U.S. patent application number 16/665104 was filed with the patent office on 2020-05-21 for influenza virus reassortment.
This patent application is currently assigned to Seqirus UK Limited. The applicant listed for this patent is Seqirus UK Limited Synthetic Genomics, Inc.. Invention is credited to Philip Ralph DORMITZER, Peter MASON, Pirada SUPHAPHIPHAT, Heidi TRUSHEIM.
Application Number | 20200155666 16/665104 |
Document ID | / |
Family ID | 49713076 |
Filed Date | 2020-05-21 |
United States Patent
Application |
20200155666 |
Kind Code |
A1 |
MASON; Peter ; et
al. |
May 21, 2020 |
INFLUENZA VIRUS REASSORTMENT
Abstract
New influenza donor strains for the production of reassortant
influenza A viruses are provided.
Inventors: |
MASON; Peter; (Cambridge,
MA) ; DORMITZER; Philip Ralph; (Cambridge, MA)
; TRUSHEIM; Heidi; (Marburg, DE) ; SUPHAPHIPHAT;
Pirada; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Seqirus UK Limited
Synthetic Genomics, Inc. |
Berkshire
La Jolla |
CA |
GB
US |
|
|
Assignee: |
; Seqirus UK Limited
Berkshire
CA
Synthetic Genomics, Inc.
La Jolla
|
Family ID: |
49713076 |
Appl. No.: |
16/665104 |
Filed: |
October 28, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15621270 |
Jun 13, 2017 |
10500266 |
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16665104 |
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14648886 |
Jun 1, 2015 |
9708585 |
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PCT/EP2013/075294 |
Dec 2, 2013 |
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15621270 |
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61732809 |
Dec 3, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2760/16152
20130101; C12N 2760/16151 20130101; C12N 2760/16134 20130101; A61K
39/12 20130101; A61K 2039/5252 20130101; A61K 39/145 20130101; A61P
31/16 20180101; C12N 2760/16121 20130101; C12N 7/00 20130101 |
International
Class: |
A61K 39/145 20060101
A61K039/145; A61K 39/12 20060101 A61K039/12; C12N 7/00 20060101
C12N007/00 |
Goverment Interests
[0001] This invention was made in part with Government support
under grant no. HHSO10020100061 C awarded by the Biomedical
Advanced Research and Development Authority (BARDA). The Government
has certain rights in the invention.
Claims
1-24. (canceled)
25. A reassortant influenza A virus comprising an HA segment, an NA
segment and backbone segments PA, PB1, PB2, NP, NS and M, wherein
the backbone segments are from two or more donor strains, wherein
(a) the HA segment and the PB1 segment are from different influenza
A strains with the same influenza virus HA subtype, (b) wherein the
HA segment and the PB1 segment are from different influenza A
strains with different influenza virus HA subtypes, wherein the PB1
segment is not from an influenza virus with a H3 HA subtype and/or
wherein the HA segment is not from an influenza virus with a H1 or
H5 HA subtype, or (c) at least one backbone segment is from the
A/California/07/09 influenza strain.
26. The reassortant influenza A virus of claim 25, wherein the HA
segment and the PB1 segment are from a H1 influenza strain.
27. The reassortant influenza A virus of claim 25, wherein the
reassortant influenza A virus comprises (b) and the PB1 segment is
from a H1 virus and/or wherein the HA segment is from a H3
influenza vims.
28. The reassortant influenza A virus of claim 25, wherein the
reassortant influenza A virus comprises (c) and the at least one
backbone segment is the PB1 segment.
29. The reassortant influenza A virus of claim 28, wherein the PB1
segment has at least 95%, at least 99% identity, or 100% identity
with the sequence of SEQ ID NO: 16.
30. The reassortant influenza A virus of claim 25, wherein the
reassortant influenza A virus comprises (c) and the HA segment is
from a H1 influenza strain.
31. The reassortant influenza A virus of claim 25, wherein the PB1
segment and the PB2 segment are from the same donor strain.
32. The reassortant influenza A virus of claim 25, wherein the
segments are selected from the group consisting of: a) the PA
segment having at least 95% or 99% identity to the sequence of SEQ
ID NO: 1; b) the PB2 segment having at least 95% or 99% identity to
the sequence of SEQ ID NO: 3; c) the M segment having at least 95%
or 99% identity to the sequence of SEQ ID NO: 5; d) the NP segment
having at least 95% or 99% identity to the sequence of SEQ ID NO:
4; and/or e) the NS segment having at least 95% or 99% identity to
the sequence of SEQ ID NO: 6.
33. The reassortant influenza A virus of claim 32, wherein the PA
segment has 95% identity to the sequence of SEQ ID NO: 1, the PB2
segment has 95% identity to the sequence of SEQ ID NO: 3, the M
segment has 95% identity to the sequence of SEQ ID NO: 5, the NP
segment has 95% identity to the sequence of SEQ ID NO: 4 and the NS
segment has 95% identity to the sequence of SEQ ID NO: 6.
34. The reassortant influenza A virus of claim 33, wherein the PA
segment has the sequence of SEQ ID NO: 1, the PB2 segment has the
sequence of SEQ ID NO: 3, the M segment has the sequence of SEQ ID
NO: 5, the NP segment has the sequence of SEQ ID NO: 4 and the NS
segment has the sequence of SEQ ID NO: 6.
35. The reassortant influenza A virus of claim 25 comprising
backbone segments (i) from two, three or four donor strains,
wherein each donor strain provides more than one backbone segment,
(ii) from two or more donor strains, wherein the PB1 segment is not
from the A/Texas/1/77 influenza strain, or (iii) from two or more
donor strains, wherein at least the PA, NP, or M segment are not
from A/Puerto Rico/8/34.
36. The reassortant influenza A virus of claim 25, wherein at least
one of the backbone segments is selected from the group consisting
of: a) the PB2 segment which has lysine in the position
corresponding to amino acid 389 of SEQ ID NO: 3 when aligned to SEQ
ID NO: 3, using a pairwise alignment algorithm; and/or b) the PB2
segment which has asparagine in the position corresponding to amino
acid 559 of SEQ ID NO: 3 when aligned to SEQ ID NO: 3, using a
pairwise alignment algorithm; and/or c) the PA segment which has
lysine in the position corresponding to amino acid 327 of SEQ ID
NO: 1 when aligned to SEQ ID NO: 1, using a pairwise alignment
algorithm; and/or d) the PA segment which has aspartic acid in the
position corresponding to amino acid 444 of SEQ ID NO: 1 when
aligned to SEQ ID NO: 1, using a pairwise alignment algorithm;
and/or e) the PA segment which has aspartic acid in the position
corresponding to amino acid 675 of SEQ ID NO: 1 when aligned to SEQ
ID NO: 1, using a pairwise alignment algorithm; and/or f) the NP
segment which has threonine in the position corresponding to amino
acid 27 of SEQ ID NO: 4 when aligned to SEQ ID NO: 4 using a
pairwise alignment algorithm; and/or g) the NP segment which has
asparagine in the position corresponding to amino acid 375 of SEQ
ID NO: 4 when aligned to SEQ ID NO: 4, using a pairwise alignment
algorithm.
37. The reassortant influenza A strain of claim 36, wherein (i) the
PB2 segment has lysine in the position corresponding to amino acid
389 of SEQ ID NO: 3 and asparagine in the position corresponding to
amino acid 559 of SEQ ID NO: 3 when aligned to SEQ ID NO: 3, using
a pairwise alignment algorithm, (ii) the PA segment has lysine in
the position corresponding to amino acid 327: aspartic acid in the
position corresponding to amino acid 444 of SEQ ID NO: 1 and
aspartic acid in the position corresponding to amino acid 675 when
aligned to SEQ ID NO: 1, using a pairwise alignment algorithm, or
(iii) the NP genome segment has threonine in the position
corresponding to amino acid 27 of SEQ ID NO: 4 and asparagine in
the position corresponding to amino acid 375 when aligned to SEQ ID
NO: 4. using a pairwise alignment algorithm, or (iv) the influenza
A strain is a H1 strain.
38. The reassortant influenza A strain of claim 25, wherein the PB2
segment has lysine in the position corresponding to amino acid 389
of SEQ ID NO: 3 and asparagine in the position corresponding to
amino acid 559 of SEQ ID NO: 3 when aligned to SEQ ID NO: 3, using
a pairwise alignment algorithm, PA genome segment has lysine in the
position corresponding to amino acid 327; aspartic acid in the
position corresponding to amino acid 444 of SEQ ID NO: 1 and
aspartic acid in the position corresponding to amino acid 675 when
aligned to SEQ ID NO: 1, using a pairwise alignment algorithm, and
an NP genome segment has threonine in the position corresponding to
amino acid 27 of SEQ ID NO: 4 and asparagine in the position
corresponding to amino acid 375 when aligned to SEQ ID NO: 4, using
a pairwise alignment algorithm
39. A method of preparing a reassortant influenza A virus of claim
25 comprising steps of (i) introducing into a culture host one or
more expression construct(s) which encode(s) the viral segments
required to produce an influenza A virus wherein the expression
construct(s) encode the backbone segments from two or more donor
strains and wherein the HA and PB1 genome segments are from
different influenza strains which have the same influenza HA
subtype; and (ii) culturing the culture host in order to produce
the reassortant influenza A virus of claim 25, wherein the
reassortant influenza A virus comprises (a).
40. The method of claim 39, wherein (1) the expression construct(s)
do/does not encode the PB1 segment from the A/Texas/1/77 influenza
strain, (2) the at least one expression construct comprises a
sequence having at least 90% or 100% identity with the sequence of
SEQ ID NO: 22, (3) the expression construct(s) further comprise(s)
one or more of the sequences having at least 90% identity or 100%
identity with the sequences of SEQ ID Nos.: 9 and/or 11 to 14, or
(4) further comprising the step (iii) of purifying the reassortant
virus obtained in step (ii).
41. A method of preparing a reassortant influenza A virus of claim
28 comprising steps of (i) introducing into a culture host one or
more expression construct(s) which encode(s) the viral segments
required to produce an influenza A virus wherein the expression
construct(s) encode the backbone segments from two or more donor
strains and wherein the PB1 backbone viral segment is from
A/California/07/09; and (ii) culturing the culture host in order to
produce the reassortant influenza A virus of claim 28.
42. The method of claim 39, wherein the expression construct(s) are
(3) and comprise(s) all of the sequences having at least 90%
identity or 100% identity with the sequences of SEQ ID Nos.: 9 and
11 to 14.
43. The method of claim 40, wherein the HA segment is from a H1
influenza virus.
44. A method for producing influenza viruses comprising steps of
(a) infecting a culture host with the reassortant influenza virus
of claim 25; (b) culturing the host from step (a) to produce the
virus; and optionally (c) purifying the virus produced in step
(b).
45. A method of preparing a vaccine, comprising the steps of (a)
preparing a virus by the method of claim 44 and (b) preparing a
vaccine from the virus.
46. The method of claim 44, wherein the culture host is an
embryonated hen egg.
47. The method of claim 44, wherein the culture host is a mammalian
cell, optionally an MDCK (such as MDCK 33016 (DSM ACC2219)), Vero
or PerC6 cell.
48. The method of claim 47, wherein the cell grows adherently or in
suspension.
49. The method of claim 45, wherein step (b) involves inactivating
the virus.
50. The method of claim 45, wherein the vaccine is a whole virion
vaccine, a split virion vaccine. a surface antigen vaccine, or a
virosomal vaccine.
51. The method of claim 45, wherein the vaccine contains less than
10 ng of residual host cell DNA per dose.
52. The method of claim 45, wherein at least one of the influenza
strains is of the H1, H2, H5, H7 or H9 subtype.
53. An expression system comprising one or more expression
construct(s) comprising the vRNA encoding segments of an influenza
A virus wherein the expression construct(s) encode(s) the backbone
viral segments from two or more influenza donor strains, wherein
(i) the HA and PB1 segments are from two different influenza
strains with the same influenza HA subtype, (ii) the HA and PB1
segments are from two different influenza strains with different
influenza virus HA subtypes, wherein the expression construct(s)
do(es) not encode the PB1 segment from an influenza virus with a H3
HA subtype and/or wherein the expression construct(s) do(es) not
encode the HA segment from an influenza virus with a H1 or H5 HA
subtype. or (iii) wherein the PB1 segment is from
A/California/07/09.
54. The expression system of claim 53, wherein (a) the expression
construct(s) may further comprise the vRNAs which encode the PB2,
NP, NS, M and PA segments from PR8-X,(b) wherein the at least one
expression construct comprises a sequence having at least 90%, at
least 95%, at least 99% or 100% identity with the sequence of SEQ
ID NO: 22, (c) the expression construct(s) further comprise(s) one
or more of the sequences having at least 90%, at least 95%, at
least 99% or 100% identity with the sequences of SEQ ID Nos.: 9,
and/or 11 to 14. or (d) the expression construct(s) comprise(s) all
of the sequences having at least 90%, at least 95%, at least 99% or
100% identity with the sequences of SEQ ID Nos.: 9 and 11 to
14.
55. A host cell comprising the expression system of claim 53.
56. The host cell of claim 55, wherein the host cell is a mammalian
cell, optionally an MDCK, Vero or PerC6 cell.
Description
TECHNICAL FIELD
[0002] This invention is in the field of influenza A virus
reassortment. Furthermore, it relates to manufacturing vaccines for
protecting against influenza A viruses.
BACKGROUND ART
[0003] The most efficient protection against influenza infection is
vaccination against circulating strains and it is important to
produce influenza viruses for vaccine production as quickly as
possible.
[0004] Wild-type influenza viruses often grow to low titres in eggs
and cell culture. In order to obtain a better-growing virus strain
for vaccine production it is currently common practice to reassort
the circulating vaccine strain with a faster-growing high-yield
donor strain. This can be achieved by co-infecting a culture host
with the circidating influenza strain (the vaccine strain) and the
high-yield donor strain and selecting for reassortant viruses which
contain the hemagglutinin (HA) and neuraminidase (NA) segments from
the vaccine strain and the other viral segments (i.e. those
encoding PB1, PB2, PA, NP, M.sub.1, M.sub.2, NS.sub.1 and NS.sub.2)
from the donor strain. Another approach is to reassort the
influenza viruses by reverse genetics (see, for example references
1 and 2).
[0005] Reference 3 reports that a reassortant influenza virus
containing a PB1 gene segment from A/Texas/1/77, the HA and NA
segments from A/New Caledonia/20/99, a modified PA segment derived
from A/Puerto Rico/8/34 and the remaining viral segments from
A/Puerto Rico/8/34 shows increased growth in cells.
[0006] There are currently only a limited number of donor strains
for reassorting influenza viruses for vaccine manufacture, and the
strain most commonly used is the A/Puerto Rico/8/34 (A/PR/8/34)
strain. However, reassortant influenza viruses comprising A/PR/8/34
backbone segments do not always grow sufficiently well to ensure
efficient vaccine manufacture. Thus, there is a need in the art to
provide further and improved donor strains for influenza virus
reassortment.
SUMMARY OF PREFERRED EMBODIMENTS
[0007] The inventors have now surprisingly discovered that
influenza viruses which comprise backbone segments from two or more
influenza donor strains can grow faster in a culture host
(particularly in cell culture) compared with reassortant influenza
A viruses which contain all backbone segments from the same donor
strain. In particular, the inventors have found that influenza
viruses which comprise backbone segments derived from two different
high-yield donor strains can produce higher yield reassortants with
target vaccine-relevant HA/NA genes than reassortants made with
either of the two original donor strains alone.
[0008] Reassortant influenza A viruses with backbone segments from
two or more influenza donor strains may comprise the HA segment and
the PB1 segment from different influenza A strains. In these
reassortant influenza viruses the PB1 segment is preferably from
donor viruses with the same influenza virus HA subtype as the
vaccine strain. For example, the PB1 segment and the HA segment may
both be from influenza viruses with a H1 subtype. The reassortant
influenza A viruses may also comprise the HA segment and the PB1
segment from different influenza A strains with different influenza
virus HA subtypes, wherein the PB1 segment is not from an influenza
virus with a H3 HA subtype and/or wherein the HA segment is not
from an influenza virus with a H1 or H5 HA subtype. For example,
the PB1 segment may be from a H1 virus and/or the HA segment may be
from a H3 influenza virus.
[0009] The invention also provides reassortant influenza A viruses
with backbone segments from two or more influenza donor strains in
which the PB1 segment is from the A/California/07/09 influenza
strain. This segment may have at least 95% identity or 100%
identity with the sequence of SEQ ID NO: 22. The reassortant
influenza A virus may have the H1 HA subtype. It will be understood
that a reassortant influenza virus according to this aspect of the
invention will not comprise the HA and/or NA segments from
A/California/07/09.
[0010] Where the reassortant influenza A virus comprises backbone
segments from two or three donor strains, each donor strain may
provide more than one of the backbone segments of the reassortant
influenza A virus, but one or two of the donor strains can also
provide only a single backbone segment.
[0011] Where the reassortant influenza A virus comprises backbone
segments from two, three, four or five donor strains, one or two of
the donor strains may provide more than one of the backbone
segments of the reassortant influenza A virus. In general the
reassortant influenza A virus cannot comprise more than six
backbone segments. Accordingly, for example, if one of the donor
strains provides five of the viral segments, the reassortant
influenza A virus can only comprise backbone segments from a total
of two different donor strains.
[0012] Where a reassortant influenza A virus comprises the PB1
segment from A/Texas/1/77, it preferably does not comprise the PA,
NP or M segment from A/Puerto Rico/8/34. Where a reassortant
influenza A virus comprises the PA, NP or M segment from A/Puerto
Rico/8/34, it preferably does not comprise the FBI segment from
A/Texas/1/77. In some embodiments, the invention does not encompass
reassortant influenza A viruses which have the PB1 segment from
A/Texas/1/77 and the PA, NP and M segments from A/Puerto Rico/8/34.
The PB1 segment from A/Texas/1/77 may have the sequence of SEQ ID
NO: 27 and the PA, NP or M segments from A/Puerto Rico/8/34 may
have the sequence of SEQ ID NOs. 28, 29 or 30, respectively.
[0013] Influenza A virus strains of the invention can grow to
higher viral titres in MDCK cells and/or in eggs in the same time
and under the same growth conditions compared with reassortant
influenza strains that comprise all backbone segments from the same
influenza donor strain.
[0014] The invention also provides a reassortant influenza A virus
comprising at least one backbone viral segment from a donor strain,
wherein the donor strain is the A/California/07/09 influenza
strain. When the at least one backbone viral segment is the PA
segment it may have a sequence having at least 95% or at least 99%
identity with the sequence of SEQ ID NO: 15. When the at least one
backbone viral segment is the PB1 segment, it may have a sequence
having at least 95% or at least 99% identity with the sequence of
SEQ ID NO: 16. When the at least one backbone viral segment is the
PB2 segment, it may have a sequence having at least 95% or at least
99% identity with the sequence of SEQ ID NO: 17. When the at least
one backbone viral segment is the NP segment it may have a sequence
having at least 95% or at least 99% identity with the sequence of
SEQ ID NO: 18. When the at least one backbone viral segment is the
M segment it may have a sequence having at least 95% or at least
99% identity with the sequence of SEQ ID NO: 19. When the at least
one backbone viral segment is the NS segment it may have a sequence
having at least 95% or at least 99% identity with the sequence of
SEQ ID NO: 20.
[0015] At least one backbone segment may be derived from the
A/California/07/09 influenza strain, as discussed in the previous
paragraph. Preferred reassortant influenza A viruses comprise the
PB1 segment from the A/California/07/09 influenza strain. The
inventors have shown that reassortant influenza A viruses
comprising this backbone segment grow well in culture hosts. The
reassortant influenza A viruses may comprise all other backbone
segments from an influenza virus which is not
A/California/07/09.
[0016] The reassortant influenza A viruses may comprise the PB1
segment from A/California/07/09 and all other backbone segments
from the influenza strain PR8-X. The segments of PR8-X have the
sequences of SEQ ID NO: 1 (PA), SEQ ID NO: 2 (PB1), SEQ ID NO: 3
(PB2), SEQ ID NO: 4 (NP), SEQ ID NO: 5 (M), SEQ ID NO: 6 (NS), SEQ
ID NO: 7 (HA) or SEQ ID NO: 8 (NA). Thus, the influenza viruses of
the invention may comprise one or more genome segments selected
from: a PA segment having at least 95% or 99% identity to the
sequence of SEQ TD NO: 1, a PB2 segment having at least 95% or 99%
identity to the sequence of SEQ ID NO: 3, a M segment having at
least 95% or 99% identity to the sequence of SEQ ID NO: 5, a NP
segment having at least 95% or 99% identity to the sequence of SEQ
ID NO: 4, and/or a NS segment having at least 95% or 99% identity
to the sequence of SEQ ID NO: 6. The reassortant influenza A
viruses may also comprise one or more viral segments which have the
sequence of SEQ ID NOs.: 1, and/or 3-6. In preferred embodiments,
the reassortant influenza strain comprises all of the genome
segments mentioned in this paragraph. This embodiment is preferred
because the inventors have found that such reassortant influenza A
viruses grow particularly well in cell culture and in embryonated
hens eggs.
[0017] In general a reassortant influenza virus will contain only
one of each backbone segment. For example, when the influenza virus
comprises the PB1 segment from A/California/07/09 it will not at
the same time comprise the PB1 segment from another influenza A
donor strain.
[0018] The backbone viral segments may be optimized for culture in
the specific culture host. For example, where the reassortant
influenza viruses are cultured in mammalian cells, it is
advantageous to adapt at least one of the viral segments for
optimal growth in the culture host. For example, where the
expression host is a canine cell, such as a MDCK cell line, the
viral segments may have a sequence which optimises viral growth in
the cell. Thus, the reassortant influenza viruses of the invention
may comprise a PB2 genome segment which has lysine in the position
corresponding to amino acid 389 of SEQ ID NO: 3 when aligned to SEQ
ID NO: 3 using a pairwise alignment algorithm, and/or asparagine in
the position corresponding to amino acid 559 of SEQ ID NO: 3 when
aligned to SEQ ID NO: 3 using a pairwise alignment algorithm. Also
provided are reassortant influenza viruses in accordance with the
invention in which the PA genome segment has lysine in the position
corresponding to amino acid 327 of SEQ ID NO: 1 when aligned to SEQ
ID NO: 1 using a pairwise alignment algorithm, and/or aspartic acid
in the position corresponding to amino acid 444 of SEQ ID NO: 1
when aligned to SEQ ID NO: 1, using a pairwise alignment algorithm,
and/or aspartic acid in the position corresponding to amino acid
675 of SEQ ID NO: 1 when aligned to SEQ ID NO: 1, using a pairwise
alignment algorithm. The reassortant influenza strains of the
invention may also have a NP genome segment with threonine in the
position corresponding to amino acid 27 of SEQ ID NO: 4 when
aligned to SEQ ID NO: 4 using a pairwise alignment algorithm,
and/or asparagine in the position corresponding to amino acid 375
of SEQ ID NO: 4 when aligned to SEQ ID NO: 4, using a pairwise
alignment algorithm. Variant influenza strains may also comprise
two or more of these mutations. It is preferred that the variant
influenza virus contains a variant PB2 segment with both of the
amino acids changes identified above, and/or a PA which contains
all three of the amino acid changes identified above, and/or a NP
segment which contains both of the amino acid changes identified
above. The influenza A virus may be a H1 strain.
[0019] Alternatively, or in addition, the reassortants influenza
viruses may comprise a PB1 segment which has isoleucine in the
position corresponding to amino acid 200 of SEQ ID NO: 2 when
aligned to SEQ ID NO: 2 using a pairwise alignment algorithm,
and/or asparagine in the position corresponding to amino acid 338
of SEQ ID NO: 2 when aligned to SEQ ID NO: 2 using a pairwise
alignment algorithm, and/or isoleucine in the position
corresponding to amino acid 529 of SEQ ID NO: 2 when aligned to SEQ
ID NO: 2 using a pairwise alignment algorithm, and/or isoleucine in
the position corresponding to amino acid 591 of SEQ TD NO: 2 when
aligned to SEQ TD NO: 2 using a pairwise alignment algorithm,
and/or histidine in the position corresponding to amino acid 687 of
SEQ ID NO: 2 when aligned to SEQ ID NO: 2 using a pairwise
alignment algorithm, and/or lysine in the position corresponding to
amino acid 754 of SEQ ID NO: 2 when aligned to SEQ ID NO: 2 using a
pairwise alignment algorithm.
[0020] The preferred pairwise alignment algorithm is the
Needleman-Wunsch global alignment algorithm [4], using default
parameters (e.g. with Gap opening penalty=10.0, and with Gap
extension penalty=0.5, using the EBLOSUM62 scoring matrix). This
algorithm is conveniently implemented in the needle tool in the
EMBOSS package [5].
[0021] The invention provides a method of preparing the reassortant
influenza A viruses of the invention. These methods comprise steps
of (i) introducing into a culture host one or more expression
construct(s) which encode(s) the viral segments required to produce
an influenza A virus wherein the backbone viral segments are from
two or more influenza strains; and (ii) culturing the culture host
in order to produce reassortant virus and optionally (iii)
purifying the virus obtained in step (ii). In these methods, the HA
and the PB1 segment may be from different influenza strains which
have the same influenza HA subtype or the HA and PB1 segments may
be from different influenza strains with different HA subtypes
provided that the PB1 segment is not from an influenza virus with a
H3 HA subtype and/or the HA segment is not from an influenza virus
with a H1 or H5 HA subtype. The PB1 backbone viral segment may be
from A/California/07/09. The one or more expression constructs may
further encode one or more of the PB2, PA, NP, M, or NS segments
from PR8-X or segments having at least 90% or 100% identity to SEQ
ID NOs.: 9, and/or 11 to 14. The expression construct(s) may not
encode the HA and/or NA segments from A/Cali for nia/07/09 when the
PB1 segment is from A/California/07/09.
[0022] The at least one expression construct may comprise a
sequence having at least 90%, at least 95%, at least 99% or 100%
identity with the sequence of SEQ ID NO: 22.
[0023] In some embodiments, the at least one expression construct
does not encode the PB1 segment from the A/Texas/1/77 influenza
strain.
[0024] The methods may further comprise steps of: (iv) infecting a
culture host with the virus obtained in step (ii) or step (iii);
(v) culturing the culture host from step (iv) to produce further
virus; and optionally (vi) purifying the virus obtained in step
(v).
[0025] The invention also provides a method for producing influenza
viruses comprising steps of (a) infecting a culture host with a
reassortant virus of the invention; (b) culturing the host from
step (a) to produce the virus; and optionally (c) purifying the
virus obtained in step (b).
[0026] The invention also provides a method of preparing a vaccine,
comprising steps of (d) preparing a virus by the methods of any one
of the embodiments described above and (e) preparing vaccine from
the virus.
[0027] The invention provides an expression system comprising one
or more expression construct(s) comprising the vRNA encoding
segments of an influenza A virus wherein the expression
construct(s) encode(s) the HA and PB1 segments from two different
influenza strains with the same influenza HA subtype or which
encodes the HA and PB1 segments from two different influenza
strains with different influenza virus HA subtypes, wherein the PB1
segment is not from an influenza virus with a H3 HA subtype and/or
the HA segment is not from an influenza virus with a H1 or H5 HA
subtype.
[0028] The invention also provides an expression system comprising
one or more expression construct(s) comprising the vRNA encoding
segments of an influenza A virus wherein the expression
construct(s) encode(s) the FBI segment of A/California/07/09. The
expression construct(s) may further comprise the vRNAs which encode
one or more of the PB2, NP, NS, M and/or PA segments from PR8-X.
Thus, the expression construct(s) may comprise one or more
nucleotide sequences having at least 90% identity, at least 95%
identity, at least 99% identity or 100% identity with the sequences
of SEQ ID Nos.: 9 and/or 11-14. It is preferred that the expression
construct(s) encode(s) all of the PB2, NP, NS, M and PA segments
from PR8-X.
[0029] The invention also provides a host cell comprising the
expression systems of the invention. These host cells can express
an influenza A virus from the expression construct(s) in the
expression system.
[0030] Expression constructs which can be used in the expression
systems of the invention arc also provided. For example, the
invention provides an expression construct which encodes the
backbone segments of the reassortant influenza strains according to
the invention on the same construct.
Donor Strains
[0031] Influenza donor strains are strains which typically provide
the backbone segments in a reassortant influenza virus, even though
they may sometimes also provide the NA segment of the virus.
Usually, however, both the HA and the NA segment in a reassortant
influenza virus will be from the vaccine strain which is the
influenza strain that provides the HA segment.
[0032] The inventors have surprisingly discovered that reassortant
influenza A viruses which comprise the HA segment and the PB1
segment from different influenza A strains with the same HA subtype
can grow much faster in culture hosts compared with reassortant
influenza viruses which comprise the HA and PB1 segments from
viruses with different HA subtypes. These reassortant influenza
viruses preferably have backbone segments from at least two donor
strains.
[0033] The PB1 segments of influenza viruses with the same HA
subtype will usually have a higher level of identity than the PB1
segments of influenza viruses with different HA subtypes. For
example, a Blast search using the PB1 segment of the H1 strain
A/California/07/09 showed that only influenza strains with the H1
HA subtype had a high identity in the PB1 segment. Likewise, a
Blast search using the PB1 segment of the H3 strain
A/Wisconsin/67/2005 showed that only influenza viruses with the H3
HA subtype had a high level of identity to the PB1 segment of this
virus.
[0034] The inventors have further discovered that reassortant
influenza A viruses which have backbone segments from at least two
donor strains and comprise the PB1 segment from A/California/07/09
grow particularly well in culture hosts. These reassortant
influenza viruses preferably have backbone segments from at least
two different donor strains. The reassortant influenza viruses may
comprise the PB1 segment from A/California/07/09 and the HA segment
of an influenza virus with the H1 subtype.
[0035] Influenza strains which contain one, two, three, four five,
six or seven of the segments of the A/California/07/09 strain can
also be used as donor strains.
[0036] The invention can be practised with donor strains having a
viral segment that has at least about 70%, at least about 75%, at
least about 80%, at least about 85%, at least about 90%, at least
about 95% or at least about 99% identity to a sequence of SEQ ID
Nos. 9-14 or 21-26. For example, due to the degeneracy of the
genetic code, it is possible to have the same polypeptide encoded
by several nucleic acids with different sequences. Thus, the
invention may be practised with viral segments that encode the same
polypeptides as the sequences of SEQ ID Nos. 1-8 or 15-20. For
example, the nucleic acid sequences of SEQ ID Nos.: 31 and 32 have
only 73% identity even though they encode the same viral
protein.
[0037] The invention may also be practised with viral segments that
encode polypeptides that have at least 80%, at least 85%, at least
90%, at least 95% or at least 99% identity to the polypeptide
sequences encoded by SEQ ID Nos. 9-22.
[0038] Variations in the DNA and the amino acid sequence may also
stem from spontaneous mutations which can occur during passaging of
the viruses. Such variant influenza strains can also be used in the
invention.
Reassortant Viruses
[0039] The invention provides reassortant influenza viruses which
comprise backbone segments from two or more influenza donor
strains. These reassortant influenza viruses may comprise the HA
segment and the PB1 segment from different influenza A strains
provided that the HA and the PB1 segments arc from influenza
viruses with the same influenza virus HA subtype. They may also
comprise the HA segment and the PB1 segment from different
influenza A strains with different influenza virus HA subtypes,
provided that the PB1 segment is not from an influenza virus with a
H3 HA subtype and/or the HA segment is not from an influenza virus
with a H1 or H5 HA subtype.
[0040] Further provided are reassortant influenza viruses with
backbone segments from two or more different donor strains which
comprise the PB1 segment from A/California/07/09.
[0041] The PB1 and PB2 segments may be from the same donor
strain.
[0042] Influenza viruses are segmented negative strand RNA viruses.
Influenza A and B viruses have eight segments (NP, M, NS, PA, PB1,
HA and NA) whereas influenza C virus has seven. The reassortant
viruses of the invention contain the backbone segments from two or
more donor strains, or at least one (i.e. one, two, three, four,
five or six) backbone viral segment from A/California/07/09. The
backbone viral segments are those which do not encode HA or NA.
Thus, backbone segments will typically encode the PB1, PB2, PA, NP,
M.sub.1, M.sub.2, NS.sub.1 and NS.sub.2 polypeptides of the
influenza virus.
[0043] The viruses may also contain an NS segment that does not
encode a functional NS protein as described, for example, in
reference 6. The reassortant viruses will not typically contain the
segments encoding HA and NA from the donor strains even though
reassortant viruses which comprise either the HA or the NA but not
both from the donor strains of the invention are also
envisioned.
[0044] When the reassortant viruses are reassortants comprising the
backbone segments from a single donor strain, the reassortant
viruses will generally include segments from the donor strain and
the vaccine strain in a ratio of 1:7, 2:6, 3:5, 4:4, 5:3, 6:2 or
7:1. Having a majority of segments from the donor strain, in
particular a ratio of 6:2, is typical. When the reassortant viruses
comprise backbone segments from two donor strains, the reassortant
virus will generally include segments from the first donor strain,
the second donor strain and the vaccine strain in a ratio of 1:1:6,
1:2:5, 1:3:4, 1:4:3, 1:5:2, 1:6:1, 2:1:5, 2:2:4, 2:3:3, 2:4:2,
2:5:1, 3:1:2, 3:2:1, 4:1:3, 4:2:2, 4:3:1, 5:1:2, 5:2:1 or
6:1:1.
[0045] Preferably, the reassortant viruses do not contain the HA
segment of the donor strain as this encodes the main vaccine
antigens of the influenza virus and should therefore come from the
vaccine strain. The reassortant viruses of the invention therefore
preferably have at least the HA segment and typically the HA and NA
segments from the vaccine strain.
[0046] The invention also encompasses reassortants which comprise
viral segments from more than one vaccine strain provided that the
reassortant comprises a backbone according to the present
invention. For example, the reassortant influenza viruses may
comprise the HA segment from one donor strain and the NA segment
from a different donor strain.
[0047] The reassortant viruses of the invention can grow to higher
viral titres than the wild-type vaccine strain from which some of
the viral segment(s) of the reassortant virus are derived in the
same time (for example 12 hours, 24 hours, 48 hours or 72 hours)
and under the same growth conditions. The viral titre can be
determined by standard methods known to those of skill in the art.
The reassortant viruses of the invention can achieve a viral titre
which is at least 10% higher, at least 20% higher, at least 50%
higher, at least 100% higher, at least 200% higher, at least 500%
higher, or at least 1000% higher than the viral titre of the wild
type vaccine strain in the same time frame and under the same
conditions.
[0048] The invention is suitable for reassorting pandemic as well
as inter-pandemic (seasonal) influenza vaccine strains. The
reassortant influenza strains may contain the influenza A virus HA
subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13,
H14, H15 or H16. They may contain the influenza A virus NA subtypes
N1, N2, N3, N4, N5, N6, N7, N8 or N9. Where the vaccine strain used
in the reassortant influenza viruses of the invention is a seasonal
influenza strain, the vaccine strain may have a H1 or H3 subtype.
In one aspect of the invention the vaccine strain is a H1N1 or H3N2
strain. The reassortants influenza strains may also contain the HA
segment of an influenza B strain.
[0049] The vaccine strains for use in the invention may also be
pandemic strains or potentially pandemic strains. The
characteristics of an influenza strain that give it the potential
to cause a pandemic outbreak arc: (a) it contains a new
hemagglutinin compared to the hemagglutinins in
currently-circulating human strains, i.e. one that has not been
evident in the human population for over a decade (e.g. H2), or has
not previously been seen at all in the human population (e.g. H5,
H6 or H9, that have generally been found only in bird populations),
such that the human population will be immunologically naive to the
strain's hemagglutinin; (b) it is capable of being transmitted
horizontally in the human population; and (c) it is pathogenic to
humans. A vaccine strain with a H5 hemagglutinin type is preferred
where the reassortant virus is used in vaccines for immunizing
against pandemic influenza, such as a H5N1 strain. Other possible
strains include H5N3, H9N2, H2N2, H7N1 and H7N7, and any other
emerging potentially pandemic strains. The invention is
particularly suitable for producing reassortant viruses for use in
a vaccine for protecting against potential pandemic virus strains
that can or have spread from a non-human animal population to
humans, for example a swine-origin H1N1 influenza strain.
[0050] The reassortant influenza strain of the invention may
comprise the HA segment and/or the NA segment from an
A/California/4/09 strain.
[0051] Strains which can be used as vaccine strains include strains
which are resistant to antiviral therapy (e.g. resistant to
oseltamivir [7] and/or zanamivir), including resistant pandemic
strains [8].
[0052] Reassortant viruses which contain an NS segment that does
not encode a functional NS protein are also within the scope of the
present invention. NS1 knockout mutants are described in reference
6. These NS 1-mutant virus strains are particularly suitable for
preparing live attenuated influenza vaccines.
[0053] The `second influenza strain` used in the methods of the
invention is different to the donor strain which is used.
Reverse Genetics
[0054] The invention is particularly suitable for producing the
reassortant influenza virus strains of the invention through
reverse genetics techniques. In these techniques, the viruses are
produced in culture hosts using an expression system.
[0055] In one aspect, the expression system may encode the HA and
PB1 segment from different influenza strains with the same HA
subtype. It may also encode the HA and PB1 segments from different
influenza strains with different HA subtypes provided that the PB1
segment is not from an influenza virus with a H3 HA subtype and/or
the HA segment is not from an influenza virus with a H1 or H5 HA
subtype. The expression system may encode the PB1 segment from
A/California/07/09. In these embodiments, the system may encode at
least one of the segments NP, M, NS, PA, and/or PB2 from another
influenza donor strain, for example PR8-X.
[0056] Reverse genetics for influenza A and B viruses can be
practised with 12 plasmids to express the four proteins required to
initiate replication and transcription (PB1, PB2, PA and
nucleoprotein) and all eight viral genome segments. To reduce the
number of constructs, however, a plurality of RNA polymerase I
transcription cassettes (for viral RNA synthesis) can be included
on a single plasmid (e.g. sequences encoding 1, 2, 3, 4, 5, 6, 7 or
all 8 influenza vRNA segments), and a plurality of protein-coding
regions with RNA polymerase II promoters on another plasmid (e.g.
sequences encoding 1, 2, 3, 4, 5, 6, 7 or 8 influenza mRNA
transcripts) [9]. It is also possible to include one or more
influenza vRNA segments under control of a pol I promoter and one
or more influenza protein coding regions under control of another
promoter, in particular a pol II promoter, on the same plasmid.
This is preferably done by using bi-directional plasmids.
[0057] Preferred aspects of the reference 9 method involve: (a)
PB1, PB2 and PA mRNA-encoding regions on a single expression
construct; and (b) all 8 vRNA encoding segments on a single
expression construct. Including the neuraminidase (NA) and
hemagglutinin (HA) segments on one expression construct and the six
other viral segments on another expression construct is
particularly preferred as newly emerging influenza virus strains
usually have mutations in the NA and/or HA segments. Therefore, the
advantage of having the HA and/or NA segments on a separate
expression construct is that only the vector comprising the HA and
NA sequence needs to be replaced. Thus, in one aspect of the
invention the NA and/or HA segments of the vaccine strain may be
included on one expression construct and the vRNA encoding segments
from the donor strain(s) of the invention, excluding the HA and/or
NA segment(s), are included on a different expression construct.
The invention thus provides an expression construct comprising one,
two, three, four, five or six vRNA encoding backbone viral segments
of a donor strain of the invention. The expression construct may
not comprise HA and/or NA viral segments that produce a functional
HA and/or NA protein.
[0058] Known reverse genetics systems involve expressing DNA
molecules which encode desired viral RNA (vRNA) molecules from poI
1 promoters, bacterial RNA polymerase promoters, bacteriophage
polymerase promoters, etc. As influenza viruses require the
presence of viral polymerase to initiate the life cycle, systems
may also provide these proteins e.g. the system further comprises
DNA molecules that encode viral polymerase proteins such that
expression of both types of DNA leads to assembly of a complete
infectious virus. It is also possible to supply the viral
polymerase as a protein.
[0059] Where reverse genetics is used for the expression of
influenza vRNA, it will be evident to the person skilled in the art
that precise spacing of the sequence elements with reference to
each other is important for the polymerase to initiate replication.
It is therefore important that the DNA molecule encoding the viral
RNA is positioned correctly between the poI 1 promoter and the
termination sequence, but this positioning is well within the
capabilities of those who work with reverse genetics systems.
[0060] In order to produce a recombinant virus, a cell must express
all segments of the viral genome which are necessary to assemble a
virion. DNA cloned into the expression constructs of the present
invention preferably provides all of the viral RNA and proteins,
but it is also possible to use a helper virus to provide some of
the RNA and proteins, although systems which do not use a helper
virus are preferred. As the influenza virus is a segmented virus,
the viral genome will usually be expressed using more than one
expression construct in the methods of the invention. It is also
envisioned, however, to combine one or more segments or even all
segments of the viral genome on a single expression construct.
[0061] In some embodiments an expression construct will also be
included which leads to expression of an accessory protein in the
host cell. For instance, it can be advantageous to express a
non-viral serine protease (e.g. trypsin) as part of a reverse
genetics system.
Expression Constructs
[0062] Expression constructs used in the expression systems of the
invention may be uni-directional or bi-directional expression
constructs. Where more than one transgene is used in the methods
(whether on the same or different expression constructs) it is
possible to use uni-directional and/or bi-directional
expression.
[0063] As influenza viruses require a protein for infectivity, it
is generally preferred to use bi-directional expression constructs
as this reduces the total number of expression constructs required
by the host cell. Thus, the method of the invention may utilise at
least one bi-directional expression construct wherein a gene or
cDNA is located between an upstream pol II promoter and a
downstream non-endogenous pol I promoter. Transcription of the gene
or cDNA from the pol II promoter produces capped positive-sense
viral mRNA which can be translated into a protein, while
transcription from the non-endogenous pol I promoter produces
negative-sense vRNA. The bi-directional expression construct may be
a bi-directional expression vector.
[0064] Bi-directional expression constructs contain at least two
promoters which drive expression in different directions (i.e. both
5' to 3' and 3' to 5') from the same construct. The two promoters
can be operably linked to different strands of the same double
stranded DNA. Preferably, one of the promoters is a pol I promoter
and at least one of the other promoters is a pol II promoter. This
is useful as the pol I promoter can be used to express uncapped
vRNAs while the pol II promoter can be used to transcribe mRNAs
which can subsequently be translated into proteins, thus allowing
simultaneous expression of RNA and protein from the same construct.
Where more than one expression construct is used within an
expression system, the promoters may be a mixture of endogenous and
non-endogenous promoters.
[0065] The pol I and pol II promoters used in the expression
constructs may be endogenous to an organism from the same taxonomic
order from which the host cell is derived. Alternatively, the
promoters can be derived from an organism in a different taxonomic
order than the host cell. The term "order" refers to conventional
taxonomic ranking, and examples of orders are primates, rodentia,
carnivora, marsupialia, cetacean, etc. Humans and chimpanzees are
in the same taxonomic order (primates), but humans and dogs are in
different orders (primates vs. carnivora). For example, the human
pol I promoter can be used to express viral segments in canine
cells (e.g. MDCK cells) [10].
[0066] The expression construct will typically include an RNA
transcription termination sequence. The termination sequence may be
an endogenous termination sequence or a termination sequence which
is not endogenous to the host cell. Suitable termination sequences
will be evident to those of skill in the art and include, but are
not limited to, RNA polymerase I transcription termination
sequence, RNA polymerase II transcription termination sequence, and
ribozymes. Furthermore, the expression constructs may contain one
or more polyadenylation signals for mRNAs, particularly at the end
of a gene whose expression is controlled by a pol II promoter.
[0067] An expression system may contain at least two, at least
three, at least four, at least five, at least six, at least seven,
at least eight, at least nine, at least ten, at least eleven or at
least twelve expression constructs.
[0068] An expression construct may be a vector, such as a plasmid
or other episomal construct. Such vectors will typically comprise
at least one bacterial and/or eukaryotic origin of replication.
Furthermore, the vector may comprise a selectable marker which
allows for selection in prokaryotic and/or eukaryotic cells.
Examples of such selectable markers are genes conferring resistance
to antibiotics, such as ampicillin or kanamycin. The vector may
further comprise one or more multiple cloning sites to facilitate
cloning of a DNA sequence.
[0069] As an alternative, an expression construct may be a linear
expression construct. Such linear expression constructs will
typically not contain any amplification and/or selection sequences.
However, linear constructs comprising such amplification and/or
selection sequences are also within the scope of the present
invention. Reference 11 describes a linear expression construct
which describes individual linear expression constructs for each
viral segment. It is also possible to include more than one, for
example two, three four, five or six viral segments on the same
linear expression construct. Such a system has been described, for
example, in reference 12.
[0070] Expression constructs can be generated using methods known
in the art. Such methods were described, for example, in reference
13. Where the expression construct is a linear expression
construct, it is possible to linearise it before introduction into
the host cell utilising a single restriction enzyme site.
Alternatively, it is possible to excise the expression construct
from a vector using at least two restriction enzyme sites.
Furthermore, it is also possible to obtain a linear expression
construct by amplifying it using a nucleic acid amplification
technique (e.g. by PCR).
[0071] The expression constructs used in the systems of the
invention may be non-bacterial expression constructs. This means
that the construct can drive expression in a eukaryotic cell of
viral RNA segments encoded therein, but it does not include
components which would be required for propagation of the construct
in bacteria. Thus the construct will not include a bacterial origin
of replication (ori), and usually will not include a bacterial
selection marker (e.g. an antibiotic resistance marker). Such
expression constructs are described in reference 14 which is
incorporated by reference.
[0072] The expression constructs may be prepared by chemical
synthesis. The expression constructs may either be prepared
entirely by chemical synthesis or in part. Suitable methods for
preparing expression constructs by chemical synthesis are
described, for example, in reference 14 which is incorporated by
reference.
[0073] The expression constructs of the invention can be introduced
into host cells using any technique known to those of skill in the
art. For example, expression constructs of the invention can be
introduced into host cells by employing electroporation,
DEAE-dextran, calcium phosphate precipitation, liposomes,
microinjection, or microparticle-bombardment.
Cells
[0074] The culture host for use in the present invention can be any
eukaryotic cell that can produce the virus of interest. The
invention will typically use a cell line although, for example,
primary cells may be used as an alternative. The cell will
typically be mammalian or avian. Suitable mammalian cells include,
but are not limited to, hamster, cattle, primate (including humans
and monkeys) and dog cells. Various cell types may be used, such as
kidney cells, fibroblasts, retinal cells, lung cells, etc. Examples
of suitable hamster cells are the cell lines having the names BHK21
or HKCC. Suitable monkey cells are e.g. African green monkey cells,
such as kidney cells as in the Vero cell line [15-17]. Suitable dog
cells are e.g. kidney cells, as in the CLDK and MDCK cell
lines.
[0075] Further suitable cells include, but are not limited to: CHO;
293T; BHK; MRC 5; PER.C6 [18]; FRhL2; WI-38; etc. Suitable cells
are widely available e.g. from the American Type Cell Culture
(ATCC) collection [19], from the Coriell Cell Repositories [20], or
from the European Collection of Cell Cultures (ECACC). For example,
the ATCC supplies various different Vero cells under catalogue
numbers CCL 81, CCL 81.2, CRL 1586 and CRE-1587, and it supplies
MDCK cells under catalogue number CCL 34. PER.C6 is available from
the ECACC under deposit number 96022940.
[0076] Preferred cells for use in the invention are MDCK cells
[21-23], derived from Madin Darby canine kidney. The original MDCK
cells are available from the ATCC as CCL 34. It is preferred that
derivatives of MDCK cells are used. Such derivatives were
described, for instance, in reference 21 which discloses MDCK cells
that were adapted for growth in suspension culture (`MDCK 33016` or
`33016-PF`, deposited as DSM ACC 2219; see also ref. 21).
Furthermore, reference 24 discloses MDCK-derived cells that grow in
suspension in serum free culture (`B-702`, deposited as FERM
BP-7449). In some embodiments, the MDCK cell line used may be
tumorigenic. It is also envisioned to use non-tumorigenic MDCK
cells. For example, reference 25 discloses non tumorigenic MDCK
cells, including `MDCK-S` (ATCC PTA-6500), `MDCK-SF101` (ATCC
PTA-6501), `MDCK-SF102` (ATCC PTA-6502) and `MDCK-SF103` (ATCC
PTA-6503). Reference 26 discloses MDCK cells with high
susceptibility to infection, including `MDCK.5F1` cells (ATCC CRL
12042).
[0077] It is possible to use a mixture of more than one cell type
to practise the methods of the present invention. However, it is
preferred that the methods of the invention are practised with a
single cell type e.g. with monoclonal cells. Preferably, the cells
used in the methods of the present invention are from a single cell
line. Furthermore, the same cell line may be used for reassorting
the virus and for any subsequent propagation of the virus.
[0078] Preferably, the cells are cultured in the absence of serum,
to avoid a common source of contaminants. Various serum-free media
for eukaryotic cell culture are known to the person skilled in the
art (e.g. Iscove's medium, ultra CHO medium (BioWhittaker), EX-CELL
(JRH Biosciences)). Furthermore, protein-free media may be used
(e.g. PF-CHO (JRH Biosciences)). Otherwise, the cells for
replication can also be cultured in the customary serum-containing
media (e.g. MEM or DMEM medium with 0.5% to 10% of fetal calf
serum).
[0079] The cells may be in adherent culture or in suspension.
Conventional Reassortment
[0080] Traditionally, influenza viruses are reassorted by
co-infecting a culture host, usually eggs, with a donor strain and
a vaccine strain. Reassortant viruses are selected by adding
antibodies with specificity for the HA and/or NA proteins of the
donor strain in order to select for reassortant viruses that
contain the vaccine strain's HA and/or NA proteins. Over several
passages of this treatment one can select for fast growing
reassortant viruses containing the vaccine strain's HA and/or NA
segments.
[0081] The invention is suitable for use in these methods. It can
be easier to use vaccine strains with a different HA and/or NA
subtype compared to the donor strain(s) as this facilitates
selection for reassortant viruses. It is also possible, however, to
use vaccine strains with the same HA and/or NA subtype as the donor
strain(s) and in some aspects of the invention this preferred. In
this case, antibodies with preferential specificity for the HA
and/or NA proteins of the donor strain(s) should be available.
Virus Preparation
[0082] In one embodiment, the invention provides a method for
producing influenza viruses comprising steps of (a) infecting a
culture host with a reassortant virus of the invention; (b)
culturing the host from step (a) to produce the virus; and
optionally (c) purifying the virus produced in step (b).
[0083] The culture host may be cells or embryonated hen eggs. Where
cells are used as a culture host in this aspect of the invention,
it is known that cell culture conditions (e.g. temperature, cell
density, pH value, etc.) are variable over a wide range subject to
the cell line and the virus employed and can be adapted to the
requirements of the application. The following information
therefore merely represents guidelines.
[0084] As mentioned above, cells are preferably cultured in
serum-free or protein-free media.
[0085] Multiplication of the cells can be conducted in accordance
with methods known to those of skill in the art. For example, the
cells can be cultivated in a perfusion system using ordinary
support methods like centrifugation or filtration. Moreover, the
cells can be multiplied according to the invention in a fed-batch
system before infection. In the context of the present invention, a
culture system is referred to as a fed-batch system in which the
cells are initially cultured in a batch system and depletion of
nutrients (or part of the nutrients) in the medium is compensated
by controlled feeding of concentrated nutrients. It can be
advantageous to adjust the pH value of the medium during
multiplication of cells before infection to a value between pH 6.6
and pH 7.8 and especially between a value between pH 7.2 and pH
7.3. Culturing of cells preferably occurs at a temperature between
30 and 40.degree. C. When culturing the infected cells (step b),
the cells are preferably cultured at a temperature of between
30.degree. C. and 36.degree. C. or between 32.degree. C. and
34.degree. C. or at 33.degree. C. This is particularly preferred,
as it has been shown that incubation of infected cells in this
temperature range results in production of a virus that results in
improved efficacy when formulated into a vaccine [27].
[0086] Oxygen partial pressure can be adjusted during culturing
before infection preferably at a value between 25% and 95% and
especially at a value between 35% and 60%. The values for the
oxygen partial pressure stated in the context of the invention are
based on saturation of air. Infection of cells occurs at a cell
density of preferably about 8-25.times.10.sup.5 cells/mL in the
batch system or preferably about 5-20.times.10.sup.6 cells/mL in
the perfusion system. The cells can be infected with a viral dose
(MOI value, "multiplicity of infection"; corresponds to the number
of virus units per cell at the time of infection) between 10.sup.-8
and 10, preferably between 0.0001 and 0.5.
[0087] Virus may be grown on cells in adherent culture or in
suspension. Microcarrier cultures can be used. In some embodiments,
the cells may thus be adapted for growth in suspension.
[0088] The methods according to the invention also include
harvesting and isolation of viruses or the proteins generated by
them. During isolation of viruses or proteins, the cells are
separated from the culture medium by standard methods like
separation, filtration or ultrafiltration. The viruses or the
proteins are then concentrated according to methods sufficiently
known to those skilled in the art, like gradient centrifugation,
filtration, precipitation, chromatography, etc., and then purified.
It is also preferred according to the invention that the viruses
are inactivated during or after purification. Vims inactivation can
occur, for example, by .beta.-propiolactone or formaldehyde at any
point within the purification process.
[0089] The culture host may be eggs. The current standard method
for influenza virus growth for vaccines uses embryonated SPF hen
eggs, with virus being purified from the egg contents (allantoic
fluid). It is also possible to passage a virus through eggs and
subsequently propagate it in cell culture and vice versa.
Vaccine
[0090] The invention utilises virus produced according to the
method to produce vaccines.
[0091] Vaccines (particularly for influenza virus) are generally
based either on live virus or on inactivated virus. Inactivated
vaccines may be based on whole virions, `split` virions, or on
purified surface antigens. Antigens can also be presented in the
form of virosomes. The invention can be used for manufacturing any
of these types of vaccine.
[0092] Where an inactivated virus is used, the vaccine may comprise
whole virion, split virion, or purified surface antigens (for
influenza, including hemagglutinin and, usually, also including
neuraminidase). Chemical means for inactivating a virus include
treatment with an effective amount of one or more of the following
agents: detergents, formaldehyde, .beta.-propiolactone, methylene
blue, psoralen, carboxyfullerene (C60), binary ethylamine, acetyl
ethyleneimine, or combinations thereof. Non-chemical methods of
viral inactivation are known in the art, such as for example UV
light or gamma irradiation.
[0093] Virions can be harvested from virus-containing fluids, e.g.
allantoic fluid or cell culture supernatant, by various methods.
For example, a purification process may involve zonal
centrifugation using a linear sucrose gradient solution that
includes detergent to disrupt the virions. Antigens may then be
purified, after optional dilution, by diafiltration.
[0094] Split virions are obtained by treating purified virions with
detergents (e.g. ethyl ether, polysorbate 80, deoxycholate,
tri-N-butyl phosphate, Triton .times.-100, Triton N101,
cetyltrimethylammonium bromide, Tergitol NP9, etc.) to produce
subvirion preparations, including the `Tween-ether` splitting
process. Methods of splitting influenza viruses, for example are
well known in the art e.g. see refs. 28-33, etc. Splitting of the
virus is typically carried out by disrupting or fragmenting whole
virus, whether infectious or non-infectious with a disrupting
concentration of a splitting agent. The disruption results in a
full or partial solubilisation of the virus proteins, altering the
integrity of the virus. Preferred splitting agents are non-ionic
and ionic (e.g. cationic) surfactants e.g. alkylglycosides,
alkylthioglycosides, acyl sugars, sulphobetaines, betains,
polyoxyethylenealkylethers, N,N-dialkyl-Glucamides, Hecameg,
alkylphenoxy-polyethoxycthanols, NP9, quaternary ammonium
compounds, sarcosyl, CTABs (cetyl trim ethyl ammonium bromides),
tri-N-butyl phosphate, Cetavlon, myristyltrimethylammonium salts,
lipofectin, lipofectamine, and DOT-MA, the octyl- or nonylphenoxy
polyoxyethanols (e.g. the Triton surfactants, such as Triton
.times.-100 or Triton N101), polyoxyethylene sorbitan esters (the
Tween surfactants), polyoxyethylene ethers, polyoxyethlene esters,
etc. One useful splitting procedure uses the consecutive effects of
sodium deoxycholate and formaldehyde, and splitting can take place
during initial virion purification (e.g. in a sucrose density
gradient solution). Thus a splitting process can involve
clarification of the virion-containing material (to remove
non-virion material), concentration of the harvested virions (e.g.
using an adsorption method, such as CaHPO.sub.4 adsorption),
separation of whole virions from non-virion material, splitting of
virions using a splitting agent in a density gradient
centrifugation step (e.g. using a sucrose gradient that contains a
splitting agent such as sodium deoxycholate), and then filtration
(e.g. ultrafiltration) to remove undesired materials. Split virions
can usefully be resuspended in sodium phosphate-buffered isotonic
sodium chloride solution. Examples of split influenza vaccines are
the BEGRIVAC.TM., FLUARIX.TM., FLUZONE.TM. and FLU SHIELD.TM.
products.
[0095] Purified influenza virus surface antigen vaccines comprise
the surface antigens hemagglutinin and, typically, also
neuraminidase. Processes for preparing these proteins in purified
form are well known in the art. The FLUVIRIN.TM. AGRIPPAL.TM. and
INFLUVAC.TM. products are influenza subunit vaccines.
[0096] Another form of inactivated antigen is the virosome [34]
(nucleic acid free viral-like liposomal particles). Virosomes can
be prepared by solubilization of virus with a detergent followed by
removal of the nucleocapsid and reconstitution of the membrane
containing the viral glycoproteins. An alternative method for
preparing virosomes involves adding viral membrane glycoproteins to
excess amounts of phospholipids, to give liposomes with viral
proteins in their membrane.
[0097] The methods of the invention may also be used to produce
live vaccines. Such vaccines are usually prepared by purifying
virions from virion-containing fluids. For example, the fluids may
be clarified by centrifugation, and stabilized with buffer (e.g.
containing sucrose, potassium phosphate, and monosodium glutamate).
Various forms of influenza virus vaccine are currently available
(e.g. see chapters 17 & 18 of reference 35). Live virus
vaccines include MedImmune's FLUMIST.TM. product (trivalent live
virus vaccine).
[0098] The virus may be attenuated. The virus may be
temperature-sensitive. The virus may be cold-adapted. These three
features are particularly useful when using live virus as an
antigen.
[0099] HA is the main immunogen in current inactivated influenza
vaccines, and vaccine doses are standardised by reference to HA
levels, typically measured by SRID. Existing vaccines typically
contain about 15 .mu.g of HA per strain, although lower doses can
be used e.g. for children, or in pandemic situations, or when using
an adjuvant. Fractional doses such as 1/2 (i.e. 7.5 .mu.g HA per
strain), 1/4 and c have been used, as have higher doses (e.g.
3.times. or 9.times. doses [36,37]). Thus vaccines may include
between 0.1 and 150 .mu.g of HA per influenza strain, preferably
between 0.1 and 50 .mu.g e.g. 0.1-20 .mu.g, 0.1-15 .mu.g, 0.1-10
.mu.g, 0.1-7.5 .mu.g, 0.5-5 .mu.g, etc. Particular doses include
e.g. about 45, about 30, about 15, about 10, about 7.5, about 5,
about 3.8, about 3.75, about 1.9, about 1.5, etc. per strain.
[0100] For live vaccines, dosing is measured by median tissue
culture infectious dose (TCID.sub.50) rather than HA content, and a
TCID.sub.50 of between 10.sup.6 and 10.sup.8 (preferably between
10.sup.6.5-10.sup.7.5) per strain is typical.
[0101] Influenza strains used with the invention may have a natural
HA as found in a wild-type virus, or a modified HA. For instance,
it is known to modify HA to remove determinants (e.g. hyper-basic
regions around the HA1/HA2 cleavage site) that cause a virus to be
highly pathogenic in avian species. The use of reverse genetics
facilitates such modifications.
[0102] As well as being suitable for immunizing against
inter-pandemic strains, the compositions of the invention are
particularly useful for immunizing against pandemic or
potentially-pandemic strains. The invention is suitable for
vaccinating humans as well as non-human animals.
[0103] Other strains whose antigens can usefully be included in the
compositions are strains which are resistant to antiviral therapy
(e.g. resistant to oseltamivir [38] and/or zanamivir), including
resistant pandemic strains [39].
[0104] Compositions of the invention may include antigen(s) from
one or more (e.g. 1, 2, 3, 4 or more) influenza virus strains,
including influenza A virus and/or influenza B virus provided that
at least one influenza strain is a reassortant influenza strain of
the invention. Compositions wherein at least two, at least three or
all of the antigens are from reassortant influenza strains of the
invention are also envisioned. Where a vaccine includes more than
one strain of influenza, the different strains are typically grown
separately and are mixed after the viruses have been harvested and
antigens have been prepared. Thus a process of the invention may
include the step of mixing antigens from more than one influenza
strain. A trivalent vaccine is typical, including antigens from two
influenza A virus strains and one influenza B virus strain. A
tetravalent vaccine is also useful [40], including antigens from
two influenza A virus strains and two influenza B virus strains, or
three influenza A virus strains and one influenza B virus
strain.
Pharmaceutical Compositions
[0105] Vaccine compositions manufactured according to the invention
are pharmaceutically acceptable. They usually include components in
addition to the antigens e.g. they typically include one or more
pharmaceutical carrier(s) and/or excipient(s). As described below,
adjuvants may also be included. A thorough discussion of such
components is available in reference 41.
[0106] Vaccine compositions will generally be in aqueous form.
However, some vaccines may be in dry form, e.g. in the form of
injectable solids or dried or polymerized preparations on a
patch.
[0107] Vaccine compositions may include preservatives such as
thiomersal or 2-phenoxyethanol. It is preferred, however, that the
vaccine should be substantially free from (i.e. less than 5
.mu.g/ml) mercurial material e.g. thiomersal-free [32,42]. Vaccines
containing no mercury are more preferred. An .alpha.-tocopherol
succinate can be included as an alternative to mercurial compounds
[32], Preservative-free vaccines are particularly preferred.
[0108] To control tonicity, it is preferred to include a
physiological salt, such as a sodium salt. Sodium chloride (NaCl)
is preferred, which may be present at between 1 and 20 mg/ml. Other
salts that may be present include potassium chloride, potassium
dihydrogen phosphate, disodium phosphate dehydrate, magnesium
chloride, calcium chloride, etc.
[0109] Vaccine compositions will generally have an osmolality of
between 200 mOsm/kg and 400 mOsm/kg, preferably between 240-360
mOsm/kg, and will more preferably fall within the range of 290-310
mOsm/kg. Osmolality has previously been reported not to have an
impact on pain caused by vaccination [43], but keeping osmolality
in this range is nevertheless preferred.
[0110] Vaccine compositions may include one or more buffers.
Typical buffers include: a phosphate buffer; a Tris buffer; a
borate buffer; a succinate buffer; a histidine buffer (particularly
with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers
will typically be included in the 5-20 mM range.
[0111] The pH of a vaccine composition will generally be between
5.0 and 8.1, and more typically between 6.0 and 8.0 e.g. 6.5 and
7.5, or between 7.0 and 7.8. A process of the invention may
therefore include a step of adjusting the pH of the bulk vaccine
prior to packaging.
[0112] The vaccine composition is preferably sterile. The vaccine
composition is preferably non-pyrogenic e.g. containing <1 EU
(endotoxin unit, a standard measure) per dose, and preferably
<0.1 EU per dose. The vaccine composition is preferably
gluten-free.
[0113] Vaccine compositions of the invention may include detergent
e.g. a polyoxyethylene sorbitan ester surfactant (known as
`Tweens`), an octoxynol (such as octoxynol-9 (Triton .times.-100)
or t-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium
bromide (`CTAB`), or sodium deoxycholate, particularly for a split
or surface antigen vaccine. The detergent may be present only at
trace amounts. Thus the vaccine may include less than 1 mg/ml of
each of octoxynol-10 and polysorbate 80. Other residual components
in trace amounts could be antibiotics (e.g. neomycin, kanamycin,
polymyxin B).
[0114] A vaccine composition may include material for a single
immunisation, or may include material for multiple immunisations
(i.e. a `multidose` kit). The inclusion of a preservative is
preferred in multidose arrangements. As an alternative (or in
addition) to including a preservative in multidose compositions,
the compositions may be contained in a container having an aseptic
adaptor for removal of material.
[0115] Influenza vaccines are typically administered in a dosage
volume of about 0.5 ml, although a half dose (i.e. about 0.25 ml)
may be administered to children.
[0116] Compositions and kits are preferably stored at between
2.degree. C. and 8.degree. C. They should not be frozen. They
should ideally be kept out of direct light.
Host Cell DNA
[0117] Where virus has been isolated and/or grown on a cell line,
it is standard practice to minimize the amount of residual cell
line DNA in the final vaccine, in order to minimize any potential
oncogenic activity of the DNA.
[0118] Thus a vaccine composition prepared according to the
invention preferably contains less than 10 ng (preferably less than
1 ng, and more preferably less than 100 pg) of residual host cell
DNA per dose, although trace amounts of host cell DNA may be
present.
[0119] It is preferred that the average length of any residual host
cell DNA is less than 500 bp e.g. less than 400 bp, less than 300
bp, less than 200 bp, less than 100 bp, etc.
[0120] Contaminating DNA can be removed during vaccine preparation
using standard purification procedures e.g. chromatography, etc.
Removal of residual host cell DNA can be enhanced by nuclease
treatment e.g. by using a DNase. A convenient method for reducing
host cell DNA contamination is disclosed in references 44 & 45,
involving a two-step treatment, first using a DNase (e.g.
Benzonase), which may be used during viral growth, and then a
cationic detergent (e.g. CTAB), which may be used during virion
disruption. Treatment with an alkylating agent, such as
(.beta.-propiolactone, can also be used to remove host cell DNA,
and advantageously may also be used to inactivate virions [46].
Adjuvants
[0121] Compositions of the invention may advantageously include an
adjuvant, which can function to enhance the immune responses
(humoral and/or cellular) elicited in a subject who receives the
composition. Preferred adjuvants comprise oil-in-water emulsions.
Various such adjuvants are known, and they typically include at
least one oil and at least one surfactant, with the oil(s) and
surfactant(s) being biodegradable (metabolisable) and
biocompatible. The oil droplets in the emulsion are generally less
than 5 .mu.m in diameter, and ideally have a sub-micron diameter,
with these small sizes being achieved with a microfluidiser to
provide stable emulsions. Droplets with a size less than 220 nm are
preferred as they can be subjected to filter sterilization.
[0122] The emulsion can comprise oils such as those from an animal
(such as fish) or vegetable source. Sources for vegetable oils
include nuts, seeds and grains. Peanut oil, soybean oil, coconut
oil, and olive oil, the most commonly available, exemplify the nut
oils. Jojoba oil can be used e.g. obtained from the jojoba bean.
Seed oils include safflower oil, cottonseed oil, sunflower seed
oil, sesame seed oil and the like. In the grain group, com oil is
the most readily available, but the oil of other cereal grains such
as wheat, oats, rye, rice, teff, triticale and the like may also be
used. 6-10 carbon fatty acid esters of glycerol and
1,2-propanediol, while not occurring naturally in seed oils, may be
prepared by hydrolysis, separation and esterification of the
appropriate materials starting from the nut and seed oils. Fats and
oils from mammalian milk are metabolizable and may therefore be
used in the practice of this invention. The procedures for
separation, purification, saponification and other means necessary
for obtaining pure oils from animal sources are well known in the
art. Most fish contain metabolizable oils which may be readily
recovered. For example, cod liver oil, shark liver oils, and whale
oil such as spermaceti exemplify several of the fish oils which may
be used herein. A number of branched chain oils are synthesized
biochemically in 5-carbon isoprene units and are generally referred
to as terpenoids. Shark liver oil contains a branched, unsaturated
terpenoids known as squalene,
2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, which
is particularly preferred herein. Squalanc, the saturated analog to
squalene, is also a preferred oil. Fish oils, including squalene
and squalane, are readily available from commercial sources or may
be obtained by methods known in the art. Another preferred oil is
.alpha.-tocopherol (see below).
[0123] Mixtures of oils can be used.
[0124] Surfactants can be classified by their `HLB`
(hydrophile/lipophile balance). Preferred surfactants of the
invention have a HLB of at least 10, preferably at least 15, and
more preferably at least 16. The invention can be used with
surfactants including, but not limited to: the polyoxyethylene
sorbitan esters surfactants (commonly referred to as the Tweens),
especially polysorbate 20 and polysorbate 80; copolymers of
ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide
(BO), sold under the DOWFAX.TM. tradename, such as linear EO/PO
block copolymers; octoxynols, which can vary in the number of
repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9
(Triton .times.-100, or t-octylphenoxypolyethoxyethanol) being of
particular interest; (octylphenoxy)poly ethoxy ethanol (IGEPAL
CA-630/N P-40); phospholipids such as phosphatidylcholine
(lecithin); nonylphenol ethoxy lates, such as the Tergitol.TM. NP
series; polyoxy ethylene fatty ethers derived from lauryl, cetyl,
stearyl and oleyl alcohols (known as Brij surfactants), such as
triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters
(commonly known as the SPANs), such as sorbitan trioleate (Span 85)
and sorbitan monolaurate. Non-ionic surfactants are preferred.
Preferred surfactants for including in the emulsion are Tween 80
(polyoxyethylene sorbitan monooleate), Span 85 (sorbitan
trioleate), lecithin and Triton .times.-100.
[0125] Mixtures of surfactants can be used e.g. Tween 80/Span 85
mixtures. A combination of a polyoxyethylene sorbitan ester such as
polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol
such as t-octylphenoxypolyethoxyethanol (Triton .times.-100) is
also suitable. Another useful combination comprises laureth 9 plus
a polyoxyethylene sorbitan ester and/or an octoxynol.
[0126] Preferred amounts of surfactants (% by weight) are:
polyoxyethylene sorbitan esters (such as Tween 80) 0.01 to 1%, in
particular about 0.1%; octyl- or nonylphenoxy polyoxyethanols (such
as Triton .times.100, or other detergents in the Triton series)
0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers
(such as laureth 9) 0.1 to 20%, preferably 0.1 to 10% and in
particular 0.1 to 1% or about 0.5%.
[0127] Where the vaccine contains a split virus, it is preferred
that it contains free surfactant in the aqueous phase. This is
advantageous as the free surfactant can exert a `splitting effect`
on the antigen, thereby disrupting any unsplit virions and/or
virion aggregates that might otherwise be present. This can improve
the safety of split virus vaccines [47].
[0128] Preferred emulsions have an average droplets size of <1
.mu.m e.g. .ltoreq.750 nm, .ltoreq.500 nm, .ltoreq.400 nm, 300 nm,
.ltoreq.250 nm, .ltoreq.220 nm, .ltoreq.200 nm, or smaller. These
droplet sizes can conveniently be achieved by techniques such as
microfluidisation.
[0129] Specific oil-in-water emulsion adjuvants useful with the
invention include, but are not limited to:
[0130] A submicron emulsion of squalene, Tween 80, and Span 85. The
composition of the emulsion by volume can be about 5% squalene,
about 0.5% polysorbate 80 and about 0.5% Span 85. In weight terms,
these ratios become 4.3% squalene, 0.5% polysorbate 80 and 0.48%
Span 85. This adjuvant is known as `MF59` [48-50], as described in
more detail in Chapter 10 of ref. 51 and chapter 12 of ref. 52. The
MF59 emulsion advantageously includes citrate ions e.g. 10 mM
sodium citrate buffer.
[0131] An emulsion comprising squalene, a tocopherol, and
polysorbate 80. The emulsion may include phosphate buffered saline.
These emulsions may have by volume from 2 to 10% squalene, from 2
to 10% tocopherol and from 0.3 to 3% polysorbate 80, and the weight
ratio of squalene:tocopherol is preferably <1 (e.g. 0.90) as
this can provide a more stable emulsion. Squalene and polysorbate
80 may be present in a volume ratio of about 5:2 or at a weight
ratio of about 11:5. Thus the three components (squalene,
tocopherol, polysorbate 80) may be present at a weight ratio of
1068:1186:485 or around 55:61:25. One such emulsion (`AS03`) can be
made by dissolving Tween 80 in PBS to give a 2% solution, then
mixing 90 ml of this solution with a mixture of (5 g of DL a
tocopherol and 5 ml squalene), then microfluidising the mixture.
The resulting emulsion may have submicron oil droplets e.g. with an
average diameter of between 100 and 250 nm, preferably about 180
nm. The emulsion may also include a 3-de-O-acylated monophosphoryl
lipid A (3d MPL). Another useful emulsion of this type may
comprise, per human dose, 0.5-10 mg squalene, 0.5-11 mg tocopherol,
and 0.1-4 mg polysorbate 80 [53] e.g. in the ratios discussed
above.
[0132] An emulsion of squalene, a tocopherol, and a Triton
detergent (e.g. Triton .times.100). The emulsion may also include a
3d-MPL (see below). The emulsion may contain a phosphate
buffer.
[0133] An emulsion comprising a polysorbate (e.g. polysorbate 80),
a Triton detergent (e.g. Triton .times.100) and a tocopherol (e.g.
an .alpha.-tocopherol succinate). The emulsion may include these
three components at a mass ratio of about 75:11:10 (e.g. 750
.mu.g/ml polysorbate 80, 110 .mu.g/ml Triton .times.100 and 100
.mu.g/ml .alpha.-tocopherol succinate), and these concentrations
should include any contribution of these components from antigens.
The emulsion may also include squalene. The emulsion may also
include a 3d-MPL (see below). The aqueous phase may contain a
phosphate buffer.
[0134] An emulsion of squalane, polysorbate 80 and poloxamer 401
("Pluronic.TM. L121"). The emulsion can be formulated in phosphate
buffered saline, pH 7.4. This emulsion is a useful delivery vehicle
for muramyl dipeptides, and has been used with threonyl-MDP in the
"SAF-1" adjuvant [54] (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic
L121 and 0.2% polysorbatc 80). It can also be used without the
Thr-MDP, as in the "AF" adjuvant [55] (5% squalane, 1.25% Pluronic
L121 and 0.2% polysorbate 80). Microfluidisation is preferred.
[0135] An emulsion comprising squalene, an aqueous solvent, a
polyoxyethylene alkyl ether hydrophilic nonionic surfactant (e.g.
polyoxy ethylene (12) cetostearyl ether) and a hydrophobic nonionic
surfactant (e.g. a sorbitan ester or mannidc ester, such as
sorbitan monoleate or `Span 80`). The emulsion is preferably
thermoreversible and/or has at least 90% of the oil droplets (by
volume) with a size less than 200 nm [56]. The emulsion may also
include one or more of: alditol; a cryoprotective agent (e.g. a
sugar, such as dodecylmaltoside and/or sucrose); and/or an
alkylpolyglycoside. The emulsion may include a TLR4 agonist [57],
Such emulsions may be lyophilized.
[0136] An emulsion of squalene, poloxamer 105 and Abil-Care [58].
The final concentration (weight) of these components in adjuvanted
vaccines are 5% squalene, 4% poloxamer 105 (pluronic polyol) and 2%
Abil-Care 85 (Bis-PEG/PPCi-16/16 PEG/PPG-16/16 dimethicone;
caprylic/capric triglyceride).
[0137] An emulsion having from 0.5-50% of an oil, 0.1-10% of a
phospholipid, and 0.05-5% of a non-ionic surfactant. As described
in reference 59, preferred phospholipid components are
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, phosphatidylglycerol, phosphatidic acid,
sphingomyelin and cardiolipin. Submicron droplet sizes are
advantageous.
[0138] A submicron oil-in-water emulsion of a non-metabolisable oil
(such as light mineral oil) and at least one surfactant (such as
lecithin, Tween 80 or Span 80). Additives may be included, such as
QuilA saponin, cholesterol, a saponin-lipophile conjugate (such as
GPI-0100, described in reference 60, produced by addition of
aliphatic amine to desacylsaponin via the carboxyl group of
glucuronic acid), dimethyidioctadecylammonium bromide and/or
N,N-dioctadecyl-N,N-bis (2-hydroxyethyl)propanediamine.
[0139] An emulsion in which a saponin (e.g. QuilA or QS21) and a
sterol (e.g. a cholesterol) are associated as helical micelles
[61].
[0140] An emulsion comprising a mineral oil, a non-ionic lipophilic
ethoxylated fatty alcohol, and a non-ionic hydrophilic surfactant
(e.g. an ethoxylated fatty alcohol and/or
polyoxyethylene-polyoxypropylene block copolymer) [62].
[0141] An emulsion comprising a mineral oil, a non-ionic
hydrophilic ethoxylated fatty alcohol, and a non-ionic lipophilic
surfactant (e.g. an ethoxylated fatty alcohol and/or
polyoxyethylene-polyoxypropylene block copolymer) [62].
[0142] In some embodiments an emulsion may be mixed with antigen
extemporaneously, at the time of delivery, and thus the adjuvant
and antigen may be kept separately in a packaged or distributed
vaccine, ready for final formulation at the time of use. In other
embodiments an emulsion is mixed with antigen during manufacture,
and thus the composition is packaged in a liquid adjuvanted form.
The antigen will generally be in an aqueous form, such that the
vaccine is finally prepared by mixing two liquids. The volume ratio
of the two liquids for mixing can vary (e.g. between 5:1 and 1:5)
but is generally about 1:1. Where concentrations of components are
given in the above descriptions of specific emulsions, these
concentrations are typically for an undiluted composition, and the
concentration after mixing with an antigen solution will thus
decrease.
Packaging of Vaccine Compositions
[0143] Suitable containers for compositions of the invention (or
kit components) include vials, syringes (e.g. disposable syringes),
nasal sprays, etc. These containers should be sterile.
[0144] Where a composition/component is located in a vial, the vial
is preferably made of a glass or plastic material. The vial is
preferably sterilized before the composition is added to it. To
avoid problems with latex-sensitive patients, vials are preferably
sealed with a latex-free stopper, and the absence of latex in all
packaging material is preferred. The vial may include a single dose
of vaccine, or it may include more than one dose (a `multidose`
vial) e.g. 10 doses. Preferred vials are made of colourless
glass.
[0145] A vial can have a cap (e.g. a Luer lock) adapted such that a
pre-filled syringe can be inserted into the cap, the contents of
the syringe can be expelled into the vial (e.g. to reconstitute
lyophilised material therein), and the contents of the vial can be
removed back into the syringe. After removal of the syringe from
the vial, a needle can then be attached and the composition can be
administered to a patient. The cap is preferably located inside a
seal or cover, such that the seal or cover has to be removed before
the cap can be accessed. A vial may have a cap that permits aseptic
removal of its contents, particularly for multidose vials.
[0146] Where a component is packaged into a syringe, the syringe
may have a needle attached to it. If a needle is not attached, a
separate needle may be supplied with the syringe for assembly and
use. Such a needle may be sheathed. Safety needles are preferred.
1-inch 23-gauge, 1-inch 25-gauge and 5/8-inch 25-gauge needles are
typical. Syringes may be provided with peel-off labels on which the
lot number, influenza season and expiration date of the contents
may be printed, to facilitate record keeping. The plunger in the
syringe preferably has a stopper to prevent the plunger from being
accidentally removed during aspiration. The syringes may have a
latex rubber cap and/or plunger. Disposable syringes contain a
single dose of vaccine. The syringe will generally have a tip cap
to seal the tip prior to attachment of a needle, and the tip cap is
preferably made of a butyl rubber. If the syringe and needle are
packaged separately then the needle is preferably fitted with a
butyl rubber shield. Preferred syringes are those marketed under
the trade name "Tip-Lok".TM..
[0147] Containers may be marked to show a half-dose volume e.g. to
facilitate delivery to children. For instance, a syringe containing
a 0.5 ml dose may have a mark showing a 0.25 ml volume.
[0148] Where a glass container (e.g. a syringe or a vial) is used,
then it is preferred to use a container made from a borosilicate
glass rather than from a soda lime glass.
[0149] A kit or composition may be packaged (e.g. in the same box)
with a leaflet including details of the vaccine e.g. instructions
for administration, details of the antigens within the vaccine,
etc. The instructions may also contain warnings e.g. to keep a
solution of adrenaline readily available in case of anaphylactic
reaction following vaccination, etc.
Methods of Treatment, and Administration of the Vaccine
[0150] The invention provides a vaccine manufactured according to
the invention. These vaccine compositions are suitable for
administration to human or non-human animal subjects, such as pigs
or birds, and the invention provides a method of raising an immune
response in a subject, comprising the step of administering a
composition of the invention to the subject. The invention also
provides a composition of the invention for use as a medicament,
and provides the use of a composition of the invention for the
manufacture of a medicament for raising an immune response in a
subject.
[0151] The immune response raised by these methods and uses will
generally include an antibody response, preferably a protective
antibody response. Methods for assessing antibody responses,
neutralising capability and protection after influenza virus
vaccination are well known in the art. Human studies have shown
that antibody titers against hemagglutinin of human influenza virus
are correlated with protection (a serum sample
hemagglutination-inhibition titer of about 30-40 gives around 50%
protection from infection by a homologous virus) [63]. Antibody
responses are typically measured by hemagglutination inhibition, by
microneutralisation, by single radial immunodiffusion (SRID),
and/or by single radial hemolysis (SRH). These assay techniques are
well known in the art.
[0152] Compositions of the invention can be administered in various
ways. The most preferred immunisation route is by intramuscular
injection (e.g. into the arm or leg), but other available routes
include subcutaneous injection, intranasal [64-66], oral [67],
intradermal [68,69], transcutaneous, transdermal [70], etc.
[0153] Vaccines prepared according to the invention may be used to
treat both children and adults. Influenza vaccines are currently
recommended for use in pediatric and adult immunisation, from the
age of 6 months. Thus a human subject may be less than 1 year old,
1-5 years old, 5-15 years old, 15-55 years old, or at least 55
years old. Preferred subjects for receiving the vaccines are the
elderly (e.g. .gtoreq.50 years old, .gtoreq.60 years old, and
preferably .gtoreq.65 years), the young (e.g. .ltoreq.5 years old),
hospitalised subjects, healthcare workers, armed service and
military personnel, pregnant women, the chronically ill,
immunodeficient subjects, subjects who have taken an antiviral
compound (e.g. an oseltamivir or zanamivir compound; see below) in
the 7 days prior to receiving the vaccine, people with egg
allergies and people travelling abroad. The vaccines are not
suitable solely for these groups, however, and may be used more
generally in a population. For pandemic strains, administration to
all age groups is preferred.
[0154] Preferred compositions of the invention satisfy 1, 2 or 3 of
the CPMP criteria for efficacy. In adults (18-60 years), these
criteria are: (1) .gtoreq.70% seroprotection; (2) .gtoreq.40%
seroconversion; and/or (3) a GMT increase of .gtoreq.2.5-fold. In
elderly (>60 years), these criteria are: (1) .gtoreq.60%
seroprotection; (2).gtoreq.30% seroconversion; and/or (3) a GMT
increase of .gtoreq.2-fold. These criteria are based on open label
studies with at least 50 patients.
[0155] Treatment can be by a single dose schedule or a multiple
dose schedule. Multiple doses may be used in a primary immunisation
schedule and/or in a booster immunisation schedule. In a multiple
dose schedule the various doses may be given by the same or
different routes e.g. a parenteral prime and mucosal boost, a
mucosal prime and parenteral boost, etc. Administration of more
than one dose (typically two doses) is particularly useful in
immunologically naive patients e.g. for people who have never
received an influenza vaccine before, or for vaccinating against a
new HA subtype (as in a pandemic outbreak). Multiple doses will
typically be administered at least 1 week apart (e.g. about 2
weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks,
about 10 weeks, about 12 weeks, about 16 weeks, etc.).
[0156] Vaccines produced by the invention may be administered to
patients at substantially the same time as (e.g. during the same
medical consultation or visit to a healthcare professional or
vaccination centre) other vaccines e.g. at substantially the same
time as a measles vaccine, a mumps vaccine, a rubella vaccine, a
MMR vaccine, a varicella vaccine, a MMRV vaccine, a diphtheria
vaccine, a tetanus vaccine, a pertussis vaccine, a DTP vaccine, a
conjugated H. influenzae type b vaccine, an inactivated poliovirus
vaccine, a hepatitis B virus vaccine, a meningococcal conjugate
vaccine (such as a tetravalent A-C-W135-Y vaccine), a respiratory
syncytial virus vaccine, a pneumococcal conjugate vaccine, etc.
Administration at substantially the same time as a pneumococcal
vaccine and/or a meningococcal vaccine is particularly useful in
elderly patients.
[0157] Similarly, vaccines of the invention may be administered to
patients at substantially the same time as (e.g. during the same
medical consultation or visit to a healthcare professional) an
antiviral compound, and in particular an antiviral compound active
against influenza virus (e.g. oseltamivir and/or zanamivir). These
antivirals include neuraminidase inhibitors, such as a
(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carbox-
ylic acid or
5-(acetylamino)-4-[(aminoiminomethyl)-amino]-2,6-anhydro-3,4,5-trideoxy-D-
-glycero-D-galactonon-2-enonic acid, including esters thereof (e.g.
the ethyl esters) and salts thereof (e.g. the phosphate salts). A
preferred antiviral is
(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carbox-
ylic acid, ethyl ester, phosphate (1:1), also known as oseltamivir
phosphate (TAMIFLU.TM.).
General
[0158] The term "comprising" encompasses "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional e.g. X+Y.
[0159] The word "substantially" does not exclude "completely" e.g.
a composition which is "substantially free" from Y may be
completely free from Y. Where necessary, the word "substantially"
may be omitted from the definition of the invention.
[0160] The term "about" in relation to a numerical value x is
optional and means, for example, x.+-.10%.
[0161] Unless specifically stated, a process comprising a step of
mixing two or more components does not require any specific order
of mixing. Thus components can be mixed in any order. Where there
are three components then two components can be combined with each
other, and then the combination may be combined with the third
component, etc.
[0162] The various steps of the methods may be carried out at the
same or different times, in the same or different geographical
locations, e.g. countries, and by the same or different people or
entities.
[0163] Where animal (and particularly bovine) materials are used in
the culture of cells, they should be obtained from sources that are
free from transmissible spongiform encephalopathies (TSEs), and in
particular free from bovine spongiform enceplialopathy (BSE).
Overall, it is preferred to culture cells in the total absence of
animal-derived materials.
[0164] Where a compound is administered to the body as part of a
composition then that compound may alternatively be replaced by a
suitable prodrug.
[0165] References to a percentage sequence identity between two
amino acid sequences means that, when aligned, that percentage of
amino acids are the same in comparing the two sequences. This
alignment and the percent homology or sequence identity can be
determined using software programs known in the art, for example
those described in section 7.7.18 of reference 71. A preferred
alignment is determined by the Smith-Waterman homology search
algorithm using an affine gap search with a gap open penalty of 12
and a gap extension penalty of 2, BLOSUM matrix of 62. The
Smith-Waterman homology search algorithm is taught in reference
72.
[0166] References to a percentage sequence identity between two
nucleic acid sequences mean that, when aligned, that percentage of
bases are the same in comparing the two sequences. This alignment
and the percent homology or sequence identity can be determined
using software programs known in the art, for example those
described in section 7.7.18 of reference 71. A preferred alignment
program is GCG Gap (Genetics Computer Group, Wisconsin, Suite
Version 10.1), preferably using default parameters, which are as
follows: open gap=3; extend gap=1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0167] FIG. 1(A) and FIG. 1(B) compare the HA content (determined
by lectin-capture ELISA) of sucrose gradient-purified viruses
harvested at 60 h post-infection from MDCK cell cultures infected
with reverse genetics-derived 6:2 reassortants containing either
the PR8-X or #21 backbone with the HA and NA segments from a
pandemic-like H1 strain (strain 1; FIG. 1(A)) or a second
pandemic-like strain (strain 2; FIG. 1(B)). In FIG. 1(A) and FIG.
1(B), the white bar represents a reference vaccine strain (derived
from WHO-Collaborating Centre-supplied strain) as control, the
dotted bar represents a reassortant virus containing the PR8-X
backbone, and the checked bar represents a reassortant virus
containing the #21 backbone. The y-axis indicates HA yield in
.mu.g/ml.
[0168] FIG. 2(A) and FIG. 2(B) compare the HA content (determined
by a lectin-capture ELISA) of unpurified viruses harvested at 60 h
post-infection from MDCK cell cultures infected with reverse
genetics-derived 6:2 reassortants containing either the PR8-X or
#21 backbone with the HA and NA segments from a pre-pandemic H1
strain (strain 1; FIG. 2(A)) and a second pre-pandemic H1 strain
(strain 2; FIG. 2(B)). In FIG. 2(A) and FIG. 2(B), the white bar
represents a reference vaccine strain (derived from
WHO-Collaborating Centre-supplied strain) as control, the dotted
bar represents a reassortant virus containing the PR8-X backbone,
and the checked bar represents a reassortant virus containing the
#21 backbone. The y-axis indicates HA yield in .mu.g/ml.
[0169] FIG. 3 compares the HA yield (determined by HPLC) of
sucrose-purified viruses harvested at 60 h post-infection from MDCK
cell cultures infected with reverse genetics-derived 6:2
reassortants containing either the PR8-X or #21 backbone with the
HA and NA segments from an H3 strain (strain 1). The white bar
represents a reference vaccine strain (derived from
WHO-Collaborating Centre-supplied strain) as control, the dotted
bar represents a reassortant virus containing the PR8-X backbone,
and the checked bar represents a reassortant virus containing the
#21 backbone. The y-axis indicates HA yield in .mu.g/ml.
[0170] FIG. 4(A) and FIG. 4(B) compare virus titers (determined by
focus formation assay (FFA); FIG. 4(A)) and HA titers (determined
by lectin-capture ELISA;
[0171] FIG. 4(B)) of viruses harvested from embyronated chicken
eggs at 60 h post-infection with a reference vaccine strain or
reverse genetics-derived 6:2 reassortant viruses made with either
the PR8-X or #21 backbone and the HA and NA segments from a
pandemic-like H1 strain (strain 2). In FIG. 4(A), the individual
dots represent data from single eggs. The line represents the
average of the individual data points. The y-axis indicates
infectious units/ml. In FIG. 4(B), the white bar represents the
reference vaccine strain (derived from WHO-Collaborating
Centre-supplied strain), the dotted bar represents a reassortant
virus containing the PR8-X backbone, and the checked bar represents
a reassortant virus containing the #21 backbone. The y-axis
indicates HA yield in .mu.g/ml for pooled egg samples
[0172] FIG. 5(A) and FIG. 5(B) compare virus titers (determined by
FFA; FIG. 5(A)) and HA titers (determined by lectin-capture ELISA;
FIG. 5(B)) from viruses harvested at 60 h post-infection from MDCK
cells infected with a reference vaccine strain or reverse
genetics-derived 6:2 reassortant viruses made with either the #21
or #21C backbone and the HA and NA segments from a pandemic-like H1
strain (strain 2). In both figures, the white bar represents a
reference vaccine strain (derived from WHO-Collaborating
Centre-supplied strain) as control, the dotted bar represents a
reassortant virus made with the #21 backbone, and the checked bar
represents a reassortant virus made with a modified #21 backbone
(#21C) containing two canine-adapted mutations (R389K, T559N) in
the PR8-X PB2 segment that comprises the backbone. The y-axis in
FIG. 5(A) and FIG. 5(B) indicates infectious units/ml and HA yield
in .mu.g/ml, respectively.
[0173] FIG. 6 compares virus titers (determined by FFA) from
viruses harvested at 60 h post-infection from MDCK cells infected
with reverse genetics-derived 6:2 reassortant viruses made with
either the PR8-X, #21 or #21C backbone and the HA and NA segments
from a different pandemic-like H1 strain (strain 1). The white bar
represents the PR8-X backbone, the dotted bar represents the #21
backbone, and the checked bar represents the #21C backbone
containing two canine-adapted mutations (R389K, T559N) in the PR8-X
PB2 segment that comprises the backbone. The y-axis indicates
infectious units/ml.
[0174] FIG. 7 compares HA titers (determined by red blood cell
hemagglutination assay) from viruses harvested at 60 h
post-infection from embryonated chicken eggs infected with a
reference vaccine strain (derived from WHO-Collaborating
Centre-supplied strain) or reverse genetics-derived 6:2 reassortant
viruses containing either the PR8-X or #21C backbone and the HA and
NA segments from a pandemic-like H1 strain (strain 1). The
individual dots represent data from a single egg. The line
represents the average of the individual data points. The y-axis
indicates HA units.
[0175] FIG. 8(A) and FIG. 8(B) compare infectious titers
(determined by FFA) of viruses harvested at different time points
post-infection of MDCK cells infected with reverse genetics-derived
6:2 reassortants made with either a PR8-X backbone or a modified
PR8-X backbone containing canine-adapted polymerase mutations and
the HA and NA segments from a pandemic-like H1 strain (strain 1).
In FIG. 8(A), the dotted line with triangle markers indicates the
PR8-X backbone and the solid line with square markers indicates a
modified PR8-X backbone "PRS-X(cPA)" containing three
canine-adapted mutations (E327K, N444D, and N675D) in the PR8-X PA
segment. In FIG. 8(B), the dotted line with triangle markers
indicates the PR8-X backbone and the solid line with open circle
markers indicates a modified PR8-X backbone "PR8-X(cNP)" containing
two canine-adapted mutations (A27T, E375N) in the PR8-X NP segment.
In both figures, the x-axis indicates hours post-infection and the
y-axis indicates infectious units/ml.
[0176] FIG. 9(A) and FIG. 9(B) compare infectious titers
(determined by FFA; FIG. 9(A)) and HA titers (determined by red
blood cell hemagglutination assay; FIG. 9(B)) of virus harvested at
different times post-infection from MDCK cells infected with a
reference vaccine strain or reverse genetics-derived 6:2
reassortant viruses made with either the PR8-X or modified PR8-X
backbones containing canine-adapted mutations and the HA and NA
segments from an H3 strain (strain 2). In FIG. 9(A), the dotted
line with x markers indicates the reference vaccine strain (derived
from WHO-Collaborating Centre-supplied strain), the dotted line
with triangle markers indicates the PR8-X backbone, the solid line
with square markers indicates a modified PR8-X backbone "PR8-X
(cPA)" containing three canine-adapted mutations (E327K, N444D, and
N675D) in the PR8-X PA segment, and the solid line with open circle
markers indicates a modified PR8-X backbone "PR8-X (cNP)"
containing two canine-adapted mutations a in the PR8-X NP segment.
The y-axis represents infectious units/ml and the x-axis represents
hours post-infection. In FIG. 9(B), the white bar indicates the
reference vaccine strain (derived from WHO-Collaborating
Centre-supplied strain), the dotted bar indicates the PR8-X
backbone, the checked bar indicates the PRB-X(cPA) backbone and the
cross-hatched bar indicates the PR8-X(cNP) backbone. The y-axis
represents HA units from the 60 h post-infection time-point.
[0177] FIG. 10(A), FIG. 10(B), and FIG. 10(C) compare the HA
content (determined by lectin-capture ELISA) of sucrose
gradient-purified viruses harvested at 60 h post-infection from
MDCK cell cultures infected with reverse genetics-derived 6:2
reassortants containing either the PR8-X or #21 backbone with the
HA and NA segments from an H3 (strain 2; FIG. 10(A)) or a second H3
strain (strain 3; FIG. 10(B)) or a third H3 strain (strain 4; FIG.
10(C)). In FIG. 10(A) and FIG. 10(B), the white bar represents a
reference vaccine strain (derived from WHO-Collaborating
Centre-supplied strain) as control, the dotted bar represents a
reassortant virus containing the PR8-X backbone, and the checked
bar represents a reassortant virus containing the #21 backbone. The
y-axis indicates HA yield in .mu.g/ml.
MODES FOR CARRYING OUT THE INVENTION
Development of New Donor Strains
[0178] In order to provide high-growth donor strains, the inventors
found that a reassortant influenza virus comprising the PB1 segment
of A/California/07/09 and all other backbone segments from PR8-X
shows improved growth characteristics compared with reassortant
influenza viruses which contain all backbone segments from PR8-X.
This influenza backbone is referred to as #21.
Focus-Forming Assays (FFA)
[0179] For the FFA, uninfected MDCK cells are plated at a density
of 1.8.times.104 cells/well in 96 well plates in 100 pi of DMEM
with 10% FCS. The next day, medium is aspirated and cells are
infected with viruses in a volume of 50 pi (viruses diluted in
DMEM+1% FCS). The cells are incubated at 37.degree. C. until the
next day.
[0180] At several time points after infection, the medium is
aspirated and the cells washed once with PBS. 50 .mu.l of ice-cold
50%/50% acetone-methanol is added to each well followed by
incubation at -20.degree. C. for 30 minutes. The acetone mix is
aspirated and the cells washed once with PBST (PBS+0.1% Tween). 50
.mu.l of 2% BSA in PBS is added to each well followed by incubation
at room temperature (RT) for 30 minutes. 50 .mu.l of a 1:6000
dilution of anti-NP is added in blocking buffer followed by
incubation at RT for 1 hours. The antibody solution is aspirated
and the cells washed three times with PBST. Secondary antibody
(goat anti mouse) is added at a dilution 1:2000 in 50 .mu.l
blocking buffer and the plate is incubated at RT for 1 hours. The
antibody solution is aspirated and the cells washed three times
with PBST. 50 .mu.l of KPL True Blue is added to each well and
incubated for 10 minutes. The reaction is stopped by aspirating the
True-Blue and washing once with dH.sub.2O. The water is aspirated
and the cells are left to dry.
Growth Characteristics of Reassortant Viruses Containing PR8-X or
#21 Backbones
[0181] In order to test the suitability of the #21 strain as a
donor strain for virus reassortment, reassortant influenza viruses
are produced by reverse genetics which contain the HA and NA
proteins from various influenza strains (including zoonotic,
seasonal, and pandemic-like strains) and the other viral segments
from either PR8-X or the #21 backbone. The HA content, HA yield and
the viral titres of these reassortant viruses are determined. As a
control a reference vaccine strain which does not contain any
backbone segments from PR8-X or A/California/07/09 is used. These
viruses are cultured either in embyronated chicken eggs or in MDCK
cells.
[0182] The results indicate that reassortant viruses which contain
the #21 backbone consistently give higher viral titres and HA
yields compared with the control virus and the virus which contains
all backbone segments from PR8-X in both eggs and cell culture.
This difference is due to the PB1 segment because this is the only
difference between #21 reassortants and PR8-X reassortants (see
FIG. 1(A), FIG. 1(B), FIG. 2(A), FIG. 2(B), FIG. 3, FIG. 4(A), and
FIG. 4(B)).
Growth Characteristics of Reassortant Viruses Containing PR8-X or
Canine Adapted PR8-X Backbones
[0183] In order to test the effect of canine-adapted mutations on
the growth characteristics of PR8-X, the inventors introduce
mutations into the PA segment (E327K, N444D, and N675D), or the NP
segment (A27T, E375N) of PR8-X. These backbones are referred to as
PR8-X(cPA) and PR8-X(cNP), respectively. Reassortant influenza
viruses are produced containing the PRB-X(cPA) and PR8-X(cNP)
backbones and the HA and NA segments of a pandemic-like H1
influenza strain (strain 1) or a H3 influenza strain (strain 2). As
a control a reference vaccine strain which does not contain any
backbone segments from PR8-X is used. The reassortant influenza
viruses are cultured in MDCK cells.
[0184] The results show that reassortant influenza viruses which
contain canine-adapted backbone segments consistently grow to
higher viral titres compared with reassortant influenza viruses
which contain unmodified PR8-X backbone segments (see FIG. 8(A),
FIG. 8(B), FIG. 9(A), and FIG. 9(B)).
Growth Characteristics of Reassortant Viruses Containing PR8-X, #21
or #21C Backbones
[0185] In order to test whether canine-adapted mutations in the
backbone segments improve the growth characteristics of the #21
backbone, the inventors modify the #21 backbone by introducing
mutations into the PR8-X PB2 segment (R389K, T559N). This backbone
is referred to as #21C. Reassortant influenza viruses are produced
by reverse genetics which contain the HA and NA proteins from two
different pandemic-like H1 strains (strains 1 and 2) and the other
viral segments from either PR8-X, the #21 backbone or the #21C
backbone. As a control a reference vaccine strain which docs not
contain any backbone segments from PR8-X or A/California/07/09 is
used. These viruses are cultured in MDCK cells. The virus yield of
these reassortant viruses is determined. For reassortant influenza
viruses containing the HA and NA segments from the pandemic-like H1
strain (strain 1) and the PR8-X or #21C backbones the FIA titres
are also determined.
[0186] The results show that reassortant influenza viruses which
contain the #21C backbone consistently grow to higher viral titres
compared with reassortant influenza viruses which contain only
PR8-X backbone segments or the #21 backbone (see FIG. 5(A), FIG.
5(B), FIG. 6, and FIG. 7). Reassortant influenza viruses comprising
the #21C backbone also show higher HA titres compared with PR8-X
reassortants.
[0187] It will be understood that the invention has been described
by way of example only and modifications may be made whilst
remaining within the scope and spirit of the invention.
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Sequence CWU 1
1
321716PRTUnknownInfluenza 1Met Glu Asp Phe Val Arg Gln Cys Phe Asn
Pro Met Ile Val Glu Leu1 5 10 15Ala Glu Lys Thr Met Lys Glu Tyr Gly
Glu Asp Leu Lys Ile Glu Thr 20 25 30Asn Lys Phe Ala Ala Ile Cys Thr
His Leu Glu Val Cys Phe Met Tyr 35 40 45Ser Asp Phe His Phe Ile Asn
Glu Gln Gly Glu Ser Ile Ile Val Glu 50 55 60Leu Gly Asp Pro Asn Ala
Leu Leu Lys His Arg Phe Glu Ile Ile Glu65 70 75 80Gly Arg Asp Arg
Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn 85 90 95Thr Thr Gly
Ala Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr 100 105 110Lys
Glu Asn Arg Phe Ile Glu Ile Gly Val Thr Arg Arg Glu Val His 115 120
125Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Lys Thr His
130 135 140Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys
Ala Asp145 150 155 160Tyr Thr Leu Asp Glu Glu Ser Arg Ala Arg Ile
Lys Thr Arg Leu Phe 165 170 175Thr Ile Arg Gln Glu Met Ala Ser Arg
Gly Leu Trp Asp Ser Phe Arg 180 185 190Gln Ser Glu Arg Gly Glu Glu
Thr Ile Glu Glu Arg Phe Glu Ile Thr 195 200 205Gly Thr Met Arg Lys
Leu Ala Asp Gln Ser Leu Pro Pro Asn Phe Ser 210 215 220Ser Leu Glu
Asn Phe Arg Ala Tyr Val Asp Gly Phe Glu Pro Asn Gly225 230 235
240Tyr Ile Glu Gly Lys Leu Ser Gln Met Ser Lys Glu Val Asn Ala Arg
245 250 255Ile Glu Pro Phe Leu Lys Thr Thr Pro Arg Pro Leu Arg Leu
Pro Asn 260 265 270Gly Pro Pro Cys Ser Gln Arg Ser Lys Phe Leu Leu
Met Asp Ala Leu 275 280 285Lys Leu Ser Ile Glu Asp Pro Ser His Glu
Gly Glu Gly Ile Pro Leu 290 295 300Tyr Asp Ala Ile Lys Cys Met Arg
Thr Phe Phe Gly Trp Lys Glu Pro305 310 315 320Asn Val Val Lys Pro
His Glu Lys Gly Ile Asn Pro Asn Tyr Leu Leu 325 330 335Ser Trp Lys
Gln Val Leu Ala Glu Leu Gln Asp Ile Glu Asn Glu Glu 340 345 350Lys
Ile Pro Lys Thr Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp 355 360
365Ala Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Asp Asp Cys
370 375 380Lys Asp Val Gly Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro
Glu Leu385 390 395 400Arg Ser Leu Ala Ser Trp Ile Gln Asn Glu Phe
Asn Lys Ala Cys Glu 405 410 415Leu Thr Asp Ser Ser Trp Ile Glu Leu
Asp Glu Ile Gly Glu Asp Val 420 425 430Ala Pro Ile Glu His Ile Ala
Ser Met Arg Arg Asn Tyr Phe Thr Ser 435 440 445Glu Val Ser His Cys
Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr 450 455 460Ile Asn Thr
Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Asp Phe465 470 475
480Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg
485 490 495Lys Thr Asn Leu Tyr Gly Phe Ile Ile Lys Gly Arg Ser His
Leu Arg 500 505 510Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu
Phe Ser Leu Thr 515 520 525Asp Pro Arg Leu Glu Pro His Lys Trp Glu
Lys Tyr Cys Val Leu Glu 530 535 540Ile Gly Asp Met Leu Ile Arg Ser
Ala Ile Gly Gln Val Ser Arg Pro545 550 555 560Met Phe Leu Tyr Val
Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys 565 570 575Trp Gly Met
Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile 580 585 590Glu
Ser Met Ile Glu Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr 595 600
605Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser
610 615 620Pro Lys Gly Val Glu Glu Ser Ser Ile Gly Lys Val Cys Arg
Thr Leu625 630 635 640Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala
Ser Pro Gln Leu Glu 645 650 655Gly Phe Ser Ala Glu Ser Arg Lys Leu
Leu Leu Ile Val Gln Ala Leu 660 665 670Arg Asp Asn Leu Glu Pro Gly
Thr Phe Asp Leu Gly Gly Leu Tyr Glu 675 680 685Ala Ile Glu Glu Cys
Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala 690 695 700Ser Trp Phe
Asn Ser Phe Leu Thr His Ala Leu Ser705 710
7152757PRTUnknownInfluenza 2Met Asp Val Asn Pro Thr Leu Leu Phe Leu
Lys Val Pro Thr Gln Asn1 5 10 15Ala Ile Ser Thr Thr Phe Pro Tyr Thr
Gly Asp Pro Pro Tyr Ser His 20 25 30Gly Thr Gly Thr Gly Tyr Thr Met
Asp Thr Val Asn Arg Thr His Gln 35 40 45Tyr Ser Glu Lys Gly Arg Trp
Thr Thr Asn Thr Glu Thr Gly Ala Pro 50 55 60Gln Leu Asn Pro Ile Asp
Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser65 70 75 80Gly Tyr Ala Gln
Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 85 90 95Glu Ser His
Pro Gly Ile Phe Glu Asn Ser Cys Ile Glu Thr Met Glu 100 105 110Val
Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr 115 120
125Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala
130 135 140Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr Ala Asn
Glu Ser145 150 155 160Gly Arg Leu Ile Asp Phe Leu Lys Asp Val Met
Glu Ser Met Asn Lys 165 170 175Glu Glu Met Gly Ile Thr Thr His Phe
Gln Arg Lys Arg Arg Val Arg 180 185 190Asp Asn Met Thr Lys Lys Met
Ile Thr Gln Arg Thr Met Gly Lys Lys 195 200 205Lys Gln Arg Leu Asn
Lys Arg Ser Tyr Leu Ile Arg Ala Leu Thr Leu 210 215 220Asn Thr Met
Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala225 230 235
240Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu
245 250 255Thr Leu Ala Arg Ser Ile Cys Glu Lys Leu Glu Gln Ser Gly
Leu Pro 260 265 270Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn
Val Val Arg Lys 275 280 285Met Met Thr Asn Ser Gln Asp Thr Glu Leu
Ser Phe Thr Ile Thr Gly 290 295 300Asp Asn Thr Lys Trp Asn Glu Asn
Gln Asn Pro Arg Met Phe Leu Ala305 310 315 320Met Ile Thr Tyr Met
Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val 325 330 335Leu Ser Ile
Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly 340 345 350Lys
Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile 355 360
365Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser
370 375 380Thr Arg Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Ile Glu
Gly Thr385 390 395 400Ala Ser Leu Ser Pro Gly Met Met Met Gly Met
Phe Asn Met Leu Ser 405 410 415Thr Val Leu Gly Val Ser Ile Leu Asn
Leu Gly Gln Lys Arg Tyr Thr 420 425 430Lys Thr Thr Tyr Trp Trp Asp
Gly Leu Gln Ser Ser Asp Asp Phe Ala 435 440 445Leu Ile Val Asn Ala
Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp 450 455 460Arg Phe Tyr
Arg Thr Cys Lys Leu Leu Gly Ile Asn Met Ser Lys Lys465 470 475
480Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe
485 490 495Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro
Ser Phe 500 505 510Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser
Ile Gly Val Thr 515 520 525Val Ile Lys Asn Asn Met Ile Asn Asn Asp
Leu Gly Pro Ala Thr Ala 530 535 540Gln Met Ala Leu Gln Leu Phe Ile
Lys Asp Tyr Arg Tyr Thr Tyr Arg545 550 555 560Cys His Arg Gly Asp
Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Ile 565 570 575Lys Lys Leu
Trp Glu Gln Thr Arg Ser Lys Ala Gly Leu Leu Val Ser 580 585 590Asp
Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu 595 600
605Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Gln Gly Arg Leu
610 615 620Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu
Ser Met625 630 635 640Asn Asn Ala Val Met Met Pro Ala His Gly Pro
Ala Lys Asn Met Glu 645 650 655Tyr Asp Ala Val Ala Thr Thr His Ser
Trp Ile Pro Lys Arg Asn Arg 660 665 670Ser Ile Leu Asn Thr Ser Gln
Arg Gly Val Leu Glu Asp Glu Gln Met 675 680 685Tyr Gln Arg Cys Cys
Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser 690 695 700Tyr Arg Arg
Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser705 710 715
720Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys
725 730 735Lys Glu Glu Phe Thr Glu Ile Met Lys Ile Cys Ser Thr Ile
Glu Glu 740 745 750Leu Arg Arg Gln Lys 7553759PRTUnknownInfluenza
3Met Glu Arg Ile Lys Glu Leu Arg Asn Leu Met Ser Gln Ser Arg Thr1 5
10 15Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile
Lys 20 25 30Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg
Met Lys 35 40 45Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys
Arg Ile Thr 50 55 60Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr
Leu Trp Ser Lys65 70 75 80Met Asn Asp Ala Gly Ser Asp Arg Val Met
Val Ser Pro Leu Ala Val 85 90 95Thr Trp Trp Asn Arg Asn Gly Pro Ile
Thr Asn Thr Val His Tyr Pro 100 105 110Lys Ile Tyr Lys Thr Tyr Phe
Glu Arg Val Glu Arg Leu Lys His Gly 115 120 125Thr Phe Gly Pro Val
His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg 130 135 140Val Asp Ile
Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln145 150 155
160Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile
165 170 175Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys
Glu Glu 180 185 190Leu Gln Asp Cys Lys Ile Ser Pro Leu Met Val Ala
Tyr Met Leu Glu 195 200 205Arg Glu Leu Val Arg Lys Thr Arg Phe Leu
Pro Val Ala Gly Gly Thr 210 215 220Ser Ser Val Tyr Ile Glu Val Leu
His Leu Thr Gln Gly Thr Cys Trp225 230 235 240Glu Gln Met Tyr Thr
Pro Gly Gly Glu Val Arg Asn Asp Asp Val Asp 245 250 255Gln Ser Leu
Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Ala Val 260 265 270Ser
Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln 275 280
285Ile Gly Gly Ile Arg Met Val Asp Ile Leu Arg Gln Asn Pro Thr Glu
290 295 300Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg
Ile Ser305 310 315 320Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys
Arg Thr Ser Gly Ser 325 330 335Ser Val Lys Arg Glu Glu Glu Val Leu
Thr Gly Asn Leu Gln Thr Leu 340 345 350Lys Ile Arg Val His Glu Gly
Tyr Glu Glu Phe Thr Met Val Gly Arg 355 360 365Arg Ala Thr Ala Ile
Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu 370 375 380Ile Val Ser
Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val385 390 395
400Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly
405 410 415Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro
Met His 420 425 430Gln Leu Leu Arg His Phe Gln Lys Asp Ala Arg Val
Leu Phe Gln Asn 435 440 445Trp Gly Val Glu Pro Ile Asp Asn Val Met
Gly Met Ile Gly Ile Leu 450 455 460Pro Asp Met Thr Pro Ser Ile Glu
Met Ser Met Arg Gly Val Arg Ile465 470 475 480Ser Lys Met Gly Val
Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val 485 490 495Ser Ile Asp
Arg Phe Leu Arg Ile Arg Asp Gln Arg Gly Asn Val Leu 500 505 510Leu
Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr 515 520
525Ile Thr Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser
530 535 540Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu
Thr Val545 550 555 560Lys Ile Gln Trp Ser Gln Asn Pro Thr Met Leu
Tyr Asn Lys Met Glu 565 570 575Phe Glu Pro Phe Gln Ser Leu Val Pro
Lys Ala Ile Arg Gly Gln Tyr 580 585 590Ser Gly Phe Val Arg Thr Leu
Phe Gln Gln Met Arg Asp Val Leu Gly 595 600 605Thr Phe Asp Thr Ala
Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala 610 615 620Pro Pro Lys
Gln Ser Arg Met Gln Phe Ser Ser Phe Thr Val Asn Val625 630 635
640Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe
645 650 655Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys
Asp Ala 660 665 670Gly Thr Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala
Gly Val Glu Ser 675 680 685Ala Val Leu Arg Gly Phe Leu Ile Leu Gly
Lys Glu Asp Lys Arg Tyr 690 695 700Gly Pro Ala Leu Ser Ile Asn Glu
Leu Ser Asn Leu Ala Lys Gly Glu705 710 715 720Lys Ala Asn Val Leu
Ile Gly Gln Gly Asp Val Val Leu Val Met Lys 725 730 735Arg Lys Arg
Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys 740 745 750Arg
Ile Arg Met Ala Ile Asn 7554498PRTUnknownInfluenza 4Met Ala Ser Gln
Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp1 5 10 15Gly Glu Arg
Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Lys Met 20 25 30Ile Gly
Gly Ile Gly Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys 35 40 45Leu
Ser Asp Tyr Glu Gly Arg Leu Ile Gln Asn Ser Leu Thr Ile Glu 50 55
60Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu65
70 75 80Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro
Ile 85 90 95Tyr Arg Arg Val Asn Gly Lys Trp Met Arg Glu Leu Ile Leu
Tyr Asp 100 105 110Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn
Asn Gly Asp Asp 115 120 125Ala Thr Ala Gly Leu Thr His Met Met Ile
Trp His Ser Asn Leu Asn 130 135 140Asp Ala Thr Tyr Gln Arg Thr Arg
Ala Leu Val Arg Thr Gly Met Asp145 150 155 160Pro Arg Met Cys Ser
Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser 165 170 175Gly Ala Ala
Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu 180 185 190Leu
Val Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg 195 200
205Gly Glu Asn Gly Arg Lys Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn
210 215 220Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Lys Ala Met
Met Asp225
230 235 240Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Phe Glu
Asp Leu 245 250 255Thr Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly
Ser Val Ala His 260 265 270Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly
Pro Ala Val Ala Ser Gly 275 280 285Tyr Asp Phe Glu Arg Glu Gly Tyr
Ser Leu Val Gly Ile Asp Pro Phe 290 295 300Arg Leu Leu Gln Asn Ser
Gln Val Tyr Ser Leu Ile Arg Pro Asn Glu305 310 315 320Asn Pro Ala
His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala 325 330 335Ala
Phe Glu Asp Leu Arg Val Leu Ser Phe Ile Lys Gly Thr Lys Val 340 345
350Leu Pro Arg Gly Lys Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn
355 360 365Glu Asn Met Glu Thr Met Glu Ser Ser Thr Leu Glu Leu Arg
Ser Arg 370 375 380Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr
Asn Gln Gln Arg385 390 395 400Ala Ser Ala Gly Gln Ile Ser Ile Gln
Pro Thr Phe Ser Val Gln Arg 405 410 415Asn Leu Pro Phe Asp Arg Thr
Thr Ile Met Ala Ala Phe Asn Gly Asn 420 425 430Thr Glu Gly Arg Thr
Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met 435 440 445Glu Ser Ala
Arg Pro Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe 450 455 460Glu
Leu Ser Asp Glu Lys Ala Ala Ser Pro Ile Val Pro Ser Phe Asp465 470
475 480Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu
Tyr 485 490 495Asp Asn5252PRTUnknownInfluenza 5Met Ser Leu Leu Thr
Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro1 5 10 15Ser Gly Pro Leu
Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe 20 25 30Ala Gly Lys
Asn Thr Asp Leu Glu Val Leu Met Glu Trp Leu Lys Thr 35 40 45Arg Pro
Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe 50 55 60Thr
Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val65 70 75
80Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Lys Ala
85 90 95Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly
Ala 100 105 110Lys Glu Ile Ser Leu Ser Tyr Ser Ala Gly Ala Leu Ala
Ser Cys Met 115 120 125Gly Leu Ile Tyr Asn Arg Met Gly Ala Val Thr
Thr Glu Val Ala Phe 130 135 140Gly Leu Val Cys Ala Thr Cys Glu Gln
Ile Ala Asp Ser Gln His Arg145 150 155 160Ser His Arg Gln Met Val
Thr Thr Thr Asn Pro Leu Ile Arg His Glu 165 170 175Asn Arg Met Val
Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met 180 185 190Ala Gly
Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln 195 200
205Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser
210 215 220Ser Ser Ala Gly Leu Lys Asn Asp Leu Leu Glu Asn Leu Gln
Ala Tyr225 230 235 240Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe
Lys 245 2506230PRTUnknownInfluenza 6Met Asp Pro Asn Thr Val Ser Ser
Phe Gln Val Asp Cys Phe Leu Trp1 5 10 15His Val Arg Lys Arg Val Ala
Asp Gln Glu Leu Gly Asp Ala Pro Phe 20 25 30Leu Asp Arg Leu Arg Arg
Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser 35 40 45Thr Leu Gly Leu Asp
Ile Lys Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60Val Glu Arg Ile
Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr65 70 75 80Met Ala
Ser Val Pro Ala Ser Arg Tyr Leu Thr Asp Met Thr Leu Glu 85 90 95Glu
Met Ser Arg Asp Trp Ser Met Leu Ile Pro Lys Gln Lys Val Ala 100 105
110Gly Pro Leu Cys Ile Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile
115 120 125Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Asp Arg Leu Glu
Thr Leu 130 135 140Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Ile
Val Gly Glu Ile145 150 155 160Ser Pro Leu Pro Ser Leu Pro Gly His
Thr Ala Glu Asp Val Lys Asn 165 170 175Ala Val Gly Val Leu Ile Gly
Gly Leu Glu Trp Asn Asp Asn Thr Val 180 185 190Arg Val Ser Glu Thr
Leu Gln Arg Phe Ala Trp Arg Ser Ser Asn Glu 195 200 205Asn Gly Arg
Pro Pro Leu Thr Pro Lys Gln Lys Arg Glu Met Ala Gly 210 215 220Thr
Ile Arg Ser Glu Val225 2307565PRTUnknownInfluenza 7Met Lys Ala Asn
Leu Leu Val Leu Leu Cys Ala Leu Ala Ala Ala Asp1 5 10 15Ala Asp Thr
Ile Cys Ile Gly Tyr His Thr Asn Asn Ser Thr Asp Thr 20 25 30Val Asp
Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45Leu
Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile 50 55
60Ala Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly65
70 75 80Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser Tyr
Ile 85 90 95Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr Pro Gly
Asp Phe 100 105 110Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
Val Ser Ser Phe 115 120 125Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser
Ser Trp Pro Asn His Asn 130 135 140Thr Asn Gly Val Thr Ala Ala Cys
Ser His Glu Gly Lys Ser Ser Phe145 150 155 160Tyr Arg Asn Leu Leu
Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175Leu Lys Asn
Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180 185 190Trp
Gly Ile His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Leu Tyr 195 200
205Gln Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr Asn Arg
210 215 220Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val Arg Asp
Gln Ala225 230 235 240Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Lys
Pro Gly Asp Thr Ile 245 250 255Ile Phe Glu Ala Asn Gly Asn Leu Ile
Ala Pro Met Tyr Ala Phe Ala 260 265 270Leu Ser Arg Gly Phe Gly Ser
Gly Ile Ile Thr Ser Asn Ala Ser Met 275 280 285His Glu Cys Asn Thr
Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Ser 290 295 300Ser Leu Pro
Tyr Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro305 310 315
320Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn
325 330 335Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala
Gly Phe 340 345 350Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly Trp
Tyr Gly Tyr His 355 360 365His Gln Asn Glu Gln Gly Ser Gly Tyr Ala
Ala Asp Gln Lys Ser Thr 370 375 380Gln Asn Ala Ile Asn Gly Ile Thr
Asn Lys Val Asn Thr Val Ile Glu385 390 395 400Lys Met Asn Ile Gln
Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu 405 410 415Glu Lys Arg
Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu 420 425 430Asp
Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 435 440
445Arg Thr Leu Glu Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys
450 455 460Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn
Gly Cys465 470 475 480Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys
Met Glu Ser Val Arg 485 490 495Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr
Ser Glu Glu Ser Lys Leu Asn 500 505 510Arg Glu Lys Val Asp Gly Val
Lys Leu Glu Ser Met Gly Ile Tyr Gln 515 520 525Ile Leu Ala Ile Tyr
Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val 530 535 540Ser Leu Gly
Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln545 550 555
560Cys Arg Ile Cys Ile 5658454PRTUnknownInfluenza 8Met Asn Pro Asn
Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys Leu Val1 5 10 15Val Gly Leu
Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30Trp Ile
Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile 35 40 45Cys
Asn Gln Asn Ile Ile Thr Tyr Lys Asn Ser Thr Trp Val Lys Asp 50 55
60Thr Thr Ser Val Ile Leu Thr Gly Asn Ser Ser Leu Cys Pro Ile Arg65
70 75 80Gly Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser
Lys 85 90 95Gly Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser
His Leu 100 105 110Glu Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu
Leu Asn Asp Lys 115 120 125His Ser Ser Gly Thr Val Lys Asp Arg Ser
Pro Tyr Arg Ala Leu Met 130 135 140Ser Cys Pro Val Gly Glu Ala Pro
Ser Pro Tyr Asn Ser Arg Phe Glu145 150 155 160Ser Val Ala Trp Ser
Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu 165 170 175Thr Ile Gly
Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys 180 185 190Tyr
Asn Gly Ile Ile Thr Glu Thr Ile Lys Ser Trp Arg Lys Lys Ile 195 200
205Leu Arg Thr Gln Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe
210 215 220Thr Ile Met Thr Asp Gly Pro Ser Asp Gly Leu Ala Ser Tyr
Lys Ile225 230 235 240Phe Lys Ile Glu Lys Gly Lys Val Thr Lys Ser
Ile Glu Leu Asn Ala 245 250 255Pro Asn Ser His Tyr Glu Glu Cys Ser
Cys Tyr Pro Asp Thr Asp Lys 260 265 270Val Met Cys Val Cys Arg Asp
Asn Trp His Gly Ser Asn Arg Pro Trp 275 280 285Val Ser Phe Asp Gln
Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser 290 295 300Gly Val Phe
Gly Asp Asn Pro Arg Pro Glu Asp Gly Thr Gly Ser Cys305 310 315
320Gly Pro Val Tyr Val Asp Gly Ala Asn Gly Val Lys Gly Phe Ser Tyr
325 330 335Arg Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser His
Ser Ser 340 345 350Arg His Gly Phe Glu Met Ile Trp Asp Pro Asn Gly
Trp Thr Glu Thr 355 360 365Asp Ser Lys Phe Ser Val Arg Gln Asp Val
Val Ala Met Thr Asp Trp 370 375 380Ser Gly Tyr Ser Gly Ser Phe Val
Gln His Pro Glu Leu Thr Gly Leu385 390 395 400Asp Cys Met Arg Pro
Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro 405 410 415Lys Glu Lys
Thr Ile Trp Thr Ser Ala Ser Ser Ile Ser Phe Cys Gly 420 425 430Val
Asn Ser Asp Thr Val Asp Trp Ser Trp Pro Asp Gly Ala Glu Leu 435 440
445Pro Phe Ser Ile Asp Lys 45092233DNAUnknownInfluenza 9agcgaaagca
ggtactgatc caaaatggaa gattttgtgc gacaatgctt caatccgatg 60attgtcgagc
ttgcggaaaa aacaatgaaa gagtatgggg aggacctgaa aatcgaaaca
120aacaaatttg cagcaatatg cactcacttg gaagtatgct tcatgtattc
agattttcac 180ttcatcaatg agcaaggcga gtcaataatc gtagaacttg
gtgatccaaa tgcacttttg 240aagcacagat ttgaaataat cgagggaaga
gatcgcacaa tggcctggac agtagtaaac 300agtatttgca acactacagg
ggctgagaaa ccaaagtttc taccagattt gtatgattac 360aaggagaata
gatttatcga aattggagta acaaggagag aagttcacat atactatctg
420gaaaaggcca ataaaattaa atctgagaaa acacacatcc acattttctc
gttcactggg 480gaagaaatgg ccacaaaggc agactacact ctcgatgaag
aaagcagggc taggatcaaa 540accagactat tcaccataag acaagaaatg
gccagcagag gcctctggga ttcctttcgt 600cagtccgaga gaggagaaga
gacaattgaa gaaaggtttg aaatcacagg aacaatgcgc 660aagcttgccg
accaaagtct cccgccgaac ttctccagcc ttgaaaattt tagagcctat
720gtggatggat tcgaaccgaa cggctacatt gagggcaagc tgtctcaaat
gtccaaagaa 780gtaaatgcta gaattgaacc ttttttgaaa acaacaccac
gaccacttag acttccgaat 840gggcctccct gttctcagcg gtccaaattc
ctgctgatgg atgccttaaa attaagcatt 900gaggacccaa gtcatgaagg
agagggaata ccgctatatg atgcaatcaa atgcatgaga 960acattctttg
gatggaagga acccaatgtt gttaaaccac acgaaaaggg aataaatcca
1020aattatcttc tgtcatggaa gcaagtactg gcagaactgc aggacattga
gaatgaggag 1080aaaattccaa agactaaaaa tatgaagaaa acaagtcagc
taaagtgggc acttggtgag 1140aacatggcac cagaaaaggt agactttgac
gactgtaaag atgtaggtga tttgaagcaa 1200tatgatagtg atgaaccaga
attgaggtcg cttgcaagtt ggattcagaa tgagtttaac 1260aaggcatgcg
aactgacaga ttcaagctgg atagagctcg atgagattgg agaagatgtg
1320gctccaattg aacacattgc aagcatgaga aggaattatt tcacatcaga
ggtgtctcac 1380tgcagagcca cagaatacat aatgaagggg gtgtacatca
atactgcctt gcttaatgca 1440tcttgtgcag caatggatga tttccaatta
attccaatga taagcaagtg tagaactaag 1500gagggaaggc gaaagaccaa
cttgtatggt ttcatcataa aaggaagatc ccacttaagg 1560aatgacaccg
acgtggtaaa ctttgtgagc atggagtttt ctctcactga cccaagactt
1620gaaccacata aatgggagaa gtactgtgtt cttgagatag gagatatgct
tataagaagt 1680gccataggcc aggtttcaag gcccatgttc ttgtatgtga
gaacaaatgg aacctcaaaa 1740attaaaatga aatggggaat ggagatgagg
cgttgcctcc tccagtcact tcaacaaatt 1800gagagtatga ttgaagctga
gtcctctgtc aaagagaaag acatgaccaa agagttcttt 1860gagaacaaat
cagaaacatg gcccattgga gagtccccca aaggagtgga ggaaagttcc
1920attgggaagg tctgcaggac tttattagca aagtcggtat tcaacagctt
gtatgcatct 1980ccacaactag aaggattttc agctgaatca agaaaactgc
ttcttatcgt tcaggctctt 2040agggacaacc ttgaacctgg gacctttgat
cttggggggc tatatgaagc aattgaggag 2100tgcctgatta atgatccctg
ggttttgctt aatgcttctt ggttcaactc cttccttaca 2160catgcattga
gttagttgtg gcagtgctac tatttgctat ccatactgtc caaaaaagta
2220ccttgtttct act 2233102341DNAUnknownInfluenza 10agcgaaagca
ggcaaaccat ttgaatggat gtcaatccga ccttactttt cttaaaagtg 60ccaacacaaa
atgctataag cacaactttc ccttatactg gagaccctcc ttacagccat
120gggacaggaa caggatacac catggatact gtcaacagga cacatcagta
ctcagaaaag 180ggaagatgga caacaaacac cgaaactgga gcaccgcaac
tcaacccgat tgatgggcca 240ctgccagaag acaatgaacc aagtggttat
gcccaaacag attgtgtatt ggaggcgatg 300gctttccttg aggaatccca
tcctggtatt tttgaaaact cgtgtattga aacgatggag 360gttgttcagc
aaacacgagt agacaagctg acacaaggcc gacagaccta tgactggact
420ctaaatagaa accaacctgc tgcaacagca ttggccaaca caatagaagt
gttcagatca 480aatggcctca cggccaatga gtctggaagg ctcatagact
tccttaagga tgtaatggag 540tcaatgaaca aagaagaaat ggggatcaca
actcattttc agagaaagag acgggtgaga 600gacaatatga ctaagaaaat
gataacacag agaacaatgg gtaaaaagaa gcagagattg 660aacaaaagga
gttatctaat tagagcattg accctgaaca caatgaccaa agatgctgag
720agagggaagc taaaacggag agcaattgca accccaggga tgcaaataag
ggggtttgta 780tactttgttg agacactggc aaggagtata tgtgagaaac
ttgaacaatc agggttgcca 840gttggaggca atgagaagaa agcaaagttg
gcaaatgttg taaggaagat gatgaccaat 900tctcaggaca ccgaactttc
tttcaccatc actggagata acaccaaatg gaacgaaaat 960cagaatcctc
ggatgttttt ggccatgatc acatatatga ccagaaatca gcccgaatgg
1020ttcagaaatg ttctaagtat tgctccaata atgttctcaa acaaaatggc
gagactggga 1080aaagggtata tgtttgagag caagagtatg aaacttagaa
ctcaaatacc tgcagaaatg 1140ctagcaagca tcgatttgaa atatttcaat
gattcaacaa gaaagaagat tgaaaaaatc 1200cgaccgctct taatagaggg
gactgcatca ttgagccctg gaatgatgat gggcatgttc 1260aatatgttaa
gcactgtatt aggcgtctcc atcctgaatc ttggacaaaa gagatacacc
1320aagactactt actggtggga tggtcttcaa tcctctgacg attttgctct
gattgtgaat 1380gcacccaatc atgaagggat tcaagccgga gtcgacaggt
tttatcgaac ctgtaagcta 1440cttggaatca atatgagcaa gaaaaagtct
tacataaaca gaacaggtac atttgaattc 1500acaagttttt tctatcgtta
tgggtttgtt gccaatttca gcatggagct tcccagtttt 1560ggggtgtctg
ggatcaacga gtcagcggac atgagtattg gagttactgt catcaaaaac
1620aatatgataa acaatgatct tggtccagca acagctcaaa tggcccttca
gttgttcatc 1680aaagattaca ggtacacgta ccgatgccat agaggtgaca
cacaaataca aacccgaaga 1740tcatttgaaa taaagaaact gtgggagcaa
acccgttcca aagctggact gctggtctcc 1800gacggaggcc caaatttata
caacattaga aatctccaca ttcctgaagt ctgcctaaaa 1860tgggaattga
tggatgagga ttaccagggg cgtttatgca acccactgaa cccatttgtc
1920agccataaag aaattgaatc aatgaacaat gcagtgatga tgccagcaca
tggtccagcc 1980aaaaacatgg agtatgatgc tgttgcaaca acacactcct
ggatccccaa aagaaatcga 2040tccatcttga atacaagtca aagaggagta
cttgaggatg aacaaatgta ccaaaggtgc 2100tgcaatttat ttgaaaaatt
cttccccagc agttcataca gaagaccagt cgggatatcc 2160agtatggtgg
aggctatggt ttccagagcc cgaattgatg cacggattga tttcgaatct
2220ggaaggataa agaaagaaga gttcactgag atcatgaaga tctgttccac
cattgaagag 2280ctcagacggc aaaaatagtg aatttagctt gtccttcatg
aaaaaatgcc ttgtttctac 2340t 2341112341DNAUnknownInfluenza
11agcgaaagca ggtcaattat attcaatatg gaaagaataa aagaactaag aaatctaatg
60tcgcagtctc gcacccgcga gatactcaca aaaaccaccg tggaccatat ggccataatc
120aagaagtaca catcaggaag acaggagaag aacccagcac ttaggatgaa
atggatgatg 180gcaatgaaat atccaattac agcagacaag aggataacgg
aaatgattcc tgagagaaat 240gagcaaggac aaactttatg gagtaaaatg
aatgatgccg gatcagaccg agtgatggta 300tcacctctgg ctgtgacatg
gtggaatagg aatggaccaa taacaaatac agttcattat 360ccaaaaatct
acaaaactta ttttgaaaga gtagaaaggc taaagcatgg aacctttggc
420cctgtccatt ttagaaacca agtcaaaata cgtcggagag ttgacataaa
tcctggtcat 480gcagatctca gtgccaagga ggcacaggat gtaatcatgg
aagttgtttt ccctaacgaa 540gtgggagcca ggatactaac atcggaatcg
caactaacga taaccaaaga gaagaaagaa 600gaactccagg attgcaaaat
ttctcctttg atggttgcat acatgttgga gagagaactg 660gtccgcaaaa
cgagattcct cccagtggct ggtggaacaa gcagtgtgta cattgaagtg
720ttgcatttga ctcaaggaac atgctgggaa cagatgtata ctccaggagg
ggaagtgagg 780aatgatgatg ttgatcaaag cttgattatt gctgctagga
acatagtgag aagagctgca 840gtatcagcag atccactagc atctttattg
gagatgtgcc acagcacaca gattggtgga 900attaggatgg tagacatcct
taggcagaac ccaacagaag agcaagccgt ggatatatgc 960aaggctgcaa
tgggactgag aattagctca tccttcagtt ttggtggatt cacatttaag
1020agaacaagcg gatcatcagt caagagagag gaagaggtgc ttacgggaaa
tcttcaaaca 1080ttgaagataa gagtgcatga gggatatgaa gagttcacaa
tggttgggag aagagcaaca 1140gccatactca gaaaagcaac caggagattg
attcagctga tagtgagtgg gagagacgaa 1200cagtcgattg ccgaagcaat
aattgtggcc atggtatttt cacaagagga ttgtatgata 1260aaagcagtca
gaggtgatct gaatttcgtc aatagggcga atcagcgatt gaatcctatg
1320catcaacttt taagacattt tcagaaggat gcgagagtgc tttttcaaaa
ttggggagtt 1380gaacctatcg acaatgtgat gggaatgatt gggatattgc
ccgacatgac tccaagcatc 1440gagatgtcaa tgagaggagt gagaatcagc
aaaatgggtg tagatgagta ctccagcacg 1500gagagggtag tggtgagcat
tgaccgtttt ttgagaatcc gggaccaacg aggaaatgta 1560ctactgtctc
ccgaggaggt cagtgaaaca cagggaacag agaaactgac aataacttac
1620tcatcgtcaa tgatgtggga gattaatggt cctgaatcag tattggtcaa
tacctatcaa 1680tggatcatca gaaactggga aactgttaaa attcagtggt
cccagaaccc tacaatgcta 1740tacaataaaa tggaatttga accatttcag
tctttagtac ctaaggccat tagaggccaa 1800tacagtgggt ttgtaagaac
tctgttccaa caaatgaggg atgtgcttgg gacatttgat 1860accgcacaga
taataaaact tcttcccttc gcagccgctc caccaaagca aagtagaatg
1920cagttctcct catttactgt gaatgtgagg ggatcaggaa tgagaatact
tgtaaggggc 1980aattctcctg tattcaacta taacaaggcc acgaagagac
tcacagttct cggaaaggat 2040gctggcactt taactgaaga cccagatgaa
ggcacagctg gagtggagtc cgctgttctg 2100aggggattcc tcattctggg
caaagaagac aagagatatg ggccagcact aagcatcaat 2160gaactgagca
accttgcgaa aggagagaag gctaatgtgc taattgggca aggagacgtg
2220gtgttggtaa tgaaacggaa acgggactct agcatactta ctgacagcca
gacagcgacc 2280aaaagaattc ggatggccat caattagtgt cgaatagttt
aaaaacgacc ttgtttctac 2340t 2341121565DNAUnknownInfluenza
12agcaaaagca gggtagataa tcactcactg agtgacatca aaatcatggc gtctcaaggc
60accaaacgat cttacgaaca gatggagact gatggagaac gccagaatgc cactgaaatc
120agagcatccg tcggaaaaat gattggtgga attggacgat tctacatcca
aatgtgcacc 180gaactcaaac tcagtgatta tgagggacgg ttgatccaaa
acagcttaac aatagagaga 240atggtgctct ctgcttttga cgaaaggaga
aataaatacc ttgaagaaca tcccagtgcg 300ggaaaagatc ctaagaaaac
tggaggacct atatacagga gagtaaacgg aaagtggatg 360agagaactca
tcctttatga caaagaagaa ataaggcgaa tctggcgcca agctaataat
420ggtgacgatg caacggctgg tctgactcac atgatgatct ggcattccaa
tttgaatgat 480gcaacttatc agaggacaag agctcttgtt cgcaccggaa
tggatcccag gatgtgctct 540ctgatgcaag gttcaactct ccctaggagg
tctggagccg caggtgctgc agtcaaagga 600gttggaacaa tggtgatgga
attggtcaga atgatcaaac gtgggatcaa tgatcggaac 660ttctggaggg
gtgagaatgg acgaaaaaca agaattgctt atgaaagaat gtgcaacatt
720ctcaaaggga aatttcaaac tgctgcacaa aaagcaatga tggatcaagt
gagagagagc 780cggaacccag ggaatgctga gttcgaagat ctcacttttc
tagcacggtc tgcactcata 840ttgagagggt cggttgctca caagtcctgc
ctgcctgcct gtgtgtatgg acctgccgta 900gccagtgggt acgactttga
aagggaggga tactctctag tcggaataga ccctttcaga 960ctgcttcaaa
acagccaagt gtacagccta atcagaccaa atgagaatcc agcacacaag
1020agtcaactgg tgtggatggc atgccattct gccgcatttg aagatctaag
agtattaagc 1080ttcatcaaag ggacgaaggt gctcccaaga gggaagcttt
ccactagagg agttcaaatt 1140gcttccaatg aaaatatgga gactatggaa
tcaagtacac ttgaactgag aagcaggtac 1200tgggccataa ggaccagaag
tggaggaaac accaatcaac agagggcatc tgcgggccaa 1260atcagcatac
aacctacgtt ctcagtacag agaaatctcc cttttgacag aacaaccatt
1320atggcagcat tcaatgggaa tacagagggg agaacatctg acatgaggac
cgaaatcata 1380aggatgatgg aaagtgcaag accagaagat gtgtctttcc
aggggcgggg agtcttcgag 1440ctctcggacg aaaaggcagc gagcccgatc
gtgccttcct ttgacatgag taatgaagga 1500tcttatttct tcggagacaa
tgcagaggag tacgacaatt aaagaaaaat acccttgttt 1560ctact
1565131027DNAUnknownInfluenza 13agcaaaagca ggtagatatt gaaagatgag
tcttctaacc gaggtcgaaa cgtacgtact 60ctctatcatc ccgtcaggcc ccctcaaagc
cgagatcgca cagagacttg aagatgtctt 120tgcagggaag aacaccgatc
ttgaggttct catggaatgg ctaaagacaa gaccaatcct 180gtcacctctg
actaagggga ttttaggatt tgtgttcacg ctcaccgtgc ccagtgagcg
240aggactgcag cgtagacgct ttgtccaaaa tgcccttaat gggaacgggg
atccaaataa 300catggacaaa gcagttaaac tgtataggaa gctcaagagg
gagataacat tccatggggc 360caaagaaatc tcactcagtt attctgctgg
tgcacttgcc agttgtatgg gcctcatata 420caacaggatg ggggctgtga
ccactgaagt ggcatttggc ctggtatgtg caacctgtga 480acagattgct
gactcccagc atcggtctca taggcaaatg gtgacaacaa ccaatccact
540aatcagacat gagaacagaa tggttttagc cagcactaca gctaaggcta
tggagcaaat 600ggctggatcg agtgagcaag cagcagaggc catggaggtt
gctagtcagg ctagacaaat 660ggtgcaagcg atgagaacca ttgggactca
tcctagctcc agtgctggtc tgaaaaatga 720tcttcttgaa aatttgcagg
cctatcagaa acgaatgggg gtgcagatgc aacggttcaa 780gtgatcctct
cactattgcc gcaaatatca ttgggatctt gcacttgaca ttgtggattc
840ttgatcgtct ttttttcaaa tgcatttacc gtcgctttaa atacggactg
aaaggagggc 900cttctacgga aggagtgcca aagtctatga gggaagaata
tcgaaaggaa cagcagagtg 960ctgtggatgc tgacgatggt cattttgtca
gcatagagct ggagtaaaaa actaccttgt 1020ttctact
102714890DNAUnknownInfluenza 14agcaaaagca gggtgacaaa aacataatgg
atccaaacac tgtgtcaagc tttcaggtag 60attgctttct ttggcatgtc cgcaaacgag
ttgcagacca agaactaggt gatgccccat 120tccttgatcg gcttcgccga
gatcagaaat ccctaagagg aaggggcagt actctcggtc 180tggacatcaa
gacagccaca cgtgctggaa agcagatagt ggagcggatt ctgaaagaag
240aatccgatga ggcacttaaa atgaccatgg cctctgtacc tgcgtcgcgt
tacctaactg 300acatgactct tgaggaaatg tcaagggact ggtccatgct
catacccaag cagaaagtgg 360caggccctct ttgtatcaga atggaccagg
cgatcatgga taagaacatc atactgaaag 420cgaacttcag tgtgattttt
gaccggctgg agactctaat attgctaagg gctttcaccg 480aagagggagc
aattgttggc gaaatttcac cattgccttc tcttccagga catactgctg
540aggatgtcaa aaatgcagtt ggagtcctca tcggaggact tgaatggaat
gataacacag 600ttcgagtctc tgaaactcta cagagattcg cttggagaag
cagtaatgag aatgggagac 660ctccactcac tccaaaacag aaacgagaaa
tggcgggaac aattaggtca gaagtttgaa 720gaaataagat ggttgattga
agaagtgaga cacaaactga agataacaga gaatagtttt 780gagcaaataa
catttatgca agccttacat ctattgcttg aagtggagca agagataaga
840actttctcgt ttcagcttat ttagtactaa aaaacaccct tgtttctact
89015716PRTUnknownInfluenza Virus Reassortment 15Met Glu Asp Phe
Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu1 5 10 15Ala Glu Lys
Ala Met Lys Glu Tyr Gly Glu Asp Pro Lys Ile Glu Thr 20 25 30Asn Lys
Phe Ala Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr 35 40 45Ser
Asp Phe His Phe Ile Asp Glu Arg Gly Glu Ser Ile Ile Val Glu 50 55
60Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu65
70 75 80Gly Arg Asp Arg Ile Met Ala Trp Thr Val Val Asn Ser Ile Cys
Asn 85 90 95Thr Thr Gly Val Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr
Asp Tyr 100 105 110Lys Glu Asn Arg Phe Ile Glu Ile Gly Val Thr Arg
Arg Glu Val His 115 120 125Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile
Lys Ser Glu Lys Thr His 130 135 140Ile His Ile Phe Ser Phe Thr Gly
Glu Glu Met Ala Thr Lys Ala Asp145 150 155 160Tyr Thr Leu Asp Glu
Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe 165 170 175Thr Ile Arg
Gln Glu Met Ala Ser Arg Ser Leu Trp Asp Ser Phe Arg 180 185 190Gln
Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Lys Phe Glu Ile Thr 195 200
205Gly Thr Met Arg Lys Leu Ala Asp Gln Ser Leu Pro Pro Asn Phe Pro
210 215 220Ser Leu Glu Asn Phe Arg Ala Tyr Val Asp Gly Phe Glu Pro
Asn Gly225 230 235 240Cys Ile Glu Gly Lys Leu Ser Gln Met Ser Lys
Glu Val Asn Ala Lys 245 250 255Ile Glu Pro Phe Leu Arg Thr Thr Pro
Arg Pro Leu Arg Leu Pro Asp 260 265 270Gly Pro Leu Cys His Gln Arg
Ser Lys Phe Leu Leu Met Asp Ala Leu 275 280 285Lys Leu Ser Ile Glu
Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu 290 295 300Tyr Asp Ala
Ile Lys Cys Met Lys Thr Phe Phe Gly Trp Lys Glu Pro305 310 315
320Asn Ile Val Lys Pro His Glu Lys Gly Ile Asn Pro Asn Tyr Leu Met
325 330 335Ala Trp Lys Gln Val Leu Ala Glu Leu Gln Asp Ile Glu Asn
Glu Glu 340 345 350Lys Ile Pro Arg Thr Lys Asn Met Lys Arg Thr Ser
Gln Leu Lys Trp 355 360 365Ala Leu Gly Glu Asn Met Ala Pro Glu Lys
Val Asp Phe Asp Asp Cys 370 375 380Lys Asp Val Gly Asp Leu Lys Gln
Tyr Asp Ser Asp Glu Pro Glu Pro385 390 395 400Arg Ser Leu Ala Ser
Trp Val Gln Asn Glu Phe Asn Lys Ala Cys Glu 405 410 415Leu Thr Asp
Ser Ser Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val 420 425 430Ala
Pro Ile Glu His Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala 435 440
445Glu Val Ser His Cys Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr
450 455 460Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp
Asp Phe465 470 475 480Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr
Lys Glu Gly Arg Arg 485 490 495Lys Thr Asn Leu Tyr Gly Phe Ile Ile
Lys Gly Arg Ser His Leu Arg 500 505 510Asn Asp Thr Asp Val Val Asn
Phe Val Ser Met Glu Phe Ser Leu Thr 515 520 525Asp Pro Arg Leu Glu
Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu 530 535 540Ile Gly Asp
Met Leu Leu Arg Thr Ala Ile Gly Gln Val Ser Arg Pro545 550 555
560Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys
565 570 575Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln
Gln Ile 580 585 590Glu Ser Met Ile Glu Ala Glu Ser Ser Val Lys Glu
Lys Asp Met Thr 595 600 605Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr
Trp Pro Ile Gly Glu Ser 610 615 620Pro Arg Gly Val Glu Glu Gly Ser
Ile Gly Lys Val Cys Arg Thr Leu625 630 635 640Leu Ala Lys Ser Val
Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu 645 650 655Gly Phe Ser
Ala Glu Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu 660 665 670Arg
Asp Asn Leu Glu Pro Gly Thr Phe Asp Leu Gly Gly Leu Tyr Glu 675 680
685Ala Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala
690 695 700Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Lys705 710
71516757PRTUnknownInfluenza 16Met Asp Val Asn Pro Thr Leu Leu Phe
Leu Lys Ile Pro Ala Gln Asn1 5 10 15Ala Ile Ser Thr Thr Phe Pro Tyr
Thr Gly Asp Pro Pro Tyr Ser His 20 25 30Gly Thr Gly Thr Gly Tyr Thr
Met Asp Thr Val Asn Arg Thr His Gln 35 40 45Tyr Ser Glu Lys Gly Lys
Trp Thr Thr Asn Thr Glu Thr Gly Ala Pro 50 55 60Gln Leu Asn Pro Ile
Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser65 70 75 80Gly Tyr Ala
Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 85 90 95Glu Ser
His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu 100 105
110Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr
115 120 125Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala
Leu Ala 130 135 140Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr
Ala Asn Glu Ser145 150 155 160Gly Arg Leu Ile Asp Phe Leu Lys Asp
Val Met Glu Ser Met Asn Lys 165 170 175Glu Glu Ile Glu Ile Thr Thr
His Phe Gln Arg Lys Arg Arg Val Arg 180 185 190Asp Asn Met Thr Lys
Lys Met Val Thr Gln Arg Thr Ile Gly Lys Lys 195 200 205Lys Gln Arg
Leu Asn Lys Arg Gly Tyr Leu Ile Arg Ala Leu Thr Leu 210 215 220Asn
Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala225 230
235 240Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val
Glu 245 250 255Thr Leu Ala Arg Ser Ile Cys Glu Lys Leu Glu Gln Ser
Gly Leu Pro 260 265 270Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala
Asn Val Val Arg Lys 275 280 285Met Met Thr Asn Ser Gln Asp Thr Glu
Ile Ser Phe Thr Ile Thr Gly 290 295 300Asp Asn Thr Lys Trp Asn Glu
Asn Gln Asn Pro Arg Met Phe Leu Ala305 310 315 320Met Ile Thr Tyr
Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Ile 325 330 335Leu Ser
Met Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly 340 345
350Lys Gly Tyr Met Phe Glu Ser Lys Arg Met Lys Ile Arg Thr Gln Ile
355 360 365Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn
Glu Ser 370 375 380Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu
Ile Asp Gly Thr385 390 395 400Ala Ser Leu Ser Pro Gly Met Met Met
Gly Met Phe Asn Met Leu Ser 405 410 415Thr Val Leu Gly Val Ser Ile
Leu Asn Leu Gly Gln Lys Lys Tyr Thr 420 425 430Lys Thr Ile Tyr Trp
Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala 435 440 445Leu Ile Val
Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp 450 455 460Arg
Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys465 470
475 480Lys Ser Tyr Ile Asn Lys Thr Gly Thr Phe Glu Phe Thr Ser Phe
Phe 485 490 495Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu
Pro Ser Phe 500 505 510Gly Val Ser Gly Val Asn Glu Ser Ala Asp Met
Ser Ile Gly Val Thr 515 520 525Val Ile Lys Asn Asn Met Ile Asn Asn
Asp Leu Gly Pro Ala Thr Ala 530 535 540Gln Met Ala Leu Gln Leu Phe
Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg545 550 555 560Cys His Arg Gly
Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu 565 570 575Lys Lys
Leu Trp Asp Gln Thr Gln Ser Lys Val Gly Leu Leu Val Ser 580 585
590Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu
595 600 605Val Cys Leu Lys Trp Glu Leu Met Asp Asp Asp Tyr Arg Gly
Arg Leu 610 615 620Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu
Ile Asp Ser Val625 630 635 640Asn Asn Ala Val Val Met Pro Ala His
Gly Pro Ala Lys Ser Met Glu 645 650 655Tyr Asp Ala Val Ala Thr Thr
His Ser Trp Ile Pro Lys Arg Asn Arg
660 665 670Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu
Gln Met 675 680 685Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe
Pro Ser Ser Ser 690 695 700Tyr Arg Arg Pro Val Gly Ile Ser Ser Met
Val Glu Ala Met Val Ser705 710 715 720Arg Ala Arg Ile Asp Ala Arg
Val Asp Phe Glu Ser Gly Arg Ile Lys 725 730 735Lys Glu Glu Phe Ser
Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu 740 745 750Leu Arg Arg
Gln Lys 75517759PRTUnknownInfluenza 17Met Glu Arg Ile Lys Glu Leu
Arg Asp Leu Met Ser Gln Ser Arg Thr1 5 10 15Arg Glu Ile Leu Thr Lys
Thr Thr Val Asp His Met Ala Ile Ile Lys 20 25 30Lys Tyr Thr Ser Gly
Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys 35 40 45Trp Met Met Ala
Met Arg Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met 50 55 60Asp Met Ile
Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys65 70 75 80Thr
Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val 85 90
95Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Val His Tyr Pro
100 105 110Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys
His Gly 115 120 125Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys
Ile Arg Arg Arg 130 135 140Val Asp Thr Asn Pro Gly His Ala Asp Leu
Ser Ala Lys Glu Ala Gln145 150 155 160Asp Val Ile Met Glu Val Val
Phe Pro Asn Glu Val Gly Ala Arg Ile 165 170 175Leu Thr Ser Glu Ser
Gln Leu Ala Ile Thr Lys Glu Lys Lys Glu Glu 180 185 190Leu Gln Asp
Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu 195 200 205Arg
Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr 210 215
220Gly Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys
Trp225 230 235 240Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn
Asp Asp Val Asp 245 250 255Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile
Val Arg Arg Ala Ala Val 260 265 270Ser Ala Asp Pro Leu Ala Ser Leu
Leu Glu Met Cys His Ser Thr Gln 275 280 285Ile Gly Gly Val Arg Met
Val Asp Ile Leu Arg Gln Asn Pro Thr Glu 290 295 300Glu Gln Ala Val
Asp Ile Cys Lys Ala Ala Ile Gly Leu Arg Ile Ser305 310 315 320Ser
Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser 325 330
335Ser Val Lys Lys Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu
340 345 350Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val
Gly Arg 355 360 365Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg
Leu Ile Gln Leu 370 375 380Ile Val Ser Gly Arg Asp Glu Gln Ser Ile
Ala Glu Ala Ile Ile Val385 390 395 400Ala Met Val Phe Ser Gln Glu
Asp Cys Met Ile Lys Ala Val Arg Gly 405 410 415Asp Leu Asn Phe Val
Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His 420 425 430Gln Leu Leu
Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn 435 440 445Trp
Gly Ile Glu Ser Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu 450 455
460Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Ile Arg
Val465 470 475 480Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu
Arg Val Val Val 485 490 495Ser Ile Asp Arg Phe Leu Arg Val Arg Asp
Gln Arg Gly Asn Val Leu 500 505 510Leu Ser Pro Glu Glu Val Ser Glu
Thr Gln Gly Thr Glu Lys Leu Thr 515 520 525Ile Thr Tyr Ser Ser Ser
Met Met Trp Glu Ile Asn Gly Pro Glu Ser 530 535 540Val Leu Val Asn
Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Ile Val545 550 555 560Lys
Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Met Glu 565 570
575Phe Glu Pro Phe Gln Ser Leu Val Pro Lys Ala Thr Arg Ser Arg Tyr
580 585 590Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val
Leu Gly 595 600 605Thr Phe Asp Thr Val Gln Ile Ile Lys Leu Leu Pro
Phe Ala Ala Ala 610 615 620Pro Pro Glu Gln Ser Arg Met Gln Phe Ser
Ser Leu Thr Val Asn Val625 630 635 640Arg Gly Ser Gly Leu Arg Ile
Leu Val Arg Gly Asn Ser Pro Val Phe 645 650 655Asn Tyr Asn Lys Ala
Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala 660 665 670Gly Ala Leu
Thr Glu Asp Pro Asp Glu Gly Thr Ser Gly Val Glu Ser 675 680 685Ala
Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asp Lys Arg Tyr 690 695
700Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Asn Leu Ala Lys Gly
Glu705 710 715 720Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val
Leu Val Met Lys 725 730 735Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp
Ser Gln Thr Ala Thr Lys 740 745 750Arg Ile Arg Met Ala Ile Asn
75518319PRTUnknownInfluenza 18Met Ala Ser Gln Gly Thr Lys Arg Ser
Tyr Glu Gln Met Glu Thr Gly1 5 10 15Gly Glu Arg Gln Asp Ala Thr Glu
Ile Arg Ala Ser Val Gly Arg Met 20 25 30Ile Gly Gly Ile Gly Arg Phe
Tyr Ile Gln Met Cys Thr Glu Leu Lys 35 40 45Leu Ser Asp Tyr Asp Gly
Arg Leu Ile Gln Asn Ser Ile Thr Ile Glu 50 55 60Arg Met Val Leu Ser
Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu65 70 75 80Glu His Pro
Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile 85 90 95Tyr Arg
Arg Val Asp Gly Lys Trp Met Arg Glu Leu Ile Leu Tyr Asp 100 105
110Lys Glu Glu Ile Arg Arg Val Trp Arg Gln Ala Asn Asn Gly Glu Asp
115 120 125Ala Thr Ala Gly Leu Thr His Ile Met Ile Trp His Ser Asn
Leu Asn 130 135 140Asp Ala Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg
Thr Gly Met Asp145 150 155 160Pro Arg Met Cys Ser Leu Met Gln Gly
Ser Thr Leu Pro Arg Arg Ser 165 170 175Gly Ala Ala Gly Ala Ala Val
Lys Gly Val Gly Thr Ile Ala Met Glu 180 185 190Leu Ile Arg Met Ile
Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg 195 200 205Gly Glu Asn
Gly Arg Arg Thr Arg Val Ala Tyr Glu Arg Met Cys Asn 210 215 220Ile
Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp225 230
235 240Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp
Leu 245 250 255Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser
Val Ala His 260 265 270Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu
Ala Val Ala Ser Gly 275 280 285His Asp Phe Glu Arg Glu Gly Tyr Ser
Leu Val Gly Ile Asp Pro Phe 290 295 300Lys Leu Leu Gln Asn Ser Gln
Val Val Ser Leu Met Arg Pro Asn305 310 31519252PRTUnknownInfluenza
19Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro1
5 10 15Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Ser Val
Phe 20 25 30Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu
Lys Thr 35 40 45Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly
Phe Val Phe 50 55 60Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg
Arg Arg Phe Val65 70 75 80Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro
Asn Asn Met Asp Arg Ala 85 90 95Val Lys Leu Tyr Lys Lys Leu Lys Arg
Glu Ile Thr Phe His Gly Ala 100 105 110Lys Glu Val Ser Leu Ser Tyr
Ser Thr Gly Ala Leu Ala Ser Cys Met 115 120 125Gly Leu Ile Tyr Asn
Arg Met Gly Thr Val Thr Thr Glu Ala Ala Phe 130 135 140Gly Leu Val
Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg145 150 155
160Ser His Arg Gln Met Ala Thr Thr Thr Asn Pro Leu Ile Arg His Glu
165 170 175Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu
Gln Met 180 185 190Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu
Val Ala Asn Gln 195 200 205Thr Arg Gln Met Val His Ala Met Arg Thr
Ile Gly Thr His Pro Ser 210 215 220Ser Ser Ala Gly Leu Lys Asp Asp
Leu Leu Glu Asn Leu Gln Ala Tyr225 230 235 240Gln Lys Arg Met Gly
Val Gln Met Gln Arg Phe Lys 245 25020219PRTUnknownInfluenza 20Met
Asp Ser Asn Thr Met Ser Ser Phe Gln Val Asp Cys Phe Leu Trp1 5 10
15His Ile Arg Lys Arg Phe Ala Asp Asn Gly Leu Gly Asp Ala Pro Phe
20 25 30Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Lys Gly Arg Gly
Asn 35 40 45Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Leu Val Gly Lys
Gln Ile 50 55 60Val Glu Trp Ile Leu Lys Glu Glu Ser Ser Glu Thr Leu
Arg Met Thr65 70 75 80Ile Ala Ser Val Pro Thr Ser Arg Tyr Leu Ser
Asp Met Thr Leu Glu 85 90 95Glu Met Ser Arg Asp Trp Phe Met Leu Met
Pro Arg Gln Lys Ile Ile 100 105 110Gly Pro Leu Cys Val Arg Leu Asp
Gln Ala Ile Met Glu Lys Asn Ile 115 120 125Val Leu Lys Ala Asn Phe
Ser Val Ile Phe Asn Arg Leu Glu Thr Leu 130 135 140Ile Leu Leu Arg
Ala Phe Thr Glu Glu Gly Ala Ile Val Gly Glu Ile145 150 155 160Ser
Pro Leu Pro Ser Leu Pro Gly His Thr Tyr Glu Asp Val Lys Asn 165 170
175Ala Val Gly Val Leu Ile Gly Gly Leu Glu Trp Asn Gly Asn Thr Val
180 185 190Arg Val Ser Glu Asn Ile Gln Arg Phe Ala Trp Arg Asn Cys
Asp Glu 195 200 205Asn Gly Arg Pro Ser Leu Pro Pro Glu Gln Lys 210
215212151DNAUnknownInfluenza 21atggaagact ttgtgcgaca atgcttcaat
ccaatgatcg tcgagcttgc ggaaaaggca 60atgaaagaat atggggaaga tccgaaaatc
gaaactaaca agtttgctgc aatatgcaca 120catttggaag tttgtttcat
gtattcggat ttccatttca tcgacgaacg gggtgaatca 180ataattgtag
aatctggtga cccgaatgca ctattgaagc accgatttga gataattgaa
240ggaagagacc gaatcatggc ctggacagtg gtgaacagta tatgtaacac
aacaggggta 300gagaagccta aatttcttcc tgatttgtat gattacaaag
agaaccggtt cattgaaatt 360ggagtaacac ggagggaagt ccacatatat
tacctagaga aagccaacaa aataaaatct 420gagaagacac acattcacat
cttttcattc actggagagg agatggccac caaagcggac 480tacacccttg
acgaagagag cagggcaaga atcaaaacta ggcttttcac tataagacaa
540gaaatggcca gtaggagtct atgggattcc tttcgtcagt ccgaaagagg
cgaagagaca 600attgaagaaa aatttgagat tacaggaact atgcgcaagc
ttgccgacca aagtctccca 660ccgaacttcc ccagccttga aaactttaga
gcctatgtag atggattcga gccgaacggc 720tgcattgagg gcaagctttc
ccaaatgtca aaagaagtga acgccaaaat tgaaccattc 780ttgaggacga
caccacgccc cctcagattg cctgatgggc ctctttgcca tcagcggtca
840aagttcctgc tgatggatgc tctgaaatta agtattgaag acccgagtca
cgagggggag 900ggaataccac tatatgatgc aatcaaatgc atgaagacat
tctttggctg gaaagagcct 960aacatagtca aaccacatga gaaaggcata
aatcccaatt acctcatggc ttggaagcag 1020gtgctagcag agctacagga
cattgaaaat gaagagaaga tcccaaggac aaagaacatg 1080aagagaacaa
gccaattgaa gtgggcactc ggtgaaaata tggcaccaga aaaagtagac
1140tttgatgact gcaaagatgt tggagacctt aaacagtatg acagtgatga
gccagagccc 1200agatctctag caagctgggt ccaaaatgaa ttcaataagg
catgtgaatt gactgattca 1260agctggatag aacttgatga aataggagaa
gatgttgccc cgattgaaca tatcgcaagc 1320atgaggagga actattttac
agcagaagtg tcccactgca gggctactga atacataatg 1380aagggagtgt
acataaatac ggccttgctc aatgcatcct gtgcagccat ggatgacttt
1440cagctgatcc caatgataag caaatgtagg accaaagaag gaagacggaa
aacaaacctg 1500tatgggttca ttataaaagg aaggtctcat ttgagaaatg
atactgatgt ggtgaacttt 1560gtaagtatgg agttctcact cactgacccg
agactggagc cacacaaatg ggaaaaatac 1620tgtgttcttg aaataggaga
catgctcttg aggactgcga taggccaagt gtcgaggccc 1680atgttcctat
atgtgagaac caatggaacc tccaagatca agatgaaatg gggcatggaa
1740atgaggcgct gccttcttca gtctcttcag cagattgaga gcatgattga
ggccgagtct 1800tctgtcaaag agaaagacat gaccaaggaa ttctttgaaa
acaaatcgga aacatggcca 1860atcggagagt cacccagggg agtggaggaa
ggctctattg ggaaagtgtg caggacctta 1920ctggcaaaat ctgtattcaa
cagtctatat gcgtctccac aacttgaggg gttttcggct 1980gaatctagaa
aattgcttct cattgttcag gcacttaggg acaacctgga acctggaacc
2040ttcgatcttg gggggctata tgaagcaatc gaggagtgcc tgattaatga
tccctgggtt 2100ttgcttaatg catcttggtt caactccttc ctcacacatg
cactgaagta g 2151222341DNAUnknownInfluenza 22agcgaaagca ggcaaaccat
ttgaatggat gtcaatccga ctctactttt cctaaaaatt 60ccagcgcaaa atgccataag
caccacattc ccttatactg gagatcctcc atacagccat 120ggaacaggaa
caggatacac catggacaca gtaaacagaa cacaccaata ctcagaaaag
180ggaaagtgga cgacaaacac agagactggt gcaccccagc tcaacccgat
tgatggacca 240ctacctgagg ataatgaacc aagtgggtat gcacaaacag
actgtgttct agaggctatg 300gctttccttg aagaatccca cccaggaata
tttgagaatt catgccttga aacaatggaa 360gttgttcaac aaacaagggt
agataaacta actcaaggtc gccagactta tgattggaca 420ttaaacagaa
atcaaccggc agcaactgca ttggccaaca ccatagaagt ctttagatcg
480aatggcctaa cagctaatga gtcaggaagg ctaatagatt tcttaaagga
tgtaatggaa 540tcaatgaaca aagaggaaat agagataaca acccactttc
aaagaaaaag gagagtaaga 600gacaacatga ccaagaagat ggtcacgcaa
agaacaatag ggaagaaaaa acaaagactg 660aataagagag gctatctaat
aagagcactg acattaaata cgatgaccaa agatgcagag 720agaggcaagt
taaaaagaag ggctatcgca acacctggga tgcagattag aggtttcgta
780tactttgttg aaactttagc taggagcatt tgcgaaaagc ttgaacagtc
tgggctccca 840gtagggggca atgaaaagaa ggccaaactg gcaaatgttg
tgagaaagat gatgactaat 900tcacaagaca cagagatttc tttcacaatc
actggggaca acactaagtg gaatgaaaat 960caaaatcctc gaatgttcct
ggcgatgatt acatatatca ccagaaatca acccgagtgg 1020ttcagaaaca
tcctgagcat ggcacccata atgttctcaa acaaaatggc aagactaggg
1080aaagggtaca tgttcgagag taaaagaatg aagattcgaa cacaaatacc
agcagaaatg 1140ctagcaagca ttgacctgaa gtacttcaat gaatcaacaa
agaagaaaat tgagaaaata 1200aggcctcttc taatagatgg cacagcatca
ctgagtcctg ggatgatgat gggcatgttc 1260aacatgctaa gtacggtctt
gggagtctcg atactgaatc ttggacaaaa gaaatacacc 1320aagacaatat
actggtggga tgggctccaa tcatccgacg attttgctct catagtgaat
1380gcaccaaacc atgagggaat acaagcagga gtggacagat tctacaggac
ctgcaagtta 1440gtgggaatca acatgagcaa aaagaagtcc tatataaata
agacagggac atttgaattc 1500acaagctttt tttatcgcta tggatttgtg
gctaatttta gcatggagct acccagcttt 1560ggagtgtctg gagtaaatga
atcagctgac atgagtattg gagtaacagt gataaagaac 1620aacatgataa
acaatgacct tggacctgca acggcccaga tggctcttca attgttcatc
1680aaagactaca gatacacata taggtgccat aggggagaca cacaaattca
gacaagaaga 1740tcatttgagt taaagaagct gtgggatcaa acccaatcaa
aggtagggct attagtatca 1800gatggaggac caaacttata caatatacgg
aatcttcaca ttcctgaagt ctgcttaaaa 1860tgggagctaa tggatgatga
ttatcgggga agactttgta atcccctgaa tccctttgtc 1920agtcataaag
agattgattc tgtaaacaat gctgtggtaa tgccagccca tggtccagcc
1980aaaagcatgg aatatgatgc cgttgcaact acacattcct ggattcccaa
gaggaatcgt 2040tctattctca acacaagcca aaggggaatt cttgaggatg
aacagatgta ccagaagtgc 2100tgcaatctat tcgagaaatt tttccctagc
agttcatata ggagaccggt tggaatttct 2160agcatggtgg aggccatggt
gtctagggcc cggattgatg ccagggtcga cttcgagtct 2220ggacggatca
agaaagaaga gttctctgag atcatgaaga tctgttccac cattgaagaa
2280ctcagacggc aaaaataatg aatttaactt gtccttcatg aaaaaatgcc
ttgtttctac 2340t 2341232280DNAUnknownInfluenza 23atggagagaa
taaaagaact gagagatcta atgtcgcagt cccgcactcg cgagatactc 60actaagacca
ctgtggacca tatggccata atcaaaaagt acacatcagg aaggcaagag
120aagaaccccg cactcagaat gaagtggatg atggcaatga gatacccaat
tacagcagac 180aagagaataa tggacatgat tccagagagg aatgaacaag
gacaaaccct ctggagcaaa 240acaaacgatg ctggatcaga ccgagtgatg
gtatcacctc tggccgtaac atggtggaat 300aggaatggcc caacaacaag
tacagttcat taccctaagg tatataaaac ttatttcgaa
360aaggtcgaaa ggttgaaaca tggtaccttc ggccctgtcc acttcagaaa
tcaagttaaa 420ataaggagga gagttgatac aaaccctggc catgcagatc
tcagtgccaa ggaggcacag 480gatgtgatta tggaagttgt tttcccaaat
gaagtggggg caagaatact gacatcagag 540tcacagctgg caataacaaa
agagaagaaa gaagagctcc aggattgtaa aattgctccc 600ttgatggtgg
cgtacatgct agaaagagaa ttggtccgta aaacaaggtt tctcccagta
660gccggcggaa caggcagtgt ttatattgaa gtgttgcact taacccaagg
gacgtgctgg 720gagcagatgt acactccagg aggagaagtg agaaatgatg
atgttgacca aagtttgatt 780atcgctgcta gaaacatagt aagaagagca
gcagtgtcag cagacccatt agcatctctc 840ttggaaatgt gccacagcac
acagattgga ggagtaagga tggtggacat ccttagacag 900aatccaactg
aggaacaagc cgtagacata tgcaaggcag caatagggtt gaggattagc
960tcatctttca gttttggtgg gttcactttc aaaaggacaa gcggatcatc
agtcaagaaa 1020gaagaagaag tgctaacggg caacctccaa acactgaaaa
taagagtaca tgaagggtat 1080gaagaattca caatggttgg gagaagagca
acagctattc tcagaaaggc aaccaggaga 1140ttgatccagt tgatagtaag
cgggagagac gagcagtcaa ttgctgaggc aataattgtg 1200gccatggtat
tctcacagga ggattgcatg atcaaggcag ttaggggcga tctgaacttt
1260gtcaataggg caaaccagcg actgaacccc atgcaccaac tcttgaggca
tttccaaaaa 1320gatgcaaaag tgcttttcca gaactgggga attgaatcca
tcgacaatgt gatgggaatg 1380atcggaatac tgcccgacat gaccccaagc
acggagatgt cgctgagagg gataagagtc 1440agcaaaatgg gagtagatga
atactccagc acggagagag tggtagtgag tattgaccga 1500tttttaaggg
ttagagatca aagagggaac gtactattgt ctcccgaaga agtcagtgaa
1560acgcaaggaa ctgagaagtt gacaataact tattcgtcat caatgatgtg
ggagatcaat 1620ggccctgagt cagtgctagt caacacttat caatggataa
tcaggaactg ggaaattgtg 1680aaaattcaat ggtcacaaga tcccacaatg
ttatacaaca aaatggaatt tgaaccattt 1740cagtctcttg tccctaaggc
aaccagaagc cggtacagtg gattcgtaag gacactgttc 1800cagcaaatgc
gggatgtgct tgggacattt gacactgtcc aaataataaa acttctcccc
1860tttgctgctg ccccaccaga acagagtagg atgcaatttt cctcattgac
tgtgaatgtg 1920agaggatcag ggttgaggat actggtaaga ggcaattctc
cagtattcaa ttacaacaag 1980gcaaccaaac gacttacagt tcttggaaag
gatgcaggtg cattgactga agatccagat 2040gaaggcacat ctggggtgga
gtctgctgtc ctgagaggat ttctcatttt gggcaaagaa 2100gacaagagat
atggcccagc attaagcatc aatgaactga gcaatcttgc aaaaggagag
2160aaggctaatg tgctaattgg gcaaggggac gtagtgttgg taatgaaacg
aaaacgggac 2220tctagcatac ttactgacag ccagacagcg accaaaagaa
ttcggatggc catcaattag 228024958DNAUnknownInfluenza 24atggcgtctc
aaggcaccaa acgatcatat gaacaaatgg agactggtgg ggagcgccag 60gatgccacag
aaatcagagc atctgtcgga agaatgattg gtggaatcgg gagattctac
120atccaaatgt gcactgaact caaactcagt gattatgatg gacgactaat
ccagaatagc 180ataacaatag agaggatggt gctttctgct tttgatgaga
gaagaaataa atacctagaa 240gagcatccca gtgctgggaa ggaccctaag
aaaacaggag gacccatata tagaagagta 300gacggaaagt ggatgagaga
actcatcctt tatgacaaag aagaaataag gagagtttgg 360cgccaagcaa
acaatggcga agatgcaaca gcaggtctta ctcatatcat gatttggcat
420tccaacctga atgatgccac atatcagaga acaagagcgc ttgttcgcac
cggaatggat 480cccagaatgt gctctctaat gcaaggttca acacttccca
gaaggtctgg tgccgcaggt 540gctgcggtga aaggagttgg aacaatagca
atggagttaa tcagaatgat caaacgtgga 600atcaatgacc gaaatttctg
gaggggtgaa aatggacgaa ggacaagggt tgcttatgaa 660agaatgtgca
atatcctcaa aggaaaattt caaacagctg cccagagggc aatgatggat
720caagtaagag aaagtcgaaa cccaggaaac gctgagattg aagacctcat
tttcctggca 780cggtcagcac tcattctgag gggatcagtt gcacataaat
cctgcctgcc tgcttgtgtg 840tatgggcttg cagtagcaag tgggcatgac
tttgaaaggg aagggtactc actggtcggg 900atagacccat tcaaattact
ccaaaacagc caagtggtca gcctgatgag accaaatg
95825982DNAUnknownInfluenza 25atgagtcttc taaccgaggt cgaaacgtac
gttctttcta tcatcccgtc aggccccctc 60aaagccgaga tcgcgcagag actggaaagt
gtctttgcag gaaagaacac agatcttgag 120gctctcatgg aatggctaaa
gacaagacca atcttgtcac ctctgactaa gggaatttta 180ggatttgtgt
tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc
240caaaatgccc taaatgggaa tggggacccg aacaacatgg atagagcagt
taaactatac 300aagaagctca aaagagaaat aacgttccat ggggccaagg
aggtgtcact aagctattca 360actggtgcac ttgccagttg catgggcctc
atatacaaca ggatgggaac agtgaccaca 420gaagctgctt ttggtctagt
gtgtgccact tgtgaacaga ttgctgattc acagcatcgg 480tctcacagac
agatggctac taccaccaat ccactaatca ggcatgaaaa cagaatggtg
540ctggctagca ctacggcaaa ggctatggaa cagatggctg gatcgagtga
acaggcagcg 600gaggccatgg aggttgctaa tcagactagg cagatggtac
atgcaatgag aactattggg 660actcatccta gctccagtgc tggtctgaaa
gatgaccttc ttgaaaattt gcaggcctac 720cagaagcgaa tgggagtgca
gatgcagcga ttcaagtgat cctctcgtca ttgcagcaaa 780tatcattggg
atcttgcacc tgatattgtg gattactgat cgtctttttt tcaaatgtat
840ttatcgtcgc tttaaatacg gtttgaaaag agggccttct acggaaggag
tgcctgagtc 900catgagggaa gaatatcaac aggaacagca gagtgctgtg
gatgttgacg atggtcattt 960tgtcaacata gagctagagt aa
98226865DNAUnknownInfluenza 26atggactcca acaccatgtc aagctttcag
gtagactgtt tcctttggca tatccgcaag 60cgatttgcag acaatggatt gggtgatgcc
ccattccttg atcggctccg ccgagatcaa 120aagtccttaa aaggaagagg
caacaccctt ggcctcgata tcgaaacagc cactcttgtt 180gggaaacaaa
tcgtggaatg gatcttgaaa gaggaatcca gcgagacact tagaatgaca
240attgcatctg tacctacttc gcgctacctt tctgacatga ccctcgagga
aatgtcacga 300gactggttca tgctcatgcc taggcaaaag ataataggcc
ctctttgcgt gcgattggac 360caggcgatca tggaaaagaa catagtactg
aaagcgaact tcagtgtaat ctttaaccga 420ttagagacct tgatactact
aagggctttc actgaggagg gagcaatagt tggagaaatt 480tcaccattac
cttctcttcc aggacatact tatgaggatg tcaaaaatgc agttggggtc
540ctcatcggag gacttgaatg gaatggtaac acggttcgag tctctgaaaa
tatacagaga 600ttcgcttgga gaaactgtga tgagaatggg agaccttcac
tacctccaga gcagaaatga 660aaagtggcga gagcaattgg gacagaaatt
tgaggaaata aggtggttaa ttgaagaaat 720gcggcacaga ttgaaagcga
cagagaatag tttcgaacaa ataacattta tgcaagcctt 780acaactactg
cttgaagtag aacaagagat aagagctttc tcgtttcagc ttatttaatg
840ataaaaaaca cccttgtttc tactg 86527758PRTUnknownInfluenza 27Met
Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Ile Pro Ala Gln Asn1 5 10
15Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His
20 25 30Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His
Gln 35 40 45Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Thr Gly
Ala Pro 50 55 60Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn
Glu Pro Ser65 70 75 80Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala
Met Ala Phe Leu Glu 85 90 95Glu Ser His Pro Gly Ile Phe Glu Asn Ser
Cys Leu Glu Thr Met Glu 100 105 110Val Val Gln Gln Thr Arg Val Asp
Arg Leu Thr Gln Gly Arg Gln Thr 115 120 125Tyr Asp Trp Thr Leu Asn
Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala 130 135 140Asn Thr Ile Glu
Val Phe Arg Ser Asn Gly Leu Thr Ala Asn Glu Ser145 150 155 160Gly
Arg Leu Ile Asp Phe Leu Lys Asp Val Met Glu Ser Met Asp Lys 165 170
175Glu Glu Ile Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg
180 185 190Asp Asn Met Thr Lys Lys Met Val Thr Gln Arg Thr Ile Gly
Lys Lys 195 200 205Lys Gln Arg Val Asn Lys Arg Ser Tyr Leu Ile Arg
Ala Leu Thr Leu 210 215 220Asn Thr Met Thr Lys Asp Ala Glu Arg Gly
Lys Leu Lys Arg Arg Ala225 230 235 240Ile Ala Thr Pro Gly Met Gln
Ile Arg Gly Phe Val Tyr Phe Val Glu 245 250 255Thr Leu Ala Arg Ser
Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro 260 265 270Val Gly Gly
Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys 275 280 285Met
Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly 290 295
300Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu
Ala305 310 315 320Met Ile Thr Tyr Ile Thr Lys Asn Gln Pro Glu Trp
Phe Arg Asn Ile 325 330 335Leu Ser Ile Ala Pro Ile Met Phe Ser Asn
Lys Met Ala Arg Leu Gly 340 345 350Lys Gly Tyr Met Phe Glu Ser Lys
Arg Met Lys Leu Arg Thr Gln Ile 355 360 365Pro Ala Glu Met Leu Ala
Ser Ile Asp Leu Lys Tyr Phe Asn Glu Ser 370 375 380Thr Arg Lys Lys
Ile Glu Lys Ile Arg Pro Leu Leu Ile Asp Gly Thr385 390 395 400Ala
Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser 405 410
415Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Lys Lys Tyr Thr
420 425 430Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp
Phe Ala 435 440 445Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln
Ala Gly Val Asp 450 455 460Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly
Ile Asn Met Ser Lys Lys465 470 475 480Lys Ser Tyr Ile Asn Arg Thr
Gly Thr Phe Glu Phe Thr Ser Phe Phe 485 490 495Tyr Arg Tyr Gly Phe
Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe 500 505 510Gly Val Ser
Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr 515 520 525Val
Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala 530 535
540Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr
Arg545 550 555 560Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg
Ser Phe Glu Leu 565 570 575Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys
Ala Gly Leu Leu Val Ser 580 585 590Asp Gly Gly Pro Asn Leu Tyr Asn
Ile Arg Asn Leu His Ile Pro Glu 595 600 605Val Cys Leu Lys Trp Glu
Leu Met Asp Glu Asp Tyr Gln Gly Arg Leu 610 615 620Cys Asn Pro Leu
Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val625 630 635 640Asn
Asn Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu 645 650
655Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg
660 665 670Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu
Gln Met 675 680 685Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe
Pro Ser Ser Ser 690 695 700Tyr Arg Arg Pro Val Gly Ile Ser Ser Met
Val Glu Ala Met Val Ser705 710 715 720Arg Ala Arg Ile Asp Ala Arg
Ile Asp Phe Glu Ser Gly Arg Ile Lys 725 730 735Lys Glu Glu Phe Ser
Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu 740 745 750Leu Arg Arg
Gln Lys Gln 75528716PRTUnknownInfluenza 28Met Glu Asp Phe Val Arg
Gln Cys Phe Asn Pro Met Ile Val Glu Leu1 5 10 15Ala Glu Lys Thr Met
Lys Glu Tyr Gly Glu Asp Leu Lys Ile Glu Thr 20 25 30Asn Lys Phe Ala
Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr 35 40 45Ser Asp Phe
His Phe Ile Asn Glu Gln Gly Glu Ser Ile Ile Val Glu 50 55 60Leu Gly
Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu65 70 75
80Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn
85 90 95Thr Thr Gly Ala Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp
Tyr 100 105 110Lys Glu Asn Arg Phe Ile Glu Ile Gly Val Thr Arg Arg
Glu Val His 115 120 125Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys
Ser Glu Lys Thr His 130 135 140Ile His Ile Phe Ser Phe Thr Gly Glu
Glu Met Ala Thr Lys Ala Asp145 150 155 160Tyr Thr Leu Asp Glu Glu
Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe 165 170 175Thr Ile Arg Gln
Glu Met Ala Ser Arg Gly Leu Trp Asp Ser Phe Arg 180 185 190Gln Ser
Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr 195 200
205Gly Thr Met Arg Lys Leu Ala Asp Gln Ser Leu Pro Pro Asn Phe Ser
210 215 220Ser Leu Glu Asn Phe Arg Ala Tyr Val Asp Gly Phe Glu Pro
Asn Gly225 230 235 240Tyr Ile Glu Gly Lys Leu Ser Gln Met Ser Lys
Glu Val Asn Ala Arg 245 250 255Ile Glu Pro Phe Leu Lys Thr Thr Pro
Arg Pro Leu Arg Leu Pro Asn 260 265 270Gly Pro Pro Cys Ser Gln Arg
Ser Lys Phe Leu Leu Met Asp Ala Leu 275 280 285Lys Leu Ser Ile Glu
Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu 290 295 300Tyr Asp Ala
Ile Lys Cys Met Arg Thr Phe Phe Gly Trp Lys Glu Pro305 310 315
320Asn Val Val Lys Pro His Glu Lys Gly Ile Asn Pro Asn Tyr Leu Leu
325 330 335Ser Trp Lys Gln Val Leu Ala Glu Leu Gln Asp Ile Glu Asn
Glu Glu 340 345 350Lys Ile Pro Lys Thr Lys Asn Met Lys Lys Thr Ser
Gln Leu Lys Trp 355 360 365Ala Leu Gly Glu Asn Met Ala Pro Glu Lys
Val Asp Phe Asp Asp Cys 370 375 380Lys Asp Val Gly Asp Leu Lys Gln
Tyr Asp Ser Asp Glu Pro Glu Leu385 390 395 400Arg Ser Leu Ala Ser
Trp Ile Gln Asn Glu Phe Asn Lys Ala Cys Glu 405 410 415Leu Thr Asp
Ser Ser Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val 420 425 430Ala
Pro Ile Glu His Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ser 435 440
445Glu Val Ser His Cys Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr
450 455 460Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp
Asp Phe465 470 475 480Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr
Lys Glu Gly Arg Arg 485 490 495Lys Thr Asn Leu Tyr Gly Phe Ile Ile
Lys Gly Arg Ser His Leu Arg 500 505 510Asn Asp Thr Asp Val Val Asn
Phe Val Ser Met Glu Phe Ser Leu Thr 515 520 525Asp Pro Arg Leu Glu
Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu 530 535 540Ile Gly Asp
Met Leu Ile Arg Ser Ala Ile Gly Gln Val Ser Arg Pro545 550 555
560Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys
565 570 575Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln
Gln Ile 580 585 590Glu Ser Met Ile Glu Ala Glu Ser Ser Val Lys Glu
Lys Asp Met Thr 595 600 605Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr
Trp Pro Ile Gly Glu Ser 610 615 620Pro Lys Gly Val Glu Glu Ser Ser
Ile Gly Lys Val Cys Arg Thr Leu625 630 635 640Leu Ala Lys Ser Val
Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu 645 650 655Gly Phe Ser
Ala Glu Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu 660 665 670Arg
Asp Asn Leu Glu Pro Gly Thr Phe Asp Leu Gly Gly Leu Tyr Glu 675 680
685Ala Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala
690 695 700Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Ser705 710
71529326PRTUnknownInfluenza 29Met Ala Ser Gln Gly Thr Lys Arg Ser
Tyr Glu Gln Met Glu Thr Asp1 5 10 15Gly Glu Arg Gln Asn Ala Thr Glu
Ile Arg Ala Ser Val Gly Lys Met 20 25 30Ile Gly Gly Ile Gly Arg Phe
Tyr Ile Gln Met Cys Thr Glu Leu Lys 35 40 45Leu Ser Asp Tyr Glu Gly
Arg Leu Ile Gln Asn Ser Leu Thr Ile Glu 50 55 60Arg Met Val Leu Ser
Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu65 70 75 80Glu His Pro
Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile 85 90 95Tyr Arg
Arg Val Asn Gly Lys Trp Met Arg Glu Leu Ile Leu Tyr Asp 100 105
110Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Asp Asp
115 120 125Ala Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn
Leu Asn 130 135 140Asp Ala Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg
Thr Gly Met Asp145 150 155 160Pro Arg Met Cys Ser Leu Met Gln Gly
Ser Thr Leu Pro Arg Arg Ser 165 170 175Gly Ala Ala Gly Ala Ala Val
Lys Gly Val Gly Thr Met Val Met Glu 180 185
190Leu Val Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg
195 200 205Gly Glu Asn Gly Arg Lys Thr Arg Ile Ala Tyr Glu Arg Met
Cys Asn 210 215 220Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Lys
Ala Met Met Asp225 230 235 240Gln Val Arg Glu Ser Arg Asp Pro Gly
Asn Ala Glu Phe Glu Asp Leu 245 250 255Thr Phe Leu Ala Arg Ser Ala
Leu Ile Leu Arg Gly Ser Val Ala His 260 265 270Lys Ser Cys Leu Pro
Ala Cys Val Tyr Gly Pro Ala Val Ala Ser Gly 275 280 285Tyr Asp Phe
Glu Arg Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe 290 295 300Arg
Leu Leu Gln Asn Ser Gln Val Tyr Ser Leu Ile Arg Pro Asn Glu305 310
315 320Asn Pro Ala His Lys Ser 32530252PRTUnknownInfluenza 30Met
Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro1 5 10
15Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe
20 25 30Ala Gly Lys Asn Thr Asp Leu Glu Val Leu Met Glu Trp Leu Lys
Thr 35 40 45Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe
Val Phe 50 55 60Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg
Arg Phe Val65 70 75 80Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn
Asn Met Asp Lys Ala 85 90 95Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu
Ile Thr Phe His Gly Ala 100 105 110Lys Glu Ile Ser Leu Ser Tyr Ser
Ala Gly Ala Leu Ala Ser Cys Met 115 120 125Gly Leu Ile Tyr Asn Arg
Met Gly Ala Val Thr Thr Glu Val Ala Phe 130 135 140Gly Leu Val Cys
Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg145 150 155 160Ser
His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu 165 170
175Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met
180 185 190Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala
Ser Gln 195 200 205Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly
Thr His Pro Ser 210 215 220Ser Ser Ala Gly Leu Lys Asn Asp Leu Leu
Glu Asn Leu Gln Ala Tyr225 230 235 240Gln Lys Arg Met Gly Val Gln
Met Gln Arg Phe Lys 245 250312299DNAUnknownInfluenza 31aatatggaaa
gaataaaaga gctaaggaat ctgatgtcac aatctcgcac tcgcgagata 60cttacaaaaa
ctactgtaga ccacatggcc ataatcaaga aatacacatc aggaagacag
120gagaaaaacc catcacttag aatgaaatgg atgatggcaa tgaaataccc
aattacagca 180gataaaagga taacggaaat gattcctgaa agaaatgagc
aaggacagac attatggagt 240aaagtgaatg atgccggatc agaccgagtg
atgatatcac ccctggctgt gacatggtgg 300aacagaaatg gaccagtggc
aagtactatt cactatccaa aaatctacaa aacttacttt 360gaaaaggttg
aaaggttaaa acatggaacc tttggccctg tacactttag aaaccaagtc
420aaaatacgcc gaagagtcga cataaatcct ggtcatgcag acctcagcgc
caaggaggca 480caggatgtaa ttatggaagt tgttttccct aatgaagtgg
gagccagaat actaacatca 540gaatcgcaat taacgataac caaggagaaa
aaagaagaac tccagaattg caaaatttcc 600cctttgatgg ttgcatacat
gttagagagg gaacttgtcc gcaaaacgag atttctcccg 660gttgctggtg
gaacaagcag tgtgtacatt gaagttttgc atttaacaca ggggacatgc
720tgggagcaga tgtacactcc aggtggggag gtgaggaatg atgatgttga
tcaaagccta 780attattgctg ctaggaacat agtgagaaga gctgcagtat
cagcagatcc actagcatct 840ttattagaaa tgtgccatag cacacagatt
ggtgggacaa ggatggtgga tattctcagg 900caaaatccaa cagaagaaca
agctgtggat atatgcaaag cagcaatggg gctgagaatc 960agttcatcct
tcagttttgg cggattcaca tttaagagaa caagtggatc atcagtcaaa
1020agggaggaag aagtgctcac gggcaatctg caaacattga agctaactgt
gcatgaggga 1080tatgaagagt tcacaatggt tgggaaaagg gcaacagcta
tactcagaaa agcaaccagg 1140agattgattc aactaatagt gagtggaaga
gacgaacagt caatagtcga agcaatagtt 1200gtagcaatgg tattctcaca
agaagattgc atggtaaaag cagttagagg tgatctgaat 1260ttcgttaata
gagcgaatca gcggttgaat cccatgcatc aacttttgag acattttcag
1320aaggatgcta aagtactttt cttaaattgg ggaattgaac ctatcgacaa
tgtgatggga 1380atgattggga tattacctga tatgactcca agtaccgaga
tgtcaatgag aggagtgaga 1440gtcagcaaaa tgggtgtaga tgaatactcc
aatgctgaaa gggtagtggt gagcattgac 1500cgttttttga gagtccggga
ccaaagagga aatgtactac tgtctccaga ggaagtcagt 1560gaaacacagg
gaacagagaa actgacaata acttactctt catcaatgat gtgggagatt
1620aatggccctg agtcagtgtt gatcaatacc tatcagtgga tcatcagaaa
ctgggagact 1680gttaaaattc agtggtctca gaaccctaca atgctataca
ataaaatgga attcgagcca 1740tttcagtctc tagtccctaa ggccattaga
ggccaataca gtgggtttgt tagaactcta 1800tttcaacaaa tgagggatgt
gcttgggacc tttgacacaa ctcagataat aaaacttctt 1860ccctttgcag
ccgctccacc aaagcaaagt agaatgcaat tctcatcatt gactgtgaat
1920gtgaggggat caggaatgag aatacttgta aggggtaatt ctccagtatt
caactacaac 1980aagaccacta agagactcac agtcctcgga aaggatgctg
gcactttaac tgaagaccca 2040gatgaaggca cagctggagt ggaatctgct
gttctaaggg gattcctcat tctaggcaaa 2100gaagatagaa gatatgggcc
agcattaagc atcaatgaat tgagcaacct tgcgaaaggg 2160gaaaaagcta
atgtgctaat tgggcaaggg gacgtagtgt tggtaatgaa acgaaaacgg
2220gactctagca tacttactga cagccagaca gcgaccaaaa gaattcggat
ggccatcaat 2280taatttcgaa taatttaaa 2299322277DNAUnknownInfluenza
32atggaacgca ttaaagaact gcgcaacctg atgagccaga gccgcacccg cgaaattctg
60accaaaacca ccgtggatca tatggcgatt attaaaaaat ataccagcgg ccgccaggaa
120aaaaacccga gcctgcgcat gaaatggatg atggcgatga aatatccgat
taccgcggat 180aaacgcatta ccgaaatgat tccggaacgc aacgaacagg
gccagaccct gtggagcaaa 240gtgaacgatg cgggcagcga tcgcgtgatg
attagcccgc tggcggtgac ctggtggaac 300cgcaacggcc cggtggcgag
caccattcat tatccgaaaa tttataaaac ctattttgaa 360aaagtggaac
gcctgaaaca tggcaccttt ggcccggtgc attttcgcaa ccaggtgaaa
420attcgccgcc gcgtggatat taacccgggc catgcggatc tgagcgcgaa
agaagcgcag 480gatgtgatta tggaagtggt gtttccgaac gaagtgggcg
cgcgcattct gaccagcgaa 540agccagctga ccattaccaa agaaaaaaaa
gaagaactgc agaactgcaa aattagcccg 600ctgatggtgg cgtatatgct
ggaacgcgaa ctggtgcgca aaacccgctt tctgccggtg 660gcgggcggca
ccagcagcgt gtatattgaa gtgctgcatc tgacccaggg cacctgctgg
720gaacagatgt ataccccggg cggcgaagtg cgcaacgatg atgtggatca
gagcctgatt 780attgcggcgc gcaacattgt gcgccgcgcg gcggtgagcg
cggatccgct ggcgagcctg 840ctggaaatgt gccatagcac ccagattggc
ggcacccgca tggtggatat tctgcgccag 900aacccgaccg aagaacaggc
ggtggatatt tgcaaagcgg cgatgggcct gcgcattagc 960agcagcttta
gctttggcgg ctttaccttt aaacgcacca gcggcagcag cgtgaaacgc
1020gaagaagaag tgctgaccgg caacctgcag accctgaaac tgaccgtgca
tgaaggctat 1080gaagaattta ccatggtggg caaacgcgcg accgcgattc
tgcgcaaagc gacccgccgc 1140ctgattcagc tgattgtgag cggccgcgat
gaacagagca ttgtggaagc gattgtggtg 1200gcgatggtgt ttagccagga
agattgcatg gtgaaagcgg tgcgcggcga tctgaacttt 1260gtgaaccgcg
cgaaccagcg cctgaacccg atgcatcagc tgctgcgcca ttttcagaaa
1320gatgcgaaag tgctgtttct gaactggggc attgaaccga ttgataacgt
gatgggcatg 1380attggcattc tgccggatat gaccccgagc accgaaatga
gcatgcgcgg cgtgcgcgtg 1440agcaaaatgg gcgtggatga atatagcaac
gcggaacgcg tggtggtgag cattgatcgc 1500tttctgcgcg tgcgcgatca
gcgcggcaac gtgctgctga gcccggaaga agtgagcgaa 1560acccagggca
ccgaaaaact gaccattacc tatagcagca gcatgatgtg ggaaattaac
1620ggcccggaaa gcgtgctgat taacacctat cagtggatta ttcgcaactg
ggaaaccgtg 1680aaaattcagt ggagccagaa cccgaccatg ctgtataaca
aaatggaatt tgaaccgttt 1740cagagcctgg tgccgaaagc gattcgcggc
cagtatagcg gctttgtgcg caccctgttt 1800cagcagatgc gcgatgtgct
gggcaccttt gataccaccc agattattaa actgctgccg 1860tttgcggcgg
cgccgccgaa acagagccgc atgcagttta gcagcctgac cgtgaacgtg
1920cgcggcagcg gcatgcgcat tctggtgcgc ggcaacagcc cggtgtttaa
ctataacaaa 1980accaccaaac gcctgaccgt gctgggcaaa gatgcgggca
ccctgaccga agatccggat 2040gaaggcaccg cgggcgtgga aagcgcggtg
ctgcgcggct ttctgattct gggcaaagaa 2100gatcgccgct atggcccggc
gctgagcatt aacgaactga gcaacctggc gaaaggcgaa 2160aaagcgaacg
tgctgattgg ccagggcgat gtggtgctgg tgatgaaacg caaacgcgat
2220agcagcattc tgaccgatag ccagaccgcg accaaacgca ttcgcatggc gattaac
2277
* * * * *
References