caa act 1061
Thr Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr
330 335 340
aga ggc cta ttc agc gca ata gca ggt ttc ata gaa aat ggt tgg gag 1109
Arg Gly Leu Phe Ser Ala lle Ala Gly Phe Ile Glu Asn Gly Trp Glu
345 350 355 360
gga atg ata gac ggt tgg tac ggt ttc agg cat caa aat tct gag ggc 1157
Gly Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly
365 370 375
aca gga caa gca gca gat ctt aaa agc act caa gca gcc atc gac caa 1205
Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln
380 385 390
atc aat ggg aaa ctg aat agg gta atc gag aag acg aac gag aaa ttc 1253
Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe
395 400 405
cat caa atc gaa aag gaa ttc tca gaa gta gaa ggg aga att cag gac 1301
His Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp
410 415 420
ctc gag aaa tac gtt gaa gac act aaa ata gat ctc tgg tct tac aat 1349
Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn
425 430 435 440
gcg gag ctt ctt gtc get ctg gag aac caa cat aca att gat ctg act 1397
Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr
445 450 455
gac tcg gaa atg aac aaa ctg ttt gaa aaa aca agg agg caa ctg agg 1445
Asp Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg
460 465 470
gaa aat get gag gac atg ggc aat ggt tgc ttc aaa ata tac cac aaa 1493
Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys
475 480 485
tgt gac aat get tgc ata ggg tca atc aga aat ggg act tat gac cat 1541
Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His
490 495 500
CA 02410284 2002-11-20
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gat gta tac aga gac gaa gca tta aac aac cgg ttt cag atc aaa ggt 1589
Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly
505 510 5'15 520
gtt gaa ctg aag tca gga tac aaa gac tgg atc ctg tgg att tcc ttt 1637
Val Glu Leu.Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe
525 530 535
gcc ata tca tgc ttt ttg ctt tgt gtt gtt ttg ctg ggg ttc atc atg 1685
Ala Bile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met
540 545 550
tgg gcc tgc cag aaa ggc aac att agg tgc aac att tgc att 1727
Trp~Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile
555 560 565
tgagtgtatt agtaattaaa aacaccctgt ttctact 1764 ..
<210> 22
<211> 566
<212> PRT
<213> Influenza A virus
<400> 22
Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Leu Gly
1 5 10 15
Gln Asp Phe Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly
20 25 30
His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile Thr Asn Asp
35 40 45
Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr
50 55 60
Gly Lys Ile Cys Asn Asn Pro His Arg Ile Leu Asp Gly Ile Asp Cys
65 70 75 80
Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Gly Phe Gln
85 90 95
Asn Glu Thr Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Phe Ser Asn
100 105 110
Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val
115 120 125
Ala Ser Ser Gly Thr Leu Glu Phe Ile Ser Glu Gly Phe Thr Trp Thr
130 135 140
Gly Val Thr Gln Asn Gly Gly Ser Asn Ala Cys Lys Arg Gly Pro Asp
145 150 155 160
Ser Gly Phe Phe Ser Arg Leu Asn Trp Leu Tyr Lys Ser Gly Ser Thr
165 170 175
Tyr Pro Val Leu Asn Val Thr Met Pro Asn Asn Asp Asn Phe Asp Lys
180 185 190
CA 02410284 2002-11-20
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Leu Tyr Ile Trp Gly Val His His Pro Ser Thr Asp Gln Glu Gln Thr
195 200 205
Ser Leu Tyr Val Gln Ala Ser Gly Arg Val Thr Val Ser Thr Lys Arg
210 215 220
Ser Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg
225 230 235 240
Gly Leu Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly
245 250 255
Asp Ile Leu Val Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro Arg Gly
260 265 270
Tyr Phe Lys Met Arg Thr Gly Lys Ser Ser Ile Met Arg Ser Asp Ala
275 280 285
Pro Ile Gly Thr Cys Ile Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile
290 295 300
Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Ile Thr Tyr Gly Ala
305 310 315 320
Cys Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met
325 330 335
Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Leu Phe Ser Ala,Ile Ala
340 345 350
Gly Phe Ile G1u Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly
355 360 365
Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys
370 375 380
Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val
385 390 395 400
Ile Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser
405 410 415
Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr
420 425 430
Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu
435 440 445
Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe
450 455 460
Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn
465 470 475 480
Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser
485 490 495
Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu
500 505 510
CA 02410284 2002-11-20
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Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys
515 520 525
Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys
530 535 540
Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile
545 550 555 560
Arg Cys Asn Ile Cys Ile
565
<210> 23
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 23
tatggaaaga ataaaagaac tacggaa 27
<210> 24
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 24
tcgtttttaa actattcaac at 22
<210> 25
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 25
agcaggtcaa ttatattcaa tatg 24
<210> 26
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:Primer ,
<400> 26
aacaaggtcg tttttaaact attc 24
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<210> 27
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 27
agaactctat tccaacaaat g 21
<210> 28
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 28
aatcggatat ttcattgcca t 21
<210> 29
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 29
agaactctat tccaacaaat gagggatgta gtt 33
<210> 30
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 30
aatcggatat ttcattgcca tcatccattt cat 33
<210> 31
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 31
agacgtggtg ttggtaatga a 21
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<210> 32
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 32
cgaagagttg acataaaccc t 21
<210> 33
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 33
tcatccctca tcccctcaca t 21
<210> 34
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 34
attttctgtt atcctcttgt ca 22
<210> 35
<211> 20
<212> DNA a
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 35
gtggagtccg ctgttttgag 20
<210> 36
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 36
aggatggtgg acattcttag g 21
<210> 37
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 37
ccgatcaatg ctaaccacta c 21
<210> 38
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 38
agcaaaagca ggtcaattat attca 25
<210> 39
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 39
agtagaaaca aggtcgtttt taaac , 25
<210> 40
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 40
agcaaaagca ggcaaaccat 20
<210> 41
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 41
agtagaaaca aggcattttt t 21
<210> 42
<211> 20
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 42
tcgagctgaa gaagctatgg 20
<210> 43
<211> 20
<2l2> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 43
gttctgttga ctgtgtccat 20
<210> 44
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 44
tcgagctgaa gaagctatgg gagcagaccc gt 32
<210> 45
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 45
gttctgttga ctgtgtccat ggtgtatcc 29
<210> 46
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 46
agcgaaagca ggcaaaccat ttgaatggat gtcaa 35
<210> 47
<211> 31
<212> DNA
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 47
attaaaaaca aggcattttt tcatgaagga c 31
<210> 48
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 48
aatttccagc atggtggagg ccatggtg 28
<210> 49
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 49
gggatctttg aaaactcgtg 20
<210> 50
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 50
cagcattgtt tacagactc 19
<210> 51
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 51
accaaagatg cagaaagagg 20
<210> 52
<211> 20
<212> DNA
<213> Artificial Sequence
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<220>
<223> Description of Artificial Sequence: Primer
<400> 52
ctcatattga ttccgactaa 20
<210> 53
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 53
tgtCClCttC CCtttttCtg a 21
<210> 54
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 54
atttttcccc agtagttcat ac 22
<210> 55
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 55
acgctgttgc aactacacac tcctg 25
<210> 56
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 56
agcaaaagca ggtactgatt cgaga 25
<210> 57
<211> 25
<212> DNA
<213> Artificial Sequence
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<220>
<223> Description of Artificial Sequence: Primer
<400> 57
agtagaaaca aggtactttt ttgga 25
<210> 58
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 58
gaacctggaa cctttgatct t 21
<210> 59
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 59
attcaccact gtccaggcca t 21
<210> 60
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 60
gaacctggaa cctttgatct tgaggggcta 30
<210> 61
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 61
attcaccact gtccaggcca ttgacgcgtc 30
<210> 62
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<223> Description of Artificial Sequence: Primer
<400> 62
agcaaaagca ggtagtgata g 21
<220> 63
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 63
agtagaaaca aggta 15
<210> 64
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer
<400> 64
agcgaaagca ggtagtgatt cgagatgga 29
<210> 65
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 65
agtagaaaca aggtactttt ttggacag 28
<210> 66
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 66
tcgatttgtt ggagtgactg a 21
<210> 67
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<400> 67
gccgaacttc tcctgccttg a 21
<210> 68
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 68
gtattcaata gcctgtatg 19
<210> 69
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 69
gacttctctc cttgtcactc 20
<210> 70
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 70
acgagtcagc taaagtgggc a 21
<210> 71
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 71
gacacctctg ctgtgaagta a 21
<210> 72
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<400> 72
ggagctgaga aaccgaagtt t 21
<210> 73
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 73
gacttggcca ataaagtcct a 21
<210> 74
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 74
gcctgcgcat agttcctgtg a ' 21
<210> 75
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 75
tctaccacta ttgactcgcc 20
<210> 76
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 76
agcaaaagca ggggataatt cta 23
<210> 77
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 77
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
agtagaaaca agggtgtttt taa 23
<210> 78
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 78
acagtttgtt catttccgag 20
<210> 79
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 79
acttcagggt gttttgctta ~ 20
<210> 80
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 80
gaacccccca aatgtatagt 20
<210> 81
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 81
tgaggaactc tgaaccagct 20
<210> 82 '
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 82
gtcactagtt gcctcgtcag 20
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<210> 83
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 83
ccgggagaca tactggtaat 20
<210> 84
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 84
caaatcaatg ggaaactgaa 20
<210> 85
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 85
tgaactgaag tcaggataca 20
<210> 86
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 86
agcaaaagca gggttaataa tcac 24
<210> 87
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 87
agtagaaaca agggtatttt tcct 24
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<210> 88
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 88
ttgcaccttc catcatcctt 20
<210> 89
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 89
gtctattccc actaaagagt 20
<210> 90
<211> 20
<212 > DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 90
tgcatcagag agcacatcct 20
<210> 91
<211> 20
<212> DNA '
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 91
gaactcgtcc tttatgacaa 20
<210> 92
<211> 17 _
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 92
agagcaatgg atcaagt 17
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<210> 93
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 93
tactatggaa tcaagtactc . 20
<210> 94
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 94
atcaatcatc ttcccgac 18
<210> 95
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 95
agtgtccttc cgtgggcg ~ 18
<210> 96
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 96
agcaaaagca ggagtgaaga tga 23
<210> 97
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 97
agtagaaaca aggagttttt tcta 24
<210> 98
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 98
tcgttgtttc tgggtgtgtc 20
<210> 99
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 99
ttatcatacc cagtgacaca 20
<210> 100
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 100
attattattg gttcacacgg 20
<210> 101
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:Primer
<400> 101
ttatgtgtca tgcgatcctg 20
<210> 102
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 102
attgttcata ttagcccatt 20
<210> 103
<211> 20
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 103
ataggtcagg ttattctggt 20
<210> 104
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 104
agcaaaagca ggtag 15
<210> 105
<211> 15 '
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 105
agtagaaaca aggta 15
<210> 106
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 106
tttactccag ctctatgctg acaa 24
<210> 107
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 107
gatccagcca tttgctccat 20
<210> 108
<211> 20
<212> DNA
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 108
gaggtgacag gattggtctt 20
<210> 109
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 109
catggacaga gcagttaaac 20
<210> 110
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 110
gcgagtatca ttgggatctt 20
<210> 111
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 111
agcaaaagca gggtgacaaa gaca 24
<210> 112
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 112
agtagaaaca agggtgtttt ttat 24
<210> 113
<211> 24
<212> DNA
<213> Artificial Sequence
CA 02410284 2002-11-20
WO 02/00884 PCT/USO1/19826
<220>
<223> Description of Artificial Sequence: Primer
<400> 113
ttttttatca ttaaataagc tgaa 24
<210> 114
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 114
aattgcattt ttgacatcct 20
<210> 115
<211> 20
<212> DNA
<213> Artificial Sequence
<220> ,
<223> Description of Artificial Sequence: Primer
<400> 115
atcttctcta ctatctgctt 20
<210> 116
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 116
caagcaatca tggataagaa 20
<210> 117
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 117
ggcgagaaca gctaggtcaa 20
